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WO2024220874A2 - Otub1 small-molecule binders and otub1-recruiting deubiquitinase-targeting chimeras (dubtacs) - Google Patents

Otub1 small-molecule binders and otub1-recruiting deubiquitinase-targeting chimeras (dubtacs) Download PDF

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Publication number
WO2024220874A2
WO2024220874A2 PCT/US2024/025509 US2024025509W WO2024220874A2 WO 2024220874 A2 WO2024220874 A2 WO 2024220874A2 US 2024025509 W US2024025509 W US 2024025509W WO 2024220874 A2 WO2024220874 A2 WO 2024220874A2
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optionally substituted
cycloalkyl
membered
alkyl
ring
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PCT/US2024/025509
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French (fr)
Inventor
Jian Jin
Wenyi WEI
Yan Xiong
Xiangyang Song
Qiong Wu
Chao Qian
Zhijie Deng
Jing Liu
Xufen YU
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Icahn School Of Medicine At Mount Sinai
Beth Israel Deaconess Medical Center, Inc.
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Publication of WO2024220874A2 publication Critical patent/WO2024220874A2/en

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  • the present disclosure is directed to compounds that are irreversible small-molecule binders of OTUB1 (also known as Otubain 1), and bivalent compounds (e.g. bi-functional small molecule compounds) which recruit OTUB1 through the irreversible small-molecule binders of OTUB1 to de-ubiquitinylate and stabilize proteins such as AMPK, cGAS and CFTR.
  • the present disclosure is also related to methods for the treatment of AMPK, cGAS and CFTR-mediated diseases in a subject in need thereof.
  • the disclosure also relates to methods for designing such compounds including irreversible small-molecule binders of OTUB1 and related bivalent compounds as deubiquitinase-targeting chimeras (DUBTACs) for targeted protein stabilization (TPS).
  • DUBTACs deubiquitinase-targeting chimeras
  • Protein ubiquitination plays a critical role in a variety of cellular process, like protein degradation, quality control, trafficking, and signaling. Therefore, controlling the ubiquitination level of disease related proteins can achieve substantial therapeutic effects. For example, inducing the ubiquitination of oncoproteins such as Myc and Ras to lead to their degradation would greatly benefit related cancer therapy. Similarly, deubiquitination and stabilization of proteins such as tumor suppressors would also be greatly beneficial for the treatment of relevant diseases including cancer.
  • AMPK 5' adenosine monophosphate-activated protein kinase
  • AMPK 5' adenosine monophosphate-activated protein kinase
  • the protein stabilization strategy could also be applied to other targets, such as cyclic GMP-AMP (cGAMP) synthase (also known as cGAS).
  • cGAMP cyclic GMP-AMP
  • STING cyclic GMP-AMP
  • the cGAS-stimulator of interferon genes (STING) pathway is a component of the innate immune system. cGAS detects the presence of cytosolic DNA and, in response, triggers expression of inflammatory genes that can lead to senescence or to the activation of defense mechanisms. In tumor cells, the cGAS-STING pathway exerts its antitumor effects through the induction of a robust type I IFN response, which activates immune cells, particularly dendritic cells (DCs), in the tumor micro environment (TME) (Samson and Ablasser, 2022).
  • DCs dendritic cells
  • Cystic fibrosis transmembrane conductance regulator (CFTR) is another suitable protein target for stabilization.
  • CFTR AF508 mutation destabilizes CFTR, and leads to the cystic fibrosis phenotype (Ward et al., 1995). Therefore, deubiquitination of AF508 mutant CFTR could be an effective strategy to prevent the cystic fibrosis process.
  • TPD targeted protein degradation
  • PROTAC PROteolysis TArgeting Chimera
  • DUBTAC Deubiquitinase-targeting chimera
  • DUBTAC which hijacks a cellular deubiquitinase to remove the polyubiquitin attached to the target protein by inducing close proximity between the target protein and deubiquitinase, recently emerged as an effective technology to improve target protein stability by reducing the proteasomal degradation of the target protein
  • DUBTACs provide a novel therapeutic approach for treating various diseases.
  • the present disclosure includes two parts: (A) small molecules that covalently bind the deubiquitinase OTUB1, and (B) heterobifunctional small molecules that stabilize AMPK, cGAS, or CFTR by recruiting OTUB 1.
  • OTUB1 (also known as OUT domain-containing ubiquitin aldehyde-binding protein 1) is a K48 linkage-specific deubiquitinating enzyme.
  • EN523 Henning et al., 2022
  • ML364 WO2020223551A1
  • EN523 has low binding affinity to OTUB1
  • compound 61 and ML364 inhibit the enzymatic activity of OTUB1. Therefore, development of more effective OTUB1 small-molecule binders that lead to effective DUBTACs is needed.
  • Described herein are small molecules that covalently modify OTUB1.
  • the present disclosure features small molecules that covalently modify OTUB1 of Formula (A-I) below:
  • a D is selected from N, or CR D 2 ;
  • Ring C D is absent, or selected from C 3 -C 12 cycloalkyl, 3-12-membered heterocyclic, C 6 - C 10 aryl, and 5-10 membered heteroaryl;
  • Ring D D is a saturated or partially unsaturated 4-12 membered heterocyclic
  • FD is selected from N, or CRD 2 ;
  • LD 1 is a bond, or a bivalent group selected from -O-, -NRD 5 -, -C(O)-, -C(O)O-, -C(O)NRD 5 -, -NRD 5 C(O)-, -OC(O)NRD 5 -, -NRD 5 C(O)O-, -NR D 6 C(O)NR D 5 -, -S(O)-, -S(O)2-, -S(O)NRD 5 -, - S(O)2NRD 5 -, CI-C 6 alkylene, C 1 -C 6 haloalkylene, C 1 -C 6 heteroalkylene, C 2 -C 6 alkenylene, and C 2 -C 6 alkynylene;
  • LD 2 is selected from -C(O)-, -S(O)-, -S(O)2-, -NRD 5 C(O)-, and C 1 -C 6 alkylene;
  • LD 3 is a bond, or a bivalent group selected from C 1 -C 6 alkylene, C 1 -C 6 haloalkylene, C 1 -C 6 heteroalkylene, C 2 -C 6 alkenylene, and C 2 -C 6 alkynylene;
  • RD 1 is selected from C 1 -C 6 haloalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkenyl, C2- C 6 , heteroalkynyl, partially unsaturated C4-C cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD 7 , NRD 7 RD 8 , C(O)NRD 7 RD 8 , C(O)ORD 7 , tri(Ci-C3alkyl) silyl, C 1 -C 6 alkyl, C 1 -
  • RD 4 is R D 4a or R D 4b ;
  • R D 4a is selected from hydrogen, ORD 7 , NRD 7 RD 8 , C(O)RD 7 , C(O)ORD 7 , C(O)NRD 7 RD 8 , - OC(O)NRD 7 RD 8 , -NRD 9 C(O)NRD 7 RD 8 -, S(O)RD 7 , S(O) 2 RD 7 , S(O)NRD 7 RD 8 , S(O) 2 NR D 7 RD 8 , C 1 -C 6 alkyl, Ci-Ce haloalkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12- membered heterocyclic, Ce-Cw aryl, and 5-10 membered heteroaryl;
  • Ro 4b is a bivalent group that connect to the Linker moiety in Formula (B), and Ro 4b is selected from a bond, -O-, -N-, -C(O)-, -C(O)O-, -C(O)NR D 7 -, -OC(O)NR D 7 -, -NR D 9 C(O)NR D 7 -, -S(O)-, -S(O) 2 -, -S(O)NRD 7 -, -S(O) 2 NRD 7 -, CI-C 6 alkylene, Ci-C 6 haloalkylene, Ci-C 6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, C3-C12 cycloalkylene, 3-12-membered heterocyclicene, Ce-Cio arylene, and 5-10 membered heteroarylene; each RD 5 and each RD 6 are independently selected from hydrogen, Ci-Ce alky
  • RD 7 and RD 8 together with the atom(s) to which they are connected, optionally form 3-12- membered heterocyclic ring; m D is an integer of 0-8; and n D is an integer of 0-8.
  • LD 1 is a bond, or a bivalent group selected from -C(O)-, -NRD 5 C(O)-, and -C(O)1STRD :> - In some embodiments, LD 1 is a bond. In some embodiments, LD 1 is -C(O)- In some embodiments, LD 1 is -C(O)NRD 5 -. In some embodiments, LD 1 is -NRD 5 C(O)-. In some embodiments, LD 3 is a bond, or a bivalent group selected from -CH2-, or -CH2CH2-. In some embodiments, LD 3 is a bond. In some embodiments, LD 3 is a bond -CH2-.
  • the small molecules that covalently modify OTUB 1 are selected from Formulae (A-I-a), (A-I-b), (A-I-c), or (A-I-d):
  • AD is N. In some embodiments, AD is CRD 2 . In some embodiments, AD in Formula (A-I-a) is N. In some embodiments, AD in Formula (A-I-b) is CRD 2 . In some embodiments, AD in Formula (A-I-c) is N. In some embodiments, AD in Formula (A-I-d) is CRD 2 .
  • the small molecules that covalently modify OTUB1 are selected from Formulae (A-I-al), (A-I-bl), (A-I-cl), or (A-I-dl):
  • the small molecules that covalently modify OTUB1 are selected from Formulae (A-I-al), (A-I-bl), (A-I-cl), and (A-I-dl). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I-al). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I-bl). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I-cl). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I- dl).
  • Ring DD in Formulae (A-I-a), (A-I-al), (A-I-bl), (A-I-cl), and (A- I-dl) is selected from 6-8 membered monocyclic heterocyclic, 7-12 membered spiro heterocyclic, 7-12 membered bridged heterocyclic, and 6-12 membered fused heterocyclic.
  • the small molecules that covalently modify OTUB1 are selected from Formulae (A-I-a2), (A-I-a3), (A-I-a4), (A-I-a5), (A-I-b2), (A-I-b3), (A-I-b4), (A-I-b5), (A-I- c2), (A-I-c3), (A-I-c4), (A-I-c5), (A-I-d2), (A-I-d3), (A-I-b4), and (A-I-d5):
  • OD 1 is selected from an integer of 1-4;
  • OD 2 , OD 3 , OD 4 , and OD 5 are independent selected from an integer of 0-4.
  • the small molecules that covalently modify OTUB1 comprise Formulae (A-I-a3), (A-I-b2), and (A-I-c4). In some embodiments, the small molecules that covalently modify OTUB 1 comprise Formula (A-I-a3). In some embodiments, the small molecules that covalently modify 0TUB1 comprise Formula (A-I-b2). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I-c4).
  • each ED 1 , each ED 2 , each ED 3 , each ED 4 , and each ED 5 are independently selected from null, CO, and CRD 2 RD 2 . In some embodiments, each ED 1 , each ED 2 , each ED 3 , each ED 4 , and each ED 5 are independently selected from CO and CRD 2 RD 2 .
  • ED 1 , ED 2 , ED 3 , ED 4 , and ED 3 in Formulae (A-I-a2), (A-I-a3), (A-I- a4), (A-I-a5), (A-I-b2), (A-I-b3), (A-I-b4), (A-I-b5), (A-I-c2), (A-I-c3), (A-I-c4), (A-I-c5), (A-I- d2), (A-I-d3), (A-I-b4), and (A-I-d5) are CRD 2 RD 2 .
  • the small molecules that covalently modify OTUB1 are selected from Formulae (A-I-a6), (A-I-a7), (A-I-a8), (A-I-a9), (A-I-b6), (A-I-b7), (A-I-b8), (A-I-b9), (A-I- c6), (A-I-c7), (A-I-c8), (A-I-c9), (A-I-d6), (A-I-d7), (A-I-d8), and (A-I-d9):
  • RD 2 is a substituent that can attach to anywhere on the monocyclic and bicyclic rings.
  • FD is selected from N, CH, or CMe. In some embodiments, FD is selected from N. In some embodiments, FD is selected from CH. In some embodiments, FD is selected from CMe.
  • LD 3 is selected from a bond or -CH 2 -. In some embodiments, LD 3 is selected from a bond. In some embodiments, LD 3 is selected from -CH 2 -.
  • the small molecules that covalently modify OTUB1 comprise Formulae (A-I-a6), (A-I-a7), (A-I-b6), (A-I-c8), and (A-I-d6). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I-a6). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I-a7).
  • the small molecules that covalently modify OTUB1 comprise Formula (A-I-b6). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I- c8). In some embodiments, the small molecules that covalently modify OTUB 1 comprise Formula (A-I-d6).
  • Ring D D in Formulae (A-I), (A-I-a), (A-I-al), (A-I-b), (A-I-bl), (A- I-c), (A-I-cl), (A-I-d), and (A-I-dl) is selected from Ring D D al , Ring D D D a2 , Ring D D b1 , Ring D D b2 , Ring D DC 1 , and Ring D D C2 .
  • Ring D D in Formulae (A-I-a), (A-I-al), (A-I-c), and (A-I-cl) is Ring D D al comprising RD 2 substituted groups of:
  • Ring D D in Formulae (A-I-a), (A-I-al), (A-I-c), and (A-I-cl) is Ring D D a2 comprising RD 2 substituted groups of:
  • Ring D D in Formulae (A-I-a) and (A-I-al) is selected from
  • the small molecules that covalently modify OTUB1 comprise
  • the small molecules that covalently modify OTUB1 comprise Formula (A-I-al 1):
  • Ring D D in Formulae (A-I-c) and (A-I-cl) is
  • the small molecules that covalently modify OTUB1 comprise Formula (A-I-clO):
  • Ring D D in Formulae (A-I-b), (A-I-bl), (A-I-d), and (A-I-dl) is Ring D D b1 comprising RD 2 substituted groups of:
  • Ring D D in Formulae (A-I-b), (A-I-bl), (A-I-d), and (A-I-dl), is Ring D D b2 comprising R D 2 substituted groups of:
  • Ring D D in Formulae (A-I-b), (A-I-bl), (A-I-d), and (A-I-dl) is
  • the small molecules that covalently modify OTUB1 comprise
  • the small molecules that covalently modify OTUB1 comprise
  • Ring D D in Formulae (A-I-b), (A-I-bl), (A-I-d), and (A-I-dl) is Ring D D b1 comprising RD 2 substituted groups of:
  • Ring D D in Formulae (A-I-b), (A-I-bl), (A-I-d), and (A-I-dl), is
  • Ring D D b2 comprising RD 2 substituted groups of:
  • Ring D D in Formulae (A-I-b), (A-I-bl), (A-I-d), and (A-I-dl) is
  • the small molecules that covalently modify OTUB1 comprise Formula (A-I-b 11):
  • the small molecules that covalently modify OTUB1 comprise
  • ED 1 in Formulae (A-I-c2), (A-I-c3), (A-I-c4), and (A-I-c5) that is adjacent to N atom is -C(O)-; and the rest of the E D 1 , E D 2 , E D 3 , E D 4 , and E D 5 are CR D 2 R D 2 .
  • the small molecules that covalently modify OTUB1 comprise Formulae (A-I-cl l), (A-I-cl2), (A-I- cl 3), and (A-I-C14): wherein
  • OD 1 -1 is selected from an integer of 0-3.
  • the small molecules that covalently modify OTUB1 comprise Formulae (A-I-cl l) and (A-I-cl2).
  • the small molecules that covalently modify OTUB1 comprise Formulae (A-I-cl 1).
  • the small molecules that covalently modify OTUB1 comprise Formulae (A-I-cl2).
  • Ring D D in Formulae (A-I-c) and (A-I-cl) is Ring D D C1 comprising R D 2 substituted groups of:
  • Ring D D in Formulae (A-I-c) and (A-I-cl) is Ring D D C2 comprising R D 2 substituted groups of:
  • Ring D D in Formulae (A-I-c) and (A-I-cl) is
  • the small molecules that covalently modify OTUB1 comprise
  • Ring CD is selected from C 3 -C 12 cycloalkyl, 3-12-membered heterocyclic, C 6 -C 10 aryl, and 5-10 membered heteroaryl.
  • Ring C D is C 3 - C 12 cycloalkyl.
  • Ring CD is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
  • Ring CD is cyclopentyl.
  • Ring CD is 3-12-membered heterocyclic.
  • Ring CD is selected from azetidinyl, pyrrolidinyl, piperidinyl, and piperazinyl.
  • Ring CD is C 6 -
  • Ring CD is phenyl. In some embodiments, Ring CD is 5-10 membered heteroaryl. In some embodiments, Ring CD is selected from thiophenyl, benzothiophenyl, tetrahydrobenzothiophenyl, thiazolyl, imidazolyl, furanyl, pyrrolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, benzofuranyl, indolyl, and indazolyl.
  • Ring CD is selected from thiophenyl, benzothiophenyl, tetrahydrobenzothiophenyl, benzofuranyl, and indolyl. In some embodiments, Ring CD is thiophenyl. In some embodiments, Ring CD is In some embodiments, Ring CD is benzothiophenyl. In some embodiments, Ring some embodiments, Ring C D is tetrahydrobenzothiophenyl. In some embodiments, Ring
  • LD 2 is selected from -C(O)-, C 1 -C 6 alkylene, and -NRD 5 C(O)-. In some embodiments, LD 2 is selected from -C(O)-, -NHC(O)-, and methylene. In some embodiments, LD 2 is -C(O)-. In some embodiments, -NHC(O)-. LD 2 is In some embodiments, LD 2 is methylene.
  • LD 2 is -C(O)-; and RD 1 is selected from C 1 -C 6 haloalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroal kynyl, partially unsaturated C 4 -C 8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroal kynyl, partially unsaturated C 4 -C 8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD 7 , NR D 7 R D 8 , C(O)NR D 7 R D 8 , C(O)OR D 7 , tri(Ci-
  • LD 2 is C 1 -C 6 alkylene; and RD 1 is selected from C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, partially unsaturated C 4 -C 8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, partially unsaturated C 4 -C 8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD 7 , NR D 7 R D 8 , C(O)NR D 7 R D 8 , C(O)OR D 7 , tri(C 1 - C 3 alkyl) silyl, C 1
  • LD 2 is methylene; and RD 1 is selected from C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, partially unsaturated C 4 -C 8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, partially unsaturated C 4 -C 8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD 7 , NR D 7 R D 8 , C(O)NR D 7 R D 8 , C(O)OR D 7 , tri(C 1 -C 3 alkyl) silyl, C 1 -C 6 h
  • LD 2 is -NHC(O)-; and RD 1 is selected from C 1 -C 6 haloalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, partially unsaturated C 4 -C 8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, partially unsaturated C 4 -C 8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD 7 , NR D 7 R D 8 , C(O)NR D 7 R D 8 , C(O)OR D 7 , tri(Ci-C
  • LD 2 is C 1 -C 6 alkylene; and RD 1 is selected from C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, partially unsaturated C 4 -C 8 cycloalkyl, and partially unsaturated 4-8 memberedheterocyclic, where each said C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, partially unsaturated C 4 -C 8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD 7 , NR D 7 R D 8 , C(O) NR D 7 R D 8 , C(O)ORD 7 , tri(Ci- C 3 alkyl) silyl, C
  • LD 2 is methylene; and R D 1 is selected from C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, partially unsaturated C 4 -C 8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, partially unsaturated C 4 -C 8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD 7 , NR D 7 R D 8 , C(O)NR D 7 R D 8 , C(O)OR D 7 , tri(Ci-C 3 alkyl) silyl, aClk 1 -y
  • D 1 is C 1 -C 6 haloalkyl. In some embodiments, R D 1 is halomethyl. In some embodiment Rs, D 1 is CH 2 C1 or CH 2 F. In some embodiments, R D 1 is selected from C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, partially unsaturated C 4 -C 8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, partially unsaturated C 4 -C 8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD 7 , NR D 7 R D 8 , C(
  • D 1 is selected from C 2 -C 3 alkenyl, C 2 -C 3 alkynyl, partially unsaturated C 4 -C 6 cycloalkyl, and partially unsaturated 4-6 membered heterocyclic, where each said C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, partially unsaturated C 4 -C 6 cycloalkyl, and partially unsaturated 4-6 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, NR D 7 R D 8 , C(O)NR D 7 R D 8 , C(O)ORD 7 , tri(C 1 -C 3 alkyl) silyl, C 1 -C 6 alkyl, 1 1 -C 6 heteroalkyl, and 3-8 membered heterocyclic.
  • D 1 is selected from vinyl, propylenyl, ethynyl, propargyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, dihydrofuranyl, dihydropyrrolyl, dihydropyranyl, and tertrahydopyridinyl, where each said vinyl, propylenyl, ethynyl, propargyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, dihydrofuranyl, dihydropyrrolyl, dihydropyranyl, and tertrahydopyridinyl were optionally substituted with hydrogen, halogen, cyano, NR D 7 R D 8 , C(O)NR D 7 R D 8 , C(O)ORD 7 , trimethyl silyl, C 1 -C 3 alkyl, C 1 -C 3 heteroalkyl, and 3-6 membered hetero
  • D 1 is selected from vinyl and propylenyl, where each said vinyl and propylenyl were optionally substituted with hydrogen, halogen, cyano, NR D 7 R D 8 , C(O)NR D 7 R D 8 , C(O)OR D 7 , trimethyl silyl, C 1 -C 3 alkyl, C 1 -C 3 heteroalkyl, and 3-6 membered heterocyclic.
  • R D 1 is selected from vinyl and propylenyl, where each said vinyl and propylenyl were optionally substituted with hydrogen, halogen, cyano, NR D 7 R D 8 , C(O)NR D 7 R D 8 , C(O)OR D 7 , trimethyl silyl, C 1 -C 3 alkyl, C 1 -C 3 heteroalkyl, and 3-6 membered heterocyclic.
  • R D 1 is selected from
  • - LD 2 -R D 1 is selected from
  • - LD 2 -R D 1 is selected from
  • each R D 2 is independently selected from hydrogen, halogen, cyano, OR D 7 , NR D 7 R D 8 , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 heteroalkenyl, C 1 -C 6 alkynyl, C 2 -C 6 heteroalkynyl, C 3 -C 8 cycloalkyl, and 3-8 membered heterocyclic.
  • each R D 2 is selected from hydrogen, halogen, hydroxy, C 1 -C 6 alkoxy, amino, C 1 -C 6 alkyl amino, (C 1 -C 6 alkyl)( C 1 -C 6 alkyl) amino, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C3-C8 cycloalkyl, 3-8 membered heterocyclic.
  • each R D 2 is selected from hydrogen, halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 3 -C 8 cycloalkyl, 3-8 membered heterocyclic.
  • R D 2 is hydrogen.
  • R D 2 is halogen.
  • R D 2 is C 1 -C 6 alkyl.
  • two R D 2 groups, together with the atom(s) to which they are connected optionally form a C 3 -C 12 cycloalkyl ring or 3-12-membered heterocyclic ring, where each said C 3 -C 12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring D D , can optionally form fused rings or bridged rings.
  • two R D 2 groups, together with the atom(s) to which they are connected and Ring D D optionally form a bridged 3-12-membered heterocyclic ring.
  • two R D 2 groups, together with the atom(s) to which they are connected and Ring D D optionally form a bridged 7-membered heterocyclic ring. In some embodiments, two R D 2 groups, together with the atom(s) to which they are connected and Ring D D , optionally form a bridged 8-membered heterocyclic ring.
  • each R D 3 is independently selected from hydrogen, halogen, cyano, OR D 7 , NR D 7 R D 8 , C(O)OR D 7 , C(O)NR D 7 R D 8 , -OC(O)NR D 7 R D 8 , - NR D 9 C(O)NR D 7 R D 8 -, - S(O)NR D 7 R D 8 -, -S(O) 2 NR D 7 R D 8 -, CI-C 6 alkyl, C h 1 a-Clo 6 alkyl, C he 1 -tCer 6 oalkyl, C 2 -C 6 alkenyl, C 1 -C 6 alkynyl, C 3 -C 12 cycloalkyl, 3-12-membered heterocyclic, C 6 -C 10 aryl, and 5-10 membered heteroaryl.
  • each R D 3 is selected from hydrogen, halogen, cyano, OR D 7 , NR D 7 R D 8 , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, 3-12-membered heterocyclic, C 6 -C 10 aryl, and 5-10 membered heteroaryl.
  • each R D 3 is selected from methyl, ethyl, isopropyl, piperidinyl, piperazinyl, and phenyl.
  • two R D 3 groups together with the atom(s) to which they are connected, optionally form a C 3 -C 12 cycloalkyl ring, or 3-12-membered heterocyclic ring, where each said C 3 -C 12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring CD, optionally form fused rings or bridged rings.
  • R D 4 is R D 4a
  • R D 4a is selected from hydrogen, OR D 7 , NR D 7 R D 8 , C(O)NR D 7 R D 8 , S(O) 2 NR D 7 R D 8 , CI-C 6 alkylene, C 1 - hCa 6 loalkylene, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 12 cycloalkyl, 3-12-membered heterocyclic, Ce-Cio aryl, and 5-10 membered heteroaryl.
  • R D 4 is Ro 4b .
  • R D 4 is a bivalent group that connect to the Linker moiety in Formula (B), and Ro 4b is selected from a bond, -O-, -N-, -C(O)-, -C(O)O-, -C(O)NR D 7 -, -S(O) 2 -, -S(O) 2 NR D 7 -, C 1 -C al 6 kylene, Ci-C 6 haloalkylene, C 1 -C 6 heteroalkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, C 3 -C 12 cycloalkylene, 3- 12-membered heterocyclicene, C 6 -C 10 arylene, and 5-10 membered heteroarylene.
  • R D 41 is a bond.
  • each R D 5 and each R D 6 are independently selected from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic.
  • each R D 7 , each R D 8 and each R D 9 are independently selected from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic.
  • R D 7 and R D 8 together with the atom(s) to which they are connected, optionally form 4-6-membered heterocyclic ring.
  • m D is an integer of 0-4. In some embodiments, m D is an integer of 1-3. In some embodiments, OD 1 is selected from an integer of 1-3. In some embodiments, O D 2 is selected from an integer of 0-3. In some embodiments, OD 3 is selected from an integer of 0-3. In some embodiments, OD 4 is selected from an integer of 0-3. In some embodiments, OD 5 is selected from an integer of 0-3.
  • the small molecules that covalently modify OTUB1 are selected from the structures of Formulae (A-I), (A-I-a), (A-I-al), (A-I-a2), (A-I-a3), (A-I-a4), (A-I-a5), (A- I-a6), (A-I-a7), (A-I-a8), (A-I-a9), (A-I-alO), (A-I-al 1), (A-I-b), (A-I-bl), (A-I-b2), (A-I-b3), (A- I-b4), (A-I-b5), (A-I-b6), (A-I-b7), (A-I-b8), (A-I-b9), (A-I-blO), (A-I-bl l), (A-I-c), (A-I-cl), (A- Lc2), (A-I-c3), (A-I-I-
  • the small molecules that covalently modify OTUB1 are selected from XS154-91, XS154-130, XS154-148, XS154-114, XS154-149, XS159-13, XS159-107, XS165-30, XS159-90, XS165-53, XS165-38, XS165-33, XS165-54, XS154-184, XS165-75, XS165-77, XS165-127, XS165-106, XS165-112, XS165-97, XS165-118, XS165-110, XS165-119, XS165-123, XS165-126, XS165-120, XS165-121, XS165-100, XS165-99, XS165-113, XS165- 109, XS165-117, XS165-105, XS165-154
  • the small molecules that covalently modify OTUB1 are N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the ubiquitination and degradation of many proteins is the cause of several classes of diseases. Therefore, de-ubiquitination and stabilization of these kinds of proteins may provide a novel therapeutic strategy.
  • this disclosure provides a method of treating AMPK, cGAS, or AF508-CFTR mediated diseases, the method including administering one or more AMPK, cGAS, or CFTR DUBTACs to a subject who has an AMPK, cGAS, or AF508-CFTR -mediated disease, the AMPK, cGAS, or CFTR DUBTACs being bivalent compounds including an AMPK, cGAS, or CFTR ligand conjugated to a de-ubiquitination tag via a linker, which would stabilize AMPK, cGAS, or CFTR.
  • the AMPK, cGAS, or AF508-CFTR -mediated disease can be a disease resulting from AMPK, cGAS, or CFTR destabilization.
  • the AMPK, cGAS, or AF508-CFTR-mediated disease can have reduced AMPK, cGAS, or CFTR expression relative to a wild-type tissue of the same species and tissue type.
  • the present disclosure provides a bivalent compound including an AMPK, cGAS, or CFTR ligand conjugated to OTUB1 recruiter via a linker.
  • the AMPK, cGAS, or CFTR DUBTACs have the form “PI-Linker- OTUB1 recruiter”, as shown below:
  • PI a ligand for a “protein of interest,” i.e., the protein to be subjected to de-ubiquitination
  • OTUB1 Recruiter comprises a de-ubiquitination tag (e g., a ligand for OTUB1).
  • PI a ligand for a “protein of interest,” i.e., the protein to be subjected to de-ubiquitination
  • OTUB1 Recruiter comprises a de-ubiquitination tag (e g., a ligand for OTUB1).
  • PI exemplary AMPK, cGAS, or CFFTR ligands
  • OTUB1 recruiters exemplary linkers (Linker) are illustrated below:
  • the AMPK ligand can be an AMPK activator or AMPK inhibitor which potently binds to AMPK.
  • the AMPK ligand comprises Metformin (Xiao et al., 2020), AICAR (Nakamaru et al., 2005), ZMP, AMP, A-769662 (Sanders et al., 2007), 991 (Ngoei et al., 2018), PF-06685249 (Edmonds et al., 2018), PXL770 (Cusi et al., 2021), MK- 8722 (Myers et al., 2017), PF-739 (Aledavood et al., 2021), 4-azaindole, RSVA405 (Vingtdeux et al., 2011), COH-SR4 (Figarola and Rahbar, 2013), B10 (Sun et al., 2020), Dorsomorphin (Xiao et
  • the AMPK ligand is a compound disclosed in one or more of WO2009124636, W02009100130, W02010036613, WO2011029855, WO2011080277, WO2011032320, WO2013116491, W02014133008, W02016008404, W02016001224, each of which is incorporated by reference in its entirety.
  • the AMPK ligand is an AMPK inhibitor. In some embodiments, the AMPK ligand is an ATP-competitive AMPK inhibitor. In some embodiments, the AMPK ligand is an AMPK activator. In some embodiments, the AMPK ligand is an AMPK activator, where the AMPK activator binds to an allosteric binding site. In some embodiments, the AMPK activator binds to an allosteric binding site located at the ⁇ / ⁇ subunit interface. In some embodiments, the
  • AMPK activator has different AMPK isoform selectivity.
  • the AMPK activator is a pan-AMPK isoform activator.
  • the AMPK ligand comprises the structure of Formula (B-I):
  • AA is selected from
  • B A is selected from denotes the point of attachment to Linker in Formula (B);
  • CA is selected from N or CR A 4 ;
  • Ring DA and Ring E A are independently selected from null, C 3 -C 12 cycloalkyl, 3-12- membered heterocyclic, C 6 -C 10 aryl, and 5-10 membered heteroaryl;
  • AA is N; and BA is . In some embodiments, AA and BA is N. In some embodiments,
  • the AMPK ligand comprises the structure of Formulae (B-I-a) and Formulae (B-I-b) :
  • XA is selected from -O-, -S-, -NR A 9 -, -C(O)-, C 1 -C 6 alkylene, C 1 - C 6 , haloalkylene, C 1 -C 6 heteroalkylene, C 6 -C 10 arylene, and 5-10 membered heteroarylene.
  • XA is selected from -O-, -S-, -NR A 9 -, -C(O)-, methylene, and halomethylene, In some embodiments, X A is selected from -O-, -S-, -NH-, -C(O)-, CH 2 , and CF 2 , In some embodiments, XA is -O-.
  • YA is selected from a bond, -C(O)-, -C(O)O-, -C(O)NR A 9 -, -S(O)-, -S(O) 2 -, -S(O)NR A 9 -, -S(O)2NR A 9 -.
  • YA is selected from a bond, -C(O)-, - C(O)NR A 9 -, and -S(O) 2 NR A 9 -.
  • YA is -C(O)NR A 9 -.
  • the AMPK ligand comprise the structure of Formulae (B-I-al), (B- I-bl), and (B-I-b2):
  • AA is N. In some embodiments, AA is CR A 4 . In some embodiments, BA is N. In some embodiments, BA is CR A 4 . In some embodiments, CA is N. In some embodiments, CA is CR A 4 . In some embodiments, Ring D A is selected from null, C 3 -C 12 cycloalkyl, 3-12-membered heterocyclic, C 6 -C 10 aryl, and 5-10 membered heteroaryl. In some embodiments, Ring D A is C 3 - C 12 cycloalkyl.
  • Ring D A is selected from cyclobutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
  • Ring DA is 3-12- membered heterocyclic.
  • Ring DA is selected from tetrahydrofuryl, hexahydrofuro[3,2-Z>]furyl, azetidinyl, pyrrolidinyl, piperidinyl, and piperazinyl.
  • Ring DA is C 6 -C 10 aryl.
  • Ring DA is phenyl.
  • Ring DA is 5-10 membered heteroaryl. In some embodiments, Ring DA is selected from thiophenyl, benzothiophenyl, tetrahydrobenzothiophenyl, thiazolyl, imidazolyl, furanyl, pyrrolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, benzofuranyl, indolyl, and indazolyl.
  • Ring EA selected from null, C 3 -C 12 cycloalkyl, 3-12-membered heterocyclic, C 6 -C 10 aryl, and 5-10 membered heteroaryl. In some embodiments, Ring EA selected from nullC, 6 -C 10 aryl, and 5-10 membered heteroaryl. In some embodiments, Ring EA selected from null. In some embodiments, Ring E A ISC 6 -C 10 aryl. In some embodiments, Ring EAis phenyl. . In some embodiments, Ring EA IS 5-10 membered heteroaryl.
  • Ring EA selected from thiophenyl, benzothiophenyl, tetrahydrobenzothiophenyl, thiazolyl, imidazolyl, furanyl, pyrrolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, benzofuranyl, indolyl, and indazolyl.
  • the AMPK ligand comprises the structure of Formulae (B-I-a2), (B- I-a3), (B-I-a4), (B-I-b3), and (B-I-b4):
  • FA is selected from N or CR A 3 ;
  • GA is selected from N or CR A 7
  • R A 3 is selected from C 3 -C 12 cycloalkyl, 3-12 membered heterocyclic, C 6 -C 10 aryl, and 5-10 membered heteroaryl. In some embodiments, R A 3 is selected from C 6 -C 10 aryl and 5-10 membered heteroaryl.
  • the AMPK ligand comprises the structure of Formulae (B-I-a5), (B- I-a6), (B-I-a7), (B-I-b5), and (B-I-b6):
  • OA is selected from 0, 1, 2, 3, 4, 5, or 6.
  • ArA is selected from C 6 -C 1 a 0 ryl and 5-10 membered heteroaryl.
  • ArA is selected from phenyl, naphthalene, indane, 5,6, 7,8- tetrahydronaphthalene, biphenyl, pyrazole, pyridine, pyrazine, pyrimidine, thiazole, thiophene, benzoimidazole, quinoline, isoquinoline, indole, indazole, carbazole, benzotriazole, benzofuran, benzothiazole, benzo[b]thiophene, benzo[d]isooxazole, 3,4-dihydro-2H-benzo[l,4]oxazine, benzo[l,3]dioxole, benzo[ 1,4] di oxane, l//-pyrrolo[2,3-b]pyridine, [l,2,4]triazol
  • R A 6 is selected from a bond, -O-, -NR A 13 -, -C(O)-, -C(O)O-, - C(O)NR A 13 -, -S(O)2NR A 13 -, C 1 -C 6 alkylene, C 1 -C 6 haloalkylene, and C 1 -C 6 heteroalkylene.
  • R A 6 is selected from -C(O)-, -C( 3 -.
  • R A 6 is -C(O)NR A 13 -.
  • the N atom of the amide attaches two Linker-OTUBl recruiter moieties.
  • R A 8 is selected from a bond, -O-, -NR A 13 -, -C(O)-, -C(O)O-, - C(O)NR A 13 -, -S(O)2NR A 13 -, C 1 -C 6 alkylene, C 1 -C 6 haloalkylene, and C 1 -C 6 heteroalkylene.
  • R A 1 is selected from H, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, Ci-C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, R A 1 is selected from H and CH3. In some embodiments, R A 1 is H. In some embodiments, R A 1 is CH3.
  • R A 2 is selected from H, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, R A 2 is selected from halogen. In some embodiments, R A 2 selected from F or Cl. In some embodiments, R A 2 is F. In some embodiments, R A 2 is Cl.
  • each R A 4 is selected from H, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, R A 4 is H.
  • each R A 5 is selected from H, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, each R A 5 is selected from H, halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl. In some embodiments, R A 5 is H, CH 3 , CH 2 CH 3 , and CH 2 OH.
  • each R A 7 is selected from H, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, each R A 7 is selected from H, halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl. In some embodiments, R A 7 is H, CH 3 , and CH 2 CH 3 .
  • each R A 9 is independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic, C 6 - aCry 1 l 0 , and 5-10 membered heteroaryl.
  • each R A 9 is independently selected from H, Ci- C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic.
  • R A 9 is H.
  • each R A 10 is independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic, C 6 - aCry 10 l, and 5-10 membered heteroaryl. In some embodiments, each R A 10 is independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, R A 10 is H.
  • each R A 11 and each R A 12 are independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic, C 6 - C 10 aryl, and 5-10 membered heteroaryl.
  • each R A 11 and each R A 12 are independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic.
  • R A 11 is selected from H and CH 3 .
  • R A 12 is selected from H and CH 3 .
  • R A 11 and R A 12 together with the atoms to which they are attached, optionally form 3-12 membered heterocyclic.
  • each R A 13 is independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic, C 6 - aCry 1 l 0 , and 5-10 membered heteroaryl.
  • each R A 13 is independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic.
  • each R A 14 is independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic, C 6 - aCry 1 l 0 , and 5-10 membered heteroaryl. In some embodiments, each R A 14 is independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic.
  • each R A 15 is independently selected from each R A 15 is independently selected from H, halogen, cyano, OR A 11 , NR A 11 A 12 , C(O)OR A 11 , C(O)NR A 11 R A 12 , S(O)2NR A ' 'R A 12 , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 12 cycloalkyl, 3-12 membered heterocyclic, C 6 -C 1 a 0 ryl and 5-10 membered heteroaryl.
  • mA is selected from 0, 1, 2, 3, and 4. In some embodiments, mA is selected from 0, 1, and 2. In some embodiments, nA is selected from 0, 1, 2, 3, and 4. In some embodiments, nA is selected from 0, 1, and 2. In some embodiments, OA is selected from 0, 1, 2, 3, 4, and 5.
  • the AMPK ligand comprise the structure of Formulae (B-I), (B-I- a), (B-I-al), (B-I-a2) and (B-I-a5).
  • the AMPK ligand comprise a derivative of following compounds:
  • ⁇ * denotes the point of attachment to Linker.
  • the cGAS ligand is seleted from a cGAS activator or cGAS inhibitor which potently binds with cGAS. In some embodiments, the cGAS ligand is cGAS activator. In some embodiments, the cGAS ligand is cGAS inhibitor.
  • the cGAS ligand comprises hydroxychloroquinine (HCQ), Quinacrine (QC), X6, RU114757, RU191752, RU100840, RU.365, RU.521, J001, G001, G108, G150, GMO, CPD-25, aspirin, CPD-C, PF- 06928215(Zhao et al., 2022).
  • the cGAS ligand is a compound disclosed in one or more of U.S. 62/318,435, U.S. 62/355,403, U.S. 2018/0230115, WO2019241787,
  • the cGAS ligand comprise the structure of Formula (C-I):
  • ⁇ ⁇ connects to either RB lb or RB 311 , with the proviso that when ** connects to RB lb ,
  • RB 3 is RB 3a ; and when ** connects to Rs 3b , RB 1 is RB 1S ;
  • AB is selected from N and CRB 4 ;
  • BB is selected from N, O, S, CRB 5 and NRB 5 ;
  • CB is selected from N and C
  • DB is selected from N and C
  • X B is X B a -X B b ;
  • X B a is selected from a bond, -C(O)-, -C(O)O-, -C(O)NR B 6 -, -S(O)-, -S(O) 2 -, -S(O)NR B 6 -, - S(O) 2 NR B 6 -;
  • X B b is selected from a bond, C 1 -C 6 alkylene, C 1 -C 6 haloalkylene, C 1 -C 6 heteroalkylene, C3- C12 cycloalkylene, and 3-12 membered heterocyclicene, C 6 -C 10 arylene, and 5-10 membered heteroarylene;
  • R B 1 is selected from monovalent group R B 1a and bivalent group R B lb ;
  • R B la is selected from H, halogen, cyano, OR B 7 , NR B 7 R B 8 , C(O)OR B 7 , C(O)NR B 7 R B 8 , S(O) 2 NR B 7 R B 8 , C 1 -C 6 alkyl, C 1 -C ha 6 loalkyl, C 1 - hCet 6 eroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 - C 12 cycloalkyl, 3-12 membered heterocyclic, C 6 -C 10 aryl, and 5-10 membered heteroaryl;
  • R B lb is connected to the Linker, and is selected from a bond, -O-, -NR B 7 -, -C(O)O-, - C(O)NR B 7 -, -S(O) 2 NR B 7 -, C 1 -C 6 alkylene, C 1 -C 6 haloalkylene, C 1 -C 6 heteroalkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, C 3 -C 12 cycloalkylene, 3-12 membered heterocyclicene, C 6 -C 10 arylene, and 5-10 membered heteroarylene; each R B 2 is independently selected from H, halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, or two R B 2 , together with the atoms to which they are attached, optionally form C 3
  • R B 3a is selected from H, halogen, OR B 9 , NR B 9 R B 10 , C(O)OR B 9 , C(O)NR B 9 R B 10 , S(O) 2 NR B 9 R B 10 , C 1 -C 6 alkyl, C 1 - hCal 6 oalkyl, C h 1 -eCte 6 roalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 - Ci 2 cycloalkyl, and 3-12 membered heterocyclic;
  • R B 3b is connected to the Linker, and is selected from a bond, -O-, -NR B 9 -, -C(O)O-, - C(O)NR B 9 -, -S(O) 2 NR B 9 -, C 1 -C 6 alkylene, C 1 -C 6 haloalkylene, C 1 -C 6 heteroalkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, C3-Ci 2 cycloalkylene, and 3-12 membered heterocyclicene; each R B 4 is independently selected from H, halogen, cyano, OR B 7 , NR B 7 R B 8 , C(O)OR B 7 , C(O)NR B 7 R B 8 , S(O) 2 NR B 7 R B 8 , C 1 -C 3 alkyl, C 1 -C 3 haloalkyl, C 1 -C 3 heteroalkyl, C 2 -
  • OB is selected from 0, 1, 2, 3, 4, 5 and 6.
  • * connects to R B 1b , R B 3 is R B 3a . In some embodiments, ** connects to R B 3b , R B 1 is R B 1 1
  • the cGAS ligand comprises the structures of Formulae (C-I-a) and (C-I-b):
  • AB is CR B 4
  • BB is NR B 5 .
  • CB is C.
  • DB is C.
  • the cGAS ligand comprises the structures of Formulae (C-I-al) and (C-I-bl):
  • R B 1a is selected from H, halogen, C 3 -C 12 cycloalkyl, 3-12 membered heterocyclic,C 6 -C 10 aryl, and 5-10 membered heteroaryl.
  • R B 13 is selected from C 3 -C 12 cycloalkyl, 3-12 membered heterocyclic, C 6 -C a 1 r 0 yl, and 5-10 membered heteroaryl.
  • R B 1a is selected from C 6 -C ar 10 yl, and 5-10 membered heteroaryl. In some embodiments, R B 1a is 5-10 membered heteroaryl.
  • R B 13 is selected from furyl, thiophenyl, pyrrolyl, pyrazolyl, oxazolyl, oxadiazolyl, thiazolyl, isoxazolyl, isothiazolyl, imidazolyl, triazolyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl.
  • R B 13 is pyrazolyl.
  • R B lb is selected from C 3 -C 12 cycloalkylene, 3-12 membered heterocyclicene, C 6 -C 10 arylene, and 5-10 membered heteroarylene.
  • R B 11 is selected from C 6 -C 10 arylene, and 5-10 membered heteroarylene.
  • the cGAS ligand comprises the structures of Formulae (C-I-a2) and (C-I-b2):
  • ArB is selected from C 6 -C 10 aryl, and 5-10 membered heteroaryl; each R B 1 1 is independently selected from H, halogen, cyano, OR B 7 , NR B 7 R B 8 , C(O)OR B 7 , C(O)NR B 7 R B 8 , S(O) 2 NR B 7 R B 8 , C 1 -C 6 alkyl, C h 1 a-lCo 6 alkyl, hCe 1 t-eCr 6 oalkyl, C 2 -C 6 alkenyl, C 2 - C 6 , alkynyl, C 3 -C 12 cycloalkyl, 3-12 membered heterocyclic, C 6 -C 10 aryl, and 5-10 membered heteroaryl; or two R B 11 , together with the atoms to which they are attached, optionally form partially unsaturated C 3 -C 12 cycloalkyl, partially unsaturated 3-12 membered heterocyclic,
  • PB is selected from 0, 1, 2, 3, 4, and 5;
  • PB-I is selected from 0, 1, 2, 3, and 4.
  • XB 3 is selected from -C(O)-, -S(O)-, and -S(O)2-.
  • XB a is -C(O)-.
  • XB b is selected from C 1 -C 6 alkylene, C 1 -C 6 haloalkylene, C 1 -C 6 heteroalkylene, C 3 -C 12 cycloalkylene, and 3-12 membered heterocyclicene.
  • the cGAS ligand comprises the structures of Formulae (C-I-a3) and (C-I-b3):
  • ArB is 5-10 membered heteroaryl.
  • ArB is selected from furyl, thiophenyl, pyrrolyl, pyrazolyl, oxazolyl, oxadiazolyl, thiazolyl, isoxazolyl, isothiazolyl, imidazolyl, triazolyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl.
  • ArB is pyrazolyl.
  • ArB is pyridinyl.
  • the cGAS ligand comprises the structures of Formulae (C-I-a4) and (C-I-b4):
  • each R B 2 is independently selected from H, C 1 -C 3 alkyl, C 1 -C 3 haloalkyl, C 1 -C 3 heteroalkyl, C 3 -C 6 cycloalkyl.
  • R B 2 is H.
  • R B 2 is C 1 -C 3 alkyl.
  • R B 2 is CH 3 .
  • two R B 2 together with the atoms to which they are attached, optionally form C 3 -C 6 cycloalkyl or 3-6 membered heterocyclic.
  • two R B 2 together with the atoms to which they are attached, optionally form C3-C5 cycloalkyl.
  • Ru 3a is selected from H, halogen, OR B 9 , NR B 9 R B 10 , C 1 -C 6 alkyl, Ci- Cg haloalkyl, Ci-Cg heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic.
  • R B 3a is selected from H, OR B 9 and NR B 9 R B 10 .
  • R B 3a is selected from H, OH, OCH 3 , OCH 2 CH 2 OCH 3 , NH 2 , NHCH 3 , and NHC(O)CH 2 NH 2 .
  • R B 3a is OH.
  • R B 3b is selected from a bond, -O-, -NR B 9 -, Ci-Cg alkylene, C 1 -C 6 haloalkylene, Ci-Cg heteroalkylene, C 3 -C 12 cycloalkylene, and 3-12 membered heterocyclicene.
  • R B 31) is selected from a bond, -O-, -NR B 9 -.
  • R B 3b is selected from a bond, -O-, -NH-, and -N(CH 3 )-. In some embodiments, R B 3b is -O-.
  • each R B 4 is independently selected from H, halogen, cyano, OR B 7 , NR B 7 R B 8 , C 1 -C 3 alkyl, C 1 -C 3 haloalkyl, C 1 -C 3 heteroalkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl, and 3-6 membered heterocyclic.
  • each R B 4 is independently selected from H.
  • each R B 4 is independently selected from cyano.
  • each R B 4 is independently selected from halogen.
  • each R B 4 is independently selected from F.
  • each R B 4 is independently selected from Cl. In some embodiments, each R B 4 is independently selected from methyl, ethyl, propyl, isopropyl, cyclopropyl, trifluoromethyl, difluoromethyl, methoxy, ethoxy, cyclopropoxy, and azetidinyl. In some embodiments, each R B 4 is independently selected from H. In some embodiments, each R B 4 is independently selected from cyano. In some embodiments, each R B 4 is independently selected from halogen. In some embodiments, each R B 4 is independently selected from F. In some embodiments, each R B 4 is independently selected from Cl. In some embodiments, each R B 4 is independently selected from Br.
  • two R B 4 together with the atoms to which they are attached, optionally form partially unsaturated C 3 -C 6 cycloalkyl, partially unsaturated 3-6 membered heterocyclic,C 6 -C 10 aryl, or 5-10 membered heteroaryl.
  • R B 5 is selected from H, C 1 -C 3 alkyl, C 1 -C 3 haloalkyl, C 1 -C 3 heteroalkyl, C 3 -C 6 cycloalkyl, and 3-6 membered heterocyclic. In some embodiments, R B 5 is selected from H and C 1 -C 3 alkyl. In some embodiments, R B 5 is H. In some embodiments, R B 5 is CH 3 .
  • R B 6 , each R B 7 , each R B 8 , each R B 9 , and each R B 10 are independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic.
  • R B 6 , each R B 7 , each R B 8 , each R B 9 , and each R B 10 are independently selected from H, methyl, ethyl, propyl, isopropyl, cyclopropyl, trifluoromethyl, and difluoromethyl. In some embodiments, R B 6 , each R B 7 , each R B 8 , each R B 9 , and each R B 10 , are H. In some embodiments, R B 6 , each R B 7 , each R B 8 , each R B 9 , and each R B 10 , are methyl.
  • each R B 11 is independently selected from H, halogen, cyano, OR B 7 , NR B 7 R B 8 , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 12 cycloalkyl, 3-12 membered heterocyclic.
  • R B 11 is H.
  • each R B 11 is CH 3 .
  • two R B 11 together with the atoms to which they are attached, optionally form partially unsaturated C 3 -C 12 cycloalkyl, 3-12 membered partially unsaturated heterocyclic,C 6 -C 10 aryl, or 5-10 membered heteroaryl.
  • each R B 12 is independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, each R B 12 is independently selected from H, and C 1 -C 6 alkyl. In some embodiments, R B 12 is H. In some embodiments, two R B 12 , R B 12 and R B 3a , and R B 12 and R B 3b , together with the atoms to which they are attached, optionally form C 3 -C 6 cycloalkyl, 3-6 membered heterocyclic.
  • m B is 1. In some embodiments, nB is 1. In some embodiments, nB is 2. In some embodiments, OB is 0. In some embodiments, OB is 1. In some embodiments, OB is 2. In some embodiments, PB is 0. In some embodiments, PB is 1. In some embodiments, PB is 2. In some embodiments, PB-I is 0. In some embodiments, PB-I is 1. In some embodiments, qB is 0. In some embodiments, q ⁇ is 1. In some embodiments, q ⁇ is 2.
  • the cGAS ligand comprises the structures of Formulae (C-I), (C-I- a), (C-I-al), (C-I-a2), (C-I-a3), and (C-I-a4).
  • the cGAS ligand may be a derivative of following compounds:
  • ⁇ * denotes the point of attachment to Linker.
  • the CFTR ligand can be a CFTR potentiator which potently binds with either wild type CFTR or mutant CFTR.
  • the CFTR ligand comprises ivacftor, lumacaftor, tezacaftor, elexacafor, or icenticaftor, or derivatives thereof.
  • the CFTR ligand is a compound disclosed in one or more of U.S. Patent No. 7,999,113; U.S. Patent No. 8,247,436; U.S. 8,410,274; WO 2011/133953; and WO2018/037350, each of which is incorporated by reference in its entirety.
  • the CFTR ligand comprises the structures of Formula (D-I):
  • Ac and Cc are independently selected from O, S, or C(Rc 8 )(Rc 9 );
  • Bc is C(Rc 10 )(Rc 11 ) or NR c 12 ;
  • Arc 1 and Arc 2 are independently selected from null, C 6 -C 10 aryl and 5-10 membered heteroaryl;
  • Rc 1 and Rc 2 are independently selected from H, halo, ORc 13 , NRc 13 Rc 14 , C(O)ORc 13 , C(O)NRC 13 RC 14 , S(O) 2 NRC 13 RC 14 , C 1 -C 6 alkyl, hCal 1 o-Cal 6 kyl, heCte 1 -rCoa 6 lkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic, or
  • Rc 1 and Rc 2 together with the atoms to which they are attached, optionally form C 3 -C 12 cycloalkyl;
  • Rc 8 , Rc 9 , Rc 10 and Rc 11 are independently selected from H, halogen, ORc 13 , NRc 13 Rc 14 , C(O)ORc 13 , C(O)NRc 13 Rc 14 , S(O) 2 NR C 13 RC 14 , CI-C 6 alkyl, C h 1 a-lCo 6 alkyl, C h 1 e-tCe 6 roalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic, or
  • Rc 8 and Rc 9 , and Rc 10 and Rc 11 together with the atoms to which they are attached, optionally form C 3 -C 12 cycloalkyl and 3-12 membered heterocyclic;
  • Rc 12 is selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic;
  • Rc 13 and Rc 14 are independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic, or
  • Rc 13 and Rc 14 together with the atoms to which they are attached, optionally form 3-12 membered heterocyclic; me is 0, 1 , 2, or 3; nc is 0, 1, 2, or 3; and oc is 0, 1, 2, or 3.
  • Arc 1 is 5-10 membered heteroaryl. In some embodiments, Arc 1 is selected from furyl, thiophenyl, pyrrolyl, pyrazolyl, oxazolyl, oxadiazolyl, thiazolyl, isoxazolyl, isothiazolyl, imidazolyl, triazolyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl. In some embodiments, ArB is pyridinyl. In some embodiments, Arc 2 is C 6 -C 1 a 0 ryl. In some embodiments, Arc 2 is phenyl.
  • the CFTR ligand comprises the structures of Formula (D-I-a):
  • the CFTR ligand comprises the structures of Formula (D-I-al):
  • Ac is O.
  • Be is C(Rc 10 )(Rc 11 ).
  • Cc is O.
  • the CFTR ligand comprises the structures of Formula (D-I-a2):
  • Rc 1 is selected from H, C 1 -C 3 alkyl, C 2 -C 3 alkenyl, C 2 -C 3 alkynyl, C3-C5 cycloalkyl, and 3-5 membered heterocyclic.
  • Rc 2 is selected from H, C 1 -C 3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C3-C5 cycloalkyl, and 3-5 membered heterocyclic.
  • Rc 1 and Rc 2 together with the atoms to which they are attached, optionally form C 3 -C 6 cycloalkyl.
  • Rc 1 and Rc 2 together with the atoms to which they are attached, optionally form ]cyclopropyl.
  • Rc 3 is selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, and C 1 -C 6 heteroalkyl. In some embodiments, Rc 3 is H.
  • each Rc 4 is independently selected from H, halo, cyano, ORc 13 , NRC 13 RC 14 , C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, each Rc 4 is independetly selected from H and C 1 -C 6 alkyl. In some embodiments, each Rc 4 is independently selected from H and CH 3 .
  • each Rc 5 is independently selected from H, halo, cyano, ORc 13 , NRC 13 RC 14 , C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic.
  • each Rc 3 is independently selected from H and C 1 -C 6 alkyl.
  • Rc 5 is H.
  • Rc 6 is selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, and C 1 -C 6 heteroalkyl. In some embodiments, Rc 6 is H. In some embodiments, Rc 3 is CH 3 .
  • each Rc 7 is independently selected from H, halogen, cyano, ORc 13 , NRC 13 RC 14 , C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, each Rc 7 is independently selected from H and C 1 -C 6 alkyl. In some embodiments, Rc 7 is H.
  • Rc 8 and Rc 9 are independently selected from H, halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, Rc 8 and Rc 9 are independently selected from H and C 1 -C 6 alkyl,
  • Rc 10 and Rc 11 are independently selected from H, halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, Rc 10 and Rc 11 are independently selected from H, halogen and C 1 -C 6 alkyl. In some embodiments. Rc 10 and Rc 11 are independently selected from H, F, Cl, CH 3 , and CF3. In some embodiments, both Rc 10 and Rc 11 are F.
  • Rc 12 is selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic.
  • Rc 13 and Rc 14 are independently selected from H, C 1 -C 6 alkyl, Ci- C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, Rc 13 and Rc 14 are independently selected from H and C 1 -C 6 alkyl. In some embodiments, Rc 13 and Rc 14 are independently selected from H and CH 3 . In some embodiments, Rc 13 and Rc 14 together with the atoms to which they are attached, optionally form 3-6 membered heterocyclic.
  • me is 1. In some embodiments, nc is 0. In some embodiments, oc is 0.
  • the CFTR ligand comprises the structures of Formulae (D-I-a3) and (D-I-a4):
  • the CFTR ligand may be a derivative of following compounds:
  • the AMPK, cGAS or CFTR ligand is conjugated to the de-ubiquitination tag through a linker.
  • the linker can include, for example, acyclic or cyclic saturated or unsaturated carbon, ethylene glycol, amide, amino, ether, urea, carbamate, aromatic, heteroaromatic, heterocyclic and/or carbonyl containing groups with different lengths.
  • the linker can be a moiety of Formula (E):
  • A, W and B, at each occurrence, are independently selected from null, or bivalent moiety and R NR 1 S(O) 2 N(R 2 )R , wherein
  • R and R are independently selected from a bond, optionally substituted R r -(C 1 -C 8 alkyl), or a moiety comprising of optionally substituted C 1 -C 8 alkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 1 -C8 a lkoxyC 1 -C8alkyl, optionally substituted C 1 -C 8 alkylaminoC 1 -C 8 alkyl, optionally substitutedC 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 alkylene, optionally substituted C 2 -C 8 alkenylene, optionally substituted C 2 -C 8 alkynylene, optionally substituted C 1 -C 8 hydroxy alkylene, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkylene, optionally
  • R r is selected from optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted C 3 -C 13 fused cycloalkyl, optionally substituted C 3 -C 13 fused heterocyclic, optionally substituted C 3 -C 13 bridged cycloalkyl, optionally substituted C 3 -C 13 bridged heterocyclic, optionally substituted C 3 -C 13 spiro cycloalkyl, optionally substituted C 3 -C 13 spiro heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
  • R 1 and R 2 are independently selected from hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituteCd 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 1 -C 8 alkoxyalkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substitute
  • the linker moiety is of Formula (E-l)
  • R 1 , R 2 , R 3 and R 4 are independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, optionally substituted C 1 -C 8 alkyl, optionally substituteC 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkoxyalkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 1 -C 8 alkylamino, and optionally substituted C 1 -C 8 alkylaminoC 1 -C 8 alkyl, optionally substituted 3-10 membered carbocyclic, optionally substituted 3-8 membered cycloalkoxy, optionally substituted 3-10 membered carbocyclicamino, optionally substituted 4-8 membered membered heterocyclic,
  • R 1 and R 2 , R 3 and R 4 together with the atom to which they are connected form a 3-20 membered cycloalkyl or 4-20 membered heterocyclic ring;
  • A, W and B, at each occurrence, are independently selected from null, or bivalent moiety selected from R -R ”, R COR , R CO 2 R , R C(O)N(R 5 )R , R’C(S)N(R 5 )R”, R OR”, R OC(O)R”, R OC(O)OR ”, R OCONR 5 R”, R SR ”, R SOR”, R SO 2 R , R SO 2 N(R 5 )R”, RN(R 5 )R”, RNR 5 COR ”, RNR 5 C(O)OR’ , RNR 5 CON(R 6 )R”, R NR 5 C(S)R ”, RNR 5 S(O)R ”, RNR 5 S(O) 2 R ”, and R NR 5 S(O) 2 N
  • R and R are independently selected from null, optionally substituted R r -(C 1 -C 8 alkyl), or a moiety comprising of optionally substituted C 1 -C 8 alkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkylaminoC 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 alkylene, optionally substituted C 2 -C 8 alkenylene, optionally substituted C 2 -C 8 alkynylene, optionally substituted C 1 -C 8 hydroxy alkylene, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkylene, optionally substituted C 1
  • R r is selected from optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted C 3 -C 13 fused cycloalkyl, optionally substituted C 3 -C 13 fused heterocyclic, optionally substituted C 3 -C 13 bridged cycloalkyl, optionally substituted C 3 -C 13 bridged heterocyclic, optionally substituted C 3 -C 13 spiro cycloalkyl, optionally substituted C 3 -C 13 spiro heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
  • R 5 and R 6 are independently selected from hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 1 -C 8 alkoxyalkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 1 -C 8 alkylaminoC 1 -C 8 alkyl, optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl; R and R , R 5 and R 6 , R and R 5 , R andR 6 together with the atom to which they are connected form a 3-20 membered cycloalkyl or 4-20 membered heterocyclic ring; m is 0 to 15; n
  • linker moiety is of Formula (E-2)
  • R 1 and R 2 are independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, and optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkoxy C 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 1 -C 8 alkylamino, Ci- CsalkylaminoC 1 -C 8 alkyl, optionally substituted 3-10 membered carbocyclic, optionally substituted 3-8 membered cycloalkoxy, optionally substituted 3-10 membered carbocyclicamino, optionally substituted 4-10 membered heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl, or
  • R 1 and R 2 together with the atom to which they are connected form a 3-20 membered cycloalkyl or 4-20 membered heterocyclic ring;
  • a and B are independently selected from null, or bivalent moiety selected from R -R , R COR , R CO 2 R , R C(O)NR 3 R’ , R C(S)NR 3 R”, R’OR”, R OC(O)R’ , R OC(O)OR , R OCON(R 3 )R , R SR , R SOR , R SO 2 R , R SO 2 N(R 3 )R , R N(R 3 )R , R’NR 3 COR”, RNR 3 C(O)OR ”, R’NR 3 CON(R 4 )R’, R’NR 3 C(S)R ”, R’NR 3 S(O)R”, R’NR 3 S(O) 2 R”, and R NR 3 S(O) 2 N(R 4 )R ”, wherein
  • R and R are independently selected from null, optionally substituted R r -(C 1 -C 8 alkyl), or a moiety comprising of optionally substituted C 1 -C 8 alkyl, optionally substituted C 2 -C 8 alkenyl, optionally substitutedC 2 -C 8 alkynyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkylaminoC 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 alkylene, optionally substituted C 2 -C 8 alkenylene, optionally substituted C 2 -C 8 alkynylene, optionally substituted C 1 -C 8 hydroxyalkylene, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkylene, optionally substituted C 1
  • R r is selected from optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted C 3 -C 13 fused cycloalkyl, optionally substituted C 3 -C 13 fused heterocyclic, optionally substituted C 3 -C 13 bridged cycloalkyl, optionally substituted C 3 -C 13 bridged heterocyclic, optionally substituted C 3 -C 13 spiro cycloalkyl, optionally substituted C 3 -C 13 spiro heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
  • R 3 and R 4 are independently selected from hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 1 -C 8 alkoxyalkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 1 -C 8 alkylaminoC 1 -C 8 alkyl, optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
  • linker moiety is of Formula (E-3):
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, optionally substituted C 1 -C 8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkoxy C 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 1 -C 8 alkylamino, optionally substituted C 1 -C 8 alkylaminoC 1 -C 8 alkyl, optionally substituted 3-10 membered carbocyclic, optionally substituted 3-8 membered cycloalkoxy, optionally substituted 4-10 membered heterocyclic, optionally substituted
  • a and B are independently selected from null, or bivalent moiety selected from R -R , R COR , R CO2R ”, R C(O)N(R 8 )R’ , R C(S)N(R 8 )R’ , R OR , R OC(O)R”, R OC(O)OR”, R OCON(R 8 )R , R SR , R SOR , R SO 2 R , R SO 2 N(R 8 )R , R N(R 8 )R , R NR 8 COR , R’NR 8 C(O)OR”, R’NR 8 CON(R 9 )R”, R’NR 8 C(S)R”, R’NR 8 S(O)R”, R’NR 8 S(O) 2 R”, and RNR 8 S(O) 2 N(R 9 )R ’, wherein
  • R and R are independently selected from null, optionally substituted R r -(C 1 -C 8 alkyl), or a moiety comprising of optionally substituted C 1 -C 8 alkyl, optionally substituted C 2 -C 8 alkenyl, optionally substitutedC 2 -C 8 alkynyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkylaminoC 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 alkylene, optionally substituted C 2 -C 8 alkenylene, optionally substituted C 2 -C 8 alkynylene, optionally substituted C 1 -C 8 hydroxyalkylene, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkylene, optionally substituted C 1
  • R r is selected from optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted C 3 -C 13 fused cycloalkyl, optionally substituted C 3 -C 13 fused heterocyclic, optionally substituted C 3 -C 13 bridged cycloalkyl, optionally substituted C 3 -C 13 bridged heterocyclic, optionally substituted C 3 -C 13 spiro cycloalkyl, optionally substituted C 3 -C 13 spiro heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
  • R 7 , R 8 and R 9 are independently selected from hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 1 -C 8 alkoxyalkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 1 -C 8 alkylaminoC 1 -C 8 alkyl, optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
  • R and R , R 8 and R 9 , R and R 8 , R and R 9 , R and R 8 , R and R 9 together with the atom to which they are connected form a 3-20 membered cycloalkyl or 4-20 membered heterocyclic ring; m, at each occurrence, is 0 to 15; n, at each occurrence, is 0 to 15; o is 0 to 15; and p is 0 to 15.
  • n and p is 0 or 1;
  • X is selected from O and NH
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently selected from hydrogen, and optionally substituted C 1 -C 6 alkyl.
  • the linker moiety comprises a ring selected from the group consisting of a 3 to 13 membered ring, a 3 to 13 membered fused ring, a 3 to 13 membered bridged ring, and a 3 to 13 membered spiro ring.
  • the linker moiety comprises a ring selected from the group consisting of Formula Fl, F2, F3, F4 and F5:
  • X' and Y' are independently selected from N and CR b ;
  • a 1 , B 1 , C 1 and D 1 are independently selected from null, O, CO, SO, SO 2 , NR b , and CR b R c ;
  • a 2 , B 2 , C 2 and D 2 are independently selected from N and CR b ;
  • a 3 , B 3 , C 3 , D 3 , and E 3 at each occurrence, are independently selected from N, O, S, NR b , and CR b ;
  • R b and R c are independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, optionally substituted C 1 -C 8 alkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkoxyalkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 1 -C 8 alkylamino, optionally substituted C 1 -C 8 alkylaminoCi- C 8 alkyl, optionally substituted 3-10 membered carbocyclic, optionally substituted 3-10 membered cycloalkoxy, optionally substituted 3-10 membered carbocyclicamino, optionally substituted 3-8 membered heterocyclic, optionally substituted aryl, and optional
  • A, B, and W, at each occurrence, are independently selected from null, optionally substituted -(CH 2 )o-8-, optionally substituted -(CH 2 )o-8-CO-(CH 2 )o-8-, optionally substituted -(CH 2 )o-8-NH-(CH 2 )o-s-, optionally substituted -(CH 2 )o-8-NH-CO-(CH 2 )o-8-, optionally substituted -(CH 2 )o-8-CO-NH-(CH 2 )o-8-, optionally substituted -(CH 2 )o-3-NH-(CH 2 )o-3-CO-NH- (CH 2 )O-8-, optionally substituted -(CH 2 )O-3-NH-(CH 2 )I-3-NH-CO-(CH 2 )O-8-, optionally substituted - (CH 2 )O-8-CO-NH-(CH 2 )I-3-NH-(CH 2 )O-3-, optionally substituted
  • R r is of Formula Fl, F2, F3, F4, or F5.
  • R r is selected from
  • the length of the linker is 0 to 40 atoms.
  • the length of the linker is 0 to 20 atoms.
  • the length of the linker is 0 to 10 atoms.
  • the length of the linker is 0 to 40 atoms.
  • the linker is selected from null, optionally substituted -CO-(CH 2 )o- io-, optionally substituted -(CH 2 )O-IO-, optionally substituted -(CH 2 ) 1-2 -(CO)NH-(CH 2 )o-io-, optionally substituted -(CH 2 )I-2-(CO)NH-(CH 2 )I-3-(OCH 2 CH 2 )I-7-, optionally substituted -(CH 2 )o- I-CO-(CH 2 ) 1-3 -(OCH 2 CH 2 ) 1-7 -, optionally substituted -CO-(CH 2 ) 0-3 -(alkenylene)-(CH 2 ) 0-3 -, optionally substituted -CO-(CH 2 ) 0-3 -(alkynylene)-(CH 2 ) 0-3 -, optionally substituted -CO-(CH 2 ) 0-3 - (3-8 membered carb
  • the linker can also be a moiety of:
  • the linker can also be a moiety of:
  • Formula (E-2-b), Formula (E-3-b) A is -C(O)N-, B is -C(O)N-, m is 0-16, n is 0-6, o is 0-6, n-1 is 0-6, and o-l is 0-6.
  • the DUB recruiter comprises the structures of Formula (A-I):
  • the DUB recruiters comprise the structures of Formulae (A-I-a), (A-I-al), (A-I-a2), (A-I-a3), (A-I-a4), (A-I-a5), (A-I-a6), (A-I-a7), (A-I-a8), (A-I-a9), (A-I-alO), (A-I-al l), (A-I-b), (A-I-bl), (A-I-b2), (A-I-b3), (A-I-b4), (A-I-b5), (A-I-b6), (A-I-b7), (A-I-b8), (A-I-b9), (A-I-blO), (A-I-bl l), (A-I-c), (A-I-cl), (A-I-c2), (A-I-c3), (A-I-c4), (A-I-c5)
  • R D 41) is a bond
  • the DUB recruiters comprise:
  • the AMPK based bivalent compound is a compound selected from the following compounds, as identified in Table 3 below: XF137-81, XF137-82, XF137-83, XF137-84, XF137-85, XF137-86, XF137-87, XF137-88, XF137-89, XF137-90, XF137-91, XF137-92, XF137-93, XF137-94, QC179-047, QC179-048, QC137-049, QC179-050, QC179- 051, QC179-052, QC179-053, QC179-054, QC137-055, and examples 367 - 380, or analogs thereof.
  • the cGAS based bivalent compound is a compound selected from the following compounds, as identified in table 4 below: ZD178-22, ZD178-23, ZD178-24, ZD178-25, ZD178-26, ZD178-27, ZD178-28, ZD178-29, ZD178-30, ZD178-31, ZD178-32, ZD178-33, ZD178-34, ZD178-35, ZD178-58-1, ZD178-58-2, ZD178-58-3, ZD178-58-4, ZD178- 58-5, ZD178-58-6, ZD178-58-7, ZD178-58-8, ZD178-58-9, ZD178-58-10, ZD178-58-11, ZD178-58-12, ZD178-58-13, ZD178-58-14, ZD178-63-1, ZD178-63-2, ZD178-63-3, ZD178-63- 4, ZD178-63-5, ZD178-63-6, ZD178-63
  • the CFTR based bivalent compound is a compound selected from the following compounds, as identified in Table 5 below: QC166-130, QC166-131, QC166-132, QC166-133, QC166-134, QC166-135, QC166-136, QC166-137, QC166-138, QC166-139,
  • this disclosure provides a method of treating AMPK, cGAS or CFTR-mediated disease, the method including administering to a subject in need thereof one or more bivalent compounds including a AMPK, cGAS, or CFTR ligand conjugated to a 0TUB1 binder via a linker.
  • the AMPK, cGAS, CFTR-mediated disease can be a disease resulting from AMPK, cGAS or CFTR degradation.
  • the AMPK, cGAS or CFTR-mediated disease can have decrease CFTR expression relative to a wild-type tissue of the same species and tissue type.
  • the bivalent compounds can be administered, e.g., orally, parenterally, intradermally, subcutaneously, topically, and/or rectally.
  • This disclosure additionally provides a method for identifying a bivalent compound which mediates de-ubiquitination/stabilization of AMPK, cGAS, or CFTR, the method including providing a heterobifunctional test compound including a AMPK, cGAS, or CFTR ligand conjugated to a de-ubiquitination tag via a linker, contacting the heterobifunctional test compound with a cell (e.g., a cell such as a AMPK, cGAS or CFTR-mediated disease cell) including a deubiquitinase (e.g., 0TUB1) and AMPK, cGAS or CFTR protein.
  • a cell e.g., a cell such as a AMPK, cGAS or CFTR-mediated disease cell
  • a deubiquitinase e.g., 0TUB1
  • the terms “about” and “approximately” are defined as being within plus or minus 10% of a given value or state, preferably within plus or minus 5% of said value or state.
  • the terms “bivalent” and “bi-functional” are used interchangeably herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
  • Figure 1 Effect of exemplary compounds on modifying OTUB1 in mass spectrometry-based assays.
  • Figure 4 Effect of exemplary AMPK based bivalent compounds in stabilization AMPK protein level in HEK293T cells in multiple concentrations.
  • Figure 5 Effect of exemplary cGAS based bivalent compounds in stabilization cGAS protein level in Hela cells.
  • Figure 9 Effect of exemplary CFTR based bivalent compounds on stabilizing CFTR protein levels in human cystic fibrosis bronchial epithelial cells.
  • Figure 10 Effect of exemplary CFTR or cGAS based bivalent compounds on modifying OTUB1 mass spectrometry-based assays.
  • novel synthesized covalent OTUB1 binder and bivalent AMPK-OTUBl, cGAS-OTUBl, CFTR-OTUB1 compounds can be assessed using standard biophysical assays (e g., isothermal titration calorimetry (ITC), surface plasmon resonance (SPR)) and mass spectrometry -based assays. Cellular assays can then be used to assess the bivalent CFTR-OTUB1 compound’s ability to stabilize AMPK, cGAS or CFTR proteins level.
  • standard biophysical assays e g., isothermal titration calorimetry (ITC), surface plasmon resonance (SPR)
  • mass spectrometry -based assays e g., cellular assays can then be used to assess the bivalent CFTR-OTUB1 compound’s ability to stabilize AMPK, cGAS or CFTR proteins level.
  • Assays suitable for use in any or all of these steps are known in the art, and include, e.g., Western blotting, quantitative mass spectrometry (MS) analysis, flow cytometry, enzymatic inhibition, ITC, SPR, cell growth inhibition and xenograft and PDX models.
  • MS mass spectrometry
  • isotopic variations of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (substituting appropriate reagents with appropriate isotopic variations of those reagents).
  • an isotopic variation is a compound in which at least one atom is replaced by an atom having the same atomic number, but an atomic mass different from the atomic mass usually found in nature.
  • Useful isotopes are known in the art and include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine. Exemplary isotopes thus include, e.g., 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, 32 P, 35 S, 18 F, and 36 C1.
  • Isotopic variations e.g., isotopic variations containing 2 H
  • certain isotopic variations can be used in drug or substrate tissue distribution studies.
  • the radioactive isotopes tritium ( 3 H) and carbon-14 ( 14 C) are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
  • solvates of the compounds disclosed herein are contemplated.
  • a solvate can be generated, e.g., by substituting a solvent used to crystallize a compound disclosed herein with an isotopic variation (e.g., D2O in place of H2O, ⁇ -acetone in place of acetone, or de- DMSO in place of DMSO).
  • an isotopic variation e.g., D2O in place of H2O, ⁇ -acetone in place of acetone, or de- DMSO in place of DMSO.
  • a fluorinated variation is a compound in which at least one hydrogen atom is replaced by a fluoro atom. Fluorinated variations can provide therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements.
  • prodrugs of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (e.g., converting hydroxyl groups or carboxylic acid groups to ester groups).
  • a prodrug refers to a compound that can be converted via some chemical or physiological process (e.g., enzymatic processes and metabolic hydrolysis) to a therapeutic agent.
  • prodrug also refers to a precursor of a biologically active compound that is pharmaceutically acceptable.
  • a prodrug may be inactive when administered to a subject, i.e.
  • prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in an organism.
  • prodrug is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a subject.
  • Prodrugs of an active compound may be prepared by modifying functional groups present in the active compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent active compound.
  • Prodrugs include compounds wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the active compound is administered to a subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively.
  • Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of an alcohol or acetamide, formamide and benzamide derivatives of an amine functional group in the active compound and the like.
  • Alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation.
  • An alkyl may comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms.
  • an alkyl comprises one to fifteen carbon atoms (e.g., C1-C15 alkyl).
  • an alkyl comprises one to thirteen carbon atoms (e.g., C1-C13 alkyl).
  • an alkyl comprises one to eight carbon atoms (e.g., C 1 -C 8 alkyl).
  • an alkyl comprises five to fifteen carbon atoms (e.g., C5-C15 alkyl). In other embodiments, an alkyl comprises five to eight carbon atoms (e.g., Cs-Cs alkyl).
  • the alkyl is attached to the rest of the molecule by a single bond, for example, methyl (Me), ethyl (Et), n-propyl, 1 -methylethyl (/.w-propyl), //-butyl, //-pentyl, 1 , 1 -dimethylethyl (/-butyl), pentyl, 3 -methylhexyl,
  • Alkylene refers to a bivalent saturated aliphatic radical (such as ethylene) regarded as derived from an alkene by opening of the double bond or from an alkane by removal of two hydrogen atoms from different carbon atoms.
  • Alkenyl 1 ' refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond.
  • An alkenyl may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms.
  • an alkenyl comprises two to twelve carbon atoms (e.g, C 2 -C 12 alkenyl).
  • an alkenyl comprises two to eight carbon atoms (e.g., C 2 -C 8 alkenyl).
  • an alkenyl comprises two to six carbon atoms (e.g., C 2 - , alkenyl).
  • an alkenyl comprises two to four carbon atoms (e.g., C 2 -C 4 alkenyl).
  • the alkenyl is attached to the rest of the molecule by a single bond, for example, ethenyl (i.e., vinyl), prop-l-enyl (i.e., allyl), but-l-enyl, pent-l-enyl, penta- 1,4-dienyl, and the like.
  • allyl as used herein, means a -CH 2 CI-GCH 2 group.
  • alkynyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond.
  • An alkynyl may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms.
  • an alkynyl comprises two to twelve carbon atoms (e.g., C2-C12 alkynyl).
  • an alkynyl comprises two to eight carbon atoms (e.g., C 2 -C 8 alkynyl).
  • an alkynyl has two to six carbon atoms (e.g., C 2 -C 6 alkynyl). In other embodiments, an alkynyl has two to four carbon atoms (e.g., C 2 -C 4 alkynyl).
  • the alkynyl is attached to the rest of the molecule by a single bond. Examples of such groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1 -pentynyl, 2-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, and the like.
  • alkoxy means an alkyl group as defined herein witch is attached to the rest of the molecule via an oxygen atom.
  • alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propyloxy, iso-propyloxy, n-butoxy, iso-butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.
  • Heteroalkyl refers to a substituted or unsubstituted alkyl group which has one or more skeletal chain atoms selected from an atom other than carbon
  • Exemplary skeletal chain atoms selected from an atom other tha n carbon include , e.g., O, N, P, Si, S, or combinations thereof, wherein the nitrogen, phosphorus, and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized .
  • a numberical range refers to the chain length in total. For example, a 3- to 8- membered heteroalkyl has a chain length of 3 to 8 atoms.
  • connection to the rest of the molecule may be through either a heteroatom or a carbon in the heteroalkyl chain.
  • a heteroalkyl group is optionally substituted by one or more substituents such as those substituents described herein.
  • aryl refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom.
  • the aromatic monocyclic or multicyclic hydrocarbon ring system contains only hydrogen and carbon atoms.
  • An aryl may comprise from six to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) 7t-electron system in accordance with the Hiickel theory.
  • an aryl comprises six to fourteen carbon atoms (Ce-Cu aryl).
  • an aryl comprises six to ten carbon atoms (C 6 -Cio aryl).
  • groups include, but are not limited to, phenyl, fluorenyl and naphthyl.
  • arylene means a divalent aromatic hydrocarbon radical of 6-20 carbon atoms (C 6 -C 20 ) derived by the removal of two hydrogen atom from two carbon atoms of a parent aromatic ring system. Some arylene groups are represented in the exemplary structures as “Ar”. Arylene includes bicyclic radicals comprising an aromatic ring fused to a saturated, partially unsaturated ring, or aromatic carbocyclic ring.
  • Typical arylene groups include, but not limited to, radicals derived from benzene (phenylene), substituted benzenes, naphthalene, anthracene, biphenylene indenylene, indaylene, 1,2-dihydronaphthalene, 1,2, 3, 4, -tetrahydronaphthyl, and the like.
  • Arylene groups are optionally substituted with one or more substituents described herein.
  • heteroaryl refers to a radical derived from a 3- to 18-membered aromatic ring radical that comprises two to seventeen carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur.
  • the heteroaryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) ⁇ -electron system in accordance with the Hiickel theory.
  • Heteroaryl includes fused or bridged ring systems.
  • a heteroaryl refers to a radical derived from a 3- to 10-membered aromatic ring radical (3-10 membered heteroaryl). In certain embodiments, a heteroaryl refers to a radical derived from 5- to 7-membered aromatic ring (5-7 membered heteroaryl). Heteroaryl includes fused or bridged ring systems. The heteroatom(s) in the heteroaryl radical is optionally oxidized. One or more nitrogen atoms, if present, are optionally quatemized. The heteroaryl is attached to the rest of the molecule through any atom of the ring(s).
  • Examples of such groups include, but not limited to, pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl
  • an heteroaryl is attached to the rest of the molecule via a ring carbon atom. In certain embodiments, an heteroaryl is attached to the rest of the molecule via a nitrogen atom (N-attached) or a carbon atom (C-attached).
  • N-attached nitrogen atom
  • C-attached carbon atom
  • a group derived from pyrrole may be pyrrol-l-yl (N-attached) or pyrrol-3-yl (C-attached).
  • a group derived from imidazole may be imidazol-l-yl (N-attached) or imidazol-3-yl (C-attached).
  • heterocyclic means a non-aromatic, monocyclic, bicyclic, tricyclic, or tetracyclic radical having a total of from 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 atoms in its ring system, and containing from 3 to 12 carbon atoms and from 1 to 4 heteroatoms each independently selected from O, S and N, and with the proviso that the ring of said group does not contain two adjacent O atoms or two adjacent S atoms.
  • a heterocyclic group may include fused, bridged or spirocyclic ring systems. In certain embodiments, a heterocyclic group comprises 3 to 10 ring atoms (3-10 membered heterocyclic).
  • a heterocyclic group comprises 3 to 8 ring atoms (3-8 membered heterocyclic). In certain embodiments, a heterocyclic group comprises 4 to 8 ring atoms (4-8 membered heterocyclic). In certain embodiments, a heterocyclic group comprises 3 to 6 ring atoms (3-6 membered heterocyclic).
  • a heterocyclic group may contain an oxo substituent at any available atom that will result in a stable compound. For example, such a group may contain an oxo atom at an available carbon or nitrogen atom. Such a group may contain more than one oxo substituent if chemically feasible.
  • heterocyclic group when such a heterocyclic group contains a sulfur atom, said sulfur atom may be oxidized with one or two oxygen atoms to afford either a sulfoxide or sulfone.
  • An example of a 4 membered heterocyclic group is azetidinyl (derived from azetidine).
  • An example of a 5 membered cycloheteroalkyl group is pyrrolidinyl.
  • An example of a 6 membered cycloheteroalkyl group is piperidinyl.
  • An example of a 9 membered cycloheteroalkyl group is indolinyl.
  • An example of a 10 membered cycloheteroalkyl group is 4H-quinolizinyl.
  • Such heterocyclic groups include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3- pyrrolinyl, indolinyl, 2H-pyranyl, 477- pyranyl, di
  • a heteroaryl group may be attached to the rest of molecular via a carbon atom (C-attached) or a nitrogen atom (N-attached).
  • a group derived from piperazine may be piperazin- 1-yl (N-attached) or piperazin-2-yl (C-attached).
  • cycloalkyl or “carbocyclic” means a saturated, monocyclic, bicyclic, tricyclic, or tetracyclic radical having a total of from 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 carbon atoms in its ring system.
  • a cycloalkyl may be fused, bridged or spirocyclic.
  • a cycloalkyl comprises 3 to 8 carbon ring atoms (C 3 -C 8 cycloalkyl).
  • a cycloalkyl comprises 3 to 6 carbon ring atoms ( C 3 -C 6 cycloalkyl).
  • Examples of such groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptyl, adamantyl, and the like.
  • cycloalkylene or “carbocyclicene” is a bidentate radical obtained by removing a hydrogen atom from a cycloalkyl ring as defined above.
  • examples of such groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentyl ene, cyclopentenylene, cyclohexylene, cycloheptylene, and the like.
  • spirocyclic as used herein has its conventional meaning, that is, any ring system containing two or more rings wherein two of the rings have one ring carbon in common.
  • Each ring of the spirocyclic ring system independently comprises 3 to 20 ring atoms. Preferably, they have 3 to 10 ring atoms.
  • Non-limiting examples of a spirocyclic system include spiro[3.3]heptane, spiro[3.4] octane, and spiro[4.5]decane.
  • aldehyde refers to a -C(O)H group.
  • alkoxy refers to both an -O-alkyl, as defined herein.
  • alkoxycarbonyl refers to a -C(O)-alkoxy, as defined herein.
  • alkylaminoalkyl refers to an -alkyl-NR-alkyl group, as defined herein.
  • alkylsulfonyl refer to a -SChalkyl, as defined herein.
  • amino refers to an optionally substituted -NH 2 .
  • aminoalkyl refers to an -alky-amino group, as defined herein.
  • aminocarbonyl refers to a -C(O)-amino, as defined herein.
  • arylalkyl refers to -alkylaryl, where alkyl and aryl are defined herein.
  • aryloxy refers to both an -O-aryl and an -O-heteroaryl group, as defined herein.
  • aryloxycarbonyl refers to -C(O)-aryloxy, as defined herein.
  • aryl sulfonyl refers to a -SCharyl, as defined herein.
  • carbonyl refers to a -C(O)- group, as defined herein.
  • a “carboxylic acid” group refers to a -C(O)OH group.
  • cycloalkoxy refers to a -O-cycloalkyl group, as defined herein.
  • halo or halogen group refers to fluorine, chlorine, bromine or iodine.
  • haloalkyl group refers to an alkyl group substituted with one or more halogen atoms.
  • a "hydroxy” group refers to an -OH group.
  • a “nitro” group refers to a -NO 2 group.
  • trihalomethyl refers to a methyl substituted with three halogen atoms.
  • substituted means that the specified group or moiety bears one or more substituents independently selected from C 1 -C 4 alkyl, aryl, heteroaryl, aryl-C 1 -C 4 alkyl-, heteroaryl-C 1 -C 4 alkyl-, C 1 -C 4 haloalkyl, -OC 1 -C 4 alkyl, -OC 1 -C 4 alkylphenyl, -C 1 -C 4 alkyl-OH, -OC 1 -C 4 haloalkyl, halo, -OH, -NH 2 , -C 1 -C 4 alkyl-NH 2 , -N(C 1 -C 4 alkyl)(C 1 -C 4 alkyl), -NH(C 1 -C 4 alkyl), -N(C 1 -C 4 alkyl)(C 1 -C 4 alkylphenyl), -NH(C 1 -C 4 alkyl), -N(C
  • null means the absence of an atom or moiety, and there is a bond between adjacent atoms in the structure.
  • Ce aryl group also called “phenyl” herein
  • phenyl substituted with one additional substituent
  • one of ordinary skill in the art would understand that such a group has 4 open positions left on carbon atoms of the C 6 aryl ring (6 initial positions, minus one at which the remainder of the compound of the present invention is attached to and an additional substituent, remaining 4 positions open).
  • the remaining 4 carbon atoms are each bound to one hydrogen atom to fill their valencies.
  • a Ce aryl group in the present compounds is said to be “di substituted,” one of ordinary skill in the art would understand it to mean that the C 6 aryl has 3 carbon atoms remaining that are unsubstituted.
  • Those three unsubstituted carbon atoms are each bound to one hydrogen atom to fill their valencies.
  • the same symbol in a different FORMULA may have a different definition, for example, the definition of R1 in FORMULA 1 is as defined with respect to FORMULA 1 and the definition of R1 in FORMULA 6 is as defined with respect to FORMULA 6.
  • m is 0 to 15
  • “Pharmaceutically acceptable salt” includes both acid and base addition salts.
  • a pharmaceutically acceptable salt of any one of the bivalent compounds described herein is intended to encompass any and all pharmaceutically suitable salt forms.
  • Preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts that are formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl -substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and. aromatic sulfonic acids, etc.
  • acetic acid tri fluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
  • Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like.
  • salts of amino acids such as arginates, gluconates, and galacturonates
  • Acid addition salts of basic compounds may be prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.
  • “Pharmaceutically acceptable base addition salt” refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N '.N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, A-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, A-ethylpiperidine, polyamine resins and the like. See Berge e
  • compositions and methods described herein include the manufacture and use of pharmaceutical compositions and medicaments that include one or more bivalent compounds as disclosed herein. Also included are the pharmaceutical compositions themselves.
  • compositions disclosed herein can include other compounds, drugs, or agents used for the treatment of cancer.
  • pharmaceutical compositions disclosed herein can be combined with one or more (e.g., one, two, three, four, five, or less than ten) compounds.
  • additional compounds can include, e.g., conventional chemotherapeutic agents known in the art.
  • 0TUB1 covalent binders or AMPK, cGAS or CFTR based bivalent compounds disclosed herein can operate in conjunction with conventional chemotherapeutic agents to produce mechanistically additive or synergistic therapeutic effects.
  • the pH of the compositions disclosed herein can be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the 0TUB1 covalent binders or AMPK, cGAS or CFTR based bivalent compounds or its delivery form.
  • compositions typically include a pharmaceutically acceptable carrier, adjuvant, or vehicle.
  • pharmaceutically acceptable refers to molecular entities and compositions that are generally believed to be physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • a pharmaceutically acceptable carrier, adjuvant, or vehicle is a composition that can be administered to a patient, together with a compound of the invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.
  • Exemplary conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles include saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • pharmaceutically acceptable carriers, adjuvants, and vehicles that can be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SED D S) such as d- a-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, wax
  • the OTUB 1 covalent binders or AMPK, cGAS or CFTR based bivalent compounds disclosed herein are defined to include pharmaceutically acceptable derivatives or prodrugs thereof.
  • a “pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, solvate, or prodrug, e.g., carbamate, ester, phosphate ester, salt of an ester, or other derivative of a compound or agent disclosed herein, which upon administration to a recipient is capable of providing (directly or indirectly) a compound described herein, or an active metabolite or residue thereof.
  • Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds disclosed herein when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species.
  • Preferred prodrugs include derivatives where a group that enhances aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein. Such derivatives are recognizable to those skilled in the art without undue experimentation. Nevertheless, reference is made to the teaching of Burger’s Medicinal Chemistry and Drug Discovery, 5 th Edition, Vol. 1: Principles and Practice, which is incorporated herein by reference to the extent of teaching such derivatives.
  • the OTUB1 covalent binders or AMPK, cGAS or CFTR based bivalent compounds disclosed herein include pure enantiomers, mixtures of enantiomers, pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates, mixtures of diastereoisomeric racemates and the meso-form and pharmaceutically acceptable salts, solvent complexes, morphological forms, or deuterated derivatives thereof.
  • pharmaceutically acceptable salts of the OTUB1 covalent binders or AMPK, cGAS or CFTR based bivalent compounds disclosed herein include, e.g., those derived from pharmaceutically acceptable inorganic and organic acids and bases.
  • Suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecyl sulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate, trifluoromethylsulfonate, and undecanoate.
  • Salts derived from appropriate bases include, e.g., alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N- (alkyl)4+ salts.
  • alkali metal e.g., sodium
  • alkaline earth metal e.g., magnesium
  • ammonium and N- (alkyl)4+ salts e.g., sodium
  • alkaline earth metal e.g., magnesium
  • ammonium and N- (alkyl)4+ salts e.g., alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N- (alkyl)4+ salts.
  • the invention also envisions the quaternization of any basic nitrogen-containing groups of the OTUB1 covalent binders or AMPK, cGAS and CFTR based bivalent compounds disclosed herein. Water or oil-soluble or dispersible products can be obtained by such qua
  • phrases “effective amount” and “effective to treat,” as used herein, refer to an amount or a concentration of one or more compounds or a pharmaceutical composition described herein utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome (e.g., treatment or prevention of cell growth, cell proliferation, or cancer).
  • pharmaceutical compositions can further include one or more additional compounds, drugs, or agents used for the treatment of cancer (e.g., conventional chemotherapeutic agents) in amounts effective for causing an intended effect or physiological outcome (e g., treatment or prevention of cell growth, cell proliferation, or cancer).
  • compositions disclosed herein can be formulated for sale in the United States, import into the United States, or export from the United States.
  • compositions disclosed herein can be formulated or adapted for administration to a subject via any route, e.g., any route approved by the Food and Drug Administration (FDA).
  • FDA Food and Drug Administration
  • Exemplary methods are described in the FDA Data Standards Manual (DSM) (available at http://www.fda.gov/Drugs/DevelopmentApprovalProcess/ FormsSubmissionRequirements/ElectronicSubmissions/DataStandardsManualmonographs).
  • DSM Food and Drug Administration
  • the pharmaceutical compositions can be formulated for and administered via oral, parenteral, or transdermal delivery.
  • parenteral includes subcutaneous, intracutaneous, intravenous, intramuscular, intraperitoneal, intra-articular, intra-arterial, intrasynovial, intrastemal, intrathecal, intralesional, and intracranial injection or infusion techniques.
  • compositions disclosed herein can be administered, e.g., topically, rectally, nasally (e.g., by inhalation spray or nebulizer), buccally, vaginally, subdermally (e.g., by injection or via an implanted reservoir), or ophthalmically.
  • compositions of this invention can be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.
  • carriers which are commonly used include lactose and com starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried com starch.
  • compositions of this invention can be administered in the form of suppositories for rectal administration.
  • These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
  • suitable non-irritating excipient include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.
  • compositions of this invention can be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, or other solubilizing or dispersing agents known in the art.
  • compositions of this invention can be administered by injection (e.g., as a solution or powder).
  • Such compositions can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, e.g., as a solution in 1,3 -butanediol.
  • acceptable vehicles and solvents that may be employed are mannitol, water, Ringer’s solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed, including synthetic mono- or diglycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, e.g., olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions.
  • Other commonly used surfactants such as Tweens, Spans, or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation.
  • an effective dose of a pharmaceutical composition of this invention can include, but is not limited to, e.g., about 0.00001, 0.0001, 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2500, 5000, or 10000 mg/kg/day, or according to the requirements of the particular pharmaceutical composition.
  • compositions disclosed herein include a combination of a compound of the formulae described herein (e.g., an OTUB1 covalent binder or an AMPK, cGAS or CFTR based bivalent compound) and one or more additional compounds (e.g., one or more additional compounds, drugs, or agents used for the treatment of cancer or any other condition or disease, including conditions or diseases known to be associated with or caused by cancer), both the compound and the additional compound should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen.
  • the additional agents can be administered separately, as part of a multiple dose regimen, from the compounds of this invention. Alternatively, those agents can be part of a single dosage form, mixed together with the compounds of this invention in a single composition.
  • compositions disclosed herein can be included in a container, pack, or dispenser together with instructions for administration.
  • the methods disclosed herein contemplate administration of an effective amount of a compound or composition to achieve the desired or stated effect.
  • the compounds or compositions of the invention will be administered from about 1 to about 6 times per day or, alternately or in addition, as a continuous infusion. Such administration can be used as a chronic or acute therapy.
  • the amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • a typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such preparations can contain from about 20% to about 80% active compound.
  • the present disclosure provides methods for using a composition comprising an 0TUB1 covalent binder or AMPK, cGAS or CFTR based bivalent compound, including pharmaceutical compositions (indicated below as ‘X’) disclosed herein in the following methods:
  • Substance X for use as a medicament in the treatment of one or more diseases or conditions disclosed herein e.g., cancer, referred to in the following examples as ‘Y’).
  • the methods disclosed include the administration of a therapeutically effective amount of one or more of the compounds or compositions described herein to a subject (e.g., a mammalian subject, e.g., a human subject) who is in need of, or who has been determined to be in need of, such treatment.
  • a subject e.g., a mammalian subject, e.g., a human subject
  • the methods disclosed include selecting a subject and administering to the subject an effective amount of one or more of the compounds or compositions described herein, and optionally repeating administration as required for the prevention or treatment of cancer.
  • subject selection can include obtaining a sample from a subject (e.g., a candidate subject) and testing the sample for an indication that the subject is suitable for selection.
  • the subject can be confirmed or identified, e.g. by a health care professional, as having had or having a condition or disease.
  • suitable subjects include, for example, subjects who have or had a condition or disease but that resolved the disease or an aspect thereof, present reduced symptoms of disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), or that survive for extended periods of time with the condition or disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), e.g., in an asymptomatic state (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease).
  • exhibition of a positive immune response towards a condition or disease can be made from patient records, family history, or detecting an indication of a positive immune response.
  • multiple parties can be included in subject selection.
  • a first party can obtain a sample from a candidate subject and a second party can test the sample.
  • subjects can be selected or referred by a medical practitioner (e.g., a general practitioner).
  • subject selection can include obtaining a sample from a selected subject and storing the sample or using the in the methods disclosed herein. Samples can include, e.g., cells or populations of cells.
  • methods of treatment can include a single administration, multiple administrations, and repeating administration of one or more compounds disclosed herein as required for the prevention or treatment of the disease or condition from which the subject is suffering (e.g., AMPK, cGAS or CFTR-related disease).
  • methods of treatment can include assessing a level of disease in the subject prior to treatment, during treatment, or after treatment. In some aspects, treatment can continue until a decrease in the level of disease in the subject is detected.
  • subject refers to any animal. In some instances, the subject is a mammal. In some instances, the term “subject,” as used herein, refers to a human (e.g., a man, a woman, or a child).
  • administer refers to implanting, ingesting, injecting, inhaling, or otherwise absorbing a compound or composition, regardless of form.
  • methods disclosed herein include administration of an effective amount of a compound or composition to achieve the desired or stated effect.
  • treat refers to partially or completely alleviating, inhibiting, ameliorating, or relieving the disease or condition from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder (e.g., cancer) are ameliorated or otherwise beneficially altered.
  • amelioration of the symptoms of a particular disorder refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with treatment by the compositions and methods of the present invention.
  • treatment can promote or result in, for example, a decrease in the number of tumor cells (e.g., in a subject) relative to the number of tumor cells prior to treatment; a decrease in the viability (e.g., the average/mean viability) of tumor cells (e.g., in a subject) relative to the viability of tumor cells prior to treatment; a decrease in the rate of growth of tumor cells; a decrease in the rate of local or distant tumor metastasis; or reductions in one or more symptoms associated with one or more tumors in a subject relative to the subject’s symptoms prior to treatment.
  • a decrease in the number of tumor cells e.g., in a subject
  • a decrease in the viability e.g., the average/mean viability
  • the rate of growth of tumor cells e.g., in a subject
  • a decrease in the rate of local or distant tumor metastasis e.g., the rate of local or distant tumor metastasis
  • prevent shall refer to a decrease in the occurrence of a disease or decrease in the risk of acquiring a disease or its associated symptoms in a subject.
  • the prevention may be complete, e.g., the total absence of disease or pathological cells in a subject.
  • the prevention may also be partial, such that the occurrence of the disease or pathological cells in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the present invention.
  • Exemplary AMPK, cGAS or CFTR-mediated diseases that can be treated with AMPK, cGAS or CFTR based DUBTACs include, for example, cystic fibrosis, breast cancer, ovarian cancer, prostate cancer, colon cancer, pancreatic cancer, bladder cancer, liver cancer and cervical cancer.
  • the term “preventing a disease” in a subject means for example, to stop the development of one or more symptoms of a disease in a subject before they occur or are detectable, e.g., by the patient or the patient’s doctor.
  • the disease e.g., cancer
  • the disease does not develop at all, i.e., no symptoms of the disease are detectable.
  • it can also result in delaying or slowing of the development of one or more symptoms of the disease.
  • it can result in the decreasing of the severity of one or more subsequently developed symptoms.
  • Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient’s disposition to the disease, condition or symptoms, and the judgment of the treating physician.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • a therapeutically effective amount of a therapeutic compound depends on the therapeutic compounds selected.
  • treatment of a subject with a therapeutically effective amount of the compounds or compositions described herein can include a single treatment or a series of treatments.
  • effective amounts can be administered at least once.
  • the compositions can be administered from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present.
  • the subject can be evaluated to detect, assess, or determine their level of disease.
  • treatment can continue until a change (e.g., reduction) in the level of disease in the subject is detected.
  • a maintenance dose of a compound, or composition disclosed herein can be administered, if necessary.
  • the dosage or frequency of administration, or both can be reduced, e.g., as a function of the symptoms, to a level at which the improved condition is retained.
  • Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • Example 1 4-acryloyl-l-(5-methylthiophen-2-yl)piperazin-2-one (XS154-91) To a solution of 2-bromo-5-methylthiophene (50.0 mg, 0.28 mmol) dissolved in dioxane (1 mL), N,N’- dimethylethylenediamine ( 9 pL, 0.0846 mmol, 0.3 eq), K 2 CO 3 ( 116.9 mg, 0.846 mmol, 3.0 eq), Cui ( 5.4 mg, 0.028 mmol, O.leq ) followed by tert-butyl 3 -oxopiperazine- 1 -carboxylate (84.7 mg, 0.42 mmol, 1.5 eq).
  • Example 2 4-acryloyl-l-(p-tolyl)piperazin-2-one (XS154-130).
  • Example 2 was synthesized following similar procedure for preparing example 1. White solid, 32% yield.
  • Example 4 4-acryloyl-l-(2-methylpyrimidin-4-yl)piperazin-2-one (XS154-149).
  • Example 4 was synthesized following similar procedure for preparing example 1. White solid, 46% yield.
  • MS (ESI) [M+H] + 247.3.
  • Example 5 l-(4-(»/-tolyl)piperazin-l-yl)prop-2-en-l-one (XS159-19).
  • Xantphos 17.3 mg, 0.3 mmol, 0.3 eq
  • Cs 2 CO 3 67%
  • Pd2(dba) 3 91.6 mg, 0.1 mmol, 0.1 eq
  • /cvZ-butyl 3 -oxopiperazine- 1 -carboxylate 279.6 mg, 1.5 mmol, 1.5 eq.
  • the reaction mixture was stirred at 100 °C under nitrogen atmosphere overnight. After cooling down to rt, resulting crude mixtures were purified via silica gel column chromatography to yield intermediate 2.
  • Example 6 3-(3-(4-acryloyl-2-oxopiperazin-l-yl)phenyl)propanoic acid (XS159-13).
  • N,N’ -dimethylethylenediamine 32 pL, 0.3 mmol, 0.3 eq
  • K 2 CO 3 414.6 mg, 3.0 mmol, 3.0 eq
  • Cui 19.1 mg, 0.1 mmol, 0.
  • Example 7 l-acryloyl-4-(5-methylfuran-2-yl)-l,4-diazepan-5-one (XS159-107).
  • Example 7 was synthesized following similar procedure for preparing example 1. White solid, 27% yield.
  • MS (ESI) [M+H] + 249.2.
  • Example 8 4-:icryloyl-l-( L2-diinethyl-l//-iniidazol-5-yl)piperazin-2-one (XS165-30).
  • Example 8 was synthesized following similar procedure for preparing example 1. White solid, 19% yield.
  • MS (ESI) [M+H] + 235.3.
  • Example 9 4-acryloyl-l-(6-methylpyridin-2-yl)piperazin-2-one (XS159-90) .
  • Example 9 was synthesized following similar procedure for preparing example 1. White solid, 53% yield.
  • Example 12 1 -(3-(zM-tolyl)-3,8-diazabicyclo[3.2.1 ] octan-8-yl)prop-2-en-1 -one(XS 165-33).
  • Example 13 l-(8-(m-tolyl)-3,8-diazabicydo[3.2.1 ]octan-3-yl)prop-2-en-l-one (XS165-54).
  • Example 13 was synthesized following similar procedure for preparing example 5. White solid, 17% yield.
  • Example 14 4-acryloyl-l-(2-methylthiazol-5-yl)piperazin-2-one (XS154-184).
  • Example 13 was synthesized following similar procedure for preparing example 1. White solid, 43% yield.
  • Example 15 4-(but-2-ynoyl)-l-(m-tolyl)piperazin-2-one (XS165-75).
  • Example 15 was synthesized following similar procedure for preparing example 1. Colorless oil, 19% yield.
  • Example 17 4-(but-2-ynoyl)-l-(5-methylthiophen-2-yl)piperazin-2-one (XS165-127).
  • Example 17 was synthesized following similar procedure for preparing example 1. Colorless oil, 14% yield.
  • MS (ESI) [M+H] + 263.1.
  • Example 19 4-(2-fluoroacryloyl)-l-(5-methylthiazol-2-yl)piperazin-2-one (XS165-112).
  • Example 19 was synthesized following similar procedure for preparing example 18. White solid, 40% yield.
  • 1 H NMR (400 MHz, Methanol-d4) 8 7.15 (s, 1H), 5.44 - 5.29 (m, 1H), 5.29 - 5.24 (m,
  • Example 20 4-(2-fluoroacryloyl)-l-(m-tolyl)piperazin-2-one (XS165-97).
  • Example 20 was synthesized following similar procedure for preparing example 18. White solid, 56% yield.
  • Example 21 2-fluoro-l-(3-(6-methylpyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)prop-2- en-l-one (XS165-118).
  • Example 21 was synthesized following similar procedure for preparing example 18. White solid, 45% yield.
  • Example 22 4-(2-fluoroacryloyl)-l-(6-methylpyridin-2-yl)piperazin-2-one (XS165-110).
  • Example 23 4-(2-chloroacetyl)-l-(5-methylthiophen-2-yl)piperazin-2-one (XS165-119).
  • Example 26 4-(2-chloroacetyl)-l-(6-methylpyridin-2-yl)piperazin-2-one (XS165-120).
  • Example 26 was synthesized following similar procedure for preparing example 18. White solid, 35% yield.
  • MS (ESI) [M+H] + 268.1.
  • Example 27 2-chloro-l-(3-(6-methylpyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)ethan-l- one (XS165-121).
  • Example 27 was synthesized following similar procedure for preparing example 18. White solid, 47% yield.
  • Example 28 (£)-4-(4-(dimethylamino)but-2-enoyl)-l-(5-methylfuran-2-yl)piperazin-2-one (XS165-100).
  • 2-bromo-5-methylfuran(50.0 mg, 0.28 mmol) dissolved in dioxane (1 mL) N,N’ -dimethylethylenediamine ( 9 ⁇ L, 0.0846 mmol, 0.3 eq)
  • K2CO3 116.9 mg, 0.846 mmol, 3.0 eq
  • Cui 5.4 mg, 0.028 mmol, O.
  • Example 29 was synthesized following similar procedure for preparing example 28. White solid, 54% yield.
  • MS (ESI) [M+H] + 302.1.
  • Example 31 (£)-4-(4-(dimethylamino)but-2-enoyl)-l-(6-methylpyridin-2-yl)piperazin-2-one (XS165-109).
  • Example 31 was synthesized following similar procedure for preparing example 28. White solid, 47% yield.
  • Example 34 (E')-l-(5-methylthiophen-2-yl)-4-(4-(pyrrolidin-l-yl)but-2-enoyl)piperazin-2- one (XS165-154).
  • Example 34 was synthesized following similar procedure for preparing example 28. White solid, 58% yield.
  • Example 35 (E)-l-(5-methylthiophen-2-yl)-4-(4-(piperidin-l-yl)but-2-enoyl)piperazin-2-one (XS165-170).
  • Example 35 was synthesized following similar procedure for preparing example 28. White solid, 55% yield.
  • Example 37 (E')-4-(dimethylamino)-l-(4-(5-methylthiophen-2-yl)piperazin-l-yl)but-2-en-l- one (XS165-172).
  • Example 37 was synthesized following similar procedure for preparing example 28. White solid, 33% yield.
  • MS (ESI) [M+H] + 294.4.
  • Example 39 (E)-4,4-dimethyl-2-(4-(5-methylthiophen-2-yl)-3-oxopiperazine-l- carbonyl)pent-2-enenitrile (XS165-177).
  • Example 39 was synthesized following similar
  • Example 46 (E)-4-(4-(dimethylamino)but-2-enoyl)-l -(3-methylthiophen-2-yl)piperazin-2- one (XS175-68).
  • Example 46 was synthesized following similar procedure for preparing example 28. White solid, 51% yield.
  • Example 47 (£)-4-(4-(dimethylamino)but-2-enoyl)-l-(4,5-dimethylthiophen-2-yl)piperazin- 2-one (XS175-70).
  • Example 47 was synthesized following similar procedure for preparing example 28. White solid, 53% yield.
  • Example 48 (E)-l-(4-(dimethylamino)but-2-enoyl)-W-(5-methylthiophen-2-yl)azetidine-3- carboxamide (XS175-71) .
  • Example 48 was synthesized following similar procedure for preparing example 28. White solid, 24% yield.
  • Example 49 (E)-4-(dimethylamino)-JV-(2-((5-methylthiophen-2-yl)amino)-2-oxoethyl)but-2- enamide (XS175-76).
  • Example 49 was synthesized following similar procedure for preparing example 28. White solid, 20% yield.
  • MS (ESI) [M+H] + 282.5.
  • Example 52 l-(2-chloroacetyl)-JV-(5-methylthiophen-2-yl)azetidine-3-carboxamide (XS175- 126).
  • Example 52 was synthesized following similar procedure for preparing example 28. White solid, 33% yield.
  • MS (ESI) [M+H] + 273.1.
  • Example 53 (E)-l-(4-(dimethylamino)but-2-enoyl)-A-(5-methylthiophen-2-yl)piperidine-4- carboxamide (XS175-132). ). To a solution of the 5-methylthiophen-2-amine (113.2 mg, 1 mmol) dissolved in DMF (2 mL), HATU (418.2 mg, 1.1 mmol, 1.1 eq), DIPEA (0.18 mL, 2.0 mmol, 2.0 eq), followed by l-(terZ-butoxycarbonyl)piperidine-4-carboxylic acid (229.3 mg, 1.0 mmol, 1.0 eq). The reaction mixture was stirred at rt 3 h.
  • Example 54 was synthesized following similar procedure for preparing example 53. White solid, 29% yield.
  • 'H NMR 400 MHz, Methanol-d 4 ) 8 6.73 - 6.56 (m, 2H), 6.40 (s, 2H), 3.85 (s, 2H), 3.83 - 3.37 (m, 4H), 3.20 - 3.04 (m, 1H), 2.80 (s, 6H), 2.27 (s, 3H), 2.25 - 1.94 (m, 2H).
  • MS (ESI) [M+H] + 322.
  • Example 56 was synthesized following similar procedure for preparing example 53. White solid, 37% yield.
  • MS (ESI) [M+H] + 336.2.
  • Example 57 (£)-l-(4-(dimethylamino)but-2-enoyl)-/V-(5-methylthiophen-2-yl)piperidine-2- carboxamide (XS175-148).
  • Example 57 was synthesized following similar procedure for preparing example 53. White solid, 25% yield.
  • Example 58 (E)-2-(4-(dimethylamino)but-2-enoyl)-JV-(5-methylthiophen-2-yl)-2- azaspiro[3.3]heptane-6-carboxamide (XS175-149).
  • Example 58 was synthesized following similar procedure for preparing example 53. White solid, 16% yield.
  • 'H NMR 400 MHz, Methanol- ⁇ ) 5 6.70 - 6.53 (m, 1H), 6.49 - 6.28 (m, 3H), 4.39 - 4.18 (m, 2H), 4.09 - 3.95 (m,
  • Example 61 (E)-4-(dimethylamino)-2V-methyl-iV-(3-(methyl(5-methylthiophen-2-yl)amino)- 3-oxopropyl)but-2-enamide (XS175-160) .
  • Example 61 was synthesized following similar procedure for preparing example 28. White solid, 23% yield.
  • J H NMR (400 MHz, Methanol-d 4 ) 8 6.86 - 6.70 (m, 2H), 6.64 (s, 1H), 6.40 (d, J 15.4 Hz, 1H), 4.
  • Example 62 (E)-l-(4-(dimethylamino)but-2-enoyl)-JV-(4-methylthiophen-2-yl)piperidine-4- carboxamide (XS175-173).
  • Example 62 was synthesized following similar procedure for preparing example 28. White solid, 46% yield.
  • Example 63 (E)-l-(4-(dimethylamino)but-2-enoyl)-JV,7V-bis(5-methylthiophen-2- yl)piperidine-4-carboxamide (XS175-174).
  • Example 63 was synthesized following similar procedure for preparing example 28. White solid, 13% yield.
  • Example 64 (E)-l-(4-(dimethylamino)but-2-enoyl)-W ⁇ V-bis(5-isopropylthiophen-2- yl)piperidine-4-carboxamide (XS175-175).
  • Example 64 was synthesized following similar procedure for preparing example 28. White solid, 19% yield.
  • Example 65 (£)-l-(4-(dimethylamino)but-2-enoyl)-A f ⁇ V-bis(5-isopropylthiophen-2- yl)pyrrolidine-3-carboxamide (XS175-176).
  • Example 65 was synthesized following similar procedure for preparing example 28. White solid, 20% yield.
  • Example 66 (E)-l-(4-(dimethylamino)but-2-enoyl)-/V-(5-isopropylthiophen-2-yl)piperidine- 4-carboxamide (XS175-178).
  • Example 66 was synthesized following similar procedure for preparing example 28. White solid, 49% yield.
  • Example 67 (E)-l-(benzo[Z>]thiophen-2-yl)-4-(4-(dimethylamino)but-2-enoyl)piperazin-2- one (XS175-179).
  • Example 67 was synthesized following similar procedure for preparing example 28. White solid, 46% yield.
  • MS (ESI) [M+H] + 344.4.
  • Example 70 (E)-4-(dimethylamino)-l-(6-(5-methylthiophen-2-yl)-2,6- diazaspiro[3.3]heptan-2-yl)but-2-en-l-one (XS186-3).
  • Example 70 was synthesized following similar procedure for preparing example 28. White solid, 23% yield.
  • MS (ESI) [M+H] + 306.2.
  • Example 73 l-(2-chloroacetyl)-A L (5-isopropylthiophen-2-yl)piperidine-4-carboxamide (XS185-6).
  • Example 73 was synthesized following similar procedure for preparing example 28. White solid, 30% yield.
  • Example 74 (£)-4-(4-(dimethylamino)but-2-enoyl)-l-(5-(piperidin-4-yl)thiophen-2- yl)piperazin-2-one (XS185-24)To a solution of tert-butyl 4-(5-brom othi ophen-2 -yl)piperidine-l- carboxylate (123.0 mg, 0.35 mmol) dissolved in dioxane (2 mL), N,N’-dimethylethylenediamine ( 9 pL, 0.105 mmol, 0.3 eq), K 2 CO 3 ( 147.2 mg, 1.05 mmol, 3.0 eq), Cui ( 6.8 mg, 0.035 mmol, O.leq ) followed by benzyl 3 -oxopiperazine- 1 -carboxylate (125.1 mg, 0.54 mmol, 1.5 eq).
  • reaction mixture was stirred at 100 °C under nitrogen atmosphere overnight. After cooling down to rt, resulting crude mixtures were purified via silica gel column then the product dissolved in MeOH (3 mL), and the Pd/C( 20 mg) were added. The reaction mixture stirred at hydrogen atmosphere. MeOH was removed then purified via silica gel column chromatography to yield intermediate 8.
  • Example 75 was synthesized following similar procedure for preparing example 28. White solid, 19% yield.
  • MS (ESI) [M+H] + 320.2.
  • Example 76 ( J E)-4-(4-(dimethylamino)but-2-enoyl)-l-(lH-indol-3-yl)piperazin-2-one (XS185-43).
  • Example 76 was synthesized following similar procedure for preparing example 28. White solid, 32% yield.
  • Example 77 was synthesized following similar procedure for preparing example 28. White solid, 17% yield.
  • MS (ESI) [M+H] + 342.4.
  • Example 78 was synthesized following similar procedure for preparing example 28. White solid, 19% yield.
  • 'H NMR 400 MHz, Methanol-tL
  • MS (ESI) [M+H] + 370.2.
  • Example 79 (E)-4-(dimethylamino)-l-(4-(5-methylthiophene-2-carbonyl)piperazin-l- yl)but-2-en-l-one (XS185-64).
  • 5-methylthiophene-2-carboxylic acid 75 mg, 0.5 mmol, 1.0 eq
  • HATU 209 mg, 0.55 mmol, 1.1 eq
  • DIPEA (0.18 mL, 2.0 eq
  • Example 80 (E)-4-(dimethylamino)-JV-(l-(5-methylthiophene-2-carbonyl)piperidin-4- yl)but-2-enamide (XS185-65).
  • Example 80 was synthesized following similar procedure for preparing example 79. White solid, 46% yield.
  • Example 82 (E')-4-(dimethylamino)-l-(6-(5-methylthiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-3-yl)but-2-en-l-one (XS185-67).
  • Example 82 was synthesized following similar procedure for preparing example 79. White solid, 57% yield.
  • Example 83 (E)-4-(dimethylamino)-l-(6-(5-methylthiophene-2-carbonyl)-2,6- diazaspiro[3.3]heptan-2-yl)but-2-en-l-one (XS185-69).
  • Example 83 was synthesized following similar procedure for preparing example 79. White solid, 19% yield.
  • Example 84 (E)-4-(dimethylamino)-l-(3-(5-methylthiophene-2-carbonyl)-3,8- diazabicyclo[3.2.1]octan-8-yl)but-2-en-l-one (XS185-70).
  • Example 84 was synthesized following similar procedure for preparing example 79. White solid, 46% yield. 1 H NMR (400
  • Example 85 (E)-4-(dimethylamino)-l-(8-(5-methylthiophene-2-carbonyl)-3,8- diazabicyclo [3.2. l]octan-3-yl)but-2-en- 1-one (XS185-71).
  • Example 85 was synthesized following similar procedure for preparing example 79. White solid, 49% yield.
  • Example 86 (E)-4-(dimethylamino)-l-(3-(5-methylthiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS185-72). White solid, 54% yield.
  • Example 86 was synthesized following similar procedure for preparing example 79.
  • Example 88 (£)-l-(benzofuran-2-yl)-4-(4-(dimethylamino)but-2-enoyl)piperazin-2-one (XS185-78)
  • Example 88 was synthesized following similar procedure for preparing example 28. White solid, 36% yield.
  • Example 89 (£)-l-(benzo[Z»]thiophen-3-yl)-4-(4-(dimethylamino)but-2-enoyl)piperazin-2- one (XS185-86).
  • Example 89 was synthesized following similar procedure for preparing example 28. White solid, 40% yield.
  • Example 92 (E)-/V-(benzo
  • Example 92 was synthesized following similar procedure for preparing example 53. White solid, 48% yield. 'H NMR (400 MHz,
  • Example 93 (E')-4-(dimethylamino)-l-(7-(5-methylthiophene-2-carbonyl)-2,7- diazaspiro[3.5]nonan-2-yl)but-2-en-l-one (XS185-96).
  • Example 93 was synthesized following similar procedure for preparing example 79. White solid, 16% yield.
  • Example 94 (E')-4-(dimethylamino)-l-(6-(5-methylthiophene-2-carbonyl)-2,6- diazaspiro[3.4]octan-2-yl)but-2-en-l-one (XS185-97).
  • Example 94 was synthesized following similar procedure for preparing example 79. White solid, 12% yield.
  • Example 95 (E)-4-(dimethylamino)-1-(7-(5-methylthiophen-2-yl)-2,7-diazaspiro[3.5]nonan- 2-yl)but-2-en-l-one (XS185-101).
  • Example 95 was synthesized following similar procedure for preparing example 28. White solid, 18% yield.
  • Example 97 (£)-4-(dimethylamino)-l-(2-(5-methylthiophen-2-yl)-2,7-diazaspiro[3.5]nonan- 7-yl)but-2-en-l-one (XS185-113).
  • Example 97 was synthesized following similar procedure for preparing example 53. White solid, 43% yield.
  • Example 98 (E)-2-(4-(dimethylamino)but-2-enoyl)-JV-(4,5,6,7-tetrahydrobenzo[/»]thiophen- 2-yl)-2-azaspiro[3.3]heptane-6-carboxamide (XS185-114).
  • Example 98 was synthesized following similar procedure for preparing example 53. White solid, 49% yield.
  • Example 99 (E)-4-(4-(dimethylamino)but-2-enoyl)-l-(lH-indol-2-yl)piperazin-2-one (XS185-116).
  • Example 99 was synthesized following similar procedure for preparing example 28. White solid, 37% yield.
  • Example 103 (E)-JN,N-dimethyl-4-(4-(5-methylthiophen-2-yl)-3-oxopiperazin-l-yl)but-2- enamide (XS185-134).
  • Example 103 was synthesized following similar procedure for preparing example 90. White solid, 19% yield.
  • MS (ESI) [M+H] 1 308.2.
  • Example 104 (2-(4-(5-methylthiophen-2-yl)-3-oxopiperazin-l-yl)-2-oxoethyl methylsulfamate (XS185-135).
  • Intermediate 1 (20 mg, 0.102 mmol) dissolved in DMF (1 mL), and the HATU( 42.7 mg, 0.112 mmol, 1.1 eq), DIPEA(0.1 mL), 2-hydroxyacetic acid(9.4 mg, 0.102 mmol, 1.0 eq) were added.
  • the reaction mixture stirred at rt 1 h, the crude mixtures were purified by HPLC yield the intermediate 10.
  • Example 105 was synthesized following similar procedure for preparing example 90. White solid, 30% yield.
  • MS (ESI) [M+Hf 262.1.
  • Example 106 l-(5-methylthiophen-2-yl)-4-(2-(phenylsulfonyl)acetyl)piperazin-2-one (XS185-140).
  • Example 106 was synthesized following similar procedure for preparing example 104. White solid, 35% yield.
  • Example 107 was synthesized following similar procedure for preparing example 28. White solid, 41% yield.
  • MS (ESI) [M+H] + 370.2.
  • Example 109 was synthesized following similar procedure for preparing example 28. White solid, 30% yield.
  • Example 110 (E)-7V-(l-(4-(dimethylamino)but-2-enoyl)azetidin-3-yl)-5-methylthiophene-2- carboxamide (XS185-171).
  • Example 110 was synthesized following similar procedure for preparing example 104.
  • Example 111 (E')-A / -(l-(4-(dimethylamino)but-2-enoyl)azetidin-3-yl)-5-methylthiophene-2- carboxamide (XS190-9).
  • Example 111 was synthesized following similar procedure for preparing example 79.
  • Example 112 (E)- ⁇ -( l-(4-(dimethylamin())biit-2-enoyl)azetidin-3-yl)-V.5- dimethylthiophene-2-carboxamide (XS190-27).
  • Example 112 was synthesized following similar procedure for preparing example 79.
  • Example 113 (E)-3-(benzo[b]thiopheii-2-yl)-8-(4-(dimethylamino)but-2-enoyl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS190-38).
  • Example 113 was synthesized following similar procedure for preparing example 28. White solid, 25% yield 1 H NMR (400 MHz, Methanol-d 4 )
  • Example 114 (E)-3-(benzo[/>]thiophen-3-yl)-8-(4-(dimethylamino)but-2-enoyl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS190-44).
  • Example 114 was synthesized following similar procedure for preparing example 28.
  • Example 115 (£)-8-(4-(dimethylamino)but-2-enoyl)-3-(5-phenylthiophen-2-yl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS190-45).
  • Example 115 was synthesized following similar procedure for preparing example 28.
  • Example 116 (£)-l-(3-(benzo[6]thiophene-2-carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)- 4-(dimethylamino)but-2-en-l-one (XS190-46).
  • Example 116 was synthesized following similar procedure for preparing example 79.
  • Example 117 (E)-l-(3-(benzo[Z>]thiophene-3-carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)- 4-(dimethylamino)but-2-en-l-one (XS190-47).
  • Example 117 was synthesized following similar procedure for preparing example 79.
  • Example 118 (E)-4-(dimethylamino)-l-(3-(4,5,6,7-tetrahydrobenzob]thiophene-2- carbonyl)-3,6-diazabicydo[3.1.1]heptan-6-yl)but-2-en-l-one (XS190-48).
  • Example 118 was synthesized following similar procedure for preparing example 79. White solid, 46% yield 1 H NMR (400 MHz, Methanol-d 4 ) 8 7.
  • Example 119 (E)-4-(dimethylamino)-l-(3-(4,5,6,7-tetrahydrobenzo[b]thiophene-3- carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS190-49).
  • Example 119 was synthesized following similar procedure for preparing example 79.
  • Example 120 (E)-l-(3-(5,6-dihydro-4//-cyclopenta[Z»]thiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS190-50).
  • Example 120 was synthesized following similar procedure for preparing example 79. White solid, 55% yield
  • Example 121 (£)-JV-(2-(6-(4-(dimethylamino)but-2-enoyl)-3,6-diazabicyclo[3.1.1Jheptane- 3-carbonyl)benzo[Z»]thiophen-5-yl)acetamide (XS190-59).
  • Example 121 was synthesized following similar procedure for preparing example 79.
  • Example 122 (E')-4-(dimethylamino)-l-(3-(5-(methylamino)benzo[B]thiophene-2- carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS190-68).
  • Example 122 was synthesized following similar procedure for preparing example 79.
  • Example 123 (£)-l-(3-(5-acetyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS190-69).
  • Example 123 was synthesized following similar procedure for preparing example 79.
  • Example 124 was synthesized following similar procedure for preparing example 79. White solid, 43% yield ’H NMR (400 MHz, Methanol-d) 4 ⁇ 6.94 - 6.63 (m, 2H), 6.47 - 6.32 (m, 1H), 5.10 - 4.94 (m, 1H), 4.46 (s, 2H), 4.03 - 3.83 (m, 3H), 3.81 - 3.75 (m, 1H), 3.75 - 3.69 (m, 1H), 3.69 - 3.53 (m, 2H),
  • Example 125 (E)-l-(4-(dimethylamino)but-2-enoyl)-Wmethyl-W(5-methylthiophen-2- yl)azetidine-3-carboxamide (XS190-75).
  • Example 125 was synthesized following similar procedure for preparing example 79.
  • Example 127 (E)-4-(dimethylamino)-l-(5-(5-methylthiophene-2-carbonyl)-2,5- diazabicyclo[2.2.2]octan-2-yl)but-2-en-l-one (XS190-77).
  • Example 127 was synthesized following similar procedure for preparing example 79.
  • Example 128 (£)-4-(dimethylamino)-l-((1S,4S)-5-(5-methylthiophene-2-carbonyl)-2,5- diazabicyclo[2.2.1]heptan-2-yl)but-2-en-l-one (XS190-80).
  • Example 128 was synthesized following similar procedure for preparing example 79.
  • Example 129 (E')-4-(dimethylamino)-l-((lR,4R)-5-(5-methylthiophene-2-carbonyl)-2,5- diazabicyclo[2.2.1]heptan-2-yl)but-2-en-l-one (XS190-81).
  • Example 129 was synthesized following similar procedure for preparing example 79.
  • Example 131 (£)-JV-((l-(4-(dimethylamino)but-2-enoyl)azetidin-2-yl)methyl)-5- methylthiophene-2-carboxamide (XS190-127).
  • Example 131 was synthesized following similar procedure for preparing example 79.
  • Example 132 (E)-l-(3-(cyclopentanecarbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)-4- (dimethylamino)but-2-en-l-one (XS190-128).
  • Example 132 was synthesized following similar procedure for preparing example 79.
  • Example 133 (E)-l-(3-(cyclohexanecarbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)-4- (dimethylamino)but-2-en-l-one (XS190-129).
  • Example 133 was synthesized following similar procedure for preparing example 79.
  • Example 134 (E)-.-N-((5-(6-(4-(dimethylamino)but-2-enoyl)-3,6-diazabicyclo[3.1.1]heptane-3- carbonyl)thiophen-2-yl)methyl)acetamide (XS190-130).
  • Example 134 was synthesized following similar procedure for preparing example 79.
  • Example 135 (E)-4-(dimethylamino)-l-(3-(5-methyloxazole-4-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS190-137).
  • Example 135 was synthesized following similar procedure for preparing example 79.
  • Example 136 (E)-4-(dimethylamino)-l-(3-(5-(methoxymethyl)thiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS190-138).
  • Example 136 was synthesized following similar procedure for preparing example 79.
  • Example 137 (E)-3-cyclopentyl-8-(4-(dimethylamino)but-2-enoyl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS190-144).
  • Example 137 was synthesized following similar procedure for preparing example 28.
  • Example 138 (E)-4-(dimethylamino)-l-(3-(5-methylfuran-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS190-157).
  • Example 138 was synthesized following similar procedure for preparing example 104.
  • Example 139 (E')-4-(dimethylamino)-l-(3-((5-methylthiophen-2-yl)methyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS190-158).
  • Example 139 was synthesized following similar procedure for preparing example 79.
  • Example 140 (E)-4-(dimethylamino)-l-(3-(3-methylcyclopentane-l-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS190-170).
  • Example 40 was synthesized following similar procedure for preparing example 79.
  • Example 141 (E)-8-(4-(dimethyIamino)but-2-enoyl)-3-(6-methylpyridin-2-yl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS190-181).
  • Example 141 was synthesized following similar procedure for preparing example 28.
  • Example 142 (E)-8-(4-(dimethylamino)but-2-enoyl)-3-(m-tolyl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS190-182).
  • Example 142 was synthesized following similar procedure for preparing example 28.
  • Example 143 (E')-8-(4-(dimethylamino)but-2-enoyl)-3-(5-methylthiazol-2-yl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS190-183).
  • Example 143 was synthesized following similar procedure for preparing example 28.
  • Example 146 (£)-8-(4-(dimethylamino)but-2-enoyl)-3-methyl-3,8-diazabicyclo[3.2.1]octan- 2-one (XS197-4).
  • Example 146 was synthesized following similar procedure for preparing example 28.
  • Example 147 (E)-8-(4-(dimethylamino)but-2-enoyl)-3-methyl-3,8-diazabicyclo[3.2.1]octan- 2-one (XS197-5).
  • Example 147 was synthesized following similar procedure for preparing example 79.
  • Example 148 (E)-l-(3-(l-acetylpiperidine-4-carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)-4- (dimethylamino)but-2-en-l-one (XS197-6).
  • Example 148 was synthesized following similar procedure for preparing example 79.
  • Example 149 (E)-8-(4-(dimethylamino)but-2-enoyl)-3-(5-methyl-lH-pyrrol-2-yl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS197-14).
  • Example 149 was synthesized following similar procedure for preparing example 28.
  • Example 151 (E)-l-(3-(cyclobutanecarbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)-4- (dimethylamino)but-2-en-l-one (XS197-38).
  • Example 151 was synthesized following similar procedure for preparing example 79.
  • Example 152 (E)-4-(dimethylamino)-l-(3-(5-isopropylthiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS197-49).
  • Example 152 was synthesized following similar procedure for preparing example 79.
  • Example 153 (£')-4-(dimethylamino)-l-(3-isobutyryl-3,6-diazabicyclo[3.1.1]heptan-6-yl)but- 2-en-l-one (XS197-50).
  • Example 153 was synthesized following similar procedure for preparing example 79.
  • Example 154 (£)-3-cyclobutyl-8-(4-(dimethylamino)but-2-enoyl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS197-70).
  • Example 154 was synthesized following similar procedure for preparing example 79.
  • Example 155 (£)-l-(3-(5-(l-acetylpyrrolidin-3-yl)thiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS197-71).
  • Example 155 was synthesized following similar procedure for preparing example 79.
  • Example 156 (£)-l-(3-(5-(l-acetylpiperidin-4-yl)thiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS197-72).
  • Example 156 was synthesized following similar procedure for preparing example 79.
  • Example 157 (£)-4-(dimethylamino)-l-(3-(5-(l-methylpyrrolidin-3-yl)thiophene-2- carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS197-73).
  • Example 157 was synthesized following similar procedure for preparing example 79.
  • Example 158 (£)-4-(dimethylamino)-l-(3-(5-(l-methylpiperidin-4-yl)thiophene-2-carbonyl)- 3,6-diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS197-74).
  • Example 158 was synthesized following similar procedure for preparing example 79.
  • Example 159 (£)-4-(dimethylamino)-l-(3-(l-methylazetidine-3-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS197-85).
  • Example 159 was synthesized following similar procedure for preparing example 79.
  • Example 160 (E')-4-(dimethylamino)-l-(3-(5-(3-methoxyprop-l-yn-l-yl)thiophene-2- carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS197-93).
  • Example 160 was synthesized following similar procedure for preparing example 79. White solid, 44% yield !
  • Example 161 (£)-4-(dimethylamino)-l-(3-(5-(3-hydroxyprop-l-yn-l-yl)thiophene-2- carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS197-94).
  • Example 161 was synthesized following similar procedure for preparing example 79.
  • Example 162 (£)-3-(5-(6-(4-(dimethylamino)but-2-enoyl)-3,6-diazabicyclo[3.1.1]heptane-3- carbonyl)thiophen-2-yl)prop-2-yn-l-yl acetate (XS197-96).
  • Example 162 was synthesized following similar procedure for preparing example 79.
  • Example 163 (E)-l-(3-(2-acetyl-2-azaspiro[3.3]heptane-6-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS197-133).
  • Example 164 (E)-3-(5-(l-acetylpiperidin-4-yl)thiophen-2-yl)-8-(4-(dimethylamino)but-2- enoyl)-3,8-diazabicyclo[3.2.1]octan-2-one (XS197-145).
  • Example 164 was synthesized following similar procedure for preparing example 28.
  • Example 166 8-(4-(dimethylamino)but-2-ynoyl)-3-(5-methylthiophen-2-yl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS197-185).
  • Example 166 was synthesized following similar procedure for preparing example 28.
  • Example 167 was synthesized following similar procedure for preparing example 79.
  • Example 168 (£)-l-(4-(dimethylamino)but-2-enoyl)-3-methyl-W-(5-methylthiophen-2- yl)azetidine-3-carboxamide (XS209-21).
  • Example 168 was synthesized following similar procedure for preparing example 53.
  • Example 169 (E')-l-(4-(dimethylamino)but-2-enoyl)-3-hydroxy-AN-(5-methylthiophen-2- yl)azetidine-3-carboxamide (XS209-22).
  • Example 169 was synthesized following similar procedure for preparing example 53.
  • Example 170 (/:)-l-(4-(dimetliylamiiio)biit-2-enioyl)-3-methyl-A-(5-methyloxazol-2- yl)azetidine-3-carboxamide (XS209-23).
  • Example 170 was synthesized following similar procedure for preparing example 53.
  • Example 174 (E)-4-(dimethylamino)-2V-(l-(5-methylthiophen-2-yl)-2-oxopiperidin-4-yl)but- 2-enamide (XS209-27).
  • Example 174 was synthesized following similar procedure for preparing example 28.
  • Example 175 (£)-4-(dimethylamino)-l-(3-propionyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)but- 2-en-l-one (XS209-28).
  • Example 175 was synthesized following similar procedure for preparing example 79.
  • Example 176 (£)-3-(cyclohex-l-en-l-yl)-8-(4-(dimethylamino)but-2-enoyl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS209-30).
  • Example 176 was synthesized following similar procedure for preparing example 28.
  • Example 177 (E)-l-(4-(dimethylamino)but-2-enoyl)-3-methyl-N-(5-methylthiazol-2- yl)azetidine-3-carboxamide (XS209-38).
  • Example 177 was synthesized following similar procedure for preparing example 53.
  • Example 178 (E)-l-(4-(dimethylamino)but-2-enoyl)-3-hydroxy-JV-(5-methylthiazol-2- yl)azetidine-3-carboxamide (XS209-39).
  • Example 178 was synthesized following similar procedure for preparing example 53.
  • Example 179 (E)-l-(3-butyryl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2- en-l-one (XS209-40).
  • Example 179 was synthesized following similar procedure for preparing example 79.
  • Example 180 (£)-l-(3-(2-acetyl-2-azaspiro[4.5]decane-8-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS209-53).
  • Example 180 was synthesized following similar procedure for preparing example 79.
  • Example 181 (E)-l-(3-(2-acetyl-2-azaspiro[3.5]nonane-7-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS209-54).
  • Example 181 was synthesized following similar procedure for preparing example 79. White solid, 21% yield !
  • Example 182 was synthesized following similar procedure for preparing example 79.
  • Example 183 (£')-4-(dimethylamino)-l-(5-(methylamino)isoindolin-2-yl)but-2-en-l-one (XS209-57).
  • Example 183 was synthesized following similar procedure for preparing example 79.
  • Example 184 (E)-4-(dimethylamino)-l-(5-(methylamino)isoindolin-2-yl)but-2-en-l-one (XS209-60).
  • Example 184 was synthesized following similar procedure for preparing example 79.
  • Example 185 was synthesized following similar procedure for preparing example 79.
  • Example 186 (E)-4-(dimethylamino)-l-(3-(5-methylthiazole-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS209-65).
  • Example 186 was synthesized following similar procedure for preparing example 79.
  • Example 187 8-(4-(dimethylamino)-4-methylpent-2-ynoyl)-3-(5-methylthiophen-2-yl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS209-74).
  • Example 187 was synthesized following similar procedure for preparing example 28.
  • Example 188 4-(dimethylamino)-4-methyl-l-(3-(5-methylthiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)pent-2-yn-l-one (XS209-75).
  • Example 188 was synthesized following similar procedure for preparing example 79. White solid, 34% yield !
  • Example 189 (£)-4-(dimethylamino)-l-(3-(5-methyloxazole-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS209-92).
  • Example 189 was synthesized following similar procedure for preparing example 79.
  • Example 190 l-(4-(dimethylamino)but-2-ynoyl)-3-hydroxy-/V-(5-methylthiophen-2- yl)azetidine-3-carboxamide (XS209-99).
  • Example 190 was synthesized following similar procedure for preparing example 53.
  • Example 193 (E)-8-(4-(dimethylamino)but-2-enoyl)-N-(5-methylthiophen-2-yl)-8- azabicyclo[3.2.1]octane-3-carboxamide (XS209-122).
  • Example 193 was synthesized following similar procedure for preparing example 53.
  • Example 194 (£)-JV-(8-(4-(dimethylamino)but-2-enoyl)-8-azabicyclo[3.2.1]octan-3-yl)-5- methylthiophene-2-carboxamide (XS209-139).
  • Example 194 was synthesized following similar procedure for preparing example 79.
  • Example 196 (E)-W-(4-(4-(dimethylamino)but-2-enamido)phenyl)-5-methylthiophene-2- carboxamide (XS209-165).
  • MS (ESI) [M+H] + 344.1.
  • Example 197 l-(3-butyryl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-yn- 1-one (XS209-166).
  • Example 197 was synthesized following similar procedure for preparing example 79.
  • Example 198 l-(3-acetyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)-4- methylpent-2-yn-l-one (XS209-167).
  • Example 198 was synthesized following similar procedure for preparing example 79.
  • Example 199 1 -(3-butyryl-3,6-diazabicyclo[3.1.1 ]heptan-6-yl)-4-(dimethylamino)-4- methylpent-2-yn-l-one (XS209-168).
  • Example 199 was synthesized following similar procedure for preparing example 79.
  • Example 200 l-(3-butyryl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)-4- methylpent-2-yn-l-one (XS209-174).
  • Example 200 was synthesized following similar procedure for preparing example 53.
  • Example 201 N-(benzo[b]thiophen-2-yl)-l-(4-(dimethylamino)but-2-ynoyl)-3- methylazetidine-3-carboxamide (XS209-175).
  • Example 201 was synthesized following similar procedure for preparing example 53.
  • Example 202 (E)-l-(4-(dimethylamino)but-2-enoyl)-3-hydroxy-iV-(4, 5,6,7- tetrahydrobenzo[Z>]thiophen-2-yl)azetidine-3-carboxamide (XS209-176).
  • Example 202 was synthesized following similar procedure for preparing example 53.
  • Example 203 (E)-l-(3-(5-(4-acetylpiperazin-l-yl)thiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS209-184).
  • Example 203 was synthesized following similar procedure for preparing example 79. White solid, 28% yield
  • Example 204 (E)-4-(dimethylamino)-l-(3-(5-(4-methylpiperazin-l-yl)thiophene-2- carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS209-185).
  • Example 204 was synthesized following similar procedure for preparing example 79.
  • Example 205 (E)-JV-(4-(4-(dimethylamino)but-2-enamido)cyclohexyl)-5-methylthiophene- 2-carboxamide (XS224-6).
  • Example 205 was synthesized following similar procedure for preparing example 79.
  • Example 206 2V-(benzo[/>]thiophen-2-yl)-l-(4-(dimethylamino)but-2-ynoyl)azetidine-3- carboxamide (XS224-106).
  • Example 206 was synthesized following similar procedure for preparing example 53.
  • Example 207 /V-(benzo
  • Example 207 was synthesized following similar procedure for preparing example 53.
  • Example 208 l-(4-(dimethylamino)but-2-ynoyl)-/V-(4,5,6,7-tetrahydrobenzo[Z>]thiophen-2- yl)azetidine-3-carboxamide (XS224-108).
  • Example 208 was synthesized following similar procedure for preparing example 53.
  • Example 210 l-(4-(dimethylamino)but-2-ynoyl)-A z -(5-methylthiophen-2-yl)azetidine-3- carboxamide (XS224-110).
  • Example 210 was synthesized following similar procedure for preparing example 53.
  • Example 212 l-(4-(dimethylamino)biit-2-ynoyl)- V-(5-isopropylthiophen-2-yl)azetidine-3- carboxamide (XS224-116).
  • Example 212 was synthesized following similar procedure for preparing example 53.
  • Example 213 l-(4-(dimethylamino)-4-methylpent-2-ynoyl)-/V-(5-isopropylthiophen-2- yl)azetidine-3-carboxamide (XS224-117).
  • Example 213 was synthesized following similar procedure for preparing example 53.
  • Example 216 (E')-4-(dimethylamino)-N -(l-(5-methylthiophene-2-carbonyl)azetidin-3- yl)but-2-enamide (XS224-143).
  • Example 216 was synthesized following similar procedure for preparing example 79.
  • Example 217 4-(dimethylamino)-2V-(l-(5-methylthiophene-2-carbonyl)azetidin-3-yl)but-2- ynamide (XS224-144).
  • Example 217 was synthesized following similar procedure for preparing example 79.
  • White solid, 29% yield 'HNMR (400 MHz, Methanol-d) 4 5 7.29 (d, J 3.2 Hz, 1H), 6.79 (s, 1H), 4.81 - 4.60 (m, 2H), 4.47 - 4.20 (m, 4H), 3.99 (s, 1H), 2.94 (s, 6H), 2.45 (s, 3H).
  • MS (ESI) [M+H] + 306.2.
  • Example 219 (E)-W-(3-(4-(dimethylamino)but-2-enamido)cyclobutyl)-5-methylthiophene-2- carboxamide (XS224-147).
  • Example 219 was synthesized following similar procedure for preparing example 79.
  • Example 223 (£)-N-(l-(4-(dimethylamino)but-2-enoyl)azetidin-3-yl)benzo[6]thiophene-2- carboxamide (XS224-154).
  • Example 223 was synthesized following similar procedure for preparing example 79.
  • Example 224 JV-(l-(4-(dimethylamino)but-2-ynoyl)azetidin-3-yl)benzo[/>]thiophene-2- carboxamide (XS224-155).
  • Example 224 was synthesized following similar procedure for preparing example 79.
  • Example 225 7V-(l-(4-(dimethylamino)-4-methylpent-2-ynoyl)azetidin-3- yl)benzo[/>]thiophene-2-carboxamide (XS224-156).
  • Example 225 was synthesized following similar procedure for preparing example 79.
  • Example 226 (E)-2V-(1-(4-(dimethylamino)but-2-enoyl)azetidin-3-yl)-4, 5,6,7- tetrahydrobenzo[Z>]thiophene-2-carboxamide (XS224-157).
  • Example 226 was synthesized following similar procedure for preparing example 79.
  • Example 227 2V-(l-(4-(dimethylamino)but-2-ynoyl)azetidin-3-yl)-4, 5,6,7- tetrahydrobenzo[Z>]thiophene-2-carboxamide (XS224-158).
  • Example 227 was synthesized following similar procedure for preparing example 79.
  • Example 228 /V-(l-(4-(dimethylamino)-4-methylpent-2-ynoyl)azetidin-3-yl)-4, 5,6,7- tetrahydrobenzo[b]thiophene-2-carboxamide (XS224-159).
  • Example 228 was synthesized following similar procedure for preparing example 79.
  • linkers 1-14 were synthesized following the reported procedures (Liu et al., 2022)
  • Example 229 A-(2-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)propanamido)ethyl)-5- ((6-chloro-5-(l-methyl-l/7-indol-5-yl)-l/7-benzo[c/]imidazol-2-yl)oxy)-2- methylbenzamide(XF137-81).
  • linker 1 (16.3 mg, 0.048 mmol, 1.0 equiv
  • EDCI 14 mg, 0.072 mmol, 1.5 equiv
  • HOAt 10 mg, 0.072 mmol, 1.5 equiv
  • NMM 15 mg, 0.14 mmol, 3.0 equiv
  • Example 230 JV-(3-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)propanamido)propyl)- 5-((6-chloro-5-( 1-methyl- l//-indol-5-yl)- 1H-benzo[d/]imidazol-2-yl)oxy)-2-methylbenzamide (XF137-82).
  • Example 230 was synthesized following the standard procedure for preparing example 229 from activator 991 (28 mg, 0.06 mmol) and linker 2 (23 mg, 0.06 mmol, 1.0 equiv). Brown solid (15.4 mg, yield 34%).
  • Example 231 7V-(4-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)propanamido)butyl)-5- ((6-chloro-5-(l-methyl-l//-indol-5-yl)-lH -benzo[ ⁇ 7
  • Example 231 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and liner 3 (17 mg, 0.048 mmol, 1.0 equiv). Brown solid (7.6 mg, yield 20%).
  • Example 233 JV-(6-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)propanamido)hexyl)-5- ((6-chloro-5-(l-methyl-lH -indol-5-yl)-lH-benzo[d]imidazol-2-yl)oxy)-2-methylbenzamide (XF137-85).
  • Example 233 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 5 (18.7 mg, 0.048 mmol, 1.0 equiv).
  • Example 234 2V-(7-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)propanamido)heptyl)- 5-((6-chloro-5-(l-methyl-l//-indol-5-yl)-l//-benzo[t/
  • Example 234 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 6 (19 mg, 0.048 mmol, 1.0 equiv). Brown solid (12.6 mg, yield 32%).
  • Example 235 JV-(8-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)propanamido)octyl)-5- ((6-chloro-5-(l-methyl-17/-indol-5-yl)-177-benzo[7]imidazol-2-yl)oxy)-2-methylbenzamide (XF137-87).
  • Example 235 was synthesized following the standard procedure for preparing Example 229 from activator 991 (21 mg, 0.048 mmol) and linker 7 (20 mg, 0.048 mmol, 1.0 equiv). Brown solid (9.6 mg, yield 24%).
  • Example 236 JV-(9-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)propanamido)nonyl)-5- ((6-chloro-5-(l-methyl-177-indol-5-yl)-177-benzo[ ⁇ 7
  • Example 236 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 8 (21 mg, 0.048 mmol, 1.0 equiv). Brown solid (12.9 mg, yield 32%).
  • Example 237 2V-(10-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yI)propanamido)decyl)- 5-((6-chloro-5-(l-methyl-17/-indol-5-yl)-177-benzo[t/]imidazol-2-yl)oxy)-2-methylbenzamide (XF137-89).
  • Example 237 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 9 (21 mg, 0.048 mmol, 1.0 equiv). Brown solid (12.8 mg, yield 31%).
  • Example 238 2V-(2-(2-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2- yl)propanamido)ethoxy)ethyl)-5-((6-chloro-5-(l-methyl-l/7-indol-5-yl)-l//- benzo [d/
  • Example 238 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 10 (18.1 mg, 0.048 mmol, 1.0 equiv).
  • Example 239 JV-(2-(2-(2-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2- yl)propanamido)ethoxy)ethoxy)ethyl)-5-((6-chloro-5-(l-methyl-l/7-indol-5-yl)-EH- benzo[d]imidazol-2-yl)oxy)-2-methylbenzamide (XF137-91).
  • Example 239 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 11 (20.2 mg, 0.048 mmol, 1.0 equiv). Brown solid (21.8 mg, yield 54%).
  • Example 240 JV-(15-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)-13-oxo-3,6,9-trioxa-12- azapentadecyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-17/-benzo[t/
  • Example 240 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 12 (22 mg, 0.048 mmol, 1.0 equiv). Brown solid (22.9 mg, yield 61%).
  • Example 241 JV-(18-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)-16-oxo-3,6,9,12- tetraoxa-15-azaoctadecyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzo[d ]imidazol-2- yl)oxy)-2-methylbenzamide (XF137-93).
  • Example 241 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 13 (24 mg, 0.048 mmol, 1.0 equiv).
  • Example 242 A-(21-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)-19-oxo-3,6,9,12,15- pentaoxa-18-azahenicosyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-177-benzo[d
  • Example 242 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 14 (27 mg, 0.048 mmol, 1.0 equiv).
  • Linker 15 (5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzo[d]imidazol-2-yl)oxy)-2- methylbenzoyl)glycine
  • activator 991 50 mg, 0.12 mmol
  • DMF dimethylaminopropyl
  • EDCI l-ethyl-3-(3- dimethylaminopropyl)carbodiimide
  • EDCI 34.3 mg, 0.18 mmol, 1.5 equiv
  • l-hydroxy-7- azabenzo-triazole HO At, 24.5 mg, 0.18 mmol, 1.5 equiv
  • NMM N-methylmorpholine
  • Linker 17 4-(5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzo[d
  • imidazol-2-yl)oxy)-2- methylbenzamido)butanoic acid MS (ESI) [M+H] + 517.4.
  • Linker 19 6-(5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzod/
  • imidazol-2-yl)oxy)-2- methylbenzamido)hexanoic acid MS (ESI) [M+H] + 545.3.
  • Example 110 (E)-N -(l-(4-(dimethylamino)but-2-enoyl)azetidin-3-yl)-5-methylthiophene-2- carboxamide (XS185-171).
  • Example 110 was synthesized following similar procedure for preparing example 104.
  • Linker 20 7-(5-((6-chloro-5-(l-methyl-1H-indol-5-yl)- 1H-benzo[d
  • imidazol-2-yl)oxy)-2- methylbenzamido)heptanoic acid MS (ESI) [M+H] + 559.4.
  • Linker 23 3-(2-(5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-l/7-benzo[ ⁇ Z
  • iniidazol-2-yl)oxy)-2- methylbenzamido)ethoxy)propanoic acid MS (ESI) [M+H] + 548.6.
  • Linker 24 3-(2-(2-(5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzo[d/
  • iniidazol-2-yl)oxy)- 2-methylbenzamido)ethoxy)ethoxy)propanoic acid MS (ESI) [M+H] + 591.4.
  • Linker 25 l-(5-((6-chloro-5-(l-methyl-l//-indol-5-yl)-177-benzo[d
  • iniidazol-2-yl)oxy)-2- methylphenyl)-l-oxo-5,8,ll-trioxa-2-azatetradecan-14-oic acid MS (ESI) [M+H] + 635.4.
  • Linker 26 l-(5-((6-chloro-5-(l-methyl-l//-indol-5-yl)-177-benzo[d
  • iniidazol-2-yl)oxy)-2- methylphenyl)-l-oxo-5,8,ll,14-tetraoxa-2-azaheptadecan-17-oic acid MS (ESI) [M+H] + 679.4.
  • Linker 28 JV-(2-aminoethyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-lH-benzo[d ]imidazol- 2-yl)oxy)-2-methylbenzamide (XH168-164).
  • Linker 29-41 was synthesized following the same procedure for preparing linker 28.
  • Linker 29 N -(3-aminopropyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl )-1H- benzo[*/]imidazol-2-yl)oxy)-2-methylbenzamide (XH168-165). Pink solid, 52 mg, 61% yield.
  • Linker 33 N-(7-aminoheptyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H- benzo[d
  • Linker 36 JV-(10-aminodecyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-EH- benzo[rf
  • imidazol-2-yl)oxy)-2-methylbenzamide (XH168-172) Yellow solid, 75 mg, 87% yield. MS (ESI) [M+H] + 586.5.
  • Linker 37 N -(2-(2-aminoethoxy)ethyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H- benzo
  • d]imidazol-2-yl)oxy)-2-methylbenzamide (XH168-173). Yellow solid, 65 mg, 73% yield. MS (ESI) [M+H] + 518.1.
  • Example 244 (E)-5-((6-chloro-5-(l-methyl-1H/-indol-5-yl)-1H-benzo[d
  • Example 244 was synthesized following the same procedure for preparing example 243 from linker 21. White solid, 9 mg, 50% yield.
  • Example 245 (E)-5-((6-chloro-5-(l-methyl-1Hindol-5-yl)-1Hbenzo[d
  • Example 245 was synthesized following the same procedure for preparing example 243 from linker 22. White solid, 9 mg, 45% yield.
  • Example 246 (E)-l-(5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-l//-benzo[d
  • Example 246 was synthesized following the same procedure for preparing example 243 from linker 27. White solid, 8 mg, 40% yield.
  • Example 248 (E)-A-(2-((5-((6-chloro-5-(l-methyl-17/-indol-5-yl)-1H-benzo[cZ
  • Example 248 was synthesized following the same procedure for preparing example 247 from linker 33. White solid 7 mg, 43% yield.
  • Example 249 (E)-N-(2-((5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzo[d
  • Example 249 was synthesized following the same procedure for preparing example 247 from linker 34. White solid, 10 mg, 48% yield.
  • Example 250 (E)-N-(2-((5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzo[d
  • Example 250 was synthesized following the same procedure for preparing example 247 from linker 35. White solid 9 mg, 50% yield.
  • Example 251 (E )-N-(2-((5-((6-chloro-5-(1-methyl-1H-indol-5-yl)-1H-benzo
  • Example 251 was synthesized following the same procedure for preparing example 247 from linker 36. White solid 7 mg, 40% yield.
  • Linker 48 10-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3- b]indol-9-yl)-l/Z-pyrazol-l-yl)ethyl)amino)-10-oxodecanoic acid. Brown solid, yield 60%.
  • Linker 49 1 l-((2-(3-(6.7-dichloro-2-(2-hydroxya cetyl)-2.3.4.5-tetrahydro-l//-pyrido
  • Linker 50 12-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3- />Jindol-9-yl)-l//-pyrazol-l-yl)ethyl)amino)-12-oxododecanoic acid. Brown solid, yield 45%.
  • Linker 51 3-(3-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3- b]indol-9-yl)-1H-pyrazol-l-yl)ethyl)amino)-3-oxopropoxy)propanoic acid. Brown solid, yield 46%.
  • Linker 52 3-(2-(3-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-6]indol-9-yl)-l//-pyrazol-l-yl)ethyl)amino)-3-oxopropoxy)ethoxy)propanoic acid Brown solid, yield 49%.
  • Linker 53 l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-i]indol- 9-yl)-1H-pyrazol-l-yl)-4-oxo-7,10,13-trioxa-3-azahexadecan-16-oic acid.
  • Brown solid yield .40 (m, Linker 54: l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3-b]indol- 9-yl)-1H-pyrazol-l-yl)-4-oxo-7,10,13,16-tetraoxa-3-azanonadecan-19-oic acid. Brown solid, yield 53 %.
  • Linker 55 l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3-/>]indol- 9-yl)-lJ/-pyrazol-l-yl)-4-oxo-7,10,13,16,19-pentaoxa-3-azadocosan-22-oic acid. Brown solid, yield 47 %.
  • Linker 56 2-amino-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indoI-9-yl)-l/7-pyrazol-l-yl)ethyl)acetamide To a solution of intermediate 19 (65 mg, 0.
  • Linker 57 - 70 were synthesized following the same procedure for preparing linker 56.

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Abstract

Disclosed are compounds that are irreversible small-molecule binders of OTUB1 (also known as Otubain 1), and bivalent compounds (e.g. bi-functional small molecule compounds) which recruit OTUB1 through the irreversible small-molecule binders of OTUB1 to de-ubiquitinylate and stabilize proteins such as AMPK, cGAS and CFTR. Also disclosed are methods for the treatment of AMPK, cGAS and CFTR-mediated diseases in a subject in need thereof. Also disclosed are methods for designing such compounds including irreversible small-molecule binders of OTUB1 and related bivalent compounds as de-ubiquitinase-targeting chimeras (DUBTACs) for targeted protein stabilization.

Description

OTUB1 Small-Molecule Binders and OTUBl-recruiting Deubiquitinase- targeting Chimeras (DUBTACs)
STATEMENT REGARDING GOVERNMENT FUNDING
This invention was made with government support under R35CA253027 awarded by the National Institutes of Health. The government has certain rights in the invention.
CROSS-REFERENCE TO RELATED APPLICATION
The present application claims priority to U.S. Provisional Application Serial No. 63/497,651, filed on April 21, 2023, the contents of which are hereby incorporated by reference in their entirety.
TECHNICAL FIELD
The present disclosure is directed to compounds that are irreversible small-molecule binders of OTUB1 (also known as Otubain 1), and bivalent compounds (e.g. bi-functional small molecule compounds) which recruit OTUB1 through the irreversible small-molecule binders of OTUB1 to de-ubiquitinylate and stabilize proteins such as AMPK, cGAS and CFTR. The present disclosure is also related to methods for the treatment of AMPK, cGAS and CFTR-mediated diseases in a subject in need thereof. The disclosure also relates to methods for designing such compounds including irreversible small-molecule binders of OTUB1 and related bivalent compounds as deubiquitinase-targeting chimeras (DUBTACs) for targeted protein stabilization (TPS).
BACKGROUND
Protein ubiquitination plays a critical role in a variety of cellular process, like protein degradation, quality control, trafficking, and signaling. Therefore, controlling the ubiquitination level of disease related proteins can achieve substantial therapeutic effects. For example, inducing the ubiquitination of oncoproteins such as Myc and Ras to lead to their degradation would greatly benefit related cancer therapy. Similarly, deubiquitination and stabilization of proteins such as tumor suppressors would also be greatly beneficial for the treatment of relevant diseases including cancer.
5' adenosine monophosphate-activated protein kinase (also known as AMPK) is an enzyme that plays key roles in cellular energy homeostasis. Recent studies identified AMPK as regulating diverse metabolic and physiological processes. However, it is downregulated in major chronic diseases, such as obesity, inflammation, diabetes and cancer. Therefore, stabilization of AMPK in the cellular context may enhance AMPK activation, and has the potential to provide a novel therapeutic strategy to treat such diseases (Jeon, 2016).
The protein stabilization strategy could also be applied to other targets, such as cyclic GMP-AMP (cGAMP) synthase (also known as cGAS). The cGAS-stimulator of interferon genes (STING) pathway is a component of the innate immune system. cGAS detects the presence of cytosolic DNA and, in response, triggers expression of inflammatory genes that can lead to senescence or to the activation of defense mechanisms. In tumor cells, the cGAS-STING pathway exerts its antitumor effects through the induction of a robust type I IFN response, which activates immune cells, particularly dendritic cells (DCs), in the tumor micro environment (TME) (Samson and Ablasser, 2022).
In addition to AMPK and cGAS, Cystic fibrosis transmembrane conductance regulator (CFTR) is another suitable protein target for stabilization. CFTR AF508 mutation destabilizes CFTR, and leads to the cystic fibrosis phenotype (Ward et al., 1995). Therefore, deubiquitination of AF508 mutant CFTR could be an effective strategy to prevent the cystic fibrosis process.
The targeted protein degradation (TPD) field has made great progress over the past 10 years. PROteolysis TArgeting Chimera (PROTAC) technology has been widely utilized in the drug discovery field (Dale et al., 2022). Deubiquitinase-targeting chimera (DUBTAC), which hijacks a cellular deubiquitinase to remove the polyubiquitin attached to the target protein by inducing close proximity between the target protein and deubiquitinase, recently emerged as an effective technology to improve target protein stability by reducing the proteasomal degradation of the target protein (Henning et al., 2022; Liu et al., 2022). DUBTACs provide a novel therapeutic approach for treating various diseases.
SUMMARY The present disclosure includes two parts: (A) small molecules that covalently bind the deubiquitinase OTUB1, and (B) heterobifunctional small molecules that stabilize AMPK, cGAS, or CFTR by recruiting OTUB 1.
A. OTUB1 irreversible small-molecule binders
OTUB1 (also known as OUT domain-containing ubiquitin aldehyde-binding protein 1) is a K48 linkage-specific deubiquitinating enzyme. There are very few small molecules reported to bind OTUB1, including the covalent binder EN523 (Henning et al., 2022), and reversible inhibitor compound 61 (Tan et al., 2022), ML364 (WO2020223551A1). However, EN523 has low binding affinity to OTUB1, and compound 61 and ML364 inhibit the enzymatic activity of OTUB1. Therefore, development of more effective OTUB1 small-molecule binders that lead to effective DUBTACs is needed.
Described herein are small molecules that covalently modify OTUB1.
In one aspect, the present disclosure features small molecules that covalently modify OTUB1 of Formula (A-I) below:
Figure imgf000005_0001
Formula (A-I) or a tautomer thereof, or a pharmaceutically acceptable salt of the compound or the tautomer, wherein
AD is selected from N, or CRD 2;
BD is selected from C=O, CH2, CH RD2, or C(RD2)2;
Ring CD is absent, or selected from C3-C12 cycloalkyl, 3-12-membered heterocyclic, C6- C10 aryl, and 5-10 membered heteroaryl;
Ring DD is a saturated or partially unsaturated 4-12 membered heterocyclic;
FD is selected from N, or CRD2;
LD1 is a bond, or a bivalent group selected from -O-, -NRD5-, -C(O)-, -C(O)O-, -C(O)NRD5-, -NRD5C(O)-, -OC(O)NRD5-, -NRD5C(O)O-, -NRD 6C(O)NRD 5-, -S(O)-, -S(O)2-, -S(O)NRD5-, - S(O)2NRD5-, CI-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, and C2-C6 alkynylene;
LD2 is selected from -C(O)-, -S(O)-, -S(O)2-, -NRD5C(O)-, and C1-C6 alkylene;
LD3 is a bond, or a bivalent group selected from C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, and C2-C6 alkynylene;
RD1 is selected from C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2- C6, heteroalkynyl, partially unsaturated C4-C cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD7, NRD7RD8, C(O)NRD7RD8, C(O)ORD7, tri(Ci-C3alkyl) silyl, C1-C6 alkyl, C1-C6 heteroalkyl, and 3-8 membered heterocyclic; each RD2 is independently selected from hydrogen, halogen, cyano, ORD7, NRD7RD8, Ci- C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C1-C6 alkynyl, C2-C6 heteroalkynyl, C3-C8 cycloalkyl, and 3-8 membered heterocyclic; or two RD2 groups, together with the atom(s) to which they are connected, optionally form a C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, where each said C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring DD, can optionally form fused rings or bridged rings; each RD3 is selected from hydrogen, halogen, cyano, ORD7, NRD7RD8, C(O)ORD7, C(O)NRD7RD8, -OC(O)NRD7RD8, - NRD9C(O)NRD7RD8-, -S(O)NRD 7RD8-, -S(O)2NRD7RD8-, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C1-C6 alkynyl, C3-C12 cycloalkyl, 3-12- membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; or two RD 3 groups, together with the atom(s) to which they are connected, optionally form a C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, where each said C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring CD, optionally form fused rings or bridged rings;
RD4 is RD 4a or RD 4b;
RD 4a is selected from hydrogen, ORD7, NRD7RD8, C(O)RD7, C(O)ORD7, C(O)NRD7RD8, - OC(O)NRD7RD8, -NRD9C(O)NRD7RD8-, S(O)RD7, S(O)2RD7, S(O)NRD7RD8, S(O)2NRD 7RD8, C1-C6 alkyl, Ci-Ce haloalkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12- membered heterocyclic, Ce-Cw aryl, and 5-10 membered heteroaryl;
Ro4b is a bivalent group that connect to the Linker moiety in Formula (B), and Ro4b is selected from a bond, -O-, -N-, -C(O)-, -C(O)O-, -C(O)NRD 7-, -OC(O)NRD 7-, -NRD 9C(O)NRD 7-, -S(O)-, -S(O)2-, -S(O)NRD7-, -S(O)2NRD7-, CI-C6 alkylene, Ci-C6 haloalkylene, Ci-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, C3-C12 cycloalkylene, 3-12-membered heterocyclicene, Ce-Cio arylene, and 5-10 membered heteroarylene; each RD5 and each RD6 are independently selected from hydrogen, Ci-Ce alkyl, Ci-Ce haloalkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, Ce-Cio aryl, and 5-10 membered heteroaryl; each RD7, each RD8 and each RD9 are independently selected from hydrogen, Ci-Ce alkyl, Ci-Ce haloalkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, Ce-Cio aryl, and 5-10 membered heteroaryl; or
RD7 and RD8, together with the atom(s) to which they are connected, optionally form 3-12- membered heterocyclic ring; mD is an integer of 0-8; and nD is an integer of 0-8.
In some embodiments, LD1 is a bond, or a bivalent group selected from -C(O)-, -NRD5C(O)-, and -C(O)1STRD:>- In some embodiments, LD1 is a bond. In some embodiments, LD1 is -C(O)- In some embodiments, LD1 is -C(O)NRD5-. In some embodiments, LD1 is -NRD5C(O)-. In some embodiments, LD3 is a bond, or a bivalent group selected from -CH2-, or -CH2CH2-. In some embodiments, LD3 is a bond. In some embodiments, LD3 is a bond -CH2-.
In some embodiments, the small molecules that covalently modify OTUB 1 are selected from Formulae (A-I-a), (A-I-b), (A-I-c), or (A-I-d):
Figure imgf000007_0001
Formula (A-I-a), Formula (A-I-b),
5
Figure imgf000008_0001
Formula (A-I-c), or Formula (A-I-d).
In some embodiments, AD is N. In some embodiments, AD is CRD2. In some embodiments, AD in Formula (A-I-a) is N. In some embodiments, AD in Formula (A-I-b) is CRD2. In some embodiments, AD in Formula (A-I-c) is N. In some embodiments, AD in Formula (A-I-d) is CRD2.
In some embodiments, the small molecules that covalently modify OTUB1 are selected from Formulae (A-I-al), (A-I-bl), (A-I-cl), or (A-I-dl):
Figure imgf000008_0002
Formula (A-I-c 1), or Formula (A-I-d 1)
In some embodiments, the small molecules that covalently modify OTUB1 are selected from Formulae (A-I-al), (A-I-bl), (A-I-cl), and (A-I-dl). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I-al). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I-bl). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I-cl). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I- dl).
6 In some embodiments, Ring DD in Formulae (A-I-a), (A-I-al), (A-I-bl), (A-I-cl), and (A- I-dl) is selected from 6-8 membered monocyclic heterocyclic, 7-12 membered spiro heterocyclic, 7-12 membered bridged heterocyclic, and 6-12 membered fused heterocyclic.
In some embodiments, The small molecules that covalently modify OTUB1 are selected from Formulae (A-I-a2), (A-I-a3), (A-I-a4), (A-I-a5), (A-I-b2), (A-I-b3), (A-I-b4), (A-I-b5), (A-I- c2), (A-I-c3), (A-I-c4), (A-I-c5), (A-I-d2), (A-I-d3), (A-I-b4), and (A-I-d5):
Figure imgf000009_0001
Figure imgf000010_0001
Formula (A-I-d4), Formula (A-I-d5), wherein each ED1, each ED2, each ED3, each ED4, and each ED5 are independently selected from null, O, CO, SO, S(O)2, NRD2, and CRD2RD2, with the proviso that no two oxygen atoms are connected to each other;
OD1 is selected from an integer of 1-4; and
OD2, OD3, OD4, and OD5 are independent selected from an integer of 0-4.
In some embodiments, the small molecules that covalently modify OTUB1 comprise Formulae (A-I-a3), (A-I-b2), and (A-I-c4). In some embodiments, the small molecules that covalently modify OTUB 1 comprise Formula (A-I-a3). In some embodiments, the small molecules that covalently modify 0TUB1 comprise Formula (A-I-b2). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I-c4).
In some embodiments, each ED1, each ED2, each ED3, each ED4, and each ED5 are independently selected from null, CO, and CRD2RD2. In some embodiments, each ED1, each ED2, each ED3, each ED4, and each ED5 are independently selected from CO and CRD2RD2.
In some embodiments, ED1, ED2, ED3, ED4, and ED3 in Formulae (A-I-a2), (A-I-a3), (A-I- a4), (A-I-a5), (A-I-b2), (A-I-b3), (A-I-b4), (A-I-b5), (A-I-c2), (A-I-c3), (A-I-c4), (A-I-c5), (A-I- d2), (A-I-d3), (A-I-b4), and (A-I-d5) are CRD2RD2.
In some embodiments, the small molecules that covalently modify OTUB1 are selected from Formulae (A-I-a6), (A-I-a7), (A-I-a8), (A-I-a9), (A-I-b6), (A-I-b7), (A-I-b8), (A-I-b9), (A-I- c6), (A-I-c7), (A-I-c8), (A-I-c9), (A-I-d6), (A-I-d7), (A-I-d8), and (A-I-d9):
Figure imgf000011_0001
Figure imgf000012_0001
Formula (A-I-d8), and Formula (A-I-d9), wherein
RD2 is a substituent that can attach to anywhere on the monocyclic and bicyclic rings.
In some embodiments, FD is selected from N, CH, or CMe. In some embodiments, FD is selected from N. In some embodiments, FD is selected from CH. In some embodiments, FD is selected from CMe.
In some embodiments, LD3 is selected from a bond or -CH2-. In some embodiments, LD3 is selected from a bond. In some embodiments, LD3 is selected from -CH2-. In some embodiments, the small molecules that covalently modify OTUB1 comprise Formulae (A-I-a6), (A-I-a7), (A-I-b6), (A-I-c8), and (A-I-d6). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I-a6). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I-a7). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I-b6). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I- c8). In some embodiments, the small molecules that covalently modify OTUB 1 comprise Formula (A-I-d6).
In some embodiments, BD is selected from C=O and C(RD2)2. In some embodiments, BD is C=O. In some embodiments, BD is C(RD2)2.
In some embodiments, Ring DD in Formulae (A-I), (A-I-a), (A-I-al), (A-I-b), (A-I-bl), (A- I-c), (A-I-cl), (A-I-d), and (A-I-dl) is selected from Ring DD al, Ring DD a2, Ring DD b1, Ring DD b2, Ring DDC 1, and Ring DD C2.
In some embodiments, Ring DD in Formulae (A-I-a), (A-I-al), (A-I-c), and (A-I-cl) is Ring DD al comprising RD2 substituted groups of:
Figure imgf000013_0001
In some embodiments, Ring DD in Formulae (A-I-a), (A-I-al), (A-I-c), and (A-I-cl) is Ring DD a2 comprising RD2 substituted groups of:
Figure imgf000013_0002
Figure imgf000014_0001
In some embodiments, Ring DD in Formulae (A-I-a) and (A-I-al) is selected from
Figure imgf000014_0003
In some embodiments, the small molecules that covalently modify OTUB1 comprise
Formula (A-I-alO):
Figure imgf000014_0002
Formula (A-I-alO).
In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I-al 1):
Figure imgf000015_0001
In some embodiments, Ring DD in Formulae (A-I-c) and (A-I-cl) is
Figure imgf000015_0002
The small molecules that covalently modify OTUB1 comprise Formula (A-I-clO):
Figure imgf000015_0003
Formula (A-I-c 10).
In some embodiments, Ring DD in Formulae (A-I-b), (A-I-bl), (A-I-d), and (A-I-dl) is Ring DD b1 comprising RD2 substituted groups of:
Figure imgf000015_0004
In some embodiments, Ring DD in Formulae (A-I-b), (A-I-bl), (A-I-d), and (A-I-dl), is Ring DD b2 comprising RD 2 substituted groups of:
Figure imgf000015_0005
Figure imgf000016_0001
In some embodiments, Ring DD in Formulae (A-I-b), (A-I-bl), (A-I-d), and (A-I-dl) is
Figure imgf000016_0002
In some embodiments, the small molecules that covalently modify OTUB1 comprise
Formula (A-I-b 10):
Figure imgf000016_0003
In some embodiments, the small molecules that covalently modify OTUB1 comprise
Formula (A-I-dlO):
Figure imgf000016_0004
In some embodiments, Ring DD in Formulae (A-I-b), (A-I-bl), (A-I-d), and (A-I-dl) is Ring DD b1 comprising RD2 substituted groups of:
Figure imgf000017_0001
In some embodiments, Ring DD in Formulae (A-I-b), (A-I-bl), (A-I-d), and (A-I-dl), is
Ring DD b2 comprising RD2 substituted groups of:
Figure imgf000017_0002
In some embodiments, Ring DD in Formulae (A-I-b), (A-I-bl), (A-I-d), and (A-I-dl) is
Figure imgf000017_0003
In some embodiments, the small molecules that covalently modify OTUB1 comprise Formula (A-I-b 11):
Figure imgf000018_0001
In some embodiments, the small molecules that covalently modify OTUB1 comprise
Formula (A-I-dl 1):
Figure imgf000018_0002
In some embodiments, ED1 in Formulae (A-I-c2), (A-I-c3), (A-I-c4), and (A-I-c5) that is adjacent to N atom is -C(O)-; and the rest of the ED 1, ED 2, ED 3, ED 4, and ED 5 are CRD 2RD 2. The small molecules that covalently modify OTUB1 comprise Formulae (A-I-cl l), (A-I-cl2), (A-I- cl 3), and (A-I-C14):
Figure imgf000018_0003
wherein
OD1-1 is selected from an integer of 0-3. In some embodiments, the small molecules that covalently modify OTUB1 comprise Formulae (A-I-cl l) and (A-I-cl2). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formulae (A-I-cl 1). In some embodiments, the small molecules that covalently modify OTUB1 comprise Formulae (A-I-cl2).
In some embodiments, Ring DD in Formulae (A-I-c) and (A-I-cl) is Ring DD C1 comprising RD 2 substituted groups of:
Figure imgf000019_0001
In some embodiments, Ring DD in Formulae (A-I-c) and (A-I-cl) is Ring DD C2 comprising RD 2 substituted groups of:
Figure imgf000019_0002
In some embodiments, Ring DD in Formulae (A-I-c) and (A-I-cl) is
Figure imgf000019_0003
Figure imgf000019_0004
In some embodiments, the small molecules that covalently modify OTUB1 comprise
Formulae (A-I-C15) and (A-I-C16):
Figure imgf000019_0005
Formula (A-I-c 15), and Formula (A-I-c 16).
In some embodiments, Ring CD is selected from C3-C12 cycloalkyl, 3-12-membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl. In some embodiments, Ring CD is C3- C12 cycloalkyl. In some embodiments, Ring CD is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. In some embodiments, Ring CD is cyclopentyl. In some embodiments, Ring CD is 3-12-membered heterocyclic. In some embodiments, Ring CD is selected from azetidinyl, pyrrolidinyl, piperidinyl, and piperazinyl. In some embodiments, Ring CD is C6-
C10 aryl. In some embodiments, Ring CD is phenyl. In some embodiments, Ring CD is 5-10 membered heteroaryl. In some embodiments, Ring CD is selected from thiophenyl, benzothiophenyl, tetrahydrobenzothiophenyl, thiazolyl, imidazolyl, furanyl, pyrrolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, benzofuranyl, indolyl, and indazolyl. In some embodiments, Ring CD is selected from thiophenyl, benzothiophenyl, tetrahydrobenzothiophenyl, benzofuranyl, and indolyl. In some embodiments, Ring CD is thiophenyl. In some embodiments,
Figure imgf000020_0001
In some embodiments, Ring CD is
Figure imgf000020_0002
In some embodiments, Ring CD is benzothiophenyl. In some embodiments, Ring
Figure imgf000020_0003
Figure imgf000020_0004
some embodiments, Ring CD is tetrahydrobenzothiophenyl. In some embodiments, Ring
Figure imgf000020_0005
Figure imgf000020_0006
In some embodiments, LD2 is selected from -C(O)-, C1-C6 alkylene, and -NRD5C(O)-. In some embodiments, LD2 is selected from -C(O)-, -NHC(O)-, and methylene. In some embodiments, LD2 is -C(O)-. In some embodiments, -NHC(O)-. LD2 is In some embodiments, LD2 is methylene.
In some embodiments, LD2 is -C(O)-; and RD1 is selected from C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroal kynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroal kynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD7, NRD 7RD 8, C(O)NRD 7RD 8, C(O)ORD 7, tri(Ci-C3alkyl) silyl, C1-C6 alkyl, Ci- Ce heteroalkyl, and 3-8 membered heterocyclic. In some embodiments, LD2 is C1-C6 alkylene; and RD1 is selected from C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD7, NRD 7RD 8, C(O)NRD 7RD 8, C(O)ORD 7, tri(C1- C3alkyl) silyl, C1-C6 alkyl, C1-C6 heteroalkyl, and 3-8 membered heterocyclic. In some embodiments, LD2 is methylene; and RD1 is selected from C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD7, NRD 7RD 8, C(O)NRD 7RD 8, C(O)ORD 7, tri(C1-C3alkyl) silyl, C1-C6 hCet1e-Cro6alkyl, and 3-8 membered heterocyclic.
In some embodiments, LD2 is -NHC(O)-; and RD1 is selected from C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD7, NRD 7RD 8, C(O)NRD 7RD 8, C(O)ORD 7, tri(Ci-C3alkyl) silyl, C1-C6 alkyl, C1- C6 heteroalkyl, and 3-8 membered heterocyclic. In some embodiments, LD2 is C1-C6 alkylene; and RD1 is selected from C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 memberedheterocyclic, where each said C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD7, NRD 7RD 8, C(O) NRD 7RD 8, C(O)ORD7, tri(Ci- C3alkyl) silyl, C1-C6 alkyl, C1-C6 heteroalkyl, and 3-8 membered heterocyclic. In some embodiments, LD2 is methylene; and RD 1 is selected from C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD7, NRD 7RD 8, C(O)NRD 7RD 8, C(O)ORD 7, tri(Ci-C3alkyl) silyl, aClk1-yCl,6 hCet1e-Cro6alkyl, and 3-8 membered heterocyclic.
In some embodiment Rs,D 1 is C1-C6 haloalkyl. In some embodiments, RD 1 is halomethyl. In some embodiment Rs,D 1 is CH2C1 or CH2F. In some embodiments, RD 1 is selected from C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD7, NRD 7RD 8, C(O)NRD 7RD 8, C(O)ORD 7, tri(Ci-C3alkyl) silyl, C1-C6 alkyl, C1- C6 heteroalkyl, and 3-8 membered heterocyclic. In some embodiment Rs,D 1 is selected from C2-C3 alkenyl, C2-C3 alkynyl, partially unsaturated C4-C6 cycloalkyl, and partially unsaturated 4-6 membered heterocyclic, where each said C2-C6 alkenyl, C2-C6 alkynyl, partially unsaturated C4-C6 cycloalkyl, and partially unsaturated 4-6 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, NRD 7RD 8, C(O)NRD 7RD 8, C(O)ORD7, tri(C1-C3alkyl) silyl, C1-C6 alkyl, 11-C6 heteroalkyl, and 3-8 membered heterocyclic. In some embodiment Rs,D 1 is selected from vinyl, propylenyl, ethynyl, propargyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, dihydrofuranyl, dihydropyrrolyl, dihydropyranyl, and tertrahydopyridinyl, where each said vinyl, propylenyl, ethynyl, propargyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, dihydrofuranyl, dihydropyrrolyl, dihydropyranyl, and tertrahydopyridinyl were optionally substituted with hydrogen, halogen, cyano, NRD 7RD 8, C(O)NRD 7RD 8, C(O)ORD7, trimethyl silyl, C1-C3 alkyl, C1-C3 heteroalkyl, and 3-6 membered heterocyclic. In some embodiment Rs,D 1 is selected from vinyl and propylenyl, where each said vinyl and propylenyl were optionally substituted with hydrogen, halogen, cyano, NRD 7RD 8, C(O)NRD 7RD 8, C(O)ORD 7, trimethyl silyl, C1-C3 alkyl, C1-C3 heteroalkyl, and 3-6 membered heterocyclic. In some embodiments, RD 1 is selected from
Figure imgf000023_0001
Figure imgf000023_0003
In some embodiments, - LD2-RD 1 is selected from
Figure imgf000023_0002
Figure imgf000024_0001
Figure imgf000025_0001
In some embodiments, - LD2-RD 1 is selected from
Figure imgf000025_0002
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000027_0002
, D . In some embodiments, - LD2-
Figure imgf000027_0003
Figure imgf000027_0004
In some embodiments, each RD 2 is independently selected from hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C1-C6 alkynyl, C2-C6 heteroalkynyl, C3-C8 cycloalkyl, and 3-8 membered heterocyclic. In some embodiments, each RD 2 is selected from hydrogen, halogen, hydroxy, C1-C6 alkoxy, amino, C1-C6 alkyl amino, (C1-C6 alkyl)( C1-C6 alkyl) amino, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, 3-8 membered heterocyclic. In some embodiments, each RD 2 is selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C8 cycloalkyl, 3-8 membered heterocyclic. In some embodiments, RD 2 is hydrogen. In some embodiments, RD 2 is halogen. In some embodiments, RD 2 is C1-C6 alkyl.
In some embodiments, two RD 2 groups, together with the atom(s) to which they are connected, optionally form a C3-C 12 cycloalkyl ring or 3-12-membered heterocyclic ring, where each said C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring DD, can optionally form fused rings or bridged rings. In some embodiments, two RD 2 groups, together with the atom(s) to which they are connected and Ring DD, optionally form a bridged 3-12-membered heterocyclic ring. In some embodiments, two RD 2 groups, together with the atom(s) to which they are connected and Ring DD, optionally form a bridged 7-membered heterocyclic ring. In some embodiments, two RD 2 groups, together with the atom(s) to which they are connected and Ring DD, optionally form a bridged 8-membered heterocyclic ring.
In some embodiments, each RD 3 is independently selected from hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C(O)ORD 7, C(O)NRD 7RD 8, -OC(O)NRD 7RD 8, - NRD 9C(O)NRD 7RD 8-, - S(O)NRD 7RD 8-, -S(O)2NRD 7RD 8-, CI-C6 alkyl, C h1a-Clo6alkyl, C he1-tCer6oalkyl, C2-C6 alkenyl, C1-C6 alkynyl, C3-C12 cycloalkyl, 3-12-membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl. In some embodiments, each RD 3 is selected from hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, 3-12-membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl. In some embodiments, each RD 3 is selected from methyl, ethyl, isopropyl, piperidinyl, piperazinyl, and phenyl.
In some embodiments, two RD 3 groups, together with the atom(s) to which they are connected, optionally form a C3-C12 cycloalkyl ring, or 3-12-membered heterocyclic ring, where each said C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring CD, optionally form fused rings or bridged rings. In some embodiments, RD 4 is RD 4a In some embodiments, RD 4a is selected from hydrogen, ORD 7, NRD 7RD 8, C(O)NRD 7RD 8, S(O)2NRD 7RD 8, CI-C6 alkylene, C1- hCa6loalkylene, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12-membered heterocyclic, Ce-Cio aryl, and 5-10 membered heteroaryl. In some embodiments, RD 4 is Ro4b. In some embodiments, RD 4 is a bivalent group that connect to the Linker moiety in Formula (B), and Ro4b is selected from a bond, -O-, -N-, -C(O)-, -C(O)O-, -C(O)NRD 7-, -S(O)2-, -S(O)2NRD 7-, C1-C al6kylene, Ci-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, C3 -C12 cycloalkylene, 3- 12-membered heterocyclicene, C6-C10 arylene, and 5-10 membered heteroarylene. In some embodiments, RD 41 is a bond.
In some embodiments, each RD 5 and each RD 6 are independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
In some embodiments, each RD 7, each RD 8 and each RD 9 are independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
In some embodiments, RD 7 and RD 8, together with the atom(s) to which they are connected, optionally form 4-6-membered heterocyclic ring.
In some embodiments, mD is an integer of 0-4. In some embodiments, mD is an integer of 1-3. In some embodiments, OD1 is selected from an integer of 1-3. In some embodiments, OD 2 is selected from an integer of 0-3. In some embodiments, OD3 is selected from an integer of 0-3. In some embodiments, OD4 is selected from an integer of 0-3. In some embodiments, OD5 is selected from an integer of 0-3.
In some embodiments, the small molecules that covalently modify OTUB1 are selected from the structures of Formulae (A-I), (A-I-a), (A-I-al), (A-I-a2), (A-I-a3), (A-I-a4), (A-I-a5), (A- I-a6), (A-I-a7), (A-I-a8), (A-I-a9), (A-I-alO), (A-I-al 1), (A-I-b), (A-I-bl), (A-I-b2), (A-I-b3), (A- I-b4), (A-I-b5), (A-I-b6), (A-I-b7), (A-I-b8), (A-I-b9), (A-I-blO), (A-I-bl l), (A-I-c), (A-I-cl), (A- Lc2), (A-I-c3), (A-I-c4), (A-I-c5), (A-I-c6), (A-I-c7), (A-I-c8), (A-I-c9), (A-I-clO), (A-I-cl l), (A- I-cl2), (A-I-C13), (A-I-C14), (A-I-C15), (A-I-C16), (A-I-d), (A-I-dl), (A-I-d2), (A-I-d3), (A-I-d4), (A-I-d5), (A-I-d6), (A-I-d7), (A-I-d8), (A-I-d9), (A-I-dlO), and (A-I-dl 1), wherein RD 4 is RD 4a In some embodiments, the small molecules that covalently modify OTUB1 are selected from the structures of Formulae (A-I), (A-I-a), (A-I-al), (A-I-a2), (A-I-a3), (A-I-a4), (A-I-a5), (A-I-a6), (A- I-a7), (A-I-a8), (A-I-a9), (A-I-alO), (A-I-al l), (A-I-b), (A-I-b 1), (A-I-b2), (A-I-b3), (A-I-b4), (A- I-b5), (A-I-b6), (A-I-b7), (A-I-b8), (A-I-b9), (A-I-b 10), (A-I-b 11), (A-I-c), (A-I-cl), (A-I-c2), (A- I-c3), (A-I-C4), (A-I-C5), (A-I-C6), (A-I-c7), (A-I-c8), (A-I-c9), (A-I-c 10), (A-I-c 11), (A-I-c 12), (A-I-C13), (A-I-C14), (A-I-C15), (A-I-d), (A-I-dl), (A-I-d2), (A-I-d3), (A-I-d4), (A-I-b5), (A-I-b6), (A-I-b 10), (A-I-d 11), wherein RD 4 is Ro4a; and exclude
Figure imgf000030_0001
In some embodiments, the small molecules that covalently modify OTUB1 are selected from XS154-91, XS154-130, XS154-148, XS154-114, XS154-149, XS159-13, XS159-107, XS165-30, XS159-90, XS165-53, XS165-38, XS165-33, XS165-54, XS154-184, XS165-75, XS165-77, XS165-127, XS165-106, XS165-112, XS165-97, XS165-118, XS165-110, XS165-119, XS165-123, XS165-126, XS165-120, XS165-121, XS165-100, XS165-99, XS165-113, XS165- 109, XS165-117, XS165-105, XS165-154, XS165-170, XS165-155, XS165-172, XS165-169, XS165-177, XS175-45, XS175-46, XS175-59, XS175-63, XS175-64, XS175-67, XS175-68, XS175-70, XS175-71, XS175-76, XS175-110, XS175-120, XS175-126, XS175-132, XS175-133, XS175-137, XS175-143, XS175-148, XS175-149, XS175-158, XS175-159, XS175-160, XS175- 173, XS175-174, XS175-175, XS175-176, XS175-178, XS175-179, XS175-180, XS175-186, XS186-3, XS185-4, XS186-5, XS185-6, XS185-24, XS185-29, XS185-43, XS185-46, XS185-59, XS185-64, XS185-65, XS185-66, XS185-67, XS185-69, XS185-70, XS185-71, XS185-72, XS185-75, XS185-78, XS185-86, XS185-87, XS185-90, 91, XS185-96, XS185-97, XS185-101, XS185-109, XS185-113, XS185-114, XS185-116, XS185-118, XS185-122, XS185-131, XS185- 134, XS185-135, XS185-138, XS185-140, XS185-147, XS185-149, XS185-150, XS185-171, XS190-9, XS190-27, XS190-38, XS190-44, XS190-45, XS190-46, XS190-47, XS190-48, XS190-49, XS190-50, XS190-59, XS190-68, XS190-69, XS190-70, XS190-75, XS190-75-2, XS190-77, XS190-80, XS190-81, XS190-126, XS190-127, XS190-128, XS190-129, XS190-130, XS190-137, XS190-138, XS190-144, XS190-157, XS190-158, XS190-170, XS190-181, XS190- 182, XS190-183, XS190-186, XS197-3, XS197-4, XS197-5, XS197-6, XS197-14, XS197-29, XS197-38, XS197-49, XS197-50, XS197-70, XS197-71, XS197-72, XS197-73, XS197-74, XS197-85, XS197-93, XS197-94, XS197-96, XS197-133, XS197-145, XS197-176, XS197-185, XS197-186, XS209-21 , XS209-22, XS209-23, XS209-24, XS209-25, XS209-26, XS209-27, XS209-28, XS209-30, XS209-38, XS209-39, XS209-40, XS209-53, XS209-54, XS209-55, XS209-57, XS209-60, XS209-62, XS209-65, XS209-74, XS209-75, XS209-92, XS209-99, XS209-100, XS209-101, XS209-122, XS209-139, XS209-140, XS209-165, XS209-166, XS209- 167, XS209-168, XS209-174, XS209-175, XS209-176, XS209-184, XS209-185, XS224-6, XS224-106, XS224-107, XS224-108, XS224-109, XS224-110, XS224-111, XS224-116, XS224- 117, XS224-118, XS224-119, XS224-143, XS224-144, XS224-145, XS224-147, XS224-148, XS224-149, XS224-150, XS224-154, XS224-155, XS224-156, XS224-157, XS224-158, XS224- 159, or analogs thereof.
In some embodiments, the small molecules that covalently modify OTUB1 are
Figure imgf000031_0001
Figure imgf000032_0001
B. OTUBl-recruiting DUBTACs
The ubiquitination and degradation of many proteins is the cause of several classes of diseases. Therefore, de-ubiquitination and stabilization of these kinds of proteins may provide a novel therapeutic strategy.
In an aspect, this disclosure provides a method of treating AMPK, cGAS, or AF508-CFTR mediated diseases, the method including administering one or more AMPK, cGAS, or CFTR DUBTACs to a subject who has an AMPK, cGAS, or AF508-CFTR -mediated disease, the AMPK, cGAS, or CFTR DUBTACs being bivalent compounds including an AMPK, cGAS, or CFTR ligand conjugated to a de-ubiquitination tag via a linker, which would stabilize AMPK, cGAS, or CFTR. The AMPK, cGAS, or AF508-CFTR -mediated disease can be a disease resulting from AMPK, cGAS, or CFTR destabilization. The AMPK, cGAS, or AF508-CFTR-mediated disease can have reduced AMPK, cGAS, or CFTR expression relative to a wild-type tissue of the same species and tissue type.
More specifically, the present disclosure provides a bivalent compound including an AMPK, cGAS, or CFTR ligand conjugated to OTUB1 recruiter via a linker.
In some aspects, the AMPK, cGAS, or CFTR DUBTACs have the form “PI-Linker- OTUB1 Recruiter”, as shown below:
Figure imgf000033_0001
Formula (B) wherein PI (a ligand for a “protein of interest,” i.e., the protein to be subjected to de-ubiquitination) (e.g., a AMPK, cGAS, or CFTR ligand), and OTUB1 Recruiter comprises a de-ubiquitination tag (e g., a ligand for OTUB1). Exemplary AMPK, cGAS, or CFFTR ligands (PI), exemplary OTUB1 binders (OTUB1 Recruiters), and exemplary linkers (Linker) are illustrated below:
AMPK ligand
In some embodiments, the AMPK ligand can be an AMPK activator or AMPK inhibitor which potently binds to AMPK. In some embodiments, the AMPK ligand comprises Metformin (Xiao et al., 2020), AICAR (Nakamaru et al., 2005), ZMP, AMP, A-769662 (Sanders et al., 2007), 991 (Ngoei et al., 2018), PF-06685249 (Edmonds et al., 2018), PXL770 (Cusi et al., 2021), MK- 8722 (Myers et al., 2017), PF-739 (Aledavood et al., 2021), 4-azaindole, RSVA405 (Vingtdeux et al., 2011), COH-SR4 (Figarola and Rahbar, 2013), B10 (Sun et al., 2020), Dorsomorphin (Kim et al., 2011), BAY-3827 (Lemos et al., 2021), GSK-690693 (Rhodes et al., 2008) or a derivative thereof. In some embodiments, the AMPK ligand is a compound disclosed in one or more of WO2009124636, W02009100130, W02010036613, WO2011029855, WO2011080277, WO2011032320, WO2013116491, W02014133008, W02016008404, W02016001224, each of which is incorporated by reference in its entirety.
In some embodiments, the AMPK ligand is an AMPK inhibitor. In some embodiments, the AMPK ligand is an ATP-competitive AMPK inhibitor. In some embodiments, the AMPK ligand is an AMPK activator. In some embodiments, the AMPK ligand is an AMPK activator, where the AMPK activator binds to an allosteric binding site. In some embodiments, the AMPK activator binds to an allosteric binding site located at the α /β subunit interface. In some embodiments, the
AMPK activator has different AMPK isoform selectivity. In some embodiments, the AMPK activator is a pan-AMPK isoform activator.
In some embodiments, the AMPK ligand comprises the structure of Formula (B-I):
Figure imgf000034_0001
Formula (B-I), or a pharmaceutically acceptable salt of the compound or the tautomer thereof, wherein
AA is selected from
Figure imgf000034_0002
BA is selected from
Figure imgf000034_0003
Figure imgf000034_0004
denotes the point of attachment to Linker in Formula (B);
CA is selected from N or CRA 4;
Ring DA and Ring EA are independently selected from null, C3-C12 cycloalkyl, 3-12- membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl;
XA and YA are independently selected from a bond, -O-, -S-, -NRA 9-, -C(O)-, -C(O)O-, - C(O)NRA 9-, -O-C(O)NRA 9-, -NRA 10C(O)NRA 9-, -S(O)-, -S(O)2-, -S(O)NRA 9-, -S(O)2NRA 9-, C1- C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, and C2-C6 alkynylene; RA 1, RA 2, each RA 4, each RA 5, and each RA 7 are independently selected from H, halogen, cyano, ORA 11, NRA HRA 12, C(O)ORA U, C(O)NRA URA 12, S(O)2NRA URA 12, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic; RA 3 is selected from H, halogen, cyano, ORA 11, NRA HRA 12, C(O)ORA U, C(O)NRA HRA 12, S(O)2NRA HRA 12, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, Ce-Cio aryl, and 5-10 membered heteroaryl; or RA 1 and RA 2, RA 2 and RA 3, RA 3 and RA 4, two RA 5, or two RA 7, together with the atoms to which they are attached, optionally form C4-C6 cycloalkyl or 5-7 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; RA 6 and RA 8 are independently selected from a bond, -O-, -NRA 13-, -C(O)-, -C(O)O-, - C(O)NRA 13-, -O-C(O)NRA 13-, -NRA 14C(O)NRA 13-, -S(O)-, -S(O)2-, -S(O)NRA 13-, -S(O)2NRA 13-, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, C6-C10 arylene, and 5-10 membered heteroarylene; each RA 9, each RA 10, each RA 11, each RA 12, each RA 13, and each RA 14 are independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic, Ce-Cio aryl, and 5-10 membered heteroaryl; or RA 11 and RA 12 together with the atoms to which they are attached, optionally form 3-12 membered heterocyclic; and mA and nA are independently selected from 0, 1, 2, 3, and 4.
In some embodiments, AA is N; and BA is
Figure imgf000035_0001
. In some embodiments, AA
Figure imgf000035_0002
and BA is N. In some embodiments,
Figure imgf000035_0003
In some embodiments, the AMPK ligand comprises the structure of Formulae (B-I-a) and Formulae (B-I-b) :
Figure imgf000036_0001
Formula (B-I-a), and Formula (B-I-b), wherein
AA and are BA independently selected from N, CRA 4
In some embodiments, XA is selected from -O-, -S-, -NRA 9-, -C(O)-, C1-C6 alkylene, C1- C6, haloalkylene, C1-C6 heteroalkylene, C6-C10 arylene, and 5-10 membered heteroarylene. In some embodiments, XA is selected from -O-, -S-, -NRA 9-, -C(O)-, methylene, and halomethylene, In some embodiments, XA is selected from -O-, -S-, -NH-, -C(O)-, CH2, and CF2, In some embodiments, XA is -O-.
In some embodiments, YA is selected from a bond, -C(O)-, -C(O)O-, -C(O)NRA 9-, -S(O)-, -S(O)2-, -S(O)NRA 9-, -S(O)2NRA 9-. In some embodiments, YA is selected from a bond, -C(O)-, - C(O)NRA 9-, and -S(O)2NRA 9-. In some embodiments, YA is -C(O)NRA 9-.
In some embodiments, the AMPK ligand comprise the structure of Formulae (B-I-al), (B- I-bl), and (B-I-b2):
Figure imgf000036_0002
Formula (B-I-al), Formula (B-I-b 1), and Formula (B-I-b2).
In some embodiments, AA is N. In some embodiments, AA is CRA 4. In some embodiments, BA is N. In some embodiments, BA is CRA 4. In some embodiments, CA is N. In some embodiments, CA is CRA 4. In some embodiments, Ring DA is selected from null, C3-C12 cycloalkyl, 3-12-membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl. In some embodiments, Ring DA is C3- C12 cycloalkyl. In some embodiments, Ring DA is selected from cyclobutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. In some embodiments, Ring DA is 3-12- membered heterocyclic. In some embodiments, Ring DA is selected from tetrahydrofuryl, hexahydrofuro[3,2-Z>]furyl, azetidinyl, pyrrolidinyl, piperidinyl, and piperazinyl. In some embodiments, Ring DA is C6-C10 aryl. In some embodiments, Ring DA is phenyl. In some embodiments, Ring DA is 5-10 membered heteroaryl. In some embodiments, Ring DA is selected from thiophenyl, benzothiophenyl, tetrahydrobenzothiophenyl, thiazolyl, imidazolyl, furanyl, pyrrolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, benzofuranyl, indolyl, and indazolyl.
In some embodiments, Ring EA selected from null, C3-C12 cycloalkyl, 3-12-membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl. In some embodiments, Ring EA selected from nullC, 6-C10 aryl, and 5-10 membered heteroaryl. In some embodiments, Ring EA selected from null. In some embodiments, Ring EAISC6-C10 aryl. In some embodiments, Ring EAis phenyl. . In some embodiments, Ring EA IS 5-10 membered heteroaryl. In some embodiments, Ring EA IS selected from thiophenyl, benzothiophenyl, tetrahydrobenzothiophenyl, thiazolyl, imidazolyl, furanyl, pyrrolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, benzofuranyl, indolyl, and indazolyl.
In some embodiments, the AMPK ligand comprises the structure of Formulae (B-I-a2), (B- I-a3), (B-I-a4), (B-I-b3), and (B-I-b4):
Figure imgf000037_0001
Formula (B-I-a2), Formula (B-I-a3), Formula (B-I-a4),
Figure imgf000038_0001
Formula (B-I-b3), Formula (B-I-b4), wherein
FA is selected from N or CRA 3; and
GA is selected from N or CRA 7
In some embodiments, RA 3 is selected from C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl. In some embodiments, RA 3 is selected from C6-C10 aryl and 5-10 membered heteroaryl.
In some embodiments, the AMPK ligand comprises the structure of Formulae (B-I-a5), (B- I-a6), (B-I-a7), (B-I-b5), and (B-I-b6):
Figure imgf000038_0002
Formula (B-I-b5), Formula (B-I-b6), wherein
ArA is selected from C6-C10 aryl, or 5-10 membered heteroaryl; each RA 15 is independently selected from H, halogen, cyano, ORA 11, NRA HRA 12, C(O)ORA 11 C(O)NRA 11R1 A 12, S(O)2NRA 11RA 12, -N=S(O)RA 11RA 12, CI-C6 alkyl, haClo1a-Clk6yl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl and 5-10 membered heteroaryl, or two of RA 15, together with the atoms to which they are attached, optionally form C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl or 5-10 membered heteroaryl; and
OA is selected from 0, 1, 2, 3, 4, 5, or 6.
In some embodiments, ArA is selected from C6-C1 a0ryl and 5-10 membered heteroaryl. In some embodiments, ArA is selected from phenyl, naphthalene, indane, 5,6, 7,8- tetrahydronaphthalene, biphenyl, pyrazole, pyridine, pyrazine, pyrimidine, thiazole, thiophene, benzoimidazole, quinoline, isoquinoline, indole, indazole, carbazole, benzotriazole, benzofuran, benzothiazole, benzo[b]thiophene, benzo[d]isooxazole, 3,4-dihydro-2H-benzo[l,4]oxazine, benzo[l,3]dioxole, benzo[ 1,4] di oxane, l//-pyrrolo[2,3-b]pyridine, [l,2,4]triazolo[4,3-a] pyridine, 3,4 dihydropyrido[3,2-b][l,4]oxazine, 3,4-dihydro-2H-l,4-benzoxazine, 2,3-dihydro-lH-indole, 2,3-dihydro-lH-isoindole, 2,3 -dihydrobenzoimidazole, 1,2-dihydroquinoline, 1, 2,3,4- tetrahydroisoqumoline, l,2,3,4-tetrahydrocyclopenta[b] indole, 1,2,3,4-tetrahydroquinoxaHne, 1,2,3,6-tetrahydropyridine. In some embodiments, ArA is selected from phenyl, pyridine, biphenyl, and indole. In some embodiments, ArA is indole.
In some embodiments, RA 6 is selected from a bond, -O-, -NRA 13-, -C(O)-, -C(O)O-, - C(O)NRA 13-, -S(O)2NRA 13-, C1-C6 alkylene, C1-C6 haloalkylene, and C1-C6 heteroalkylene. In some embodiments, RA 6 is selected from -C(O)-, -C( 3-. In some embodiments, RA 6 is -C(O)NRA 13-. In some embodiments,
Figure imgf000039_0001
the N atom of the amide attaches two Linker-OTUBl Recruiter moieties.
In some embodiments, RA 8 is selected from a bond, -O-, -NRA 13-, -C(O)-, -C(O)O-, - C(O)NRA 13-, -S(O)2NRA 13-, C1-C6 alkylene, C1-C6 haloalkylene, and C1-C6 heteroalkylene.
In some embodiments, RA 1 is selected from H, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, Ci-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, RA 1 is selected from H and CH3. In some embodiments, RA 1 is H. In some embodiments, RA 1 is CH3.
In some embodiments, RA 2 is selected from H, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, RA 2 is selected from halogen. In some embodiments, RA 2 selected from F or Cl. In some embodiments, RA 2 is F. In some embodiments, RA 2 is Cl.
In some embodiments, each RA 4 is selected from H, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, RA 4 is H.
In some embodiments, each RA 5 is selected from H, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, each RA 5 is selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl. In some embodiments, RA 5 is H, CH3, CH2CH3, and CH2OH.
In some embodiments, each RA 7 is selected from H, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, each RA 7 is selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl. In some embodiments, RA 7 is H, CH3, and CH2 CH3.
In some embodiments, each RA 9 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic, C6- aCry1l0, and 5-10 membered heteroaryl. In some embodiments, each RA 9 is independently selected from H, Ci- C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, RA 9 is H.
In some embodiments, each RA 10 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic, C6- aCry10l, and 5-10 membered heteroaryl. In some embodiments, each RA 10 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, RA 10 is H.
In some embodiments, each RA 11 and each RA 12 are independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic, C6- C10 aryl, and 5-10 membered heteroaryl. In some embodiments, each RA 11 and each RA 12 are independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, RA 11 is selected from H and CH3. In some embodiments, RA 12 is selected from H and CH3.
In some embodiments, RA 11 and RA 12 together with the atoms to which they are attached, optionally form 3-12 membered heterocyclic. In some embodiments, each RA 13 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic, C6- aCry1l0, and 5-10 membered heteroaryl. In some embodiments, each RA 13 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
In some embodiments, each RA 14 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic, C6- aCry1l0, and 5-10 membered heteroaryl. In some embodiments, each RA 14 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
In some embodiments, each RA 15 is independently selected from each RA 15 is independently selected from H, halogen, cyano, ORA 11, NRA 11 A 12, C(O)ORA 11, C(O)NRA 11RA 12, S(O)2NRA' 'RA 12, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C1 a0ryl and 5-10 membered heteroaryl.
In some embodiments, each RA 15 is independently selected from each RA 15 is independently selected from H, halogen, cyano, ORA 11, NRA’ 'RA 12, S(O)2NRA 1 1RA 12, -N=S(O)RA 11RA 12, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl and 5-10 membered heteroaryl. In some embodiments, each RA 15 is independently selected from each RA 15 is independently selected from H, CN, CH3 , cyclopropyl, hydroxymethyl cyclopropyl, OCH3, dimethyl amino, -N=S(O)(CH3)2, and -Ph-N=S(O)(CH3)2.
In some embodiments, mA is selected from 0, 1, 2, 3, and 4. In some embodiments, mA is selected from 0, 1, and 2. In some embodiments, nA is selected from 0, 1, 2, 3, and 4. In some embodiments, nA is selected from 0, 1, and 2. In some embodiments, OA is selected from 0, 1, 2, 3, 4, and 5.
In some embodiments, the AMPK ligand comprise the structure of Formulae (B-I), (B-I- a), (B-I-al), (B-I-a2) and (B-I-a5).
In some embodiments, the AMPK ligand comprise a derivative of following compounds:
Figure imgf000042_0001
Figure imgf000043_0001
Wherein, ♦* denotes the point of attachment to Linker. cGAS ligand
In some embodiments, the cGAS ligand is seleted from a cGAS activator or cGAS inhibitor which potently binds with cGAS. In some embodiments, the cGAS ligand is cGAS activator. In some embodiments, the cGAS ligand is cGAS inhibitor. In some embodiments, the cGAS ligand comprises hydroxychloroquinine (HCQ), Quinacrine (QC), X6, RU114757, RU191752, RU100840, RU.365, RU.521, J001, G001, G108, G150, GMO, CPD-25, aspirin, CPD-C, PF- 06928215(Zhao et al., 2022). In some embodiments, the cGAS ligand is a compound disclosed in one or more of U.S. 62/318,435, U.S. 62/355,403, U.S. 2018/0230115, WO2019241787,
W02019153002, WO2022140387, W02023004437, each of which is incorporated herein by reference in its entirety.
In some embodiments, the cGAS ligand comprise the structure of Formula (C-I):
Figure imgf000043_0002
Formula (C-I), or a pharmaceutically acceptable salt of the compound or the tautomer thereof, wherein denotes the point of attachment to Linker in Formula (B);
♦ ♦
Figure imgf000043_0003
connects to either RBlb or RB311, with the proviso that when ** connects to RBlb,
Figure imgf000043_0004
RB3 is RB3a; and when ** connects to Rs3b, RB1 is RB1S;
AB is selected from N and CRB4;
BB is selected from N, O, S, CRB5 and NRB5;
CB is selected from N and C; DB is selected from N and C;
XB is XB a-XB b;
XB a is selected from a bond, -C(O)-, -C(O)O-, -C(O)NRB 6-, -S(O)-, -S(O)2-, -S(O)NRB 6-, - S(O)2NRB 6-;
XB b is selected from a bond, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C3- C12 cycloalkylene, and 3-12 membered heterocyclicene, C6-C10 arylene, and 5-10 membered heteroarylene; RB 1 is selected from monovalent group RB 1a and bivalent group RB lb;
RB la is selected from H, halogen, cyano, ORB 7, NRB 7RB 8, C(O)ORB 7, C(O)NRB 7RB 8, S(O)2NRB 7RB 8, C1-C6 alkyl, C1-C ha6loalkyl, C1- hCet6eroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3- C12 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl;
RB lb is connected to the Linker, and is selected from a bond, -O-, -NRB 7-, -C(O)O-, - C(O)NRB 7-, -S(O)2NRB 7-, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, C3-C12 cycloalkylene, 3-12 membered heterocyclicene, C6-C10 arylene, and 5-10 membered heteroarylene; each RB 2 is independently selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, or two RB 2, together with the atoms to which they are attached, optionally form C3-C12 cycloalkyl or 3-12 membered heterocyclic; RB 3 is selected from monovalent group RB 3a and bivalent group RB 3b;
RB 3a is selected from H, halogen, ORB 9, NRB 9RB 10, C(O)ORB 9, C(O)NRB 9RB 10, S(O)2NRB 9RB 10, C1-C6 alkyl, C1- hCal6oalkyl, C h1-eCte6roalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3- Ci2 cycloalkyl, and 3-12 membered heterocyclic;
RB 3b is connected to the Linker, and is selected from a bond, -O-, -NRB 9-, -C(O)O-, - C(O)NRB 9-, -S(O)2NRB 9-, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, C3-Ci2 cycloalkylene, and 3-12 membered heterocyclicene; each RB 4 is independently selected from H, halogen, cyano, ORB 7, NRB 7RB 8, C(O)ORB 7, C(O)NRB 7RB 8, S(O)2NRB 7RB 8, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 heteroalkyl, C2-C4 alkenyl, C2- C4 alkynyl, C3-C6 cycloalkyl, and 3-6 membered heterocyclic, or two RB 4, together with the atoms to which they are attached, optionally form partially unsaturated C3-C12 cycloalkyl, partially unsaturated 3-12 membered heterocyclic, C6-C ar10ylene, or 5-10 membered heteroarylene; RB 5 is selected from H, halogen, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 heteroalkyl, C3-C6 cycloalkyl, and 3-6 membered heterocyclic; RB 6, each RB 7, each RB 8, each RB 9, and each RB 10, are independently selected from H, C1- C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic, or RB 7 and RB 8, and RB 9 and RB 10 together with the atoms to which they are attached, optionally form 3-12 membered heterocyclic; mB is selected from 1 and 2; riB is selected from 0, 1, 2 and 3; and
OB is selected from 0, 1, 2, 3, 4, 5 and 6.
Figure imgf000045_0001
In some embodiments, * connects to RB 1b, RB 3 is RB 3a. In some embodiments, ** connects to RB 3b, RB 1 is RB 1 1
In some embodiments, the cGAS ligand comprises the structures of Formulae (C-I-a) and (C-I-b):
Figure imgf000045_0002
Formula (C-I-a), Formula (C-I-b).
In some embodiments, AB is CRB 4 In some embodiments, BB is NRB 5. In some embodiments, CB is C. In some embodiments, DB is C.
In some embodiments, the cGAS ligand comprises the structures of Formulae (C-I-al) and (C-I-bl):
Figure imgf000046_0001
Formula (C-I-al), Formula (C-I-bl).
In some embodiments, RB 1a is selected from H, halogen, C3-C12 cycloalkyl, 3-12 membered heterocyclic,C6-C10 aryl, and 5-10 membered heteroaryl. In some embodiments, RB 13 is selected from C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C a1r0yl, and 5-10 membered heteroaryl. In some embodiments, RB 1a is selected from C6-C ar10yl, and 5-10 membered heteroaryl. In some embodiments, RB 1a is 5-10 membered heteroaryl. In some embodiments, RB 13 is selected from furyl, thiophenyl, pyrrolyl, pyrazolyl, oxazolyl, oxadiazolyl, thiazolyl, isoxazolyl, isothiazolyl, imidazolyl, triazolyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl. In some embodiments, RB 13 is pyrazolyl. In some embodiments, RB 11 is = pyridinyl.
In some embodiments, RB lb is selected from C3-C12 cycloalkylene, 3-12 membered heterocyclicene, C6-C10 arylene, and 5-10 membered heteroarylene. In some embodiments, RB 11 is selected from C6-C10 arylene, and 5-10 membered heteroarylene.
In some embodiments, the cGAS ligand comprises the structures of Formulae (C-I-a2) and (C-I-b2):
Figure imgf000046_0002
Formula (C-I-a2), Formula (C-I-b2), wherein
ArB is selected from C6-C10 aryl, and 5-10 membered heteroaryl; each RB 1 1 is independently selected from H, halogen, cyano, ORB 7, NRB 7RB 8, C(O)ORB 7, C(O)NRB 7RB 8, S(O)2NRB 7RB 8, C1-C6 alkyl, C h1a-lCo6alkyl, hCe1t-eCr6oalkyl, C2-C6 alkenyl, C2- C6, alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; or two RB 11, together with the atoms to which they are attached, optionally form partially unsaturated C3-C12 cycloalkyl, partially unsaturated 3-12 membered heterocyclic, C6-C a1r0yl, or 5-10 membered heteroaryl;
PB is selected from 0, 1, 2, 3, 4, and 5; and
PB-I is selected from 0, 1, 2, 3, and 4.
In some embodiments, XB3 is selected from -C(O)-, -S(O)-, and -S(O)2-. In some embodiments, XBa is -C(O)-. In some embodiments, XBb is selected from C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C3-C12 cycloalkylene, and 3-12 membered heterocyclicene.
In some embodiments, the cGAS ligand comprises the structures of Formulae (C-I-a3) and (C-I-b3):
Figure imgf000047_0001
Formula (C-I-a3), Formula (C-I-b3), wherein each RB 12 is independently selected from H, halogen, ORB 7, NRB 7RB 8, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic; or two RB 12, RB 12 and RB 3a, and RB 12 and RB b, together with the atoms to which they are attached, optionally form C3-C12 cycloalkyl, 3-12 membered heterocyclic; and qB is sleeted from 0, 1, 2, 3, 4, and 5.
In some embodiments, ArB is 5-10 membered heteroaryl. In some embodiments, ArB is selected from furyl, thiophenyl, pyrrolyl, pyrazolyl, oxazolyl, oxadiazolyl, thiazolyl, isoxazolyl, isothiazolyl, imidazolyl, triazolyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl. In some embodiments, ArB is pyrazolyl. In some embodiments, ArB is pyridinyl.
In some embodiments, the cGAS ligand comprises the structures of Formulae (C-I-a4) and (C-I-b4):
Figure imgf000048_0001
Formula (C-I-a4), Formula (C-I-b4).
In some embodiments, each RB 2 is independently selected from H, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 heteroalkyl, C3-C6 cycloalkyl. In some embodiments, RB 2 is H. In some embodiments, RB 2 is C1-C3 alkyl. In some embodiments, RB 2 is CH3. In some embodiments, two RB 2, together with the atoms to which they are attached, optionally form C3-C6 cycloalkyl or 3-6 membered heterocyclic. In some embodiments, two RB 2, together with the atoms to which they are attached, optionally form C3-C5 cycloalkyl.
In some embodiments, Ru3a is selected from H, halogen, ORB 9, NRB 9RB 10, C1-C6 alkyl, Ci- Cg haloalkyl, Ci-Cg heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, RB 3a is selected from H, ORB 9 and NRB 9RB 10. In some embodiments, RB 3a is selected from H, OH, OCH3, OCH2CH2OCH3, NH2, NHCH3, and NHC(O)CH2NH2. In some embodiments, RB 3a is OH.
In some embodiments, RB 3b is selected from a bond, -O-, -NRB 9-, Ci-Cg alkylene, C1-C6 haloalkylene, Ci-Cg heteroalkylene, C3-C12 cycloalkylene, and 3-12 membered heterocyclicene. In some embodiments, RB 31) is selected from a bond, -O-, -NRB 9-. In some embodiments, RB 3b is selected from a bond, -O-, -NH-, and -N(CH3)-. In some embodiments, RB 3b is -O-.
In some embodiments, each RB 4 is independently selected from H, halogen, cyano, ORB 7, NRB 7RB 8, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 heteroalkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, and 3-6 membered heterocyclic. In some embodiments, each RB 4 is independently selected from H. In some embodiments, each RB 4 is independently selected from cyano. In some embodiments, each RB 4 is independently selected from halogen. In some embodiments, each RB 4 is independently selected from F. In some embodiments, each RB 4 is independently selected from Cl. In some embodiments, each RB 4 is independently selected from methyl, ethyl, propyl, isopropyl, cyclopropyl, trifluoromethyl, difluoromethyl, methoxy, ethoxy, cyclopropoxy, and azetidinyl. In some embodiments, each RB 4 is independently selected from H. In some embodiments, each RB 4 is independently selected from cyano. In some embodiments, each RB 4 is independently selected from halogen. In some embodiments, each RB 4 is independently selected from F. In some embodiments, each RB 4 is independently selected from Cl. In some embodiments, each RB 4 is independently selected from Br.
In some embodiments, two RB 4, together with the atoms to which they are attached, optionally form partially unsaturated C3-C6 cycloalkyl, partially unsaturated 3-6 membered heterocyclic,C6-C10 aryl, or 5-10 membered heteroaryl.
In some embodiments, RB 5 is selected from H, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 heteroalkyl, C3-C6 cycloalkyl, and 3-6 membered heterocyclic. In some embodiments, RB 5 is selected from H and C1-C3 alkyl. In some embodiments, RB 5 is H. In some embodiments, RB 5 is CH3.
In some embodiments, RB 6, each RB 7, each RB 8, each RB 9, and each RB 10, are independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, RB 6, each RB 7, each RB 8, each RB 9, and each RB 10, are independently selected from H, methyl, ethyl, propyl, isopropyl, cyclopropyl, trifluoromethyl, and difluoromethyl. In some embodiments, RB 6, each RB 7, each RB 8, each RB 9, and each RB 10, are H. In some embodiments, RB 6, each RB 7, each RB 8, each RB 9, and each RB 10, are methyl.
In some embodiments, RB 7 and RB 8, and RB 9 and RB 10 together with the atoms to which they are attached, optionally form 3-6 membered heterocyclic.
In some embodiments, each RB 11 is independently selected from H, halogen, cyano, ORB 7, NRB 7RB 8, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic. In some embodiments, RB 11 is H. In some embodiments, each RB 11 is CH3.
In some embodiments, two RB 11, together with the atoms to which they are attached, optionally form partially unsaturated C3-C12 cycloalkyl, 3-12 membered partially unsaturated heterocyclic,C6-C10 aryl, or 5-10 membered heteroaryl.
In some embodiments, each RB 12 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, each RB 12 is independently selected from H, and C1-C6 alkyl. In some embodiments, RB 12 is H. In some embodiments, two RB 12, RB 12 and RB 3a, and RB 12 and RB 3b, together with the atoms to which they are attached, optionally form C3-C6 cycloalkyl, 3-6 membered heterocyclic.
In some embodiments, mB is 1. In some embodiments, nB is 1. In some embodiments, nB is 2. In some embodiments, OB is 0. In some embodiments, OB is 1. In some embodiments, OB is 2. In some embodiments, PB is 0. In some embodiments, PB is 1. In some embodiments, PB is 2. In some embodiments, PB-I is 0. In some embodiments, PB-I is 1. In some embodiments, qB is 0. In some embodiments, qβ is 1. In some embodiments, qβ is 2.
In some embodiments, the cGAS ligand comprises the structures of Formulae (C-I), (C-I- a), (C-I-al), (C-I-a2), (C-I-a3), and (C-I-a4).
In some embodiments, the cGAS ligand may be a derivative of following compounds:
Figure imgf000051_0001
Wherein, ♦* denotes the point of attachment to Linker.
CFTR Ligand
In some embodiments, the CFTR ligand can be a CFTR potentiator which potently binds with either wild type CFTR or mutant CFTR. In some embodiments, the CFTR ligand comprises ivacftor, lumacaftor, tezacaftor, elexacafor, or icenticaftor, or derivatives thereof. In some embodiments, the CFTR ligand is a compound disclosed in one or more of U.S. Patent No. 7,999,113; U.S. Patent No. 8,247,436; U.S. 8,410,274; WO 2011/133953; and WO2018/037350, each of which is incorporated by reference in its entirety.
In some embodiments, the CFTR ligand comprises the structures of Formula (D-I):
Figure imgf000052_0001
Formula (D-I), or a tautomer thereof, or a pharmaceutically acceptable salt of the compound or the tautomer, wherein
Figure imgf000052_0002
denotes the point of attachment to Linker in Formula (B).
Ac and Cc are independently selected from O, S, or C(Rc8)(Rc9);
Bc is C(Rc10)(Rc11) or NRc 12;
Arc1 and Arc2 are independently selected from null, C6-C10 aryl and 5-10 membered heteroaryl;
Rc1 and Rc2 are independently selected from H, halo, ORc13, NRc13Rc14, C(O)ORc13, C(O)NRC13RC14, S(O)2NRC13RC14, C1-C6 alkyl, hCal1o-Cal6kyl, heCte1-rCoa6lkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic, or
Rc1 and Rc2, together with the atoms to which they are attached, optionally form C3-C12 cycloalkyl; Rc3 and Rc6 are independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic; each Rc4, each Rc5, and each Rc7 are independently selected from H, halogen, cyano, ORc13, NRc13Rc14, C(O)ORc13, C(O)NRC 13RC14, S(O)2NRC 13RC14, CI-C6 alkyl, hCal1o-aClk6yl, Ci-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic, two Rc4, two Rc5, or two Rc7, together with the atoms to which they are attached, optionally form partially unsaturated C3-C12 cycloalkyl, partially unsaturated 3-12 membered heterocyclic, C6-C10 aryl and 5-10 membered heteroaryl;
Rc8, Rc9, Rc10 and Rc11 are independently selected from H, halogen, ORc13, NRc13Rc14, C(O)ORc13, C(O)NRc13Rc14, S(O)2NRC 13RC14, CI-C6 alkyl, C h1a-lCo6alkyl, C h1e-tCe6roalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic, or
Rc8 and Rc9, and Rc10 and Rc11, together with the atoms to which they are attached, optionally form C3-C12 cycloalkyl and 3-12 membered heterocyclic;
Rc12 is selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic;
Rc13 and Rc14 are independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic, or
Rc13 and Rc14 together with the atoms to which they are attached, optionally form 3-12 membered heterocyclic; me is 0, 1 , 2, or 3; nc is 0, 1, 2, or 3; and oc is 0, 1, 2, or 3.
In some embodiments, Arc1 is 5-10 membered heteroaryl. In some embodiments, Arc1 is selected from furyl, thiophenyl, pyrrolyl, pyrazolyl, oxazolyl, oxadiazolyl, thiazolyl, isoxazolyl, isothiazolyl, imidazolyl, triazolyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl. In some embodiments, ArB is pyridinyl. In some embodiments, Arc2 is C6-C1 a0ryl. In some embodiments, Arc2 is phenyl.
In some embodiments, the CFTR ligand comprises the structures of Formula (D-I-a):
Figure imgf000054_0001
Formula (D-I-a), or a tautomer thereof, or a pharmaceutically acceptable salt of the compound or the tautomer.
In some embodiments, the CFTR ligand comprises the structures of Formula (D-I-al):
Figure imgf000054_0002
Formula (D-I-a 1), or a tautomer thereof, or a pharmaceutically acceptable salt of the compound or the tautomer.
In some embodiments, Ac is O. In some embodiments, Be is C(Rc10)(Rc11). In some embodiments, Cc is O.
In some embodiments, the CFTR ligand comprises the structures of Formula (D-I-a2):
Figure imgf000054_0003
Formula (D-I-a2), or a tautomer thereof, or a pharmaceutically acceptable salt of the compound or the tautomer.
In some embodiments, Rc1 is selected from H, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C3-C5 cycloalkyl, and 3-5 membered heterocyclic. In some embodiments, Rc2 is selected from H, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C3-C5 cycloalkyl, and 3-5 membered heterocyclic. In some embodiments, Rc1 and Rc2, together with the atoms to which they are attached, optionally form C3-C6 cycloalkyl. In some embodiments, Rc1 and Rc2, together with the atoms to which they are attached, optionally form ]cyclopropyl. In some embodiments, Rc3 is selected from H, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 heteroalkyl. In some embodiments, Rc3 is H.
In some embodiments, each Rc4 is independently selected from H, halo, cyano, ORc13, NRC13RC14, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, each Rc4is independetly selected from H and C1-C6 alkyl. In some embodiments, each Rc4is independently selected from H and CH3.
In some embodiments, each Rc5 is independently selected from H, halo, cyano, ORc13, NRC13RC14, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, each Rc3 is independently selected from H and C1-C6 alkyl. In some embodiments, Rc5 is H.
In some embodiments, Rc6 is selected from H, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 heteroalkyl. In some embodiments, Rc6 is H. In some embodiments, Rc3 is CH3.
In some embodiments, each Rc7is independently selected from H, halogen, cyano, ORc13, NRC13RC14, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, each Rc7 is independently selected from H and C1-C6 alkyl. In some embodiments, Rc7is H.
In some embodiments, Rc8 and Rc9 are independently selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, Rc8 and Rc9 are independently selected from H and C1-C6 alkyl,
In some embodiments, Rc10 and Rc11 are independently selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, Rc10 and Rc11 are independently selected from H, halogen and C1-C6 alkyl. In some embodiments. Rc10 and Rc11 are independently selected from H, F, Cl, CH3, and CF3. In some embodiments, both Rc10 and Rc11 are F.
In some embodiments, Rc12 is selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
In some embodiments, Rc13 and Rc14 are independently selected from H, C1-C6 alkyl, Ci- C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, Rc13 and Rc14 are independently selected from H and C1-C6 alkyl. In some embodiments, Rc13 and Rc14 are independently selected from H and CH3. In some embodiments, Rc13 and Rc14 together with the atoms to which they are attached, optionally form 3-6 membered heterocyclic.
In some embodiments, me is 1. In some embodiments, nc is 0. In some embodiments, oc is 0.
In some embodiments, the CFTR ligand comprises the structures of Formulae (D-I-a3) and (D-I-a4):
Figure imgf000056_0001
Formula (D-I-a3), Formula (D-I-a4).
In some embodiments, the CFTR ligand may be a derivative of following compounds:
Figure imgf000056_0002
Wherein, •* denotes the point of attachment to Linker.
Linkers
In all of the above-described compounds, the AMPK, cGAS or CFTR ligand is conjugated to the de-ubiquitination tag through a linker. The linker can include, for example, acyclic or cyclic saturated or unsaturated carbon, ethylene glycol, amide, amino, ether, urea, carbamate, aromatic, heteroaromatic, heterocyclic and/or carbonyl containing groups with different lengths. In some embodiments, the linker can be a moiety of Formula (E):
Figure imgf000057_0001
Formula (E) wherein
A, W and B, at each occurrence, are independently selected from null, or bivalent moiety
Figure imgf000057_0002
and R NR1S(O)2N(R2)R , wherein
R and R are independently selected from a bond, optionally substituted Rr-(C1-C8 alkyl), or a moiety comprising of optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substitutedC1-C8 haloalkyl, optionally substituted C1-C8 alkylene, optionally substituted C2-C8 alkenylene, optionally substituted C2-C8 alkynylene, optionally substituted C1-C8 hydroxy alkylene, optionally substituted C1-C8alkoxyC1-C8alkylene, optionally substituted C1-C8alkylaminoCi- Csalkylene, optionally substituted C1-C8 haloalkylene, optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclic, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclic, optionally substituted C3- C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
Rr is selected from optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclic, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclic, optionally substituted C3-C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl; R1 and R2 are independently selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituteCd2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
R and R , R1 and R2, R and R1, R andR2, R and R1, R andR2 together with the atom to which they are connected form a 3-20 membered cycloalkyl or 4-20 membered heterocyclic ring; and m is 0 to 15.
In one embodiment, the linker moiety is of Formula (E-l)
Figure imgf000058_0001
Formula (E-l), wherein
R1, R2, R3 and R4, at each occurrence, are independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, optionally substituted C1-C8 alkyl, optionally substituteC2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8 alkylamino, and optionally substituted C1-C8 alkylaminoC1-C8 alkyl, optionally substituted 3-10 membered carbocyclic, optionally substituted 3-8 membered cycloalkoxy, optionally substituted 3-10 membered carbocyclicamino, optionally substituted 4-8 membered membered heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl, or
R1 and R2, R3 and R4 together with the atom to which they are connected form a 3-20 membered cycloalkyl or 4-20 membered heterocyclic ring; A, W and B, at each occurrence, are independently selected from null, or bivalent moiety selected from R -R ”, R COR , R CO2R , R C(O)N(R5)R , R’C(S)N(R5)R”, R OR”, R OC(O)R”, R OC(O)OR ”, R OCONR5R”, R SR ”, R SOR”, R SO2R , R SO2N(R5)R”, RN(R5)R”, RNR5COR ”, RNR5C(O)OR’ , RNR5CON(R6)R”, R NR5C(S)R ”, RNR5S(O)R ”, RNR5S(O)2R ”, and R NR5S(O)2N(R6)R ”, wherein
R and R are independently selected from null, optionally substituted Rr-(C1-C8 alkyl), or a moiety comprising of optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 alkylene, optionally substituted C2-C8 alkenylene, optionally substituted C2-C8 alkynylene, optionally substituted C1-C8 hydroxy alkylene, optionally substituted C1-C8alkoxyC1-C8alkylene, optionally substituted C1-C8alkylaminoCi- Csalkylene, optionally substituted C1-C8 haloalkylene, optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclic, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclic, optionally substituted C3- C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
Rr is selected from optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclic, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclic, optionally substituted C3-C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
R5 and R6 are independently selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl; R and R , R5 and R6, R and R5, R andR6, R and R5, R andR6 together with the atom to which they are connected form a 3-20 membered cycloalkyl or 4-20 membered heterocyclic ring; m is 0 to 15; n, at each occurrence, is 0 to 15; and o is 0 to 15.
In another embodiment, the linker moiety is of Formula (E-2)
Figure imgf000060_0001
Formula (E-2), wherein
R1 and R2, at each occurrence, are independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, and optionally substituted C1-C8 alkyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8 alkoxy C1-C8 alkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8 alkylamino, Ci- CsalkylaminoC1-C8alkyl, optionally substituted 3-10 membered carbocyclic, optionally substituted 3-8 membered cycloalkoxy, optionally substituted 3-10 membered carbocyclicamino, optionally substituted 4-10 membered heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl, or
R1 and R2 together with the atom to which they are connected form a 3-20 membered cycloalkyl or 4-20 membered heterocyclic ring;
A and B, at each occurrence, are independently selected from null, or bivalent moiety selected from R -R , R COR , R CO2R , R C(O)NR3R’ , R C(S)NR3R”, R’OR”, R OC(O)R’ , R OC(O)OR , R OCON(R3)R , R SR , R SOR , R SO2R , R SO2N(R3)R , R N(R3)R , R’NR3COR”, RNR3C(O)OR ”, R’NR3CON(R4)R’, R’NR3C(S)R ”, R’NR3S(O)R”, R’NR3S(O)2R”, and R NR3S(O)2N(R4)R ”, wherein
R and R are independently selected from null, optionally substituted Rr-(C1-C8 alkyl), or a moiety comprising of optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substitutedC2-C8 alkynyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 alkylene, optionally substituted C2-C8 alkenylene, optionally substituted C2-C8 alkynylene, optionally substituted C1-C8 hydroxyalkylene, optionally substituted C1-C8alkoxyC1-C8alkylene, optionally substituted C1-C8alkylaminoCi- Csalkylene, optionally substituted C1-C8 haloalkylene, optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclic, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclic, optionally substituted C3- C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
Rr is selected from optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclic, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclic, optionally substituted C3-C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
R3 and R4 are independently selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
R and R , R3 and R4, R and R3, R and R4, R and R3, R and R4 together with the atom to which they are connected form a 3-20 membered cycloalkyl or 4-20 membered heterocyclic ring; m, at each occurrence, is 0 to 15; and n is 0 to 15.
In another embodiment, the linker moiety is of Formula (E-3):
Figure imgf000061_0001
Formula (E-3) wherein X is selected from O, NH, and NR7;
R1, R2, R3, R4, R5, and R6, at each occurrence, are independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8 alkoxy C1-C8 alkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8 alkylamino, optionally substituted C1-C8 alkylaminoC1-C8 alkyl, optionally substituted 3-10 membered carbocyclic, optionally substituted 3-8 membered cycloalkoxy, optionally substituted 4-10 membered heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
A and B are independently selected from null, or bivalent moiety selected from R -R , R COR , R CO2R ”, R C(O)N(R8)R’ , R C(S)N(R8)R’ , R OR , R OC(O)R”, R OC(O)OR”, R OCON(R8)R , R SR , R SOR , R SO2R , R SO2N(R8)R , R N(R8)R , R NR8COR , R’NR8C(O)OR”, R’NR8CON(R9)R”, R’NR8C(S)R”, R’NR8S(O)R”, R’NR8S(O)2R”, and RNR8S(O)2N(R9)R ’, wherein
R and R are independently selected from null, optionally substituted Rr-(C1-C8 alkyl), or a moiety comprising of optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substitutedC2-C8 alkynyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 alkylene, optionally substituted C2-C8 alkenylene, optionally substituted C2-C8 alkynylene, optionally substituted C1-C8 hydroxyalkylene, optionally substituted C1-C8alkoxyC1-C8alkylene, optionally substituted C1-C8alkylaminoCi- Csalkylene, optionally substituted C1-C8 haloalkylene, optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclic, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclic, optionally substituted C3- C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
Rr is selected from optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclic, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclic, optionally substituted C3-C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
R7, R8 and R9 are independently selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted 3-10 membered carbocyclic, optionally substituted 4-10 membered heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
R and R , R8 and R9, R and R8, R and R9, R and R8, R and R9 together with the atom to which they are connected form a 3-20 membered cycloalkyl or 4-20 membered heterocyclic ring; m, at each occurrence, is 0 to 15; n, at each occurrence, is 0 to 15; o is 0 to 15; and p is 0 to 15.
In some embodiments, in Formular (E-3), m and n is 0 or 1, and p is 0 to 15;
In some embodiments, in Formular (E-3), X is selected from O and NH;
In some embodiments, in Formular (E-3), R1, R2, R3, R4, R5, and R6, are independently selected from hydrogen, and optionally substituted C1-C6 alkyl.
In some embodiments, Formulae E, E-l, E-2, and E-3, the linker moiety comprises a ring selected from the group consisting of a 3 to 13 membered ring, a 3 to 13 membered fused ring, a 3 to 13 membered bridged ring, and a 3 to 13 membered spiro ring.
In some aspects of Formulae E, E-l, E-2, and E-3, the linker moiety comprises a ring selected from the group consisting of Formula Fl, F2, F3, F4 and F5:
Figure imgf000063_0001
Figure imgf000064_0001
Formula F4, Formula F5,
Wherein
X' and Y' are independently selected from N and CRb;
A1, B1, C1 and D1, at each occurrence, are independently selected from null, O, CO, SO, SO2, NRb, and CRbRc;
A2, B2, C2 and D2, at each occurrence, are independently selected from N and CRb;
A3, B3, C3, D3, and E3 at each occurrence, are independently selected from N, O, S, NRb, and CRb;
Rb and Rc , at each occurrence, are independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8alkylamino, optionally substituted C1-C8alkylaminoCi- C8 alkyl, optionally substituted 3-10 membered carbocyclic, optionally substituted 3-10 membered cycloalkoxy, optionally substituted 3-10 membered carbocyclicamino, optionally substituted 3-8 membered heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl;
Rb and Rb, or Rb and Rc together with the atom to which they are connected form a 3-8 membered carbocyclic or 3-8 membered heterocyclic ring; and m1, n1, o1 and p1 are independently selected from 0, 1, 2, 3, 4, and 5.
In one embodiment, A, B, and W, at each occurrence, are independently selected from null, optionally substituted -(CH2)o-8-, optionally substituted -(CH2)o-8-CO-(CH2)o-8-, optionally substituted -(CH2)o-8-NH-(CH2)o-s-, optionally substituted -(CH2)o-8-NH-CO-(CH2)o-8-, optionally substituted -(CH2)o-8-CO-NH-(CH2)o-8-, optionally substituted -(CH2)o-3-NH-(CH2)o-3-CO-NH- (CH2)O-8-, optionally substituted -(CH2)O-3-NH-(CH2)I-3-NH-CO-(CH2)O-8-, optionally substituted - (CH2)O-8-CO-NH-(CH2)I-3-NH-(CH2)O-3-, optionally substituted -(CH2)o-8-Rr-(CH2)o-s-, optionally substituted -(CH2)o-3-CO-(CH2)o-3- Rr-(CH2)o-3-, -(CH2)o-8-Rr-(CH2)o-8-, optionally substituted - (CH2)O-3-NH-CO-(CH2)0-3- Rr-(CH2)0-3-, optionally substituted <CH2)o-3-NH-(CH2)0-3- Rr-(CH2)o-
In one embodiment, Rr is of Formula Fl, F2, F3, F4, or F5.
In one embodiment, Rr is selected from
Figure imgf000065_0001
Figure imgf000066_0001
In another embodiment, the length of the linker is 0 to 40 atoms.
In another embodiment, the length of the linker is 0 to 20 atoms.
In another embodiment, the length of the linker is 0 to 10 atoms.
In another embodiment, the length of the linker is 0 to 40 atoms.
In another embodiment, the linker is selected from null, optionally substituted -CO-(CH2)o- io-, optionally substituted -(CH2)O-IO-, optionally substituted -(CH2)1-2-(CO)NH-(CH2)o-io-, optionally substituted -(CH2)I-2-(CO)NH-(CH2)I-3-(OCH2CH2)I-7-, optionally substituted -(CH2)o- I-CO-(CH2)1-3-(OCH2CH2)1-7-, optionally substituted -CO-(CH2)0-3-(alkenylene)-(CH2)0-3-, optionally substituted -CO-(CH2)0-3-(alkynylene)-(CH2)0-3-, optionally substituted -CO-(CH2)0-3- (3-8 membered carbocyclic)-(CH2)0-3-, optionally substituted -CO-(CH2)0-3-(3-8 membered heterocyclic)-(CH2)o-3-, optionally substituted -(CH2)o-3-(alkenylene)-(CH2)o-3-, optionally substituted -(CH2)o-3-(alkynylene)-(CH2)o-3-, optionally substituted -(CH2)o-3-(3-8 membered carbocyclic)-(CH2)o-3-, optionally substituted -(CH2)o-3-(3-8 membered heterocyclic)-(CH2)o-3-, optionally substituted -(CH2)o-s-Rr-(CH2)o-s-, optionally substituted -(CH2)o-8-Rr-(CO)-(CH2)i-8-, optionally substituted -(CH2)o-s-Rr-(CH2)i-2-(CO)-NH-(CH2)2-9-, optionally substituted -(CH2)o-8- Rr-(CH2)i-2-(CO)-NH-(CH2)i-3-(OCH2CH2)i-7-, optionally substituted -(CH2)o-s-Rr-(CH2)o-i- (CO)-(CH2)i-3-(OCH2CH2)i-7-, optionally substituted -(CH2)o-8-Rr-(CO)-(CH2)o-3-(alkenylene)- (CH2)O-3-, optionally substituted -(CH2)o-8-Rr-(CO)-(CH2)o-3-(alkynylene)-(CH2)o-3-, optionally substituted -(CH2)o-8-Rr-(CO)-(CH2)o-3-(3-8 membered carbocyclic)-(CH2)o-3-, optionally substituted -(CH2)o-8-Rr-(CO)-(CH2)o-3-(3-8 heterocyclic)-(CH2)o-3-, optionally substituted - (CH2)o-8-Rr-(CH2)o-3-(alkenylene)-(CH2)o-3-, optionally substituted -(CH2)o-8-Rr-(CH2)o-3- (alkynylene)-(CH2)o-3-, optionally substituted -(CH2)o-8-Rr-(CH2)o-3-(3-8 membered carbocyclcyl)-(CH2)o-3-, and optionally substituted -(CH2)o-8-Rr-(CH2)o-3-(heterocyclic)-(CH2)o-3--
In some embodiments, the linker can also be a moiety of:
Figure imgf000067_0001
Formula (E-2-a) Formula (E-3-a) wherein
A is C=O or CH2,
B is C=O or CH2, and m is 0-16, n is 0-6, and o is 0-6.
In some embodiments, the linker can also be a moiety of:
Figure imgf000067_0002
Formula (E-2-b), Formula (E-3-b) A is -C(O)N-, B is -C(O)N-, m is 0-16, n is 0-6, o is 0-6, n-1 is 0-6, and o-l is 0-6.
DUB Recruiter
In some aspects, the DUB recruiter comprises the structures of Formula (A-I):
Figure imgf000068_0001
Formula (A-I) or a tautomer thereof, or a pharmaceutically acceptable salt of the compound or the tautomer, wherein RD 4 is RD 4b
In some embodiments, the DUB recruiters comprise the structures of Formulae (A-I-a), (A-I-al), (A-I-a2), (A-I-a3), (A-I-a4), (A-I-a5), (A-I-a6), (A-I-a7), (A-I-a8), (A-I-a9), (A-I-alO), (A-I-al l), (A-I-b), (A-I-bl), (A-I-b2), (A-I-b3), (A-I-b4), (A-I-b5), (A-I-b6), (A-I-b7), (A-I-b8), (A-I-b9), (A-I-blO), (A-I-bl l), (A-I-c), (A-I-cl), (A-I-c2), (A-I-c3), (A-I-c4), (A-I-c5), (A-I-c6), (A-I-C7), (A-I-C8), (A-I-C9), (A-I-c 10), (A-I-c 11), (A-I-c 12), (A-I-c 13), (A-I-c 14), (A-I-c 15), (A- I-C16), (A-I-d), (A-I-d 1), (A-I-d2), (A-I-d3), (A-I-d4), (A-I-d5), (A-I-d6), (A-I-d7), (A-I-d8), (A- I-d9), (A-I-d 10), and (A-I-b 11), wherein RD 4 is RD 4b.
In some embodiments, RD 41) is a bond.
In some embodiments, the DUB recruiters comprise:
Figure imgf000068_0002
Figure imgf000068_0003
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
In some embodiments, the AMPK based bivalent compound is a compound selected from the following compounds, as identified in Table 3 below: XF137-81, XF137-82, XF137-83, XF137-84, XF137-85, XF137-86, XF137-87, XF137-88, XF137-89, XF137-90, XF137-91, XF137-92, XF137-93, XF137-94, QC179-047, QC179-048, QC137-049, QC179-050, QC179- 051, QC179-052, QC179-053, QC179-054, QC137-055, and examples 367 - 380, or analogs thereof.
In some embodiments, the cGAS based bivalent compound is a compound selected from the following compounds, as identified in table 4 below: ZD178-22, ZD178-23, ZD178-24, ZD178-25, ZD178-26, ZD178-27, ZD178-28, ZD178-29, ZD178-30, ZD178-31, ZD178-32, ZD178-33, ZD178-34, ZD178-35, ZD178-58-1, ZD178-58-2, ZD178-58-3, ZD178-58-4, ZD178- 58-5, ZD178-58-6, ZD178-58-7, ZD178-58-8, ZD178-58-9, ZD178-58-10, ZD178-58-11, ZD178-58-12, ZD178-58-13, ZD178-58-14, ZD178-63-1, ZD178-63-2, ZD178-63-3, ZD178-63- 4, ZD178-63-5, ZD178-63-6, ZD178-63-78, ZD178-63-8, ZD178-63-9, ZD178-63-10, ZD178- 63-11, ZD178-63-12, ZD178-63-13, ZD178-63-14, ZD178-63-15, ZD178-69-1, ZD178-69-2, ZD178-69-3, ZD178-69-4, ZD178-69-5, ZD178-69-6, ZD178-69-7, ZD178-69-8, ZD178-69-9, ZD178-69-10, ZD178-69-11, ZD178-69-12, ZD178-69-13, ZD178-69-14, ZD178-69-15, and examples 381 - 388, or analogs thereof.
In some embodiments, the CFTR based bivalent compound is a compound selected from the following compounds, as identified in Table 5 below: QC166-130, QC166-131, QC166-132, QC166-133, QC166-134, QC166-135, QC166-136, QC166-137, QC166-138, QC166-139,
QC166-140, QC166-141, QC166-142, QC166-143, QC166-167, QC166-174, QC166-175,
QC166-176, QC166-177, QC166-178, QC166-179, QC166-181, QC166-182, QC166-183,
QC166-184, QC166-185, QC179-104, QC179-105, QC179-106, QC179-107, QC179-108,
QC179-109, QC179-110, QC179-111, QC179-112, QC179-113, QC179-137, QC179-138,
QC179-139, QC179-140, QC179-141, QC179-154, QC179-155, QC179-156, QC179-157,
QC179-158, QC192-153, QC192-154, QC192-155, QC192-156, QC192-157, QC192-184,
QC192-178, QC192-179, QC192-180, QC192-181, QC192-182, or analogs thereof.
In some embodiments, this disclosure provides a method of treating AMPK, cGAS or CFTR-mediated disease, the method including administering to a subject in need thereof one or more bivalent compounds including a AMPK, cGAS, or CFTR ligand conjugated to a 0TUB1 binder via a linker. The AMPK, cGAS, CFTR-mediated disease can be a disease resulting from AMPK, cGAS or CFTR degradation. The AMPK, cGAS or CFTR-mediated disease can have decrease CFTR expression relative to a wild-type tissue of the same species and tissue type.
In some embodiments, the methods described herein, the bivalent compounds can be administered, e.g., orally, parenterally, intradermally, subcutaneously, topically, and/or rectally.
This disclosure additionally provides a method for identifying a bivalent compound which mediates de-ubiquitination/stabilization of AMPK, cGAS, or CFTR, the method including providing a heterobifunctional test compound including a AMPK, cGAS, or CFTR ligand conjugated to a de-ubiquitination tag via a linker, contacting the heterobifunctional test compound with a cell (e.g., a cell such as a AMPK, cGAS or CFTR-mediated disease cell) including a deubiquitinase (e.g., 0TUB1) and AMPK, cGAS or CFTR protein.
As used herein, the terms “about” and “approximately” are defined as being within plus or minus 10% of a given value or state, preferably within plus or minus 5% of said value or state. The terms “bivalent” and “bi-functional” are used interchangeably herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Effect of exemplary compounds on modifying OTUB1 in mass spectrometry-based assays.
Figure 2. Discovery of improved OTUB1 ligand MS5105.
Figure 3. Effect of exemplary AMPK based bivalent compounds in stabilization AMPK protein levels in Hela and HEK293T cells
Figure 4. Effect of exemplary AMPK based bivalent compounds in stabilization AMPK protein level in HEK293T cells in multiple concentrations.
Figure 5. Effect of exemplary cGAS based bivalent compounds in stabilization cGAS protein level in Hela cells.
Figure 6. Discovery of first-in-class cGAS DUBTACs.
Figure 7. cGAS DUBTACs MS7829 and mS8588 activate the cGAS/STING/IRF3 signaling.
Figure 8. The MS5105-based DUBTAC MS6178 increases CFTR-AF508 protein level much more effectively than the EN523-based CFTR DUBTAC NJH-2-057.
Figure 9. Effect of exemplary CFTR based bivalent compounds on stabilizing CFTR protein levels in human cystic fibrosis bronchial epithelial cells.
Figure 10. Effect of exemplary CFTR or cGAS based bivalent compounds on modifying OTUB1 mass spectrometry-based assays.
Testing of exemplary Compounds
The activity of novel synthesized covalent OTUB1 binder and bivalent AMPK-OTUBl, cGAS-OTUBl, CFTR-OTUB1 compounds can be assessed using standard biophysical assays (e g., isothermal titration calorimetry (ITC), surface plasmon resonance (SPR)) and mass spectrometry -based assays. Cellular assays can then be used to assess the bivalent CFTR-OTUB1 compound’s ability to stabilize AMPK, cGAS or CFTR proteins level. Assays suitable for use in any or all of these steps are known in the art, and include, e.g., Western blotting, quantitative mass spectrometry (MS) analysis, flow cytometry, enzymatic inhibition, ITC, SPR, cell growth inhibition and xenograft and PDX models.
By way of non-limiting example, detailed synthesis protocols are described in the Examples for specific exemplary covalent 0TUB1 binders, bivalent AMPK-0TUB1, cGAS- 0TUB1 and CFTR-OTUB1 compounds.
Pharmaceutically acceptable isotopic variations of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (substituting appropriate reagents with appropriate isotopic variations of those reagents). Specifically, an isotopic variation is a compound in which at least one atom is replaced by an atom having the same atomic number, but an atomic mass different from the atomic mass usually found in nature. Useful isotopes are known in the art and include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine. Exemplary isotopes thus include, e.g., 2H, 3H, 13C, 14C, 15N, 17O, 18O, 32P, 35 S, 18F, and 36C1.
Isotopic variations (e.g., isotopic variations containing 2H) can provide therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements. In addition, certain isotopic variations (particularly those containing a radioactive isotope) can be used in drug or substrate tissue distribution studies. The radioactive isotopes tritium (3H) and carbon-14 (14C) are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
Pharmaceutically acceptable solvates of the compounds disclosed herein are contemplated. A solvate can be generated, e.g., by substituting a solvent used to crystallize a compound disclosed herein with an isotopic variation (e.g., D2O in place of H2O, ^-acetone in place of acetone, or de- DMSO in place of DMSO).
Pharmaceutically acceptable fluorinated variations of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (substituting appropriate reagents with appropriate fluorinated variations of those reagents). Specifically, a fluorinated variation is a compound in which at least one hydrogen atom is replaced by a fluoro atom. Fluorinated variations can provide therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements. Pharmaceutically acceptable prodrugs of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (e.g., converting hydroxyl groups or carboxylic acid groups to ester groups). As used herein, a "prodrug" refers to a compound that can be converted via some chemical or physiological process (e.g., enzymatic processes and metabolic hydrolysis) to a therapeutic agent. Thus, the term "prodrug" also refers to a precursor of a biologically active compound that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject, i.e. an ester, but is converted hi vivo to an active compound, for example, by hydrolysis to the free carboxylic acid or free hydroxyl. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in an organism. The term "prodrug" is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a subject. Prodrugs of an active compound may be prepared by modifying functional groups present in the active compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent active compound. Prodrugs include compounds wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the active compound is administered to a subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of an alcohol or acetamide, formamide and benzamide derivatives of an amine functional group in the active compound and the like.
Definition of Terms
"Alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation. An alkyl may comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms. In certain embodiments, an alkyl comprises one to fifteen carbon atoms (e.g., C1-C15 alkyl). In certain embodiments, an alkyl comprises one to thirteen carbon atoms (e.g., C1-C13 alkyl). In certain embodiments, an alkyl comprises one to eight carbon atoms (e.g., C1-C8 alkyl). In other embodiments, an alkyl comprises five to fifteen carbon atoms (e.g., C5-C15 alkyl). In other embodiments, an alkyl comprises five to eight carbon atoms (e.g., Cs-Cs alkyl). The alkyl is attached to the rest of the molecule by a single bond, for example, methyl (Me), ethyl (Et), n-propyl, 1 -methylethyl (/.w-propyl), //-butyl, //-pentyl, 1 , 1 -dimethylethyl (/-butyl), pentyl, 3 -methylhexyl,
2-methylhexyl, and the like.
“Alkylene” refers to a bivalent saturated aliphatic radical (such as ethylene) regarded as derived from an alkene by opening of the double bond or from an alkane by removal of two hydrogen atoms from different carbon atoms.
"Alkenyl1' refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond. An alkenyl may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms. In certain embodiments, an alkenyl comprises two to twelve carbon atoms (e.g, C2-C12 alkenyl). In certain embodiments, an alkenyl comprises two to eight carbon atoms (e.g., C2-C8 alkenyl). In certain embodiments, an alkenyl comprises two to six carbon atoms (e.g., C2- , alkenyl). In other embodiments, an alkenyl comprises two to four carbon atoms (e.g., C2-C4 alkenyl). The alkenyl is attached to the rest of the molecule by a single bond, for example, ethenyl (i.e., vinyl), prop-l-enyl (i.e., allyl), but-l-enyl, pent-l-enyl, penta- 1,4-dienyl, and the like.
The term “alkenylene” refers to linear or branched-chain divalent hydrocarbon radical of two to eight carbon atoms (C2-C8) with at least one site of unsaturation, i.e., a carbon-carbon, SP2 double bond, wherein the alkenylene radical may be optionally substituted independently with one or more substituents described herein, and includes radicals having “cis” and “trans” orientations, or alternatively, “E” and “Z” orientations. Examples include, but are not limited to ,ethylenylene or vinylene (-CH=CH-), allyl (-CH2 CH=CH-), and the like.
The term “allyl,” as used herein, means a -CH2CI-GCH2 group.
As used herein, the term "alkynyl" refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond. An alkynyl may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms. In certain embodiments, an alkynyl comprises two to twelve carbon atoms (e.g., C2-C12 alkynyl). In certain embodiments, an alkynyl comprises two to eight carbon atoms (e.g., C2-C8 alkynyl). In other embodiments, an alkynyl has two to six carbon atoms (e.g., C2-C6 alkynyl). In other embodiments, an alkynyl has two to four carbon atoms (e.g., C2-C4 alkynyl). The alkynyl is attached to the rest of the molecule by a single bond. Examples of such groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1 -pentynyl, 2-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, and the like. The term “alkynylene” refers to a linear or branched divalent hydrocarbon radical of two to eight carbon atoms (C2-C8) with at least one site of unsaturation, i.e., a carbon-carbon, sp triple bond, wherein the alkynylene radical may be optionally substituted independently with one or more substituents described herein. Examples include, but are not limited to ,ethynylene (-C =C~), propynylene (propargylene, -CH2 C = C-), and the like.
The term " alkoxy", as used herein, means an alkyl group as defined herein witch is attached to the rest of the molecule via an oxygen atom. Examples of such groups include, but are not limited to, methoxy, ethoxy, n-propyloxy, iso-propyloxy, n-butoxy, iso-butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.
“Heteroalkyl” refers to a substituted or unsubstituted alkyl group which has one or more skeletal chain atoms selected from an atom other than carbon, Exemplary skeletal chain atoms selected from an atom other tha n carbon include , e.g., O, N, P, Si, S, or combinations thereof, wherein the nitrogen, phosphorus, and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized . If given, a numberical range refers to the chain length in total. For example, a 3- to 8- membered heteroalkyl has a chain length of 3 to 8 atoms. Connection to the rest of the molecule may be through either a heteroatom or a carbon in the heteroalkyl chain. Unless stated otherwise specifically in the specification, a heteroalkyl group is optionally substituted by one or more substituents such as those substituents described herein.
The term “aryl”, as used herein, " refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or multicyclic hydrocarbon ring system contains only hydrogen and carbon atoms. An aryl may comprise from six to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) 7t-electron system in accordance with the Hiickel theory. In certain embodiments, an aryl comprises six to fourteen carbon atoms (Ce-Cu aryl). In certain embodiments, an aryl comprises six to ten carbon atoms (C6-Cio aryl). Examples of such groups include, but are not limited to, phenyl, fluorenyl and naphthyl. The terms “Ph” and “phenyl,” as used herein, mean a -C6H5 group.
The term “arylene” means a divalent aromatic hydrocarbon radical of 6-20 carbon atoms (C6-C20) derived by the removal of two hydrogen atom from two carbon atoms of a parent aromatic ring system. Some arylene groups are represented in the exemplary structures as “Ar”. Arylene includes bicyclic radicals comprising an aromatic ring fused to a saturated, partially unsaturated ring, or aromatic carbocyclic ring. Typical arylene groups include, but not limited to, radicals derived from benzene (phenylene), substituted benzenes, naphthalene, anthracene, biphenylene indenylene, indaylene, 1,2-dihydronaphthalene, 1,2, 3, 4, -tetrahydronaphthyl, and the like. Arylene groups are optionally substituted with one or more substituents described herein.
The term “heteroaryl”, refers to a radical derived from a 3- to 18-membered aromatic ring radical that comprises two to seventeen carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, the heteroaryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) ^-electron system in accordance with the Hiickel theory. Heteroaryl includes fused or bridged ring systems. In certain embodiments, a heteroaryl refers to a radical derived from a 3- to 10-membered aromatic ring radical (3-10 membered heteroaryl). In certain embodiments, a heteroaryl refers to a radical derived from 5- to 7-membered aromatic ring (5-7 membered heteroaryl). Heteroaryl includes fused or bridged ring systems. The heteroatom(s) in the heteroaryl radical is optionally oxidized. One or more nitrogen atoms, if present, are optionally quatemized. The heteroaryl is attached to the rest of the molecule through any atom of the ring(s). Examples of such groups include, but not limited to, pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, furopyridinyl, and the like. In certain embodiments, an heteroaryl is attached to the rest of the molecule via a ring carbon atom. In certain embodiments, an heteroaryl is attached to the rest of the molecule via a nitrogen atom (N-attached) or a carbon atom (C-attached). For instance, a group derived from pyrrole may be pyrrol-l-yl (N-attached) or pyrrol-3-yl (C-attached). Further, a group derived from imidazole may be imidazol-l-yl (N-attached) or imidazol-3-yl (C-attached).
The term “heterocyclic”, as used herein, means a non-aromatic, monocyclic, bicyclic, tricyclic, or tetracyclic radical having a total of from 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 atoms in its ring system, and containing from 3 to 12 carbon atoms and from 1 to 4 heteroatoms each independently selected from O, S and N, and with the proviso that the ring of said group does not contain two adjacent O atoms or two adjacent S atoms. A heterocyclic group may include fused, bridged or spirocyclic ring systems. In certain embodiments, a heterocyclic group comprises 3 to 10 ring atoms (3-10 membered heterocyclic). In certain embodiments, a heterocyclic group comprises 3 to 8 ring atoms (3-8 membered heterocyclic). In certain embodiments, a heterocyclic group comprises 4 to 8 ring atoms (4-8 membered heterocyclic). In certain embodiments, a heterocyclic group comprises 3 to 6 ring atoms (3-6 membered heterocyclic). A heterocyclic group may contain an oxo substituent at any available atom that will result in a stable compound. For example, such a group may contain an oxo atom at an available carbon or nitrogen atom. Such a group may contain more than one oxo substituent if chemically feasible. In addition, it is to be understood that when such a heterocyclic group contains a sulfur atom, said sulfur atom may be oxidized with one or two oxygen atoms to afford either a sulfoxide or sulfone. An example of a 4 membered heterocyclic group is azetidinyl (derived from azetidine). An example of a 5 membered cycloheteroalkyl group is pyrrolidinyl. An example of a 6 membered cycloheteroalkyl group is piperidinyl. An example of a 9 membered cycloheteroalkyl group is indolinyl. An example of a 10 membered cycloheteroalkyl group is 4H-quinolizinyl. Further examples of such heterocyclic groups include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3- pyrrolinyl, indolinyl, 2H-pyranyl, 477- pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3. 1.0]hexanyl, 3-azabicyclo[4. 1.0]heptanyl, 3/7-indolyl, quinolizinyl, 3-oxopiperazinyl, 4-methylpiperazinyl, 4-ethylpiperazinyl, and l-oxo-2,8,diazaspiro[4.5]dec-8-yl. A heteroaryl group may be attached to the rest of molecular via a carbon atom (C-attached) or a nitrogen atom (N-attached). For instance, a group derived from piperazine may be piperazin- 1-yl (N-attached) or piperazin-2-yl (C-attached).
The term " cycloalkyl" or “carbocyclic” means a saturated, monocyclic, bicyclic, tricyclic, or tetracyclic radical having a total of from 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 carbon atoms in its ring system. A cycloalkyl may be fused, bridged or spirocyclic. In certain embodiments, a cycloalkyl comprises 3 to 8 carbon ring atoms (C3-C8 cycloalkyl). In certain embodiments, a cycloalkyl comprises 3 to 6 carbon ring atoms ( C3-C6 cycloalkyl). Examples of such groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptyl, adamantyl, and the like.
The term “cycloalkylene” or “carbocyclicene” is a bidentate radical obtained by removing a hydrogen atom from a cycloalkyl ring as defined above. Examples of such groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentyl ene, cyclopentenylene, cyclohexylene, cycloheptylene, and the like.
The term "spirocyclic" as used herein has its conventional meaning, that is, any ring system containing two or more rings wherein two of the rings have one ring carbon in common. Each ring of the spirocyclic ring system, as herein defined, independently comprises 3 to 20 ring atoms. Preferably, they have 3 to 10 ring atoms. Non-limiting examples of a spirocyclic system include spiro[3.3]heptane, spiro[3.4] octane, and spiro[4.5]decane.
The term cyano" refers to a -C=N group.
An "aldehyde" group refers to a -C(O)H group.
An "alkoxy" group refers to both an -O-alkyl, as defined herein.
An "alkoxycarbonyl" refers to a -C(O)-alkoxy, as defined herein.
An "alkylaminoalkyl" group refers to an -alkyl-NR-alkyl group, as defined herein.
An "alkylsulfonyl" group refer to a -SChalkyl, as defined herein.
An "amino" group refers to an optionally substituted -NH2.
An "aminoalkyl" group refers to an -alky-amino group, as defined herein.
An "aminocarbonyl" refers to a -C(O)-amino, as defined herein.
An "arylalkyl" group refers to -alkylaryl, where alkyl and aryl are defined herein.
An "aryloxy" group refers to both an -O-aryl and an -O-heteroaryl group, as defined herein.
An "aryloxycarbonyl" refers to -C(O)-aryloxy, as defined herein.
An "aryl sulfonyl" group refers to a -SCharyl, as defined herein.
A "carbonyl" group refers to a -C(O)- group, as defined herein.
A "carboxylic acid" group refers to a -C(O)OH group.
A “cycloalkoxy” refers to a -O-cycloalkyl group, as defined herein.
A "halo" or "halogen" group refers to fluorine, chlorine, bromine or iodine.
A "haloalkyl" group refers to an alkyl group substituted with one or more halogen atoms.
A "hydroxy" group refers to an -OH group.
A "nitro" group refers to a -NO2 group. An “oxo” group refers to the =0 substituent.
A "trihalomethyl" group refers to a methyl substituted with three halogen atoms.
The term “substituted,” means that the specified group or moiety bears one or more substituents independently selected from C1-C4 alkyl, aryl, heteroaryl, aryl-C1-C4 alkyl-, heteroaryl-C1-C4 alkyl-, C1-C4 haloalkyl, -OC1-C4 alkyl, -OC1-C4 alkylphenyl, -C1-C4 alkyl-OH, -OC1-C4 haloalkyl, halo, -OH, -NH2, -C1-C4 alkyl-NH2, -N(C1-C4 alkyl)(C1-C4 alkyl), -NH(C1-C4 alkyl), -N(C1-C4 alkyl)(C1-C4 alkylphenyl), -NH(C1-C4 alkylphenyl), cyano, nitro, oxo, -C02H, -C(O)OC1-C4 alkyl, -CON(C1-C4 alkyl)(C1-C4 alkyl), -CONH(C1-C4 alkyl), -C0NH2, -NHC(O)(C1-C4 alkyl), -NHC(O)(phenyl), -N(C1-C4 alkyl)C(O)(C1-C4 alkyl), -N(C1-C4 alkyl)C(O)(phenyl), -C(O)C1-C4 alkyl, -C(O)C1-C4 alkylphenyl, -C(O)C1-C4 haloalkyl, -OC(O)C1-C4 alkyl, -SO2(C1-C4 alkyl), -SO2(phenyl), -SO2(C1-C4 haloalkyl), -SO2NH2, -SO2NH(C1-C4 alkyl), -SO2NH(phenyl), -NHSO2(C1-C4 alkyl), -NHSO2(phenyl), and -NHSO2(C1-C4 haloalkyl).
The term “null” means the absence of an atom or moiety, and there is a bond between adjacent atoms in the structure.
The term “optionally substituted” means that the specified group may be either unsubstituted or substituted by one or more substituents as defined herein. It is to be understood that in the compounds of the present invention when a group is said to be “unsubstituted,” or is “substituted” with fewer groups than would fill the valencies of all the atoms in the compound, the remaining valencies on such a group are filled by hydrogen. For example, if a Ce aryl group, also called “phenyl” herein, is substituted with one additional substituent, one of ordinary skill in the art would understand that such a group has 4 open positions left on carbon atoms of the C6 aryl ring (6 initial positions, minus one at which the remainder of the compound of the present invention is attached to and an additional substituent, remaining 4 positions open). In such cases, the remaining 4 carbon atoms are each bound to one hydrogen atom to fill their valencies. Similarly, if a Ce aryl group in the present compounds is said to be “di substituted,” one of ordinary skill in the art would understand it to mean that the C6 aryl has 3 carbon atoms remaining that are unsubstituted. Those three unsubstituted carbon atoms are each bound to one hydrogen atom to fill their valencies. As used herein, the same symbol in a different FORMULA may have a different definition, for example, the definition of R1 in FORMULA 1 is as defined with respect to FORMULA 1 and the definition of R1 in FORMULA 6 is as defined with respect to FORMULA 6.
As used herein, when m (or n or u or v or w) is defined by a range, for example, “m is 0 to 15” or “m = 0-3” means that m is an integer from 0 to 15 (i.e. m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) or m is an integer from 0 to 3(i.e. m is 0, 1,2, or 3) or is any integer in the defined range.
"Pharmaceutically acceptable salt" includes both acid and base addition salts. A pharmaceutically acceptable salt of any one of the bivalent compounds described herein is intended to encompass any and all pharmaceutically suitable salt forms. Preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
"Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts that are formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl -substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and. aromatic sulfonic acids, etc. and include, for example, acetic acid, tri fluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like. Also contemplated are salts of amino acids, such as arginates, gluconates, and galacturonates (see, for example, Berge S.M. et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, 66: 1-19 (1997), which is hereby incorporated by reference in its entirety). Acid addition salts of basic compounds may be prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.
"Pharmaceutically acceptable base addition salt" refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N '.N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, A-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, A-ethylpiperidine, polyamine resins and the like. See Berge et al., supra.
Pharmaceutical Compositions
In some aspects, the compositions and methods described herein include the manufacture and use of pharmaceutical compositions and medicaments that include one or more bivalent compounds as disclosed herein. Also included are the pharmaceutical compositions themselves.
In some aspects, the compositions disclosed herein can include other compounds, drugs, or agents used for the treatment of cancer. For example, in some instances, pharmaceutical compositions disclosed herein can be combined with one or more (e.g., one, two, three, four, five, or less than ten) compounds. Such additional compounds can include, e.g., conventional chemotherapeutic agents known in the art. When co-administered, 0TUB1 covalent binders or AMPK, cGAS or CFTR based bivalent compounds disclosed herein can operate in conjunction with conventional chemotherapeutic agents to produce mechanistically additive or synergistic therapeutic effects. In some aspects, the pH of the compositions disclosed herein can be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the 0TUB1 covalent binders or AMPK, cGAS or CFTR based bivalent compounds or its delivery form.
Pharmaceutical compositions typically include a pharmaceutically acceptable carrier, adjuvant, or vehicle. As used herein, the phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are generally believed to be physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. A pharmaceutically acceptable carrier, adjuvant, or vehicle is a composition that can be administered to a patient, together with a compound of the invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound. Exemplary conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles include saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
In particular, pharmaceutically acceptable carriers, adjuvants, and vehicles that can be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDD S) such as d- a-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene polyoxypropyl ene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as a-, -, and γ-cyclodextrin, may also be advantageously used to enhance delivery of compounds of the formulae described herein.
As used herein, the OTUB 1 covalent binders or AMPK, cGAS or CFTR based bivalent compounds disclosed herein are defined to include pharmaceutically acceptable derivatives or prodrugs thereof. A “pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, solvate, or prodrug, e.g., carbamate, ester, phosphate ester, salt of an ester, or other derivative of a compound or agent disclosed herein, which upon administration to a recipient is capable of providing (directly or indirectly) a compound described herein, or an active metabolite or residue thereof. Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds disclosed herein when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Preferred prodrugs include derivatives where a group that enhances aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein. Such derivatives are recognizable to those skilled in the art without undue experimentation. Nevertheless, reference is made to the teaching of Burger’s Medicinal Chemistry and Drug Discovery, 5th Edition, Vol. 1: Principles and Practice, which is incorporated herein by reference to the extent of teaching such derivatives.
The OTUB1 covalent binders or AMPK, cGAS or CFTR based bivalent compounds disclosed herein include pure enantiomers, mixtures of enantiomers, pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates, mixtures of diastereoisomeric racemates and the meso-form and pharmaceutically acceptable salts, solvent complexes, morphological forms, or deuterated derivatives thereof.
In particular, pharmaceutically acceptable salts of the OTUB1 covalent binders or AMPK, cGAS or CFTR based bivalent compounds disclosed herein include, e.g., those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecyl sulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate, trifluoromethylsulfonate, and undecanoate. Salts derived from appropriate bases include, e.g., alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N- (alkyl)4+ salts. The invention also envisions the quaternization of any basic nitrogen-containing groups of the OTUB1 covalent binders or AMPK, cGAS and CFTR based bivalent compounds disclosed herein. Water or oil-soluble or dispersible products can be obtained by such quaternization. In some aspects, the pharmaceutical compositions disclosed herein can include an effective amount of one or more OTUB1 covalent binders or AMPK, cGAS and CFTR based bivalent compounds. The terms “effective amount” and “effective to treat,” as used herein, refer to an amount or a concentration of one or more compounds or a pharmaceutical composition described herein utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome (e.g., treatment or prevention of cell growth, cell proliferation, or cancer). In some aspects, pharmaceutical compositions can further include one or more additional compounds, drugs, or agents used for the treatment of cancer (e.g., conventional chemotherapeutic agents) in amounts effective for causing an intended effect or physiological outcome (e g., treatment or prevention of cell growth, cell proliferation, or cancer).
In some aspects, the pharmaceutical compositions disclosed herein can be formulated for sale in the United States, import into the United States, or export from the United States.
Administration of Pharmaceutical Compositions
The pharmaceutical compositions disclosed herein can be formulated or adapted for administration to a subject via any route, e.g., any route approved by the Food and Drug Administration (FDA). Exemplary methods are described in the FDA Data Standards Manual (DSM) (available at http://www.fda.gov/Drugs/DevelopmentApprovalProcess/ FormsSubmissionRequirements/ElectronicSubmissions/DataStandardsManualmonographs). In particular, the pharmaceutical compositions can be formulated for and administered via oral, parenteral, or transdermal delivery. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraperitoneal, intra-articular, intra-arterial, intrasynovial, intrastemal, intrathecal, intralesional, and intracranial injection or infusion techniques.
For example, the pharmaceutical compositions disclosed herein can be administered, e.g., topically, rectally, nasally (e.g., by inhalation spray or nebulizer), buccally, vaginally, subdermally (e.g., by injection or via an implanted reservoir), or ophthalmically.
For example, pharmaceutical compositions of this invention can be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried com starch. When aqueous suspensions or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added.
For example, the pharmaceutical compositions of this invention can be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.
For example, the pharmaceutical compositions of this invention can be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, or other solubilizing or dispersing agents known in the art.
For example, the pharmaceutical compositions of this invention can be administered by injection (e.g., as a solution or powder). Such compositions can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, e.g., as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, e.g., olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions. Other commonly used surfactants such as Tweens, Spans, or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation.
In some aspects, an effective dose of a pharmaceutical composition of this invention can include, but is not limited to, e.g., about 0.00001, 0.0001, 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2500, 5000, or 10000 mg/kg/day, or according to the requirements of the particular pharmaceutical composition.
When the pharmaceutical compositions disclosed herein include a combination of a compound of the formulae described herein (e.g., an OTUB1 covalent binder or an AMPK, cGAS or CFTR based bivalent compound) and one or more additional compounds (e.g., one or more additional compounds, drugs, or agents used for the treatment of cancer or any other condition or disease, including conditions or diseases known to be associated with or caused by cancer), both the compound and the additional compound should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen. The additional agents can be administered separately, as part of a multiple dose regimen, from the compounds of this invention. Alternatively, those agents can be part of a single dosage form, mixed together with the compounds of this invention in a single composition.
In some aspects, the pharmaceutical compositions disclosed herein can be included in a container, pack, or dispenser together with instructions for administration.
Methods of Treatment
The methods disclosed herein contemplate administration of an effective amount of a compound or composition to achieve the desired or stated effect. Typically, the compounds or compositions of the invention will be administered from about 1 to about 6 times per day or, alternately or in addition, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such preparations can contain from about 20% to about 80% active compound. In some aspects, the present disclosure provides methods for using a composition comprising an 0TUB1 covalent binder or AMPK, cGAS or CFTR based bivalent compound, including pharmaceutical compositions (indicated below as ‘X’) disclosed herein in the following methods:
Substance X for use as a medicament in the treatment of one or more diseases or conditions disclosed herein (e.g., cancer, referred to in the following examples as ‘Y’). Use of substance X for the manufacture of a medicament for the treatment of Y; and substance X for use in the treatment of Y.
In some aspects, the methods disclosed include the administration of a therapeutically effective amount of one or more of the compounds or compositions described herein to a subject (e.g., a mammalian subject, e.g., a human subject) who is in need of, or who has been determined to be in need of, such treatment. In some aspects, the methods disclosed include selecting a subject and administering to the subject an effective amount of one or more of the compounds or compositions described herein, and optionally repeating administration as required for the prevention or treatment of cancer.
In some aspects, subject selection can include obtaining a sample from a subject (e.g., a candidate subject) and testing the sample for an indication that the subject is suitable for selection. In some aspects, the subject can be confirmed or identified, e.g. by a health care professional, as having had or having a condition or disease. In some aspects, suitable subjects include, for example, subjects who have or had a condition or disease but that resolved the disease or an aspect thereof, present reduced symptoms of disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), or that survive for extended periods of time with the condition or disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), e.g., in an asymptomatic state (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease). In some aspects, exhibition of a positive immune response towards a condition or disease can be made from patient records, family history, or detecting an indication of a positive immune response. In some aspects, multiple parties can be included in subject selection. For example, a first party can obtain a sample from a candidate subject and a second party can test the sample. In some aspects, subjects can be selected or referred by a medical practitioner (e.g., a general practitioner). In some aspects, subject selection can include obtaining a sample from a selected subject and storing the sample or using the in the methods disclosed herein. Samples can include, e.g., cells or populations of cells.
In some aspects, methods of treatment can include a single administration, multiple administrations, and repeating administration of one or more compounds disclosed herein as required for the prevention or treatment of the disease or condition from which the subject is suffering (e.g., AMPK, cGAS or CFTR-related disease). In some aspects, methods of treatment can include assessing a level of disease in the subject prior to treatment, during treatment, or after treatment. In some aspects, treatment can continue until a decrease in the level of disease in the subject is detected.
The term “subject,” as used herein, refers to any animal. In some instances, the subject is a mammal. In some instances, the term “subject,” as used herein, refers to a human (e.g., a man, a woman, or a child).
The terms “administer,” “administering,” or “administration,” as used herein, refer to implanting, ingesting, injecting, inhaling, or otherwise absorbing a compound or composition, regardless of form. For example, the methods disclosed herein include administration of an effective amount of a compound or composition to achieve the desired or stated effect.
The terms “treat”, “treating,” or “treatment,” as used herein, refer to partially or completely alleviating, inhibiting, ameliorating, or relieving the disease or condition from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder (e.g., cancer) are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular disorder (e.g., cancer) refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with treatment by the compositions and methods of the present invention. In some embodiments, treatment can promote or result in, for example, a decrease in the number of tumor cells (e.g., in a subject) relative to the number of tumor cells prior to treatment; a decrease in the viability (e.g., the average/mean viability) of tumor cells (e.g., in a subject) relative to the viability of tumor cells prior to treatment; a decrease in the rate of growth of tumor cells; a decrease in the rate of local or distant tumor metastasis; or reductions in one or more symptoms associated with one or more tumors in a subject relative to the subject’s symptoms prior to treatment.
The terms “prevent,” “preventing,” and “prevention,” as used herein, shall refer to a decrease in the occurrence of a disease or decrease in the risk of acquiring a disease or its associated symptoms in a subject. The prevention may be complete, e.g., the total absence of disease or pathological cells in a subject. The prevention may also be partial, such that the occurrence of the disease or pathological cells in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the present invention. Exemplary AMPK, cGAS or CFTR-mediated diseases that can be treated with AMPK, cGAS or CFTR based DUBTACs include, for example, cystic fibrosis, breast cancer, ovarian cancer, prostate cancer, colon cancer, pancreatic cancer, bladder cancer, liver cancer and cervical cancer.
As used herein, the term “preventing a disease” (e g., preventing cancer) in a subject means for example, to stop the development of one or more symptoms of a disease in a subject before they occur or are detectable, e.g., by the patient or the patient’s doctor. Preferably, the disease (e.g., cancer) does not develop at all, i.e., no symptoms of the disease are detectable. However, it can also result in delaying or slowing of the development of one or more symptoms of the disease. Alternatively, or in addition, it can result in the decreasing of the severity of one or more subsequently developed symptoms.
Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient’s disposition to the disease, condition or symptoms, and the judgment of the treating physician.
An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. Moreover, treatment of a subject with a therapeutically effective amount of the compounds or compositions described herein can include a single treatment or a series of treatments. For example, effective amounts can be administered at least once. The compositions can be administered from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present.
Following administration, the subject can be evaluated to detect, assess, or determine their level of disease. In some instances, treatment can continue until a change (e.g., reduction) in the level of disease in the subject is detected. Upon improvement of a patient’s condition (e.g., a change (e.g., decrease) in the level of disease in the subject), a maintenance dose of a compound, or composition disclosed herein can be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, can be reduced, e.g., as a function of the symptoms, to a level at which the improved condition is retained. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
The present disclosure is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiment or aspect described herein. Indeed, many modifications and variations may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.
EXAMPLES
Procedures for the synthesis of OTUB1 covalent binders
Scheme 1. Synthesis of example 1
Figure imgf000099_0001
Example 1
Example 1: 4-acryloyl-l-(5-methylthiophen-2-yl)piperazin-2-one (XS154-91) To a solution of 2-bromo-5-methylthiophene (50.0 mg, 0.28 mmol) dissolved in dioxane (1 mL), N,N’- dimethylethylenediamine ( 9 pL, 0.0846 mmol, 0.3 eq), K2CO3 ( 116.9 mg, 0.846 mmol, 3.0 eq), Cui ( 5.4 mg, 0.028 mmol, O.leq ) followed by tert-butyl 3 -oxopiperazine- 1 -carboxylate (84.7 mg, 0.42 mmol, 1.5 eq). The reaction mixture was stirred at 100 °C under nitrogen atmosphere overnight. After cooling down to rt, resulting crude mixtures were purified via silica gel column chromatography to yield intermediate 1. To a solution of intermediate 1 (20.4 mg, 0.069 mmol) dissolved in DCM (0.5 mL), and the TFA( 0.5 mL) were added. The reaction mixture stirred at rt 1 h. Excess TFA was removed, resulting crude product was re-dissovled in DCM (1 mL), Et3N (13 pL, 0.089 mmol, 2.0 eq) was added. At 0 °C the acryloyl chloride (4.8 pL, 0.053 mmol, 1.2 eq.) was added, the reaction mixture stirred at 0 °C for 30 mins. Resulting crude mixtures were purified via prep-HPLC to yield title compound (White solid, 55% yield) 'HNMR (400 MHz, Methanol-d4) 8 6.92 - 6.68 (m, 1H), 6.68 - 6.54 (m, 2H), 6.31 (d, J = 16.7 Hz, 1H), 5.90 - 5.76 (m, 1H), 4.48 (d, J = 28.0 Hz, 2H), 4.15 - 3.81 (m, 4H), 2.42 (d, J= 2.2 Hz, 3H). MS (ESI) [M+H]+ =251.0.
Figure imgf000100_0001
Example 2: 4-acryloyl-l-(p-tolyl)piperazin-2-one (XS154-130). Example 2 was synthesized following similar procedure for preparing example 1. White solid, 32% yield. TH NMR (400 MHz,
Methanol-^) 6 7.31 - 7.20 (m, 4H), 6.92 - 6.69 (m, 1H), 6.33 (dt, J= 16.7, 2.4 Hz, 1H), 5.85 (d,
J= 10.6 Hz, 1H), 4.54 - 4.30 (m, 2H), 4.12 - 3.97 (m, 2H), 3.88 - 3.74 (m, 2H), 2.37 (d, J= 2.2
Figure imgf000100_0002
Example 4: 4-acryloyl-l-(2-methylpyrimidin-4-yl)piperazin-2-one (XS154-149). Example 4 was synthesized following similar procedure for preparing example 1. White solid, 46% yield. 1 H NMR (400 MHz, Chloroform-d/) 8 8.68 (d, J= 6.3 Hz, 1H), 8.35 (s, 1H), 6.54 (t, J= 13.2 Hz, 1H), 6.44 (d, J = 16.4 Hz, 1H), 5.85 (dd, J = 10.0, 2.6 Hz, 1H), 4.64 - 4.45 (m, 2H), 4.44 - 4.26 (m, 2H), 4.05 - 3.87 (m, 2H), 2.77 (d, J= 2.2 Hz, 3H). MS (ESI) [M+H]+ =247.3.
Scheme 2. Synthesis of example 5
Figure imgf000101_0001
Example 5: l-(4-(»/-tolyl)piperazin-l-yl)prop-2-en-l-one (XS159-19). To a solution of the 1- iodo-3 -methylbenzene (218.0 mg, 1 mmol) dissolved in dioxane (3 mL), Xantphos (173 mg, 0.3 mmol, 0.3 eq), Cs2CO3 (655.6 mg, 2.0 eq), Pd2(dba)3 (91.6 mg, 0.1 mmol, 0.1 eq), followed by /cvZ-butyl 3 -oxopiperazine- 1 -carboxylate (279.6 mg, 1.5 mmol, 1.5 eq). The reaction mixture was stirred at 100 °C under nitrogen atmosphere overnight. After cooling down to rt, resulting crude mixtures were purified via silica gel column chromatography to yield intermediate 2.
To a solution of intermediate 2 (260 mg, 0.94 mmol) dissolved in DCM (1 mL), and the TFA( 1 mL) were added. The reaction mixture stirred at rt 1 h. Excess TFA was removed, resulting crude product was re-dissovled in DCM (1 mL), Et3N (380 pL, 2.68 mmol, 3.0 eq) was added. At 0 °C the acryloyl chloride (87 pL, 1.07 mmol, 1.2 eq.) was added, the reaction mixture stirred at 0 °C for 30 mins. Resulting crude mixtures were purified via prep-HPLC to yield title compound (White solid, 17% yield) *HNMR (400 MHz, DMSO-d6) 87.21 - 7.07 (m, 1H), 6.91 - 6.76 (m, 3H), 6.71 (d, J = 7.6 Hz, 1H), 6.14 (dd, J = 16.6, 3.1 Hz, 1H), 5.71 (dd, J = 10.4, 3.0 Hz, 1H), 3.78 - 3.65 (m, 4H), 3.17 (s, 4H), 2.26 (d, J= 2.3 Hz, 3H). MS (ESI) [M+H]+ =231.1
Scheme 3. Synthesis of example 6
Figure imgf000101_0002
Example 6: 3-(3-(4-acryloyl-2-oxopiperazin-l-yl)phenyl)propanoic acid (XS159-13). To a solution of tert-butyl 3-(3-iodophenyl)propanoate (290.1 mg, 1 mmol) dissolved in dioxane (3 mL), N,N’ -dimethylethylenediamine ( 32 pL, 0.3 mmol, 0.3 eq), K2CO3 ( 414.6 mg, 3.0 mmol, 3.0 eq), Cui ( 19.1 mg, 0.1 mmol, 0. leq ) followed by tert-butyl 3 -oxopiperazine- 1 -carboxylate (300.3 mg, 1.5 mmol, 1.5 eq). The reaction mixture was stirred at 100 °C under nitrogen atmosphere overnight. After cooling down to rt, resulting crude mixtures were purified via silica gel column chromatography to yield intermediate 3.
To a solution of intermediate 3 (130 mg, 0.36 mmol) dissolved in DCM (0.5 mL), and the TFA( 0.5 mL) were added. The reaction mixture stirred at rt 1 h. Excess TFA was removed, resulting crude product was re-dissovled in DCM (1 mL), Et3N (68 pL, 0.483 mmol, 3.0 eq) was added. At 0 °C the acryloyl chloride (16 pL, 0.194 mmol, 1.2 eq.) was added, the reaction mixture stirred at 0 °C for 30 mins. Resulting crude mixtures were purified via prep-HPLC to yield title compound (White solid, 37% yield) 'H NMR (400 MHz, Methanol-d 4 7.35 (t, J= 6.3 Hz, 1H), 7.24 - 7.16 (m, 3H), 6.90 - 6.66 (m, 1H), 6.32 (dd, J= 16.9, 3.7 Hz, 1H), 5.84 (d, J= 10.5 Hz, 1H), 4.43 (d, J = 25.3 Hz, 2H), 4.04 (t, J= 5.9 Hz, 2H), 3.90 - 3.77 (m, 2H), 2.95 (q, J= 1A, 6.5 Hz, 2H), 2.63 (q, J= 7.5, 5.6 Hz, 2H). MS (ESI) [M+H]+ =303.0.
Figure imgf000102_0001
Example 7: l-acryloyl-4-(5-methylfuran-2-yl)-l,4-diazepan-5-one (XS159-107). Example 7 was synthesized following similar procedure for preparing example 1. White solid, 27% yield. 'H NMR (400 MHz, Methanol-^) 8 7.43 - 6.67 (m, 2H), 6.23 (d, J= 17.0 Hz, 1H), 6.03 (d, J= 13.7 Hz, 1H), 5.79 (s, 1H), 3.92 - 3.76 (m, 6H), 2.73 - 2.63 (m, 2H), 2.25 (s, 3H). MS (ESI) [M+H]+ =249.2.
Figure imgf000102_0002
Example 8: 4-:icryloyl-l-( L2-diinethyl-l//-iniidazol-5-yl)piperazin-2-one (XS165-30).
Example 8 was synthesized following similar procedure for preparing example 1. White solid, 19% yield. JH NMR (400 MHz, Chloroform-d/) δ 7.38 (d, J= 20.7 Hz, 1H), 6.65 - 6.41 (m, 1H), 6.34 (d, J= 16.6 Hz, 1H), 5.79 (d, J= 10.4 Hz, 1H), 4.41 (s, 2H), 3.98 (d, J= 19.4 Hz, 4H), 3.69 (s, 3H), 2.58 (s, 3H). MS (ESI) [M+H]+ =235.3.
Figure imgf000103_0001
Example 9: 4-acryloyl-l-(6-methylpyridin-2-yl)piperazin-2-one (XS159-90) . Example 9 was synthesized following similar procedure for preparing example 1. White solid, 53% yield. 'H NMR (400 MHz, Chloroform -d) 8 7.82 - 7.65 (m, 1H), 7.60 (t, J= 7.8 Hz, 1H), 6.98 (d, J= 7.5 Hz, 1H), 6.63 - 6.46 (m, 1H), 6.40 (dd, J = 16.7, 1.9 Hz, 1H), 5.89 - 5.69 (m, 1H), 4.44 (d, J = 27.3 Hz, 2H), 4.20 (d, J= 29.6 Hz, 2H), 3.94 (d, J= 25.4 Hz, 2H), 2.49 (s, 3H). MS (ESI) [M+H]+ =246.4.
Figure imgf000103_0002
Example 12: 1 -(3-(zM-tolyl)-3,8-diazabicyclo[3.2.1 ] octan-8-yl)prop-2-en-1 -one(XS 165-33). Example 12 was synthesized following similar procedure for preparing example 5. White solid, 13% yield. ’H NMR (400 MHz, DMSO-t76) 8 7.05 (t, J= 7.8 Hz, 1H), 6.80 - 6.70 (m, 1H), 6.69 - 6.60 (m, 2H), 6.56 (d, J= 7.5 Hz, 1H), 6. 17 (dt, J= 16.5, 2.3 Hz, 1H), 5.69 (dt, J= 10.2, 2.3 Hz, 1H), 4.73 - 4.50 (m, 2H), 3.52 (t, J= 13.1 Hz, 2H), 2.71 (dd, J= 25.4, 11.2 Hz, 2H), 2.54 - 2.45 (m, 4H), 2.22 (s, 3H), 1.88 (dd, J= 17.7, 7.5 Hz, 2H), 1.79 (d, J= 8.3 Hz, 2H). MS (ESI) [M+H]- =257.5.
Figure imgf000104_0001
Example 13: l-(8-(m-tolyl)-3,8-diazabicydo[3.2.1 ]octan-3-yl)prop-2-en-l-one (XS165-54). Example 13 was synthesized following similar procedure for preparing example 5. White solid, 17% yield. *H NMR (400 MHz, Methanol-^) 8 7.15 - 7.06 (m, 1H), 6.82 (s, 1H), 6.77 (d, J= 8.3 Hz, 1H), 6.69 - 6.55 (m, 2H), 6.15 (dt, J= 16.8, 2.0 Hz, 1H), 5.68 (dd, J= 10.6, 2.0 Hz, 1H), 4.31 (d, J= 5.3 Hz, 2H), 4.21 (d, J= 13.5 Hz, 1H), 3.75 (d, J= 13.0 Hz, 1H), 3.53 (d, J= 13.1 Hz, 1H), 3.10 (d, J = 13.4 Hz, 1H), 2.25 (s, 3H), 1.96 (t, J= 6.3 Hz, 2H), 1.77 - 1.63 (m, 2H). MS (ESI)
Figure imgf000104_0002
Example 14: 4-acryloyl-l-(2-methylthiazol-5-yl)piperazin-2-one (XS154-184). Example 13 was synthesized following similar procedure for preparing example 1. White solid, 43% yield. 'H NMR (400 MHz, Methanol-d4) 8 7.66 (d, J= 44.8 Hz, 1H), 6.94 - 6.68 (m, 1H), 6.32 (d, J= 16.6
Hz, 1H), 5.85 (d, .7= 9.2 Hz, 1H), 4.74 - 4.48 (m, 3H), 4.16 - 3.93 (m, 4H), 2.67 (s, 3H). MS (ESI) [M+H]+ =252.0.
Figure imgf000104_0003
Example 15: 4-(but-2-ynoyl)-l-(m-tolyl)piperazin-2-one (XS165-75). Example 15 was synthesized following similar procedure for preparing example 1. Colorless oil, 19% yield. ’H NMR (400 MHz, Methanol-^) 6 7.36 - 7.25 (m, 1H), 7.20 - 7.03 (m, 3H), 4.54 (d, J= 1.9 Hz, 1H), 4.32 (d, J= 1.8 Hz, 1H), 4.16 (td, J= 5.6, 1.9 Hz, 1H), 4.00 - 3.90 (m, 1H), 3.83 - 3.79 (m, 1H), 3.78 - 3.72 (m, 1H), 2.36 (d, J= 1.8 Hz, 3H), 2.07 (dd, J= 8.9, 1.8 Hz, 3H). MS (ESI) [M+H]+ =252.5.
Figure imgf000105_0002
Example 17: 4-(but-2-ynoyl)-l-(5-methylthiophen-2-yl)piperazin-2-one (XS165-127).
Example 17 was synthesized following similar procedure for preparing example 1. Colorless oil, 14% yield. 'H NMR (400 MHz, Chloroform-c/) 5 6.55 (q, J= 3.6, 3.2 Hz, 1H), 6.47 (dt, J = 8.8, 2.8 Hz, 1H), 4.59 (d, J = 2.1 Hz, 1H), 4.43 (d, J= 2.0 Hz, 1H), 4.18 - 4.07 (m, 1H), 4.04 - 3.93 (m, 1H), 3.91 - 3.84 (m, 1H), 3.84 - 3.75 (m, 1H), 2.42 (d, J= 2.4 Hz, 3H), 2.04 (dd, J= 5.0, 2.1 Hz, 3H). MS (ESI) [M+H]+ =263.1.
Scheme 4. Synthesis of example 18
Figure imgf000105_0001
Example 18: 4-(2-fluoroacryloyl)-l-(5-methylthiophen-2-yl)piperazin-2-one (XS165-106). To a solution of 2-bromo-5-methylthiophene (50.0 mg, 0.28 mmol) dissolved in dioxane (1 mL),
N,N’ -dimethyl ethylenediamine ( 9 pL, 0.0846 mmol, 0.3 eq), K2CO3 ( 116.9 mg, 0.846 mmol, 3.0 eq), Cui ( 5.4 mg, 0.028 mmol, O.leq ) followed by tert'Z-butyl 3 -oxopiperazine- 1 -carboxylate (84.7 mg, 0.42 mmol, 1.5 eq). The reaction mixture was stirred at 100 °C under nitrogen atmosphere overnight. After cooling down to rt, resulting crude mixtures were purified via silica gel column chromatography to yield intermediate 1.
To a solution of intermediate 1 (40 mg, 0.135 mmol) dissolved in DCM (0.5 mL), and the TFA(
O.5 mL) were added. The reaction mixture stirred at rt 1 h. Excess TFA was removed, resulting crude product was re-dissovled in DMF (1 mL), EDCI (58.8 mg, 0.306 mmol, 2.0 eq) and HOAT (41.7 mg, 0.306 mmol, 2.0 eq) were added. At rt the 2-fluoroacrylic acid (13.8 mg, 0.153 mmol, 1.0 eq.) was added, the reaction mixture stirred at rt for 2 h. Resulting crude mixtures were purified via prep-HPLC to yield title compound (White solid, 39% yield) 'H NMR (400 MHz, MethanoL d4) 8 6.67 - 6.62 (m, 1H), 6.60 (d, J= 4.0 Hz, 1H), 5.50 - 5.35 (m, 1H), 5.35 - 5.29 (m, 1H), 4.44 (s, 2H), 4.05 (t, J = 5.5 Hz, 2H), 3.93 (t, J = 5.5 Hz, 2H), 2.41 (s, 3H). MS (ESI) [2(M-1)+H]’ =535.4.
Figure imgf000106_0001
Example 19: 4-(2-fluoroacryloyl)-l-(5-methylthiazol-2-yl)piperazin-2-one (XS165-112).
Example 19 was synthesized following similar procedure for preparing example 18. White solid, 40% yield. 1H NMR (400 MHz, Methanol-d4) 8 7.15 (s, 1H), 5.44 - 5.29 (m, 1H), 5.29 - 5.24 (m,
1H), 4.47 (s, 2H), 4.19 (d, J = 5.7 Hz, 2H), 4.00 (t, J= 5.7 Hz, 2H), 2.36 (d, J = 2.0 Hz, 3H). MS
(ESI) [M+H]+ =309.1.
Figure imgf000106_0002
Example 20: 4-(2-fluoroacryloyl)-l-(m-tolyl)piperazin-2-one (XS165-97). Example 20 was synthesized following similar procedure for preparing example 18. White solid, 56% yield. 'H NMR (400 MHz, Chloroform-;/) 5 7.34 - 7.28 (m, 1H), 7.15 - 7.09 (m, 2H), 7.06 (d, J= 8.0 Hz,
1H), 5.57 - 5.35 (m, 1H), 5.34 - 5.14 (m, 1H), 4.44 (s, 2H), 3.98 (t, J= 5.7 Hz, 2H), 3.78 (t, J = 4.8 Hz, 2H), 2.37 (s, 3H). MS (ESI) [M+H]+ =263.7.
Figure imgf000107_0001
Example 21: 2-fluoro-l-(3-(6-methylpyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)prop-2- en-l-one (XS165-118). Example 21 was synthesized following similar procedure for preparing example 18. White solid, 45% yield. 'H NMR (400 MHz, Methanol-^) 5 8.01 - 7.87 (m, 1H), 7.17 (d, J = 9.2 Hz, 1H), 6.90 (d, J = 7.3 Hz, 1H), 5.49 (dt, J = 47.5, 2.9 Hz, 1H), 5.31 (dt, J = 16.9, 2.9 Hz, 1H), 4.87 (s, 16H), 4.02 (d, J= 12.1 Hz, 2H), 3.51 - 3.39 (m, 2H), 2.58 (d, J= 2.1 Hz, 3H), 2.08 (s, 2H), 1.93 (d, J= 8.7 Hz, 2H). MS (ESI) [M+H]+ =276.2.
Figure imgf000107_0002
Example 22: 4-(2-fluoroacryloyl)-l-(6-methylpyridin-2-yl)piperazin-2-one (XS165-110).
Example 22 was synthesized following similar procedure for preparing example 18. White solid, 49% yield. 'H NMR (400 MHz, Methanol-d4) 5 7.80 (td, J = 8.0, 1.8 Hz, 1H), 7.59 (d, J= 8.1 Hz,
1H), 7.20 (dd, J= 7.6, 2.1 Hz, 1H), 5.51 - 5.25 (m, 2H), 4.42 (s, 2H), 4.16 - 4.05 (m, 2H), 3.99 (s, 2H), 2.52 (d, J= 2.1 Hz, 3H). MS (ESI) [M+H]+ =264.1.
Figure imgf000107_0003
Example 23: 4-(2-chloroacetyl)-l-(5-methylthiophen-2-yl)piperazin-2-one (XS165-119).
Example 23 was synthesized following similar procedure for preparing example 1. White solid, 32% yield. *HNMR (400 MHz, Methanol-d4) 8 6.64 (d, J= 3.9 Hz, 1H), 6.60 (d, J= 3.9 Hz, 1H), 4.48 - 4.30 (m, 4H), 4.08 - 3.84 (m, 4H), 2.41 (s, 3H). MS (ESI) [M+H]+ =273.1. hloroacetyl)-l-(5-methylthiazol-2-yl)piperazin-2-one (XS165-123) . esized following similar procedure for preparing example 1. White solid, 400 MHz, Chloroform-^/) 8 7.16 (s, 1H), 4.48 (d, J= 6.3 Hz, 2H), 4.34 (d, d, ./ = 5.4 Hz, 1H), 4.12 (d, ,/ = 8.6 Hz, 2H), 4.04 - 3.88 (m, 2H), 2.41 (s, =273.4. luoroacetyl)-l-(5-methylthiophen-2-yl)piperazin-2-one (XS165-126). esized following similar procedure for preparing example 18. White solid, 00 MHz, Chloroform-d/) 8 6.56 (d, J = 3.7 Hz, 1H), 6.49 (d, J = 5.5 Hz, Hz, 1H), 4.99 (d, J= 10.2 Hz, 1H), 4.40 (d, J= 34.5 Hz, 2H), 4.09 - 3.77 1 Hz, 3H). MS (ESI) [M+H]+ =257.1.
Figure imgf000108_0001
Example 26: 4-(2-chloroacetyl)-l-(6-methylpyridin-2-yl)piperazin-2-one (XS165-120).
Example 26 was synthesized following similar procedure for preparing example 18. White solid, 35% yield. 'H NMR (400 MHz, Chloroform-d/) δ 7.72 (dd, J= 23.9, 8.2 Hz, 1H), 7.61 (t, J= 7.9 Hz, 1H), 6.99 (t, J= 6.7 Hz, 1H), 4.40 (d, J= 9.7 Hz, 2H), 4.30 - 4.23 (m, 1H), 4.17 (d, J= 5.7 Hz, 1H), 4.12 (d, J= 2.9 Hz, 2H), 3.97 - 3.84 (m, 2H), 2.50 (s, 3H). MS (ESI) [M+H]+ =268.1.
Figure imgf000109_0001
Example 27: 2-chloro-l-(3-(6-methylpyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)ethan-l- one (XS165-121). Example 27 was synthesized following similar procedure for preparing example 18. White solid, 47% yield. 'H NMR (400 MHz, Chloroform-tT) 8 7.44 - 7.30 (m, 1H), 6.52 (d, J = 7.3 Hz, 1H), 6.35 (d, J= 8.5 Hz, 1H), 4.82 (d, J = 6.4 Hz, 1H), 4.37 (d, J= 6.5 Hz, 1H), 4.20 (d, J= 12.2 Hz, 1H), 4.13 - 4.01 (m, 2H), 3.92 (d, J= 12.0 Hz, 1H), 3.08 (dd, J= 12.1, 2.9 Hz, 2H), 2.39 (s, 3H), 2.10 - 1.77 (m, 4H). MS (ESI) [M+H|+ =280.1.
Scheme 5. Synthesis of example 28
Figure imgf000109_0002
Example 28: (£)-4-(4-(dimethylamino)but-2-enoyl)-l-(5-methylfuran-2-yl)piperazin-2-one (XS165-100). To a solution of 2-bromo-5-methylfuran(50.0 mg, 0.28 mmol) dissolved in dioxane (1 mL), N,N’ -dimethylethylenediamine ( 9 μL, 0.0846 mmol, 0.3 eq), K2CO3 ( 116.9 mg, 0.846 mmol, 3.0 eq), Cui ( 5.4 mg, 0.028 mmol, O. leq ) followed by tert -butyl 3 -oxopiperazine- 1- carboxylate (84.7 mg, 0.42 mmol, 1.5 eq). The reaction mixture was stirred at 100 °C under nitrogen atmosphere overnight. After cooling down to rt, resulting crude mixtures were purified via silica gel column chromatography to yield intermediate 5.
To a solution of intermediate 1 (30 mg, 0.107 mmol) dissolved in DCM (0.5 mL), and the TFA( 0.5 mL) were added. The reaction mixture stirred at rt 1 h. Excess TFA was removed, resulting crude product was re-dissovled in DMF (1 mL), EDCI (41.1 mg, 0.214 mmol, 2.0 eq) and HOAT (29.2 mg, 0.214 mmol, 2.0 eq) were added. At rt the (£)-4-(dimethylamino)but-2-enoic acid (17.7 mg, 0.107 mmol, 1.0 eq.) was added, the reaction mixture stirred at rt for 2 h. Resulting crude mixtures were purified via prep-HPLC to yield title compound (White solid, 31% yield) 'H NMR (400 MHz, Methanol-d4) δ 7.02 - 6.87 (m, 1H), 6.78 (q, J= 9.6, 8.5 Hz, 1H), 6.22 (t, J= 2.7 Hz, 1H), 6.08 - 6.00 (m, 1H), 4.44 (d, J= 28.2 Hz, 2H), 4.05 - 3.96 (m, 4H), 3.93 - 3.83 (m, 2H), 2.93 (d, J= 2.1 Hz, 6H), 2.26 (d, J= 1.8 Hz, 3H). MS (ESI) [M+H]+ =292.2.
Figure imgf000110_0001
Example 29: (£)-4-(4-(dimethylamino)but-2-enoyl)-l-(//i-tolyl)piperazin-2-one (XS165-99).
Example 29 was synthesized following similar procedure for preparing example 28. White solid, 54% yield. 1HMR C400Hz, Methanol-^) 87.36 - 7.24 (m, 1H), 7.12 (d, J= 5.7 Hz, 2H), 7.08 (d, 7 = 8.0 Hz, 1H), 6.92 (dd, 7= 22.6, 15.1 Hz, 1H), 6.75 (dt, 7= 14.8, 7.1 Hz, 1H), 4.40 (d, J = 26.9 Hz, 2H), 4.00 (p, J= 5.2 Hz, 2H), 3.94 (d, J= 7.2 Hz, 2H), 3.88 - 3.72 (m, 2H), 2.88 (d, J = 1.8 Hz, 6H), 2.33 (d, J= 1.8 Hz, 3H). MS (ESI) [M+H]+ =302.1.
Figure imgf000110_0002
Example 31: (£)-4-(4-(dimethylamino)but-2-enoyl)-l-(6-methylpyridin-2-yl)piperazin-2-one (XS165-109). Example 31 was synthesized following similar procedure for preparing example 28. White solid, 47% yield. ’H NMR (400 MHz, Methanol-d4) 8 7.74 (t, 7 = 7.9 Hz, 1H), 7.59 (t, 7 = 9.7 Hz, 1H), 7.16 (d, J = 7.5 Hz, 1H), 6.95 (t, J = 16.4 Hz, 1H), 6.85 - 6.72 (m, 1H), 4.48 (d, 7 = 27.1 Hz, 2H), 4.16 (d, J= 5.6 Hz, 1H), 4.09 (t, J= 5.0 Hz, 1H), 4.06 - 3.93 (m, 4H), 2.93 (d, J = 2.0 Hz, 6H), 2.52 (d, J= 2. 1 Hz, 3H). MS (ESI) [M+H]+ =303.2.
Figure imgf000111_0001
Example 34: (E')-l-(5-methylthiophen-2-yl)-4-(4-(pyrrolidin-l-yl)but-2-enoyl)piperazin-2- one (XS165-154). Example 34 was synthesized following similar procedure for preparing example 28. White solid, 58% yield. 'H NMR (400 MHz, Methanol-^) 8 6.90 (dd, J = 36.2, 15.2 Hz, 1H), 6.81 - 6.68 (m, 1H), 6.65 - 6.44 (m, 2H), 4.43 (d, J= 30.7 Hz, 2H), 4.00 (d, J= 7.1 Hz, 4H), 3.94 - 3.78 (m, 2H), 3.62 (s, 2H), 3.12 (s, 2H), 2.37 (d, J= 2.3 Hz, 3H), 2.08 (d, J= 48.5 Hz, 4H). MS (ESI) [M+H]+ =334.2.
Figure imgf000112_0001
Example 35: (E)-l-(5-methylthiophen-2-yl)-4-(4-(piperidin-l-yl)but-2-enoyl)piperazin-2-one (XS165-170). Example 35 was synthesized following similar procedure for preparing example 28. White solid, 55% yield. 1HNMR(400 MHz, Methanol-d4) δ 6.91 (dd, J= 34.6, 15.2 Hz, 1H), 6.82 - 6.67 (m, 1H), 6.57 (dt, J= 11.4, 3.5 Hz, 2H), 4.43 (d, J= 29.1 Hz, 2H), 4.02 (q, J= 8.4, 6.8 Hz,
2H), 3.88 (dd, J= 25.4, 6.4 Hz, 4H), 3.51 (d, J= 12.4 Hz, 2H), 2.94 (t, J= 12.5 Hz, 2H), 2.37 (d,
J = 2.3 Hz, 3H), 1.95 (d, J= 14.4 Hz, 2H), 1.88 - 1.63 (m, 3H), 1.59 - 1.40 (m, 1H). MS (ESI) [M+H]+ =348.3.
Figure imgf000112_0002
Example 37: (E')-4-(dimethylamino)-l-(4-(5-methylthiophen-2-yl)piperazin-l-yl)but-2-en-l- one (XS165-172). Example 37 was synthesized following similar procedure for preparing example 28. White solid, 33% yield. 'H NMR (400 MHz, Methanol-^) 8 7.00 - 6.72 (m, 2H), 6.73 - 6.30 (m, 2H), 4.12 - 3.84 (m, 8H), 3.76 - 3.69 (m, 2H), 2.89 - 2.85 (m, 9H). MS (ESI) [M+H]+ =294.4.
Scheme 6. Synthesis of example 38
Figure imgf000113_0001
Example 38: (£)-4-(dimethylamino)-l-(4-(5-methylthiophen-2-yl)piperidin-l-yl)but-2-en-l- one (XS165-169). ). To a solution of the 2-bromo-5-methylthiophene (177.1 mg, 1 mmol) dissolved in dioxane/H2O = 4: 1 (3 mL), K3PO4 (424.6 mg, 2 mmol, 2.0 eq), Pd (PPh3)2C12 (70.2 mg, 0.1 mmol, 0.1 eq), followed by tert-butyl 4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-3,6- dihydropyridine-l(277)-carboxylate (618.4 mg, 2.0 mmol, 2.0 eq). The reaction mixture was stirred at 90 °C under nitrogen atmosphere overnight. After cooling down to rt, resulting crude mixtures were purified via silica gel column chromatography to yield intermediate 5.
To a solution of intermediate 5 (200 mg, 0.72 mmol) dissolved in MeOH (3 mL), and the Pd/C( 20 mg) were added. The reaction mixture stirred at hydrogen atmosphere. MeOH was removed, resulting crude product was re-dissovled in DCM (1 mL), TFA (1 mL) was added. The reaction mixture stirred at rt for 1 h. Excess TFA was removed, resulting crude product was re-dissovled in DMF (1 mL), EDCI (272 mg, 1.42 mmol, 2.0 eq) and HOAT (187.4 mg, 1.42 mmol, 2.0 eq) were added. At rt the (E)-4-(dimethylamino)but-2-enoic acid (117.6 mg, 0.71 mmol, 1.0 eq.) was added, the reaction mixture stirred at rt for 2 h. Resulting crude mixtures were purified via prep- HPLC to yield title compound (White solid, 43% yield) 1 H NMR (400 MHz, Methanol-d4) 5 6.95 (d, J= 15.1 Hz, 1H), 6.72 - 6.58 (m, 2H), 6.55 (d, J= 2.8 Hz, 1H), 4.59 (d, J= 13.4 Hz, 1H), 4.13 (d, J= 13.9 Hz, 1H), 3.92 (dd, J= 7.3, 2.2 Hz, 2H), 3.24 (d, J= 13.1 Hz, 1H), 3.14 - 3.00 (m, 1H), 2.95 - 2.76 (m, 7H), 2.38 (d, J= 2.3 Hz, 3H), 2.13 - 1.96 (m, 2H), 1.74 - 1.42 (m, 2H). MS (ESI) [M+H]+ =293.4.
Figure imgf000113_0002
Example 39: (E)-4,4-dimethyl-2-(4-(5-methylthiophen-2-yl)-3-oxopiperazine-l- carbonyl)pent-2-enenitrile (XS165-177). Example 39 was synthesized following similar
I l l procedure for preparing example 28. White solid, 47% yield. 1 H NMR (400 MHz, Methanol-^) 8 7.12 - 6.97 (m, 1H), 6.67 - 6.43 (m, 2H), 4.38 (s, 2H), 4.08 - 3.76 (m, 4H), 2.39 (s, 3H), 1.32
Figure imgf000114_0001
Figure imgf000115_0001
Example 46: (E)-4-(4-(dimethylamino)but-2-enoyl)-l -(3-methylthiophen-2-yl)piperazin-2- one (XS175-68). Example 46 was synthesized following similar procedure for preparing example 28. White solid, 51% yield. 'H NMR (400 MHz, Methanol-d4) δ 7.23 (d, J= 5.3 Hz, 1H), 7.03 - 6.87 (m, 1H), 6.84 (d, J= 5.4 Hz, 1H), 6.77 (dt, J= 14.8, 7.2 Hz, 1H), 4.46 (d, J= 30.5 Hz, 2H), 4.04 (q, J= 5.8 Hz, 2H), 4.00 - 3.87 (m, 2H), 3.82 - 3.64 (m, 2H), 2.90 (s, 6H), 2.06 (s, 3H). MS
Figure imgf000116_0001
Example 47 : (£)-4-(4-(dimethylamino)but-2-enoyl)-l-(4,5-dimethylthiophen-2-yl)piperazin- 2-one (XS175-70). Example 47 was synthesized following similar procedure for preparing example 28. White solid, 53% yield. 'H NMR (400 MHz, Methanol-d4 ) 8 6.93 (dd, J = 36.6, 15.2 Hz, 1H), 6.76 (dt, J= 15.0, 7.4 Hz, 1H), 6.50 (s, 1H), 4.43 (d, J= 29.5 Hz, 2H), 4.03 (s, 2H), 3.95 (d, J= 7.3 Hz, 2H), 3.93 - 3.77 (m, 2H), 2.90 (s, 6H), 2.24 (s, 3H), 2.07 (s, 3H). MS (ESI) [M+H]- =322.2.
Figure imgf000116_0002
Example 48: (E)-l-(4-(dimethylamino)but-2-enoyl)-W-(5-methylthiophen-2-yl)azetidine-3- carboxamide (XS175-71) . Example 48 was synthesized following similar procedure for preparing example 28. White solid, 24% yield. 'H NMR (400 MHz, Methanol-d4 ) 8 6.80 - 6.62 (m, 1H), 6.53 - 6.37 (m, 3H), 4.54 - 4.39 (m, 2H), 4.29 - 4.20 (m, 1H), 4.19 - 4.11 (m, 1H), 3.95 - 3.85 (m, 2H), 3.63 - 3.51 (m, 1H), 2.87 (s, 6H), 2.35 (s, 3H). MS (ESI) [M+H]+ =308.3
Figure imgf000116_0003
Example 49: (E)-4-(dimethylamino)-JV-(2-((5-methylthiophen-2-yl)amino)-2-oxoethyl)but-2- enamide (XS175-76). Example 49 was synthesized following similar procedure for preparing example 28. White solid, 20% yield. 'H NMR (400 MHz, Methanol-d4) 8 6.74 - 6.56 (m, 1H), 6.46 - 6.32 (m, 3H), 4.04 - 3.95 (m, 2H), 3.90 - 3.81 (m, 2H), 2.81 (s, 6H), 2.28 (s, 3H). MS (ESI) [M+H]+ =282.5.
Figure imgf000117_0001
Example 52: l-(2-chloroacetyl)-JV-(5-methylthiophen-2-yl)azetidine-3-carboxamide (XS175- 126). Example 52 was synthesized following similar procedure for preparing example 28. White solid, 33% yield. JH NMR (400 MHz, Methanol-d4) 8 6.56 - 6.37 (m, 2H), 4.55 - 4.40 (m, 2H), 4.29 - 4.12 (m, 2H), 4.09 - 4.00 (m, 2H), 3.66 - 3.53 (m, 1H), 2.38 (s, 3H). MS (ESI) [M+H]+ =273.1.
Scheme 7. Synthesis of example 53
Figure imgf000118_0001
Example 53: (E)-l-(4-(dimethylamino)but-2-enoyl)-A-(5-methylthiophen-2-yl)piperidine-4- carboxamide (XS175-132). ). To a solution of the 5-methylthiophen-2-amine (113.2 mg, 1 mmol) dissolved in DMF (2 mL), HATU (418.2 mg, 1.1 mmol, 1.1 eq), DIPEA (0.18 mL, 2.0 mmol, 2.0 eq), followed by l-(terZ-butoxycarbonyl)piperidine-4-carboxylic acid (229.3 mg, 1.0 mmol, 1.0 eq). The reaction mixture was stirred at rt 3 h. The resulting crude mixtures were purified via silica gel column chromatography. The resulting crude product was re-dissovled in DCM (1 mL), TFA (1 mL) was added. The reaction mixture stirred at rt for 1 h. Excess TFA was removed, resulting crude product was were purified via silica gel column chromatography to yield intermediate 7.
To a solution of intermediate 7 (25 mg, 0.116 mmol) dissolved in DMF (1 mL), HATU (50.9 mg, 0.14 mmol, 1.2 eq), DIPEA (0.043 mL, 0.24 mmol, 2.0 eq), followed by (E)-4-
(dimethylamino)but-2-enoic acid (18.5 mg, 0. 116 mmol, 1.0 eq) the reaction mixture stirred at rt for 2 h. Resulting crude mixtures were purified via prep-HPLC to yield title compound (White solid, 43% yield) 1H NMR (400 MHz, Methanol-d)48 6.88 (d, J= 15.1 Hz, 1H), 6.65 - 6.50 (m, 1H), 6.41 (s, 2H), 4.49 (d, J = 12.9 Hz, 1H), 4.07 (d, J= 13.5 Hz, 1H), 3.85 (d, J= 6.7 Hz, 2H),
3.16 (t, J = 12.9 Hz, 1H), 2.82 (s, 7H), 2.59 (t, J = 9.8 Hz, 1H), 2.28 (s, 3H), 1.84 (d, J= 12.4 Hz, 2H), 1.61 (p, J= 13.0 Hz, 2H). MS (ESI) [M+H]+ =336.2.
Figure imgf000118_0002
Example 54: (E)-l-(4-(dimethylamino)but-2-enoyl)-/V-(5-methylthiophen-2-yl)pyrrolidine-
3-carboxamide (XS175-133). Example 54 was synthesized following similar procedure for preparing example 53. White solid, 29% yield. 'H NMR (400 MHz, Methanol-d4) 8 6.73 - 6.56 (m, 2H), 6.40 (s, 2H), 3.85 (s, 2H), 3.83 - 3.37 (m, 4H), 3.20 - 3.04 (m, 1H), 2.80 (s, 6H), 2.27 (s, 3H), 2.25 - 1.94 (m, 2H). MS (ESI) [M+H]+ =322.
Figure imgf000119_0002
Example 56: (£)-l-(4-(dimethylamino)but-2-enoyl)-7V-(5-isopropylthiophen-2-yl)azetidine-
3-carboxamide (XS175-143). Example 56 was synthesized following similar procedure for preparing example 53. White solid, 37% yield. 'H NMR (400 MHz, Methanol-d4) 8 6.75 - 6.59 (m, 1H), 6.57 - 6.40 (m, 3H), 4.56 - 4.36 (m, 2H), 4.24 (t, J= 9.4 Hz, 1H), 4.19 - 4.09 (m, 1H), 3.90 (d, J= 6.7 Hz, 2H), 3.63 - 3.50 (m, 1H), 3.03 (h, J= 6.5, 5.6 Hz, 1H), 2.86 (s, 6H), 1.25 (d, J= 6.5 Hz, 6H). MS (ESI) [M+H]+ =336.2.
Figure imgf000119_0001
Example 57: (£)-l-(4-(dimethylamino)but-2-enoyl)-/V-(5-methylthiophen-2-yl)piperidine-2- carboxamide (XS175-148). Example 57 was synthesized following similar procedure for preparing example 53. White solid, 25% yield. 'H NMR (400 MHz, Methanol-d4) δ 7.07 - 6.79 (m, 1H), 6.72 - 6.43 (m, 3H), 5.23 (s, 1H), 4.10 - 3.82 (m, 3H), 3.61 - 3.44 (m, 1H), 2.89 (s, 6H), 2.36 (s, 3H), 2.29 - 2.14 (m, 1H), 1.87 - 1.64 (m, 3H), 1.63 - 1.41 (m, 2H). MS (ESI) [M+Hf =336.2.
Figure imgf000120_0001
Example 58: (E)-2-(4-(dimethylamino)but-2-enoyl)-JV-(5-methylthiophen-2-yl)-2- azaspiro[3.3]heptane-6-carboxamide (XS175-149). Example 58 was synthesized following similar procedure for preparing example 53. White solid, 16% yield. 'H NMR (400 MHz, Methanol-^) 5 6.70 - 6.53 (m, 1H), 6.49 - 6.28 (m, 3H), 4.39 - 4.18 (m, 2H), 4.09 - 3.95 (m,
2H), 3.94 - 3.80 (m, 2H), 3.13 - 2.97 (m, 1H), 2.84 (s, 6H), 2.44 (d, J= 7.9 Hz, 4H), 2.31 (s, 3H).
MS (ESI) [M+H]+ =348.3.
Figure imgf000120_0002
Example 61 : (E)-4-(dimethylamino)-2V-methyl-iV-(3-(methyl(5-methylthiophen-2-yl)amino)- 3-oxopropyl)but-2-enamide (XS175-160) . Example 61 was synthesized following similar procedure for preparing example 28. White solid, 23% yield. JH NMR (400 MHz, Methanol-d4) 8 6.86 - 6.70 (m, 2H), 6.64 (s, 1H), 6.40 (d, J= 15.4 Hz, 1H), 4. 12 (d, J= 7.2 Hz, 2H), 3.52 - 3.43 (m, 2H), 3.23 (s, 3H), 3.15 (s, 9H), 2.45 (s, 3H). MS (ESI) [M+H]+ =324.2.
Figure imgf000121_0001
Example 62: (E)-l-(4-(dimethylamino)but-2-enoyl)-JV-(4-methylthiophen-2-yl)piperidine-4- carboxamide (XS175-173). Example 62 was synthesized following similar procedure for preparing example 28. White solid, 46% yield. 'H NMR (400 MHz, Methanol-6/4) δ 6.93 (d, J = 15.1 Hz, 1H), 6.62 (dt, J= 14.6, 6.8 Hz, 1H), 6.51 (s, 1H), 4.54 (d, J= 12.9 Hz, 1H), 4.12 (d, J = 13.4 Hz, 1H), 3.90 (d, .7= 6.8 Hz, 2H), 3.21 (t, ,7= 12.9 Hz, 1H), 2.87 (s, 7H), 2.70 - 2.58 (m, 1H), 2.13 (s, 3H), 1.89 (d, J= 12.5 Hz, 2H), 1.78 - 1.52 (m, 2H). MS (ESI) [M+H]+ =336.2.
Figure imgf000121_0002
Example 63: (E)-l-(4-(dimethylamino)but-2-enoyl)-JV,7V-bis(5-methylthiophen-2- yl)piperidine-4-carboxamide (XS175-174). Example 63 was synthesized following similar procedure for preparing example 28. White solid, 13% yield. 'H NMR (400 MHz, Methanol-6/4) 8 7.17 - 6.88 (m, 2H), 6.77 (s, 1H), 6.70 - 6.58 (m, 1H), 6.47 (s, 1H), 6.21 (s, 1H), 4.53 (d, J = 13.1 Hz, 1H), 4.09 (d, .7= 13.5 Hz, 1H), 3.93 (d, .7= 7.0 Hz, 2H), 3.06 (t, ,7= 12.8 Hz, 1H), 3.01 - 2.94 (m, 1H), 2.94 - 2.86 (m, 6H), 2.66 (t, J= 12.9 Hz, 1H), 2.45 (d, J= 49.1 Hz, 6H), 1.89 (d, J = 13.0 Hz, 2H), 1.82 - 1.59 (m, 2H). MS (ESI) [M+H]+ =432.2.
Figure imgf000121_0003
Example 64: (E)-l-(4-(dimethylamino)but-2-enoyl)-W^V-bis(5-isopropylthiophen-2- yl)piperidine-4-carboxamide (XS175-175). Example 64 was synthesized following similar procedure for preparing example 28. White solid, 19% yield. 'H NMR (400 MHz, Methanol-d4) 8 7.18 - 6.89 (m, 2H), 6.81 (d, J= 18.9 Hz, 1H), 6.70 - 6.60 (m, 1H), 6.60 - 6.38 (m, 1H), 6.26 (s, 1H), 4.54 (d, J= 13.4 Hz, 1H), 4.10 (d, J= 13.9 Hz, 1H), 3.98 - 3.86 (m, 2H), 3.29 - 3.02 (m, 4H), 2.92 (s, 7H), 2.76 - 2.57 (m, 1H), 1.91 (d, J = 13.3 Hz, 2H), 1.84 - 1.64 (m, 2H), 1.34 (s,
Figure imgf000122_0001
Example 65: (£)-l-(4-(dimethylamino)but-2-enoyl)-Af^V-bis(5-isopropylthiophen-2- yl)pyrrolidine-3-carboxamide (XS175-176). Example 65 was synthesized following similar procedure for preparing example 28. White solid, 20% yield. 'H NMR (400 MHz, Methanol-d4) 8 7.14 - 6.94 (m, 1H), 6.83 (s, 1H), 6.73 (s, 2H), 6.64 - 6.46 (m, 1H), 6.28 (s, 1H), 3.95 (s, 2H), 3.90 - 3.72 (m, 2H), 3.66 (t, J= 7.5 Hz, 1H), 3.57 - 3.37 (m, 2H), 3.27 - 3.02 (m, 2H), 2.92 (s, 6H), 2.40 - 2.09 (m, 2H), 1.38 - 1.28 (m, 12H). MS (ESI) [M+H]+ =474.3.
Figure imgf000122_0002
Example 66: (E)-l-(4-(dimethylamino)but-2-enoyl)-/V-(5-isopropylthiophen-2-yl)piperidine- 4-carboxamide (XS175-178). Example 66 was synthesized following similar procedure for preparing example 28. White solid, 49% yield. 'H NMR (400 MHz, Methanol-d4) 8 6.95 (d, J = 15.0 Hz, 1H), 6.71 - 6.57 (m, 1H), 6.51 (d, J= 9.7 Hz, 2H), 4.57 (d, J = 12.9 Hz, 1H), 4.14 (d, J = 13.4 Hz, 1H), 3.93 (d, J= 6.8 Hz, 2H), 3.23 (d, J= 12.9 Hz, 1H), 3.11 - 2.99 (m, 1H), 2.90 (s, 7H), 2.74 -2.56 (m, 1H), 1.92 (d, J= 13.5 Hz, 2H), 1.80 - 1.57 (m, 2H). MS (ESI) [M+H]+ =364.7.
Figure imgf000122_0003
Example 67: (E)-l-(benzo[Z>]thiophen-2-yl)-4-(4-(dimethylamino)but-2-enoyl)piperazin-2- one (XS175-179). Example 67 was synthesized following similar procedure for preparing example 28. White solid, 46% yield. 'H NMR (400 MHz, Methanol-d)48 7.83 - 7.60 (m, 2H), 7.40 - 7.20 (m, 2H), 7.11 - 6.91 (m, 2H), 6.79 (d, J= 14.6 Hz, 1H), 4.54 (d, J= 30.3 Hz, 2H), 4.23 - 3.86 (m, 6H), 2.93 (s, 6H). MS (ESI) [M+H]+ =344.4.
Figure imgf000123_0001
Example 70: (E)-4-(dimethylamino)-l-(6-(5-methylthiophen-2-yl)-2,6- diazaspiro[3.3]heptan-2-yl)but-2-en-l-one (XS186-3). Example 70 was synthesized following similar procedure for preparing example 28. White solid, 23% yield. 'H NMR (400 MHz, Methanol- r) 8 6.69 (s, 2H), 6.55 - 6.32 (m, 2H), 4.50 (s, 2H), 4.22 (d, J = 3.2 Hz, 2H), 4.01 - 3.91 (m, 6H), 2.90 - 2.88 (m, 6H), 2.32 (d, J= 3.4 Hz, 3H). MS (ESI) [M+H]+ =306.2.
Figure imgf000124_0001
Example 73: l-(2-chloroacetyl)-AL(5-isopropylthiophen-2-yl)piperidine-4-carboxamide (XS185-6). Example 73 was synthesized following similar procedure for preparing example 28. White solid, 30% yield. ’H NMR (400 MHz, Methanol-d)48 6.59 - 6.45 (m, 2H), 4.60 - 4.43 (m, 1H), 4.36 - 4.18 (m, 2H), 4.08 - 3.95 (m, 1H), 3.25 (t, J= 13.1 Hz, 1H), 3.14 - 3.00 (m, 1H), 2.83 (t, J= 12.7 Hz, 1H), 2.71 - 2.58 (m, 1H), 2.02 - 1.77 (m, 3H), 1.76 - 1.62 (m, 1H), 1.34 - 1.23 (m, 6H). MS (ESI) [M+H]+ =329.1.
Scheme 8. Synthesis of example 74
Figure imgf000125_0001
Example 74
Example 74: (£)-4-(4-(dimethylamino)but-2-enoyl)-l-(5-(piperidin-4-yl)thiophen-2- yl)piperazin-2-one (XS185-24)To a solution of tert-butyl 4-(5-brom othi ophen-2 -yl)piperidine-l- carboxylate (123.0 mg, 0.35 mmol) dissolved in dioxane (2 mL), N,N’-dimethylethylenediamine ( 9 pL, 0.105 mmol, 0.3 eq), K2CO3 ( 147.2 mg, 1.05 mmol, 3.0 eq), Cui ( 6.8 mg, 0.035 mmol, O.leq ) followed by benzyl 3 -oxopiperazine- 1 -carboxylate (125.1 mg, 0.54 mmol, 1.5 eq). The reaction mixture was stirred at 100 °C under nitrogen atmosphere overnight. After cooling down to rt, resulting crude mixtures were purified via silica gel column then the product dissolved in MeOH (3 mL), and the Pd/C( 20 mg) were added. The reaction mixture stirred at hydrogen atmosphere. MeOH was removed then purified via silica gel column chromatography to yield intermediate 8.
To a solution of intermediate 8 was dissovled in DMF (1 mL), HATU (59.3 mg, 0.144 mmol, 1.1 eq) and DIPEA (0.1 mL, 2.0 eq) were added. At rt the (E)-4-(dimethylamino)but-2-enoic acid (17.7 mg, 0.107 mmol, 1.0 eq.) was added, the reaction mixture stirred at rt for 2 h. Resulting crude mixtures were purified via prep-HPLC then re-dissolved in DCM (0.5 mL), and the TFA( 0.5 mL) were added. The reaction mixture stirred at rt 1 h. Excess TFA was removed, resulting crude product was purified via prep-HPLC to yield title compound (White solid, 19% yield) 'H NMR (400 MHz, Methanol-d4) δ 6.95 (dd, J= 38.8, 15.4 Hz, 1H), 6.86 - 6.62 (m, 3H), 4.48 (d, J = 29.9 Hz, 2H), 4.16 - 4.01 (m, 2H), 4.01 - 3.85 (m, 4H), 3.46 (d, ,7= 13.0 Hz, 2H), 3.12 (t, J = 12.5 Hz, 4H), 2.91 (s, 6H), 2.22 (d, J= 14.2 Hz, 2H), 1.89 (q, J = 13.1 Hz, 2H), 1.45 - 1.12 (m,
Figure imgf000125_0002
Example 75: (E)-4-(dimethylamino)-l-(3-(5-methylthiophen-2-yl)-3,8- diazabicyclo[3.2.1]octan-8-yl)but-2-en-l-one (XS185-29). Example 75 was synthesized following similar procedure for preparing example 28. White solid, 19% yield. 'H NMR (400 MHz, Methanol-d4) 8 6.94 - 6.74 (m, 3H), 6.36 (s, 1H), 4.78 - 4.40 (m, 3H), 3.99-3.95 (m, 4H), 2.92 (m, 8H), 2.31 (s, 3H), 2.09 - 2.03 (m, 2H), 1.98 (td, J= 14.5, 12.6, 8.6 Hz, 3H). MS (ESI) [M+H]+ =320.2.
Figure imgf000126_0001
Example 76: (JE)-4-(4-(dimethylamino)but-2-enoyl)-l-(lH-indol-3-yl)piperazin-2-one (XS185-43). Example 76 was synthesized following similar procedure for preparing example 28. White solid, 32% yield. 'H NMR (400 MHz, Methanol-t/4) 8 7.40 (t, J= 9.8 Hz, 2H), 7.32 (d, J = 3.1 Hz, 1H), 7.14 (d, J = 9.3 Hz, 1H), 7.07 (d, J= 8.6 Hz, 1H), 7.02 - 6.87 (m, 1H), 6.81 - 6.64 (m, 1H), 4.64 - 4.38 (m, 2H), 4.09 (t, J= 5.4 Hz, 2H), 4.02 - 3.79 (m, 4H), 2.88 (s, 6H). MS (ESI) [M+H]+ =327.2.
Figure imgf000126_0002
Example 77 : (E)- l-(6-(benzo [b] thiophen-2-yl)-2,6-diazaspiro [3.3] heptan-2-yl)-4-
(dimethylamino)but-2-en-l-one (XS185-46). Example 77 was synthesized following similar procedure for preparing example 28. White solid, 17% yield. 1 H NMR (400 MHz, Methanol - h) 8 7.57 (t, J= 6.2 Hz, 1H), 7.43 (t, J= 6.1 Hz, 1H), 7.19 (q, J= 6.8, 6.2 Hz, 1H), 7.05 (t, J= 6.6 Hz, 1H), 6.78 - 6.39 (m, 3H), 4.53 (d, J = 4.7 Hz, 2H), 4.32 - 4.21 (m, 4H), 4.11 (t, J = 3.0 Hz, 3H), 3.94 (t, J= 5.7 Hz, 2H), 2.90 (s, 6H). MS (ESI) [M+H]+ =342.4.
Figure imgf000126_0003
Example 78: (E)-l-(2-(benzo[Z>]thiophen-2-yl)-2,7-diazaspiro[3.5]nonan-7-yl)-4-
(dimethylamino)but-2-en-l-one (XS185-59). Example 78 was synthesized following similar procedure for preparing example 28. White solid, 19% yield. 'H NMR (400 MHz, Methanol-tL) 8 7.59 - 7.49 (m, 1H), 7.44 - 7.34 (m, 1H), 7.17 (d, J = 9.1 Hz, 1H), 7.06 - 6.89 (m, 2H), 6.76 - 6.49 (m, 2H), 3.94 (t, J= 4.2 Hz, 4H), 3.78 - 3.77 (m, 2H), 3.72 - 3.62 (m, 4H), 2.91 (s, 7H), 1.94 - 1.85 (m, 4H). MS (ESI) [M+H]+ =370.2.
Scheme 9. Synthesis of example 79
Figure imgf000127_0001
Example 79: (E)-4-(dimethylamino)-l-(4-(5-methylthiophene-2-carbonyl)piperazin-l- yl)but-2-en-l-one (XS185-64). To a solution of 5-methylthiophene-2-carboxylic acid(75 mg, 0.5 mmol, 1.0 eq) was dissovled in DMF (1 mL), HATU (209 mg, 0.55 mmol, 1.1 eq) and DIPEA (0.18 mL, 2.0 eq) were added. At rt the tert-butyl piperazine- 1 -carboxylate (93.3 mg, 0.5 mmol, 1.0 eq.) was added, the reaction mixture stirred at rt for 2 h. Resulting crude mixtures were purified via prep-HPLC then re-dissolved in DCM (0.5 mL), and the TFA( 0.5 mL) were added. The reaction mixture stirred at rt 1 h. Excess TFA was removed, resulting crude product was purified via prep-HPLC to yield intermediate 9.
To a solution of intermediate 9(20 mg, 0.095 mmol) was dissovled in DMF (1 mL), HATU (39.8 mg, 0.1 mmol, 1.1 eq) and DIPEA (0.1 mL, 2.0 eq) were added. At rt the (£)-4- (dimethylamino)but-2-enoic acid (15.8 mg, 0.095 mmol, 1.0 eq.) was added, the reaction mixture stirred at rt for 2 h. Resulting crude mixtures were purified via prep-HPLC to yield title compound (White solid, 30% yield) 'H NMR (400 MHz, Methanol-d4) 8 7.28 - 7.18 (m, 1H), 7.01 - 6.86 (m, 1H), 6.78 (d, J= 4.5 Hz, 1H), 6.74 - 6.60 (m, 1H), 4.02 - 3.86 (m, 2H), 3.86 - 3.75 (m, 4H), 3.75 - 3.62 (m, 4H), 2.87 (s, 6H), 2.48 (s, 3H). MS (ESI) [M+H]+ =322.2.
Figure imgf000127_0002
Example 80: (E)-4-(dimethylamino)-JV-(l-(5-methylthiophene-2-carbonyl)piperidin-4- yl)but-2-enamide (XS185-65). Example 80 was synthesized following similar procedure for preparing example 79. White solid, 46% yield. 'H NMR (400 MHz, Methanol-r/i) 8 7.11 (t, J = 4.0 Hz, 1H), 6.72 (d, J= 4.8 Hz, 1H), 6.69 - 6.56 (m, 1H), 6.35 - 6.19 (m, 1H), 4.28 (d, J= 13.5 Hz, 2H), 4.06 - 3.89 (m, 1H), 3.84 (t, J= 5.5 Hz, 2H), 3.20 - 3.03 (m, 2H), 2.82 (s, 6H), 2.42 (s, 3H), 1.90 (d, J= 12.9 Hz, 2H), 1.54 - 1.35 (m, 2H). MS (ESI) [M+H]+ =336.2.
Figure imgf000128_0002
Example 82: (E')-4-(dimethylamino)-l-(6-(5-methylthiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-3-yl)but-2-en-l-one (XS185-67). Example 82 was synthesized following similar procedure for preparing example 79. White solid, 57% yield. 'H NMR (400 MHz, Methanol-d4) 5 7.48 - 7.31 (m, 1H), 6.88 - 6.73 (m, 2H), 6.73 - 6.60 (m, 1H), 5.12 - 4.89 (m, 1H), 4.71 - 4.42 (m, 1H), 4.35 - 4.00 (m, 1H), 3.96 - 3.75 (m, 4H), 3.73 - 3.60 (m, 1H), 2.88 - 2.76 (m, 7H), 2.46 (s, 3H), 1.69 - 1.57 (m, 1H). MS (ESI) [M+H]+ =334.2.
Figure imgf000128_0001
Example 83: (E)-4-(dimethylamino)-l-(6-(5-methylthiophene-2-carbonyl)-2,6- diazaspiro[3.3]heptan-2-yl)but-2-en-l-one (XS185-69). Example 83 was synthesized following similar procedure for preparing example 79. White solid, 19% yield. 'H NMR (400 MHz, Methanol-d4) 8 7.27 (d, J= 4.1 Hz, 1H), 6.75 (d, J= 4.6 Hz, 1H), 6.73 - 6.54 (m, 1H), 6.49 - 6.33 (m, 1H), 4.59 (s, 2H), 4.50 - 4.39 (m, 2H), 4.32 - 4.12 (m, 4H), 3.85 (t, J = 5.4 Hz, 2H), 2.81 (s, 6H), 2.42 (s, 3H). MS (ESI) [M+H]+ =334.2.
Figure imgf000129_0001
Example 84: (E)-4-(dimethylamino)-l-(3-(5-methylthiophene-2-carbonyl)-3,8- diazabicyclo[3.2.1]octan-8-yl)but-2-en-l-one (XS185-70). Example 84 was synthesized following similar procedure for preparing example 79. White solid, 46% yield. 1 H NMR (400
MHz, Methanol-d4) 8 7.30 - 7.17 (m, 1H), 7.01 - 6.88 (m, 1H), 6.88 - 6.72 (m, 2H), 4.76 (s, 1H), 4.61 (t, J= 4.8 Hz, 1H), 4.38 (d, J= 15.7 Hz, 2H), 3.99 (t, J= 5.1 Hz, 2H), 3.34 (s, 3H), 2.94 (s, 6H), 2.53 (s, 3H), 2.22 - 1.70 (m, 4H). MS (ESI) [M+H]+ =348.2.
Figure imgf000129_0002
Example 85: (E)-4-(dimethylamino)-l-(8-(5-methylthiophene-2-carbonyl)-3,8- diazabicyclo [3.2. l]octan-3-yl)but-2-en- 1-one (XS185-71). Example 85 was synthesized following similar procedure for preparing example 79. White solid, 49% yield. 'H NMR (400 MHz, Methanol-d4) 8 7.38 - 7.29 (m, 1H), 6.94 - 6.82 (m, 1H), 6.80 (d, J= 4.6 Hz, 1H), 6.76 -
6.57 (m, 1H), 4.74 (s, 2H), 4.48 - 4.30 (m, 1H), 4.00 - 3.82 (m, 3H), 3.56 - 3.41 (m, 1H), 3.09 - 2.98 (m, 1H), 2.86 (s, 6H), 2.47 (s, 3H), 2.05 - 1.83 (m, 2H), 1.83 - 1.59 (m, 2H). MS (ESI)
Figure imgf000129_0003
Example 86: (E)-4-(dimethylamino)-l-(3-(5-methylthiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS185-72). White solid, 54% yield. Example 86 was synthesized following similar procedure for preparing example 79. 1 H NMR (400 MHz, Methanol-c/4) 8 7.35 (d, J= 3.8 Hz, 1H), 6.75 (s, 1H), 6.73 - 6.63 (m, 1H), 6.60 - 6.44 (m, 1H), 4.73 (s, 1H), 4.49 (s, 1H), 4.43 - 3.74 (m, 6H), 2.82 (s, 7H), 2.43 (s, 3H), 1.77 - 1.54 (m, 1H). MS (ESI) [M+H]+ =334.6.
Figure imgf000130_0002
Example 88: (£)-l-(benzofuran-2-yl)-4-(4-(dimethylamino)but-2-enoyl)piperazin-2-one (XS185-78) Example 88 was synthesized following similar procedure for preparing example 28. White solid, 36% yield. 'H NMR (400 MHz, Methanol-d4) δ 7.55 (d, J = 5.3 Hz, 1H), 7.44 (s, 1H), 7.32 - 7.20 (m, 2H), 6.91 (d, J= 3.0 Hz, 1H), 6.78 (s, 2H), 4.54 (d, J= 28.2 Hz, 2H), 4.14 (d, J = 37.0 Hz, 4H), 3.99 (d, J = 8.4 Hz, 3H), 3.87 - 3.73 (m, 1H), 2.93 (s, 6H). MS (ESI) [M+Hf =328.2.
Figure imgf000130_0001
Example 89: (£)-l-(benzo[Z»]thiophen-3-yl)-4-(4-(dimethylamino)but-2-enoyl)piperazin-2- one (XS185-86). Example 89 was synthesized following similar procedure for preparing example 28. White solid, 40% yield. 'H NMR (400 MHz, Methanol^) 6 7.93 (dt, J = 6.2, 3.8 Hz, 1H), 7.78 - 7.58 (m, 2H), 7.52 - 7.37 (m, 2H), 7.02 (t, J= 17.5 Hz, 1H), 6.89 - 6.75 (m, 1H), 4.57 (d, J= 26.5 Hz, 2H), 4.16 (t, J= 5.3 Hz, 2H), 4.06 - 3.97 (m, 2H), 3.95 - 3.80 (m, 2H), 2.94 (s, 6H). MS (ESI) [M+H]+ =344.1
Scheme 10. Synthesis of example 90
Figure imgf000131_0001
Example 90: 2-((4-(5-methylthiophen-2-yl)-3-oxopiperazin-l-yl)methyl)acrylonitrile
(XS185-87). To a solution of 2-bromo-5-methylthiophene (50.0 mg, 0.28 mmol) dissolved in dioxane (1 mL), N,N’ -dimethyl ethylenediamine ( 9 μL, 0.0846 mmol, 0.3 eq), K2CO3 ( 116.9 mg,
0.846 mmol, 3.0 eq), Cui ( 5.4 mg, 0.028 mmol, O. leq ) followed by tert rtoxopiperazine- 1-carboxylate (84.7 mg, 0.42 mmol, 1.5 eq). The reaction mixture was stirred at 100 °C under nitrogen atmosphere overnight. After cooling down to rt, resulting crude mixtures were purified via silica gel column chromatography to yield intermediate 1.
To a solution of intermediate 1 (20 mg, 0.102 mmol) dissolved in DCM (0.5 mL), and the TFA( 0.5 mL) were added. The reaction mixture stirred at rt 1 h. Excess TFA was removed, resulting crude product was re-dissolved in DMF (1 mL), K2CO3 (28.2 mg, 0.2 mmol, 2.0 eq) and KI (16.9 mg, 1.0 eq) were added. At rt the 2-(bromomethyl)acrylonitrile (17.9 mg, 0.124 mmol, 1.2 eq.) was added, the reaction mixture stirred at rt for 5 h. Resulting crude mixtures were purified via prep-HPLC to yield title compound (White solid, 24% yield) XH NMR (400 MHz, Methanol-d4) 8 6.62 (d, J= 14.6 Hz, 2H), 6.36 - 6.13 (m, 2H), 3.92 (d, J= 5.2 Hz, 2H), 3.55 (t, J= 5.1 Hz, 4H), 3.15 (d, J = 5.4 Hz, 2H), 2.41 (s, 3H). MS (ESI) [M+H]+ =262.1.
Figure imgf000131_0002
Example 92: (E)-/V-(benzo|b ]thiophen-2-yl)-2-(4-(dimethylamino)but-2-enoyl)-2- azaspiro[3.3]heptane-6-carboxamide (XS185-91). Example 92 was synthesized following similar procedure for preparing example 53. White solid, 48% yield. 'H NMR (400 MHz,
Methanol-^) 8 7.74 - 7.64 (m, 1H), 7.63 - 7.50 (m, 1H), 7.24 (d, J= 8.4 Hz, 1H), 7.18 (d, J= 8.5 Hz, 1H), 6.88 (d, J= 3.6 Hz, 1H), 6.74 - 6.59 (m, 1H), 6.51 - 6.36 (m, 1H), 4.41 - 4.26 (m, 2H), 4.14 - 4.01 (m, 2H), 3.99 - 3.85 (m, 2H), 3.24 - 3.09 (m, 1H), 2.86 (s, 6H), 2.51 (d, J= 5.0 Hz, 4H). MS (ESI) [M+H]+ =384.2.
Figure imgf000132_0001
Example 93: (E')-4-(dimethylamino)-l-(7-(5-methylthiophene-2-carbonyl)-2,7- diazaspiro[3.5]nonan-2-yl)but-2-en-l-one (XS185-96). Example 93 was synthesized following similar procedure for preparing example 79. White solid, 16% yield. 'H NMR (400 MHz, Methanol-^) 8 7.15 (d, J = 3.1 Hz, 1H), 6.78 - 6.73 (m, 1H), 6.73 - 6.63 (m, 1H), 6.46 (d, J = 15.3 Hz, 1H), 4.06 (s, 2H), 3.96 - 3.87 (m, 2H), 3.81 (s, 2H), 3.74 - 3.58 (m, 4H), 2.85 (s, 6H), 2.50 - 2.42 (m, 3H), 1.89 - 1.71 (m, 4H). MS (ESI) [M+H]+ =362.2.
Figure imgf000132_0002
Example 94: (E')-4-(dimethylamino)-l-(6-(5-methylthiophene-2-carbonyl)-2,6- diazaspiro[3.4]octan-2-yl)but-2-en-l-one (XS185-97). Example 94 was synthesized following similar procedure for preparing example 79. White solid, 12% yield. 'H NMR (400 MHz, Methanol-^) 8 7.40 (d, J= 3.8 Hz, 1H), 6.85 - 6.76 (m, 1H), 6.76 - 6.59 (m, 1H), 6.56 - 6.31 (m, 1H), 4.39 - 4.15 (m, 2H), 4.13 - 3.95 (m, 3H), 3.88 (t, J= 10.4 Hz, 3H), 3.69 (d, J= 53.8 Hz, 2H), 2.85 (s, 6H), 2.51 - 2.43 (m, 3H), 2.32 - 2.08 (m, 2H). MS (ESI) [M+H]+ =348.2.
Figure imgf000132_0003
Example 95: (E)-4-(dimethylamino)-1-(7-(5-methylthiophen-2-yl)-2,7-diazaspiro[3.5]nonan- 2-yl)but-2-en-l-one (XS185-101). Example 95 was synthesized following similar procedure for preparing example 28. White solid, 18% yield. 1 H NMR (400 MHz, Methanol-64) 8 6.82 - 6.68 (m, 1H), 6.64 - 6.58 (m, 1H), 6.58 - 6.47 (m, 2H), 4.84 (s, 2H), 4.60 - 4.50 (m, 1H), 4.41 - 4.27 (m, 2H), 4.09 (d, J= 10.5 Hz, 1H), 4.01 - 3.94 (m, 2H), 3.93 - 3.84 (m, 2H), 2.93 (s, 6H), 2.64 - 2.57 (m, 2H), 2.43 (s, 3H). MS (ESI) [M+H]+ =334.2.
Figure imgf000133_0002
Example 97: (£)-4-(dimethylamino)-l-(2-(5-methylthiophen-2-yl)-2,7-diazaspiro[3.5]nonan- 7-yl)but-2-en-l-one (XS185-113). Example 97 was synthesized following similar procedure for preparing example 53. White solid, 43% yield. ’H NMR (400 MHz, Methanol-6/4) 8 6.76 - 6.61 (m, 1H), 6.50 - 6.39 (m, 1H), 6.34 (s, 1H), 4.51 - 4.37 (m, 2H), 4.22 (t, J= 9.6 Hz, 1H), 4.18 - 4.08 (m, 1H), 3.96 - 3.83 (m, 2H), 3.61 - 3.49 (m, 1H), 2.86 (s, 6H), 2.60 (t, J= 5.9 Hz, 2H), 2.53 - 2.40 (m, 2H), 1.85 - 1.63 (m, 4H). MS (ESI) [M+H]+ =348.2.
Figure imgf000133_0001
Example 98: (E)-2-(4-(dimethylamino)but-2-enoyl)-JV-(4,5,6,7-tetrahydrobenzo[/»]thiophen- 2-yl)-2-azaspiro[3.3]heptane-6-carboxamide (XS185-114). Example 98 was synthesized following similar procedure for preparing example 53. White solid, 49% yield. JH NMR (400 MHz, Methanol-d4) 5 6.68 - 6.51 (m, 1H), 6.40 - 6.30 (m, 1H), 6.23 (s, 1H), 4.22 (d, J= 26.3 Hz, 2H), 3.96 (d, ,7 = 22.1 Hz, 2H), 3.89 - 3.79 (m, 2H), 3.12 - 2.94 (m, 1H), 2.79 (s, 6H), 2.54 (d, J = 5.7 Hz, 2H), 2.46 - 2.25 (m, 6H), 1.81 - 1.58 (m, 4H). MS (ESI) [M+H]+ =388.2.
Figure imgf000134_0001
Example 99: (E)-4-(4-(dimethylamino)but-2-enoyl)-l-(lH-indol-2-yl)piperazin-2-one (XS185-116). Example 99 was synthesized following similar procedure for preparing example 28. White solid, 37% yield. ’H NMR (400 MHz, Methanol-d4) 8 7.47 - 7.36 (m, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.22 - 7.00 (m, 2H), 7.00 - 6.91 (m, 1H), 6.91 - 6.62 (m, 2H), 4.54 - 4.14 (m, 2H), 4.03 (q, J= 7.1, 6.2 Hz, 1H), 3.99 - 3.83 (m, 3H), 3.83 - 3.65 (m, 2H), 2.88 - 2.78 (m, 6H). MS (ESI) [M+H]+ =327.2.
Figure imgf000134_0002
53. White solid, 21% yield. 'H NMR (400 MHz, Methanol-d)48 6.78 - 6.65 (m, 1H), 6.52 - 6.43 (m, 1H), 4.50 - 4.31 (m, 2H), 4.25 - 4.04 (m, 3H), 3.98 - 3.87 (m, 2H), 3.49 - 3.34 (m, 1H), 2.89 (s, 6H), 2.02 - 1.84 (m, 2H), 1.81 - 1.53 (m, 4H), 1.52 - 1.39 (m, 2H). MS (ESI) [M+H]+ =280.2.
Figure imgf000135_0001
Example 103: (E)-JN,N-dimethyl-4-(4-(5-methylthiophen-2-yl)-3-oxopiperazin-l-yl)but-2- enamide (XS185-134). Example 103 was synthesized following similar procedure for preparing example 90. White solid, 19% yield. ’H NMR (400 MHz, Methanol-d4) 8 6.98 (d, J = 15.0 Hz, 1H), 6.74 - 6.59 (m, 3H), 4.16 - 4.02 (m, 6H), 3.72 (t, J= 5.7 Hz, 2H), 3.17 (s, 3H), 3.03 (s, 3H), 2.41 (s, 3H). MS (ESI) [M+H] 1 =308.2.
Scheme 11. Synthesis of example 104
Figure imgf000135_0002
Example 104: (2-(4-(5-methylthiophen-2-yl)-3-oxopiperazin-l-yl)-2-oxoethyl methylsulfamate (XS185-135). To a solution of intermediate 1 (20 mg, 0.102 mmol) dissolved in DMF (1 mL), and the HATU( 42.7 mg, 0.112 mmol, 1.1 eq), DIPEA(0.1 mL), 2-hydroxyacetic acid(9.4 mg, 0.102 mmol, 1.0 eq) were added. The reaction mixture stirred at rt 1 h, the crude mixtures were purified by HPLC yield the intermediate 10.
Then the intermediate 10 was re-dissovled in DCM (1 mL), EtsN (0.1 mL, 2.0 eq) and methyl sulfamoyl chloride (9.3 mg , 1.2 eq) were added. The reaction mixture stirred at rt for 5 h. Resulting crude mixtures were purified by HPLC yield the title example 104. White solid, 29% yield. ’H NMR (400 MHz, Methanol-d4) 8 6.75 - 6.51 (m, 2H), 4.91 (s, 2H), 4.38 (s, 2H), 3.95 (m, 4H), 2.75 (s, 3H), 2.41 (s, 3H). MS (ESI) [M+H]+ =348.1.
Figure imgf000135_0003
Example 105: (E)-4-(4-(5-methylthiophen-2-yl)-3-oxopiperazin-l-yl)but-2-enenitrile
(XS185-138). Example 105 was synthesized following similar procedure for preparing example 90. White solid, 30% yield. XH NMR (400 MHz, Methanol^) 8 6.84 - 6.61 (m, 1H), 6.57 (d, J = 3.9 Hz, 1H), 6.55 - 6.48 (m, 1H), 5.99 - 5.77 (m, 1H), 3.93 - 3.86 (m, 2H), 3.80 - 3.72 (m, 1H), 3.66 (d, J= 6.5 Hz, 2H), 3.64 - 3.59 (m, 1H), 3.34 - 3.24 (m, 2H), 2.34 (s, 3H). MS (ESI) [M+Hf =262.1.
Figure imgf000136_0001
Example 106: l-(5-methylthiophen-2-yl)-4-(2-(phenylsulfonyl)acetyl)piperazin-2-one (XS185-140). Example 106 was synthesized following similar procedure for preparing example 104. White solid, 35% yield. 'H NMR (400 MHz, Methanol-d4) 8 8.03 - 7.90 (m, 2H), 7.82 - 7.53 (m, 3H), 6.68 - 6.51 (m, 2H), 4.58 (d, J= 18.3 Hz, 2H), 4.36 (d, J= 22.2 Hz, 2H), 4.06 (t, J = 5.4 Hz, 1H), 4.00 - 3.89 (m, 2H), 3.78 (t, J = 5.5 Hz, 1H), 2.42 (s, 3H). MS (ESI) [M+H]+ =380.1.
Figure imgf000136_0002
Example 107: (E)-l-(7-(benzo[Z>]thiophen-2-yl)-2,7-diazaspiro[3.5]nonan-2-yl)-4-
(dimethylamino)but-2-en-l-one (XS185-147). Example 107 was synthesized following similar procedure for preparing example 28. White solid, 41% yield. 'H NMR (400 MHz, Methanol-d4) 8 7.54 (d, J= 7.9 Hz, 1H), 7.41 (d, J= 7.9 Hz, 1H), 7.16 (t, J= 7.6 Hz, 1H), 7.07 - 6.95 (m, 1H), 6.79 - 6.63 (m, 1H), 6.55 - 6.40 (m, 1H), 4.06 (s, 2H), 3.94 - 3.89 (m, 2H), 3.81 (s, 2H), 3.26 - 3.16 (m, 4H), 2.88 (s, 7H), 1.93 (t, J= 5.6 Hz, 4H). MS (ESI) [M+H]+ =370.2.
Figure imgf000136_0003
Example 108: (E)-l-(6-(benzo[Z»]thiophen-2-yl)-2,6-diazaspiro[3.4]octan-2-yl)-4-
(dimethylamino)but-2-en-l-one (XS185-149). Example 108 was synthesized following similar procedure for preparing example 28. White solid, 30% yield. ’H NMR (400 MHz, Methanol- d4) 8 7.51 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H), 7.13 (t, J= 7.6 Hz, 1H), 6.93 (t, J= 7.6 Hz, 1H), 6.79 - 6.37 (m, 3H), 4.35 - 4.21 (m, 2H), 4.15 - 3.96 (m, 2H), 3.96 - 3.85 (m, 2H), 3.58 - 3.47 (m, 2H), 3.40 (t, J= 7.0 Hz, 2H), 2.86 (s, 7H), 2.27 (t, J= 6.8 Hz, 2H).MS (ESI) [M+Hf =356.3.
Figure imgf000137_0001
Exam pie 109: (E)- l-(2-(benzo [b] thiophen-2-yl)-2,6-diazaspiro [3.4] octan-6-yl)-4-
(dimethylamino)but-2-en-l-one (XS185-150). Example 109 was synthesized following similar procedure for preparing example 28. White solid, 30% yield. *HNMR (400 MHz, Methanol-)d 84 7.56 (d, J = 8.0 Hz, 1H), 7.42 (d, J = 7.9 Hz, 1H), 7.23 - 7.14 (m, 1H), 7.03 (t, J= 7.6 Hz, 1H), 6.83 - 6.64 (m, 2H), 4.24 - 4.01 (m, 2H), 3.99 - 3.86 (m, 5H), 3.79 - 3.68 (m, 2H), 3.65 - 3.53 (m, 1H), 2.95 - 2.84 (m, 6H), 2.38 - 2.15 (m, 2H).MS (ESI) [M+H]+ =356.3.
Figure imgf000137_0002
Example 110: (E)-7V-(l-(4-(dimethylamino)but-2-enoyl)azetidin-3-yl)-5-methylthiophene-2- carboxamide (XS185-171). Example 110 was synthesized following similar procedure for preparing example 104. White solid, 15% yield 'H NMR (400 MHz, D SO-d6) 8 8.54 - 8.35 (m, 1H), 7.34 (d, J= 4.1 Hz, 4H), 7.29 - 7.20 (m, 1H), 6.67 - 6.49 (m, 2H), 4.81 (d, J= 21.1 Hz, 2H),
4.25 (s, 2H), 4.19 (d, J= 4.8 Hz, 2H), 3.91 - 3.82 (m, 2H), 3.82 - 3.69 (m, 2H), 2.36 (s, 3H).MS
(ESI) [M+H]+ =424.2.
Figure imgf000137_0003
Example 111: (E')-A/-(l-(4-(dimethylamino)but-2-enoyl)azetidin-3-yl)-5-methylthiophene-2- carboxamide (XS190-9). Example 111 was synthesized following similar procedure for preparing example 79. White solid, 56% yield *H NMR (400 MHz, Methanol-d4) 8 7.47 (d, J = 3.7 Hz, 1H), 6.74 (d, J= 3.0 Hz, 1H), 6.71 - 6.57 (m, 1H), 6.44 (d, J= 15.3 Hz, 1H), 4.76 - 4.63 (m, 1H), 4.58 (t, J= 8.6 Hz, 1H), 4.36 - 4.28 (m, 1H), 4.27 - 4.18 (m, 1H), 4.07 - 3.96 (m, 1H), 3.87 (d, J= 7.1 Hz, 2H), 2.83 (s, 6H), 2.43 (s, 3H).MS (ESI) [M+H]+ =307.4.
Figure imgf000138_0001
Example 112: (E)-\-( l-(4-(dimethylamin())biit-2-enoyl)azetidin-3-yl)-V.5- dimethylthiophene-2-carboxamide (XS190-27). Example 112 was synthesized following similar procedure for preparing example 79. White solid, 23% yield XH NMR (400 MHz, Methanol-^) 8 7.54 (d, J = 3.7 Hz, 1H), 6.89 - 6.77 (m, 2H), 6.57 (d, J = 15.2 Hz, 1H), 4.81 - 4.70 (m, 1H), 4.67 (t, J = 8.5 Hz, 1H), 4.45 - 4.35 (m, 1H), 4.34 - 4.24 (m, 1H), 4.14 (d, J = 7.2 Hz, 3H), 3.16 (s, 9H), 2.51 (s, 3H).MS (ESI) [M+H]+ =322.2.
Figure imgf000138_0002
Example 113: (E)-3-(benzo[b]thiopheii-2-yl)-8-(4-(dimethylamino)but-2-enoyl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS190-38). Example 113 was synthesized following similar procedure for preparing example 28. White solid, 25% yield 1 H NMR (400 MHz, Methanol-d4)
8 7.72 (d, J= 7.8 Hz, 1H), 7.65 (d, J= 7.6 Hz, 1H), 7.34 - 7.19 (m, 2H), 7.03 - 6.69 (m, 3H), 5.07 (t, J= 35.5 Hz, 2H), 4.12 (t, J= 1 1 .3 Hz, 1H), 3.96 (s, 2H), 3.89 - 3.71 (m, 1H), 2.89 (s, 6H), 2.51 - 2.16 (m, 3H), 2.16 - 1.95 (m, 1H).MS (ESI) [M+H]+ =370.2.
Figure imgf000138_0003
Example 114: (E)-3-(benzo[/>]thiophen-3-yl)-8-(4-(dimethylamino)but-2-enoyl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS190-44). Example 114 was synthesized following similar procedure for preparing example 28. 1 H NMR (400 MHz, Methano)l- 8d47.84 (d, J= 4.2 Hz, 1H), 7.58 (s, 1H), 7.41 - 7.28 (m, 3H), 6.97 - 6.75 (m, 2H), 5.05 - 4.88 (m, 2H), 4.08 - 3.80 (m, 3H), 3.57 - 3.39 (m, 1H), 2.83 (s, 6H), 2.52 - 2.1 1 (m, 4H). White solid, 15% yield MS (ESI) [M+H]+ =370.2.
Figure imgf000139_0001
Example 115: (£)-8-(4-(dimethylamino)but-2-enoyl)-3-(5-phenylthiophen-2-yl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS190-45). Example 115 was synthesized following similar procedure for preparing example 28. White solid, 18% yield JH NMR (400 MHz, Methanol -t) 8 7.49 - 7.41 (m, 2H), 7.23 (t, J= 7.7 Hz, 2H), 7.12 (t, J= 7.4 Hz, 1H), 7.05 (d, J= 4.1 Hz, 1H), 6.91 - 6.64 (m, 2H), 6.63 - 6.55 (m, 1H), 5.10 - 4.89 (m, 2H), 4.05 - 3.89 (m, 1H), 3.89 - 3.80 (m, 2H), 3.64 (dd, J= 25.2, 11.4 Hz, 1H), 2.80 (s, 3H), 2.78 (s, 3H), 2.38 - 2.02 (m, 3H), 2.04 - 1.81 (m, 1H).MS (ESI) [M+H]+ =396.3.
Figure imgf000139_0002
Example 116: (£)-l-(3-(benzo[6]thiophene-2-carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)- 4-(dimethylamino)but-2-en-l-one (XS190-46). Example 116 was synthesized following similar procedure for preparing example 79. White solid, 40% yield 1 H NMR (400 MHz, Methanol-d4) 6 7.79 (d, J= 7.4 Hz, 2H), 7.72 (s, 1H), 7.32 (p, J= 7.0 Hz, 2H), 6.74 - 6.57 (m, 1H), 6.56 - 6.36 (m, 1H), 4.69 (d, J= 17.5 Hz, 1H), 4.47 (d, J = 23.4 Hz, 1H), 4.13 (t, J = 8.2 Hz, 1H), 4.07 - 3.96 (m, 1H), 3.94 - 3.74 (m, 4H), 2.80 (s, 3H), 2.78 - 2.67 (m, 4H), 1.65 (d, J= 9.2 Hz, 1H). MS (ESI) [M+H]+ =370.2.
Figure imgf000139_0003
Example 117: (E)-l-(3-(benzo[Z>]thiophene-3-carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)- 4-(dimethylamino)but-2-en-l-one (XS190-47). Example 117 was synthesized following similar procedure for preparing example 79. White solid, 50% yield 1 H NMR (400 MHz, Methanol-d)4 8 7.91 - 7.81 (m, 1H), 7.75 (d, J= 6.4 Hz, 1H), 7.64 (d, J= 5.6 Hz, 1H), 7.38 - 7.26 (m, 2H), 6.75 - 6.57 (m, 1H), 6.59 - 6.28 (m, 1H), 4.73 (s, 1H), 4.50 (s, 1H), 4.31 - 4.06 (m, 1H), 4.01 - 3.81 (m, 3H), 3.78 - 3.61 (m, 2H), 3.54 (d, J= 11.8 Hz, 1H), 2.81 (s, 3H), 2.76 - 2.61 (m, 4H), 1.65 (d, J= 9.2 Hz, 1H).MS (ESI) [M+H]+ =370.2.
Figure imgf000140_0001
Example 118: (E)-4-(dimethylamino)-l-(3-(4,5,6,7-tetrahydrobenzob]thiophene-2- carbonyl)-3,6-diazabicydo[3.1.1]heptan-6-yl)but-2-en-l-one (XS190-48). Example 118 was synthesized following similar procedure for preparing example 79. White solid, 46% yield 1H NMR (400 MHz, Methanol-d4) 8 7. 15 (s, 1H), 6.70 - 6.56 (m, 1H), 6.46 (d, J= 15.2 Hz, 1H), 4.66 (s, 1H), 4.42 (s, 1H), 4.31 - 3.69 (m, 6H), 2.83 - 2.67 (m, 7H), 2.67 - 2.60 (m, 2H), 2.54 - 2.42 (m, 2H), 1.70 (dt, J= 10.4, 5.4 Hz, 4H), 1.58 (d, J= 9.2 Hz, 1H).MS (ESI) [M+H]+ =374.2.
Figure imgf000140_0002
Example 119: (E)-4-(dimethylamino)-l-(3-(4,5,6,7-tetrahydrobenzo[b]thiophene-3- carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS190-49). Example 119 was synthesized following similar procedure for preparing example 79. White solid, 39% yield 1 H NMR (400 MHz, Methanol-d4) δ 7.15 (d, J = 4.1 Hz, 1H), 6.69 - 6.54 (m, 1H), 6.52 - 6.30 (m, 1H), 4.59 (d, J= 51.2 Hz, 1H), 4.36 (d, J= 73.5 Hz, 1H), 3.88 - 3.73 (m, 4H), 3.67 - 3.52 (m, 1H), 3.45 (d, J= 12.0 Hz, 1H), 2.79 - 2.70 (m, 6H), 2.70 - 2.58 (m, 3H), 2.39 - 2.28 (m, 2H), 1.77 - 1.59 (m, 4H), 1.59 - 1.49 (m, 1H).MS (ESI) [M+H]+ =374.2.
Figure imgf000141_0001
Example 120: (E)-l-(3-(5,6-dihydro-4//-cyclopenta[Z»]thiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS190-50). Example 120 was synthesized following similar procedure for preparing example 79. White solid, 55% yield
NMR (400 MHz, Methanol-d4) 8 7.26 (s, 1H), 6.72 - 6.56 (m, 1H), 6.49 (d, J= 15.1 Hz, 1H), 4.70 (s, 1H), 4.46 (s, 1H), 4.36 - 3.72 (m, 6H), 2.86 - 2.69 (m, 9H), 2.69 - 2.57 (m, 2H), 2.41 - 2.24 (m, 2H), 1.62 (d, J= 9.2 Hz, 1H).MS (ESI) [M+H]+ =360.3.
Figure imgf000141_0002
Example 121: (£)-JV-(2-(6-(4-(dimethylamino)but-2-enoyl)-3,6-diazabicyclo[3.1.1Jheptane- 3-carbonyl)benzo[Z»]thiophen-5-yl)acetamide (XS190-59). Example 121 was synthesized following similar procedure for preparing example 79. White solid, 26% yield 1 H NMR (400 MHz, Methanol-d4) 1H NMR (400 MHz, Methanol-d)48 8.22 (s, 1H), 7.74 (d, J= 8.7 Hz, 1H), 7.69 (s, 1H), 7.39 (t, J= 9.9 Hz, 1H), 6.75 - 6.62 (m, 1H), 6.58 - 6.41 (m, 1H), 4.72 (d, J= 17.8 Hz, 1H), 4.50 (d, J = 23.8 Hz, 1H), 4.38 - 3.75 (m, 6H), 2.83 (s, 3H), 2.80 - 2.71 (m, 4H), 2.08 (s, 3H), 1.68 (d, J= 9.2 Hz, 1H).MS (ESI) [M+H]+ =427.3.
Figure imgf000141_0003
Example 122: (E')-4-(dimethylamino)-l-(3-(5-(methylamino)benzo[B]thiophene-2- carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS190-68). Example 122 was synthesized following similar procedure for preparing example 79. White solid, 33% yield 'H NMR (400 MHz, Methanol-d4) 8 8.06 - 7.98 (m, 1H), 7.91 (d, J = 7.4 Hz, 1H), 7.83 (d, J = 6.2 Hz, 1H), 7.42 (d, J= 8.6 Hz, 1H), 6.76 - 6.64 (m, 1H), 6.58 - 6.44 (m, 1H), 4.73 (d, J= 18.3 Hz, 1H), 4.50 (d, J= Tl. 1 Hz, 1H), 4.41 - 3.81 (m, 6H), 3.07 - 3.00 (m, 3H), 2.84 - 2.74 (m, 7H), 1.69 (d, J= 9.2 Hz, 1H).MS (ESI) [M+H]+ =399.2.
Figure imgf000142_0001
Example 123: (£)-l-(3-(5-acetyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS190-69). Example 123 was synthesized following similar procedure for preparing example 79. White solid, 22% yield 'H NMR (400 MHz, Methanol-d)45 7.26 (d, J= 10.6 Hz, 1H), 6.67 - 6.53 (m, 1H), 6.46 (dd, J = 14.7, 6.7 Hz, 1H), 4.66 (s, 1H), 4.53 - 4.36 (m, 3H), 4.31 - 3.94 (m, 2H), 3.93 - 3.64 (m, 6H), 2.84 - 2.78 (m, 1H), 2.78 - 2.62 (m, 8H), 2.14 - 1.99 (m, 3H), 1.58 (d, J= 9.2 Hz, 1H).MS (ESI) [M+H]+ =417.3.
Figure imgf000142_0002
Example 124: (E)-3-(5-acetyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yI)-8-(4-
(dimethylamino)but-2-enoyl)-3,8-diazabicyclo[3.2.1]octan-2-one (XS190-70). Example 124 was synthesized following similar procedure for preparing example 79. White solid, 43% yield ’H NMR (400 MHz, Methanol-d)4δ 6.94 - 6.63 (m, 2H), 6.47 - 6.32 (m, 1H), 5.10 - 4.94 (m, 1H), 4.46 (s, 2H), 4.03 - 3.83 (m, 3H), 3.81 - 3.75 (m, 1H), 3.75 - 3.69 (m, 1H), 3.69 - 3.53 (m, 2H),
2.85 - 2.78 (m, 6H), 2.77 - 2.60 (m, 2H), 2.40 - 2.15 (m, 2H), 2.11 (s, 3H), 2.07 (s, 1H), 2.03 -
1.86 (m, 1H).MS (ESI) [M+H]+ =417.3.
Figure imgf000142_0003
Example 125: (E)-l-(4-(dimethylamino)but-2-enoyl)-Wmethyl-W(5-methylthiophen-2- yl)azetidine-3-carboxamide (XS190-75). Example 125 was synthesized following similar procedure for preparing example 79. White solid, 26% yield 'H NMR (400 MHz, Methanol-tZr) 8 6.84 - 6.70 (m, 1H), 6.51 (d, J= 15.1 Hz, 1H), 6.44 (s, 2H), 4.52 - 4.39 (m, 2H), 4.23 (t, J = 9.7 Hz, 1H), 4.19 - 4.04 (m, 3H), 3.60 - 3.48 (m, 1H), 3.10 (s, 9H), 2.32 (s, 3H).MS (ESI) [M+H]+ =322.4.
Figure imgf000143_0001
(E)- N,N,N ,-trimethyl-4-(3-(methyl(5-methylthiophen-2- yl)carbamoyl)azetidin-l-yl)-4-oxobut-2-en-l-aminium (XS190-75-2). Example 126was synthesized following similar procedure for preparing example 79. White solid, 15% yield !H NMR (400 MHz, Methanol-d4) 8 6.79 - 6.68 (m, 1H), 6.67 (d, ,7 = 3.6 Hz, 1H), 6.60 - 6.55 (m,
1H), 6.43 (d, J= 15.1 Hz, 1H), 4.41 - 4.33 (m, 1H), 4.17 (t, J= 8.9 Hz, 1H), 4.11 - 4.02 (m, 3H), 3.88 (t, J = 9.7 Hz, 1H), 3.71 - 3.59 (m, 1H), 3.18 (s, 3H), 3.06 (s, 9H), 2.39 (s, 3H).MS (ESI) [M+H]+ =322.4.
Figure imgf000143_0002
Example 127: (E)-4-(dimethylamino)-l-(5-(5-methylthiophene-2-carbonyl)-2,5- diazabicyclo[2.2.2]octan-2-yl)but-2-en-l-one (XS190-77). Example 127 was synthesized following similar procedure for preparing example 79. White solid, 46% yield 1 H NMR (400 MHz, Methanol-^) 8 7.24 (d, J= 75.4 Hz, 1H), 6.91 - 6.54 (m, 3H), 4.73 (d, J = 24.1 Hz, 1H), 4.62 - 4.33 (m, 1H), 4.11 - 3.71 (m, 4H), 3.69 - 3.49 (m, 2H), 2.81 (s, 6H), 2.41 (s, 3H), 2.16 - 1.70 (m,
Figure imgf000143_0003
Example 128: (£)-4-(dimethylamino)-l-((1S,4S)-5-(5-methylthiophene-2-carbonyl)-2,5- diazabicyclo[2.2.1]heptan-2-yl)but-2-en-l-one (XS190-80). Example 128 was synthesized following similar procedure for preparing example 79. White solid, 42% yield 1 H NMR (400 MHz, Methanol-^) 8 7.31 - 7.14 (m, 1H), 6.80 - 6.34 (m, 3H), 4.86 - 4.78 (m, 2H), 3.80 (h, J= 18.8, 15.5 Hz, 3H), 3.67 - 3.31 (m, 3H), 2.73 (d, J= 7.4 Hz, 6H), 2.34 (s, 3H), 2.03 - 1.77 (m, 2H).MS (ESI) [M+H]+ =334.1.
Figure imgf000144_0001
Example 129: (E')-4-(dimethylamino)-l-((lR,4R)-5-(5-methylthiophene-2-carbonyl)-2,5- diazabicyclo[2.2.1]heptan-2-yl)but-2-en-l-one (XS190-81). Example 129 was synthesized following similar procedure for preparing example 79. White solid, 59% yield 1 H NMR (400 MHz, Methanol-^) 8 7.35 - 7.18 (m, 1H), 6.90 - 6.40 (m, 3H), 4.87 - 4.80 (m, 2H), 3.98 - 3.71 (m, 3H), 3.72 - 3.32 (m, 3H), 2.82 - 2.70 (m, 6H), 2.36 (s, 3H), 2.03 - 1.78 (m, 2H).MS (ESI) [M+H]- =334.1.
Figure imgf000144_0002
Example 131 (£)-JV-((l-(4-(dimethylamino)but-2-enoyl)azetidin-2-yl)methyl)-5- methylthiophene-2-carboxamide (XS190-127). Example 131 was synthesized following similar procedure for preparing example 79. White solid, 34% yield 'H NMR (400 MHz, Methanol-6/4) 8 7.55 - 7.42 (m, 1H), 6.85 - 6.63 (m, 2H), 6.59 - 6.41 (m, 1H), 4.78 - 4.57 (m, 1H), 4.24 (t, J = 7.8 Hz, 1H), 4.02 - 3.89 (m, 2H), 3.88 - 3.73 (m, 2H), 3.69 - 3.50 (m, 1H), 2.88 (d, J= 11.0 Hz, 6H), 2.59 - 2.41 (m, 4H), 2.24 - 2.08 (m, 1H).MS (ESI) [M+H]+ =322.2.
Figure imgf000145_0001
Example 132 (E)-l-(3-(cyclopentanecarbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)-4- (dimethylamino)but-2-en-l-one (XS190-128). Example 132 was synthesized following similar procedure for preparing example 79. White solid, 34% yield 1 H NMR (400 MHz, Methanol-d4) δ 6.78 - 6.63 (m, 1H), 6.61 - 6.48 (m, 1H), 4.75 (s, 1H), 4.52 (s, 1H), 4.20 - 3.66 (m, 6H), 3.05 - 2.94 (m, 1H), 2.88 (d, J = 2.7 Hz, 6H), 2.83 - 2.74 (m, 1H), 1.95 - 1.80 (m, 2H), 1.80 - 1.55 (m, 7H).MS (ESI) [M+Hf =306.2.
Figure imgf000145_0002
Example 133 (E)-l-(3-(cyclohexanecarbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)-4- (dimethylamino)but-2-en-l-one (XS190-129). Example 133 was synthesized following similar procedure for preparing example 79. White solid, 45% yield 'H NMR (400 MHz, Methanol-d4) 8 6.84 - 6.66 (m, 1H), 6.62 - 6.46 (m, 1H), 4.75 (s, 1H), 4.52 (s, 1H), 4.24 - 3.59 (m, 6H), 2.89 (d, J = 5.2 Hz, 6H), 2.84 - 2.74 (m, 1H), 2.63 - 2.47 (m, 1H), 1.86 - 1.58 (m, 6H), 1.54 - 1.13 (m, 5H).MS (ESI) [M+Hf =320.2.
Figure imgf000145_0003
Example 134 (E)-.-N-((5-(6-(4-(dimethylamino)but-2-enoyl)-3,6-diazabicyclo[3.1.1]heptane-3- carbonyl)thiophen-2-yl)methyl)acetamide (XS190-130). Example 134 was synthesized following similar procedure for preparing example 79. White solid, 22% yield 1 H NMR (400 MHz, Methanol-^) 8 7.53 - 7.40 (m, 1H), 7.01 (d, J = 3.3 Hz, 1H), 6.84 - 6.70 (m, 1H), 6.59 (d, J = 15.1 Hz, 1H), 4.80 (s, 1H), 4.54 (s, 3H), 4.47 - 3.86 (m, 6H), 2.98 - 2.76 (m, 7H), 1.99 (s, 3H), 1.74 (d, J= 9.2 Hz, 1H).MS (ESI) [M+H]+ =391.2.
Figure imgf000146_0001
Example 135 (E)-4-(dimethylamino)-l-(3-(5-methyloxazole-4-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS190-137). Example 135 was synthesized following similar procedure for preparing example 79. White solid, 47% yield 1 H NMR (400 MHz, Methanol-^) 8 8.05 (d, J = 3.4 Hz, 1H), 6.85 - 6.73 (m, 1H), 6.67 - 6.54 (m, 1H), 4.77 (s, 1H), 4.58 (d, J= 31.8 Hz, 1H), 4.49 - 4.23 (m, 2H), 4.09 - 3.88 (m, 4H), 2.93 (d, J= 3.0 Hz, 6H), 2.85 (d, J= 5.8 Hz, 1H), 2.54 (s, 3H), 1.80 - 1.71 (m, 1H).MS (ESI) [M+H]+ =319.2.
Figure imgf000146_0002
Example 136 (E)-4-(dimethylamino)-l-(3-(5-(methoxymethyl)thiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS190-138). Example 136 was synthesized following similar procedure for preparing example 79. White solid, 38% yield 'H NMR (400 MHz, Methanol-^) 8 7.49 (d, J= 3.8 Hz, 1H), 7.05 (d, J= 3.8 Hz, 1H), 6.83 - 6.71 (m, 1H), 6.59 (d, J = 15.2 Hz, 1H), 4.81 (s, 1H), 4.63 (s, 3H), 4.47 - 3.82 (m, 6H), 3.39 (s, 3H), 2.97 - 2.78 (m, 7H), 1.74 (d, J= 9.2 Hz, 1H).MS (ESI) [M+H]+ =364.2.
Figure imgf000146_0003
Example 137 (E)-3-cyclopentyl-8-(4-(dimethylamino)but-2-enoyl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS190-144). Example 137 was synthesized following similar procedure for preparing example 28. White solid, 19% yield 'H NMR (400 MHz, Methanol-d)48 6.81 - 6.44 (tn, 2H), 4.75 - 4.28 (m, 3H), 3.78 - 3.63 (m, 2H), 3.41 - 3.25 (m, 1H), 2.95 - 2.80 (m, 1H), 2.66 (s, 6H), 2.15 - 1.75 (m, 3H), 1.70 - 1.54 (m, 2H), 1.47 (s, 3H), 1.41 - 1.21 (m, 4H).MS (ESI) [M+Hf =306.2.
Figure imgf000147_0001
Example 138 (E)-4-(dimethylamino)-l-(3-(5-methylfuran-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS190-157). Example 138 was synthesized following similar procedure for preparing example 104. White solid, 35% yield 'H NMR (400 MHz, Methanol-d4) δ 7.04 (d, J= 3.5 Hz, 1H), 6.78 - 6.65 (m, 1H), 6.58 (d, J= 15.3 Hz, 1H), 6.20 (s, 1H), 4.79 (s, 1H), 4.56 (s, 1H), 4.47 - 3.79 (m, 6H), 2.90 - 2.75 (m, 7H), 2.34 (s, 3H), 1.69 (d, J= 9.2 Hz, 1H).MS (ESI) [M+H]+ =318.2.
Figure imgf000147_0002
Example 139 (E')-4-(dimethylamino)-l-(3-((5-methylthiophen-2-yl)methyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS190-158). Example 139 was synthesized following similar procedure for preparing example 79. White solid, 23% yield 1 H NMR (400 MHz, Methanol-^) 8 7.14 (d, J = 3.5 Hz, 1H), 6.85 - 6.70 (m, 2H), 6.53 (d, J = 15.2 Hz, 1H), 4.82 - 4.53 (m, 4H), 3.96 (d, J= 1A Hz, 2H), 3.90 - 3.61 (m, 4H), 2.83 (s, 2H), 2.52 (s, 3H), 2.07 (d, J = 10.2 Hz, 1H).MS (ESI) [M+H]+ =320.2.
Figure imgf000147_0003
Example 140 (E)-4-(dimethylamino)-l-(3-(3-methylcyclopentane-l-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS190-170). Example 40 was synthesized following similar procedure for preparing example 79. White solid, 25% yield 'H NMR (400 MHz, Methanol-d4) δ 6.66 - 6.54 (m, 1H), 6.47 - 6.36 (m, 1H), 4.62 (s, 1H), 4.38 (s, 1H), 4.02 (d, J = 11.4 Hz, 1H), 3.86 - 3.69 (m, 4H), 3.57 (d, J= 10.1 Hz, 1H), 2.77 - 2.73 (m, 6H), 2.70 - 2.61 (m, 1H), 2.02 - 1.59 (m, 6H), 1.49 (d, J= 9.4 Hz, 1H), 1.24 - 1.03 (m, 2H), 0.92 - 0.86 (m, 3H).MS (ESI) [M+H]+ =320.2.
Figure imgf000148_0001
Example 141 (E)-8-(4-(dimethyIamino)but-2-enoyl)-3-(6-methylpyridin-2-yl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS190-181). Example 141 was synthesized following similar procedure for preparing example 28. White solid, 29% yield 'H NMR (400 MHz, Methanol-6/4) 5 7.84 - 7.60 (m, 1H), 7.46 - 7.07 (m, 1H), 6.99 - 6.72 (m, 2H), 6.72 - 6.47 (m, 1H), 4.97 (dd, J = 42.5, 7.7 Hz, 1H), 4.84 - 4.70 (m, 1H), 3.96 - 3.83 (m, 2H), 3.81 - 3.70 (m, 1H), 2.91 - 2.75 (m, 6H), 2.47 (d, J= 37.5 Hz, 3H), 2.39 - 2.15 (m, 3H), 2.14 - 1.79 (m, 1H).MS (ESI) [M+H]+ =329.2.
Figure imgf000148_0002
Example 142 (E)-8-(4-(dimethylamino)but-2-enoyl)-3-(m-tolyl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS190-182). Example 142 was synthesized following similar procedure for preparing example 28. White solid, 34% yield 1 H NMR (400 MHz, Methanol)-d 54 7.20 (t, J = 7.7 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H), 6.98 (s, 1H), 6.93 (d, J = 7.8 Hz, 1H), 6.90 - 6.66 (m, 2H), 4.98 - 4.88 (m, 1H), 4.83 - 4.70 (m, 1H), 3.95 - 3.75 (m, 3H), 3.39 (dd, J = 21.6, 11.8 Hz, 1H), 2.81 (s, 6H), 2.43 - 1.96 (m, 7H).MS (ESI) [M+H]+ =328.2.
Figure imgf000148_0003
Example 143 (E')-8-(4-(dimethylamino)but-2-enoyl)-3-(5-methylthiazol-2-yl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS190-183). Example 143 was synthesized following similar procedure for preparing example 28. White solid, 33% yield 'H NMR (400 MHz, Methanol-d)48 6.87 (s, 1H), 6.77 - 6.46 (m, 2H), 4.84 - 4.70 (m, 2H), 3.90 - 3.60 (m, 4H), 2.65 (s, 6H), 2.22 - 1.91 (m, 6H), 1.85 - 1.58 (m, 1H).MS (ESI) [M+H]+ =335.2.
Figure imgf000149_0001
Example 146 (£)-8-(4-(dimethylamino)but-2-enoyl)-3-methyl-3,8-diazabicyclo[3.2.1]octan- 2-one (XS197-4). Example 146 was synthesized following similar procedure for preparing example 28. White solid, 13% yield JH NMR (400 MHz, Methanol-d4) 8 6.88 - 6.62 (m, 2H), 4.82 - 4.49 (m, 2H), 3.84 (d, J= 7.4 Hz, 2H), 3.66 - 3.41 (m, 2H), 2.77 (d, J = 6.3 Hz, 6H), 2.72 (s, 3H), 2.28 - 1.70 (m, 4H).MS (ESI) [M+H]+ =252.2.
Figure imgf000150_0001
Example 147 (E)-8-(4-(dimethylamino)but-2-enoyl)-3-methyl-3,8-diazabicyclo[3.2.1]octan- 2-one (XS197-5). Example 147 was synthesized following similar procedure for preparing example 79. White solid, 25% yield 'H NMR (400 MHz, Methanol-d4) 8 6.73 - 6.56 (m, 1H), 6.54 - 6.35 (m, 1H), 4.71 - 4.51 (m, 2H), 4.42 (s, 1H), 4.12 - 3.71 (m, 4H), 3.70 - 3.38 (m, 4H),
2.83 - 2.73 (m, 6H), 2.73 - 2.63 (m, 1H), 2.25 - 2.08 (m, 1H), 2.05 - 1.79 (m, 5H), 1.78 - 1.46 (m, 2H).MS (ESI) [M+H]+ =349.2.
Figure imgf000150_0002
Example 148 (E)-l-(3-(l-acetylpiperidine-4-carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)-4- (dimethylamino)but-2-en-l-one (XS197-6). Example 148 was synthesized following similar procedure for preparing example 79. White solid, 23% yield 'H NMR (400 MHz, Methanol-t/4) 6 6.68 - 6.53 (m, 1H), 6.47 - 6.35 (m, 1H), 4.62 (d, J= 12.9 Hz, 1H), 4.38 (s, 2H), 4.11 - 3.63 (m, 6H), 3.63 - 3.51 (m, 3H), 2.74 (d, J= 6.9 Hz, 6H), 2.68 - 2.48 (m, 2H), 2.01 (d, J= 48.1 Hz, 3H), 1.73 - 1.28 (m, 5H).MS (ESI) [M+H]+ =363.2.
Figure imgf000150_0003
Example 149 (E)-8-(4-(dimethylamino)but-2-enoyl)-3-(5-methyl-lH-pyrrol-2-yl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS197-14). Example 149 was synthesized following similar procedure for preparing example 28. White solid, 34% yield 'H NMR (400 MHz, Methanol-d)45 7.55 - 6.56 (m, 2H), 6.49 (d, J= 15.2 Hz, 2H), 5.83 (s, 1H), 4.71 (s, 1H), 4.48 (s, 1H), 3.83 (d, J = 6.5 Hz, 4H), 2.84 - 2.68 (m, 8H), 2.15 (s, 3H), 1.59 (d, .1= 9.1 Hz, 1H), 1.32 - 1.16 (m, 1H).MS (ESI) [M+H]+ =317.2.
Figure imgf000151_0001
Example 150 (XS197-29). 8-(2-chloroacetyl)-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one
Example 150 was synthesized following similar procedure for preparing example 18. White solid, 33% yield 1H NMR (400 MHz, Methanol-d4) 8 4.30 - 4.06 (m, 2H), 3.75 - 3.52 (m, 2H), 3.20 - 3.00 (m, 2H), 2.80 (s, 3H), 2.34 - 1.76 (m, 4H).MS (ESI) [M+H]+ =217.1.
Figure imgf000151_0002
Example 151 (E)-l-(3-(cyclobutanecarbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)-4- (dimethylamino)but-2-en-l-one (XS197-38). Example 151 was synthesized following similar procedure for preparing example 79. White solid, 23% yield 1 H NMR (400 MHz, Methanol)-d 84 6.55 (dt, J= 14.7, 7.1 Hz, 1H), 6.41 (d, J= 15.2 Hz, 1H), 4.64 - 4.52 (m, 1H), 4.40 - 4.27 (m, 1H), 3.90 - 3.72 (m, 3H), 3.67 - 3.44 (m, 3H), 3.32 - 3.22 (m, 1H), 2.72 (s, 6H), 2.61 (t, J = 7.6 Hz, 1H), 2.14 - 1.96 (m, 4H), 1.91 - 1.81 (m, 1H), 1.73 - 1.57 (m, 1H), 1.46 (d, J= 9.2 Hz, 1H).MS (ESI) [M+H]+ =292.2.
Figure imgf000151_0003
Example 152 (E)-4-(dimethylamino)-l-(3-(5-isopropylthiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS197-49). Example 152 was synthesized following similar procedure for preparing example 79. White solid, 46% yield 'H NMR (400 MHz, Methanol-d4) 8 7.48 (d, J= 3.9 Hz, 1H), 6.91 (d, ,7 = 3.9 Hz, 1H), 6.85 - 6.72 (m, 1H), 6.62 (d, J= 15.3 Hz, 1H), 4.82 (s, 1H), 4.65 - 4.51 (m, 1H), 4.48 - 3.91 (m, 6H), 3.22 (p, J= 6.9 Hz, 1H), 2.98 - 2.78 (m, 7H), 1.74 (d, J = 9.2 Hz, 1H), 1.35 (d, J = 6.9 Hz, 6H).MS (ESI) [M+Hf =362.2.
Figure imgf000152_0001
Example 153 (£')-4-(dimethylamino)-l-(3-isobutyryl-3,6-diazabicyclo[3.1.1]heptan-6-yl)but- 2-en-l-one (XS197-50). Example 153 was synthesized following similar procedure for preparing example 79. White solid, 27% yield 'H NMR (400 MHz, Methanol-d4) 8 6.71 - 6.58 (m, 1H), 6.55 - 6.41 (m, 1H), 4.68 (s, 1H), 4.44 (s, 1H), 3.91 - 3.81 (m, 3H), 3.70 - 3.56 (m, 3H), 3.14 (q, 7.4 Hz, 1H), 2.81 (s, 6H), 2.74 - 2.68 (m, 1H), 1.59 - 1.53 (m, 1H), 1.08 - 0.96 (m, 6H).MS (ESI) [M+H]+ =280.2.
Figure imgf000152_0002
Example 154 (£)-3-cyclobutyl-8-(4-(dimethylamino)but-2-enoyl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS197-70). Example 154 was synthesized following similar procedure for preparing example 79. White solid, 21% yield 'H NMR (400 MHz, Methanol-d4) 6
6.83 - 6.55 (m, 2H), 4.81 - 4.43 (m, 3H), 3.88 - 3.70 (m, 2H), 3.52 - 3.37 (m, 1H), 3.15 - 3.07
(m, 1H), 2.75 (d, J= 6.0 Hz, 6H), 2.25 - 1.83 (m, 7H), 1.83 - 1.65 (m, 1H), 1.62 - 1.46 (m, 2H).MS
(ESI) [M+H]+ =292.2.
Figure imgf000152_0003
Example 155 (£)-l-(3-(5-(l-acetylpyrrolidin-3-yl)thiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS197-71). Example 155 was synthesized following similar procedure for preparing example 79. White solid, 26% yield ’H NMR (400 MHz, Methanol-d4) 8 7.41 (s, 1H), 6.98 - 6.88 (m, 1H), 6.74 - 6.61 (m, 1H), 6.55 - 6.42 (m, 1H), 4.72 (s, 1H), 4.47 (s, 1H), 4.11 - 3.79 (m, 6H), 3.71 - 3.52 (m, 3H), 3.40 - 3.29 (m, 1H), 3.14 (q, J = 7.4 Hz, 1H), 2.81 (s, 6H), 2.78 - 2.74 (m, 1H), 2.43 - 2.25 (m, 1H), 2.13 - 1.93 (m, 4H), 1.65 (d, J = 9.2 Hz, 1H).MS (ESI) [M+H]+ =431.2.
Figure imgf000153_0001
Example 156 (£)-l-(3-(5-(l-acetylpiperidin-4-yl)thiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS197-72). Example 156 was synthesized following similar procedure for preparing example 79. White solid, 25% yield 1 HMR400 MHz, Methanol-d4) 5 7.37 (d, J= 3.9 Hz, 1H), 6.83 (d, J= 4.0 Hz, 1H), 6.70 - 6.58 (m, 1H), 6.48 (d, J = 15.3 Hz, 1H), 4.69 (s, 1H), 4.47 (t, J = 14.4 Hz, 2H), 4.35 - 4.07 (m, 1H), 4.02 - 3.73 (m, 6H), 3.18 - 3.00 (m, 2H), 2.78 (s, 6H), 2.73 - 2.59 (m, 2H), 2.05 - 1.90 (m, 5H), 1.64 - 1.38 (m, 3H).MS (ESI) [M+H] 1 =445.2.
Figure imgf000153_0002
Example 157 (£)-4-(dimethylamino)-l-(3-(5-(l-methylpyrrolidin-3-yl)thiophene-2- carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS197-73). Example 157 was synthesized following similar procedure for preparing example 79. White solid, 20% yield 'H NMR (400 MHz, Methanol-6/4) 5 7.46 (d, J= 4.3 Hz, 1H), 7.02 (s, 1H), 6.77 - 6.64 (m, 1H), 6.53 (d, J= 15.0 Hz, 1H), 4.74 (s, 1H), 4.51 (d, J= 19.5 Hz, 1H), 4.39 - 4.12 (m, 1H), 4.09 - 3.95 (m, 2H), 3.93 - 3.72 (m, 5H), 3.68 - 3.49 (m, 1H), 3.21 - 3.08 (m, 1H), 2.95 (s, 3H), 2.83 (s, 6H), 2.81
- 2.70 (m, 2H), 2.67 - 2.45 (m, 1H), 2.35 - 2.07 (m, 1H), 1.67 (d, J = 9.2 Hz, 1H).MS (ESI) [M+H]+ =403.2.
Figure imgf000154_0001
Example 158 (£)-4-(dimethylamino)-l-(3-(5-(l-methylpiperidin-4-yl)thiophene-2-carbonyl)- 3,6-diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS197-74). Example 158 was synthesized following similar procedure for preparing example 79. White solid, 17% yield 'H NMR (400 MHz, Methanol-d4) 8 7.43 (d, J= 4.1 Hz, 1H), 6.91 (d, J= 3.9 Hz, 1H), 6.76 - 6.62 (m, 1H), 6.51
(d, J= 15.1 Hz, 1H), 4.72 (s, 1H), 4.49 (d, J= 18.1 Hz, 1H), 4.32 - 4.07 (m, 1H), 4.01 (d, J= 11.4
Hz, 1H), 3.88 - 3.82 (m, 3H), 3.58 - 3.47 (m, 2H), 3.20 - 3.02 (m, 3H), 2.85 - 2.80 (m, 8H), 2.76
- 2.70 (m, 3H), 2.21 (d, J= 14.4 Hz, 2H), 1.96 - 1.78 (m, 2H), 1.65 (d, .7 = 9.2 Hz, 1H).MS (ESI) [M+H] 1 =417.2.
Figure imgf000154_0002
Example 159 (£)-4-(dimethylamino)-l-(3-(l-methylazetidine-3-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS197-85). Example 159 was synthesized following similar procedure for preparing example 79. White solid, 19% yield 'H NMR (400
MHz, Methanol-d4) δ 6.70 - 6.56 (m, 1H), 6.48 - 6.36 (m, 1H), 4.70 - 4.61 (m, 1H), 4.47 - 4.32
(m, 3H), 4.11 - 3.98 (m, 2H), 3.94 - 3.79 (m, 4H), 3.75 - 3.65 (m, 2H), 2.86 - 2.79 (m, 4H), 2.79
- 2.75 (m, 6H), 2.73 - 2.64 (m, 1H), 1.57 (d, J= 9.2 Hz, 1H).MS (ESI) [M+H]+ =307.2.
Figure imgf000154_0003
Example 160 (E')-4-(dimethylamino)-l-(3-(5-(3-methoxyprop-l-yn-l-yl)thiophene-2- carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS197-93). Example 160 was synthesized following similar procedure for preparing example 79. White solid, 44% yield !H NMR (400 MHz, Methanol-d4) 8 7.39 (d, J = 4.0 Hz, 1H), 7.12 (d, J = 4.1 Hz, 1H), 6.69 - 6.58 (m, 1H), 6.53 - 6.39 (m, 1H), 4.69 (s, 1H), 4.46 (d, J= 16.4 Hz, 1H), 4.34 - 4.18 (m, 3H), 4.10 - 3.92 (m, 2H), 3.87 - 3.79 (m, 3H), 3.29 (s, 3H), 2.84 - 2.65 (m, 7H), 1.62 (d, J= 9.3 Hz, 1H).MS (ESI) [M+H]+ =388.2.
Figure imgf000155_0001
Example 161 (£)-4-(dimethylamino)-l-(3-(5-(3-hydroxyprop-l-yn-l-yl)thiophene-2- carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS197-94). Example 161 was synthesized following similar procedure for preparing example 79. White solid, 28% yield 'H NMR (400 MHz, Methanol-d4) δ 7.24 (d, J = 3.9 Hz, 1H), 6.94 (d, J = 4.0 Hz, 1H), 6.58 - 6.44 (m, 1H), 6.37 - 6.23 (m, 1H), 4.54 (s, 1H), 4.31 (d, J= 14.2 Hz, 1H), 4.20 - 4.07 (m, 2H), 3.97 - 3.77 (m, 2H), 3.77 - 3.58 (m, 4H), 2.67 - 2.52 (m, 7H), 1.47 (d, J= 9.2 Hz, 1H).MS (ESI) [M+Hf =374.1.
Figure imgf000155_0002
Example 162 (£)-3-(5-(6-(4-(dimethylamino)but-2-enoyl)-3,6-diazabicyclo[3.1.1]heptane-3- carbonyl)thiophen-2-yl)prop-2-yn-l-yl acetate (XS197-96). Example 162 was synthesized following similar procedure for preparing example 79. White solid, 29% yield 1 H NMR (400 MHz, Methanol-^) 8 7.45 (d, J= 4.7 Hz, 1H), 7.19 (d, J= 3.9 Hz, 1H), 6.75 - 6.64 (m, 1H), 6.51 (t, J = 14.1 Hz, 1H), 4.92 - 4.88 (m, 1H), 4.73 (s, 1H), 4.51 (d, J= 17.4 Hz, 1H), 4.33 (d, J= 11.9 Hz, 1H), 4.19 - 3.98 (m, 2H), 3.96 - 3.80 (m, 4H), 2.90 - 2.71 (tn, 7H), 2.03 (s, 3H), 1.67 (d, J= 9.2 Hz, 1H).MS (ESI) [M+H]+ =416.2.
Figure imgf000156_0001
Example 163 (E)-l-(3-(2-acetyl-2-azaspiro[3.3]heptane-6-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS197-133). Example 163 was synthesized following similar procedure for preparing example 79. White solid, 22% yield ’H NMR (400 MHz, Methanol-d4) δ 6.73 - 6.61 (m, 1H), 6.48 (d, J= 15.3 Hz, 1H), 4.67 (s, 1H), 4.46 (d, J= 11.9 Hz, 1H), 4. 18 (s, 1H), 4.05 (d, J= 4.1 Hz, 1H), 3.99 - 3.93 (m, 2H), 3.87 (d, J - 1.2 Hz, 2H), 3.80 (d, J= 8.3 Hz, 1H), 3.76 - 3.57 (m, 3H), 2.83 (d, J = 3.3 Hz, 6H), 2.78 - 2.68 (m, 2H), 2.45 - 2.27 (m, 4H), 1.77 (d, J= 6.7 Hz, 3H), 1.61 - 1.54 (m, 1H).MS (ESI) [M+H]+ =375.2.
Figure imgf000156_0002
Example 164 (E)-3-(5-(l-acetylpiperidin-4-yl)thiophen-2-yl)-8-(4-(dimethylamino)but-2- enoyl)-3,8-diazabicyclo[3.2.1]octan-2-one (XS197-145). Example 164 was synthesized following similar procedure for preparing example 28. White solid, 25% yield 1 H NMR (400 MHz, Methanol-dr) 6 6.95 - 6.65 (m, 2H), 6.61 (d, J = 3.9 Hz, 1H), 6.57 - 6.46 (m, 1H), 5.01 (d, J = 38.0 Hz, 2H), 4.51 (d, J = 13.2 Hz, 1H), 4.06 - 3.83 (m, 4H), 3.64 (dd, J = 20.1, 11.7 Hz, 1H), 3.23 - 3.10 (m, 1H), 2.97 (t, J= 13.4 Hz, 1H), 2.84 (d, J= 5.2 Hz, 6H), 2.74 - 2.61 (m, 1H), 2.40 - 2.10 (m, 3H), 2.06 (s, 3H), 2.03 - 1.85 (m, 3H), 1.70 - 1.41 (m, 2H).MS (ESI) [M+H]+ =445.2.
Figure imgf000157_0002
Example 166 8-(4-(dimethylamino)but-2-ynoyl)-3-(5-methylthiophen-2-yl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS197-185). Example 166 was synthesized following similar procedure for preparing example 28. White solid, 39% yield ’H NMR (400 MHz, Methanol-d)4 δ 6.59 (d, J= 2.0 Hz, 2H), 5.12 - 4.91 (m, 2H), 4.41 - 4.34 (m, 2H), 4.14 - 4.00 (m, 1H), 3.81 - 3.65 (m, 1H), 3.11 - 3.01 (m, 6H), 2.41 (s, 3H), 2.40 - 2.32 (m, 1H), 2.31 - 2.16 (m, 2H), 2.12 - 2.00 (m, 1H).MS (ESI) [M+H]+ =332.1.
Figure imgf000157_0001
Example 167 (£)-4-(dimethylamino)-l-(isoindolin-2-yl)but-2-en-l-one (XS197-186).
Example 167 was synthesized following similar procedure for preparing example 79. White solid , 60% yield rH NMR (400 MHz, Methanol-d4) 8 7.33 - 7.20 (m, 4H), 6.86 - 6.67 (m, 2H), 4.94 (s, 2H), 4.75 (s, 2H), 3.94 (d, J= 6.2 Hz, 2H), 2.87 (s, 6H).MS (ESI) [M+H]+ =231.2.
Figure imgf000158_0001
Example 168 (£)-l-(4-(dimethylamino)but-2-enoyl)-3-methyl-W-(5-methylthiophen-2- yl)azetidine-3-carboxamide (XS209-21). Example 168 was synthesized following similar procedure for preparing example 53. White solid, 43% yield 1H NMR (400 MHz, Methanol)-d 84 6.77 - 6.65 (m, 1H), 6.55 (d, J= 3.5 Hz, 1H), 6.52 - 6.42 (m, 2H), 4.65 (d, J= 8.9 Hz, 1H), 4.35 (d, J= 10.6 Hz, 1H), 4.12 (d, J= 9.0 Hz, 1H), 3.99 - 3.85 (m, 3H), 2.89 (s, 6H), 2.37 (s, 3H), 1.65 (s, 3H).MS (ESI) [M+H]+ =322.2.
Figure imgf000158_0002
Example 169 (E')-l-(4-(dimethylamino)but-2-enoyl)-3-hydroxy-AN-(5-methylthiophen-2- yl)azetidine-3-carboxamide (XS209-22). Example 169 was synthesized following similar procedure for preparing example 53. White solid, 29% yield 'H NMR (400 MHz, Methanol-d)48 6.84 - 6.71 (m, 1H), 6.68 (d, J = 3.6 Hz, 1H), 6.55 (d, J= 15.2 Hz, 2H), 4.71 (d, J= 9.4 Hz, 1H), 4.46 (d, J = 10.9 Hz, 1H), 4.29 (d, J= 9.4 Hz, 1H), 4.05 (d, J= 10.9 Hz, 1H), 3.97 (d, J= 7.1 Hz, 2H), 2.92 (s, 6H), 2.41 (s, 3H).MS (ESI) [M+H]+ =324.1.
Figure imgf000158_0003
Example 170 (/:)-l-(4-(dimetliylamiiio)biit-2-enioyl)-3-methyl-A-(5-methyloxazol-2- yl)azetidine-3-carboxamide (XS209-23). Example 170 was synthesized following similar procedure for preparing example 53. White solid, 38% yield ’H NMR (400 MHz, Methanol-d4) 8 6.81 (s, 1H), 6.79 - 6.66 (m, 1H), 6.50 (d, J= 15.2 Hz, 1H), 4.71 (d, J= 9.0 Hz, 1H), 4.39 (d, J = 10.6 Hz, 1H), 4.14 (d, J = 9.1 Hz, 1H), 4.00 - 3.89 (m, 3H), 2.91 (s, 6H), 2.32 (s, 3H), 1.68 (s, 3H).MS (ESI) [M+Hf =307.2.
Figure imgf000159_0001
Example 174 (E)-4-(dimethylamino)-2V-(l-(5-methylthiophen-2-yl)-2-oxopiperidin-4-yl)but- 2-enamide (XS209-27). Example 174 was synthesized following similar procedure for preparing example 28. White solid, 27% yield 'H NMR (400 MHz, Methanol-d4) 8 6.81 - 6.66 (m, 1H), 6.63 - 6.51 (m, 2H), 6.37 (d, J = 15.3 Hz, 1H), 4.37 - 4.24 (m, 1H), 3.96 - 3.76 (m, 4H), 2.89 (s, 7H), 2.58 - 2.46 (m, 1H), 2.38 (s, 3H), 2.32 - 2.21 (m, 1H), 2.11 - 2.00 (m, 1H).MS (ESI) [M+Hf =322.2.
Figure imgf000160_0001
Example 175 (£)-4-(dimethylamino)-l-(3-propionyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)but- 2-en-l-one (XS209-28). Example 175 was synthesized following similar procedure for preparing example 79. White solid, 21% yield *HNMR (400 MHz, Methanol-)d 846.76 - 6.62 (m, 1H), 6.53 (d, J= 15.2 Hz, 1H), 4.76 - 4.69 (m, 1H), 4.54 - 4.43 (m, 1H), 4.10 - 3.76 (m, 4H), 3.72 (d, J = 5.4 Hz, 1H), 3.66 (d, J= 6.9 Hz, 1H), 2.86 (s, 6H), 2.80 - 2.70 (m, 1H), 2.46 - 2.23 (m, 2H), 1.61 (d, J= 9.1 Hz, 1H), 1.06 (t, J= 7.4 Hz, 3H).MS (ESI) [M+H]+ =266.2.
Figure imgf000160_0002
Example 176 (£)-3-(cyclohex-l-en-l-yl)-8-(4-(dimethylamino)but-2-enoyl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS209-30). Example 176 was synthesized following similar procedure for preparing example 28. White solid, 11% yield 'H NMR (400 MHz, Methanol-d4) 8 6.95 - 6.71 (m, 2H), 5.64 (s, 1H), 4.97 - 4.88 (m, 1H), 4.82 - 4.64 (m, 1H), 4.00 - 3.90 (m, 2H), 3.80 - 3.62 (m, 1H), 3.27 - 3.06 (m, 1H), 2.90 (s, 6H), 2.42 - 2.04 (m, 6H), 2.03 - 1.92 (m, 1H), 1.75 - 1.64 (m, 2H), 1.64 - 1.52 (m, 2H), 1.52 - 1.32 (m, 1H).MS (ESI) [M+H]+ =318.2.
Figure imgf000160_0003
Example 177 (E)-l-(4-(dimethylamino)but-2-enoyl)-3-methyl-N-(5-methylthiazol-2- yl)azetidine-3-carboxamide (XS209-38). Example 177 was synthesized following similar procedure for preparing example 53. White solid, 44% yield 1 H NMR (400 MHz, M Methanol)-d 84 7.15 (s, 1H), 6.72 - 6.56 (m, 1H), 6.43 (d, J= 15.3 Hz, 1H), 4.65 (d, J= 9.1 Hz, 1H), 4.35 (d, J = 10.7 Hz, 1H), 4.12 (d, J= 9.2 Hz, 1H), 3.87 (d, J= 8.2 Hz, 3H), 2.83 (s, 6H), 2.35 (s, 3H), 1.62 (s, 3H).MS (ESI) [M+H]- =323.1.
Figure imgf000161_0001
Example 178 (E)-l-(4-(dimethylamino)but-2-enoyl)-3-hydroxy-JV-(5-methylthiazol-2- yl)azetidine-3-carboxamide (XS209-39). Example 178 was synthesized following similar procedure for preparing example 53. White solid, 27% yield 1 H NMR (400 MHz, Methanol)-d 84 7.13 (s, 1H), 6.81 - 6.66 (m, 1H), 6.49 (d, J = 15.3 Hz, 1H), 4.68 (d, J= 9.5 Hz, 1H), 4.43 (d, J = 11.0 Hz, 1H), 4.27 (d, J = 9.5 Hz, 1H), 4.03 (d, J= 11.0 Hz, 1H), 3.91 (d, J= 7.1 Hz, 2H), 2.87 (s, 6H), 2.37 (s, 3H).MS (ESI) [M+H]+ =325.1.
Figure imgf000161_0002
Example 179 (E)-l-(3-butyryl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2- en-l-one (XS209-40). Example 179 was synthesized following similar procedure for preparing example 79. White solid, 19% yield 1HNMR (400 MHz, Methanol-)d 846.84 - 6.69 (m, 1H), 6.57 (d, J = 15.3 Hz, 1H), 4.77 (s, 1H), 4.54 (s, 1H), 4.13 (d, J = 11.5 Hz, 1H), 4.02 - 3.87 (m, 3H), 3.81 - 3.69 (m, 2H), 2.91 (d, J= 4.4 Hz, 6H), 2.82 (q, J= 6.6 Hz, 1H), 2.47 - 2.23 (m, 2H), 1.72 - 1.58 (m, 3H), 0.98 (t, J= 13 Hz, 3H).MS (ESI) [M+H]+ =280.2.
Figure imgf000162_0001
Example 180 (£)-l-(3-(2-acetyl-2-azaspiro[4.5]decane-8-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS209-53). Example 180 was synthesized following similar procedure for preparing example 79. White solid, 18% yield 'H NMR (400 MHz, Methanol-d4) 86.67 - 6.54 (m, 1H), 6.47 (d, J= 15.2 Hz, 1H), 4.66 (s, 1H), 4.42 (s, 1H), 4.12 - 3.91 (m, 1H), 3.87 - 3.77 (m, 3H), 3.68 - 3.51 (m, 4H), 3.51 - 3.40 (m, 1H), 3.15 (d, J = 5.7 Hz, 1H), 2.78 (s, 6H), 2.74 (s, 3H), 2.71 - 2.65 (m, 1H), 2.59 - 2.46 (m, 1H), 2.13 - 2.02 (m, 3H), 1.90 - 1.81 (m, 1H), 1.63 - 1.50 (m, 5H), 1.46 - 1.37 (m, 2H).MS (ESI) [M+H]+
=417.3.
Figure imgf000162_0002
Example 181 (E)-l-(3-(2-acetyl-2-azaspiro[3.5]nonane-7-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS209-54). Example 181 was synthesized following similar procedure for preparing example 79. White solid, 21% yield !H NMR (400 MHz, Methanol-d4) δ 6.71 - 6.60 (m, 1H), 6.51 (d, J= 15.2 Hz, 1H), 4.75 - 4.64 (m, 1H), 4.49 - 4.41 (m, 1H), 4.16 - 3.92 (m, 2H), 3.90 - 3.81 (m, 4H), 3.70 (d, J = 9.3 Hz, 2H), 3.63 - 3.55 (m, 2H), 2.82 (s, 6H), 2.77 - 2.69 (m, 1H), 2.54 (t, J = 11.2 Hz, 1H), 1.98 - 1.83 (m, 5H), 1.71 (d, J= 13.3 Hz, 1H), 1.65 - 1.49 (m, 4H), 1.47 - 1.34 (m, 2H).MS (ESI) [M+H]+ =403.3.
Figure imgf000162_0003
Example 182 (/f)-V-(2-(4-(dimethylamino)biit-2-eiioyl)isoindolin-5-yl)acetamide (XS209-55).
Example 182 was synthesized following similar procedure for preparing example 79. White solid, 39% yield 'H NMR (400 MHz, Methanol-d4) 8 7.55 (d, J = 21.6 Hz, 1H), 7.33 - 7.21 (m, 1H), 7.15 (t, J= 8.1 Hz, 1H), 6.81 - 6.61 (m, 2H), 4.85 (s, 2H), 4.66 (d, J= 7.2 Hz, 2H), 3.89 (d, J = 6.5 Hz, 2H), 2.82 (s, 6H), 2.01 (s, 3H).MS (ESI) [M+H]+ =288.2.
Figure imgf000163_0001
Example 183 (£')-4-(dimethylamino)-l-(5-(methylamino)isoindolin-2-yl)but-2-en-l-one (XS209-57). Example 183 was synthesized following similar procedure for preparing example 79. White solid, 55% yield JH NMR (400 MHz, Methanol-d)48 7.49 (t, J = 9.0 Hz, 1H), 7.42 (d, J = 7.1 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 6.84 (d, J= 15.2 Hz, 1H), 6.80 - 6.66 (m, 1H), 5.04 (d, J = 10.6 Hz, 2H), 4.83 (d, J = 8.7 Hz, 2H), 3.95 (d, J= 6.8 Hz, 2H), 3.03 (s, 3H), 2.87 (s, 6H).MS (ESI) [M+H]+ =260.2.
Figure imgf000163_0002
Example 184 (E)-4-(dimethylamino)-l-(5-(methylamino)isoindolin-2-yl)but-2-en-l-one (XS209-60). Example 184 was synthesized following similar procedure for preparing example 79. White solid, 36% yield 'H NMR (400 MHz, Methanol-d4) 8 6.75 - 6.62 (m, 1H), 6.60 - 6.47 (m, 1H), 6.34 (s, 1H), 4.74 (d, J= 35.5 Hz, 1H), 4.50 (d, J= 33.5 Hz, 1H), 4.29 - 3.94 (m, 3H), 3.91 - 3.82 (m, 3H), 2.87 - 2.78 (m, 7H), 2.42 (d, J= 3.3 Hz, 3H), 1.68 (d, J= 7.1 Hz, 1H).MS (ESI)
Figure imgf000163_0003
Example 185 (E)-l-(3-(6-acetyl-6-azaspiro[3.4]octane-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS209-62). Example 185 was synthesized following similar procedure for preparing example 79. White solid, 19% yield 1H NMR (400 MHz, Methanol-d4) 8 6.72 - 6.58 (m, 1H), 6.47 (d, J= 15.2 Hz, 1H), 4.77 - 4.60 (m, 1H), 4.58 - 4.38 (m, 1H), 4.03 - 3.67 (m, 5H), 3.53 - 3.24 (m, 5H), 2.80 (s, 6H), 2.75 - 2.65 (m, 1H), 2.27 - 2.05 (m, 4H), 2.00 - 1.90 (m, 3H), 1.89 - 1.82 (m, 1H), 1.80 - 1.74 (m, 1H), 1.55 (d, J= 8.8 Hz, 1H), 1.46 - 1.40 (m, 1H).MS (ESI) [M+H]+ =389.2.
Figure imgf000164_0001
Example 186 (E)-4-(dimethylamino)-l-(3-(5-methylthiazole-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS209-65). Example 186 was synthesized following similar procedure for preparing example 79. White solid, 44% yield 1 H NMR (400 MHz, Methanol-^) 8 7.46 (d, J = 9.5 Hz, 1H), 6.65 - 6.50 (m, 1H), 6.49 - 6.34 (m, 1H), 4.65 (d, J = 14.6 Hz, 1H), 4.50 - 4.24 (m, 3H), 3.99 - 3.71 (m, 4H), 2.75 - 2.60 (m, 7H), 2.35 (s, 3H), 1.57 (d, J= 9.2 Hz, IH).MS (ESI) [M+H] 1 =335.1.
Figure imgf000164_0002
Example 187 8-(4-(dimethylamino)-4-methylpent-2-ynoyl)-3-(5-methylthiophen-2-yl)-3,8- diazabicyclo[3.2.1]octan-2-one (XS209-74). Example 187 was synthesized following similar procedure for preparing example 28. White solid, 29% yield 'H NMR (400 MHz, Methanol-d4) 8 6.49 (s, 2H), 4.99 (t, J= 5.5 Hz, 1H), 4.78 (d, J = 6.5 Hz, 1H), 4.04 - 3.90 (m, 1H), 3.73 - 3.54 (m, 1H), 2.97 - 2.88 (m, 6H), 2.40 - 2.20 (m, 5H), 2.18 - 2.08 (m, 1H), 2.04 - 1.89 (m, 1H), 1.77 - 1.64 (m, 6H).MS (ESI) [M+Hf =360.2.
Figure imgf000165_0001
Example 188 4-(dimethylamino)-4-methyl-l-(3-(5-methylthiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)pent-2-yn-l-one (XS209-75). Example 188 was synthesized following similar procedure for preparing example 79. White solid, 34% yield !H NMR (400 MHz, Methanol-^) 8 7.43 - 7.28 (m, 1H), 6.75 (t, J= 4.5 Hz, 1H), 4.71 - 4.47 (m, 2H), 4.40 - 4.20 (m, 1H), 4.20 - 4.06 (m, 1H), 4.06 - 3.78 (m, 2H), 3.13 - 3.00 (m, 1H), 2.92 - 2.85 (m, 8H), 2.74 (q, J= 7.1 Hz, 1H), 2.41 (s, 3H), 1.73 - 1.55 (m, 4H).MS (ESI) [M+H]+ =360.1.
Figure imgf000165_0002
Example 189 (£)-4-(dimethylamino)-l-(3-(5-methyloxazole-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS209-92). Example 189 was synthesized following similar procedure for preparing example 79. White solid, 29% yield 'H NMR (400 MHz, Methanol-d4) 8 6.89 (s, 1H), 6.66 - 6.54 (m, 1H), 6.52 - 6.38 (m, 1H), 4.73 - 4.63 (m, 1H), 4.48 - 4.22 (m, 3H), 3.94 - 3.72 (m, 4H), 2.77 - 2.66 (m, 7H), 2.26 (s, 3H), 1.59 (d, J= 9.2 Hz,
Figure imgf000165_0003
Example 190 l-(4-(dimethylamino)but-2-ynoyl)-3-hydroxy-/V-(5-methylthiophen-2- yl)azetidine-3-carboxamide (XS209-99). Example 190 was synthesized following similar procedure for preparing example 53. White solid, 29% yield 1 H NMR (400 MHz, Methanol-d4) 8 6.55 (d, J= 3.4 Hz, 1H), 6.41 (d, J= 4.2 Hz, 1H), 4.55 (d, J= 9.7 Hz, 1H), 4.31 (d, J= 11.0 Hz, 1H), 4.23 (s, 2H), 4.13 (d, J= 9.7 Hz, 1H), 3.90 (d, J= 10.9 Hz, 1H), 2.88 (s, 6H), 2.28 (s, 3H).
MS (ESI) [M+H]+ =322.1.
Figure imgf000166_0001
Example 193 (E)-8-(4-(dimethylamino)but-2-enoyl)-N-(5-methylthiophen-2-yl)-8- azabicyclo[3.2.1]octane-3-carboxamide (XS209-122). Example 193 was synthesized following similar procedure for preparing example 53. White solid 1 H NMR (400 MHz, Methanol-d4) 8 6.79 (d, J= 15.2 Hz, 1H), 6.72 - 6.60 (m, 1H), 6.38 (s, 2H), 4.62 (d, J= 6.6 Hz, 1H), 4.52 - 4.40 (m, 1H), 3.86 (d, J= 7.0 Hz, 2H), 3.03 - 2.90 (m, 1H), 2.82 (s, 6H), 2.26 (s, 3H), 2.13 - 1.99 (m, 1H), 1.98 - 1.59 (m, 7H). MS (ESI) [M+H]+ =362.2.
Figure imgf000167_0001
Example 194 (£)-JV-(8-(4-(dimethylamino)but-2-enoyl)-8-azabicyclo[3.2.1]octan-3-yl)-5- methylthiophene-2-carboxamide (XS209-139). Example 194 was synthesized following similar procedure for preparing example 79. White solid, 45% yield 'H NMR (400 MHz, Methanol-6/4) 6 7.34 (d, J= 3.2 Hz, 1H), 6.80 - 6.51 (m, 3H), 4.51 (s, 1H), 4.37 (s, 1H), 3.89 (s, 1H), 3.81 (d, J = 6.8 Hz, 2H), 2.76 (s, 6H), 2.35 (s, 3H), 2.15 - 2.05 (m, 1H), 2.05 - 1.91 (m, 5H), 1.90 - 1.76 (m,
Figure imgf000167_0002
Example 196 (E)-W-(4-(4-(dimethylamino)but-2-enamido)phenyl)-5-methylthiophene-2- carboxamide (XS209-165). Example 196 was synthesized following similar procedure for preparing example 79. White solid, 33% yield 1H NMR (400 MHz, Methanol-d4) 8 7.71 (d, J = 3.5 Hz, 1H), 7.65 (s, 4H), 6.93 - 6.76 (m, 2H), 6.56 (d, J= 15.2 Hz, 1H), 4.00 (d, J = 7.2 Hz, 2H), 2.94 (s, 6H), 2.55 (s, 3H). MS (ESI) [M+H]+ =344.1.
Figure imgf000168_0001
Example 197 l-(3-butyryl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-yn- 1-one (XS209-166). Example 197 was synthesized following similar procedure for preparing example 79. White solid, 18% yield 'H NMR (400 MHz, Methanol-d)48 4.62 (s, 1H), 4.59 - 4.47 (m, 1H), 4.27 (s, 2H), 4.04 - 3.93 (m, 1H), 3.87 - 3.77 (m, 1H), 3.77 - 3.62 (m, 2H), 2.94 - 2.90 (m, 6H), 2.79 - 2.64 (m, 1H), 2.40 - 2.20 (m, 2H), 1.64 - 1.52 (m, 3H), 0.97 - 0.88 (m, 3H).MS (ESI) [M+H]+ =278.2.
Figure imgf000168_0002
Example 198 l-(3-acetyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)-4- methylpent-2-yn-l-one (XS209-167). Example 198 was synthesized following similar procedure for preparing example 79. White solid, 19% yield 'H NMR (400 MHz, Methanol-d)48 4.66 (d, J = 6.2 Hz, 2H), 4.10 (d, J= 12.0 Hz, 1H), 3.94 (d, J= 13.6 Hz, 1H), 3.86 (d, J= 12. 1 Hz, 1H), 3.75 (d, J= 13.6 Hz, 1H), 3.15 - 3.04 (m, 1H), 2.93 (s, 6H), 2.92 (s, 6H), 2.12 (s, 3H), 1.81 (d, J= 9.0 Hz, 1H). MS (ESI) [M+H]+ =278.2.
Figure imgf000168_0003
Example 199 1 -(3-butyryl-3,6-diazabicyclo[3.1.1 ]heptan-6-yl)-4-(dimethylamino)-4- methylpent-2-yn-l-one (XS209-168). Example 199 was synthesized following similar procedure for preparing example 79. White solid, 20% yield 'H NMR (400 MHz, Methanol-d)48 4.64 (d, J = 5.8 Hz, 2H), 4.06 (d, J= 11.9 Hz, 1H), 3.91 (d, J= 13.5 Hz, 1H), 3.84 (d, J= 12. 1 Hz, 1H), 3.72 (d, J= 13.4 Hz, 1H), 3.07 (q, .7= 7.4 Hz, 1H), 2.92 - 2.86 (m, 12H), 2.40 - 2.29 (m, 2H), 1.76 (d, J= 7.9 Hz, 1H), 1.63 - 1.56 (m, 2H), 0.92 (t, J= 7.3 Hz, 3H). MS (ESI) [M+H]+ =306.2.
Figure imgf000169_0001
Example 200: l-(3-butyryl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)-4- methylpent-2-yn-l-one (XS209-174). Example 200 was synthesized following similar procedure for preparing example 53. White solid, 45% yield 'H NMR (400 MHz, Methanol-d)45 7.66 (d, J
= 7.8 Hz, 1H), 7.55 (d, J= 7.8 Hz, 1H), 7.21 (t, J= 7.5 Hz, 1H), 7.14 (t, J = 7.4 Hz, 1H), 7.06 (s,
1H), 6.74 - 6.61 (m, 1H), 6.47 (d, J= 15.2 Hz, 1H), 4.66 (d, J= 9.5 Hz, 1H), 4.40 (d, J= 11.0 Hz, 1H), 4.23 (d, J= 9.5 Hz, 1H), 3.98 (d, J= 11.0 Hz, 1H), 3.87 (d, J = 7.1 Hz, 2H), 2.82 (s, 6H). MS
Figure imgf000169_0002
Example 201: N-(benzo[b]thiophen-2-yl)-l-(4-(dimethylamino)but-2-ynoyl)-3- methylazetidine-3-carboxamide (XS209-175). Example 201 was synthesized following similar procedure for preparing example 53. White solid, 44% yield *HNMR (400 MHz, Methanol-d4) δ 7.67 (d, J= 7.9 Hz, 1H), 7.55 (d, J= 7.8 Hz, 1H), 7.22 (t, J= 7.4 Hz, 1H), 7.15 (t, J = 7.4 Hz, 1H), 6.94 (s, 1H), 4.61 (d, J= 9.2 Hz, 1H), 4.32 (d, J= 10.6 Hz, 1H), 4.27 (s, 2H), 4.05 (d, J= 9.2 Hz, 1H), 3.87 (d, J= 10.6 Hz, 1H), 2.93 (s, 6H), 1.63 (s, 3H).MS (ESI) [M+Hf =345.2.
Figure imgf000169_0003
Example 202: (E)-l-(4-(dimethylamino)but-2-enoyl)-3-hydroxy-iV-(4, 5,6,7- tetrahydrobenzo[Z>]thiophen-2-yl)azetidine-3-carboxamide (XS209-176). Example 202 was synthesized following similar procedure for preparing example 53. White solid, 39% yield 'H NMR (400 MHz, Methanol-d4) 8 6.70 - 6.55 (m, 1H), 6.49 - 6.36 (m, 2H), 4.58 (d, J= 9.4 Hz, 1H), 4.32 (d, J= 10.9 Hz, 1H), 4.16 (d, J = 9.4 Hz, 1H), 3.91 (d, J= 10.9 Hz, 1H), 3.84 (d, J= 1A Hz, 2H), 2.79 (s, 6H), 2.54 (t, J= 62 Hz, 2H), 2.40 (t, J= 5.9 Hz, 2H), 1.79 - 1.59 (m, 4H). MS (ESI) [M+H]+ =364.2.
Figure imgf000170_0001
Example 203: (E)-l-(3-(5-(4-acetylpiperazin-l-yl)thiophene-2-carbonyl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)-4-(dimethylamino)but-2-en-l-one (XS209-184). Example 203 was synthesized following similar procedure for preparing example 79. White solid, 28% yield
NMR (400 MHz, Methanol-d4) δ 7.46 (s, 1H), 6.84 - 6.69 (m, 1H), 6.60 (d, J= 15.3 Hz, 1H), 6.25 (d, J = 4.1 Hz, 1H), 4.82 (s, 1H), 4.60 (s, 1H), 4.02 - 3.87 (m, 3H), 3.79 - 3.63 (m, 4H), 3.38 - 3.30 (m, 7H), 2.91 (s, 6H), 2.87 - 2.79 (m, 1H), 2.17 (s, 3H), 1.74 (d, J= 9.1 Hz, 1H). MS (ESI) [M+H] 1 =446.2
Figure imgf000170_0002
Example 204: (E)-4-(dimethylamino)-l-(3-(5-(4-methylpiperazin-l-yl)thiophene-2- carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)but-2-en-l-one (XS209-185). Example 204 was synthesized following similar procedure for preparing example 79. White solid, 23% yield 'H NMR (400 MHz, Methanol-d4) 8 7.32 (s, 1H), 6.70 - 6.58 (m, 1H), 6.47 (d, J= 15.3 Hz, 1H), 6.20 (d, J= 4.1 Hz, 1H), 4.69 (s, 1H), 4.46 (s, 1H), 4.37 - 3.89 (m, 3H), 3.83 (d, J= 7.0 Hz, 3H), 3.73 (d, J = 9.8 Hz, 2H), 3.57 - 3.39 (m, 2H), 3.19 - 3.07 (m, 4H), 2.85 (s, 3H), 2.77 (s, 6H), 2.74 - 2.65 (m, 1H), 1.60 (d, J= 9.1 Hz, 1H).MS (ESI) [M+H]+ =418.2
Figure imgf000171_0001
Example 205: (E)-JV-(4-(4-(dimethylamino)but-2-enamido)cyclohexyl)-5-methylthiophene- 2-carboxamide (XS224-6). Example 205 was synthesized following similar procedure for preparing example 79. White solid, 25% yield 1 H NMR (400 MHz, Methano)l 8-d 74.53 - 7.31 (m, 1H), 6.73 (s, 1H), 6.70 - 6.53 (m, 1H), 6.29 (d, J= 15.3 Hz, 1H), 3.87 (d, J= 6.7 Hz, 2H), 3.84 - 3.62 (m, 2H), 2.84 (s, 6H), 2.44 (s, 3H), 1.94 (d, J= 9.3 Hz, 4H), 1.57 - 1.22 (m, 4H).MS (ESI)
Figure imgf000171_0002
Example 206: 2V-(benzo[/>]thiophen-2-yl)-l-(4-(dimethylamino)but-2-ynoyl)azetidine-3- carboxamide (XS224-106). Example 206 was synthesized following similar procedure for preparing example 53. White solid, 44% yield *H NMR (400 MHz, Methanol-d4) 87.72 (d, J= 7.8 Hz, 1H), 7.61 (d, J= 7.8 Hz, 1H), 7.28 (t, J= 7.5 Hz, 1H), 7.21 (t, J= 7.5 Hz, 1H), 6.95 (s, 1H), 4.49 (p, J= 9.3 Hz, 2H), 4.31 (d, J= 13.3 Hz, 3H), 4.25 - 4.17 (m, 1H), 3.67 (p, J= 7.1, 6.0 Hz, 1H), 2.98 (s, 6H). MS (ESI) [M+H]+ =342.1.
Figure imgf000171_0003
Example 207: /V-(benzo|6]thiophen-2-yl)-l-(4-(dimethylamino)-4-methylpent-2- ynoyl)azetidine-3-carboxamide (XS224-107). Example 207 was synthesized following similar procedure for preparing example 53. White solid, 39% yield 1 H NMR (400 MHz, Methanol)-d 84 7.72 (d, J= 7.8 Hz, 1H), 7.60 (d, J= 7.8 Hz, 1H), 7.28 (t, J= 7.5 Hz, 1H), 7.20 (t, J = 7.4 Hz, 1H), ΰ.95 (s, 1H), 4.48 (p, J= 8.7 Hz, 2H), 4.29 (t, J= 9.6 Hz, 1H), 4.24 - 4.16 (m, 1H), 3.67 (p, J = 7.2, 6.0 Hz, 1H), 2.98 (s, 6H), 1.75 (s, 6H). MS (ESI) [M+H]+ =370.2.
Figure imgf000172_0001
Example 208: l-(4-(dimethylamino)but-2-ynoyl)-/V-(4,5,6,7-tetrahydrobenzo[Z>]thiophen-2- yl)azetidine-3-carboxamide (XS224-108). Example 208 was synthesized following similar procedure for preparing example 53. White solid, 38% yield 'H NMR (400 MHz, Methanol-d4) δ 6.33 (s, 1H), 4.48 - 4.35 (m, 2H), 4.29 (s, 2H), 4.25 - 4.17 (m, 1H), 4.14 - 4.08 (m, 1H), 3.62 - 3.51 (m, 1H), 2.95 (s, 6H), 2.60 (t, J= 6.3 Hz, 2H), 2.45 (t, J= 5.7 Hz, 2H), 1.85 - 1.67 (m, 4H). MS (ESI) [M+H]+ =346.2.
Figure imgf000172_0002
Example 210: l-(4-(dimethylamino)but-2-ynoyl)-Az-(5-methylthiophen-2-yl)azetidine-3- carboxamide (XS224-110). Example 210 was synthesized following similar procedure for preparing example 53. White solid, 29% yield 'H NMR (400 MHz, Methanol-d4) 8 6.47 (s, 2H), 4.42 (p, J = 9.1 Hz, 2H), 4.31 (s, 2H), 4.23 (t, J = 9.7 Hz, 1H), 4.17 - 4.09 (m, 1H), 3.64 - 3.50 (m, 1H), 2.96 (s, 6H), 2.35 (s, 3H).MS (ESI) [M+H]+ =306.1.
Figure imgf000173_0002
Example 212: l-(4-(dimethylamino)biit-2-ynoyl)- V-(5-isopropylthiophen-2-yl)azetidine-3- carboxamide (XS224-116). Example 212 was synthesized following similar procedure for preparing example 53. White solid, 35% yield 1 H NMR (400 MHz, Methano)l 8-d 64.51 (d, J= 5.6 Hz, 2H), 4.43 (p, J= 8.9 Hz, 2H), 4.31 (s, 2H), 4.24 (t, J= 9.6 Hz, 1H), 4.14 (d, J= 15.8 Hz, 1H), 3.58 (p, J= 7.0, 6.2 Hz, 1H), 3.09 - 3.00 (m, 1H), 2.96 (s, 6H), 1.27 (d, J= 6.8 Hz, 6H). MS (ESI) [M+H]+ =334.2.
Figure imgf000173_0001
Example 213: l-(4-(dimethylamino)-4-methylpent-2-ynoyl)-/V-(5-isopropylthiophen-2- yl)azetidine-3-carboxamide (XS224-117). Example 213 was synthesized following similar procedure for preparing example 53. White solid, 25% yield 'H NMR (400 MHz, Methanol-d)48 6.51 (d, J= 5.9 Hz, 2H), 4.50 - 4.34 (m, 2H), 4.24 (t, J = 9.6 Hz, 1H), 4.18 - 4.11 (m, 1H), 3.58 (p, J = 7.2, 6.0 Hz, 1H), 3.11 - 3.00 (m, 1H), 2.97 (s, 6H), 1.74 (s, 6H), 1.27 (d, J= 6.8 Hz, 6H). MS (ESI) [M+H]+ =362.1.
Figure imgf000174_0001
Example 216:(E')-4-(dimethylamino)-N -(l-(5-methylthiophene-2-carbonyl)azetidin-3- yl)but-2-enamide (XS224-143). Example 216 was synthesized following similar procedure for preparing example 79. White solid, 33% yield ’H NMR (400 MHz, Methanol-d4) 87.29 (d, J= 3.1 Hz, 1H), 6.78 (s, 1H), 6.74 - 6.59 (m, 1H), 6.31 (d, J= 15.3 Hz, 1H), 4.82 - 4.61 (m, 2H), 4.35 (d, J = 38.0 Hz, 2H), 3.98 (s, 1H), 3.88 (d, J = 7.2 Hz, 2H), 2.83 (s, 6H), 2.45 (s, 3H). MS (ESI) [M+H]+ =308.2.
Figure imgf000175_0001
Example 217: 4-(dimethylamino)-2V-(l-(5-methylthiophene-2-carbonyl)azetidin-3-yl)but-2- ynamide (XS224-144). Example 217 was synthesized following similar procedure for preparing example 79. White solid, 29% yield 'HNMR (400 MHz, Methanol-d)45 7.29 (d, J= 3.2 Hz, 1H), 6.79 (s, 1H), 4.81 - 4.60 (m, 2H), 4.47 - 4.20 (m, 4H), 3.99 (s, 1H), 2.94 (s, 6H), 2.45 (s, 3H). MS (ESI) [M+H]+ =306.2.
Figure imgf000175_0002
Example 219: (E)-W-(3-(4-(dimethylamino)but-2-enamido)cyclobutyl)-5-methylthiophene-2- carboxamide (XS224-147). Example 219 was synthesized following similar procedure for preparing example 79. White solid, 45% yield 1 H NMR (400 MHz, Methanol-d4) 8 7.48 - 7.35 (m, 1H), 6.71 (s, 1H), 6.69 - 6.54 (m, 1H), 6.34 - 6.20 (m, 1H), 4.46 (p, J= 6.9, 6.4 Hz, 1H), 4.38 - 4.26 (m, 1H), 3.89 - 3.77 (m, 2H), 2.80 (s, 6H), 2.73 - 2.61 (m, 1H), 2.47 - 2.35 (m, 4H), 2.35 - 2.25 (m, 1H), 2.08 - 1.91 (m, 1H). MS (ESI) [M+H]+ =322.1.
Figure imgf000176_0001
Example 223: (£)-N-(l-(4-(dimethylamino)but-2-enoyl)azetidin-3-yl)benzo[6]thiophene-2- carboxamide (XS224-154). Example 223 was synthesized following similar procedure for preparing example 79. White solid, 36% yield 'H NMR (400 MHz, Methanol-d)45 7.98 (s, 1H), 7.88 (t, J= 6.9 Hz, 2H), 7.42 (p, J= 6.9 Hz, 2H), 6.77 - 6.62 (m, 1H), 6.50 (d, J= 15.3 Hz, 1H), 4.83 - 4.76 (m, 1H), 4.67 (t, J= 8.5 Hz, 1H), 4.42 (t, J= 9.5 Hz, 1H), 4.37 - 4.29 (m, 1H), 4.19 - 4.11 (m, 1H), 3.93 (d, J= 7.0 Hz, 2H), 2.88 (s, 6H). MS (ESI) [M+H]+ =344.2.
Figure imgf000177_0001
Example 224: JV-(l-(4-(dimethylamino)but-2-ynoyl)azetidin-3-yl)benzo[/>]thiophene-2- carboxamide (XS224-155). Example 224 was synthesized following similar procedure for preparing example 79. White solid, 29% yield 'H NMR (400 MHz, Methanol-d)48 7.99 (s, 1H), 7.90 (t, J = 6.4 Hz, 2H), 7.43 (p, J = 7.0 Hz, 2H), 4.84 - 4.78 (m, 1H), 4.66 (t, J= 8.7 Hz, 1H), 4.42 (t, ,/ = 9.5 Hz, 1H), 4.37 - 4.29 (m, 3H), 4.20 - 4.08 (m, 1H), 2.99 (s, 6H). MS (ESI) [M+H]+ =342.2.
Figure imgf000177_0002
Example 225: 7V-(l-(4-(dimethylamino)-4-methylpent-2-ynoyl)azetidin-3- yl)benzo[/>]thiophene-2-carboxamide (XS224-156). Example 225 was synthesized following similar procedure for preparing example 79. White solid, 26% yield 'H NMR (400 MHz, Methanol-^) 87.99 (s, 1H), 7.90 (t, J= 6.6 Hz, 2H), 7.43 (p, J= 6.7 Hz, 2H), 4.83 - 4.76 (m, 1H), 4.65 (t, J= 8.7 Hz, 1H), 4.42 (t, J= 9.5 Hz, 1H), 4.36 - 4.30 (m, 1H), 4.20 - 4.09 (m, 1H), 2.99 (s, 6H), 1.76 (s, 6H). MS (ESI) [M+H]+ =370.2.
Figure imgf000177_0003
Example 226: (E)-2V-(1-(4-(dimethylamino)but-2-enoyl)azetidin-3-yl)-4, 5,6,7- tetrahydrobenzo[Z>]thiophene-2-carboxamide (XS224-157). Example 226 was synthesized following similar procedure for preparing example 79. White solid, 25% yield XH NMR (400 MHz, Methanol-d4) 8 7.31 (s, 1H), 6.71 - 6.57 (m, 1H), 6.43 (d, J= 15.3 Hz, 1H), 4.73 - 4.62 (m, 1H), 4.58 (t, J= 8.5 Hz, 1H), 4.32 (t, J = 9.4 Hz, 1H), 4.26 - 4.16 (m, 1H), 4.09 - 3.97 (m, 1H), 3.87 (d, J= 7.0 Hz, 2H), 2.82 (s, 6H), 2.69 (t, J= 6.3 Hz, 2H), 2.53 (t, J= 5.8 Hz, 2H), 1.81 - 1.64 (m, 4H). MS (ESI) [M+H]+ =348.3.
Figure imgf000178_0001
Example 227: 2V-(l-(4-(dimethylamino)but-2-ynoyl)azetidin-3-yl)-4, 5,6,7- tetrahydrobenzo[Z>]thiophene-2-carboxamide (XS224-158). Example 227 was synthesized following similar procedure for preparing example 79. White solid, 23% yield 1 H NMR (400 MHz, Methanol-d4) 8 7.37 (s, 1H), 4.79 - 4.68 (m, 1H), 4.60 (t, ,7 = 8.7 Hz, 1H), 4.40 - 4.30 (m, 3H), 4.30 - 4.21 (m, 1H), 4.14 - 4.02 (m, 1H), 2.98 (s, 6H), 2.77 (t, J= 6.0 Hz, 2H), 2.61 (t, .7= 6.0 Hz, 2H), 1.93 - 1.72 (m, 4H). MS (ESI) [M+H]+ =346.2.
Figure imgf000178_0002
Example 228: /V-(l-(4-(dimethylamino)-4-methylpent-2-ynoyl)azetidin-3-yl)-4, 5,6,7- tetrahydrobenzo[b]thiophene-2-carboxamide (XS224-159). Example 228 was synthesized following similar procedure for preparing example 79. White solid, 29% yield 1 H NMR (400 MHz, Methanol-d4) 8 7.36 (s, 1H), 4.74 (p, J= 6.5, 5.9 Hz, 1H), 4.59 (t, J= 8.6 Hz, 1H), 4.36 (t, J = 9.5 Hz, 1H), 4.29 - 4.20 (m, 1H), 4.12 - 4.03 (m, 1H), 2.98 (s, 6H), 2.75 (t, J= 6.4 Hz, 2H), 2.60 (t, J = 6.0 Hz, 2H), 1.89 - 1.77 (m, 4H), 1.75 (s, 6H). MS (ESI) [M+H]+ =374.2.
Procedures for the synthesis of AMPK based bivalent compounds
Figure imgf000179_0001
Linker 10-14: n = 1-5
The linkers 1-14 were synthesized following the reported procedures (Liu et al., 2022)
Scheme 12. Syntheses of example 229
Figure imgf000179_0002
example 229
Example 229: A-(2-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)propanamido)ethyl)-5- ((6-chloro-5-(l-methyl-l/7-indol-5-yl)-l/7-benzo[c/]imidazol-2-yl)oxy)-2- methylbenzamide(XF137-81). To a solution of activator 991 (prepared according to previous published paper) (Ngoei et al., 2018) (21 mg, 0.048 mmol) in DMSO (1 mL) were added linker 1 (16.3 mg, 0.048 mmol, 1.0 equiv), EDCI (14 mg, 0.072 mmol, 1.5 equiv), HOAt (10 mg, 0.072 mmol, 1.5 equiv), and NMM (15 mg, 0.14 mmol, 3.0 equiv). After being stirred for 3 h at rt, the resulting mixture was purified by preparative HPLC to afford titled compound as brown solid. (6.7 mg, yield 19%). 1H NMR (400 MHz, CD3OD) δ 7.62 - 7.48 (m, 3H), 7.48 - 7.35 (m, 5H), 7.27 - 7.18 (m, 2H), 6.75 - 6.59 (m, 1H), 6.30 - 6.25 (m, 1H), 6.16 (d, J= 3.1 Hz, 1H), 6.00 (d, J= 3.2 Hz, 1H), 5.80 (d, J= 10.5 Hz, 1H), 4.42 - 4.21 (m, 2H), 3.91 - 3.82 (m, 5H), 3.81 - 3.69 (m, 2H), 3.53 - 3.38 (m, 4H), 2.80 (t, J= 7.5 Hz, 2H), 2.47 (s, 5H). MS (ESI) [M+H]+ = 748.3.
Figure imgf000180_0001
Example 230: JV-(3-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)propanamido)propyl)- 5-((6-chloro-5-( 1-methyl- l//-indol-5-yl)- 1H-benzo[d/]imidazol-2-yl)oxy)-2-methylbenzamide (XF137-82). Example 230 was synthesized following the standard procedure for preparing example 229 from activator 991 (28 mg, 0.06 mmol) and linker 2 (23 mg, 0.06 mmol, 1.0 equiv). Brown solid (15.4 mg, yield 34%). ’H NMR (400 MHz, CD3OD) δ 7.61 - 7.47 (m, 3H), 7.45 - 7.34 (m, 5H), 7.28 - 7.14 (m, 2H), 6.83 - 6.61 (m, 1H), 6.28 (dd, J= 16.8, 1.9 Hz, 1H), 6.21 (d, J = 3.2 Hz, 1H), 6.09 (d, J= 3.2 Hz, 1H), 5.80 (dd, J= 10.5, 1.9 Hz, 1H), 4.45 - 4.27 (m, 2H), 3.93 (t, J= 5.3 Hz, 2H), 3.84 (d, J= 9.3 Hz, 5H), 3.38 - 3.34 (m, 2H), 3.27 (d, J= 6.8 Hz, 2H), 2.88 (t, J= 7.3 Hz, 2H), 2.48 (d, J= 11.3 Hz, 5H), 1.76 (t, J= 6.8 Hz, 2H). MS (ESI) [M+H]+ = 762.3.
Figure imgf000180_0002
Example 231: 7V-(4-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)propanamido)butyl)-5- ((6-chloro-5-(l-methyl-l//-indol-5-yl)-lH -benzo[<7|imidazol-2-yl)oxy)-2-methylbenzamide
(XF137-83). Example 231 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and liner 3 (17 mg, 0.048 mmol, 1.0 equiv). Brown solid (7.6 mg, yield 20%). 'HNMR (400 MHz, CD3OD) 5 7.57 (d, J= 2.5 Hz, 3H), 7.51 - 7.32 (m, 5H), 7.23 (d, J= 8.3 Hz, 2H), 6.84 - 6.57 (m, 1H), 6.29 (dd, J = 16.8, 1.9 Hz, 1H), 6.20 (d, J= 3.2 Hz, 1H), 6.08 (d, J= 3.2 Hz, 1H), 5.81 (dd, J= 10.6, 1.9 Hz, 1H), 4.44 - 4.24 (m, 2H), 3.95 - 3.89 (m, 2H), 3.87 - 3.75 (m, 5H), 3.47 - 3.35 (m, 2H), 3.21 (t, J= 6.4 Hz, 2H), 2.87 (t, J = 7.3 Hz, 2H), 2.47 (s, 5H), 1.73 - 1.47 (m, 4H). MS (ESI) [M+H]+ = 776.3.
Figure imgf000181_0001
Example 233: JV-(6-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)propanamido)hexyl)-5- ((6-chloro-5-(l-methyl-lH -indol-5-yl)-lH-benzo[d]imidazol-2-yl)oxy)-2-methylbenzamide (XF137-85). Example 233 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 5 (18.7 mg, 0.048 mmol, 1.0 equiv). Compound XF137-85 was obtained as brown solid (9.1 mg, yield 24%). 'H NMR (400 MHz, CD3OD) δ 7.58 (d, J= 3.0 Hz, 2H), 7.52 - 7.32 (m, 6H), 7.23 (d, J= 8.3 Hz, 2H), 6.85 - 6.61 (m, 1H), 6.29 (dd, J= 16.7, 2.0 Hz, 1H), 6.22 (d, J= 3.2 Hz, 1H), 6.08 (d, J= 3.2 Hz, 1H), 5.82 (dd, J = 10.5, 1.9 Hz, 1H), 4.47 - 4.26 (m, 2H), 4.03 - 3.89 (m, 2H), 3.89 - 3.74 (m, 5H), 3.43 - 3.34 (m, 2H), 3.15 (t, J= 6.8 Hz, 2H), 2.88 (t, J= 7.3 Hz, 2H), 2.47 (s, 5H), 1.69 - 1.55 (m, 2H), 1.54 - 1.27 (m, 6H). MS (ESI) [M+H]+ = 804.3.
Figure imgf000182_0001
Example 234: 2V-(7-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)propanamido)heptyl)- 5-((6-chloro-5-(l-methyl-l//-indol-5-yl)-l//-benzo[t/|imidazol-2-yl)oxy)-2-methylbenzamide (XF137-86). Example 234 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 6 (19 mg, 0.048 mmol, 1.0 equiv). Brown solid (12.6 mg, yield 32%). ’H NMR (400 MHz, CD3OD) 8 7.63 - 7.52 (m, 2H), 7.49 - 7.31 (m, 6H), 7.28 - 7.16 (m, 2H), 6.88 - 6.64 (m, 1H), 6.30 (dd, J= 16.8, 1.9 Hz, 1H), 6.22 (d, J = 3.3 Hz, 1H), 6.08 (d, 7= 3.2 Hz, 1H), 5.82 (dd, J= 10.6, 1.9 Hz, 1H), 4.47 - 4.31 (m, 2H), 4.06 - 3.94 (m, 2H), 3.86 (s, 5H), 3.41 - 3.36 (m, 2H), 3.14 (t, J= 6.9 Hz, 2H), 2.89 (t, J= 7.3 Hz, 2H), 2.52 - 2.40 (m, 5H), 1.62 (q, J= 7.1 Hz, 2H), 1.52 - 1.21 (m, 8H). MS (ESI) [M+H]+ = 818.4.
Figure imgf000182_0002
Example 235: JV-(8-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)propanamido)octyl)-5- ((6-chloro-5-(l-methyl-17/-indol-5-yl)-177-benzo[7]imidazol-2-yl)oxy)-2-methylbenzamide (XF137-87). Example 235 was synthesized following the standard procedure for preparing Example 229 from activator 991 (21 mg, 0.048 mmol) and linker 7 (20 mg, 0.048 mmol, 1.0 equiv). Brown solid (9.6 mg, yield 24%). 'H NMR (400 MHz, CD3OD) 8 'H NMR (400 MHz, MeOD) 8 7.62 - 7.52 (m, 2H), 7.49 - 7.36 (m, 6H), 7.28 - 7.18 (m, 2H), 6.82 - 6.64 (m, 1H), 6.35 - 6.26 (m, 1H), 6.25 - 6.21 (m, 1H), 6.08 (d, J= 3.2 Hz, 1H), 5.82 (dt, J= 10.6, 1.3 Hz, 1H), 4.40 (d, J = 26.3 Hz, 2H), 4.02 - 3.92 (m, 2H), 3.89 - 3.78 (m, 5H), 3.42 - 3.35 (m, 2H), 3.13 (t, J= 7.0 Hz, 2H), 2.89 (t, 7= 7.3 Hz, 2H), 2.55 - 2.39 (m, 5H), 1.62 (q, 7= 7.2 Hz, 2H), 1.52 - 1.24 (m, 10H). MS (ESI) [M+H]+ = 832.2.
Figure imgf000183_0001
Example 236: JV-(9-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)propanamido)nonyl)-5- ((6-chloro-5-(l-methyl-177-indol-5-yl)-177-benzo[<7|imidazol-2-yl)oxy)-2-methylbenzamide (XF137-88). Example 236 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 8 (21 mg, 0.048 mmol, 1.0 equiv). Brown solid (12.9 mg, yield 32%). 'H NMR (400 MHz, CD3OD) 6 7.54 (d, J= 17.1 Hz, 2H), 7.43 - 7.28 (m, 6H), 7.28 - 7.10 (m, 2H), 6.73 (d, J= 10.8 Hz, 1H), 6.30 - 6.28 (m, 1H), 6.22 (d, J = 3.2 Hz, 1H), 6.11 - 5.98 (m, 1H), 5.81 (dd, J= 10.5, 2.0 Hz, 1H), 4.46 - 4.26 (m, 2H), 3.93 (t, J = 5.4 Hz, 2H), 3.87 - 3.76 (m, 5H), 3.41 - 3.34 (m, 2H), 3.12 (t, ,7 = 7.0 Hz, 2H), 2.88 (t, ,7= 7.3 Hz, 2H), 2.51 - 2.38 (m, 5H), 1.61 (q, J = 7.2 Hz, 2H), 1.50 - 1.22 (m, 12H). MS (ESI) [M+H]+ =
846.4.
Figure imgf000183_0002
Example 237 : 2V-(10-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yI)propanamido)decyl)- 5-((6-chloro-5-(l-methyl-17/-indol-5-yl)-177-benzo[t/]imidazol-2-yl)oxy)-2-methylbenzamide (XF137-89). Example 237 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 9 (21 mg, 0.048 mmol, 1.0 equiv). Brown solid (12.8 mg, yield 31%). ’H NMR (400 MHz, CD3OD) 8 7.62 - 7.47 (m, 2H), 7.47 - 7.29 (m, 6H), 7.27 - 7.19 (m, 2H), 6.82 - 6.65 (m, 1H), 6.35 - 6.25 (m, 1H), 6.24 - 6.17 (m, 1H), 6.08 (d, .7= 3.2 Hz, 1H), 5.88 - 5.67 (m, 1H), 4.48 - 4.28 (m, 2H), 3.94 (t, J= 5.1 Hz, 2H), 3.84 (d, J= 1.4 Hz, 5H), 3.40 - 3.34 (m, 2H), 3.12 (t, J= 7.0 Hz, 2H), 2.89 (t, J= 7.3 Hz, 2H), 2.46 (d, J= 1.8 Hz, 5H), 1.62 (q, J= 7.1 Hz, 2H), 1.50 - 1.17 (m, 14H). MS (ESI) [M+H]+ = 860.3.
Figure imgf000184_0001
Example 238: 2V-(2-(2-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2- yl)propanamido)ethoxy)ethyl)-5-((6-chloro-5-(l-methyl-l/7-indol-5-yl)-l//- benzo [d/|imidazol-2-yl)oxy)-2-ni ethylbenzamide (XF137-90). Example 238 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 10 (18.1 mg, 0.048 mmol, 1.0 equiv). Brown solid (16 mg, yield 42%). 'H NMR (400 MHz, CD3OD) δ 7.60 - 7.52 (m, 2H), 7.51 - 7.36 (m, 6H), 7.26 - 7.18 (m, 2H), 6.82 - 6.59 (m, 1H), 6.28 (dd, J= 16.8, 1.9 Hz, 1H), 6.20 (d, J= 3.2 Hz, 1H), 6.03 (d, J= 3.2 Hz, 1H), 5.80
(dd, J= 10.6, 1.9 Hz, 1H), 4.41 - 4.26 (m, 2H), 3.90 (t, J= 5.4 Hz, 2H), 3.85 (s, 3H), 3.78 (s, 2H),
3.67 - 3.49 (m, 6H), 3.38 - 3.35 (m, 2H), 2.79 (t, J= 1A Hz, 2H), 2.47 (s, 3H), 2.42 (t, J= 7.4 Hz,
2H). MS (ESI) [M+H]+ = 792.2.
Figure imgf000184_0002
Example 239: JV-(2-(2-(2-(3-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2- yl)propanamido)ethoxy)ethoxy)ethyl)-5-((6-chloro-5-(l-methyl-l/7-indol-5-yl)-EH- benzo[d]imidazol-2-yl)oxy)-2-methylbenzamide (XF137-91). Example 239 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 11 (20.2 mg, 0.048 mmol, 1.0 equiv). Brown solid (21.8 mg, yield 54%). 'H NMR (400 MHz, CD3OD) δ 7.64 - 7.53 (m, 2H), 7.52 - 7.33 (m, 6H), 7.23 (d, J= 8.7 Hz, 2H), 6.80 - 6.61 (m, 1H), 6.28 (dd, J= 16.8, 1.9 Hz, 1H), 6.22 - 6.13 (m, 1H), 6.07 (d, J= 3.2 Hz, 1H), 5.81 (dt, J= 10.6, 1.6 Hz, 1H), 4.37 (d, J= 25.5 Hz, 2H), 3.92 (t, J= 5.4 Hz, 2H), 3.87 - 3.74 (m, 5H), 3.70 - 3.52 (m, 8H), 3.47 (t, J= 5.5 Hz, 2H), 3.31 - 3.26 (m, 2H), 2.85 (t, J= 7.4 Hz, 2H), 2.50 - 2.39 (m, 5H). MS (ESI) [M+H]+ = 836.3.
Figure imgf000185_0001
Example 240: JV-(15-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)-13-oxo-3,6,9-trioxa-12- azapentadecyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-17/-benzo[t/|imidazol-2-yl)oxy)-2- methylbenzamide (XF137-92). Example 240 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 12 (22 mg, 0.048 mmol, 1.0 equiv). Brown solid (22.9 mg, yield 61%). JH NMR (400 MHz, CD3OD) 5 7.59 (d, J = 7.6 Hz, 2H), 7.53 - 7.35 (m, 6H), 7.27 - 7.19 (m, 2H), 6.80 - 6.64 (m, 1H), 6.29 (dd, J= 16.7, 1.9 Hz, 1H), 6.22 (d, J= 3.3 Hz, 1H), 6.08 (d, J= 3.2 Hz, 1H), 5.81 (dd, J= 10.7, 1.9 Hz, 1H), 4.44 - 4.30 (m, 2H), 3.97 - 3.88 (m, 2H), 3.87 - 3.75 (m, 5H), 3.70 - 3.49 (m, 12H), 3.45 (t, J= 5.4 Hz, 2H), 3.30 (d, J= 5.5 Hz, 2H), 2.88 (t, J = 7.4 Hz, 2H), 2.51 - 2.44 (m, 5H). MS (ESI) [M+H]+ =
880.3.
Figure imgf000185_0002
Example 241: JV-(18-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)-16-oxo-3,6,9,12- tetraoxa-15-azaoctadecyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzo[d ]imidazol-2- yl)oxy)-2-methylbenzamide (XF137-93). Example 241 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 13 (24 mg, 0.048 mmol, 1.0 equiv). Brown solid (19.8 mg, yield 45%). 'H NMR (400 MHz, CD3OD) 8 7.58 (d, J= 5.8 Hz, 2H), 7.51 - 7.40 (m, 6H), 7.28 - 7.18 (m, 2H), 6.83 - 6.65 (m, 1H), 6.29 (dd, J = 16.8, 2.0 Hz, 1H), 6.22 (d, J= 3.2 Hz, 1H), 6.09 (d, J = 3.2 Hz, 1H), 5.81 (dd, J= 10.6, 1.9 Hz, 1H), 4.47 - 4.31 (m, 2H), 3.97 - 3.88 (m, 2H), 3.88 - 3.75 (m, 3H), 3.70 - 3.51 (m, 18H), 3.47 (t, J= 5.5 Hz, 2H), 3.38 - 3.36 (m, 2H), 2.89 (t, J= 7.4 Hz, 2H), 2.55 - 2.40 (m, 5H). MS (ESI) [M+H]+ = 924.4.
Figure imgf000186_0001
Example 242: A-(21-(5-(4-acryloyl-2-oxopiperazin-l-yl)furan-2-yl)-19-oxo-3,6,9,12,15- pentaoxa-18-azahenicosyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-177-benzo[d|imidazol-2- yl)oxy)-2-methylbenzamide (XF137-94). Example 242 was synthesized following the standard procedure for preparing example 229 from activator 991 (21 mg, 0.048 mmol) and linker 14 (27 mg, 0.048 mmol, 1.0 equiv). Brown solid (16.8 mg, yield 36%). 'H NMR (400 MHz, CD3OD) 8 7.59 (d, J= 5.8 Hz, 2H), 7.52 - 7.41 (m, 6H), 7.28 - 7.18 (m, 2H), 6.82 - 6.66 (m, 1H), 6.29 (dd, J = 16.7, 2.0 Hz, 1H), 6.23 (d, J = 3.2 Hz, 1H), 6.10 (d, J = 3.2 Hz, 1H), 5.82 (dd, J = 10.6, 1.9 Hz, 1H), 4.48 - 4.32 (m, 2H), 3.97 - 3.88 (m, 2H), 3.86 - 3.74 (m, 5H), 3.70 - 3.51 (m, 20H), 3.48 (t, J= 5.5 Hz, 2H), 3.39 - 3.36 (m, 2H), 2.90 (t, J= 7.4 Hz, 2H), 2.55 - 2.42 (m, 5H). MS (ESI) [M+H]+ = 968.5.
Scheme 13. The syntheses of linker 15
Figure imgf000186_0002
Linker 15: (5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzo[d]imidazol-2-yl)oxy)-2- methylbenzoyl)glycine To a solution of activator 991 (50 mg, 0.12 mmol) in DMF (1 mL) were added tert-butyl (2-aminoethyl)carbamate (19.2 mg, 0.12 mmol, 1.0 equiv), l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI, 34.3 mg, 0.18 mmol, 1.5 equiv), l-hydroxy-7- azabenzo-triazole (HO At, 24.5 mg, 0.18 mmol, 1.5 equiv), and N-methylmorpholine (NMM, 36.4 mg, 0.36 mmol, 3.0 equiv). After being stirred for overnight atrt, the resulting mixture was purified by prep-HPLC to afford crude product. The crude product was dissolved in DCM (1 mL) and TFA (1 mL). After being stirred at rt for 30 min, the resulting mixture was purified by prep-HPLC to afford compound titled compound as yellow solid (57 mg, yield 81%). MS (ESI) [M+H]+ = 489.3. Linker 16-27 were synthesized following the same procedure for preparing linker 15.
Figure imgf000186_0003
Linker 16: 3-(5-((6-chloro-5-(l-methyl-lH-indol-5-yl)-l/7-benzo[J]imidazol-2-yl)oxy)-2- methylbenzamido)propanoic acid MS (ESI) [M+H]+ = 503.2.
Figure imgf000187_0001
Linker 17: 4-(5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzo[d|imidazol-2-yl)oxy)-2- methylbenzamido)butanoic acid MS (ESI) [M+H]+ =517.4.
Figure imgf000187_0002
Linker 18: 5-(5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzo[d]imidazol-2-yl)oxy)-2- methylbenzamido)pentanoic acid MS (ESI) [M+H]+ = 531.3.
Figure imgf000187_0003
Linker 19: 6-(5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzod/|imidazol-2-yl)oxy)-2- methylbenzamido)hexanoic acid MS (ESI) [M+H]+ = 545.3.
Figure imgf000187_0004
Example 110: (E)-N -(l-(4-(dimethylamino)but-2-enoyl)azetidin-3-yl)-5-methylthiophene-2- carboxamide (XS185-171). Example 110 was synthesized following similar procedure for preparing example 104. White solid, 15% yield 1H NMR (400 MHz, DMSO-Jc,) 88.54 - 8.35 (m, 1H), 7.34 (d, J= 4.1 Hz, 4H), 7.29 - 7.20 (m, 1H), 6.67 - 6.49 (m, 2H), 4.81 (d, .7 = 21.1 Hz, 2H), 4.25 (s, 2H), 4.19 (d, J= 4.8 Hz, 2H), 3.91 - 3.82 (m, 2H), 3.82 - 3.69 (m, 2H), 2.36 (s, 3H).MS (ESI) [M+H]+ =424.2
Linker 20: 7-(5-((6-chloro-5-(l-methyl-1H-indol-5-yl)- 1H-benzo[d|imidazol-2-yl)oxy)-2- methylbenzamido)heptanoic acid MS (ESI) [M+H]+ = 559.4.
Figure imgf000188_0001
Linker 21 : 8-(5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzo[d/]imidazol-2-yl)oxy)-2- methylbenzamido)octanoic acid MS (ESI) [M+H]+ = 573.3.
Figure imgf000188_0002
Linker 22: 9-(5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-l//-benzo[d]imidazol-2-yl)oxy)-2- methylbenzamido)nonanoic acid MS (ESI) [M+H]+ = 587.5.
Figure imgf000188_0003
Linker 23: 3-(2-(5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-l/7-benzo[<Z|iniidazol-2-yl)oxy)-2- methylbenzamido)ethoxy)propanoic acid MS (ESI) [M+H]+ = 548.6.
Figure imgf000188_0004
Linker 24: 3-(2-(2-(5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzo[d/|iniidazol-2-yl)oxy)- 2-methylbenzamido)ethoxy)ethoxy)propanoic acid MS (ESI) [M+H]+ = 591.4.
Figure imgf000188_0005
Linker 25: l-(5-((6-chloro-5-(l-methyl-l//-indol-5-yl)-177-benzo[d |iniidazol-2-yl)oxy)-2- methylphenyl)-l-oxo-5,8,ll-trioxa-2-azatetradecan-14-oic acid MS (ESI) [M+H]+ = 635.4.
Figure imgf000188_0006
Linker 26: l-(5-((6-chloro-5-(l-methyl-l//-indol-5-yl)-177-benzo[d|iniidazol-2-yl)oxy)-2- methylphenyl)-l-oxo-5,8,ll,14-tetraoxa-2-azaheptadecan-17-oic acid MS (ESI) [M+H]+ = 679.4.
Figure imgf000189_0001
Linker 27: l-(5-((6-chIoro-5-(1-methyl-1H-indol-5-yl)-1H-benzo[d]imidazol-2-yl)oxy)-2- methylphenyl)-l-oxo-5,8,ll,14,17-pentaoxa-2-azaicosan-20-oic acid MS (ESI) [M+H]+ =
723.3.
Scheme 14. The syntheses of linker 28
Figure imgf000189_0002
activator 991 linker 28
Linker 28 : JV-(2-aminoethyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-lH-benzo[d ]imidazol- 2-yl)oxy)-2-methylbenzamide (XH168-164). To a solution of activator 991 (50 mg, 0.12 mmol) in DMF (1 mL) were added tert-butyl (2-aminoethyl)carbamate (19.2 mg, 0.12 mmol, 1.0 equiv), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 34.3 mg, 0.18 mmol, 1.5 equiv), 1- hydroxy-7-azabenzo-triazole (HO At, 24.5 mg, 0.18 mmol, 1.5 equiv), and N-methylmorpholine (NMM, 36.4 mg, 0.36 mmol, 3.0 equiv). After being stirred for overnight at rt, the resulting mixture was purified by preparative HPLC ( 10%- 100% acetonitrile / 0.1% TFA in H2O) to afford crude product. The crude product was dissolved in DCM (1 mL) and TFA (1 mL). After being stirred at rt for 30 min, the resulting mixture was purified by preparative HPLC (10%-100% acetonitrile / 0.1% TFA in H2O) to afford compound titled compound as yellow solid (57 mg, yield 81%). MS (ESI) m/z 474.4 [M+H]+.
Linker 29-41 was synthesized following the same procedure for preparing linker 28.
Figure imgf000189_0003
Linker 29: N -(3-aminopropyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl )-1H- benzo[*/]imidazol-2-yl)oxy)-2-methylbenzamide (XH168-165). Pink solid, 52 mg, 61% yield.
MS (ESI) [M+H]+= 488.3.
Figure imgf000190_0002
Linker 33: N-(7-aminoheptyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H- benzo[d |imidazol-2-yl)oxy)-2-methylbenzamide (XH168-169). Yellow solid, 73 mg, 79% yield.
MS (ESI) [M+H]+= 544.5.
Figure imgf000190_0001
Linker 34: N -(8-aminooctyl)-5-((6-chloro-5-(1-methyl-1H-indol-5-yl)-1H-benzo[d]imidazol- 2-yl)oxy)-2-methylbenzamide (XH168-170). Yellow solid, 69 mg, 73% yield. MS (ESI) [M+H]+ = 558.4.
Figure imgf000191_0003
Linker 36: JV-(10-aminodecyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-EH- benzo[rf|imidazol-2-yl)oxy)-2-methylbenzamide (XH168-172) Yellow solid, 75 mg, 87% yield. MS (ESI) [M+H]+ = 586.5.
Figure imgf000191_0001
Linker 37: N -(2-(2-aminoethoxy)ethyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H- benzo|d]imidazol-2-yl)oxy)-2-methylbenzamide (XH168-173). Yellow solid, 65 mg, 73% yield. MS (ESI) [M+H]+ = 518.1.
Figure imgf000191_0002
Linker 38: N -(2-(2-(2-aminoethoxy)ethoxy)ethyl)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl)- 1H-benzo[d]imidazol-2-yl)oxy)-2-methylbenzamide (XH168-174). Yellow solid, 79 mg, 81% yield. MS (ESI) [M+H]+ = 562.5.
Figure imgf000192_0001
Linker 41: N-(17-amino-3,6,9,12,15-pentaoxaheptadecyl)-5-((6-chloro-5-(l-methyl-l1H- indol-5-yl)-1H-benzo[d]imidazol-2-yl)oxy)-2-niethylbenzaniide (XH168-177). Yellow solid, 89 mg, 81% yield. MS (ESI) [M+Hf = 694.5.
Scheme 15. Synthesis of intermediate 13.
Figure imgf000193_0001
Intermediate 13
(E)-3-(5-(4-(4-(dimethylamino)but-2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propanoic acid (Intermediate 13) methyl 3-(5-bromothiophen-2-yl)acrylate (246 mg, 1.0 mmol, 1.0 eq), tert-butyl 3-oxopiperazine-l -carboxylate (300 mg, 1.5 mmol, 1.5 eq), Cui (85 mg, 0.5 mmol), dimethylethane-l,2-diamine (88 mg, 1.0 mmol, 1.0 eq), K2CO3 (276 mg, 2.0 mmol, 2.0 eq) were added Toluene ( 5 mL). The mixture was stirred at 110°C under N2 for 6 h. The mixture was purified by ISCO. The obtained product was then dissolved in MeOH (5 mL) followed by Pd/C (60 mg) ed. The mixture was stirred at room temperature under 1 atm H2 for 1 h then filtered to yield intermediate 12 as a white solid (200 mg, 55%). 'H NMR (400 MHz, CDCI3) 8 6.62 (s, 1H), 6.48 (s, 1H), 4.28 (s, 2H), 3.81 (s, 4H), 3.68 (s, 3H), 3.08 (t, J= 7.7 Hz, 2H), 2.68 (d, J= 7.7 Hz, 2H), 1.48 (s, 9H). MS (ESI) [M+H]+ = 369.2.
Intermediate 13 (184 mg, 0.5 mmol, 1.0 eq) was dissolved in MeOH (2 mL) and HC1 (4M in dioxane, 2 mL, 8 mmol), the mixture was stirred at room temperature for 3h, then removed all the volatiles. The resulted residue (71mg, 0.55 mmol, 1.1 eq), HATU (285 mg, 0.75 mmol, 1.5 eq), DIPEA (194 mg, 1.5 mmol, 3.0 eq) were stirred in DCM (5 mL) at room temperature for 30 min. Then removed all the volatiles, and the mixture was purified via reverse-ISCO. The purified compound (120 mg, 0.33 mmol, 1.0 eq) was dissolved in MeOH (2 mL). LiOH aq. (2M, 0.5 mL, 1 mmol) was added dropwise at 0 °C. The mixture was stirred at 0 °C for 20 min, then was purified via reverse-ISCO to yield title compound as a brown solid (100 mg, 83%).1H NMR (400 MHz, CD3OD) δ 7.08 - 6.64 (m, 4H), 4.13 - 3.89 (m, 6H), 3.15 - 3.04 (m, 2H), 2.98 - 2.87 (m, 8H), 2.73 - 2.62 (m, 2H). MS (ESI) [M+H]+ = 366.3.
Scheme 16. Synthesis of intermediate 15
Figure imgf000194_0001
(£')-l-(5-(2-aminoethyl)thiophen-2-yl)-4-(4-(dimethylamino)but-2-enoyl)piperazin-2-one (Intermediate 15) tert-butyl (2-(5-bromothiophen-2-yl)ethyl)carbamate (306 mg, 1.0 mmol, 1.0 eq), benzyl 3-oxopiperazine-l-carboxylate (350 mg, 1.5 mmol, 1.5 eq), Cui (85 mg, 0.5 mmol), N1-dimethylethane-l,2-diamine (88 mg, 1.0 mmol, 1.0 eq), K2CO3 (276 mg, 2.0 mmol, 2.0 eq) were added Toluene (5 mL). The mixture was stirred at 110 °C under N2 for 6 h. The mixture was purified ISCO to yield intermediate 14 as a white solid (278 mg, 61%). 'H NMR (400 MHz, CD3OD) 5 7.47 - 7.32 (m, 5H), 6.69 (s, 2H), 5.20 (s, 2H), 4.33 (s, 2H), 3.91 (s, 4H), 3.33 - 3.25 (m, 2H), 2.94 - 2.87 (m, 2H), 1.45 (s, 9H). MS (ESI) [M+H]+ = 460.4.
Intermediate 14 (459 mg, 1.0 mmol, 1.0 eq), Pd/C (120 mg), Pd(OH)2 (120 mg) were added to MeOH (5 mL), the mixture was stirred under 10 atm H2 at 60 °C for 2 h. The mixture was filtered and the product was used without further purification. MS (ESI) [M+H]+ = 326.3. The obtained crude product (160 mg, 0.5 mmol, 1.0 eq), (E)-4-(dimethylamino)but-2-enoic acid (71mg, 0.55 mmol, 1.1 eq), HATU (285 mg, 0.75 mmol, 1.5 eq), DIPEA (194 mg, 1.5 mmol, 3.0 eq) were stirred in DCM (5 mL) at room temperature for 30 min. Then removed all the volatiles, and the mixture was purified via reverse-ISCO. The obtained product was stirred with DCM/TFA (1 mL/lmL) for 20 min. Then removed all the volatiles, and the mixture was purified via reverse- ISCO to yield title compound as a white solid (150 mg, 98%). 'H NMR (400 MHz, CD3OD) 8 7.17 - 6.74 (m, 4H), 4.19 - 3.93 (m, 6H), 3.26 - 3.19 (m, 2H), 3.18 - 3.11 (m, 2H), 3.01 - 2.90 (m, 8H). MS (ESI) [M+H]+ = 337.2.
Scheme 17. The syntheses of example 243
Figure imgf000195_0001
Example 243
Example 243: (E)-5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzo[d|imidazol-2-yl)oxy)-A- (7-((2-(5-(4-(4-(dimethylamino)but-2-enoyl)-2-oxopiperazin-l-yl)thiophen-2- yl)ethyl)amino)-7-oxoheptyl)-2-methylbenzamide (QC179-047) Linker 20 (12 mg, 0.025 mmol, 1.0 eq), intermediate 15 (10 mg, 0.028 mmol, 1.1 eq), EDC HC1 (7 mg, 0.038 mmol, 1.5 eq), HOAt (5 mg, 0.038 mmol, 1.5 eq), NMM (8 mg, 0.075 mmol, 3.0 eq) were stirred in 2 mL DMF at room temperature for 2 h. Then the mixture was purified via Prep-HPLC to yield titled compound as a white solid (8 mg, 46% yield). 1H NMR (400 MHz, CD3OD) 8 7.58 (s, 1H), 7.51 (s, 1H), 7.46 - 7.32 (m, 5H), 7.27 - 7.21 (m, 2H), 6.94 (dd, J= 32.4, 15.1 Hz, 1H), 6.77 (br, 1H), 6.70 - 6.64 (m, 2H), 6.49 (s, 1H), 4.50 (s, 1H), 4.43 (s, 1H), 4.04 (s, 2H), 3.95 (s, 3H), 3.87 (s, 4H), 3.44 - 3.36 (m, 4H), 2.92 (s, 8H), 2.46 (s, 3H), 2.24 - 2.13 (m, 2H), 1.64 (s, 4H), 1.47 - 1.30 (m, 4H). MS (ESI) [M+H]+ = 877.5.
Figure imgf000195_0002
Example 244: (E)-5-((6-chloro-5-(l-methyl-1H/-indol-5-yl)-1H-benzo[d|imidazol-2-yl)oxy)-A- (8-((2-(5-(4-(4-(dimethylamino)but-2-enoyl)-2-oxopiperazin-l-yl)thiophen-2- yl)ethyl)amino)-8-oxooctyl)-2-methylbenzamide (QC179-048) Example 244 was synthesized following the same procedure for preparing example 243 from linker 21. White solid, 9 mg, 50% yield. 1H NMR (400 MHz, CD3OD) δ 7.67 - 7.11 (m, 10H), 7.02 - 6.85 (m, 1H), 6.78 (br, 1H), 6.68 - 6.56 (m, 2H), 4.52 - 4.39 (m, 2H), 4.13 - 3.86 (m, 9H), 3.40 (s, 4H), 2.91 (s, 8H), 2.45 (s, 3H), 2.15 (s, 2H), 1.61 (s, 4H), 1.46 - 1.30 (s, 6H). MS (ESI) [M+H]+ = 891.5.
Figure imgf000196_0001
Example 245: (E)-5-((6-chloro-5-(l-methyl-1Hindol-5-yl)-1Hbenzo[d|imidazol-2-yl)oxy)-N- (9-((2-(5-(4-(4-(dimethylamino)but-2-enoyl)-2-oxopiperazin-l-yl)thiophen-2- yl)ethyl)amino)-9-oxononyl)-2-methylbenzamide (QC179-049) Example 245 was synthesized following the same procedure for preparing example 243 from linker 22. White solid, 9 mg, 45% yield. 1H NMR (400 MHz, CD3OD) 8 7.58 (s, 1H), 7.51 (s, 1H), 7.44 - 7.36 (m, 5H), 7.29 - 7.20 (m, 2H), 6.95 (dd, J= 33.6, 15.2 Hz, 1H), 6.77 (br, 1H), 6.67 (s, 2H), 6.49 (s, 1H), 4.50 (s, 1H), 4.44 (s, 1H), 4.04 (s, 2H), 3.98 - 3.92 (m, 3H), 3.91 - 3.95 (m, 4H), 3.38 (s, 4H), 2.92 (s, 8H), 2.46 (s, 3H), 2.20 - 2.11 (m, 2H), 1.68 - 1.54 (m, 4H), 1.48 -1.24 (m, 8H). MS (ESI) [M+H]+ = 905.6.
Figure imgf000196_0002
Example 246: (E)-l-(5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-l//-benzo[d|imidazol-2- yl)oxy)-2-methylbenzamido)-N -(2-(5-(4-(4-(dimethylamino)but-2-enoyl)-2-oxopiperazin-l- yl)thiophen-2-yl)ethyl)-3,6,9,12,15-pentaoxaoctadecan-18-amide (QC179-050) Example 246was synthesized following the same procedure for preparing example 243 from linker 27. White solid, 8 mg, 40% yield. 1H NMR (400 MHz, CD3OD) 8 7.58 (s, 1H), 7.52 (s, 1H), 7.45 - 7.37 (m, 5H), 7.27 - 7.21 (m, 2H), 7.00 - 6.87 (m, 1H), 6.77 (s, 1H), 6.69 (s, 1H), 6.65 (s, 1H), 6.49 (s, 1H), 4.49 (s, 1H), 4.43 (s, 1H), 4.03 (s, 2H), 3.98 - 3.93 (m, 3H), 3.87 (s, 4H), 3.66 - 3.50 (m, 26H), 2.92 (s, 8H), 2.47 (s, 3H). MS (ESI) [M+H]+ = 1141.6.
Scheme 18. The syntheses of example 247
Figure imgf000197_0001
Example 247
Example 247: (£)-A-(2-((5-((6-chloro-5-(l-methyl-l//-indol-5-yl)-l//-benzo[J|imidazol-2- yl)oxy)-2-methylbenzoyl)tetramethyl-l7-azaneyl)ethyl)-3-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propenamide (QC179-051) Linker 32 (14 mg, 0.020 mmol, 1.0 eq), intermediate 13 (11 mg, 0.022 mmol, 1.1 eq), EDC HC1 (6 mg, 0.030 mmol, 1.5 eq), HOAt (4 mg, 0.030 mmol, 1.5 eq), NMM (6 mg, 0.060 mmol, 3.0 eq) were stirred in 1 mL DMF at room temperature for 2 h. Then the mixture was purified via Prep-HPLC to yield titled compound as a white solid (8 mg, 40% yield). 'H NMR (400 MHz, CD3OD) δ 7.57 (s, 1H), 7.51 (s, 1H), 7.45 - 7.36 (m, 5H), 7.27 - 7.21 (m, 2H), 6.93 (dd, J= 31.9, 14.9 Hz, 1H), 6.76 (s, 1H), 6.63 (s, 2H), 6.49 (s, 1H), 4.49 (s, 1H), 4.43 (s, 1H), 4.03 (s, 2H), 3.99 - 3.93 (m, 3H), 3.87 (s, 4H), 3.15 (s, 2H), 3.08 - 3.00 (m, 2H), 2.92 (s, 6H), 2.54 - 2.44 (m, 5H), 1.61 (s, 2H), 1.53 - 1.28 (m, 8H). MS (ESI) [M+H]+ = 877.4.
Figure imgf000197_0002
Example 248: (E)-A-(2-((5-((6-chloro-5-(l-methyl-17/-indol-5-yl)-1H-benzo[cZ|imidazol-2- yl)oxy)-2-methylbenzoyl)pentamethyl-l8-azaneyl)ethyl)-3-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propenamide (QC179-052) Example 248 was synthesized following the same procedure for preparing example 247 from linker 33. White solid 7 mg, 43% yield. 1H NMR (400 MHz, CD3OD) 8 7.58 (s, 1H), 7.51 (s, 1H), 7.43 - 7.36 (m, 5H), 7.27 - 7.21 (m, 2H), 7.01 - 7.88 (m, 1H), 6.76 (s, 1H), 6.64 (s, 2H), 6.49 (s, 1H), 4.51 - 4.37 (m, 2H), 4.08 - 4.02 (m, 2H), 3.98 - 3.93 (m, 3H), 3.87 (s, 4H), 3.18 - 3.11 (m, 2H), 3.07 -3.01 (m, 2H), 2.92 (s, 6H), 2.54 - 2.43 (m, 5H), 1.62 - 1.32 (m, 12H). MS (ESI) [M+H]+ = 891.4.
Figure imgf000198_0001
Example 249: (E)-N-(2-((5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzo[d|imidazol-2- yl)oxy)-2-methylbenzoyl)hexamethyl-l9-azaneyl)ethyl)-3-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propenamide (QC179-053) Example 249 was synthesized following the same procedure for preparing example 247 from linker 34. White solid, 10 mg, 48% yield. 'H NMR (400 MHz, CD3OD) δ 7.58 (s, 1H), 7.51 (s, 1H), 7.44 - 7.35 (m, 5H), 7.27 -7.21 (m, 2H), 6.95 (dd, J = 35.2, 15.7 Hz, 1H), 6.77 (s, 1H), 6.67 - 6.47 (m, 3H), 4.51 - 4.38 (m, 2H), 4.05 - 3.85 (m, 9H), 3.17 - 3.05 (m, 4H), 2.92 (s, 6H), 2.52 - 2.44 (m, 5H), 1.62 (s, 2H), 1.43 - 1.23 (m, 12H). MS (ESI) [M+H]+ = = 905.4.
Figure imgf000198_0002
Example 250: (E)-N-(2-((5-((6-chloro-5-(l-methyl-1H-indol-5-yl)-1H-benzo[d|imidazol-2- yl)oxy)-2-methylbenzoyl)heptamethyl-l10-azaneyl)ethyl)-3-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propenamide (QC179-054) Example 250 was synthesized following the same procedure for preparing example 247 from linker 35. White solid 9 mg, 50% yield. 'HNMR (400 MHz, CD3OD) δ 7.58 - 7.14 (m, 10H), 7.02 - 6.88 (m, 1H), 6.77 (s, 1H), 6.66 - 6.49 (m, 2H), 4.41 (s, 2H), 4.03 - 3.71 (m, 9H), 3.13 - 2.99 (m, 4H), 2.91 (s, 6H), 2.53 - 2.44 (m, 5H), 1.60 (s, 2H), 1.41 - 1.23 (m, 14H). MS (ESI) [M+H]+ = 919.3.
Figure imgf000198_0003
Example 251: (E )-N-(2-((5-((6-chloro-5-(1-methyl-1H-indol-5-yl)-1H-benzo|d|imidazol-2- yl)oxy)-2-methylbenzoyl)octamethyl-l11-azaneyl)ethyl)-3-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propenamide (QC179-055) Example 251 was synthesized following the same procedure for preparing example 247 from linker 36. White solid 7 mg, 40% yield. ’H NMR (400 MHz, CD3OD) 8 7.58 (s, 1H), 7.52 (s, 1H), 7.45 - 7.34 (m, 5H), 7.28 - 7.21 (m, 2H), 6.94 (dd, J= 34.8, 15.2 Hz, 1H), 6.78 (s, 1H), 6.67 - 6.60 (m, 2H), 6.49 (s, 1H), 4.49 (s, 1H), 4.43 (s, 1H), 4.04 (s, 2H), 3.99 - 3.85 (m, 7H), 3.16 - 3.03 (m, 4H), 2.92 (s, 6H), 2.55 - 2.42 (m, 5H), 1.64 (s, 2H), 1.47 - 1.22 (m, 16H). MS (ESI) [M+H]+ = 933.5.
Procedures for the synthesis of cGAS based bivalent compounds
Scheme 19. The syntheses of linker 42
Figure imgf000199_0001
Linker 42: 2-amino-A-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indoI-9-yl)-1H-pyrazol-l-yl)ethyl)acetamide To a solution of 3-(4, 4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)-177-pyrazole (7.3 g, 37.6 mmol, 1.0 eq) in MeCN (30ml) was added K2CO3 (15.6 g, 112.9 mmol, 3.0 eq) and tert-butyl (2-bromoethyl)carbamate (10.1 g, 45.1 mmol, 1.2 eq). The mixture was stirred at 80 °C for 6h. After that, water was added and the mixture was extracted with ethyl acetate. The organic layer was collected, and excess solvent was removed to get intermediate 16 and used for the next step directly without further purification.
To a solution of intermediate 17 (1.05 g, 2.5 mmol, 1 eq) (prepared following previous reported lit.) (Lama et al., 2019) and intermediate 16 (3.2 g, 9.5 mmol, 3.8 eq) in dioxane (40 ml) were added KOAc (981 mg, 10.0 mmol, 4.0 eq) and Pd(dppf)Cl2(408 mg, 0.5 mmol, 0.2 eq). The mixture was stirred at 100 °C for 6 h under N2 followed concentrated under reduced pressure. The resulted residue was purified by column chromatography (SiCL, DCM/MeOH = 10: 1) to give intermediate 18 as a brown oil (894 mg, 65% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.88 (s, 1H), 7.32 (d, J = 9.7 Hz, 1H), 6.70 (dd, J = 27.9, 2.3 Hz, 1H), 4.82 (d, J = 61.8 Hz, 2H), 4.68 - 4.36 (m, 4H), 3.76 (dt, J= 45.2, 5.9 Hz, 2H), 2.89 (dt, J= 46.7, 5.8 Hz, 2H), 2.07 (d, J= 1.9 Hz, 5H), 1.11 (s, 9H).
To a solution of intermediate 18 (420 mg, 0.76 mmol, 1 eq) in MeOH/H2O (10:1, 4.4 ml) was added LiOH.H2O (91 mg, 2.3 mmol, 3 eq). The mixture was stirred at rt for 10 min followed by concentrated and diluted with H2O. After that, the pH value of the solution was adjusted to 6 with HC1 aq. solution (IM) followed by concentration. The residue was re-dissolved in DCM/TFA (2: 1, 7.5 mL). After stirring at rt for Ih, excess solvent was removed and the residue was purified by prep-HPLC to give intermediate 19 as a yellow solid, (178.6 mg, 45% yield). 1 H NMR (400 MHz, CD3OD) 8 7.77 (d, J= 13.4 Hz, IH), 7.23 (dd, J = 9.9, 3.0 Hz, IH), 6.56 (d, J= 9.3 Hz, IH), 4.73 - 4.47 (m, 4H), 4.40 - 4.13 (m, 2H), 3.97 - 3.46 (m, 4H), 3.07 - 2.81 (m, 2H).
To a solution of succinic acid (35.4mg, 0.3 mmol, 2.0 eq) in DMF (0.75 mL) and DCM (18 mL) was added NMM (75.9mg, 0.75 mmol, 5.0 eq), intermediate 18 (78.3mg, 0.15 mmol, 1.0 eq), HO At (24.5mg, 0.18 mmol, 1.2 eq), and EDCI (34.5mg, 0.18 mmol, 12eq) at 0°C. The resulted reaction solution was stirred at 0 °C for 6 h before being stirred at room temperature overnight followed by concentrated. The resulted residue was purified by reverse-phase chromatography to yield the titled product (38.1 mg, 50% yield). ’H NMR (400 MHz, CD3OD) 87.78 - 7.65 (m, IH), 7.21 (d, J= 18.1 Hz, IH), 6.49 (dd, J= 23.8, 2.3 Hz, IH), 4.55 (d, J= 7.0 Hz, 2H), 4.42 - 4.16 (m, 4H), 3.99 - 3.61 (m, 4H), 2.94 (dt, J = 22.2, 5.8 Hz, 2H), 2.59 (t, J = 6.8 Hz, 2H), 2.46 (dt, J = 10.8, 6.7 Hz, 2H).
Linker 43 - 55 were synthesized following the same procedure for preparing linker 42.
Figure imgf000201_0001
J= 25.4, 2.3 Hz, 1H), 4.65 - 4.17 (m, 6H), 3.79 - 3.56 (m, 4H), 3.02 - 2.74 (m, 2H), 2.40 - 2.03 (m, 4H), 1.40 (s, 6H).
Figure imgf000202_0001
Linker 48: 10-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3- b]indol-9-yl)-l/Z-pyrazol-l-yl)ethyl)amino)-10-oxodecanoic acid. Brown solid, yield 60%. I I NMR (400 MHz, CD3OD) 6 7.65 (dd, J= 20.7, 2.3 Hz, 1H), 7.20 (d, J= 14.7 Hz, 1H), 6.48 (dd, J= 24.8, 2.3 Hz, 1H), 4.65 - 4.17 (m, 6H), 3.97 - 3.56 (m, 4H), 2.92 (d, J= 27.6 Hz, 2H), 2.18 (t, J= 59.4 Hz, 4H), 1.67 - 1.04 (m, 12H).
Figure imgf000203_0001
Linker 49: 1 l-((2-(3-(6.7-dichloro-2-(2-hydroxya cetyl)-2.3.4.5-tetrahydro-l//-pyrido|4.3- />|indol-9-yl)-l//-pyrazol-l-yl)ethyl)amino)- 11-oxoundecanoic acid Brown solid, yield 47%. *H NMR (400 MHz, CD3OD) 8 7.65 (dd, J = 20.6, 2.3 Hz, 1H), 7.21 (d, J = 13.3 Hz, 1H), 6.49 (dd, J= 24.3, 2.3 Hz, 1H), 4.70 - 4.10 (m, 6H), 3.97 - 3.59 (m, 1H), 3.05 - 2.80 (m, 2H), 2.30 - 2.03 (m, 4H), 1.64 - 0.92 (m, 14H).
Figure imgf000203_0002
Linker 50: 12-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3- />Jindol-9-yl)-l//-pyrazol-l-yl)ethyl)amino)-12-oxododecanoic acid. Brown solid, yield 45%. JH NMR (400 MHz, CD3OD) 8 7.65 (dd, J = 21.4, 2.0 Hz, 1H), 7.21 (d, J = 13.7 Hz, 1H), 6.48 (dd, J= 25.4, 2.2 Hz, 1H), 4.70 - 4.18 (m, 6H), 4.00 - 3.56 (m, 4H), 3.09 - 2.81 (m, 2H), 2.38 - 2.02 (m, 4H), 1.64 - 0.93 (m, 16H).
Figure imgf000203_0003
Linker 51: 3-(3-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3- b]indol-9-yl)-1H-pyrazol-l-yl)ethyl)amino)-3-oxopropoxy)propanoic acid. Brown solid, yield 46%. 1H NMR (400 MHz, CD3OD) 8 7.69 (dd, J= 15.3, 2.3 Hz, 1H), 7.20 (d, J= 17.8 Hz, 1H), 6.49 (dd, J = 23.4, 2.3 Hz, 1H), 4.62 - 4.17 (m, 6H), 3.97 - 3.56 (m, 8H), 3.02 - 2.82 (tn, 2H), 2.57 - 2.31 (m, 4H).
Figure imgf000204_0001
Linker 52: 3-(2-(3-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-6]indol-9-yl)-l//-pyrazol-l-yl)ethyl)amino)-3-oxopropoxy)ethoxy)propanoic acid Brown solid, yield 49%. 'H NMR (400 MHz, CD3OD) 8 7.70 (dd, J= 17.0, 2.2 Hz, 1H), 7.20 (d, J= 16.8 Hz, 1H), 6.48 (dd, J = 24.2, 2.3 Hz, 1H), 4.62 - 4.15 (m, 6H), 3.97 - 3.42 (m, 12H), 3.01 - 2.82 (m, 2H), 2.54 - 2.32 (m, 4H).
Figure imgf000204_0002
Linker 53: l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-i]indol- 9-yl)-1H-pyrazol-l-yl)-4-oxo-7,10,13-trioxa-3-azahexadecan-16-oic acid. Brown solid, yield .40 (m,
Figure imgf000204_0003
Linker 54: l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3-b]indol- 9-yl)-1H-pyrazol-l-yl)-4-oxo-7,10,13,16-tetraoxa-3-azanonadecan-19-oic acid. Brown solid, yield 53 %. ’H NMR (400 MHz, CD3OD) 5 7.80 - 7.66 (m, 1H), 7.23 (d, J= 17.3 Hz, 1H), 6.52 (dd, J= 24.5, 2.3 Hz, 1H), 4.63 - 4.17 (m, 6H), 3.79 - 3.47 (m, 20H), 3.01 - 2.86 (m, 2H), 2.63 - 2.28 (m, 4H).
Figure imgf000205_0001
Linker 55: l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3-/>]indol- 9-yl)-lJ/-pyrazol-l-yl)-4-oxo-7,10,13,16,19-pentaoxa-3-azadocosan-22-oic acid. Brown solid, yield 47 %. 'H NMR (400 MHz, CD3OD) δ 7.79 - 7.49 (m, 1H), 7.11 (d, J = 17.3 Hz, 1H), 6.40 (dd, J= 24.3, 2.3 Hz, 1H), 4.54 - 4.10 (m, 6H), 3.73 - 3.38 (m, 24H), 2.92 - 2.76 (m, 2H), 2.51 - 2.14 (m, 4H).
Scheme 20. The syntheses of linker 56
Figure imgf000205_0002
Intermediate 19 Linker 56
Linker 56: 2-amino-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indoI-9-yl)-l/7-pyrazol-l-yl)ethyl)acetamide To a solution of intermediate 19 (65 mg, 0. 12 mmol) in DMF (0.5 mL) were added A-Boc glycine (19.2 mg, 0.12 mmol, 1.0 equiv), 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 34.3 mg, 0.18 mmol, 1.5 equiv), 1- hydroxy-7-azabenzo-triazole (HOAt, 24.5 mg, 0.18 mmol, 1.5 equiv), and A-methylmorpholine (NMM, 36.4 mg, 0.36 mmol, 3.0 equiv). After being stirred overnight at rt, the resulting mixture was purified by prep-HPLC to afford crude product. The crude product was dissolved in DCM/TFA (1 :1, 2 mL) followed by stirred at rt for 30 min. The resulting mixture was purified by prep-HPLC to afford title compound as yellow solid (44 mg, 63% yield). 'H NMR (400 MHz, CD3OD) 8 7.78 - 7.65 (m, 1H), 7.21 (d, J= 17.2 Hz, 1H), 6.51 (dd, J= 26.0, 2.0 Hz, 1H), 4.60 - 4.50 (m, 2H), 4.44 - 4.30 (m, 4H), 3.00 - 2.87 (m, 6H), 7.78 - 7.65 (m, 2H).
Linker 57 - 70 were synthesized following the same procedure for preparing linker 56.
Figure imgf000206_0001
Linker 58: 4-amino-JV-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-l/7-pyrazol-l-yl)ethyl)butanamide. Brown solid, yield 41%. 1HNMR (400 MHz, CD3OD) 87.67 (dd, J= 18.2, 2.3 Hz, 1H), 7.20 (d, J= 17.4 Hz, 1H), 6.49 (dd, J= 28.0, 2.3 Hz, 1H), 4.61 - 4.30 (m, 6H), 3.81 - 3.58 (m, 4H), 3.03 - 2.77 (m, 4H), 2.35 (t, J= 7.1 Hz, 2H), 2.01 - 1.80 (m, 2H).
Figure imgf000207_0001
Linker 59: 5-amino-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-6]indol-9-yl)-1H-pyrazol-l-yl)ethyl)pentanamide. Brown solid, yield 44%. JH NMR (400 MHz, CD3OD) 5 7.78 - 7.58 (m, 1H), 7.21 (d, J= 14.0 Hz, 1H), 6.67 - 6.29 (m, 1H), 4.65 - 4.18 (m, 6H), 4.02 - 3.61 (m, 4H), 4.02 - 3.62 (m, 4H), 2.29 - 2.21 (m, 2H), 1.72 - 1.59
Figure imgf000207_0002
Linker 60: 6-amino-N -(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-6]indol-9-yl)-LH-pyrazol-l-yl)ethyl)hexanamide. Brown solid, yield 47%. ’l l NMR (400 MHz, CD3OD) 8 7.68 (dd, J = 20.8, 2.3 Hz, 1H), 7.20 (d, J = 14.7 Hz, 1H), 6.50 (dd, J = 24.4, 2.3 Hz, 1H), 4.65 - 4.30 (m, 6H), 3.75 - 3.66 (m, 4H), 2.98 - 2.84 (m, 4H), 2.30 - 2.02 (m, 2H), 1.65 - 1.53 (m, 4H), 1.44 - 1.24 (m, 2H).
Figure imgf000207_0003
Linker 61: 7-amino-N -(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-1H-pyrazol-l-yl)ethyl)heptanamide. Brown solid, yield 43%. 1H NMR (400 MHz, CD3OD) 8 7.70 - 7.60 (m, 1H), 7.20 (d, J= 14.3 Hz, 1H), 6.50 (dd, J= 23.5, 2.3 Hz, 1H), 4.70 - 4.29 (m, 6H), 3.82 - 3.60 (m, 4H), 3.04 - 2.73 (m, 4H), 2.29 - 2.06 (m, 2H), 1.78
Figure imgf000208_0001
Linker 64: 10-amino-/V-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro1H - pyrido[4,3-^]indoI-9-yl)-l/7-pyrazol-l-yl)ethyl)decanamide. Brown solid, yield 49%. 1HNMR (400 MHz, CD3OD) 87.66 (dd, J= 21.3, 2.3 Hz, 1H), 7.21 (d, J= 12.8 Hz, 1H), 6.49 (dd, J= 24.3,
2.3 Hz, 1H), 4.56 - 4.16 (m, 6H), 3.88 - 3.49 (m, 4H), 2.81 (dq, J= 28.2, 7.7, 6.7 Hz, 4H), 2.05 (dt, J= 16.3, 7.5 Hz, OH), 1.60 - 1.03 (m, 14H).
Figure imgf000209_0001
Linker 66: 3-(2-aminoethoxy)-JV-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro- l//-pyrido|4,3-B)|indol-9-yl)- l//-pyrazol-l-yl)ethyl)propenainide. Brown solid, yield 42%. 1H NMR (400 MHz, CD3OD) 8 7.69 (dd, J = 17.1, 2.3 Hz, 1H), 7.21 (d, J= 16.7 Hz, 1H), 6.51 (dd, .7= 26.1, 2.3 Hz, 1H), 4.62 - 4.29 (m, 6H), 3.83 - 3.55 (m, 8H), 3.18 - 2.85 (m, 4H), 2.61 - 2.23 (m, 2H).
Figure imgf000210_0001
Linker 67: 3-(2-(2-aminoethoxy)ethoxy)-N -(2-(3-(6,7-dichIoro-2-(2-hydroxyacetyl)-2,3,4,5- tetrahydro- 1H-pyrido[4,3-Bbindol-9-yl)-lH-pyrazol-l-yl)ethyl)propenamide. Brown solid, yield 45%. 'H NMR (400 MHz, CD3OD) 8 7.73 - 7.67 (m, 1H), 7.21 (d, J= 16.0 Hz, 1H), 6.51 (dd, J= 24.7, 2.3 Hz, 1H), 4.65 - 4.30 (m, 6H), 3.80 - 3.44 (m, 12H), 3.13 - 2.85 (m, 4H), 2.54 - 2.34 (m, 2H).
Figure imgf000210_0002
Linker 68: 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-2V-(2-(3-(6,7-dichloro-2-(2- hydroxyacetyl)-2,3,4,5-tetrahydr 0-1H-pyr ido [4 ,3-b] ind ol-9-y 1)-1H-pyrazol- 1- yl)ethyl)propenamide. Brown solid, yield 46%. 1H NMR (400 MHz, CD3OD) 6 7.73 - 7.68 (m, 1H), 7.22 (d, J= 15.6 Hz, 1H), 6.51 (dd, J= 25.8, 2.3 Hz, 1H), 4.65 - 4.31 (m, 6H), 3.78 - 3.48 (m, 16H), 3.18 - 2.87 (m, 4H), 2.52 - 2.28 (m, 2H).
Figure imgf000211_0001
Linker 69: l-amino-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-6]indol-9-yl)-1H-pyrazol-l-yl)ethyl)-3,6,9,12-tetraoxapentadecan-15-amide.
Brown solid, yield 42 %. 'H NMR (400 MHz, CD3OD) δ 7.68 (dd, J= 12.8, 2.3 Hz, 1H), 7.22 (d, J= 16.5 Hz, 1H), 6.51 (dd, J= 25.3, 2.3 Hz, 1H), 4.65 - 4.31 (m, 6H), 3.78 - 3.51 (m, 20H), 3.17 - 2.82 (m, 4H), 2.57 - 2.31 (m, 2H).
Figure imgf000211_0002
Linker 70: l-amino-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-bbindol-9-yl)-lH-pyrazol-l-yl)ethyl)-3,6,9,12,15-pentaoxaoctadecan-18-amide.
Brown solid, yield 44%. 'H NMR (400 MHz, CD3OD) 5 7.74 - 7.66 (m, 1H), 7.22 (d, J = 17.0 Hz, 1H), 6.51 (dd, J= 25.1, 2.3 Hz, 1H), 4.65 - 4.30 (m, 6H), 3.79 - 3.48 (m, 24H), 3.18 - 3.04 (m, 4H), 2.55 - 2.34 (m, 2H).
Scheme 21. The syntheses of linker 71
Figure imgf000212_0001
ethyl 2-bromoacetate
Intermediate 20
Figure imgf000212_0002
Linker 71: 7V-(2-aminoethyl)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-/>]indol-9-yl)-1H-pyrazol-l-yl)acetamide (XH168-108) To a solution of 3-(4, 4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)-l//-pyrazole (7.3 g, 37.6 mmol, 1.0 eq) in MeCN (30ml) was added K2CO3 (15.6 g, 112.9 mmol, 3.0 eq) and tert-butyl 2-bromoacetate (7.5 g, 45.1 mmol, 1.2 eq). The mixture was stirred at 80 °C for 6h. After that, water was added and the mixture was extracted with ethyl acetate. The organic layer was collected, and excess solvent was removed to get intermediate 20 and used for the next step directly without further purification.
To a solution of intermediate 17 (1.05 g, 2.5 mmol, 1 eq) and intermediate 20 (2.7 g, 9.5 mmol, 3.8 eq) in dioxane (40 ml) was added KOAc (981.4 mg, 10.0 mmol, 4.0 eq) and Pd(dppf)C12 CH2C12 (408 mg, 0.5 mmol, 0.2 eq). The mixture was stirred at 100 °C for 6 h under N2. Ater cooling down to room temperature, the reaction mixture was concentrated under reduced pressure and purified by column chromatography (SiCh, DCM/MeOH = 10: 1) to give intermediate 21 as a brown oil, 68% yield.
To a solution of intermediate 21 (840 mg, 1.7 mmol, 1 eq) in MeOH (20 ml) and H2O (5 ml) was added LiOH (214 mg, 5.1 mmol, 3 eq). After stirred at rt for 10 min, excess MeOH was removed, resulted residue was diluted with H2O (2 mL) and the pH value was adjusted to 6 with HC1 aq. (IM) followed by purified by prep-HPLC to yield intermediate 22 as a yellow solid (456 mg, 33% in 2 steps). 'H NMR (400 MHz, CD3OD) 5 7.72 (d, J= 2.3 Hz, 1H), 7.24 (s, 1H), 6.53 (d, J= 2.3 Hz, 1H), 4.89 - 4.84 (m, 2H), 4.63 - 4.49 (m, 2H), 4.37 - 4.26 (m, 2H), 4.01 - 3.70 (m, 2H), 3.01 - 2.85 (m, 2H).
To a solution of intermediate 22 (65 mg, 0.12 mmol) in DMF (0.5 mL) were added tertbutyl (2- aminoethyl)carbamate (19.2 mg, 0.12 mmol, 1.0 equiv), l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI, 34.3 mg, 0.18 mmol, 1.5 equiv), l-hydroxy-7- azabenzo-tri azole (HO At, 24.5 mg, 0.18 mmol, 1.5 equiv), and N-m ethyl morpholine (NMM, 36.4 mg, 0.36 mmol, 3.0 equiv). After being stirred for overnight atrt, the resulting mixture was purified by prep-HPLC to afford crude product. The crude product was dissolved in DCM/TFA (1 : 1, 2 mL) followed by stirred at rt for 30 min. The resulting mixture was purified by prep-HPLC to afford titled compound as yellow solid (44 mg, 63% yield). 1H NMR (400 MHz, CD3OD) 5 7.77 (s, 1H), 7.14 (s, 1H), 6.51 (s, 1H), 5.02 - 4.97 (m, 2H), 4.58 (s, 1H), 4.32 (s, 1H), 4.17 (s, 1H), 3.92 (s, 1H), 3.64 - 3.53 (m, 1H), 3.52 - 3.49 (m, 2H), ), 3.29 - 3.23 (m, 1H), 3.06 (s, 2H), 2.89 - 2.81 (m, 2H).
Linker 72 - 84 were synthesized following the same procedure for preparing linker 71.
Figure imgf000213_0001
Linker 72: N -(3-aminopropyl)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro- lH-pyrido[4,3-/>]indol-9-yl)-LH-pyrazol-l-yl)acetamide (xhl68-109). Yellow solid, 53% yield 'H NMR (400 MHz, CD3OD) δ 7.74 (s, 1H), 7.19 (s, 1H), 6.54 (s, 1H), 4.96 (s, 2H), 4.64 (s, 1H), 4.41 (s, 1H), 4.35 (s, 1H), 4.21 (s, 1H), 3.91 (s, 1H), 3.67 (s, 1H), 3.34 (s, 3H), 2.97 - 2.87 (m, 3H), 1.92 - 1.75 (m, 2H).
Figure imgf000214_0001
Linker 75: N-(6-aminohexyl)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-1H-pyrazol-l-yl)acetamide (xhl68-114). Yellow solid, 38% yield. 1H NMR (400 MHz, CD3OD) 8 7.73 (s, 1H), 7.20 (s, 1H), 6.54 (s, 1H), 4.93 (s, 2H), 4.71 - 4.57 (m,
1H), 4.47 - 4.37 (m, 1H), 4.35 - 4.28 (m, 1H), 4.28 - 4.16 (m, 1H), 4.00 - 3.79 (m, 1H), 3.70 - 3.67 (m, 1H), 3.30 - 3.21 (m, 2H), 2.92 - 2.85 (m, 4H), 1.62 - 1.51 (m, 4H), 1.38 - 1.33 (m, 4H).
Figure imgf000215_0001
Linker 76: JV-(7-aminoheptyl)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-/;|indol-9-yl)-l1H-pyrazol-1 -yl)acetaniide (xhl68-115). Yellow solid, 35% yield
NMR (400 MHz, CD3OD) δ 7.74 (s, 1H), 7.20 (s, 1H), 6.54 (s, 1H), 4.92 (s, 2H), 4.68 - 4.56
(m, 1H), 4.45 - 4.34 (m, 1H), 4.35 - 4.28 (m, 1H), 4.26 - 4.18 (m, 1H), 3.92 - 3.89 (m, 1H), 3.69 - 3.66 (m, 1H), 3.23 - 3.18 (m, 2H), 2.93 - 2.82 (m, 4H), 1.62 - 1.47 (m, 4H), 1.32 - 1.29 (m, 6H).
Figure imgf000215_0002
Linker 77: Af-(8-aminooctyl)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H pyrido[4,3-b]indol-9-yl)-1H-pyrazol-l-yl)acetamide (xhl 68-116). Yellow solid, 33% yield. 1H NMR (400 MHz, CD3OD) 8 7.73 (s, 1H), 7.19 (s, 1H), 6.53 (s, 1H), 4.92 (s, 2H), 4.67 - 4.60 (m, 1H), 4.43 - 4.37 (m, 1H), 4.36 - 4.28 (m, 1H), 4.26 - 4.18 (m, 1H), 3.99 - 3.83 (m, 1H), 3.75 - 3.62 (m, 1H), 3.27 - 3.16 (m, 2H), 2.96 - 2.74 (m, 4H), 1.71 - 1.42 (m, 4H), 1.41 - 1.16 (m, 8H).
Figure imgf000216_0001
Linker 78: JV-(9-aminononyl)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-1H-pyrazol-l-yl)acetamide (xhl68-117). Yellow solid, 79% yield. 1H NMR (400 MHz, CD3OD) 8 7.72 (s, 1H), 7.17 (s, 1H), 6.52 (s, 1H), 4.92 (s, 2H), 4.74 - 4.56 (m, 1H), 4.50 - 4.38 (m, 1H), 4.31 - 4.21 (m, 2H), 4.00 - 3.84 (m, 1H), 3.75 - 3.59 (m, 1H), 3.27 - 3.09 (m, 2H), 3.03 - 2.68 (m, 4H), 1.61 - 1.46 (m, 4H), 1.33 - 1.20 (m, 10H).
Figure imgf000216_0002
Linker 79: N-(10-aminodecyl)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido|4.3-bindol-9-yl)- 1 //-pyrazol-1 -yl)acetaniide (xhl68-118). Yellow solid, 39 mg, 47% yield. 'H NMR (400 MHz, CD3OD) 8 7.73 (s, 1H), 7.20 (s, 1H), 6.54 (s, 1H), 4.92 (s, 2H), 4.71 - 4.60 (m, 1H), 4.49 - 4.40 (m, 1H), 4.38 - 4.31 (m, 1H), 4.31 - 4.18 (m, 1H), 3.97 - 3.91 (m, 1H), 3.76 - 3.62 (m, 1H), 3.28 - 3.11 (m, 2H), 3.03 - 2.79 (m, 4H), 1.63 - 1.45 (m, 4H), 1.35 - 1.21
Figure imgf000216_0003
Linker 80: N-(2-(2-aminoethoxy)ethyl)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5- tetrahydro-1H-pyrido[4,3-^]indoI-9-yl)-1H-pyrazol-l-yl)acetamide (xhl68-119). Yellow solid, 53% yield. ’H NMR (400 MHz, CD3OD) 8 7.72 (s, 1H), 7.18 (s, 1H), 6.52 (s, 1H), 4.97 -
4.96 (m, 2H), 4.72 - 4.60 (m, 1H), 4.41 - 4.35 (m, 2H), 4.29 - 4.14 (m, 1H), 3.91 - 3.88 (m, 1H),
3.66 - 3.63 (m, 3H), 3.60 - 3.51 (m, 2H), 3.51 - 3.39 (m, 2H), 3.16 - 3.00 (m, 2H), 2.99 - 2.78
(m, 2H).
Figure imgf000217_0001
Linker 81: N -(2-(2-(2-aminoethoxy)ethoxy)ethyl)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-
2.3.4.5-tetrahydro- 1 //-pyrido|4.3-b)|indol-9-yl)-l //-pyrazol-1 -yl)acetamide (xhl68-120).
Yellow solid, 46% yield. 'H NMR (400 MHz, CD3OD) 8 7.72 (s, 1H), 7.17 (s, 1H), 6.51 (s, 1H), 4.96 - 4.94 (m, 2H), 4.68 - 4.58 (m, 1H), 4.50 - 4.39 (m, 1H), 4.39 - 4.31 (m, 1H), 4.30 - 4.22 (m, 1H), 3.91 - 3.88 (m, 1H), 3.69 - 3.48 (m, 9H), 3.47 - 3.36 (m, 2H), 3.12 - 2.97 (m, 2H), 2.95 - 2.79 (m, 2H).
Figure imgf000217_0002
Linker 82: JV-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-2-(3-(6,7-dichloro-2-(2- hydroxyacetyl)-2, 3, 4, 5- tetrahydro-1H-pyrido[4,3-b]indol-9-yl)-1H-pyrazol-l-yl)acetamide (xhl68-121). Yellow solid, 30% yield. 'H NMR (400 MHz, CD3OD) 8 7.73 (s, 1H), 7.18 (s, 1H), 6.53 (s, 1H), 4.96 - 4.90 (m, 2H), 4.68 - 4.60 (m, 1H), 4.54 - 4.43 (m, 1H), 4.38 - 4.30 (m, 1H), 4.30 - 4.19 (m, 1H), 4.00 - 3.84 (m, 1H), 3.73 - 3.45 (m, 13H), 3.43 - 3.35 (m, 2H), 3.07 - 3.03 (m, 2H), 2.92 - 2.85 (m, 2H).
Figure imgf000218_0001
Linker 84: JV-(17-amino-3,6,9,12,15-pentaoxaheptadecyl)-2-(3-(6,7-dichloro-2-(2- hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indol-9-yl)-1H-pyrazol-l-yl)acetamide (xhl68-123) Yellow solid, 32% yield. 1H NMR (400 MHz, CD3OD) 8 7.76 (s, 1H), 7.23 (s, 1H), 6.57 (s, 1H), 4.92 (s, 2H), 4.71 - 4.61 (m, 1H), 4.51 - 4.41 (m, 1H), 4.38 - 4.29 (m, 1H), 4.29 - 4.18 (m, 1H), 3.99 - 3.89 (m, 1H), 3.71 - 3.48 (m, 21H), 3.46 - 3.36 (m, 2H), 3.12 - 2.98 (m, 2H), 2.98 - 2.83 (m, 2H).
Scheme 22. The syntheses of linker 85
Figure imgf000219_0001
Intermediate 22 Linker 85
Linker 85: 7V-(2-aminoethyl)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lJ/-pyrazol-l-yl)acetamide (XH168-108). To a solution of Intermediate 22 (50 mg, 0.1 mmol) in DMF (1 mL) were added tert-butyl glycinate (13.1 mg, 0.1 mmol, 1.0 equiv), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 28.7 mg, 0.15 mmol, 1.5 equiv), l-hydroxy-7-azabenzo-triazole (HOAt, 20.4 mg, 0.15 mmol, 1.5 equiv), and N- methylmorpholine (NMM, 30.3 mg, 0.3 mmol, 3.0 equiv). After being stirred at rt overnight, the resulting mixture was puritert by prep-HPLC to afford crude product. The crude product was dissolved in DCM/TFA (1 :1, 2 mL) followed by stirred at rt for 30 min. The resulting mixture was purified by prep- HPLC to afford titled compound as white solid (16 mg, 32% yield). JH NMR (400 MHz, CD3OD) 87.76 (s, 1H), 7.21 (s, 1H), 6.55 (s, 1H), 5.07 - 4.98 (m, 2H), 4.68 - 4.56 (m, 1H), 4.49 - 4.39 (m, 1H), 4.35 - 4.24 (m, 1H), 4.25 - 4.14 (m, 1H), 4.04 - 3.86 (m, 3H), 3.81 - 3.63 (m, 1H), 3.03 - 2.75 (m, 2H).
Linkers 86 - 99 were synthesized following the same procedure for preparing linker 85.
Figure imgf000219_0002
Linker 86: 3-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3- />]indol-9-yl)-lH-pyrazol-l-yl)acetamido)propanoic acid (XH181-71). White solid, 49% yield. White solid, 49%. 1H NMR (400 MHz, CD3OD) δ 7.75 (s, 1H), 7.23 (s, 1H), 6.55 (s, 1H), 4.94 (s, 2H), 4.69 - 4.59 (m, 1H), 4.49 - 4.39 (m, 1H), 4.36 - 4.26 (m, 1H), 4.26 - 4.17 (m, 1H), 4.03 - 3.87 (m, 1H), 3.83 - 3.64 (m, 1H), 3.58 - 3.40 (m, 2H), 3.02 - 2.76 (m, 2H), 2.60 - 2.45 (m, 2H).
Figure imgf000220_0001
Linker 87: 4-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3- b ]indol-9-yl)-1H pyrazol-l-yl)acetamido)butanoic acid (XH181-74). White solid, 70% yield.
NMR (400 MHz, CD30D) 3 7.76 (s, 1H), 7.22 (s, 1H), 6.55 (s, 1H), 4.97 - 4.93 (m, 2H), 4.69 - 4.59 (m, 1H), 4.49 - 4.39 (m, 1H), 4.36 - 4.26 (m, 1H), 4.25 - 4.15 (m, 1H), 4.00 - 3.88 (m, 1H), 3.79 - 3.69 (m, 1H), 3.23 - 3.17 (m, 2H), 3.01 - 2.75 (m, 2H), 2.41 - 2.25 (m, 2H), 1.69 -
Figure imgf000220_0002
Linker 88: 5-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3- b]indol-9-yl)-l//-pyrazol-l-yl)acetamido)pentanoic acid (XH181-75). White solid, 47% yield. ’H NMR (400 MHz, CD3OD) δ 7.75 (s, 1H), 7.22 (s, 1H), 6.55 (s, 1H), 5.01 - 4.90 (m, 2H), 4.73 - 4.61 (m, 1H), 4.48 - 4.38 (m, 1H), 4.37 - 4.28 (m, 1H), 4.26 - 4.14 (m, 1H), 4.00 - 3.88 (m, 1H), 3.81 - 3.69 (m, 1H), 3.27 - 3.19 (m, 2H), 3.01 - 2.78 (m, 2H), 2.41 - 2.24 (m, 2H), 1.72 - 1.45 (m, 4H).
Figure imgf000221_0001
Linker 89: 6-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l1H-pyrido[4,3- b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)hexanoic acid (XH181-151). Yellow solid, 25% yield. ’H NMR (400 MHz, CD3OD) δ 7.74 (s, 1H), 7.21 (s, 1H), 6.55 (s, 1H), 4.99 - 4.91 (m, 2H), 4.71 - 4.60 (m, 1H), 4.50 - 4.37 (m, 1H), 4.37 - 4.26 (m, 1H),4.27 - 4.14 (m, 1H), 4.02 - 3.85 (m, 1H), 3.79 - 3.64 (m, 1H), 3.28 - 3.15 (m, 2H), 3.03 - 2.79 (m, 2H), 2.33 - 2.14 (m, 2H), 1.69 - 1.43 (m, 4H), 1.43 - 1.24 (m, 2H).
Figure imgf000221_0002
Linker 90: 7-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyI)-2,3,4,5-tetrahydro-1H-pyrido[4,3- />]indol-9-yl)-lH-pyrazol-l-yl)acetamido)heptanoic acid (XH181-152). Yellow solid, 27% yield. Yellow solid, 27%. 'H NMR (400 MHz, CD3OD) 5 7.74 (s, 1H), 7.21 (s, 1H), 6.54 (s, 1H), 4.99 - 4.90 (m, 2H), 4.72 - 4.62 (m, 1H), 4.49 - 4.39 (m, 1H), 4.37 - 4.27 (m, 1H),4.26 - 4.12 (m, 1H), 4.03 - 3.89 (m, 1H), 3.82 - 3.65 (m, 1H), 3.27 - 3.14 (m, 2H), 3.01 - 2.81 (m, 2H), 2.33 - 2.15 (m, 2H), 1.66 - 1.42 (m, 4H), 1.41 - 1.22 (m, 4H).
Figure imgf000222_0001
Linker 91: 8-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l//-pyrido[4,3- bdol-9-yl)- 1 //-pyrazol- l-yl)acetamido)octanoic acid (XH181-81). White solid, 67% yield.
NMR (400 MHz, CD30D) 6 7.75 (s, 1H), 7.23 (s, 1H), 6.55 (s, 1H), 4.99 - 4.89 (m, 2H), 4.71 - 4.59 (m, 1H), 4.48 - 4.36 (m, 1H), 4.35 - 4.26 (m, 1H), 4.24 - 4.15 (m, 1H), 4.00 - 3.87 (m, 1H), 3.82 - 3.65 (m, 1H), 3.26 - 3.16 (m, 2H), 3.01 - 2.77 (m, 2H), 2.36 - 2.18 (m, 2H), 1.67 - 1.42 (m, 4H), 1.39 - 1.26 (m, 6H).
Figure imgf000222_0002
Linker 92: 9-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido [4,3- />]indol-9-yl)-l£Z-pyrazol-l-yl)acetamido)nonanoic acid (XH181-82). White solid, 54% yield. ’H NMR (400 MHz, CD3OD) δ 7.74 (s, 1H), 7.21 (s, 1H), 6.54 (s, 1H), 5.03 - 4.90 (m, 2H), 4.71 - 4.53 (m, 1H), 4.49 - 4.38 (m, 1H), 4.37 - 4.26 (m, 1H), 4.26 - 4.12 (m, 1H), 4.02 - 3.87 (m, 1H), 3.78 - 3.63 (m, 1H), 3.27 - 3.15 (m, 2H), 2.99 - 2.81 (m, 2H), 2.26 - 2.16 (m, 2H), 1.62 - 1.43 (m, 4H), 1.42 - 1.19 (m, 8H).
Figure imgf000222_0003
Linker 93: 10-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l1H-py r ido [4,3- b>]indol-9-yl)-lH-pyrazol-l-yl)acetamido)decanoic acid Yellow solid, 50%. 'HNMR (400 MHz, CD3OD) 5 7.77 (s, 1H), 7.24 (s, 1H), 6.57 (s, 1H), 5.06 - 4.93 (m, 2H), 4.74 - 4.60 (m, 1H), 4.48 - 4.38 (m, 1H), 4.39 - 4.30 (m, 1H), 4.28 - 4.14 (m, 1H), 3.99 - 3.87 (m, 1H), 3.81 - 3.69 (m, 1H), 3.28 - 3.14 (m, 2H), 3.08 - 2.78 (m, 2H), 2.38 - 2.09 (m, 2H), 1.72 - 1.40 (m, 4H), 1.39 -
Figure imgf000223_0001
Linker 95: 3-(2-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H/-pyrido[4,3- bJindol-9-yl)-1H-pyrazol-l-yl)acetamido)ethoxy)propanoic acid (XH181-153). White solid, 35% yield. 1H NMR (400 MHz, CD3OD) δ 7.75 (s, 1H), 7.21 (s, 1H), 6.55 (s, 1H), 5.01 - 4.92 (m, 2H), 4.68 - 4.58 (m, 1H), 4.46 - 4.39 (m, 1H), 4.34 - 4.27 (m, 1H), 4.26 - 4.11 (m, 1H), 3.96 - 3.87 (m, 1H), 3.77 - 3.56 (m, 3H), 3.59 - 3.47 (m, 2H), 3.46 - 3.36 (m, 2H), 3.01 - 2.79 (m, 2H), 2.63 - 2.38 (m, 2H).
Figure imgf000224_0001
Linker 96: 3-(2-(2-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-1H-pyrazol-l-yl)acetamido)ethoxy)ethoxy)propanoic acid (XH181- 154). Yellow solid, 47% yield. 'H NMR (400 MHz, CD3OD) δ 7.75 (s, 1H), 7.22 (s, 1H), 6.55 (s, 1H), 5.04 - 4.93 (m, 2H), 4.70 - 4.56 (m, 1H), 4.50 - 4.40 (m, 1H), 4.37 - 4.26 (m, 1H), 4.24 - 4.16 (m, 1H), 4.00 - 3.89 (m, 1H), 3.76 - 3.60 (m, 3H), 3.60 - 3.45 (m, 6H), 3.45 - 3.36 (m, 2H), 3.01 - 2.80 (m, 2H), 2.62 - 2.40 (m, 2H).
Figure imgf000224_0002
Linker 97: l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3-/>]indol- 9-yl)-lJ7-pyrazol-l-yl)-2-oxo-6,9,12-trioxa-3-azapentadecan-15-oic acid (XH181-155).
Yellow solid, 71 % yield. 'H NMR (400 MHz, CD3OD) δ 7.74 (s, 1H), 7.20 (s, 1H), 6.54 (s, 1H), 5.03 - 4.95 (m, 2H), 4.79 - 4.58 (m, 1H), 4.53 - 4.36 (m, 1H), 4.35 - 4.24 (m, 1H), 4.24 - 4.11 (m, 1H), 4.01 - 3.83 (m, 1H), 3.75 - 3.60 (m, 3H), 3.60 - 3.33 (m, 12H), 3.00 - 2.72 (m, 2H), 2.60 - 2.44 (m, 2H).
Figure imgf000225_0001
Linker 98: l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l//-pyrido[4,3-6]indol-
9-yl)-1H-pyrazol-l-yl)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (XH181-156).
Yellow solid, 61% yield. 'H NMR (400 MHz, CD3OD) 8 7.74 (s, 1H), 7.21 (s, 1H), 6.55 (s, 1H),
4.98 - 4.91 (m, 2H), 4.72 - 4.60 (m, 1H), 4.52 - 4.40 (m, 1H), 4.38 - 4.27 (m, 1H),4.25 - 4.17 (m,
1H), 3.99 - 3.86 (m, 1H), 3.79 - 3.61 (m, 3H), 3.61 - 3.31 (m, 16H), 2.97 - 2.82 (m, 2H), 2.64 -
2.38 (tn, 2H).
Figure imgf000225_0002
Linker 99: l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l//-pyrido[4,3-i]indol- 9-yl)-1H-pyrazol-l-yl)-2-oxo-6,9,12,15,18-pentaoxa-3-azahenicosan-21-oic acid (XH181- 157). Yellow solid, 63% yield. Yellow solid, 71%. 1H NMR (400 MHz, CD3OD) 8 7.75 (s, 1H), 7.21 (s, 1H), 6.55 (s, 1H), 5.01 - 4.93 (m, 2H), 4.70 - 4.61 (m, 1H), 4.52 - 4.42 (m, 1H), 4.38 - 4.26 (m, 1H), 4.26 -4.13 (m, 1H), 4.00 - 3.85 (m, 1H), 3.78 - 3.64 (m, 3H), 3.62 - 3.35 (m, 20H), 2.98 - 2.77 (m, 2H), 2.65 - 2.37 (m, 2H).
Scheme 23. The syntheses of example 252
Figure imgf000226_0001
Example 252
Example 252 : (E)-N-(2-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)ethyl)-3-(5-(4-(4-(dimethylamino)but- 2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propenamide (ZD178-22). To a solution of Linker 71 (0.015 mmol, 1.0 eq) and Intermediate 15 (0.018 mmol, 1.2 eq) in dimethylformamide (1.0 mL) was added HO At (0.03 mmol, 2.0 eq), NMM (0.15 mmol, 10.0 eq), and EDCI (0.03 mmol, 2.0 eq). The resulting reaction solution was stirred at rt for 12 h. Then the resulting residue was purified by reverse-phase chromatography to yield the desired product. Brown solid, 50% yield. 1H NMR (400 MHz, CD3OD) 6 7.83 - 7.71 (m, 1H), 7.34 - 7.18 (m, 1H), 7.04 - 6.69 (m, 2H), 6.60 (d, J = 18.4 Hz, 3H), 5.05 - 4.92 (m, 2H), 4.71 - 4.17 (m, 6H), 4.08 - 3.65 (m, 8H), 3.65 - 3.43 (m, 4H), 3.09 - 2.81 (m, 10H), 2.63 - 2.41 (m, 2H). MS (ESI) [M+H]+ = 812.4.
Examples 253 - 265 were synthesized following the same procedure for preparing example 252 from related linkers and intermediate 15.
Figure imgf000227_0002
Example 254: (E)-N-(4-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)butyl)-3-(5-(4-(4-(dimethylamino)but- 2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propenamide (ZD178-24) Brown solid, 48% yield. ’H NMR (400 MHz, CD3OD) 5 7.82 - 7.72 (m, 1H), 7.40 - 7.17 (m, 1H), 7.01 - 6.64 (m, 2H), 6.68 - 6.50 (m, 3H), 4.98 - 4.90 (m, 2H), 4.72 - 4.14 (m, 6H), 4.03 - 3.63 (m, 8H), 3.25 - 2.84 (m, 14H), 2.60 - 2.36 (m, 2H), 1.93 - 1.27 (m, 4H). MS (ESI) [M+H]+ = 840.3.
Figure imgf000227_0001
Example 255 (E)-N-(5-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)pentyl)-3-(5-(4-(4-(dimethylamino)but- 2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propanamide (ZD178-25) Brown solid, 48% yield. 1H NMR (400 MHz, CD3OD) δ 7.95 - 7.69 (m, 1H), 7.48 - 7.12 (m, 1H), 7.03 - 6.67 (m, 2H), 6.71 - 6.44 (m, 3H), 5.05 - 4.90 (m, 2H), 4.74 - 4.15 (m, 6H), 4.06 - 3.65 (m, 8H), 3.25 - 2.74 (m, 14H), 2.57 - 2.39 (m, 2H), 1.64 - 1.13 (m, 6H). MS (ESI) [M+H]+ = 854.3.
Figure imgf000228_0001
Example 256 (E)-N-(6-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)hexyl)-3-(5-(4-(4-(dimethylamino)but- 2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propenamide (ZD178-26) Brown solid, 52% yield. ’H NMR (400 MHz, CD3OD) 8 8.03 - 7.69 (m, 1H), 7.42 - 7.15 (m, 1H), 7.02 - 6.65 (m, 2H), 6.67 - 6.49 (m, 3H), 5.13 - 4.92 (m, 2H), 4.75 - 4.15 (m, 6H), 4.06 - 3.57 (m, 8H), 3.26 - 2.69 (m, 14H), 2.60 - 2.26 (m, 2H), 1.74 - 0.96 (m, 8H). MS (ESI) [M+H]+ = 868.4.
Figure imgf000228_0002
Example 257 (E)-N-(7-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indoI-9-yl)-lH-pyrazol-l-yl)acetamido)heptyl)-3-(5-(4-(4-(dimethylamino)but- 2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propenamide (ZD178-27) Brown solid, 52% yield. 1H NMR (400 MHz, CD3OD) 8 7.89 - 7.67 (m, 1H), 7.38 - 7.12 (m, 1H), 7.07 - 6.67 (m, 2H), 6.73 - 6.44 (m, 3H), 5.30 - 4.90 (m, 2H), 4.77 - 4.17 (m, 6H), 4.10 - 3.61 (m, 8H), 3.27 - 2.82 (m, 14H), 2.63 - 2.35 (m, 2H), 1.62 - 1.05 (m, 10H). MS (ESI) [M+H] 1 = 882.6.
Figure imgf000228_0003
6.69-6.48 (tn, 3H), 5.07-4.90 (m, 2H), 4.74-4.17 (m, 6H), 4.14-3.57 (m, 8H), 3.27-2.71 (m, 14H), 2.59 - 2.33 (m, 2H), 1.71 - 0.92 (tn, 12H). MS (ESI) [M+H]+ = 896.3.
Figure imgf000229_0001
Example 259 (E)-N-(9-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3?4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)nonyl)-3-(5-(4-(4-(dimethylamino)but- 2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propenamide (ZD178-29, MS7829) Brown solid, 45% yield. 1H NMR (400 MHz, CD3OD) 57.89-7.61 (m, 1H), 7.33-7.13 (m, 1H), 7.03-6.67 (m, 2H), 6.67 - 6.51 (m, 3H), 4.98 - 4.91 (m, 2H), 4.70 - 4.65 (m, 1H), 4.50 - 4.41 (m, 2H), 4.41 -4.31 (m, 2H), 4.25-4.20 (m, 1H), 4.07 - 3.60 (m, 8H), 3.27 -3.17 (m, 2H), 3.15 - 3.07 (m, 2H), 3.07 - 3.00 (m, 2H), 2.95 - 2.84 (m, 8H), 2.60 - 2.40 (m, 2H), 1.70 - 0.99 (m, 14H). MS (ESI) [M+H]+ = 910.4.
Figure imgf000229_0002
Example 261 : (E)-N-(2-(2-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)ethoxy)ethyl)-3-(5-(4-(4- (dimethylamino)but-2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propenamide (ZD178-31)
Brown solid, 47% yield. 1H NMR (400 MHz, CD3OD) δ 7.85 - 7.68 (m, 1H), 7.48 - 7.14 (m, 1H), 7.05 - 6.65 (m, 2H), 6.67 - 6.40 (m, 3H), 5.09 - 4.90 (m, 2H), 4.77 - 4.17 (m, 6H), 4.10 - 3.63 (m, 8H), 3.61 - 3.36 (m, 4H), 3.27 - 3.23 (m, 4H), 3.12 - 2.71 (m, 10H), 2.59 - 2.33 (m, 2H). MS (ESI) [M+H]+ = 856.4
Figure imgf000230_0001
Example 262 : (E)-N-(2-(2-(2-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)ethoxy)ethoxy)ethyl)-3-(5-(4-(4- (dimethylamino)but-2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propenamide (ZD178-32)
Brown solid, 53% yield. 1HNMR (400 MHz, CD3OD) 5 7.89 - 7.70 (m, 1H), 7.30 - 7.14 (m, 1H), 7.00 - 6.69 (m, 2H), 6.67 - 6.49 (m, 3H), 5.10 - 4.92 (m, 2H), 4.72 - 4.15 (m, 6H), 4.05 - 3.65 (m, 8H), 3.62 - 3.35 (m, 10H), 3.29 - 3.24 (m, 2H), 3.10 - 2.83 (m, 10H), 2.57 - 2.40 (m, 2H). MS (ESI) [M+H]+ = 900.2.
Figure imgf000230_0002
Example 263 (E)-N-(l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-3-(5- (4-(4-(dimethylamino)but-2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propenamide (ZD178-33) Brown solid, 53% yield. 1H NMR (400 MHz, CD3OD) 5 8.08 - 7.65 (m, 1H), 7.36 - 7.12 (m, 1H), 7.03 - 6.69 (m, 2H), 6.68 - 6.50 (m, 3H), 5.12 - 4.91 (m, 2H), 4.76 - 4.13 (m, 6H), 4.13 - 3.68 (m, 8H), 3.63 - 3.35 (m, 16H), 3.07 - 2.80 (m, 10H), 2.59 - 2.40 (m, 2H). MS (ESI) [M+H]+ = 944.1.
Figure imgf000231_0001
Example 264 (E)-N-(l-(3-(6,7-dichIoro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-yl)- 3-(5-(4-(4-(dimethylamino)but-2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propenamide (ZD178-34) Brown solid, 51% yield. 'H NMR (400 MHz, CD3OD) δ 7.92 - 7.57 (m, 1H), 7.37 - 7.09 (m, 1H), 7.03 - 6.68 (m, 2H), 6.68 - 6.52 (m, 3H), 5.10 - 4.93 (m, 2H), 4.73 - 4.16 (m, 6H), 4.11 - 3.64 (m, 8H), 3.62 - 3.37 (m, 20H), 3.21 - 2.81 (m, 10H), 2.62 - 2.30 (m, 2H). MS (ESI) [M+H]+ = 988.3.
Figure imgf000231_0002
Example 265: (E)-N-(l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)-2-oxo-6,9,12,15,18-pentaoxa-3-azaicosan-20-yl)-3- (5-(4-(4-(dimethylamino)but-2-enoyl)-2-oxopiperazin-1-yl)thiophen-2-yl)propenamide (ZD178-35) Brown solid, 51% yield. 1H NMR (400 MHz, CD3OD) 8 7.91 - 7.58 (m, 1H), 7.37 - 7.07 (m, 1H), 7.05 - 6.70 (m, 2H), 6.72 - 6.47 (m, 3H), 5.16 - 4.93 (m, 2H), 4.78 - 4.14 (m, 6H), 4.10 - 3.66 (m, 8H), 3.61 - 3.36 (m, 24H), 3.18 - 2.82 (m, 10H), 2.70 - 2.40 (m, 2H). MS (ESI) [M+H]+ = 1032.3.
Scheme 24. The syntheses of example 266
Figure imgf000232_0001
Example 266 (E)-Nl-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-N4-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)succinamide (ZD178-58-1) To a solution of Linker 43 (0.015 mmol, 1.0 eq) and Intermediate 13 (0.018 mmol, 1.2 eq) in dimethylformamide (1.0 mL) was added HOAt (0.03 mmol, 2.0 eq), NMM (0.15 mmol, 10.0 eq), and EDCI (0.03 mmol, 2.0 eq). The resulting reaction solution was stirred at rt for 12 h. Then the resulting residue was purified by reverse-phase chromatography to yield the desired product. Brown solid, 55% yield. 1H NMR (400 MHz, CD3OD) 8 7.81 - 7.62 (m, 1H), 7.30 - 7.11 (m, 1H), 7.03 - 6.69 (m, 2H), 6.69 - 6.39 (m, 3H), 4.64 - 4.11 (m, 8H), 4.08 - 3.59 (m, 10H), 3.44 - 3.34 (m, 2H), 3.22 - 3.14 (m, 2H), 3.02 - 2.84 (m, 8H), 2.58 - 2.34 (m, 4H). MS (ESI) [M+H]+ = 826.3.
Example 267 - 279 were synthesized following the same procedure for preparing example 266 from related linkers and intermediate 13.
Figure imgf000233_0001
Example 267 (E)-Nl-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-N5-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)glutaramide (ZD178-58-2) Brown solid, 55% yield. 'H NMR (400 MHz, CD3OD) 8 7.81 - 7.60 (m, 1H), 7.35 - 7.11 (m, 1H), 7.05 - 6.72 (m, 2H), 6.68 - 6.32 (m, 3H), 4.67 - 4.08 (m, 8H), 4.12 - 3.58 (m, 10H), 3.23 - 3.10 (m, 2H), 3.02 -
2.72 (m, 10H), 2.47 - 2.05 (m, 4H), 2.01 - 1.64 (m, 2H). MS (ESI) [M+H]+ = 840.2.
Figure imgf000233_0002
Example 268 (E)-Nl-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-N6-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)adipamide (ZD178-58-3) Brown solid, 49% yield. 1H NMR (400 MHz, CD3OD) δ 7.89 - 7.55 (m, 1H), 7.41 - 7.09 (m, 1H), 7.07 - 6.68 (m, 2H), 6.72 - 6.32 (m, 3H), 4.62 - 4.09 (m, 8H), 4.09 - 3.63 (m, 10H), 3.24 - 3.09 (m, 2H), 3.03 - 2.74 (m, 10H), 2.30 - 2.05 (m, 4H), 1.68 - 1.30 (m, 4H). MS (ESI) [M+H]- = 854.9.
Figure imgf000233_0003
enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)heptanediamide (ZD178-58-4) Brown solid, 48% yield. JH NMR (400 MHz, CD3OD) 5 7.81 - 7.57 (m, 1H), 7.32 - 7.14 (m, 1H), 7.09 - 6.70 (m, 2H), 6.72 - 6.32 (m, 3H), 4.69 - 4.10 (m, 8H), 4.10 - 3.62 (m, 10H), 3.44 - 3.34 (m, 2H), 3.21 - 2.80 (m, 10H), 2.28 - 2.04 (m, 4H), 1.71 - 1.09 (m, 6H). MS (ESI) [M+H]+ = 868.3.
Figure imgf000234_0001
Example 270: (E)-Nl-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-N8-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yI)ethyl)octanediamide (ZD178-58-5) Brown solid, 50% yield. JH NMR (400 MHz, CD3OD) 5 7.79 - 7.51 (m, 1H), 7.37 - 7.12 (m, 1H), 7.07 - 6.72 (m, 2H), 6.72 - 6.30 (m, 3H), 4.71 - 4.10 (m, 8H), 4.13 - 3.58 (m, 10H), 3.46 - 3.35 (m, 2H), 3.23 - 2.74 (m, 10H), 2.37 - 2.03 (m, 4H), 1.66 - 0.90 (m, 8H).MS (ESI) [M+H]+ = 882.5.
Figure imgf000234_0002
Example 271 (E)-Nl-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-N9-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)nonanediamide (ZD178-58-6) Brown solid, 48% yield. JH NMR (400 MHz, CD3OD) 5 7.83 - 7.56 (m, 1H), 7.33 - 7.10 (m, 1H), 7.04 - 6.71 (m, 2H), 6.75 - 6.29 (m, 3H), 4.68 - 4.11 (m, 8H), 4.13 - 3.58 (m, 10H), 3.45 - 3.35 (m, 2H), 3.23 - 2.74 (m, 10H), 2.29 - 2.00 (m, 4H), 1.68 - 1.12 (m, 10H). MS (ESI) [M+H]+ = 896.4.
Figure imgf000235_0001
Example 272 (E)-Nl-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-N10-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)decanediamide (ZD178-58-7) Brown solid,
51% yield. JH NMR (400 MHz, CD3OD) 5 7.91 - 7.58 (m, 1H), 7.37 - 7.14 (m, 1H), 7.08 - 6.71 (m, 2H), 6.73 - 6.36 (m, 3H), 4.70 - 4.11 (m, 8H), 4.14 - 3.54 (m, 10H), 3.49 - 3.35 (m, 2H), 3.27 - 2.70 (m, 10H), 2.31 - 1.86 (m, 4H), 1.62 - 1.10 (m, 12H). MS (ESI) [M+H]+ = 910.5.
Figure imgf000235_0002
Example 273 (E)-Nl-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-Nll-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)undecanediamide (ZD178-58-8, MS8588)
Brown solid, 51% yield. 1H NMR (400 MHz, CD3OD) 5 7.73 - 7.57 (m, 1H), 7.27 - 7.11 (m, 1H), 7.04 - 6.69 (m, 2H), 6.66 - 6.24 (m, 3H), 4.71 - 4.20 (m, 8H), 4.07 - 3.57 (m, 10H), 3.49 - 3.35 (m, 2H), 3.07 - 2.80 (m, 10H), 2.26 - 2.02 (m, 4H), 1.62 - 1.06 (m, 14H). MS (ESI) [M+H]+ = 924.3.
Figure imgf000235_0003
Example 274 (E)-Nl-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-N12-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)dodecanediamide (ZD178-58-9) Brown solid, 46% yield. 1H NMR (400 MHz, CD3OD) 8 7.81 - 7.56 (m, 1H), 7.33 - 7.14 (m, 1H), 7.06 - 6.75 (m, 2H), 6.75 - 6.35 (m, 3H), 4.75 - 4.10 (m, 8H), 4.08 - 3.61 (m, 10H), 3.54 - 3.36 (m, 2H), 3.23 - 2.71 (m, 10H), 2.38 - 2.04 (m, 4H), 1.73 - 0.95 (m, 16H). MS (ESI) [M+H]+ = 938.1.
Figure imgf000236_0001
Example 275: (E)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-3-(3-((2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)amino)-3-oxopropoxy)propenamide (ZD178-58-10) Brown solid, 46% yield. JH NMR (400 MHz, CD3OD) δ 7.87 - 7.57 (m, 1H), 7.32
- 7.10 (m, 1H), 7.06 - 6.71 (m, 2H), 6.66 - 6.39 (m, 3H), 4.69 - 4.09 (m, 8H), 4.07 - 3.55 (m, 14H), 3.41 - 3.35 (m, 2H), 3.20 - 2.78 (m, 10H), 2.49 - 2.29 (m, 4H). MS (ESI) [M+H]+ = 870.3.
Figure imgf000236_0002
Example 276: (E)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-3-(2-(3-((2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)amino)-3-oxopropoxy)ethoxy)propenamide (ZD178-58-11) Brown solid, 53% yield. JH NMR (400 MHz, CD3OD) 87.82 - 7.55 (m, 1H), 7.34
- 7.14 (m, 1H), 7.03 - 6.69 (m, 2H), 6.69 - 6.31 (m, 3H), 4.69 - 4.09 (m, 8H), 4.05 - 3.59 (m, 14H), 3.56 - 3.45 (m, 4H), 3.40 - 3.34 (m, 2H), 3.24 - 2.77 (m, 10H), 2.53 - 2.25 (m, 4H).MS (ESI) [M+H]+ = 914.3.
Figure imgf000236_0003
Example 277 (E)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-3-(2-(2-(3-((2-(5-(4-(4-(dimethylamino)but- 2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)amino)-3- oxopropoxy)ethoxy)ethoxy)propenamide (ZD178-58-12) Brown solid, 53% yield. 1H NMR (400 MHz, CD3OD) δ 7.95 - 7.61 (m, 1H), 7.36 - 7.12 (m, 1H), 7.08 - 6.71 (m, 2H), 6.71 - 6.33 (m, 3H), 4.73 - 4. 11 (m, 8H), 4.06 - 3.60 (m, 14H), 3.58 - 3.46 (m, 8H), 3.40 - 3.34 (m, 2H), 3.23 - 2.82 (m, 10H), 2.52 - 2.33 (m, 4H). MS (ESI) [M+H]+ = 958.4.
Figure imgf000237_0001
Example 278 (E)-Nl-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-N16-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)-4,7,10,13-tetraoxahexadecanediamide (ZD178-58-13) Brown solid, 57% yield. JH NMR (400 MHz, CD3OD) 87.80 - 7.65 (m, 1H), 7.31 - 7.15 (m, 1H), 7.06 - 6.70 (m, 2H), 6.69 - 6.42 (m, 3H), 4.68 - 4.11 (m, 8H), 4.09 - 3.59 (m, 14H), 3.60 - 3.46 (m, 12H), 3.44 - 3.35 (m, 2H), 3.21 - 2.75 (m, 10H), 2.53 - 2.27 (m, 4H). MS (ESI) [M+H]+ = 1002.4.
Figure imgf000237_0002
Example 279 (E)-N1-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-N19-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)-4,7,10,13,16-pentaoxanonadecanediamide (ZD178-58-14) Brown solid, 50% yield. 1H NMR (400 MHz, CD3OD) 87.79 - 7.54 (m, 1H), 7.32
- 7.12 (m, 1H), 7.05 - 6.71 (m, 2H), 6.70 - 6.41 (m, 3H), 4.68 - 4.10 (m, 8H), 4.07 - 3.62 (m, 14H), 3.61 - 3.46 (m, 16H), 3.44 - 3.36 (m, 2H), 3.23 - 2.79 (m, 10H), 2.60 - 2.27 (m, 4H). MS (ESI) [M+H]+ = 1046.5. Scheme 25. The syntheses of example 280
Figure imgf000238_0001
Example 280 (E)-N-(2-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)amino)-2-oxoethyl)-3-(5-(4-(4-
(dimethyIamino)but-2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propenamide (ZD178-63-1) To a solution of Linker 56 (0.015 mmol, 1.0 eq) and Intermediate 15 (0.018 mmol, 1.2 eq) in dimethylformamide (1.0 mL) was added HOAt (0.03 mmol, 2.0 eq), NMM (0.15 mmol, 10.0 eq), and EDCI (0.03 mmol, 2.0 eq). The resulting reaction solution was stirred at rt for 12 h. Then the resulting residue was purified by reverse-phase chromatography to yield the desired product.. Brown solid, 54% yield. 1H NMR (400 MHz, CD3OD) 5 7.73 - 7.59 (m, 1H), 7.25 - 7.15 (m, 1H), 7.01 - 6.69 (m, 2H), 6.64 - 6.40 (m, 3H), 4.60 - 4.17 (m, 8H), 4.05 - 3.59 (m, 12H), 3.07 - 2.81 (m, 10H), 2.65 - 2.36 (m, 2H). MS (ESI) [M+H]+ = 812.4.
Example 281 - 294 were synthesized following the same procedure for preparing example 280 from related linkers and intermediate 15.
Figure imgf000239_0001
Example 281 : (E)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-3-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)- 2-oxopiperazin-l-yl)thiophen-2-yl)propanamido)propenamide (ZD178-63-2) Brown solid, 54% yield. 1H NMR (400 MHz, CD3OD) 5 7.76 - 7.61 (m, 1H), 7.29 - 7.09 (m, 1H), 7.00 - 6.69 (m, 2H), 6.62 - 6.39 (m, 3H), 4.64 - 4.15 (m, 8H), 4.05 - 3.59 (m, 10H), 3.45 - 3.34 (m, 2H), 3.13 - 2.76 (m, 10H), 2.50 - 2.25 (m, 4H). MS (ESI) [M+H]+ = 826.6.
Figure imgf000239_0002
Example 282 (E)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-4-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)- 2-oxopiperazin-l-yl)thiophen-2-yl)propanamido)butanamide (ZD178-63-3) Brown solid, 485 yield. 'H NMR (400 MHz, CD3OD) δ 7.76 - 7.62 (m, 1H), 7.28 - 7.15 (m, 1H), 7.00 - 6.67 (m, 2H), 6.64 - 6.35 (m, 3H), 4.68 - 4.15 (m, 8H), 4.07 - 3.51 (m, 10H), 3.19 - 2.78 (m, 12H), 2.55 - 2.36 (m, 2H), 2.19 - 2.03 (m, 2H), 1.77 - 1.60 (m, 2H). MS (ESI) [M+H]+ = 840.7.
Figure imgf000239_0003
2-oxopiperazin-l-yl)thiophen-2-yl)propanamido)pentanamide (ZD178-63-4) Brown solid, 43% yield. 1H NMR (400 MHz, CD3OD) δ δ 7.71 - 7.57 (m, 1H), 7.29 - 7.12 (m, 1H), 7.01 - 6.66 (m, 2H), 6.63 - 6.42 (m, 3H), 4.63 - 4.20 (m, 8H), 4.07 - 3.62 (m, 10H), 3.15 - 2.83 (m, 12H), 2.55 - 2.33 (m, 2H), 2.15 (s, 2H), 1.59 - 1.25 (m, 4H). MS (ESI) [M+H]+ = 854.9.
Figure imgf000240_0001
Example 284 (E)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-6-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)- 2-oxopiperazin-l-yl)thiophen-2-yl)propanamido)hexanamide (ZD178-63-5) Brown solid, 55% yield. 1H NMR (400 MHz, CD3OD) 5 7.78 - 7.57 (m, 1H), 7.31 - 7.14 (m, 1H), 7.03 - 6.68 (m, 2H), 6.67 - 6.42 (m, 3H), 4.73 - 4.18 (m, 8H), 4.11 - 3.57 (m, 10H), 3.16 - 2.73 (m, 12H), 2.64 - 2.38 (m, 2H), 2.26 - 2.05 (m, 2H), 1.66 - 1.05 (m, 6H). MS (ESI) [M+H]+ = 868.5.
Figure imgf000240_0002
Example 285 (E)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-7-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)- 2-oxopiperazin-l-yl)thiophen-2-yl)propanamido)heptanamide (ZD178-63-6) Brown solid, 55% yield. 1H NMR (400 MHz, CD3OD) 8 8 7.78 - 7.51 (m, 1H), 7.30 - 7.14 (m, 1H), 7.07 - 6.68 (m, 2H), 6.68 - 6.33 (m, 3H), 4.76 - 4.18 (m, 8H), 4.12 - 3.61 (m, 10H), 3.15 - 2.78 (m, 12H), 2.61 - 2.41 (m, 2H), 2.34 - 2.01 (m, 2H), 1.70 - 0.88 (m, 8H). MS (ESI) [M+H]+ = 882.5.
Figure imgf000241_0001
Example 286: (E)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-8-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)-
2-oxopiperazin-l-yl)thiophen-2-yl)propanamido)octanamide (ZD178-63-7) Brown solid, 49% yield. 'H NMR (400 MHz, CD3OD) 5 5 7.78 - 7.58 (m, 1H), 7.32 - 7. 12 (m, 1H), 7.04 - 6.68 (m, 2H), 6.66 - 6.36 (m, 3H), 4.69 - 4.11 (m, 8H), 4.08 - 3.61 (m, 10H), 3.16 - 2.75 (m, 12H), 2.57 - 2.37 (m, 2H), 2.27 - 2.03 (m, 2H), 1.65 - 1.06 (m, 10H). MS (ESI) [M+H]+ = 896.0.
Figure imgf000241_0002
Example 287: (E)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-9-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)- 2-oxopiperazin-l-yl)thiophen-2-yl)propanamido)nonanamide (ZD178-63-8) Brown solid, 49% yield. 'H NMR (400 MHz, CD3OD) 5 7.77 - 7.55 (m, 1H), 7.31 - 7.13 (m, 1H), 7.04 - 6.70 (m, 2H), 6.71 - 6.41 (m, 3H), 4.68 - 4.19 (m, 8H), 4.11 - 3.61 (m, 10H), 3.18 - 2.79 (m, 12H), 2.60 - 2.38 (m, 2H), 2.30 - 1.92 (m, 2H), 1.74 - 0.81 (m, 12H). MS (ESI) [M+H]+ = 910.1.
Figure imgf000241_0003
Example 288 (E)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indoI-9-yl)-lH-pyrazol-l-yl)ethyl)-10-(3-(5-(4-(4-(dimethyIamino)but-2-enoyl)- 2-oxopiperazin-l-yl)thiophen-2-yl)propanamido)decanamide (ZD178-63-9) Brown solid, 52% yield. 'H NMR (400 MHz, CD3OD) 8 7.80 - 7.51 (m, 1H), 7.41 - 7.07 (m, 1H), 7.09 - 6.70 (m, 2H), 6.68 - 6.34 (m, 3H), 4.71 - 4.17 (m, 8H), 4.19 - 3.54 (m, 10H), 3.27 - 2.77 (m, 12H), 2.62 - 2.41 (m, 2H), 2.30 - 1.99 (m, 2H), 1.66 - 0.97 (m, 14H). MS (ESI) [M+H]+ = 924.0.
Figure imgf000242_0001
Example 289: (E)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-ll-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)- 2-oxopiperazin-l-yl)thiophen-2-yl)propanamido)undecanamide (ZD178-63-10) Brown solid, 52% yield. 1H NMR (400 MHz, CD30D) δ 7.79 - 7.55 (m, 1H), 7.21 (dt, J= 12.6, 2.5 Hz, 1H), 7.09 - 6.70 (m, 2H), 6.68 - 6.34 (m, 3H), 4.68 - 4.18 (m, 8H), 4.10 - 3.63 (m, 10H), 3.22 - 2.80 (m, 12H), 2.61 - 2.36 (m, 2H), 2.28 - 2.05 (m, 2H), 1.70 - 0.89 (m, 16H). MS (ESI) [M+H]+ = 938.7.
Figure imgf000242_0002
Example 290 (E)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-3-(2-(3-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propanamido)ethoxy)propenamide (ZD178-63- 11) Brown solid, 54% yield. ’H NMR (400 MHz, CD3OD) 8 7.73 - 7.59 (m, 1H), 7.30 - 7.13 (m, 1H), 7.04 - 6.66 (m, 2H), 6.63 - 6.34 (m, 3H), 4.64 - 4.14 (m, 8H), 4.06 - 3.54 (m, 12H), 3.48 - 3.38 (m, 2H), 3.28 - 3.21 (m, 2H), 3.09 - 2.83 (m, 10H), 2.63 - 2.33 (m, 4H). MS (ESI) [M+Hf = 870.3.
Figure imgf000243_0001
Example 291 (E)-N-(2-(3-(6,7-dichIoro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido [4,3-b] indol-9-yl)- 1 H-pyrazol- l-yl)ethyl)-3-(2-(2-(3-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propanamido)ethoxy)ethoxy)propenamide
(ZD178-63-12) Brown solid, 40% yield. 1HNMR 400 MHz, CD3OD) δ 7.86 - 7.59 (m, 1H), 7.31
- 7.09 (m, 1H), 7.04 - 6.66 (m, 2H), 6.67 - 6.33 (m, 3H), 4.74 - 4.15 (m, 8H), 4.06 - 3.62 (m, 12H), 3.59 - 3.36 (m, 8H), 3.06 - 2.81 (m, 10H), 2.66 - 2.31 (m, 4H). MS (ESI) [M+H]+ = 914.6.
Figure imgf000243_0002
Example 292 (E)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-3-(2-(2-(3-((2-(5-(4-(4-(dimethylamino)but-
2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yI)ethyl)amino)-3- oxopropoxy)ethoxy)ethoxy)propenamide (ZD178-63-13) Brown solid, 45% yield. 1H NMR (400 MHz, CD3OD) 8 7.87 - 7.50 (m, 1H), 7.35 - 7.12 (m, 1H), 7.06 - 6.69 (m, 2H), 6.68 - 6.41 (m, 3H), 4.70 - 4.16 (m, 8H), 4.07 - 3.61 (m, 12H), 3.58 - 3.36 (m, 12H), 3.06 - 2.82 (m, 10H), 2.54 - 2.37 (m, 4H). MS (ESI) [M+H]+ = 958.7.
Figure imgf000244_0001
Example 293 (E)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-l-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)- 2-oxopiperazin-l-yl)thiophen-2-yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (ZD178-63-14) Brown solid, 46% yield. 'H NMR (400 MHz, CD3OD) 87.82 - 7.65 (m, 1H), 7.36
- 7.15 (m, 1H), 7.05 - 6.69 (m, 2H), 6.65 - 6.39 (m, 3H), 4.67 - 4.19 (m, 8H), 4.10 - 3.62 (m, 12H), 3.60 - 3.40 (m, 14H), 3.18 - 2.85 (m, 10H), 2.63 - 2.33 (m, 4H). MS (ESI) [M+H]+ = 1002.5.
Figure imgf000244_0002
Example 294 (E)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazoI-l-yl)ethyl)-l-(3-(5-(4-(4-(dimethylamino)but-2-enoyI)- 2-oxopiperazin-l-yl)thiophen-2-yl)propanamido)-3,6,9,12,15-pentaoxaoctadecan-18-amide (ZD178-63-15) Brown solid, 54% yield. 'H NMR (400 MHz, CD3OD) 87.78 - 7.64 (m, 1H), 7.30
- 7.15 (m, 1H), 7.05 - 6.67 (m, 2H), 6.68 - 6.36 (m, 3H), 4.70 - 4.10 (m, 8H), 4.09 - 3.62 (m, 12H), 3.60 - 3.41 (m, 18H), 3.12 - 2.82 (m, 10H), 2.63 - 2.32 (m, 4H). MS (ESI) [M+H]+ = 1046.4.
Scheme 26. The syntheses of example 295
Figure imgf000245_0001
Example 295 : (E)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3- b]indol-9-yl)-lH-pyrazol-l-yl)-N-(2-((2-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)ethyl)amino)-2-oxoethyl)acetamide (ZD178-69-1) To a solution of Linker 85 (0.015 mmol, 1.0 eq} and Intermediate 13 (0.018 mmol, 1.2 eq in dimethylformamide (1.0 mL) was added HOAt (0.03 mmol, 2.0 eq}, NMM (0.15 mmol, 10.0 eq}, and EDCI (0.03 mmol, 2.0 eq}. The resulting reaction solution was stirred at rt for 12 h. Then the resulting residue was purified by reverse-phase chromatography to yield the desired product. Brown solid, 46% yield. 1H NMR (400 MHz, CD3OD) 5 7.88 - 7.76 (m, 1H), 7.30 - 7.18 (m, 1H), 7.04 - 6.68 (m, 2H), 6.65 - 6.44 (m, 3H), 5.12 - 4.98 (m, 2H), 4.72 - 4.15 (m, 6H), 4.06 - 3.65 (m, 10H), 3.25 - 3.12 (m, 2H), 3.01 - 2.77 (m, 10H). MS (ESI) [M+H]+ = 798.0.
Example 296 - 309 were synthesized following the same procedure for preparing example 295 from related linkers and intermediate 13.
Figure imgf000246_0001
Example 296 (E)-3-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)-N-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)propenamide (ZD178-69-2) Brown solid, 49% yield. *H NMR (400 MHz, CD3OD) δ 7.88 - 7.60 (m, 1H), 7.37 - 7.08 (m, 1H), 7.04 - 6.68 (m, 2H), 6.68 - 6.41 (m, 3H), 5.04 - 4.90 (m, 2H), 4.73 - 4.08 (m, 6H), 4.04 - 3.65 (m, 8H), 3.56 -
3.35 (m, 4H), 3.03 - 2.71 (m, 10H), 2.51 - 2.30 (m, 2H). MS (ESI) [M+H]+ = 812.4.
Figure imgf000246_0002
Example 297 (E)-4-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)-N-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)butanamide (ZD178-69-3) Brown solid, 49% yield. 1H NMR (400 MHz, CD3OD) 8 7.83 - 7.74 (m, 1H), 7.29 - 7.17 (m, 1H), 7.02 - 6.68 (m, 2H), 6.67 - 6.50 (m, 3H), 5.03 - 4.92 (m, 2H), 4.71 - 4.14 (m, 6H), 4.05 - 3.66 (m, 8H), 3.46 - 3.35 (m, 2H), 3.27 - 3.12 (m, 4H), 3.03 - 2.80 (m, 8H), 2.34 - 2.11 (m, 2H), 1.80 (s, 2H). MS
(ESI) [M+H]+ = 826.4.
Figure imgf000246_0003
pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)acetamido)-N-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)pentanamide (ZD178-69-4) Brown solid, 51% yield. 'H NMR (400 MHz, CD3OD) 8 7.86 - 7.65 (m, 1H), 7.36 - 7.12 (m, 1H), 6.98 - 6.68 (m, 2H), 6.68 - 6.49 (m, 3H), 5.06 - 4.92 (m, 2H), 4.74 - 4.14 (m, 6H), 4.07 - 3.66 (m, 8H), 3.46 - 3.36 (m, 2H), 3.28 - 3.16 (m, 4H), 3.03 - 2.79 (m, 8H), 2.31 - 2.02 (m, 2H), 1.73 - 1.42 (m, 4H). MS (ESI) [M+H]+ = 840.4.
Figure imgf000247_0001
Example 299 (E)-6-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)-N-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)hexanamide (ZD178-69-5) Brown solid, 51% yield. 1H NMR (400 MHz, CD3OD) 1 7.86 - 7.69 (m, 1H), 7.29 - 7.14 (m, 1H), 7.03 - 6.69 (m, 2H), 6.69 - 6.53 (m, 3H), 5.02 - 4.90 (m, 2H), 4.77 - 4.11 (m, 6H), 4.08 - 3.66 (m, 8H), 3.56 - 3.35 (m, 2H), 3.26 - 3.09 (m, 4H), 3.03 - 2.79 (m, 8H), 2.27 - 2.04 (m, 2H), 1.71 - 1.12 (m, 6H). MS (ESI) [M+H]+ = 854.4.
Figure imgf000247_0002
Example 300 (E)-7-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)-N-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)heptanamide (ZD178-69-6) Brown solid, 53% yield. 1H NMR (400 MHz, CD3OD) 8 7.97 - 7.64 (m, 1H), 7.53 - 7.18 (m, 1H), 7.02 - 6.70 (m, 2H), 6.70 - 6.50 (m, 3H), 5.09 - 4.92 (m, 2H), 4.73 - 4.14 (m, 6H), 4.10 - 3.66 (m, 8H), 3.49 - 3.36 (m, 2H), 3.27 - 3.11 (m, 4H), 3.04 - 2.82 (m, 8H), 2.28 - 2.04 (m, 2H), 1.72 - 1.09 (m, 8H). MS (ESI) [M+H]+ = 868.4.
Figure imgf000248_0001
Example 301 : (E)-8-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)-N-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)octanamide (ZD178-69-7) Brown solid, 53% yield. 'H NMR (400 MHz, CD3OD) 8 7.97 - 7.58 (m, 1H), 7.47 - 7.12 (m, 1H), 7.05 - 6.70 (m, 2H), 6.74 - 6.42 (m, 3H), 5.01 - 4.91 (m, 2H), 4.71 - 4.16 (m, 6H), 4.05 - 3.64 (m, 8H), 3.48 - 3.36 (m, 2H), 3.27 - 3.17 (m, 4H), 2.99 - 2.72 (m, 8H), 2.29 - 1.99 (m, 2H), 1.71 - 1.15 (m, 1 OH). MS (ESI) [M+H] = 882.5.
Figure imgf000248_0002
Example 302: (E)-9-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)-N-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)nonaiiamide (ZD178-69-8) Brown solid, 49% yield. 'H NMR (400 MHz, CD3OD) δ 7.97 - 7.70 (m, 1H), 7.29 - 7.21 (m, 1H), 7.03 - 6.70 (m, 2H), 6.70 - 6.51 (m, 3H), 5.04 - 4.90 (m, 2H), 4.81 - 4.15 (m, 6H), 4.10 - 3.67 (m, 8H), 3.50 - 3.37 (m, 2H), 3.27 - 3.12 (m, 4H), 3.08 - 2.64 (m, 8H), 2.51 - 1.97 (m, 2H), 1.78 - 0.65 (m, 12H). MS (ESI) [M+H]+ = 896.3.
Figure imgf000249_0001
Example 303: (E)-10-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazoI-l-yl)acetamido)-N-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)decanamide (ZD178-69-9) Brown solid, 49% yield. 'H NMR (400 MHz, CD3OD) δ 7.91 - 7.67 (m, 1H), 7.35 - 7.14 (m, 1H), 7.02 - 6.70 (m, 2H), 6.71 - 6.52 (m, 3H), 5.09 - 4.90 (m, 2H), 4.78 - 4.16 (m, 6H), 4.08 - 3.61 (m, 8H), 3.51 - 3.37 (m, 2H), 3.28 - 3.17 (m, 4H), 3.07 - 2.66 (m, 8H), 2.22 - 2.02 (m, 2H), 1.67 - 1.10 (m, 14H). MS (ESI) [M+H]+ = 910.4.
Figure imgf000249_0002
Example 304 (E)-ll-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)-N-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)undecanamide (ZD178-69-9) Brown solid, 52% yield. H NMR (400 MHz, CD3OD) 8 7.87 - 7.58 (m, 1H), 7.49 - 7.20 (m, 1H), 7.03 - 6.70 (m, 2H), 6.72 - 6.43 (m, 3H), 5.01 - 4.91 (m, 2H), 4.72 - 4.15 (m, 6H), 4.08 - 3.65 (m, 8H), 3.45 - 3.37 (m, 2H), 3.27 - 3.15 (m, 4H), 3.03 - 2.79 (m, 8H), 2.34 - 2.05 (m, 2H), 1.75 - 1.02 (m, 16H).MS (ESI) [M+H]+ = 924.4.
Figure imgf000250_0001
Example 305 (E)-3-(2-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)ethoxy)-N-(2-(5-(4-(4-
(dimethyIamino)but-2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)propenamide
(ZD178-69-11) Brown solid, 47% yield. *HNMR (400 MHz, CD3OD) 8 8.04 - 7.66 (m, 1H), 7.41
- 7.16 (m, 1H), 7.07 - 6.68 (m, 2H), 6.72 - 6.43 (m, 3H), 5.11 - 4.90 (m, 2H), 4.73 - 4.12 (m, 6H), 4.04 - 3.60 (m, 10H), 3.57 - 3.35 (m, 6H), 2.97 - 2.83 (m, 10H), 2.50 - 2.31 (m, 2H). MS
(ESI) [M+H]+ = 856.9.
Figure imgf000250_0002
Example 306 : (E)-3-(2-(2-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)ethoxy)ethoxy)-N-(2-(5-(4-(4- (dimethylamino)but-2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)propenamide (ZD178-69-12) Brown solid, 50% yield. JH NMR (400 MHz, CD3OD) 87.99 - 7.45 (m, 1H), 7.43 - 7.05 (m, 1H), 6.99 - 6.70 (m, 2H), 6.69 - 6.45 (m, 3H), 5.05 - 4.92 (m, 2H), 4.74 - 4.17 (m, 6H), 4.03 - 3.59 (m, 10H), 3.59 - 3.34 (m, 10H), 3.02 - 2.81 (m, 10H), 2.52 - 2.33 (m, 2H). MS (ESI) [M+H]+ = 900.4.
Figure imgf000251_0001
Example 307 : (E)-3-(2-(2-(2-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)ethoxy)ethoxy)ethoxy)-N-(2-(5-(4-(4- (dimethylamino)but-2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)propenamide (ZD178-69-13) Brown solid, 33% yield. 1 H NMR (400 MHz, CD3OD) 87.95 - 7.56 (m, 1H), 7.45 - 7.14 (m, 1H), 7.01 - 6.70 (m, 2H), 6.70 - 6.52 (m, 3H), 5.07 - 4.91 (m, 2H), 4.71 - 4.12 (m, 6H), 4.09 - 3.62 (m, 8H), 3.60 - 3.35 (m, 16H), 3.05 - 2.76 (m, 10H), 2.53 - 2.26 (m, 2H). MS (ESI) [M+H]+ = 944.0.
Figure imgf000251_0002
Example 308 (E)-l-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)-N-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)-3,6,9,12-tetraoxapentadecan-15-amide (ZD178-69-14) Brown solid, 56% yield. JH NMR (400 MHz, CD3OD) 87.99 - 7.62 (m, 1H), 7.46
- 7.19 (m, 1H), 7.03 - 6.69 (m, 2H), 6.69 - 6.49 (m, 3H), 5.07 - 4.93 (m, 2H), 4.78 - 4.18 (m, 6H), 4.12 - 3.62 (m, 10H), 3.62 - 3.34 (m, 18H), 3.05 - 2.79 (m, 10H), 2.51 - 2.33 (m, 2H). MS (ESI) [M+H]+ = 988.1.
Figure imgf000252_0001
Example 309 (E)-l-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)-N-(2-(5-(4-(4-(dimethylamino)but-2- enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)-3,6,9,12,15-pentaoxaoctadecan-18-amide (ZD178-69-15) Brown solid, 50% yield. 'H NMR (400 MHz, CD3OD) 87.87 - 7.72 (m, 1H), 7.32 - 7.21 (m, 1H), 7.01 - 6.69 (m, 2H), 6.69 - 6.54 (m, 3H), 5.04 - 4.94 (m, 2H), 4.71 - 4.18 (m, 6H), 4.07 - 3.63 (m, 10H), 3.61 - 3.36 (m, 22H), 2.98 - 2.81 (m, 10H), 2.52 - 2.37 (m, 2H). MS (ESI) [M+H] 1 = 1032.4.
Procedures for the synthesis of CFTR based bivalent compounds
Scheme 27. Synthesis of Linker 100
Figure imgf000252_0002
Linker 100: N-(2-aminoethyl)-3-(6-(l-(2,2-difluorobenzo[J| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)benzamide 3-(6-(l-(2,2-difluorobenzo[d/][l,3]dioxol-5- yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzoic acid (100 mg, 0.22 mmol, 1.0 eq), tert-butyl (2-aminoethyl)carbamate (38 mg, 0.24 mmol, 1.1 eq), EDC HCI (63 mg, 0.33 mmol, 1.5 eq), HO At (45 mg, 0.33 mmol, 1.5 eq), NMM (67 mg, 0.66 mmol, 3.0 eq) were stirred in DMF (1.5 mL) at room temperature for 2 h. Then the mixture was purified via reverse-ISCO to get related intermediate. Then the intermediate was dissolved in MeOH (1 mL) followed by HCI in dioxane (4 M, 1 mb, 4 mmol), the mixture was stirred at room temperature for 2 h. After removed all the volatiles, the titled compound was obtained as a white solid (50 mg, 45% yield over two steps). 1H NMR (400 MHz, CD3OD) 8 8.13 (d, J= 8.8 Hz, 1H), 8.07 (d, J= 7.6 Hz, 1H), 8.02 - 7.98 (tn, 2H), 7.70 - 7.63 (m, 2H), 7.37 - 7.30 (tn, 2H), 7.23 (d, J= 10.8 Hz, 1H), 3.80 - 3.77 (tn, 2H), 3.33 - 3.29 (m, 2H), 2.36 (s, 3H), 1.77 - 1.73 (m, 2H), 1.35 - 1.31 (m, 2H).
Linkers 101 - 113 are synthesized as the same procedure for preparing linker 100.
Figure imgf000253_0003
Linker 102: N-(4-aminobutyl)-3-(6-(l-(2,2-difluorobenzo[d] [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)benzamide White solid, 60% yield. 'H NMR (400 MHz, CD3OD) 5 8.09 (d, .7= 8.4 Hz, 1H), 7.89 (d, J= 5.4 Hz, 1H), 7.84 (s, 1H), 7.80 (d, J= 8.6 Hz, 1H), 7.60 - 7.55(m, 2H), 7.40 (s, 1H), 7.34 (d, J= 8.3 Hz, 1H), 7.23 (d, J= 8.3 Hz, 1H), δ 3.37 - 3.28 (m, 2H), 2.85 (t, J= 7.7 Hz, 2H).2.26 (s, 3H), 1.77 - 1.66 (m, 6H), 1.30 - 1.20 (m, 2H).
Figure imgf000253_0001
Linker 103: JV-(5-aminopentyl)-3-(6-(l-(2,2-difluorobenzo[d | [l,3]dioxol-5-yl)cyclopropane- l-carboxamido)-3-methylpyridin-2-yl)benzamide White solid, 65% yield 1H NMR (400 MHz, CD3OD) 8 8.19 - 7.84 (m, 4H), 7.67 - 7.53 (m, 2H), 7.30 (s, 1H), 7.24 (d, J= 7.9 Hz, 1H), 7.15 (d, J= 8.9 Hz, 1H), 3.33 (t, J= 7.8 Hz, 2H), 2.84 (t, J= 7.6 Hz, 2H), 2.31 - 2.20 (m, 3H), 1.69 - 1.54 (m, 6H), 1.45 - 1.35 (m, 2H), 1.30 - 1.21 (m, 2H).
Figure imgf000253_0002
Linker 104: N-(6-aminohexyl)-3-(6-(l-(2,2-difluorobenzo[J] [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)benzamide White solid, 62% yield. 'H NMR (400 MHz, CD3OD) δ 8.39 (d, J= 8.0 Hz, 1H), 8.16 - 8.06 (m, 3H), 7.80 - 7.70 (m, 2H), 7.35 - 7.30 (m, 2H),
7.20 - 7.18 (m, 1H), 3.38 - 3.34 (m, 2H), 2.93 - 2.89 (m, 2H), 2.38 (s, 3H), 1.75 - 1.63 (m, 6H), 1.42 - 1.34 (m, 6H).
Figure imgf000254_0001
Linker 105: 7V-(7-aminoheptyl)-3-(6-(l-(2,2-difluorobenzo[J] [l,3]dioxol-5-yI)cyclopropane- l-carboxamido)-3-methylpyridin-2-yl)benzamide White solid, 64% yield. JH NMR (400 MHz, CD3OD) 5 8.38 (d, J= 8.0 Hz, 1H), 8.12 - 8.04 (m, 3H), 7.81 - 7.70 (m, 2H), 7.35 - 7.30 (m, 2H),
7.19 (d, J= 7.2 Hz, 1H), 3.37 - 3.34 (m, 2H), 2.91 - 2.87 (m, 2H), 2.38 (s, 3H), 1.74 - 1.61 (m, 6H), 1.38 - 1.34 (m, 8H).
Figure imgf000254_0002
Linker 106 : N(8-aminooctyl)-3-(6-(l-(2,2-difluorobenzo[</|[l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)benzamide White solid, 60% yield. 1 H NMR (400 MHz, CD3OD) 5 8.38 (d, J= 8.0 Hz, 1H), 8.14 - 8.04 (m, 3H), 7.81 - 7.70 (m, 2H), 7.35 - 7.30 (m, 2H), 7.19 - 7.18 (m, 1H), 3.36 - 3.34 (m, 2H), 2.89 (t, J = 7.6 Hz, 2H), 2.38 (s, 3H), 1.80 - 1.53 (m, 6H), 1.37 - 1.33 (m, 10H).
Figure imgf000254_0003
Linker 107: N-(9-aminononyl)-3-(6-(l-(2,2-difluorobenzo[</| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)benzamide White solid, 59% yield. 'H NMR (400 MHz, CD3OD) δ 8.38 (d, J= 8.0 Hz, 1H), 8.14 - 8.04 (m, 3H), 7.81 - 7.70 (m, 2H), 7.35 - 7.30 (m, 2H),
7.20 - 7.18 (m, 1H), 3.35 - 3.37 (m, 2H), 2.89 (t, J= 8.0 Hz, 2H), 2.38 (s, 3H), 1.75 (s, 2H), 1.66 - 1.58 (m, 4H), 1.34 - 1.33 (m, 12H).
Figure imgf000254_0004
Linker 108: JV-(10-aminodecyl)-3-(6-(l-(2,2-difluorobenzo[J] [l,3]dioxol-5-yI)cyclopropane- l-carboxamido)-3-methylpyridin-2-yl)benzamide White solid, 64% yield. 'H NMR (400 MHz, CD3OD) δ 8.39 (s, 1H), 8.18 - 8.05 (m, 3H), 7.82 - 7.69 (m, 2H), 7.33 - 7.30 (m, 2H), 7.18 (s, 1H), 3.34 - 7.32 (m, 2H), 2.89 (s, 2H), 2.39 (s, 3H), 1.75 - 1.59 (m, 6H), 1.33 - 1.30 (m, 14H).
Figure imgf000255_0001
Linker 109: N-(2-(2-aminoethoxy)ethyl)-3-(6-(l-(2,2-difluorobenzo[d/] [l,3]dioxol-5- yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamide White solid, 62% yield. 1H NMR (400 MHz, CD3OD) 5 8.39 (d, J = 8.4 Hz, 1H), 8.19 - 8.11 (m, 3H), 7.83 - 7.81 (m, 1H), 7.71 (t, J= 7.6 Hz, 1H), 7.34 - 7.30 (m, 2H), 7.20 - 7.18 (m, 1H), 3.72 - 3.68 (m, 4H), 3.61 - 3.55 (m, 2H), 3.14 - 3.08 (m, 2H), 2.39 (s, 3H), 1.77 - 1.73 (m, 2H), 1.36 - 1.31 (m, 2H).
Figure imgf000255_0002
Linker 110: N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(6-(l-(2,2-difluorobenzo[r/|[l,3]dioxol- 5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamide White solid, 60% yield. JH NMR (400 MHz, CD3OD) 5 8.08 (d, .7 = 8.6 Hz, 1H), 7.84 - 7.77 (m, 3H), 7.52 - 7.50 (m, 1H), 7.41 (t, J= 8.0 Hz, 1H), 7.04 - 7.00 (m, 2H), 6.91 - 6.88 (m, 1H), 3.39 - 3.36 (m, 8H), 3.28 - 3.27 (m, 2H), 2.79 - 2.75 (m, 2H), 2.09 (s, 3H), 1.48 - 1.42 (m, 2H), 1.08 - 1.02 (m, 2H).
Figure imgf000255_0003
Linker 111: W-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6-(l-(2,2- difluorobenzo[d |[1,3]dioxol-5-yl)cyclopropane-l-carboxaniido)-3-niethylpyridin-2- yl)benzamide White solid, 61% yield. ’H NMR (400 MHz, CD3OD) 5 7.70 (d, J= 8.0 Hz, 1H), 7.59 - 7.50 (m, 3H), 7.22 - 7.19 (m 2H), 6.95 - 6.91 (m, 2H), 6.83 - 6.81 (m, 1H), 3.35 - 3.31 (m,
12H), 3.25 - 3.24 (m 2H), 2.80 - 2.75 (m, 2H), 1.93 (s, 3H), 1.34 - 1.29 (m, 2H), 0.91 - 0.87 (m,
2H).
Figure imgf000255_0004
Linker 112: \-(14-ainino-3.6.9.12-tetraoxatetradecyl)-3-(6-( 1 (2.2- difluorobenzo[d/][l,3]dioxol-5-yl)cyclopropane-l-carboxaniido)-3-niethylpyridin-2- yl)benzamide White solid, 58% yield. 'H NMR (400 MHz, CD3OD) δ 7.72 (d, J= 8.0 Hz, 1H), 7.62 - 7.57 (m, 3H), 7.27 - 7.26 (m, 2H), 6.99 - 6.94 (m, 2H), 6.85 - 6.83 (m, 1H), 3.39 - 3.30 (m, 18H), 2.85 - 2.82 (m, 2H), 1.96 (s, 3H), 1.37 - 1.33 (m, 2H), 0.95 - 0.90 (m, 2H).
Figure imgf000256_0001
Linker 113: N -(17-amino-3,6,9,12,15-pentaoxaheptadecyl)-3-(6-(l-(2,2- difluorobenzo[</|[l,3]dioxol-5-yl)cyclopropane-l-carboxaniido)-3-niethylpyridin-2- yl)benzamide White solid, 63% yield. ’H NMR (400 MHz, CD3OD) δ 7.85 (d, J= 8.0 Hz, 1H), 7.73 - 7.63 (m, 3H), 7.37 - 7.33 (m, 2H), 7.11 - 7.06 (m, 2H), 6.98 - 6.96 (m, 1H), 3.53 - 3.48 (m, 2H), 3.45 - 3.40 (m, 20H), 2.96 - 2.90 (m, 2H), 2.06 (s, 3H), 1.48 - 1.44 (m, 2H), 1.05 - 1.00 (m, 2H).
Scheme 28. Synthesis of Linker 114
Figure imgf000256_0002
Linker 114: (3-(6-(l-(2,2-difluorobenzo[d ][l,3]dioxol-5-yl)cyclopropane-l-carboxaniido)-3- methylpyridin-2-yl)benzoyl)glycine 3-(6-(l-(2,2-difluorobenzo[d][l,3]dioxol-5- yl)cyclopropane- l-carboxamido)-3-methylpyri din-2 -yl)benzoic acid (100 mg, 0.22 mmol, 1.0 eq), /c/7-butyl glycinate (31 mg, 0.24 mmol, 1.1 eq) EDC HC1 (63 mg, 0.33 mmol, 1.5 eq), HOAt (45 mg, 0.33 mmol, 1.5 eq), NMM (67 mg, 0.66 mmol, 3.0 eq) were stirred in 1.5 mL DMF at room temperature for 2 h. Then the mixture was purified via RP-C 18 column [MeOH/Water (0.1%TFA)]. Then the product was dissolved in 1 mL DCM and 1 mL TFA, the mixture was stirred at room temperature for 1 h. Then removed all the volatiles and purified via RP-C 18 column [MeOH/Water (0.1%TFA)]. The product was obtained as a white solid (70 mg, 61% yield over two steps). !H NMR (400 MHz, CD3OD) δ 8.08 (d, J= 8.5 Hz, 1H), 7.96 (d, J= 6.6 Hz, 1H), 7.92 (s, 1H), 7.84 (d, J= 8.5 Hz, 1H), 7.64 - 7.57 (dt, J= 6.4, 1.6 Hz, 2H), 7.39 (s, 1H), 7.34 (d, J= 8.3 Hz, 1H), 7.23 (d, ,7= 8.3 Hz, 1H), 4.14 - 4.06 (m, 2H), 2.32 - 2.26 (s, 3H), 1.75 -1.68 (m, 2H), 1.32 - 1.23 (m, 2H).
Linkers 115 - 125 are synthesized following the same procedure for preparing linker 114.
Figure imgf000257_0003
Linker 116: 4-(3-(6-(l-(2,2-difluorobenzo[d| [l,3]dioxol-5-yl)cydopropane-l-carboxamido)- 3-methylpyridin-2-yl)benzamido)butanoic acid White solid, 63% yield. 'H NMR (400 MHz, CD3OD) 5 7.96 (d, J= 8.5 Hz, 1H), 7.83 - 7.76 (m, 3H), 7.54 - 7.43 (m, 2H), 7.27 (s, 1H), 7.22 (d, J= 8.3 Hz, 1H), 7.11 (d, J = 8.3 Hz, 1H), 3.33 (t, J= 6.9 Hz, 2H), 2.28 (t, J= 7.3 Hz, 2H), 2.18 (s, 3H), 1.85 - 1.79 (m, 2H), 1.63 - 1.57 (m, 2H), 1.20 - 1.12(m, 2H).
Figure imgf000257_0001
Linker 117: 5-(3-(6-(l-(2,2-difluorobenzo[<Z|[l,3]dioxol-5-yl)cyclopropane-l-carboxamido)- 3-methylpyridin-2-yl)benzamido)pentanoic acid White solid, 63% yield. 1H NMR (400 MHz, CD3OD) 5 8.08 (d, J= 8.6 Hz, 1H), 7.94 - 7.84 (m, 3H), 7.62 - 7.57 (m, 2H), 7.40 (s, 1H), 7.34 (d, J= 10.4 Hz, 1H), 7.24 (d, J= 8.4 Hz, 1H), 3.47 - 3.35 (m, 2H), 2.39 - 2.32 (m, 2H), 2.29 (s, 3H), 1.73 - 1.63 (m, 6H), 1.33 - 1.26 (m, 2H).
Figure imgf000257_0002
Linker 118: 6-(3-(6-(l-(2,2-difluorobenzod /| [l,3]dioxol-5-yl)cyclopropane-l-carboxamido)- 3-methylpyridin-2-yl)benzamido)hexanoic acid White solid, 65% yield. 'H NMR (400 MHz, CD3OD) 5 8.06 (d, J= 8.5 Hz, 1H), 7.88 (d, J= 6.4 Hz, 1H), 7.85 - 7.80 (m, 2H), 7.56 (d, J= 5.2 Hz, 2H), 7.37 (s, 1H), 7.31 (d, J= 8.3 Hz, 1H), 7.21 (d, J= 8.3 Hz, 1H), 3.38 (t, J= 6.3 Hz, 2H), 2.31 (t, J= 8.4 Hz, 2H), 2.27 (s, 3H), 1.71 - 1.58 (m, 6H), 1.48 - 1.37 (m, 2H), 1.30 - 1.24 (m, 2H).
Figure imgf000258_0001
Linker 119: 7-(3-(6-(l-(2,2-difluorobenzo[d| [l,3]dioxol-5-yl)cyclopropane-l-carboxamido)- 3-methylpyridin-2-yl)benzainido)heptanoic acid White solid, 61% yield. H NMR (400 MHz, CD3OD) 8 8.05 (d, J= 8.5 Hz, 1H), 7.87 (d, J= 5.1 Hz, 1H), 7.85 -7.79 (m, 2H), 7.59 - 7.51 (m, 2H), 7.35 (s, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 3.38 - 3.33 (m, 2H), 2.31 - 2.24 (m, 5H), 1.71 - 1.64 (m, 2H), 1.64 - 1.56 (m, 4H), 1.44 - 1.36 (m, 4H), 1.29 - 1.22 (m, 2H).
Figure imgf000258_0002
Linker 120: 8-(3-(6-(l-(2,2-difluorobenzo[d| [l,3]dioxol-5-yl)cydopropane-l-carboxamido)- 3-methylpyridin-2-yl)benzamido)octanoic acid White solid, 59% yield. 'H NMR (400 MHz, CD3OD) 8 8.04 (d, J= 8.5 Hz, 1H), 7.86 (d, J= 6.0 Hz, 1H), 7.83 (s, 1H), 7.79 (d, J= 8.5 Hz, 1H), 7.54 (d, J = 5.0 Hz, 2H), 7.34 (s, 1H), 7.29 (d, J= 8.1 Hz, 1H), 7.18 (d, J= 8.3 Hz, 1H), 3.38 - 3.33 (m, 2H), 2.30 - 2.22 (m, 5H), 1.70 - 1.64 (m, 2H), 1.62 - 1.53 (m, 4H), 1.42 - 1.30 (m, 6H), 1.27 - 1.20 (m, 2H).
Figure imgf000258_0003
Linker 121: 3-(2-(3-(6-(l-(2,2-difluorobenzo[</| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)benzamido)ethoxy)propanoic acid White solid, 67% yield. 1H NMR (400 MHz, CD3OD) 8 8.03 (d, J = 8.6 Hz, 1H), 7.88 (d, J = 6.8 Hz, 1H), 7.83 (s, 1H), 7.78 (d, J= 8.6 Hz, 1H), 7.57 - 7.49 (m, 2H), 7.32 (s, 1H), 7.28 (d, J= 8.3 Hz, 1H), 7.17 (d, J= 8.3 Hz, 1H), 3.71 (t, J= 6.1 Hz, 2H), 3.64 - 3.57 (m, 2H), 3.56 - 3.50 (m, 2H), 2.52 (t, J= 6.1 Hz, 2H), 2.24 (s, 3H), 1.67 - 1.64 (m, 2H), 1.25 - 1.20 (m, 2H).
Figure imgf000258_0004
Linker 122: 3-(2-(2-(3-(6-(l-(2,2-difluorobenzo[d | [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methyIpyridin-2-yI)benzamido)ethoxy)ethoxy)propanoic acid White solid, 63% yield. 1H NMR (400 MHz, CD3OD) δ 8.05 (d, J= 8.8 Hz, 1H), 7.93 - 7.89 (m, 1H), 7.86 (s, 1H), 7.83 (d, .7= 7.9 Hz, 1H), 7.60 - 7.53 (m, 2H), 7.36 (s, 1H), 7.31 (d, J= 8.2 Hz, 1H), 7.21 (d, J = 8.3 Hz, 1H), 3.71 (d, J = 5.5 Hz, 2H), 3.67 - 3.53 (m, 8H), 2.48 (t, J= 5.8 Hz, 2H), 2.26 (s, 3H), 1.70 - 1.62 (m, 2H), 1.28 - 1.22 (m, 2H).
Figure imgf000259_0001
Linker 123: l-(3-(6-(l-(2,2-difluorobenzo[d/| [l,3]dioxol-5-yl)cyclopropane-l-carboxamido)- 3-methylpyridin-2-yl)phenyl)-l-oxo-5,8,ll-trioxa-2-azatetradecan-14-oic acid White solid, 60% yield. 1H NMR (400 MHz, CD3OD) δ 8.04 (d, J = 7.8 Hz, 1H), 7.95 - 7.85 (m, 3H), 7.63 - 7.54 (m, 2H), 7.36 (s, 1H), 7.30 (d, J = 9.0 Hz, 1H), 7.20 (d, J= 7.4 Hz, 1H), 3.67 - 3.50 (m, 14H), 2.47 (t, J= 6.1 Hz, 2H), 2.27 (s, 3H), 1.72 - 1.66 (m, 2H), 1.30 - 1.24 (m, 2H).
Figure imgf000259_0002
Linker 125: l-(3-(6-(l-(2,2-difluorobenzo[d ] [l,3]dioxol-5-yl)cyclopropane-l-carboxamido)- 3-methylpyridin-2-yl)phenyl)-l-oxo-5,8,ll,14,17-pentaoxa-2-azaicosan-20-oic acid White solid, 61% yield. ’H NMR (400 MHz, CD3OD) 5 7.97 (s, 2H), 7.91 (d, J= 6.9 Hz, 1H), 7.87 (s, 1H), 7.63 - 7.53 (m, 2H), 7.31 (s, 1H), 7.25 (d, J= 8.2 Hz, 1H), 7.16 (d, J = 8.3 Hz, 1H), 3.65 - 3.56 (m, 8H), 3.55 - 3.47 (m, 14H), 2.45 (t, J= 6.0 Hz, 2H), 2.25 (s, 3H), 1.69 - 1.63 (m, 2H), 1.27 - 1.21 (m, 2H).
Scheme 29. Synthesis of example 310
Figure imgf000260_0001
Example 310
Example 310 : (E)-3-(6-(l-(2,2-difluorobenzo[J][l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-A-(2-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yI)propanamido)ethyl)benzamide Linker 100 (10 mg, 0.02 mmol, 1.0 eq), intermediate 13 (8 mg, 0.022 mmol, 1.1 eq), EDC HC1 (5.7 mg, 0.03 mmol, 1.5 eq), HO At (4.1 mg, 0.03 mmol, 1.5 eq), NMM (6.0 mg, 0.06 mmol, 3.0 eq) were stirred in 1.5 mL DMF at room temperature for 2 h. Then the mixture was purified via Prep-HPLC to yield titled compound as a white solid (7.8 mg, 46%). 'H NMR (400 MHz, CD3OD) δ 8.04 (d, J = 8.4 Hz, 1H), 7.87 - 7.77 (m, 3H), 7.61 - 7.52 (m, 2H), 7.35 (s, 1H), 7.30 (d, J= 8.1 Hz, 1H), 7.19 (d, J = 7.4 Hz, 1H), 7.00 - 6.70 (m, 2H), 6.58 - 6.51 (m, 2H), 4.44 (s, 1H), 4.36 (s, 1H), 4.02 - 3.90 (m, 4H), 3.84 (s, 1H), 3.74 (s, 1H), 3.48 - 3.36 (m, 4H), 3.05 - 2.96 (m, 2H), 2.89 (s, 6H), 2.53 - 2.46 (m, 2H), 2.25 (s, 3H), 1.74 - 1.69 (m, 2H), 1.32 - 1.26 (m, 2H). MS (ESI) [M+H]+ = 842.1.
Examples 311 - 323 are synthesized as the same procedure for preparing example 310 from related linkers and intermediate 13.
Figure imgf000260_0002
Example 311: (E)-3-(6-(l-(2,2-difluorobenzo[</|[l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(3-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)propanamido)propyl)benzamide White solid, 45% yield. 1H NMR (400 MHz, CD3OD) δ 8.06 (d, J = 7.9 Hz, 1H), 7.94 - 7.83 (m, 3H), 7.63 - 7.54 (m, 2H), 7.37 (s, 1H), 7.31 (d, J= 8.2 Hz, 1H), 7.21 (d, J= 7.8 Hz, 1H), 6.92 (dd, J= 39.4, 15.1 Hz, 1H),
6.80 - 7.70 (m, 1H), 6.70 - 6.62 (m, 2H), 4.46 (s, 1H), 4.38 (s, 1H), 4.06 - 3.82 (m, 6H), 3.27 - 3.20 (m, 2H), 3.09 - 3.02 (m, 2H), 2.99 - 2.82 (m, 8H), 2.56 - 2.49 (m, 2H), 2.28 (s, 3H), 1.78 - 1.65 (m, 4H), 1.32 - 1.26 (m, 2H). MS (ESI) [M+H]+ = 856.4.
Figure imgf000261_0001
Example 312: (£)-3-(6-(l-(2,2-difluorobenzo[</| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(4-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)propanamido)butyl)benzamide White solid, 40% yield. JH NMR (400 MHz, CD30D) 5 8.04 (d, J= 7.5 Hz, 1H), 7.91 - 7.83 (m, 3H), 7.60 - 7.53 (m, 2H), 7.35 (s, 1H), 7.30 (d, J = 8.9 Hz, 1H), 7. 19 (d, J = 8.1 Hz, 1H), 6.91 (dd, J= 38.0, 15.1 Hz, 1H),
6.79 - 6.69 (m, 1H), 6.62 - 6.59 (m, 2H), 4.46 (s, 1H), 4.38 (s, 1H), 4.06 - 3.80 (m, 6H), 3.36 - 3.31 (m, 2H), 3.21 - 3.13 (m, 2H), 3.06 - 2.97 (m, 2H), 2.89 (s, 6H), 2.51 - 2.44 (m, 2H), 2.26 (s, 3H), 1.74 - 1.69 (m, 2H), 1.59 - 1.52 (m, 4H), 1.32 - 1.26 (m, 2H). MS (ESI) [M+H]+ = 870.5.
Figure imgf000261_0002
Example 313: (£)-3-(6-(l-(2,2-difluorobenzo[d| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-N -(5-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)propanamido)pentyl)benzamide White solid, 45% yield.1H NMR (400 MHz, CD3OD) 5 8.03 (d, J = 8.2 Hz, 1H), 7.90 - 7.82 (m, 3H), 7.59 - 7.52 (m, 2H), 7.35 (s, 1H), 7.29 (d, J= 8.0 Hz, 1H), 7.19 (d, J = 7.4 Hz, 1H), 6.92 (dd, J= 36.2, 15.2 Hz, 1H),
6.79 - 7.69 (m, 1H), 6.69 - 6.60 (m, 2H), 4.47 (s, 1H), 4.39 (s, 1H), 4.10 - 4.02 (m, 2H), 3.97 -
3.80 (m, 4H), 3.35 - 3.30 (m, 2H), 3.17 -3.11(m, 2H), 3.03 - 2.96 (m, 2H), 2.89 (s, 6H), 2.45 (t, J = 7.5 Hz, 2H), 2.25 (s, 3H), 1.69 - 1.65 (m, 2H), 1.62 - 1.53 (m, 2H), 1.52 - 1.42 (m, 2H), 1.36 - 1.20 (m, 4H). MS (ESI) [M+H]+ = = 884.4.
Figure imgf000262_0001
Example 314: (E')-3-(6-(l-(2,2-difluorobenzo[</| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(6-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)propanainido)hexyl)benzamide White solid, 44% yield. 1H NMR (400 MHz, CD30D) 5 8.04 (d, J = 8.4 Hz, 1H), 7.91 - 7.83 (m, 3H), 7.60 - 7.53 (m, 2H), 7.35 (s, 1H), 7.30 (d, J= 8.1 Hz, 1H), 7.20 (d, J= 7.5 Hz, 1H), 6.93 (dd, J = 36.6, 15.1 Hz, 1H), 6.81 - 6.69 (m, 1H), 6.66 - 6.60 (m, 2H), 4.48 (s, 1H), 4.39 (s, 1H), 4.16 - 3.77 (m, 6H), 3.37 - 3.31 (m, 2H), 3.17 - 3.10 (m, 2H), 3.04 - 2.96 (m, 2H), 2.90 (s, 6H), 2.51 - 2.44 (m, 2H), 2.26 (s, 3H), 1.70 - 1.65 (m, 2H), 1.62 - 1.40 (m, 4H), 1.38 - 1.23 (m, 6H). MS (ESI) [M+Hf = 898.5.
Figure imgf000262_0002
Example 315: (E)-3-(6-(l-(2,2-difluorobenzo[</| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(7-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)propanamido)heptyl)benzamide White solid, 47% yield. 1 H NMR (400 MHz, CD3OD) 5 8.04 (d, J = 8.4 Hz, 1H), 7.91 - 7.80 (m, 3H), 7.60 - 7.53 (m, 2H), 7.35 (s, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.19 (d, J = 8.1 Hz, 1H), 6.94 (dd, J= 37.3, 15.1 Hz, 1H), 6.81 - 6.69 (m, 1H), 6.65 - 6.60 (m, 2H), 4.49 (s, 1H), 4.41 (s, 1H), 4.03 - 3.81 (m, 6H), 3.37 - 3.31 (m, 2H), 3.16 - 3.09 (m, 2H), 3.01 (t, J= 6.6 Hz, 2H), 2.90 (s, 6H), 2.51 - 2.44 (m, 2H), 2.25 (s, 3H), 1.67 - 1.54 (m, 4H), 1.49 - 1.21 (m, 10H). MS (ESI) [M+H]+ = 912.6.
Figure imgf000262_0003
Example 316: (E)-3-(6-(l-(2,2-difluorobenzo[d/][1,]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(8-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)propanamido)octyl)benzamide White solid, 43% yield. 1H NMR (400 MHz, CD3OD) 5 8.04 (d, J = 8.4 Hz, 1H), 7.91 - 7.80 (m, 3H), 7.60 - 7.52 (m, 2H), 7.36 (s, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.20 (d, J = 8.1 Hz, 1H), 6.94 (dd, J= 37.3, 15.1 Hz, 1H), 6.81 - 6.70 (m, 1H), 6.65 - 6.60 (m, 2H), 4.49 (s, 1H), 4.41 (s, 1H), 4.05 - 3.81 (m, 6H), 3.40 - 3.32 (m, 2H), 3.18 - 3.08 (m, 2H), 3.01 (t, J= 6.6 Hz, 2H), 2.90 (s, 6H), 2.51 - 2.44 (m, 2H), 2.25 (s, 3H), 1.69 - 1.54 (m, 4H), 1.49 - 1.21 (m, 12H). MS (ESI) [M+H]+ = 926.5.
Figure imgf000263_0001
Example 317: (E)-3-(6-(l-(2,2-difluorobenzo[</| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-N -(9-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)propanamido)nonyl)benzamide White solid, 48% yield. 1H NMR (400 MHz, CD30D) 5 8.05 (d, J = 8.3 Hz, 1H), 7.90 - 7.81 (m, 3H), 7.60 - 7.52 (m, 2H), 7.36 (s, 1H), 7.31 (d, J= 8.2 Hz, 1H), 7.20 (d, J= 7.8 Hz, 1H), 6.94 (dd, J= 38.1, 15.1 Hz, 1H), 6.81 - 6.71 (m, 1H), 6.63 (s, 2H), 4.49 (s, 1H), 4.41 (s, 1H), 4.03 (s, 2H), 3.98 - 3.82 (m, 4H), 3.39 - 3.33 (m, 2H), 3.14 - 3.07 (m, 2H), 3.05 - 2.99 (m, 2H), 2.91 (s, 6H), 2.52 - 2.45 (m, 2H), 2.26 (s, 3H), 1.70 - 1.55 (m, 4H), 1.46 - 1.22 (m, 14H). MS (ESI) [M+H]+ = 940.6.
Figure imgf000263_0002
Example 318: (E)-3-(6-(l-(2,2-difluorobenzo[d| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(10-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)propanamido)decyl)benzamide White solid, 45% yield. 1H NMR (400 MHz, CD3OD) 5 8.03 (d, J = 8.3 Hz, 1H), 7.91 - 7.82 (m, 3H), 7.61 - 7.51 (m, 2H), 7.35 (s, 1H), 7.29 (d, J= 8.0 Hz, 1H), 7.19 (d, J= 8.1 Hz, 1H), 6.93 (dd, J= 38.3, 15.2 Hz, 1H), 6.80 - 6.71 (m, 1H), 6.61 (s, 2H), 4.48 (s, 1H), 4.40 (s, 1H), 4.03 (s, 2H), 3.97 - 3.80 (m, 4H), 3.39 - 3.31 (m, 2H), 3.14 - 3.06 (m, 2H), 3.04 - 2.97 (m, 2H), 2.89 (s, 6H), 2.49 - 2.43 (m, 2H), 2.26 (s, 3H), 1.67 (s, 2H), 1.63 - 1.54 (m, 2H), 1.43 - 1.22 (m, 16H). MS (ESI) [M+H]+ = 954.6.
Figure imgf000264_0001
Example 319: (E)-3-(6-(l-(2,2-difluorobenzo[</| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(2-(2-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)propanamido)ethoxy)ethyl)benzamide White solid, 46% yield. JH NMR (400 MHz, CD3OD) 5 8.04 (d, J = 8.1 Hz, 1H), 7.90 - 7.78 (m, 3H), 7.60 - 7.51 (m, 2H), 7.37 (s, 1H), 7.31 (d, J= 8.2 Hz, 1H), 7.20 (d, J = 7.9 Hz, 1H), 6.94 (dd, J= 34.7, 15.1 Hz, 1H), 6.80 - 6.71 (m, 1H), 6.57 - 6.53 (m, 2H), 4.48 (s, 1H), 4.40 (s, 1H), 4.08 - 3.76 (m, 6H), 3.62 - 3.48 (m, 6H), 3.37 - 3.32 (m, 2H), 2.98 - 2.88 (m, 8H), 2.45 - 2.36 (m, 2H), 2.25 (s, 3H), 1.71 - 1.65 (m, 2H), 1.27 - 1.23 (m, 2H). MS (ESI) [M+H]+ = 886.5.
Figure imgf000264_0002
Example 320: (E')-3-(6-(l-(2,2-difluorobenzo[</| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(2-(2-(2-(3-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)propanamido)ethoxy)ethoxy)ethyl)benzamide White solid, 44% yield. 1H NMR (400 MHz, CD3OD) 5 8.02 (d, J= 8.0 Hz, 1H), 7.91 - 7.83 (m, 3H), 7.61 - 7.51 (m, 2H), 7.34 (s, 1H), 7.29 (d, J= 8.2 Hz, 1H), 7.19 (d, J= 8.2 Hz, 1H), 6.92 (dd, J= 313, 15.1 Hz, 1H), 6.80 - 6.69 (m, 1H), 6.61 - 6.57 (m, 2H), 4.47 (s, 1H), 4.39 (s, 1H), 4.07 - 3.78 (m, 6H), 3.65 - 3.51 (m, 8H), 3.47 - 3.42 (m, 2H), 3.27 -3.22 (m, 2H), 3.01 - 2.93 (m, 2H), 2.89 (s, 6H), 2.47 - 2.40 (m, 2H), 2.25 (s, 3H), 1.71 - 1.65 (m, 2H), 1.27 - 1.23 (m, 2H). MS (ESI) [M+H]+
= 930.6.
Figure imgf000264_0003
7.60 - 7.51 (m, 2H), 7.35 (s, 1H), 7.30 (d, J= 8.1 Hz, 1H), 7.19 (d, J= 7.7 Hz, 1H), 6.92 (dd, J = 37.9, 15.2 Hz, 1H), 6.80 - 6.69 (m, 1H), 6.64 - 6.56 (m, 2H), 4.47 (s, 1H), 4.39 (s, 1H), 4.04 - 3.79 (m, 6H), 3.67 - 3.47 (m, 12H), 3.44 - 3.38 (m, 2H), 3.27 -3.22 (m, 2H), 3.02 - 2.95 (m, 2H), 2.90 (s, 6H), 2.49 - 2.42 (m, 2H),2.25 (s, 3H), 1.71 - 1.65 (m, 2H), 1.27 - 1.23 (m, 2H). MS (ESI) [M+H]+ = 974.5.
Figure imgf000265_0001
Example 322: (E)-3-(6-(l-(2,2-difluorobenzo[d| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-N18-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)-16-oxo-3,6,9,12-tetraoxa-15-azaoctadecyl)benzamide
White solid, 45% yield. JH NMR (400 MHz, CD3OD) 5 8.06 (d, J= 8.4 Hz, 1H), 7.92 - 7.80 (m, 3H), 7.61 - 7.53 (m, 2H), 7.37 (s, 1H), 7.32 (d, J= 8.2 Hz, 1H), 7.21 (d, J= 7.9 Hz, 1H), 6.94 (dd, J = 37.3, 15.2 Hz, 1H), 6.83 - 6.71 (m, 1H), 6.65 - 6.58 (m, 2H), 4.49 (s, 1H), 4.42 (s, 1H), 4.09 - 3.80 (m, 6H), 3.66 - 3.42 (m, 18H), 3.30 - 3.27 (m, 2H), 3.06 -2.98 (m, 2H), 2.91 (s, 6H), 2.54
- 2.47 (m, 2H), 2.26 (s, 3H), 1.71 - 1.65 (m, 2H), 1.27 - 1.23 (m, 2H). MS (ESI) [M+H]+ =
1018.4.
Figure imgf000265_0002
Example 323: (E)-3-(6-(l-(2,2-difluorobenzo[d| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-7V-(21-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)-19-oxo-3,6,9,12,15-pentaoxa-18-azahenicosyl)benzamide
White solid, 47% yield. JH NMR (400 MHz, CD3OD) 5 8.09 (d, J= 8.1 Hz, 1H), 7.91 - 7.83 (m, 2H), 7.75 (d, J= 8.1 Hz, 1H), 7.56 (s, 2H), 7.39 (s, 1H), 7.34 (d, J= 8.1 Hz, 1H), 7.23 (d, J= 8.1 Hz, 1H), 6.96 (dd, J= 36.4, 15.1 Hz, 1H), 6.85 - 6.72 (m, 1H), 6.68 - 6.60 (m, 2H), 4.51 (s, 1H), 4.44 (s, 1H), 4.08 - 3.85 (m, 6H), 3.70 - 3.52 (m, 20H), 3.52 - 3.46 (m, 2H), 3.36 - 3.33 (m, 2H), 3.09 - 3.00 (m, 2H), 2.93 (s, 6H), 2.56 - 2.49 (m, 2H), 2.26 (s, 3H), 1.71 - 1.65 (m, 2H), 1.27 - 1.23 (m, 2H). MS (ESI) [M+H]+ = 1062.6.
Figure imgf000265_0003
Example 324: (E)-3-(6-(l-(2,2-difluorobenzo[d/][1,3dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-W-(2-((2-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)ethyl)amino)-2-oxoethyl)benzamide Example 324 was synthesized following similar procedure for preparing example 310 from intermediate 15 and linker 114. White solid, 43% yield. ’H NMR (400 MHz, CD3OD) 8 7.99 (d, J= 7.9 Hz, 1H), 7.91 - 7.73 (m, 3H), 7.57 - 7.47 (m, 2H), 7.30 (s, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.13 (d, J = 7.4 Hz, 1H), 6.86 (dd, J= 34.7, 16.4 Hz, 1H), 6.74 - 6.64 (m, 1H), 6.62 - 6.55 (m, 2H), 4.42 - 4.39 (m, 1H), 4.35 - 4.31 (m, 1H), 3.98 - 3.73 (m, 7H), 3.58 - 3.54 (m, 1H), 3.41 - 3.34 (m, 2H), 2.91 - 2.76 (m, 8H), 2.20 (s, 3H), 1.63 - 1.59 (m, 2H), 1.21 - 1.17(m, 2H). MS (ESI) [M+H]+ = 828.3.
Example 325 - 335 were synthesized following the similar procedure for preparing example 324.
Figure imgf000266_0001
Example 325: (£)-3-(6-(l-(2,2-difluorobenzo[d| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(3-((2-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)ethyl)amino)-3-oxopropyl)benzamide White solid, 42% yield. 'H NMR (400 MHz, CD3OD) 8 8.07 (d, J = 13 Hz, 1H), 7.91 - 7.78 (m, 3H), 7.60 - 7.54 (m, 2H), 7.38 (s, 1H), 7.33 (d, J= 6.8 Hz, 1H), 7.22 (d, J= 7.4 Hz, 1H), 6.94 (dd, J= 35.9, 15.3 Hz, 1H), 6.82 - 6.71 (m, 1H), 6.65 - 6.61 (m, 2H), 4.49 (s, 1H), 4.42 (s, 1H), 4.05 - 3.82 (m, 6H), 3.67 - 3.59 (m, 2H), 3.48 - 3.39 (m, 2H), 2.95 - 2.90 (m, 8H), 2.55 - 2.46 (m, 2H), 2.26 (s, 3H), 1.71 - 1.69 (m, 2H), 1.29 - 1.25 (m, 2H). MS (ESI) [M+H]+ = 842.4.
Figure imgf000266_0002
(s, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.24 (d, J= 7.6 Hz, 1H), 6.96 (dd, J= 30.3, 15.3 Hz, 1H), 6.84 - 6.72 (m, 1H), 6.65 - 6.61(m, 2H), 4.50 (s, 1H), 4.42 (s, 1H), 4.08 - 3.97 (m, 4H), 3.93 - 3.79 (m, 2H), 3.47 - 3.37 (m, 4H), 2.98 - 2.90 (m, 8H), 2.38 - 2.23 (m, 5H), 1.99 - 1.86 (m, 2H), 1.72 - 1.70 (m, 2H), 1.31 - 1.27 (m, 2H). MS (ESI) [M+H]+ = 856.3.
Figure imgf000267_0001
Example 327: (E)-3-(6-(l-(2,2-difluorobenzo[J] [l,3Jdioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(5-((2-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)ethyl)amino)-5-oxopentyl)benzamide White solid, 46% yield. ' H NMR (400 MHz, CD3OD) 8 8.06 (d, .7= 8.0 Hz, 1H), 7.93 - 7.80 (m, 3H), 7.62 -7.53 (m, 2H), 7.38 (s, 1H), 7.32 (d, J= 7.0 Hz, 1H), 7.22 (d, J= 7.4 Hz, 1H), 6.94 (dd, J = 37.1, 15.2 Hz, 1H), 6.83 - 6.70 (m, 1H), 6.68 - 6.64 (m, 2H), 4.48 (s, 1H), 4.40 (s, 1H), 4.07 - 3.81 (m, 6H), 3.47 - 3.34 (m, 4H), 2.94 - 2.90 (m, 8H), 2.31 - 2.18 (m, 5H), 1.74 - 1.55 (m, 6H), 1.29 - 1.25 (m, 2H). MS (ESI) [M+H]+ = 870.5.
Figure imgf000267_0002
Example 328: (E)-3-(6-(l-(2,2-difluorobenzod] [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(6-((2-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)ethyl)amino)-6-oxohexyl)benzamide White solid, 42% yield.
NMR (400 MHz, CD3OD) 8 8.07 (d, J= 7.1 Hz, 1H), 7.93 - 7.76 (m, 3H), 7.57 (s, 2H), 7.40 (s, 1H), 7.34 (d, J= 8.1 Hz, 1H), 7.23 (d, J= 7.5 Hz, 1H), 6.95 (dd, J= 33.8, 15.0 Hz, 1H), 6.83 - 6.71 (m, 1H), 6.68 - 6.64 (m, 2H), 4.51 (s, 1H), 4.44 (s, 1H), 4.11 - 3.83 (m, 6H), 3.43 - 3.35 (m, 4H), 2.96 - 2.85 (m, 8H), 2.30 - 2.15 (m, 5H), 1.74 - 1.55 (m, 6H), 1.43 - 1.23 (m, 4H). MS (ESI) [M+H] 1 = 884.7.
Figure imgf000268_0001
Example 329: (E)-3-(6-(l-(2,2-difluorobenzo[J][l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(7-((2-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)ethyl)amino)-7-oxoheptyl)benzamide (MS6178) White solid, 45% yield. 'H NMR (400 MHz, CD3OD) 8 8.07 (d, J= 7.8 Hz, 1H), 7.94 - 7.78 (m, 3H), 7.59 (s, 2H), 7.39 (s, 1H), 7.33 (d, J= 8.0 Hz, 1H), 7.23 (d, J= 8.0 Hz, 1H), 6.95 (dd, J= 34.8, 15.2 Hz, 1H), 6.83 - 6.72 (m, 1H), 6.69 - 6.66 (m, 2H), 4.51 (s, 1H), 4.44 (s, 1H), 4.11 - 3.85 (m, 6H), 3.45
- 3.35 (m, 4H), 2.95 - 2.90 (m, 8H), 2.27 (s, 3H), 2.21 - 2.10 (m, 2H), 1.69 - 1.67 (m, 2H), 1.66
- 1.56 (m, 4H), 1.39 - 1.35 (m, 4H), 1.29 - 1.25 (m, 2H). MS (ESI) [M+H]+ = 898.5.
Figure imgf000268_0002
Example 330: (£)-3-(6-(l-(2,2-difluorobenzo[d| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(8-((2-(5-(4-(4-(dimethylainino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)ethyl)amino)-8-oxooctyl)benzamide White solid, 43% yield.
NMR (400 MHz, CD3OD) δ 8.08 (d, J= 7.9 Hz, 1H), 7.92 - 7.77 (m, 3H), 7.58 (s, 2H), 7.39 (s, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.23 (d, J= 7.9 Hz, 1H), 6.96 (dd, J= 34.2, 15.1 Hz, 1H), 6.83 - 6.72 (m, 1H), 6.69 - 6.66 (m, 2H), 4.52 (s, 1H), 4.45 (s, 1H), 4.11 - 3.87 (m, 6H), 3.45 - 3.36 (m, 4H), 2.95 - 2.90 (m, 8H), 2.27 (s, 3H), 2.20 - 2.14 (m, 2H), 1.74 - 1.54 (m, 6H), 1.41 - 1.23 (m, 8H). MS (ESI) [M+H]+ = 912.4.
Figure imgf000268_0003
2H), 7.39 (s, 1H), 7.33 (d, J= 6.0 Hz, 1H), 7.23 (d, J= 7.3 Hz, 1H), 6.96 (dd, J= 32.6, 15.1 Hz, 1H), 6.85 - 6.72 (m, 1H), 6.61 - 6.57 (m, 2H), 4.50 (s, 1H), 4.43 (s, 1H), 4.09 - 3.82 (m, 6H), 3.77 -3.69 (m, 2H), 3.63 - 3.55 (m, 4H), 3.31 - 3.26 (m, 2H), 2.93 (s, 6H), 2.84 -2.78 (m, 2H), 2.46 - 2.41 (m, 2H), 2.26 (s, 3H), 1.71 - 1.67 (m, 2H), 1.29 - 1.27 (m, 2H). MS (ESI) [M+H]+ = 886.6.
Figure imgf000269_0001
Example 332: (E)-3-(6-(l-(2,2-difluorobenzo[d] [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(2-(2-(3-((2-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)ethyl)amino)-3-oxopropoxy)ethoxy)ethyl)benzamide White solid, 47% yield. 'H NMR (400 MHz, CD30D) δ 8.07 (d, J = 8.0 Hz, 1H), 7.93 - 7.78 (m, 3H), 7.58 (s, 2H), 7.39 (s, 1H), 7.33 (d, J= 7.9 Hz, 1H), 7.23 (d, J = 7.5 Hz, 1H), 6.96 (dd, J= 35.0, 15.2 Hz, 1H), 6.84 - 7.72 (m, 1H), 6.69 - 6.62 (m, 2H), 4.51 (s, 1H), 4.44 (s, 1H), 4.10 - 3.85 (m, 6H), 3.71 - 3.56 (m, 10H), 3.40 - 3.33 (m, 2H), 2.96 - 2.85 (m, 8H), 2.42 - 2.34 (m, 2H), 2.26 (s, 3H), 1.71 - 1.67 (m, 2H), 1.29 - 1.27 (m, 2H). MS (ESI) [M+H] 1 = 930.4.
Figure imgf000269_0002
Example 333: (E')-3-(6-(l-(2,2-difluorobenzo[d] [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(15-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)-12-oxo-3,6,9-trioxa-13-azapentadecyl)benzamide White solid, 45% yield. 'H NMR (400 MHz, CD3OD) 8 8.08 (d, ,/ = 8.0 Hz, 1H), 7.94 - 7.80 (m, 3H), 7.58 (s, 2H), 7.40 (s, 1H), 7.34 (d, J= 8.1 Hz, 1H), 7.23 (d, J = 7.9 Hz, 1H), 6.96 (dd, J= 35.3, 15.1 Hz, 1H), 6.83 - 6.73 (m, 1H), 6.68 - 6.66 (m, 2H), 4.51 (s, 1H), 4.44 (s, 1H), 4.09 - 3.87 (m, 6H), 3.69 - 3.51 (m, 14H), 3.43 - 3.36 (m, 2H), 2.98 - 2.86 (m, 8H), 2.42 - 2.36 (d, J= 7.3 Hz, 2H), 2.27 (s, 3H), 1.71 - 1.67 (m, 2H), 1.29 - 1.27 (m, 2H). MS (ESI) [M+H]+ = 974.9.
Figure imgf000269_0003
Example 334: (E)-3-(6-(l-(2,2-difluorobenzo[</| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-7V-(18-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)-15-oxo-3,6,9,12-tetraoxa-16-azaoctadecyl)benzamide White solid, 46% yield. 1H NMR (400 MHz, CD3OD) δ 8.08 (d, J= 8.1 Hz, 1H), 7.93 - 7.77 (m, 3H), 7.58 (s, 2H), 7.39 (s, 1H), 7.34 (d, J= 7.6 Hz, 1H), 7.23 (d, J = 7.4 Hz, 1H), 6.96 (dd, J = 35.4, 15.2 Hz, 1H), 6.84 - 6.74 (m, 1H), 6.69 - 6.66 (m, 2H), 4.51 (s, 1H), 4.44 (s, 1H), 4.10 - 3.86 (m, 6H), 3.73 - 3.51 (m, 18H), 3.46 - 3.37 (m, 2H), 2.95 - 2.90 (m, 8H), 2.45 - 2.38 (m, 2H), 2.27 (s, 3H), 1.71 - 1.67 (m, 2H), 1.29 - 1.27 (m, 2H). MS (ESI) [M+H]+ = 1018.6.
Figure imgf000270_0001
Example 335: (E)-3-(6-(l-(2,2-difluorobenzo[d| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-A-(21-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin-l-yl)thiophen-2-yl)-18-oxo-3,6,9,12,15-pentaoxa-19-azahenicosyl)benzamide
White solid, 48% yield. JH NMR (400 MHz, CD3OD) 5 8.07 (d, J= 1A Hz, 1H), 7.95 - 7.80 (m, 3H), 7.59 (s, 2H), 7.39 (s, 1H), 7.33 (d, J= 8.1 Hz, 1H), 7.23 (d, J = 7.5 Hz, 1H), 6.96 (dd, J = 35.8, 15.0 Hz, 1H), 6.82 - 6.72 (m, 1H), 6.69 - 6.67 (m, 2H), 4.51 (s, 1H), 4.44 (s, 1H), 4.10 - 3.87 (m, 6H), 3.72 - 3.53 (m, 22H), 3.44 - 3.36 (m, 2H), 2.95 - 2.90 (m, 8H), 2.47 - 2.39 (m, 2H), 2.28 (s, 3H), 1.71 - 1.67 (m, 2H), 1.29 - 1.27 (m, 2H). MS (ESI) [M+H]+ = 1062.5.
Scheme 30. Synthesis of intermediate 23
Figure imgf000270_0002
Intermdiate 23
Intermediate 23: 3-(6-(4-(tert-butoxycarbonyl)-2-oxopiperazin-l-yl)pyridin-2-yl)propanoic acid Intermediate 23 was synthesized following similar procedure for preparing intermediate 13. White solid, 35% yield. 1H NMR (400 MHz, CD3OD) δ 7.79 - 7.66 (m, 2H), 7.18 (d, .7= 6.7 Hz, 1H), 4.26 (s, 2H), 4.10 (s, 2H), 3.78 (s, 2H), 3.17 - 3.08 (m, 2H), 2.83 - 2.73 (m, 2H), 1.53 (s, 9H). MS (ESI) [M+H]+ = 350.1.
Scheme 31. Synthesis of Example 336
Figure imgf000271_0002
Example 336: N-(4-(3-(6-(4-(2-chloroacetyl)-2-oxopiperazin-l-yl)pyridin-2- yl)propanamido)butyl)-3-(6-(l-(2,2-difluorobeiizo[d][1,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)benzamide Linker 102 (10 mg, 0.02 mmol, 1.0 eq), intermediate 23 (8.7 mg, 0.022 mmol, 1.1 eq), EDC HCI (5.7 mg, 0.03 mmol, 1.5 eq), HOAt (4.1 mg, 0.03 mmol, 1.5 eq), NMM (6.0 mg, 0.06 mmol, 3.0 eq) were stirred in DMF (1.5 mL) at room temperature for 2 h. Then the mixture was purified via Reverse-ISCO. The purified product was then dissolved in MeOH (1 mL) and HCI solution (4 M in dioxane, 1 mL, 4 mmol), stirred for 2h. Then removed all the volatiles and re-dissolved in DCM (1 mL), followed by 2-chloroacetyl chloride (2.5 mg, 0.024 mmol, 1.2 eq) and Et3N (2.6 mg, 0.026 mmol, 1.3 eq). After stirred at rt for 5 min, removed all the volatiles, the mixture was purified via prep-HPLC to yield titled compound (2.7 mg, 16 % over 3 steps). 1H NMR (400 MHz, CD3OD) 6 8.06 (d, J= 7.5 Hz, 1H), 7.97 - 7.88 (m, 3H), 7.73 - 7.57 (m, 4H), 7.39 (s, 1H), 7.33 (d, J= 6.2 Hz, 1H), 7.23 (d, J= 7.9 Hz, 1H), 7.12 (d, J= 7.2 Hz, 1H), 4.42 (s, 1H), 4.38 - 4.31 (m, 3H), 4.24 - 4.10 (m, 2H), 3.93 (s, 2H), 3.35 (s, 2H), 3.19 (s, 2H), 3.07 (s, 2H), 2.64 (s, 2H), 2.30 (s, 3H), 1.71 (s, 2H), 1.52 (s, 4H), 1.30 (s, 2H). MS (ESI) [M+H]+ = 830.4.
Examples 337- 340 are synthesized as the same procedure for preparing example 336 from related linkers and intermediate 23.
Figure imgf000271_0001
Example 337: 7V-(5-(3-(6-(4-(2-chloroacetyl)-2-oxopiperazin-l-yl)pyridin-2- yl)propanamido)pentyl)-3-(6-(l-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)cycIopropane-l- carboxamido)-3-methylpyridin-2-yl)benzamide White solid, 15% yield. 1H NMR (400 MHz, CD30D) 5 8.08 (d, J= 7.4 Hz, 1H), 7.94 - 7.80 (m, 3H), 7.70 (s, 2H), 7.58 (s, 2H), 7.39 (s, 1H), 7.34 (d, J= 5.9 Hz, 1H), 7.23 (d, J= 7.2 Hz, 1H), 7.11 (s, 1H), 4.44 (s, 1H), 4.39 - 4.31 (m, 3H), 4.21 (s, 1H), 4.13 (s, 1H), 4.00 - 3.87 (m, 2H), 3.36 (s, 2H), 3.16 (s, 2H), 3.06 (s, 2H), 2.62 (s, 2H), 2.27 (s, 3H), 1.70 (s, 2H), 1.60 (s, 2H), 1.49 (s, 2H), 1.38 - 1.24 (m, 4H). MS (ESI) [M+H]+ = 844.9.
Figure imgf000272_0001
Example 338: N-(6-(3-(6-(4-(2-chloroacetyl)-2-oxopiperazin-l-yl)pyridin-2- yl)propanamido)hexyl)-3-(6-(l-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)benzamide White solid, 14% yield. 'H NMR (400 MHz, CD3OD) 5 8.08 (d, J= 7.6 Hz, 1H), 7.94 - 7.82 (m, 3H), 7.70 (s, 2H), 7.59 (s, 2H), 7.39 (s, 1H), 7.33 (d, J = 7.7 Hz, 1H), 7.23 (d, J= 7.6 Hz, 1H), 7.12 (s, 1H), 4.43 (s, 1H), 4.35 (s, 3H), 4.21 (s, 1H), 4.13 (s, 1H), 3.97 -3.89 (m, 2H), 3.37 (s, 2H), 3.15 (s, 2H), 3.08 (s, 2H), 2.63 (s, 2H), 2.28 (s, 3H), 1.69 (s, 2H), 1.60 (s, 2H), 1.49 - 1.26 (m, 8H). MS (ESI) [M+H]+ = 858.5.
Figure imgf000272_0002
Example 339: N-(7-(3-(6-(4-(2-chloroacetyl)-2-oxopiperazin-l-yl)pyridin-2- yl)propanamido)heptyl)-3-(6-(l-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)benzamide White solid, 12% yield. 'H NMR (400 MHz, CD3OD) 5 8.08 (d, J = 6.8 Hz, 1H), 7.88 (s, 1H), 7.85 (s, 1H), 7.79 (d, J= 7.3 Hz, 1H), 7.70 (s, 2H), 7.57 (s, 2H), 7.39 (s, 1H), 7.34 (d, ,7 = 7.9 Hz, 1H), 7.23 (d, .J = 7.3 Hz, 1H), 7.12 (s, 1H), 4.44 (s, 1H), 4.36 (s, 3H), 4.20 (s, 1H), 4.13 (s, 1H), 3.99 - 3.87 (m, 2H), 3.40 - 3.35 (d, J = 7.6 Hz, 2H), 3.15 (s, 2H), 3.08 (s, 2H), 2.63 (s, 2H), 2.27 (s, 3H), 1.69 (s, 2H), 1.61 (s, 2H), 1.45 (s, 2H), 1.41 - 1.23 (m, 8H). MS (ESI) [M+H]+ = 872.5.
Figure imgf000272_0003
Example 340: /V-(8-(3-(6-(4-(2-chloroacetyl)-2-oxopiperazin-l-yl)pyridin-2- yl)propanamido)octyl)-3-(6-(l-(2,2-difluorobenzo[d|[l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)benzamide White solid, 16% yield. 'H NMR (400 MHz, CD3OD) 5 8.08 (d, J= 7.9 Hz, 1H), 7.95 - 7.84 (m, 3H), 7.72 (s, 2H), 7.60 (s, 2H), 7.39 (s, 1H), 7.33 (d, J= 6.8 Hz, 1H), 7.23 (d, J= 8.4 Hz, 1H), 7.13 (s, 1H), 4.44 (s, 1H), 4.36 (s, 3H), 4.21 (s, 1H), 4. 13 (s, 1H), 3.98 - 3.88 (m, 2H), 3.41 - 3.35 (m, 2H), 3.17 - 3.04 (m, 4H), 2.62 (s, 2H), 2.29 (s, 3H), 1.70 (s, 2H), 1.62 (s, 2H), 1.48 - 1.21 (m, 12H). MS (ESI) [M+H]+ = 886.5.
Scheme 32. Synthesis of intermedia 25.
Figure imgf000273_0001
Intermediate 24 Intermediate 25
Intermediate 25: tert-butyl 4-(6-(2-aminoethyl)pyridin-2-yl)-3-oxopiperazine-l-carboxylate 2-(6-bromopyridin-2-yl)acetonitrile (196 mg, 1.0 mmol, 1.0 eq), tert-butyl 3 -oxopiperazine- 1- carboxylate (300 mg, 1.5 mmol, 1.5 eq), Cui (85 mg, 0.5 mmol), N1,N2-dimethylethane-l ,2- diamine (88 mg, 1.0 mmol, 1.0 eq), K2CO3 (276 mg, 2.0 mmol, 2.0 eq) were added Toluene (5 mb). The mixture was stirred at 110 °C under N2 for 6 h. The mixture was purified by ISCO to yield intermediate 17 as a white solid (205 mg, 65%).1H NMR (400 MHz, CD3OD) 5 7.96 - 7.83 (m, 2H), 7.31 (d, J = 7.9 Hz, 1H), 4.29 (s, 2H), 4.18 (s, 2H), 4.10 (s, 2H), 3.80 (s, 2H), 1.55 (s, 9H). MS (ESI) [M+H]+ = 317.4.
Intermediate 24 (160 mg, 0.5 mmol, 1.0 eq), Raney Ni (30 mg) were stirred in MeOH (2 mlL at room temperature under 1 atm H2 for 30 min. Then the mixture was purified by reverse-ISCO to yield titled compound as a white solid (120 mg, 75%). 1HMDR (400 MHz, CD3OD) δ 7.69 - 7.62 (m, 1H), 7.49 (d, J= 8.8 Hz, 1H), 7.32 (d, J= 9.9 Hz, 1H), 3.38 - 3.31 (m, 2H), 3.15 - 3.08 (m, 2H). MS (ESI) [M+H]+ = 321.5.
Example 341 - 345 were synthesized following similar procedure for preparing example 336 from related linker and intermediate 25.
Figure imgf000273_0002
Example 341 : N-(4-((2-(6-(4-(2-chloroacetyl)-2-oxopiperazin-1-yl)pyridin-2-yl)ethyl)amino)- 4-oxobutyl)-3-(6-(l-(2,2-difluorobenzo[J|[l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3- methylpyridin-2-yl)benzamide White solid, 18% yield. 'H NMR (400 MHz, CD3OD) 8 8.06 (d, J= 7.8 Hz, 1H), 7.95 - 7.86 (m, 3H), 7.77 - 7.56 (m, 4H), 7.39 (s, 1H), 7.33 (d, J= 7.3 Hz, 1H), 7.23 (d, J= 7.2 Hz, 1H), 7.12 (d, J= 7.2 Hz, 1H), 4.43 (s, 1H), 4.34 (s, 3H), 4.21 (s, 1H), 4.12 (s, 1H), 3.99 - 3.85 (m, 2H), 3.59 (s, 2H), 3.38 - 3.33 (m, 2H), 2.95 (s, 2H), 2.29 (s, 3H), 2.22 (s, 2H), 1.84 (s, 2H), 1.71 (s, 2H), 1.29 (s, 2H). MS (ESI) [M+H]+ = 816.4.
Figure imgf000274_0001
Example 342: W-(5-((2-(6-(4-(2-chloroacetyl)-2-oxopiperazin-l-yl)pyridin-2-yl)ethyl)amino)- 5-oxopentyl)-3-(6-(l-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3- methylpyridin-2-yl)benzamide White solid, 15% yield. 'H NMR (400 MHz, CD3OD) 8 8.08 (d, J= 7.7 Hz, 1H), 7.95 - 7.83 (m, 3H), 7.74 - 7.55 (m, 4H), 7.39 (s, 1H), 7.33 (d, J= 7.0 Hz, 1H), 7.23 (d, J= 7.0 Hz, 1H), 7.12 (d, J= 5.9 Hz, 1H), 4.44 (s, 1H), 4.36 (s, 3H), 4.21 (s, 1H), 4.13 (s, 1H), 3.98 - 3.88 (m, 2H), 3.61 (s, 2H), 3.38 - 3.34 (m, 2H), 2.96 (s, 2H), 2.28 (s, 3H), 2.19 (s, 2H), 1.70 (s, 2H), 1.62 (s, 2H), 1.54 (s, 2H), 1.27 (s, 2H). MS (ESI) [M+H]+ = 830.4.
Figure imgf000274_0002
Example 343: N-(6-((2-(6-(4-(2-chloroacetyl)-2-oxopiperazin-l-yl)pyridin-2-yl)ethyl)amino)- 6-oxohexyl)-3-(6-(l-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3- methylpyridin-2-yl)benzamide White solid, 17% yield. 'H NMR (400 MHz, CD3OD) 8 8.07 (d, J= 7.2 Hz, 1H), 7.94 - 7.82 (m, 3H), 7.78 - 7.66 (m, 2H), 7.60 (s, 2H), 7.40 (s, 1H), 7.34 (d, J = 7.5 Hz, 1H), 7.24 (d, J = 6.7 Hz, 1H), 7.11 (d, J = 7.5 Hz, 1H), 4.45 (s, 1H), 4.37 (s, 3H), 4.22 (s, 1H), 4.13 (s, 1H), 3.98 - 3.88 (m, 2H), 3.58 (s, 2H), 3.39 - 3.34 (m, 2H), 2.94 (s, 2H), 2.29 (s, 3H), 2.16 (s, 2H), 1.71 (s, 2H), 1.60 (s, 4H), 1.39 - 1.26 (m, 4H). MS (ESI) [M+H]+ = 844.5.
Figure imgf000274_0003
Example 344: N-(7-((2-(6-(4-(2-chloroacetyl)-2-oxopiperazin-1 -yl)pyridin-2-yl)ethyl)amino)- 7-oxoheptyl)-3-(6-(l-(2,2-difluorobenzo[</][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3- methylpyridin-2-yl)benzamide White solid, 14% yield. 'H NMR (400 MHz, CD3OD) 8 8.08 (d, J = 8.3 Hz, 1H), 7.94 - 7.81 (m, 3H), 7.76 -7.66 (m, 2H), 7.59 (s, 2H), 7.39 (s, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.23 (d, J= 6.1 Hz, 1H), 7.13 (s, 1H), 4.44 (s, 1H), 4.36 (s, 3H), 4.23 (s, 1H), 4.14 (s, 1H), 3.98 - 3.88 (m, 2H), 3.60 (s, 2H), 3.39 - 3.33 (m, 2H), 2.97 (s, 2H), 2.28 (s, 3H), 2.14 (s, 2H), 1.70 (s, 2H), 1.57 (br, 4H), 1.41 - 1.22 (m, 6H). MS (ESI) [M+H]+ = 858.5.
Figure imgf000275_0001
Example 345N: -(8-((2-(6-(4-(2-chloroacetyl)-2-oxopiperazin-l-yl)pyridin-2-yl)ethyl)amino)- 8-oxooctyl)-3-(6-(l-(2,2-difluorobenzo[r/|[l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3- methylpyridin-2-yl)benzamide White solid, 13% yield. JH NMR (400 MHz, CD3OD) 5 8.08 (d, J= 7.9 Hz, 1H), 7.92 - 7.68 (m, 5H), 7.57 (s, 2H), 7.39 (s, 1H), 7.34 (d, J= 7.8 Hz, 1H), 7.23 (d, ./ - 6,9 Hz, 1H), 7.12 (s, 1H), 4.44 (s, 1H), 4.37 (s, 3H), 4.23 (s, 1H), 4.14 (s, 1H), 3.99 - 3.89 (m, 2H), 3.60 (s, 2H), 3.41 - 3.34 (m, 2H), 2.96 (s, 2H), 2.27 (s, 3H), 2.13 (s, 2H), 1.69 (s, 2H), 1.64 - 1.52 (dm, 4H), 1.40 - 1.22 (m, 8H). MS (ESI) [M+H]+ = 872.5.
Scheme 33. Synthesis of intermedia 27
Figure imgf000275_0002
Intermediate 27: 3-(5-(l-(tert-butoxycarbonyl)azetidine-3-carboxamido)thiophen-2- yl)propanoic acid Methyl (E)-3-(5-nitrothiophen-2-yl)acrylate (416 mg, 2.0 mmol, 1.0 eq), Pd/C (120 mg), HSiEts (1.16 g, 10 mmol, 5.0 eq) and 2.5 mL MeOH were stirred in a sealed tube at 60 °C for 30 min. Then the mixture was filtered and purified with silica gel column (Hexane/Ethyl acetate = 1: 1) to afford intermediate 26 as yellow solid (200 mg, 49%). 1H NMR (400 MHz, Methanol-dr) 6 7.64 (s, 1H), 6.96 (s, 1H), 5.99 (s, 1H), 5.65 (s, 1H), 3.73 (s, 3H). LCMS m/z = 184.1.
Intermediate 26 (180 mg, 1.0 mmol, 1.0 eq), l-(ter/-butoxycarbonyl)azetidine-3-carboxylic acid (201 mg, 1.0 mmol, 1.0 eq), EDC HC1 (288 mg, 1.5 mmol, 1.5 eq), HOAt (204 mg, 1.5 mmol, 1.5 eq), NMM (300 mg, 3.0 mmol, 3.0 eq) were stirred in DMF (2.5 mL) at room temperature for 2 h. Then the mixture was purified via RP-C 18 [MeCN/Water (0.1%TFA)] to give 140 mg yellow solid without further purification. MS (ESI) [M+H]+ =367.2.
The crude product and Pd(OH)2 (70 mg) were stirred in MeOH (5 mL) at 60 °C under 1 atm H2 overnight. Then the mixture was filtered. To the filtrate, LiOH (56 mg, 2.3 mmol, 3.0 eq) and H2O (2 mL) were added, the mixture was stirred at room temperature for 1 h followed by purified via RP-C18 [MeCN/Water (0. 1%TFA)] to afford colorless solid (65 mg, 18% over 3 steps). 'H NMR (400 MHz, Methanol -d4) δ 6.60 (d, 7= 3.8 Hz, 1H), 6.54 (d, J = 3.8 Hz, 1H), 4.15 (s, 2H), 4.05 (s, 2H), 3.24 - 3.20 (m, 1H), 3.04 (t, J= 7.5 Hz, 2H), 2.56 (t, J= 7.4 Hz, 2H), 1.46 (s, 9H). MS (ESI) [M+H]+ = 355.2.
Examples 346 - 351 were synthesized following the same procedure for preparing example 336 from related linkers and intermediate 27.
Figure imgf000276_0001
Example 346: l-(2-chloroacetyl)-JV-(5-(3-((4-(3-(6-(l-(2,2-difluorobenzo[d] [l,3]dioxol-5- yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)butyl)amino)-3- oxopropyl)thiophen-2-yl)azetidine-3-carboxamide White solid, 35% yield. 1H NMR (400 MHz, Methanol-dr) δ 8.08 (d, J= 8.5 Hz, 1H), 7.93 - 7.89 (m, 1H), 7.86 (s, 1H), 7.81 (d, J= 8.5 Hz, 1H), 7.60 - 7.57 (d, J = 5.9 Hz, 2H), 7.39 (s, 1H), 7.34 (d, J = 7.9 Hz, 1H), 7.23 (d, J = 8.2 Hz, 1H), 6.58 (d, J= 3.1 Hz, 1H), 6.52 (d, J= 3.8 Hz, 1H), 4.48 - 4.42 (m, 2H), 4.24 - 4.10 (m, 2H), 4.04 (s, 2H), 3.60 - 3.54 (m, 1H), 3.38 - 3.35 (m, 2H), 3.24 - 3.19 (m, 2H), 3.05 (t, J= 7.2 Hz, 2H), 2.50 (t, J = 7.2 Hz, 2H), 2.28 (s, 3H), 1.70 (q, J = 4.0 Hz, 2H), 1.56 (s, 4H), 1.27 (q, J = 4.0 Hz, 2H). MS (ESI) [M+H]+ = 835.3.
Figure imgf000277_0001
Example 347: l-(2-chloroacetyl)-7V-(5-(3-((5-(3-(6-(l-(2,2-difluorobenzo[d][1,3]dioxol-5- yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)pentyl)amino)-3- oxopropyl)thiophen-2-yl)azetidine-3-carboxamide White solid, 36% yield. 1H MR (400 MHz, Methanol-d4 δ 8.11 - 8.06 (m, 1H), 7.97 - 7.89 (m, 3H), 7.66 - 7.59 (m, 2H), 7.41 (s, 1H), 7.39 - 7.33 (m, 1H), 7.27 - 7.23 (m, 1H), 6.57 (s, 1H), 6.56 - 6.51 (m, 1H), 4.49 - 4.43 (m, 2H), 4.25 - 4.11 (m, 2H), 4.05 (s, 2H), 3.60 (s, 1H), 3.40 - 3.36 (m, 2H), 3.23 - 3.14 (m, 2H), 3.07 - 3.00 (m, 2H), 2.52 - 2.44 (m, 2H), 2.31 (s, 3H), 1.73 (s, 2H), 1.62 (s, 2H), 1.52 (s, 2H), 1.32 (s, 4H). MS
Figure imgf000277_0002
Example 348: 1 -( 2-chloroa cetyl )- N-( 5-( 3-( ( 6-( 3-( 6-( 1 -(2,2-difluorobenzo[d| [l,3]dioxol-5- yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)hexyl)amino)-3- oxopropyl)thiophen-2-yl)azetidine-3-carboxamide White solid, 32% yield. NMR (400 MHz, Methanol-^) 8 8.09 (d, .7 = 8.5 Hz, 1H), 7.94 - 7.90 (m, 1H), 7.88 (s, 1H), 7.84 (d, ,7 = 8.5 Hz, 1H), 7.62 - 7.58 (m, 2H), 7.40 (d, J= 1.8 Hz, 1H), 7.36 - 7.32 (m, 1H), 7.24 (d, J= 8.2 Hz, 1H), 6.58 (d, .7= 3.8 Hz, 1H), 6.52 (d, .7= 3.8 Hz, 1H), 4.50 - 4.43 (m, 2H), 4.26 - 4.13 (m, 2H), 4.05 (s, 2H), 3.64 - 3.56 (m, 1H), 3.38 (t, J= 7.2 Hz, 2H), 3.16 (t, J= 6.8 Hz, 2H), 3.04 (t, J= 7.2 Hz, 2H), 2.50 (t, J= 7.3 Hz, 2H), 2.29 (s, 3H), 1.70 (q, J = 4.0 Hz, 2H), 1.64 - 1.56 (m, 2H), 1.52 - 1.44 (m, 2H), 1.40 - 1.25 (m, 6H MS (ESI) [M+H]+ = 863.3.
Figure imgf000277_0003
Example 349: l-(2-chloroacetyl)-N(5-(3-((7-(3-(6-(l-(2,2-difluorobenzo[d| [l,3]dioxol-5- yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)heptyl)amino)-3- oxopropyl)thiophen-2-yl)azetidine-3-carboxamide
White solid, 25% yield. ‘HNMR (400 MHz, Methanol-d)48 8.09 (d, J = 8.5 Hz, 1H), 7.94 - 7.88 (m, 1H), 7.87 (s, 1H), 7.83 (d, J= 8.5 Hz, 1H), 7.61 - 7.55 (m, 2H), 7.40 (d, J= 1.7 Hz, 1H), 7.34 (dd, J = 8.2, 1.8 Hz, 1H), 7.24 (d, J= 8.2 Hz, 1H), 6.57 (d, J= 3.8 Hz, 1H), 6.52 (d, J= 3.8 Hz, 1H), 4.53 - 4.43 (m, 2H), 4.27 - 4.13 (m, 2H), 4.05 (s, 2H), 3.65 - 3.57 (m, 1H), 3.38 (t, J= 7.2 Hz, 2H), 3.15 (t, J= 6.9 Hz, 2H), 3.04 (t, J= 7.3 Hz, 2H), 2.50 (t, J= 13 Hz, 2H), 2.28 (s, 3H), 1.70 (q, J= 4.0 Hz, 2H), 1.66 - 1.58 (m, 2H), 1.50 - 1.42 (m, 2H), 1.41 - 1.24 (m, 8H). MS (ESI) [M+H]+ = 877.3.
Figure imgf000278_0001
Example 350: l-(2-chloroacetyl)-N-(5-(3-((8-(3-(6-(l-(2,2-difluorobenzo[d] [l,3]dioxol-5- yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)octyl)amino)-3- oxopropyl)thiophen-2-yl)azetidine-3-carboxamide
White solid, 26% yield. 'H NMR (400 MHz, Methanol-d)48 8.09 (d, J= 8.5 Hz, 1H), 7.93 - 7.88 (m, 1H), 7.86 (s, 1H), 7.81 (d, J= 8.6 Hz, 1H), 7.61 - 7.55 (m, 2H), 7.40 (d, J= 1.8 Hz, 1H), 7.34 (dd, J= 8.3, 1.8 Hz, 1H), 7.24 (d, J= 8.3 Hz, 1H), 6.58 (d, J= 3.8 Hz, 1H), 6.53 (d, J= 3.8 Hz, 1H), 4.52 - 4.44 (m, 2H), 4.26 - 4.14 (m, 2H), 4.05 (s, 2H), 3.65 - 3.55 (m, 1H), 3.39 (t, J = 7.3 Hz, 2H), 3.14 (t, J= 6.9 Hz, 2H), 3.04 (t, J= 7.2 Hz, 2H), 2.50 (t, J= 7.3 Hz, 2H), 2.28 (s, 3H), 1.70 (q, J= 4.0 Hz, 2H), 1.66 - 1.56 (m, 2H), 1.48 - 1.23 (m, 12H). MS (ESI) [M+H]+ = 891.3.
Figure imgf000278_0002
Intermediate 28
Intermediate
Figure imgf000278_0003
3-(5-(2-(tert-butoxycarbonyl)-2-azaspiro[3.3]heptane-6- carboxamido)thiophen-2-yl)propanoic acid Intermediate 28 was synthesized following the same procedure for preparing intermediate 27. Yellow solid, 20% yield.
Figure imgf000278_0004
NMR (400 MHz, Methanol-d4) 8 6.59 (d, J= 3.8 Hz, 1H), 6.49 (d, J= 3.8 Hz, 1H), 3.98 (s, 2H), 3.89 (s, 2H), 3.12 - 3.08 (m, 1H), 3.03 (t, J= 7.5 Hz, 2H), 2.63 (t, J= 7.4 Hz, 2H), 2.49 - 2.42 (m, 4H), 1.45 (s, 9H). MS (ESI) [M+H]+ = 395.2.
Examples 351 - 355 were synthesized following the same procedure for preparing example 336 from related linkers and intermediate 28.
Figure imgf000278_0005
Example 351: 2-(2-chloroacetyl)-N-(5-(3-((4-(3-(6-(l-(2,2-difluorobenzo[d|[1,3]dioxol-5- yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)butyl)amino)-3- oxopropyl)thiophen-2-yl)-2-azaspiro[3.3]heptane-6-carboxamide
White solid, 31% yield. 'H NMR (400 MHz, Methanol-d4) 6 8.09 (d, J= 8.5 Hz, 1H), 7.92 - 7.88 (m, 1H), 7.85 (s, 1H), 7.77 (d, J= 8.5 Hz, 1H), 7.60 - 7.53 (m, 2H), 7.39 (d, J= 1.8 Hz, 1H), 7.36 - 7.29 (m, 1H), 7.23 (d, J= 8.3 Hz, 1H), 6.56 (s, 1H), 6.47 (d, J= 3.8 Hz, 1H), 4.33 (s, 1H), 4.23 (s, 1H), 4.05 (s, 1H), 4.02 (s, 1H), 4.00 (s, 1H), 3.97 (s, 1H), 3.40 - 3.36 (m, 2H), 3.24 - 3.18 (m, 2H), 3.16 - 3.12 (m, 1H), 3.04 (t, J= 7.7 Hz, 2H), 2.52 - 2.42 (m, 6H), 2.26 (s, 3H), 1.69 (q, J = 3.9 Hz, 2H), 1.56 (s, 4H), 1.26 (q, J= 4.0 Hz, 2H). MS (ESI) [M+H]+ = 875.3.
Figure imgf000279_0001
Example 352: 2-(2-chloroacetyl)-JV-(5-(3-((5-(3-(6-(l-(2,2-difluorobenzo[d| [l,3]dioxol-5- yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)pentyl)amino)-3- oxopropyl)thiophen-2-yl)-2-azaspiro[3.3]heptane-6-carboxamide
White solid, 33% yield. ‘H NMR (400 MHz, Methanol-d4) 8 8.09 (d, J= 8.5 Hz, 1H), 7.94 - 7.89 (m, 1H), 7.87 (s, 1H), 7.82 (d, J= 8.3 Hz, 1H), 7.61 - 7.55 (m, 2H), 7.40 (d, J= 1.7 Hz, 1H), 7.34 (dd, J= 8.2, 1.8 Hz, 1H), 7.24 (d, .J= 8.3 Hz, 1H), 6.57 - 6.53 (m, 1H), 6.48 (d, J= 3.7 Hz, 1H), 4.30 (s, 1H), 4.24 (s, 1H), 4.04 (s, 1H), 4.00 (s, 2H), 3.98 (s, 1H), 3.40 - 3.35 (m, 2H), 3.18 (t, J = 6.7 Hz, 2H), 3.13 - 3.07 (m, 1H), 3.02 (t, J= 7.2 Hz, 2H), 2.51 - 2.44 (m, 6H), 2.28 (s, 3H), 1.70 (q, J= 4.0 Hz, 2H), 1.60 (p, J= 7.4 Hz, 2H), 1.51 (p, J= 7.0 Hz, 2H), 1.37 - 1.25 (m, 4H). MS (ESI) [M+H]+ = 889.3.
Figure imgf000279_0002
Example 353: 2-(2-chloroacetyl)-N-(5-(3-((6-(3-(6-(l-(2,2-difluorobenzo[</] [l,3]dioxol-5- yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)hexyl)amino)-3- oxopropyl)thiophen-2-yl)-2-azaspiro[3.3]heptane-6-carboxamide
White solid, 35% yield. 1H NMR (400 MHz, Methanol-d4) 8 8.09 (d, J= 8.5 Hz, 1H), 7.94 - 7.89 (m, 1H), 7.87 (s, 1H), 7.81 (d, J= 8.6 Hz, 1H), 7.62 - 7.53 (m, 2H), 7.40 (d, J= 1.7 Hz, 1H), 7.34 (dd, J= 8.2, 1.8 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 6.56 (d, J = 3.8 Hz, 1H), 6.48 (d, J= 3.8 Hz, 1H), 4.31 (s, 1H), 4.25 (s, 1H), 4.05 (s, 1H), 4.01 (s, 2H), 3.99 (s, 1H), 3.41 - 3.36 (m, 2H), 3.19 - 2.99 (m, 5H), 2.54 -2.46 (m, 6H), 2.28 (s, 3H), 1.70 (q, J= 4.0 Hz, 2H), 1.60 (p, J= 7.1 Hz, 2H), 1.47 (p, J= 6.9 Hz, 2H), 1.39 - 1.26 (m, 6H). MS (ESI) [M+H]+ = 903.3.
Figure imgf000280_0001
Example 354: 2-(2-chloroacetyl)-N-(5-(3-((7-(3-(6-(l-(2,2-difluorobenzo[d [l,3]dioxol-5- yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)heptyl)amino)-3- oxopropyl)thiophen-2-yl)-2-azaspiro[3.3]heptane-6-carboxamide
White solid, 32% yield. 1H NMR (400 MHz, Methanol-)d48 8.09 (d, J= 8.5 Hz, 1H), 7.93 - 7.88 (m, 1H), 7.86 (s, 1H), 7.80 (d, J= 8.5 Hz, 1H), 7.60 - 7.55 (m, 2H), 7.40 (d, J= 1.7 Hz, 1H), 7.34 (dd, J= 8.3, 1.8 Hz, 1H), 7.24 (d, J= 8.3 Hz, 1H), 6.55 (d, J= 3.7 Hz, 1H), 6.47 (d, J= 3.7 Hz, 1H), 4.32 (s, 1H), 4.26 (s, 1H), 4.06 (s, 1H), 4.01 (s, 3H), 3.38 (t, J= 7.0 Hz, 2H), 3.18 - 3.00 (m, 5H), 2.52 - 2.46 (m, 6H), 2.27 (s, 3H), 1.70 (q, J= 4.0 Hz, 2H), 1.61 (p, J = 6.7 Hz, 2H), 1.50 - 1.27 (m, 10H). MS (ESI) (M+Hf = 917.3.
Figure imgf000280_0002
Example 355: 2-(2-chloroacetyl)-JV-(5-(3-((8-(3-(6-(l-(2,2-difluorobenzo[d] [l,3]dioxol-5- yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)octyl)amino)-3- oxopropyl)thiophen-2-yl)-2-azaspiro[3.3]heptane-6-carboxamide
White solid, 31% yield. 1 HMR (400 MHz, Methanol-d4) 8 8.10 (d, J= 8.5 Hz, 1H), 7.91 - 7.87 (m, 1H), 7.85 (s, 1H), 7.78 (d, J= 8.5 Hz, 1H), 7.59 - 7.55 (m, 2H), 7.40 (d, J= 1.7 Hz, 1H), 7.36
- 7.32 (m, 1H), 7.24 (d, J= 8.3 Hz, 1H), 6.55 (s, 1H), 6.48 (d, J= 3.7 Hz, 1H), 4.32 (s, 1H), 4.27 (s, 1H), 4.06 (s, 1H), 4.01 (d, ,7 = 3.6 Hz, 3H), 3.39 (t, .7 = 7.2 Hz, 2H), 3.18 - 3.00 (m, 5H), 2.55
- 2.40 (m, 6H), 2.27 (s, 3H), 1.70 (q, J= 4.0 Hz, 2H), 1.62 (p, J= 7.2 Hz, 2H), 1.49 - 1.26 (m, 12H). MS (ESI) [M+H]+ = 931.3.
Figure imgf000280_0003
Example 356: (E)-3-(6-(l -(2,2-difluorobenzo [d] [ 1 ,3] dioxol-5-yl)cyclopropane-1 - carboxamido)-3-methylpyridin-2-yl)-N-(4-(3-(5-(6-(4-(dimethylamino)but-2-enoyl)-3,6- diazabicyclo[3.1.1]heptane-3-carbonyl)thiophen-2-yl)propanamido)butyl)benzamide (QC192-153) White solid, 31% yield. 'H NMR (400 MHz, Methanol-d4) 8 8.00 (d, J = 8.5 Hz, 1H), 7.87 - 7.77 (m, 3H), 7.56 - 7.46 (m, 2H), 7.38 - 7.30 (m, 2H), 7.25 (dd, J= 8.2, 1.7 Hz, 1H),
7.15 (d, J= 8.2 Hz, 1H), 6.81 (d, .7= 3.8 Hz, 1H), 6.73 - 6.64 (m, 1H), 6.50 (d, J= 15.2 Hz, 1H), 4.70 (s, 1H), 4.46 (s, 1H), 4.35 - 3.73 (m, 6H), 3.32 - 3.26 (m, 2H), 3.12 (t, J= 6.2 Hz, 2H), 3.06 (t, J= 7.2 Hz, 2H), 2.85 - 2.69 (m, 7H), 2.47 (t, J= 7.2 Hz, 2H), 2.21 (s, 3H), 1.65 - 1.59 (m, 3H), 1.54 - 1.40 (m, 4H), 1.20 (q, J= 4.0 Hz, 2H). MS (ESI) [M+H]+ = 896.4.
Figure imgf000281_0001
Example 357: (E)-3-(6-(l-(2,2-difluorobenzo[d] [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-AL(5-(3-(5-(6-(4-(dimethylamino)but-2-enoyl)-3,6- diazabicyclo[3.1.1]heptane-3-carbonyl)thiophen-2-yl)propanamido)pentyl)benzamide (QC192-154) White solid, 28% yield. ’H NMR (400 MHz, Methanol-d4) δ 7.99 (d, J = 8.6 Hz, 1H), 7.89 - 7.77 (m, 3H), 7.60 - 7.47 (m, 2H), 7.40 - 7.29 (m, 2H), 7.25 (dd, J= 8.3, 1.7 Hz, 1H),
7.15 (d, J = 8.3 Hz, 1H), 6.79 (d, J= 3.8 Hz, 1H), 6.73 - 6.63 (m, 1H), 6.49 (d, J= 15.1 Hz, 1H), 4.70 (s, 1H), 4.47 (s, 1H), 4.37 - 3.76 (m, 6H), 3.32 - 3.26 (m, 2H), 3.12 -3.00 (m, 4H), 2.86 - 2.67 (m, 7H), 2.45 (t, J= 7.2 Hz, 2H), 2.21 (s, 3H), 1.66 - 1.58 (m, 3H), 1.58 - 1.38 (m, 4H), 1.32 - 1.18 (m, 4H). MS (ESI) [M+H]+ = 910.4.
Figure imgf000281_0002
Example 358: (E')-3-(6-(l-(2,2-difluorobenzo[d] [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(6-(3-(5-(6-(4-(dimethylamino)but-2-enoyl)-3,6- diazabicyclo[3.1.1]heptane-3-carbonyl)thiophen-2-yl)propanamido)hexyl)benzamide (QC192-155) White solid, 30% yield. 'H NMR (400 MHz, Methanol-d4) 8 8.00 (d, J = 8.6 Hz, 1H), 7.87 - 7.78 (m, 3H), 7.56 - 7.48 (m, 2H), 7.41 - 7.30 (m, 2H), 7.25 (dd, J= 8.3, 1.7 Hz, 1H),
7.15 (d, ,7 = 8.3 Hz, 1H), 6.81 (d, .7= 3.8 Hz, 1H), 6.73 - 6.64 (m, 1H), 6.50 (d, .7= 15.0 Hz, 1H), 4.70 (s, 1H), 4.47 (s, 1H), 4.37 - 3.77 (m, 6H), 3.34 - 3.27 (m, 2H), 3.1 1 - 3.02 (m, 4H), 2.87 - 2.68 (m, 7H), 2.46 (t, J= 7.2 Hz, 2H), 2.21 (s, 3H), 1.66 - 1.18 (m, 13H). MS (ESI) [M+H]+ = 924.4.
Figure imgf000282_0001
Example 359: (E)-3-(6-(l-(2,2-difluorobenzo[</| [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(7-(3-(5-(6-(4-(dimethylamino)but-2-enoyl)-3,6- diazabicyclo[3.1.1]heptane-3-carbonyl)thiophen-2-yl)propanamido)heptyl)benzamide (QC192-156) White solid, 31% yield. 'H NMR (400 MHz, Methanol^) 8 7.99 (d, J = 8.6 Hz, 1H), 7.86 - 7.78 (m, 3H), 7.56 - 7.47 (m, 2H), 7.39 - 7.29 (m, 2H), 7.25 (dd, J= 8.3, 1.7 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H), 6.80 (d, J= 3.8 Hz, 1H), 6.73 - 6.64 (m, 1H), 6.50 (d, J= 15.1 Hz, 1H), 4.71 (s, 1H), 4.47 (s, 1H), 4.37 - 3.79 (m, 6H), 3.29 (t, J= 7.2 Hz, 2H), 3.09 - 3.02 (m, 4H), 2.88 - 2.70 (m, 7H), 2.46 (t, J = 7.2 Hz, 2H), 2.20 (s, 3H), 1.66 - 1.15 (m, 15H). MS (ESI) [M+H]+ = 938.4.
Figure imgf000282_0002
Example 360: (E)-3-(6-(l-(2,2-difluorobenzo[J] [l,3Jdioxol-5-yl)cyclopropane-l- carboxamido)-3-methyIpyridin-2-yl)-JV-(8-(3-(5-(6-(4-(dimethylamino)but-2-enoyl)-3,6- diazabicydo[3.1.1]heptane-3-carbonyl)thiophen-2-yl)propanamido)octyl)benzamide (QC192-157) White solid, 27% yield. 'H NMR (400 MHz, Methanol-d4) 8 8.00 (d, ,J = 8.6 Hz, 1H), 7.86 - 7.77 (m, 3H), 7.56 - 7.48 (m, 2H), 7.41 - 7.29 (m, 2H), 7.25 (dd, J= 8.3, 1.7 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H), 6.81 (d, J= 3.8 Hz, 1H), 6.74 - 6.63 (m, 1H), 6.50 (d, J= 14.8 Hz, 1H), 4.71 (s, 1H), 4.48 (s, 1H), 4.38 - 3.78 (m, 6H), 3.29 (t, J= 7.4 Hz, 2H), 3.12 - 3.00 (m, 4H), 2.86 - 2.67 (m, 7H), 2.46 (t, J= 7.3 Hz, 2H), 2.21 (s, 3H), 1.64 - 1.50 (m, 5H), 1.40 - 1.19 (m, 12H). MS (ESI) [M+H] 1 = 952.4.
Figure imgf000283_0001
Example 361: (E)-3-(6-(l-(2,2-difluorobenzo[J][l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-N-(4-(3-(5-(8-(4-(dimethylamino)but-2-enoyl)-2-oxo- 3,8-diazabicydo[3.2.1]octan-3-yl)thiophen-2-yl)propanamido)butyl)benzamide (QC192-184) White solid, 30% yield. 'H NMR (400 MHz, Methanol-d4) δ 8.00 (d, J= 8.5 Hz, 1H), 7.86 - 7.75 (m, 3H), 7.55 - 7.48 (m, 2H), 7.32 (d, J= 1.7 Hz, 1H), 7.25 (dd, J= 8.4, 1.7 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 6.91 - 6.65 (m, 2H), 6.54 (d, J = 3.9 Hz, 1H), 6.48 - 6.40 (m, 1H), 3.98 - 3.82 (m, 3H), 3.59 (dd, J = 31.1, 11.6 Hz, 1H), 3.31 - 3.20 (m, 4H), 3.15 - 3.08 (m, 2H), 2.95 (t, J = 7.3 Hz, 2H), 2.85 - 2.79 (m, 6H), 2.41 (t, J= 7.3 Hz, 2H), 2.35 - 1.80 (m, 7H), 1.61 (q, J= 4.1 Hz, 2H), 1.54 -1.37 (m, 4H), 1.20 (q, J= 4.1 Hz, 2H). MS (ESI) [M+H]+ = 896.4.
Figure imgf000283_0002
Example 362: (E)-3-(6-(l-(2,2-difluorobenzo[d] [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(5-(3-(5-(8-(4-(dimethylamino)but-2-enoyl)-2-oxo- 3,8-diazabicyclo[3.2.1]octan-3-yl)thiophen-2-yl)propanamido)pentyl)benzamide (QC192- 178) White solid, 31% yield. ’H NMR (400 MHz, Methanol-d)48 7.99 (d, J= 8.5 Hz, 1H), 7.86 - 7.74 (m, 3H), 7.55 - 7.45 (m, 2H), 7.31 (d, J= 1.7 Hz, 1H), 7.25 (dd, J = 8.4, 1.7 Hz, 1H), 7.15 (d, J= 8.2 Hz, 1H), 6.90 - 6.65 (m, 2H), 6.55 - 6.40 (m, 2H), 4.00 - 3.81 (m, 3H), 3.61 (dd, J = 27.0, 11.5 Hz, 1H), 3.30 - 3.20 (m, 4H), 3.08 (t, J= 6.8 Hz, 2H), 2.92 (t, J= 7.3 Hz, 2H), 2.88 - 2.78 (m, 6H), 2.43 - 1.81 (m, 9H), 1.65 - 1.36 (m, 6H), 1.31 - 1.14 (m, 4H). MS (ESI) [M+H]+ =
910.4.
Figure imgf000283_0003
Example 363: (E)-3-(6-(l-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(6-(3-(5-(8-(4-(dimethylamino)but-2-enoyl)-2-oxo-
3,8-diazabicyclo[3.2.1]octan-3-yl)thiophen-2-yl)propanamido)hexyl)benzamide (QC192-179)
White solid, 30% yield. 'H NMR (400 MHz, Methanol-d4) δ 8.00 (d, J= 8.6 Hz, 1H), 7.85 - 7.73 (m, 3H), 7.56 - 7.47 (m, 2H), 7.32 (d, J= 1.8 Hz, 1H), 7.25 (dd, J= 8.4, 1.7 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 6.93 - 6.65 (m, 2H), 6.56 - 6.42 (m, 2H), 4.01 - 3.85 (m, 3H), 3.61 (dd, J = 25.6, 11.6 Hz, 1H), 3.35 - 3.20 (m, 4H), 3.07 (t, J= 6.8 Hz, 2H), 2.94 (t, J= 7.3 Hz, 2H), 2.87 - 2.79 (m, 6H), 2.45 - 1.84 (m, 9H), 1.65 - 1.16 (m, 12H). MS (ESI) [M+H]+ = 924.4.
Figure imgf000284_0001
Example 364: (£)-3-(6-(l-(2,2-difluorobenzo[d] [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(7-(3-(5-(8-(4-(dimethylamino)but-2-enoyl)-2-oxo-
3,8-diazabicyclo[3.2.1]octan-3-yl)thiophen-2-yl)propanamido)heptyl)benzamide (QC192- 180) White solid, 29% yield. ‘HNMR (400 MHz, Methanol-d)48 8.00 (d, J= 8.6 Hz, 1H), 7.87 - 7.74 (m, 3H), 7.56 - 7.46 (m, 2H), 7.32 (d, J= 1.7 Hz, 1H), 7.25 (dd, J= 8.2, 1.8 Hz, 1H), 7.16 (d, J= 8.3 Hz, 1H), 6.94 - 6.65 (m, 2H), 6.58 - 6.41 (m, 2H), 4.02 - 3.83 (m, 3H), 3.61 (dd, J = 24.3, 11.6 Hz, 1H), 3.34 - 3.20 (m, 4H), 3.05 (t, J= 6.9 Hz, 2H), 2.94 (t, J= 7.3 Hz, 2H), 2.88 - 2.78 (m, 6H), 2.45 - 1.79 (m, 9H), 1.65 - 1.47 (m, 4H), 1.42 - 1.12 (m, 10H). MS (ESI) [M+Hf = 938.4.
Figure imgf000284_0002
Example 365: (E)-3-(6-(l-(2,2-difluorobenzo[d] [l,3]dioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-N-(8-(3-(5-(8-(4-(dimethylamino)but-2-enoyl)-2-oxo-
3,8-diazabicyclo[3.2.1]octan-3-yl)thiophen-2-yl)propanamido)octyl)benzamide (QC192-181)
White solid, 29% yield. ’H NMR (400 MHz, Methanol-d4) δ 8.01 (d, J= 8.5 Hz, 1H), 7.87 - 7.73 (m, 3H), 7.56 - 7.46 (m, 2H), 7.32 (d, J = 1.7 Hz, 1H), 7.26 (dd, J = 8.3, 1.7 Hz, 1H), 7.16 (d, J = 8.2 Hz, 1H), 6.92 - 6.66 (m, 2H), 6.57 - 6.42 (m, 2H), 4.02 - 3.85 (m, 3H), 3.62 (dd, J = 24.5, 11.5 Hz, 1H), 3.35 - 3.20 (m, 4H), 3.05 (t, J = 6.9 Hz, 2H), 2.94 (t, J= 7.3 Hz, 2H), 2.87 - 2.79 (m, 6H), 2.46 - 1.84 (m, 9H), 1.67 - 1.48 (m, 4H), 1.43 - 1.12 (m, 12H). MS (ESI) [M+H]+ = 952.4.
Figure imgf000285_0001
Example 366: (E)-3-(6-(l-(2,2-difluorobenzo[J] [l,3Jdioxol-5-yl)cyclopropane-l- carboxamido)-3-methylpyridin-2-yl)-JV-(9-(3-(5-(8-(4-(dimethylamino)but-2-enoyl)-2-oxo- 3,8-diazabicyclo[3.2.1]octan-3-yl)thiophen-2-yl)propanamido)nonyl)benzamide (QC192-182)
White solid, 26% yield. 'H NMR (400 MHz M, ethanol-d4) δ 8.01 (d, ./= 8.5 Hz, 1H), 7.86 - 7.74 (m, 3H), 7.56 - 7.47 (m, 2H), 7.32 (d, J= 1.7 Hz, 1H), 7.26 (dd, J= 8.2, 1.7 Hz, 1H), 7.15 (d, J= 8.2 Hz, 1H), 6.92 - 6.65 (m, 2H), 6.57 - 6.40 (m, 2H), 4.02 - 3.80 (m, 3H), 3.61 (dd, J = 24.3, 11.5 Hz, 1H), 3.36 - 3.20 (m, 4H), 3.04 (t, J= 7.0 Hz, 2H), 2.94 (t, J= 7.4 Hz, 2H), 2.87 - 2.79 (m, 6H), 2.44 - 1.80 (m, 9H), 1.66 - 1.47 (m, 4H), 1.44 - 1.11 (m, 14H). MS (ESI) [M+H]+ = 966.4.
Examples of OTUB1 covalent binders are set forth in table 1 below.
Table 1.
Figure imgf000285_0002
Figure imgf000286_0001
Figure imgf000287_0001
Figure imgf000288_0001
Figure imgf000289_0001
Figure imgf000290_0001
Figure imgf000291_0001
Figure imgf000292_0001
Figure imgf000293_0001
Figure imgf000294_0001
Figure imgf000295_0001
Figure imgf000296_0001
Figure imgf000297_0001
Figure imgf000298_0001
Figure imgf000299_0001
Figure imgf000300_0001
Figure imgf000301_0001
Figure imgf000302_0001
Figure imgf000303_0001
Figure imgf000304_0001
Figure imgf000305_0001
Figure imgf000306_0001
Figure imgf000307_0001
Figure imgf000308_0001
Figure imgf000309_0001
Figure imgf000310_0001
Figure imgf000311_0001
Figure imgf000312_0001
Figure imgf000313_0001
Figure imgf000314_0001
Figure imgf000315_0001
Figure imgf000316_0001
Figure imgf000317_0001
Figure imgf000318_0001
Figure imgf000319_0001
Examples of AMPK based bivalent compounds are set forth in table 2 below.
Table 2.
Figure imgf000319_0002
Figure imgf000320_0001
Figure imgf000321_0001
Figure imgf000322_0001
Figure imgf000323_0001
Figure imgf000324_0001
Examples of cGAS based bivalent compounds are set forth in table 3 below.
Table 3.
Figure imgf000324_0002
Figure imgf000325_0001
Figure imgf000326_0001
Figure imgf000327_0001
Figure imgf000328_0001
Figure imgf000329_0001
3T1
Figure imgf000330_0001
Figure imgf000331_0001
Figure imgf000332_0001
Figure imgf000333_0001
Figure imgf000334_0001
Figure imgf000335_0001
Figure imgf000336_0001
Figure imgf000337_0001
Figure imgf000338_0001
Figure imgf000339_0001
Figure imgf000340_0001
Examples of CFTR based bivalent compounds are set forth in table 4 below.
Table 4.
Figure imgf000340_0002
Figure imgf000341_0001
Figure imgf000342_0001
Figure imgf000343_0001
Figure imgf000344_0001
Figure imgf000345_0001
Figure imgf000346_0001
Figure imgf000347_0001
Figure imgf000348_0001
Figure imgf000349_0001
Figure imgf000350_0001
Figure imgf000351_0001
Figure imgf000352_0001
Figure imgf000353_0001
Figure imgf000354_0001
Figure imgf000355_0002
Additional OTUB1 recruiting bivalent compounds that can be made are shown in table 5. Table 5.
Figure imgf000355_0001
Figure imgf000356_0001
Figure imgf000357_0001
Figure imgf000358_0001
Figure imgf000359_0001
Figure imgf000360_0001
As used herein, in case of discrepancy between the structure and chemical name provided for a particular compound, the structure shall control.
0TUB1 covalent binders were tested for their ability to form the covalent adduct with 0TUB1 protein using a mass-spectrometry based assay.
Table 6.
Figure imgf000360_0002
Figure imgf000361_0001
Figure imgf000362_0001
Figure imgf000363_0001
Figure imgf000364_0001
Example 389. Assessment of the covalent modification of OTUB1 protein by selected OTUB1 covalent binders using a mass spectrometry-based assay (Figure 1)
OTUB1 protein (10 pM) was incubated with selecting compounds (2500 pM) at RT for 1 h. The adduct was qualified by mass spectrometry-based assays.
Example 390. Assessment of the improved OTUB1 ligand MS5105 (Figure 2)
A) Quantification of the OTUBl-ligand adduct formed after EN523 (red) or MS5105 (blue) was incubated with WT OTUB1 at the indicated ligand:OTUBl ratio for 1 h. Data shown are the means ± SD from two independent experiments. B) Quantification of the OTUBl-ligand adduct formed after EN523 (red) or MS5105 (blue) was incubated with WT OTUB1 for the indicated time at the 100: 1 (ligand:OTUBl) ratio. Data shown are the means ± SD from two independent experiments. C) Representative HPLC spectra of EN523 (left) or MS5105 (right) at 1 M concentration after the indicated time in 0.01 M HC1 MeOH solution from two independent experiments. D) Representative images of EN523 (0.4 mg) and MS5105 (15.7 mg) in 0.5 mL water and aqueous solubility ofEN523 andMS5105. E) Representative mass spectra of the OTUB1 C23S mutant (10 pM) incubated with DMSO (top) or MS5105 (bottom) for 1 h at the 250: 1 (ligand:protein) ratio from two independent experiments. F) Representative western blot results of the tetra-ubiquitin (3.6 pM) substrate incubated with OTUB1 (1.5 pM) with or without MS5105 (750 pM) at the indicated time point (0, 0.5, or 1 h) from two independent experiments.
Example 391. Assessment of the effect of selected AMPK based bivalent compounds on stabilization of the AMPK protein level in Hela and HEK293T cells using WB assay (Figure
3)
The Hela or HEK293T cells was treated with DMSO, positive control (991) or selected bivalent compound at 10 pM for 24 h. The Western blot results showed that multiple compounds can increase AMPKβ1 protein level.
Example 392. Assessment of selected AMPK based bivalent compounds in stabilization of the AMPK protein level in HEK293T cells using WB assay in multiple concentrations (Figure
4)
The HEK293T cells was treated with DMSO, positive control (991) or selected bivalent compound at indicated concentrations (0. 1, 0.3 1, 3, and 10 pM) for 24 h. The Western blot results showed that several compounds dose-dependently increase AMPKβi protein level.
Example 393. Assessment of the effect of selected cGAS based bivalent compounds on stabilization of the cGAS protein level in Hela cells using WB assay (Figure 5)
The Hela cells was treated with DMSO, or selected bivalent compound at 10 pM for 24 h. The Western blot results showed that multiple compounds can increase cGAS and its downstream target STING proteins level.
Example 394. Assessment of the leading cGAS based bivalent compounds (Figure 6)
A) WB results of cGAS and STING in HeLa cells treated with MS7829 or MS8588 at the indicated concentration for 24 h. B) WB results of cGAS and STING in HeLa cells treated with 5 pM of MS7829 or MS8588 for the indicated time. C) RT-qPCR analysis of the cGAS mRNA level in HeLa cells treated with DMSO or 5 pM of MS7829 or MS8588 for 24 h. Data shown are the means ± SD from 2 independent experiments. D) WB results of cGAS, STING and 0TUB1 in HeLa cells infected with lentivirus of sgOTUBl to deplete 0TUB1 using CRISPR/Cas-9, followed by treatment with the indicated cGAS DUBTAC at 10 pM for 24 h. E) WB result of cGAS protein level inHela cells treated with (left) MS7829 (5 pM), cGAS antagonist G108 (25 pM), orMS5105 (25 pM), alone or in combination, for 24 h; (right) MS8588 (5 pM), cGAS antagonist G108 (25 pM), or MS5105 (25 pM), alone or in combination, for 24 h. F) cGAS co-elutes with OTUB1 in the presence of MS7829 or MS8588.
Example 395. Assessment of selected cGAS based bivalent compounds in stabilization of the cGAS protein level and activation of its downstream targets in Hela cells (Figure 7)
A) A schematic diagram of the cGAS/STING/IRF3 signaling. B) WB results of p-IRF3, IRF3, p- STING and STING in HeLa cells treated with 5 pM of the indicated compound for 24 h followed by stimulation with 1 pg/mL HT-DNA for additional 12 h. C-D. WB results of p-IRF3, IRF3 and p-STING in HeLa cells treated with 5 pM of MS7829 (C) or MS8588 (D) in the presence or absence of 20 pM of MS5105 for 24 h followed by stimulation with 1 pg/mL HT-DNA for additional 12 h. E) ELISA analysis of the cGAMP level in HeLa cells treated with 5 pM of MS7829 or MS8588 for 24 h followed by stimulation with 1 pg/mL HT-DNA for additional 12 h. Data shown are the means ± SD from 2 independent experiments. F-G) RT-qPCR analysis of CXCL10 (F) and CCL5 (G) mRNA levels in HeLa cells treated with 5 pM of MS7829 or MS8588 for 24 h followed by stimulation with 1 pg/mL HT-DNA for additional 12 h. Data shown are the means ± SD from 2 independent experiments. H) Growth curves of HeLa cells treated with 5 pM of MS7829, MS8588, (top) MS5105 or G108 (bottom). Data shown are the means ± SD from 2 independent experiments. I. Clonogenic assay results of HeLa cells (300/well) treated with 5 pM of MS7829, MS8588, MS5105 or G108 for 3 weeks. Cells were fixed and stained with crystal violet. The images shown are representative of two independent experiments. J. Quantification of the clonogenic assay results in panel I. WB results shown in panels B-D are representative of at least 2 independent experiments. For panels E-G and J, ***P < 0.001.
Example 396. Assessment of the effect of selected CFTR based bivalent compounds on stabilization of the CFTR protein level in CFBE41o- 4.7 AF508-CFTR human cystic fibrosis bronchial epithelial cells using WB assay (Figure 8)
A-B) Western blot (WB) results of the CFTR protein level in CFBE41o-4.7 AF508-CFTR cells treated with 10 pM of the indicated compound for 24 h, with NJH-2-057 as a positive control. C) RT-qPCR analysis of the CFTR mRNA level in CFBE41o-4.7 AF508-CFTR cells treated with 10 pM of MS6178 for 24 h. Data shown are the means ± SD from two independent experiments. D) WB results of the CFTR protein level in CFBE41o-4.7 AF508-CFTR cells treated with MS6178 at the indicated concentration for 24 h. E) WB results of the CFTR protein level in CFBE41o-4.7 AF508-CFTR cells treated with 10 pM of MS6178 for the indicated time. F) WB results of the CFTR protein level in CFBE41 o-4.7 AF508-CFTR cells treated with MS6178 (10 pM), Lumacaftor (50 pM), or MS5105 (50 pM), alone or in combination, for 24 h.
Example 397. Assessment of the effect of selected CFTR based bivalent compounds on stabilization of the CFTR protein level in CFBE41o- 4.7 AF508-CFTR human cystic fibrosis bronchial epithelial cells using WB assay (Figure 9)
The CFBE41O- 4.7 AF508-CFTR human cystic fibrosis bronchial epithelial cells was treated with DMSO, positive control (NJH-2-057) or selected bivalent compound at 10 pM for 24 h. The Western blot results showed that multiple compounds significantly increase CFTR protein level.
Example 398. Assessment of the covalent modification of OTUB1 protein by selected CFTR based bivalent compounds using mass spectrometry-based assay (Figure 10)
OTUB1 protein (10 pM) was incubated with selecting bivalent compounds (2500 pM) at RT for 1 h. The adduct was qualified by mass spectrometry-based assays.
Materials and Methods;
General Chemistry Methods:
All chemicals and reagents were purchased from commercial suppliers and used without further purification. HPLC spectra for all compounds were acquired using an Agilent 1200 Series system with DAD detector. Chromatography was performed on a 2.1 x 150 mm Zorbax 300SB- C18 5 pm column with water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B at a flow rate of 0.4 ml/min. The linear gradient was as follows: 1% B (0-1 min), 1-99% B (1-4 min), and 99% B (4-8 min). High-resolution mass spectra (HRMS) data were acquired in positive ion mode using an Agilent G1969A API-TOF with an electrospray ionization (ESI) source. Proton Nuclear Magnetic Resonance (1 H-NMR) spectra were recorded on a Bruker DRX-400 MHz spectrometer, and Carbon Nuclear Magnetic (13C NMR) were recorded at 150 MHz. Chemical shifts are expressed in parts per million (ppm) and reported as 8 value (chemical shift 8). Coupling constants are reported in units of hertz (J value, Hz; Integration and splitting patterns: where s = singlet, d = double, t = triplet, q = quartet, brs = broad singlet, m = multiple). Preparative HPLC was performed on Agilent Prep 1200 series with UV detector set to 254 nm. Samples were injected onto a Phenomenex Luna 75 x 30 mm, 5 pm, C 18 column at room temperature. The flow rate was 40 ml/min. A linear gradient was used with 10% (or 50%) of MeOH (A) in H2O (with 0.1 % TFA) (B) to 100% of MeOH (A). HPLC was used to establish the purity of target compounds. All compounds showed > 95% purity using the HPLC methods described above.
Expression and purification of OTUB1 proteins
Human OTUB1 wild-type (NM 017670 3) and C2.3S mutation genes were optimized based on the codon preference of E. coll, synthesized and cloned into pETl 5b vector with a N-terminal His tag by Genscript, respectively. The plasmid was transferred to BL21-CodonPlus (DE3)-RIPL Competent Cell (230280, Agilent Technologies) and the recombinant protein was induced expressed by 0.5 mM IPTG at 16 °C with shaking 200 rpm for 18 h. Induced cells were harvested by centrifugation and resuspended in cold lysis buffer (50 mM Tris-HCI pH 7.5, 150 mM NaCl, 25 mM imidazole pH 7.5, 0.01% IGEPAL, 5% Glycerol) in the presence of Pierce Protease Inhibitor tablets (PI A32963, Fisher Scientific) and 2 mM TCEP pH 7.5.
The cells were lysed by sonication, clarified by centrifugation, and the filtered supernatant loaded onto a 5 mL HisTrap HP affinity column (17524802, Cytiva) in AKTA system. The His- recombinant protein bound to column was washed using buffer A (50 mM Tris-HCI pH 7.5, 150 mM NaCl, 25 mM imidazole pH 7.5, 0.01% IGEPAL, 5% Glycerol) and eluted with buffer B (50 mM: Tris-HCI pH 7.5, 150 mM NaCl, 250 mM imidazole pH 7.5, 0.01% IGEPAL, 5% Glycerol) The eluted fractions containing OTUB1 protein were concentrated and subjected to size exclusion chromatography using a 320 mL HiLoad 26/600 Superdex 200 pg column (28989336, Cytiva) under the buffer condition (50 mM Tris-HC1 pH 7.5, 150 mM NaCl, and 2 mM TCEP pH 7.5). The eluted fractions containing OTUB 1 protein were concentrated, divided into aliquots and stored at -80 °C freezer for further usage. The final concentration of OTUB1 wild-type and OTUB1 C23S mutation proteins were 33 mg/mL and 38 mg/mL, respectively. Quality control of the OTUB1 proteins was performed by SDS-PAGE, Nanodrop and mass spectrometry.
Cell culture
The CFBE410- 4.7 AF508-CFTR human cystic fibrosis bronchial epithelial cells were purchased from Millipore Sigma (SCC159). CFBE41o- 4.7 AF508-CFTR cells are derived from the parental CFBE41O- cells by introduced the AF508-CFTR construct. CFBE41o- 4.7 AF508-CFTR cells were cultured in a-MEM media (Sigma. #M2279) supplemented with 10% FBS, 2 mM L- Glutamine, 300 μg/mL Hygromycin B, 10,000 units/mL Penicillin and 10,000 μg/mL Streptomycin. For treatment, cells were plated in 6-well plate which is pre-coated with coating mixture (10 μg/mL Fibronectin (Sigma, #F2006), 30 μg/mL PureCol Collagen (Sigma, #5006), and 100 μg/rnL BSA(Sigma. #126575)), and treated with indicated drugs when cells reach 60-70% confluency. HeLa cells were cultured in DuJbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 Units of penicillin and 100 μg/rnL streptomycin. For treatment, cells in 6-well plate were treated with indicated compounds. To activate the cGAS/STING pathways, cells were transfected with either HT-DNA (Sigma, D6898) with PEI (Polysciences, # 23966). For free ligand competition assay, the free ligand, USP7 ligand, cGAS ligand or AMPK ligand in DMSO were co-treated with indicated DUBTAC compounds.
Western blot
Cells were lysed in EBC buffer ( 120 mM NaCI, 50 mM Tris-Cl pH 8.0, 0.5%NP-40) supplemented with protease inhibitors cocktail (Pierce) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). The protein samples were resolved by 7% SDS-PAGE at 130 V for 80 min, and immunoblotted with indicated antibodies at 4°C overnight, washed four times with Tris-buffered saline with 0.1% Tween-20 (TBST) buffer, incubated with secondary' antibody in 5% non-fat milk for 1 hour at room temperature, and then washed another four times with TBST buffer. CFTR (#78335, 1 : 1,000) antibody was purchased from Cell Signaling Technologies. Anti- Actin (A2228, 1 :50,000), anti-vinculin antibody (V-4505, 1 :50,000), peroxi dase-conjugated anti- mouse secondary/ antibody (A-4416, 1 :3000) and peroxidase-conjugated anti-rabbit secondary' antibody (A-4914, 1 :3,000) were purchased from Sigma All primary antibodies were diluted in 5% bovine serum albumin (BSA) in TBST buffer, and secondary antibodies were diluted in 5% non-fat milk in TBST buffer.
Mass spectrometry-based analysis of OTUBl-covalent ligand adducts
To screen the covalent ligands, recombinant 0TUB1 protein (10 pM) was respectively incubated with DMSO, EN523 or seven novel ligands (2500 pM) in reaction buffer (50 mM Tris-HCI pH 7.5, 150 mM NaCI, and 0.5 mM TCEP pH 7.5) for 1 h at room temperature.
For quantitative analysis of the effect of different protein: ligand ratios, recombinant OTUB1 protein (10 pM) was respectively incubated with EN523 or MS5105 (200 pM, 500 pM, 1000 pM, 2500 pM) in reaction buffer for 1 h at room temperature.
To quantitatively assess the time course of the OTUB1 covalent modification by the ligands, recombinant OTUB1 protein (10 pM) was respectively incubated with EN523 or MS5105 (1000 pM) in reaction buffer for 0.5 h, 1 h, 2 h, 4 h, 8 h at room temperature.
Then, the total 500 pL reaction solution was exchanged and concentrated to 50 pL 50mM ammonium bicarbonate with a 10 kDa MWCO centrifugal filter unit (UFC901024, Millipore) and diluted in MS buffer (30% ACN, 0.2% FA) to a final concentration of 10 pM. Mass spectrometry detection was performed by an Agilent LC/MSD Time-Of-Flight (TOF) mass spectrometer (Agilent Technologies) equipped with an electrospray ion source. The data was analyzed by TOF Protein Confirmation Software. All experiments were performed in duplicate.
In vitro deubiquitinase assay
Recombinant OTUB1 protein (10 pM) was preincubated with DMSO or MS51O5 (5000 pM) in reaction buffer for 1 h at room temperature. Then, the pretreated protein was purified with a 10 kDa MWCO centrifugal filter unit (UFC901024, Millipore).
To initiate the assay, untreated or pretreated OTUB1 (1.5 pM) was incubated with K48-Linked Tetra-Ubiquitin (3.7 pM, SI-4804-0025, LifeSensors) in reaction buffer for 0 , 5, 15, and 30 min at 37°C. The reaction was terminated by adding 4x Laemmli Sample Buffer (1610747, BIO-RAD) with 100 mM TECP PH 7 5 and heating at 95 °C for 6 min.
The appearance of tetra-ubiquitin and mono-ubiquitin was monitored with western blotting. The primary antibody was Ubiquitin (P4D1) Mouse mAb (3936S, Cell Signaling Technology). The secondary antibody was IRDye 680RD Donkey anti-Mouse IgG (926-68072, LI-COR). Protein signals were detected by OdysseyCLx imaging system (LI -COR) and then analyzed by Image Studio Lite software (LI-COR).
Measurement of cGAMP level
The cGAMP level was measured using 2’3’-cGAMP ELISA kit (Cayman, #501700) following the manufacturer’s manual. Briefly, cells were lysed using the M-PERtm Mammalian Protein Extraction Reagent (ThermoFisher, #78503), and 100 pL cell lysates were used for analysis. The OD450 were measured for calculation of cGAMP level, and finally normalized with protein concentration.
RT-qPCR
Total RNAs were extracted using Qiagen RNeasy mini kit (Qiagen. #74106) and reversed transcripted into cDNA using iScriptTM Reverse Transcription Supermix (Bio-Rad, # 1708841). RT-qPCR was performed with SYBR Select Master Mix (ThermoFisher, #4472908) using indicated primers.
Clonal formation assay
HeLa cells in a 6-well plate were treated with the indicated DUBTAC for 24 hours. Two weeks later, cells were fixed in fixation buffer (acetic acid: methanol=l :7) and stained with 0.4% crystal violet in 20% ethanol. The clonal numbers were quantified using ImageJ software.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
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Figure imgf000373_0001

Claims

WHAT IS CLAIMS IS:
1. An OTUB 1 binder comprising a compound according to Formula (A-I) :
Figure imgf000374_0001
Formula (A-I) wherein
AD is selected from N or CRD 2;
BD is selected from C=O, CH2, CH RD 2, or C(RD 2)2;
Ring CD is absent, or selected from C3-C12 cycloalkyl, 3-12-membered heterocyclic, C6- C10 aryl, and 5-10 membered heteroaryl;
Ring DD is a saturated or partially unsaturated 4-12 membered heterocyclic;
FD is selected from N, or CRD 2;
LD1 is a bond, or a bivalent group selected from -O-, -NRD 5-, -C(O)-, -C(O)O-, -C(O)NRD 5-, -NRD 5C(O)-, -OC(O)NRD 5-, -NRD 5C(O)O-, -NRD 6C(O)NRD 5-, -S(O)-, -S(O)2-, -S(O)NRD 5-, - S(O)2NRD 5-, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, and C2- C6 alkynylene;
LD2 is selected from -C(O)-, -S(O)-, -S(O)2-, -NRD 5C(O)-, and C1-C6 alkylene;
LD3 is a bond, or a bivalent group selected from C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, and C2-C6 alkynylene; RD 1 is selected from C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2- C6, heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C(O)NRD 7RD 8, C(O)ORD 7, tri(Ci-C3alkyl) silyl, C1-C6 alkyl, C1-C6 heteroalkyl, and 3-8 membered heterocyclic; each RD 2 is independently selected from hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C1- C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C1-C6 alkynyl, C2-C6 heteroalkynyl, C3-C8 cycloalkyl, and 3-8 membered heterocyclic; or two RD 2 groups, together with the atom(s) to which they are connected, optionally form a C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, where each said C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring DD, can optionally form fused rings or bridged rings; each RD 3 is selected from hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C(O)ORD 7, C(O)NRD 7RD 8, -OC(O)NRD 7RD 8, - NRD 9C(O)NRD 7RD 8-, -S(O)NRD 7RD 8-, -S(O)2NRD 7RD 8-, CI-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C1-C6 alkynyl, C3-C12 cycloalkyl, 3-12- membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; or two RD 3 groups, together with the atom(s) to which they are connected, optionally form a C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, where each said C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring CD, optionally form fused rings or bridged rings; RD 4 is RD 4a or RD 4b; RD 4;I is selected from hydrogen, ORD 7, NRD 7RD 8, C(O)RD 7, C(O)ORD 7, C(O)NRD 7RD 8, - OC(O)NRD 7RD 8, -NRD 9C(O)NRD 7RD 8-, S(O)RD 7, S(O)2RD 7, S(O)NRD 7RD 8, S(O)2NRD 7RD 8, CI-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-Ci2 cycloalkyl, 3-12- membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl;
Ro4b is optionally a bivalent group for connection to a linker, and is selected from a bond, -O-, -N-, -C(O)-, -C(O)O-, -C(O)NRD 7-, -OC(O)NRD 7-, -NRD 9C(O)NRD 7-, -S(O)-, -S(O)2-, - S(O)NRD 7-, -S(O)2NRD 7-, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, C3-C12 cycloalkylene, 3-12-membered heterocyclicene, C6-C10 arylene, and 5-10 membered heteroarylene; each RD 5 and each RD 6 are independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; each RD 7, each RD 8 and each RD 9 are independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; or RD 7 and RD 8, together with the atom(s) to which they are connected, optionally form 3-12- membered heterocyclic ring; mD is an integer of 0-8; nD is an integer of 0-8; and tautomers and pharmaceutically acceptable salts thereof.
2. An 0TUB1 binder comprising a compound according to one of Formulae (A-I-a),
(A-I-b), (A-I-c) and (A-I-d):
Figure imgf000376_0001
Formula (A-I-c), or Formula (A-I-d). wherein
AD is selected from N or CRD 2;
BD is selected from C=O, CH2, CH RD 2, or C(RD 2)2;
Ring CD is absent, or selected from C3-C12 cycloalkyl, 3-12-membered heterocyclic, C6- C10 aryl, and 5-10 membered heteroaryl;
Ring DD is a saturated or partially unsaturated 4-12 membered heterocyclic;
FD is selected from N, or CRD 2;
LD2 is selected from -C(O)-, -S(O)-, -S(O)2-, -NRD 5C(O)-, and C1 a-Clk6ylene;
LD3 is a bond, or a bivalent group selected from C1-C6 alkylene, Ci-Cg haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, and C2-C6 alkynylene; RD 1 is selected from C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2- C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C(O)NRD 7RD 8, C(O)ORD 7, tri(Ci-C3alkyl) silyl, C1-C6 alkyl, C1-C6 heteroalkyl, and 3-8 membered heterocyclic; each RD 2 is independently selected from hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C1- C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C1-C6 alkynyl, C2-C6 heteroalky nyl, C3-C8 cycloalkyl, and 3-8 membered heterocyclic; or two RD 2 groups, together with the atom(s) to which they are connected, optionally form a C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, where each said C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring DD, can optionally form fused rings or bridged rings; each RD 3 is selected from hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C(O)ORD 7, C(O)NRD 7RD 8, -OC(O)NRD 7RD 8, - NRD 9C(O)NRD 7RD 8-, -S(O)NRD 7RD 8-, -S(O)2NRD 7RD 8-, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C1-C6 alkynyl, C3-C12 cycloalkyl, 3-12- membered heterocyclic, Cg-Cio aryl, and 5-10 membered heteroaryl; or two RD 3 groups, together with the atom(s) to which they are connected, optionally form a C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, where each said C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring CD. optionally form fused rings or bridged rings; RD 4 is RD 4a or RD 4b;
RD 4a is selected from hydrogen, ORD 7, NRD 7RD 8, C(O)RD 7, C(O)ORD 7, C(O)NRD 7RD 8, - OC(O)NRD 7RD 8, -NRD 9C(O)NRD 7RD 8-, S(O)RD 7, S(O)2RD 7, S(O)NRD 7RD 8, S(O)2NRD 7RD 8, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12- membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl;
RD 4b is optionally a bivalent group for connection to a linker, and is selected firoma bond, - O-, -N-, -C(O)-, -C(O)O-, -C(O)NRD 7-, -OC(O)NRD 7-, -NRD 9C(O)NRD 7-, -S(O)-, -S(O)2-, - S(O)NRD 7-, -S(O)2NRD 7-, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, C3-C12 cycloalkylene, 3-12-membered heterocyclicene, C6-C10 arylene, and 5-10 membered heteroarylene; each RD 5 and each RD 6 are independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; each RD 7, each RD 8 and each RD 9 are independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; or RD 7 and RD 8, together with the atom(s) to which they are connected, optionally form 3-12- membered heterocyclic ring; mo is an integer of 0-8; no is an integer of 0-8; and tautomers and pharmaceutically acceptable salts thereof.
3. An 0TUB1 binder comprising a compound according to one of Formulae (A-I-al), (A-I-bl), (A-I-cl) and (A-I-dl):
Figure imgf000378_0001
Formula (A-I-cl), or Formula (A-I-dl) wherein
BD is selected from C=O, CH2, CH RD 2, or C(RD 2)2;
Ring CD is absent, or selected from C3-C12 cycloalkyl, 3-12-membered heterocyclic, Ce- Cio aryl, and 5-10 membered heteroaryl;
Ring DD is a saturated or partially unsaturated 4-12 membered heterocyclic;
FD is selected from N, or CRD 2;
LD2 is selected from -C(O)-, -S(O)-, -S(O)2-, -NRD 5C(O)-, and C1-C6 alkylene;
LD3 is a bond, or a bivalent group selected from C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, and C2-C6 alkynylene; RD 1 is selected from C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2- C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C(O)NRD 7RD 8, C(O)ORD 7, tri(C1-C3alkyl) silyl, C1-C6 alkyl, C1-C6 heteroalkyl, and 3-8 membered heterocyclic; each RD 2 is independently selected from hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C1- C6, alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C1-C6 alkynyl, C2-C6 heteroalkynyl, C3-C8 cycloalkyl, and 3-8 membered heterocyclic; or two RD 2 groups, together with the atom(s) to which they are connected, optionally form a C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, where each said C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring DD, can optionally form fused rings or bridged rings; each RD 3 is selected from hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C(O)ORD 7, C(O)NRD 7RD 8, -OC(O)NRD 7RD 8, - NRD 9C(O)NRD 7RD 8-, -S(O)NRD 7RD 8-, -S(O)2NRD 7RD 8-, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 C a2lk-Cen6yl, C1-C6 alkynyl, C3-C12 cycloalkyl, 3-12- membered heterocyclic, Cg-Cio aryl, and 5-10 membered heteroaryl; or two RD 3 groups, together with the atom(s) to which they are connected, optionally form a C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, where each said C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring CD, optionally form fused rings or bridged rings; RD 4 is RD 4a or RD 4b;
RD4a is selected from hydrogen, ORD 7, NRD 7RD 8, C(O)RD 7, C(O)ORD 7, C(O)NRD 7RD 8, - OC(O)NRD 7RD 8, -NRD 9C(O)NRD 7RD 8-, S(O)RD 7, S(O)2RD 7, S(O)NRD 7RD 8, S(O)2NRD 7RD 8, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 6yl, C2-C6 alkynyl, C3-C 12 cycloalkyl, 3-12- membered heterocyclic, Cg-Cio aryl, and 5-10 membered heteroaryl; RD 41> is optionally a bivalent group for connection to a linker, and is selected from a bond, -O-, -N-, -C(O)-, -C(O)O-, -C(O)NRD 7-, -OC(O)NRD 7-, -NRD 9C(O)NRD 7-, -S(O)-, -S(O)2-, - S(O)NRD 7-, -S(O)2NRD 7-, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, C3-C12 cycloalkylene, 3-12-membered heterocyclicene, C6-C10 arylene, and 5-10 membered heteroarylene; each RD 5 and each RD 6 are independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic,C6-C10 aryl, and 5-10 membered heteroaryl; each RD 7, each RD 8 and each RD 9 are independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; or RD 7 and RD 8, together with the atom(s) to which they are connected, optionally form 3-12- membered heterocyclic ring; mo is an integer of 0-8; no is an integer of 0-8; and tautomers and pharmaceutically acceptable salts thereof.
4. An 0TUB1 binder comprising a compound according to one of
Formulae (A-I-a2), (A-I-a3), (A-I-a4), (A-I-a5), (A-I-b2), (A-I-b3), (A-I-b4), (A-I-b5), (A-
I-c2), (A-I-c3), (A-I-c4), (A-I-c5), (A-I-d2), (A-I-d3), (A-I-b4) and (A-I-d5):
Figure imgf000380_0001
Figure imgf000381_0001
Formula (A-I-d4), Formula (A-T-d5), wherein
Ring CD is absent, or selected from C3-C12 cycloalkyl, 3-12-membered heterocyclic, C6- C10 aryl, and 5-10 membered heteroaryl;
FD is selected from N, or CRD 2;
LD2 is selected from -C(O)-, -S(O)-, -S(O)2-, -NRD 3C(O)-, and C1-C6 alkylene;
LD3 is a bond, or a bivalent group selected from C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, and C2-C6 alkynylene; RD 1 is selected from C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2- C6, heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C(O)NRD 7RD 8, C(O)ORD 7, tri(Ci-C3alkyl) silyl, C1-C6 alkyl, C1-C6 heteroalkyl, and 3-8 membered heterocyclic; each RD 2 is independently selected from hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C1- C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C1-C6 alkynyl, C2-C6 heteroalkynyl, C3-C8 cycloalkyl, and 3-8 membered heterocyclic; or two RD 2 groups, together with the atom(s) to which they are connected, optionally form a C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, where each said C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring DD, can optionally form fused rings or bridged rings; each RD 3 is selected from hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C(O)ORD 7, C(O)NRD 7RD 8, -OC(O)NRD 7RD 8, - NRD 9C(O)NRD 7RD 8-, -S(O)NRD 7RD 8-, -S(O)2NRD 7RD 8-, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C1-C6 alkynyl, C3-C12 cycloalkyl, 3-12- membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; or two RD 3 groups, together with the atom(s) to which they are connected, optionally form a C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, where each said C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring CD, optionally form fused rings or bridged rings; RD 4 is RD 4a or RD 4b; RD 4a is selected from hydrogen, ORD 7, NRD 7RD 8, C(O)RD 7, C(O)ORD 7, C(O)NRD 7RD 8, - OC(O)NRD 7RD 8, -NRD 9C(O)NRD 7RD 8-, S(O)RD 7, S(O)2RD 7, S(O)NRD 7RD 8, S(O)2NRD 7RD 8, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12- membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl;
RD 4b is optionally a bivalent group for connection to a linker, and is selected from a bond, -O-, -N-, -C(O)-, -C(O)O-, -C(O)NRD 7-, -OC(O)NRD 7-, -NRD 9C(O)NRD 7-, -S(O)-, -S(O)2-, - S(O)NRD 7-, -S(O)2NRD 7-, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, C3-C12 cycloalkylene, 3-12-membered heterocyclicene, C6-C10 arylene, and 5-10 membered heteroarylene; each RD 5 and each RD 6 are independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic,C6-C10 aryl, and 5-10 membered heteroaryl; each RD 7, each RD 8 and each RD 9 are independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-Ci2 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; or RD 7 and RD 8, together with the atom(s) to which they are connected, optionally form 3-12- membered heterocyclic ring; no is an integer of 0-8; and each ED 1, each ED 2, each ED 3, each ED 4, and each ED 5 are independently selected from null, O, CO, SO, S(O)2, NRD 2, and CRD 2RD 2, with the proviso that no two oxygen atoms are connected to each other;
OD 1 is selected from an integer of 1-4;
OD 2, OD3, OD4, and oD5 are independent selected from an integer of 0-4; and tautomers and pharmaceutically acceptable salts thereof.
5. An OTUB 1 binder comprising a compound according to one of Formulae (A-I-a6),
(A-I-a7), (A-I-a8), (A-I-a9), (A-I-b6), (A-I-b7), (A-I-b8), (A-I-b9), (A-I-c6), (A-I-c7), (A-I-c8), (A-I-c9), (A-I-d6), (A-I-d7), (A-I-d8), and (A-I-d9):
Figure imgf000384_0001
Formula (A-I-c8), Formula (A-I-c9),
Figure imgf000385_0001
Formula (A-I-d8), and Formula (A-I-d9), wherein ring CD is absent, or selected from C3-C12 cycloalkyl, 3-12-membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl;
FD is selected from N, or CRD 2;
LD2 is selected from -C(O)-, -S(O)-, -S(O)2-, -NRD 5C(O)-, and C1-C6 alkylene;
LD3 is a bond, or a bivalent group selected from C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, and C2-C6 alkynylene; RD 1 is selected from C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2- C6, heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C(O)NRD 7RD 8, C(O)ORD 7, tri(Ci-C3alkyl) silyl, C1-C6 alkyl, C1-C6 heteroalkyl, and 3-8 membered heterocyclic; each RD 2 is a substituent that can attach to anywhere on the monocyclic and bicyclic rings and is independently selected from hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C1-C6 alkynyl, C2-C6 heteroalkynyl, C3-C8 cycloalkyl, and 3-8 membered heterocyclic; or two RD 2 groups, together with the atom(s) to which they are connected, optionally form a C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, where each said C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring DD, can optionally form fused rings or bridged rings; each RD 3 is selected from hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C(O)ORD 7, C(O)NRD 7RD 8, -OC(O)NRD 7RD 8, - NRD 9C(O)NRD 7RD 8-, -S(O)NRD 7RD 8-, -S(O)2NRD 7RD 8-, C1-C6 alkyl,C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C1-C6 alkynyl, C3-C12 cycloalkyl, 3-12- membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; or two RD 3 groups, together with the atom(s) to which they are connected, optionally form a C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, where each said C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring CD. optionally form fused rings or bridged rings; RD 4 is RD 4a or RD 4b;
Ro4a is selected from hydrogen, ORD 7, NRD 7RD 8, C(O)RD 7, C(O)ORD 7, C(O)NRD 7RD 8, - OC(O)NRD 7RD 8, -NRD 9C(O)NRD 7RD 8-, S(O)RD 7, S(O)2RD 7, S(O)NRD 7RD 8, S(O)2NRD 7RD 8, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12- membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl;
RD 4b is optionally a bivalent group for connection to a linker, and is selected from a bond, -O-, -N-, -C(O)-, -C(O)O-, -C(O)NRD 7-, -OC(O)NRD 7-, -NRD 9C(O)NRD 7-, -S(O)-, -S(O)2-, - S(O)NRD 7-, -S(O)2NRD 7-, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, C3-C12 cycloalkylene, 3-12-membered heterocyclicene, C6-C10 arylene, and 5-10 membered heteroarylene; each RD 5 and each RD 6 are independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; each RD 7, each RD 8 and each RD 9 are independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; or RD 7 and RD 8, together with the atom(s) to which they are connected, optionally form 3-12- membered heterocyclic ring;
OD1 is selected from an integer of 1-4;
OD 2, OD 3, OD4, and OD5 are independent selected from an integer of 0-4; mD is an integer of 0-8; nD is an integer of 0-8; and tautomers and pharmaceutically acceptable salts thereof.
6. An 0TUB1 binder according to one of claims 1 - 3, wherein Ring DD is selected from
Figure imgf000387_0001
Figure imgf000388_0001
7. An OTUB1 binder according to any one of claims 1-3, wherein Ring DD is selected from:
Figure imgf000388_0002
8. An 0TUB1 binder according to any one of claim 1-3, wherein Ring DD is selected from:
Figure imgf000389_0001
9. An 0TUB1 binder according to any one of claims 1-3, wherein Ring DD is selected from:
Figure imgf000389_0002
Figure imgf000390_0001
10. An OTUB1 binder according to any one of claim 1-5, wherein ring CD is selected from azetidinyl, pyrrolidinyl, piperidinyl, and piperazinyl thiophenyl, benzothiophenyl, tetrahydrobenzothiophenyl, thiazolyl, imidazolyl, furanyl, pyrrolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, benzofuranyl, indolyl, and indazolyl.
11. An 0TUB1 binder according to any one of claims 1-10 wherein - LD 2-RD 1 is selected from
Figure imgf000390_0002
Figure imgf000391_0001
Figure imgf000392_0001
Figure imgf000393_0001
Figure imgf000394_0001
12. An 0TUB1 binder selected from XS154-91, XS154-130, XS154-148, XS154-114, XS154-149, XS159-13, XS159-107, XS165-30, XS159-90, XS165-53, XS165-38, XS165-33, XS165-54, XS154-184, XS165-75, XS165-77, XS165-127, XS165-106, XS165-112, XS165-97, XS165-118, XS165-110, XS165-119, XS165-123, XS165-126, XS165-120, XS165-121, XS165- 100, XS165-99, XS165-113, XS165-109, XS165-117, XS165-105, XS165-154, XS165-170, XS165-155, XS165-172, XS165-169, XS165-177, XS175-45, XS175-46, XS175-59, XS175-63, XS175-64, XS175-67, XS175-68, XS175-70, XS175-71, XS175-76, XS175-110, XS175-120, XS175-126, XS175-132, XS175-133, XS175-137, XS175-143, XS175-148, XS175-149, XS175- 158, XS175-159, XS175-160, XS175-173, XS175-174, XS175-175, XS175-176, XS175-178, XS175-179, XS175-180, XS175-186, XS186-3, XS185-4, XS186-5, XS185-6, XS185-24, XS185-29, XS185-43, XS185-46, XS185-59, XS185-64, XS185-65, XS185-66, XS185-67, XS185-69, XS185-70, XS185-71, XS185-72, XS185-75, XS185-78, XS185-86, XS185-87, XS185-90, 91, XS185-96, XS185-97, XS185-101, XS185-109, XS185-113, XS185-114, XS185- 116, XS185-118, XS185-122, XS185-131, XS185-134, XS185-135, XS185-138, XS185-140,
XS185-147, XS185-149, XS185-150, XS185-171, XS190-9, XS190-27, XS190-38, XS190-44, XS190-45, XS190-46, XS190-47, XS190-48, XS190-49, XS190-50, XS190-59, XS190-68, XS190-69, XS190-70, XS190-75, XS190-75-2, XS190-77, XS190-80, XS190-81, XS190-126, XS190-127, XS190-128, XS190-129, XS190-130, XS190-137, XS190-138, XS190-144, XS190- 157, XS190-158, XS190-170, XS190-181, XS190-182, XS190-183, XS190-186, XS197-3, XS 197-4, XS 197-5, XS 197-6, XS 197- 14, XS 197-29, XS 197-38, XS 197-49, XS 197-50, XS 197- 70, XS197-71, XS197-72, XS197-73, XS197-74, XS197-85, XS197-93, XS197-94, XS197-96, XS197-133, XS197-145, XS197-176, XS197-185, XS197-186, XS209-21, XS209-22, XS209-23, XS209-24, XS209-25, XS209-26, XS209-27, XS209-28, XS209-30, XS209-38, XS209-39, XS209-40, XS209-53, XS209-54, XS209-55, XS209-57, XS209-60, XS209-62, XS209-65, XS209-74, XS209-75, XS209-92, XS209-99, XS209-100, XS209-101, XS209-122, XS209-139, XS209-140, XS209-165, XS209-166, XS209-167, XS209-168, XS209-174, XS209-175, XS209- 176, XS209-184, XS209-185, XS224-6, XS224-106, XS224-107, XS224-108, XS224-109, XS224-110, XS224-111, XS224-116, XS224-117, XS224-118, XS224-119, XS224-143, XS224- 144, XS224-145, XS224-147, XS224-148, XS224-149, XS224-150, XS224-154, XS224-155, XS224-156, XS224-157, XS224-158, XS224-159, or analogs thereof.
Figure imgf000395_0001
Figure imgf000396_0001
14. A bivalent compound comprising a ligand bound to a de-ubiquitination tag through a linker, the de-ubiquitination tag including an 0TUB1 recruiter moiety according to Formula A-I
Figure imgf000397_0001
Formula (A-I) wherein
AD is selected from N or CRD 2;
BD is selected from C=O, CH2, CH RD 2, or C(RD 2)2;
Ring CD is absent, or selected from C3-C12 cycloalkyl, 3-12-membered heterocyclic, Ce- C10 aryl, and 5-10 membered heteroaryl;
Ring DD is a saturated or partially unsaturated 4-12 membered heterocyclic;
FD is selected from N, or CRD 2;
LD1 is a bond, or a bivalent group selected from -O-, -NRD 5-, -C(O)-, -C(O)O-, -C(O)NRD 5-, -NRD 5C(O)-, -OC(O)NRD 5-, -NRD 5C(O)O-, -NRD 6C(O)NRD 5-, -S(O)-, -S(O)2-, -S(O)NRD 5-, - S(O)2NRD 5-, C1-C8 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, and C2- C6 alkynylene;
LD2 is selected from -C(O)-, -S(O)-, -S(O)2-, -NRD 5C(O)-, and C1-C6 alkylene;
LD3 is a bond, or a bivalent group selected from C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, and C2-C6 alkynylene; RD 1 is selected from C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic, where each said C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 heteroalkenyl, C2- Ce heteroalkynyl, partially unsaturated C4-C8 cycloalkyl, and partially unsaturated 4-8 membered heterocyclic were optionally substituted with hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C(O)NRD 7RD 8, C(O)ORD 7, tri(Ci-C3alkyl) silyl, C1-C6 alkyl, C1-C6 heteroalkyl, and 3-8 membered heterocyclic; each RD 2 is independently selected from hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C1- C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C1-C6 alkynyl, C2-C6 heteroalkynyl, C3-C8 cycloalkyl, and 3-8 membered heterocyclic; or two RD 2 groups, together with the atom(s) to which they are connected, optionally form a C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, where each said C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring DD, can optionally form fused rings or bridged rings; each RD 3 is selected from hydrogen, halogen, cyano, ORD 7, NRD 7RD 8, C(O)ORD 7, C(O)NRD 7RD 8, -OC(O)NRD 7RD 8, - NRD 9C(O)NRD 7RD 8-, -S(O)NRD 7RD 8-, -S(O)2NRD 7RD 8-, C1-C6 alkyl,C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C1-C6 alkynyl, C3-C12 cycloalkyl, 3-12- membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; or two RD 3 groups, together with the atom(s) to which they are connected, optionally form a C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, where each said C3-C12 cycloalkyl ring or 3-12-membered heterocyclic ring, together with Ring CD. optionally form fused rings or bridged rings; RD 4 is RD 4a or RD 4b;
RD 4a is selected from hydrogen, ORD 7, NRD 7RD 8, C(O)RD 7, C(O)ORD 7, C(O)NRD 7RD 8, - OC(O)NRD 7RD 8, -NRD 9C(O)NRD 7RD 8-, S(O)RD 7, S(O)2RD 7, S(O)NRD 7RD 8, S(O)2NRD 7RD 8, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12- membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl;
RD 4b is optionally a bivalent group for connection to a linker, and is selected from a bond, -O-, -N-, -C(O)-, -C(O)O-, -C(O)NRD 7-, -OC(O)NRD 7-, -NRD 9C(O)NRD 7-, -S(O)-, -S(O)2-, - S(O)NRD 7-, -S(O)2NRD 7-, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, C3-C12 cycloalkylene, 3-12-membered heterocyclicene, C6-C10 arylene, and 5-10 membered heteroarylene; each RD 5 and each RD 6 are independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; each RD 7, each RD 8 and each RD 9 are independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; or RD 7 and RD 8, together with the atom(s) to which they are connected, optionally form 3-12- membered heterocyclic ring; mID is an integer of 0-8; nD is an integer of 0-8; and tautomers and pharmaceutically acceptable salts thereof.
15. The bivalent compound of claim 14, wherein the linker is a moiety according to Formula (E):
Figure imgf000399_0001
Formula (E) wherein
A, W and B, at each occurrence, are independently selected from null, or bivalent moiety
Figure imgf000399_0002
RNR1S(O)2R , and R NR1S(O)2N(R2)R ”, wherein
R and R are independently selected from a bond, optionally substituted Rr-(C1-C8 alkyl), or a moiety comprising of optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted Ci-C8alkylaminoCi- Csalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 alkylene, optionally substituted C2-C8 alkenylene, optionally substituted C2-C8 alkynylene, optionally substituted C1- C8 hydroxyalkylene, optionally substituted C1-C8alkoxyC1-C8alkylene, optionally substituted C1- C8alkylaminoC1-C8alkylene, optionally substituted C1-C8 haloalkylene, optionally substituted 3- 10 membered carbocyclyl, optionally substituted 4-10 membered heterocyclyl, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclyl, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclyl, optionally substituted C3-C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
Rr is selected from optionally substituted 3-10 membered carbocyclyl, optionally substituted 4-10 membered heterocyclyl, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclyl, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclyl, optionally substituted C3-C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl; R1 and R2 are independently selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C a8lkynyl, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted 3-10 membered carbocyclyl, optionally substituted 4-10 membered heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
R and R , R1 and R2, R and R1, R andR2, R and R1, R andR2 together with the atom to which they are connected form a 3-20 membered cycloalkyl or 4-20 membered heterocyclyl ring; m is 0 to 15; and and pharmaceutically acceptable salts and the tautomers thereof.
16. The bivalent compound according to claim 14, wherein the linker is a moiety according to Formula (E-l):
Figure imgf000400_0001
Formula (E-l ), wherein
R1, R2, R3 and R4, at each occurrence, are independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8 alkylamino, and optionally substituted C1-C8 alkylaminoC1-C8 alkyl, optionally substituted 3-10 membered carbocyclyl, optionally substituted 3-8 membered cycloalkoxy, optionally substituted 3-10 membered carbocyclylamino, optionally substituted 4-8 membered heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or R1 and R2, R3 and R4 together with the atom to which they are connected form a 3-20 membered cycloalkyl or 4-20 membered heterocyclyl ring;
A, W and B, at each occurrence, are independently selected from null, or bivalent moiety selected from R -R ”, R COR ”, R CO2R ”, R C(O)N(R5)R”, R C(S)N(R5)R ”, R OR , R OC(O)R’ , R OC(O)OR , R OCONR5R”, R SR”, R SOR , R SO2R , R’SO2N(R5)R”, RN(R5)R”, RNR5COR , RNR5C(O)OR ”, R’NR5CON(R6)R , R’NR5C(S)R ”, R’NR5S(O)R , R5NR5S(O)2R”, and RNR5S(O)2N(R6)R ”, wherein
R and R are independently selected from null, optionally substituted Rr-(C1-C8 alkyl), or a moiety comprising of optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C6 alkylene, optionally substituted C2-C8 alkenylene, optionally substituted C2-C8 alkynylene, optionally substituted C1- C8 hydroxyalkylene, optionally substituted C1-C8alkoxyC1-C8alkylene, optionally substituted C1- C8alkylaminoC1-C8alkylene, optionally substituted C1-C8 haloalkylene, optionally substituted 3- 10 membered carbocyclyl, optionally substituted 4-10 membered heterocyclyl, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclyl, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclyl, optionally substituted C3-C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
Rr is selected from optionally substituted 3-10 membered carbocyclyl, optionally substituted 4-10 membered heterocyclyl, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclyl, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclyl, optionally substituted C3-C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
R5 and R6 are independently selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-Cx alkynyl, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted 3-10 membered carbocyclyl, optionally substituted 4-10 membered heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
R and R , R5 and R6, R and R5, R andR6, R and R5, R andR6 together with the atom to which they are connected form a 3-20 membered cycloalkyl or 4-20 membered heterocyclyl ring; m is 0 to 15; n, at each occurrence, is 0 to 15; and o is 0 to 15.
17. The bivalent compound according to claim 14, wherein the linker is a moiety according to Formula (E-2):
Figure imgf000402_0001
Formula (E-2), wherein
R1 and R2, at each occurrence, are independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, and optionally substituted C1-C8 alkyl, optionally substituted Ci- C8 alkoxy, optionally substituted C1-C8 alkoxy C1-C6 alkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8 alkylamino, C1-C8alkylaminoC1-C8alkyl, optionally substituted 3-10 membered carbocyclyl, optionally substituted 3-8 membered cycloalkoxy, optionally substituted 3-10 membered carbocyclylamino, optionally substituted 4-10 membered heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or
R1 and R2 together with the atom to which they are connected form a 3-20 membered cycloalkyl or 4-20 membered heterocyclyl ring;
A and B, at each occurrence, are independently selected from null, or bivalent moiety selected from R -R ”, R COR ”, R CO2R”, R C(O)NR3R”, R C(S)NR3R”, R OR , R OC(O)R ”, R OC(O)OR , R OCON(R3)R”, R SR”, R SOR , R SO2R”, R’SO2N(R3)R”, RN(R3)R”, R’NR3COR R NR3C(O)OR’ , R NR3CON(R4)R’, R NR3C(S)R’ , R NR3S(O)R ”, R’NR3S(O)2R ”, and RNR3S(O)2N(R4)R ”, wherein
R and R are independently selected from null, optionally substituted Rr-(C1-C8 alkyl), or a moiety comprising of optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 alkylene, optionally substituted C2-C8 alkenylene, optionally substituted C2-C8 alkynylene, optionally substituted C1- C8 hydroxyalkylene, optionally substituted C1-CxalkoxyC1-Cxalkylene, optionally substituted C1- C8alkylaminoC1-C8alkylene, optionally substituted C1-C8 haloalkylene, optionally substituted 3- 10 membered carbocyclyl, optionally substituted 4-10 membered heterocyclyl, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclyl, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclyl, optionally substituted C3-C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
Rr is selected from optionally substituted 3-10 membered carbocyclyl, optionally substituted 4-10 membered heterocyclyl, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclyl, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclyl, optionally substituted C3-C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
R3 and R4 are independently selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted 3-10 membered carbocyclyl, optionally substituted 4-10 membered heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
R and R , R3 and R4, R and R3, R and R4, R and R3, R and R4 together with the atom to which they are connected form a 3-20 membered cycloalkyl or 4-20 membered heterocyclyl ring; m, at each occurrence, is 0 to 15; and n is 0 to 15.
18. The bivalent compound according to claim 14, wherein the linker is a moiety according to Formula (E-3):
Figure imgf000404_0001
Formula (E-3) wherein
X is selected from O, NH, and NR7;
R1, R2, R3, R4, R3, and R6, at each occurrence, are independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8 alkoxy C1-C8 alkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8 alkylamino, optionally substituted C1-C8 alkylaminoC1-C8 alkyl, optionally substituted 3-10 membered carbocyclyl, optionally substituted 3-8 membered cycloalkoxy, optionally substituted 4-10 membered heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
A and B are independently selected from null, or bivalent moiety selected from R -R ”, R COR ”, R CO2R , RC(O)N(R8)R”, R C(S)N(R8)R”, R OR , R OC(O)R”, R OC(O)OR”, R OCON(R8)R ”, R SR ”, R SOR”, R SO2R”, R SO2N(R8)R”, RN(R8)R ”, RNR8COR”, RNR8C(O)OR”, RNR8CON(R9)R”, R’NR8C(S)R”, R’NR8S(O)R”, R’NR8S(O)2R”, and R’NR8S(O)2N(R9)R”, wherein
R and R are independently selected from null, optionally substituted Rr-(C1-C6 alkyl), or a moiety comprising of optionally substituted C1-C8 alkyl, optionally substituted C2-C a8lkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 alkylene, optionally substitutedC2-C8 alkenylene, optionally substituted C2-C a8lkynylene, optionally substituted C1- C8 hydroxy alkylene, optionally substituted C1-C8alkoxyC1-C8alkylene, optionally substituted C1- C8alkylaminoC1-C8alkylene, optionally substituted C1-C8 haloalkylene, optionally substituted 3- 10 membered carbocyclyl, optionally substituted 4-10 membered heterocyclyl, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclyl, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclyl, optionally substituted C3-C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
Rr is selected from optionally substituted 3-10 membered carbocyclyl, optionally substituted 4-10 membered heterocyclyl, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclyl, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclyl, optionally substituted C3-C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
R7, R8 and R9 are independently selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C a8lkynyl, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1- C8 hydroxyalkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted 3-10 membered carbocyclyl, optionally substituted 4-10 membered heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
R and R , R8 and R9, R and R8, R andR9, R and R8, R and R9 together with the atom to which they are connected form a 3-20 membered cycloalkyl or 4-20 membered heterocyclyl ring; m, at each occurrence, is 0 to 15; n, at each occurrence, is 0 to 15; o is 0 to 15; and p is 0 to 15.
19. The bivalent compound according to claim 14, wherein the linker is a moiety according to Formula (E-2-a) or (E-3-a):
Figure imgf000406_0001
Formula (E-2-a) Formula (E-3-a) wherein
A is C=O or CH2, B is C=O or CH2, and m is 0-16, n is 0-6, and o is 0-6.
20. The bivalent compound according to claim 13, wherein the linker is a moiety according to
Formula (E-2-b) or (E-3-b):
Figure imgf000406_0002
Formula (E-2-b) Formula (E-3-b) A is -C(O)N-, B is -C(O)N-, m is 0-16, n is 0-6, o is 0-6, n-1 is 0-6, and o-l is 0-6.
21. The bivalent compound according to any one of claims claim 14 - 20 , wherein the OTUB1 recruiter moiety is selected from:
Figure imgf000407_0001
Figure imgf000408_0001
Figure imgf000409_0001
Figure imgf000410_0001
Figure imgf000411_0001
Figure imgf000412_0001
Figure imgf000413_0001
Figure imgf000414_0001
Figure imgf000415_0001
Figure imgf000416_0001
22. A bivalent compound according to any one of claims 14 -21, wherein the ligand is an AMPK ligand selected from:
Figure imgf000417_0001
wherein, ♦* denotes the point of attachment to Linker.
23. A bivalent compound according to any one of claims 14 - 22, wherein the ligand is an AMPK ligand according to Formula (B-I):
Figure imgf000418_0001
Formula (B-I), wherein
AA is selected from
Figure imgf000418_0002
BA is selected from
Figure imgf000418_0003
Figure imgf000418_0004
denotes the point of attachment to Linker in Formula (A);
CA is selected from N or CRA 4; ring DA and ring EA are independently selected from null, C3-C12 cycloalkyl, 3-12- membered heterocyclyl, C6-C10 aryl, and 5-10 membered heteroaryl;
XA and YA are independently selected from a bond, -O-, -S-, -NRA 9-, -C(O)-, -C(O)O-, - C(O)NRA 9-, -O-C(O)NRA 9-, -NRA 10C(O)NRA 9-, -S(O)-, -S(O)2-, -S(O)NRA 9-, -S(O)2NRA 9-, C1- C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, and C2-C6 alkynylene; RA 1, RA 2, each RA 4, each RA 5, and each RA 7 are independently selected from H, halogen, cyano, ORA 11, NRA 11RA 12, C(O)ORA 11, C(O)NRA 11RA 12, S(O)2NRA 11RA 12, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclyl; RA 3 is selected from H, halogen, cyano, ORA 11, NRA 11RA 12, C(O)ORA 11, C(O)NRA 11RA 12, S(O)2NRA 11RA 12, C1-C6 alkyl, C1- hCa6loalkyl, C1 h-Cet6eroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclyl, C6-C10 aryl, and 5-10 membered heteroaryl; or RA 1 and RA 2, RA 2 and RA 3, RA 3 and RA 4, two RA 5 or two RA 7, together with the atoms to which they are attached, optionally form C4-C6 cycloalkyl or 5-7 membered heterocyclyl, C6-C10 aryl, and 5-10 membered heteroaryl; RA 6 and RA 8 are independently selected from a bond, -O-, -NRA 13-, -C(O)-, -C(O)O-, - C(O)NRA 13-, -O-C(O)NRA 13-, -NRA 14C(O)NRA 13-, -S(O)-, -S(O)2-, -S(O)NRA 13-, -S(O)2NRA 13-, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, C6-C10 arylene, and 5-10 membered heteroarylene; each RA 9, each RA 10, each RA 11, each RA 12, each RA 13, and each RA 14 are independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclyl, C6-C10 aryl, and 5-10 membered heteroaryl; or RA 11 and RA 12 together with the atoms to which they are attached, optionally form 3-12 membered heterocyclyl; and mA and nA are independently selected from 0, 1, 2, 3, and 4.
24. A bivalent compound according to any one of claims 14 - 21, wherein the ligand is a cGAS ligand according to Formula (C-I):
Figure imgf000419_0001
Formula (C-I) wherein denotes the point of attachment to Linker in Formula (A);
** connects to either RB 1B or RB 3b, with the proviso that when
Figure imgf000419_0002
connects to RB lb,
Figure imgf000419_0003
RB 3 is RB 3a; and when ** connects to RB 3b, RB 1 is RB 1a
AB is selected from N and CRB 4; BB is selected from N, O, S, CRB 5 and NRB 5;
CB is selected from N and C;
DB is selected from N and C;
XB is XB a-XB b;
XB a is selected from a bond, -C(O)-, -C(O)O-, -C(O)NRB 6-, -S(O)-, -S(O)2-, -S(O)NRB 6-, -S(O)2NRB 6-;
XB b is selected from a bond, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C3-C12 cycloalkylene, and 3-12 membered heterocyclylene, C6-C10 arylene, and 5-10 membered heteroarylene; RB 1 is selected from monovalent group RB 1 aand bivalent group RB lb;
RB la is selected from H, halogen, cyano, ORB 7, NRB 7RB 8, C(O)ORB 7, C(O)NRB 7RB 8, S(O)2NRB 7RB 8, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclyl, C6-C10 aryl, and 5-10 membered heteroaryl;
RB lb is connected to the Linker, and is selected from a bond, -O-, -NRB 7-, -C(O)O-, - C(O)NRB 7-, -S(O)2NRB 7-, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, C3-C12 cycloalkylene, 3-12 membered heterocyclylene, C6-C10 arylene, and 5-10 membered heteroarylene; each RB 2 is independently selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, or two RB 2, together with the atoms to which they are attached, optionally form C3-C12 cycloalkyl or 3-12 membered heterocyclyl; RB 3 is selected from monovalent group RB 3a and bivalent group RB 3b;
RB 3a is selected from H, halogen, ORB 9, NRB 9RB 10, C(O)ORB 9, C(O)NRB 9RB 10, S(O)2NRB 9RB 10, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclyl;
RB 3b is connected to the Linker, and is selected from a bond, -O-, -NRB 9-, -C(O)O-, - C(O)NRB 9-, -S(O)2NRB 9-, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, C3-C12 cycloalkylene, and 3-12 membered heterocyclylene; each RB 4 is independently selected from H, halogen, cyano, ORB 7, NRB 7RB 8, C(O)ORB 7, C(O)NRB 7RB 8, S(O)2NRB 7RB 8, CI-C3 alkyl, Ci-C3 haloalkyl, Ci-C3 heteroalkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, and 3-6 membered heterocyclyl, or two RB 4, together with the atoms to which they are attached, optionally form partially unsaturated C3-C12 cycloalkyl, partially unsaturated 3-12 membered heterocyclyl, C6-C ar1y0lene, or 5-10 membered heteroarylene; RB 5 is selected from H, halogen, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 heteroalkyl, C3-C6 cycloalkyl, and 3-6 membered heterocyclyl; RB 6, each RB 7, each RB 8, each RB 9, and each RB 10, are independently selected from H, C1- C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclyl, or RB 7 and RB 8, and RB 9 and RB 10 together with the atoms to which they are attached, optionally form 3-12 membered heterocyclyl; mB is selected from 1 and 2; nB is selected from 0, 1, 2 and 3; and
OB is selected from 0, 1, 2, 3, 4, 5 and 6.
25. A bivalent compound according to any one of claims 14 - 21, wherein the ligand is a cGAS ligand selected from
Figure imgf000422_0001
wherein, ♦* denotes the point of attachment to Linker.
26. A bivalent compound according to any one of claims 14-21, wherein the ligand is a CFTR ligand according to Formula (D-I):
Figure imgf000423_0001
denotes the point of attachment to the linker,
Ac and Cc are independently selected from O, S, or C(Rc8)(Rc9);
Bc is C(Rc10)(Rc11) or NRc12;
Arc1 and Arc2 are independently selected from null, C6-C1 a0ryl and 5-10 membered heteroaryl;
Rc1 and Rc2 are independently selected from H, halo, ORc13, NRc13Rc14, C(O)ORc13, C(O)NRC13RC14, S(O)2NRC13RC14, C1-C6 alkyl, hCa1lo-Cal6kyl, heCte1-rCoa6lkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclyl, or
Rc1 and Rc2, together with the atoms to which they are attached, optionally form C3-C12 cycloalkyl;
Rc3 and Rc6 are independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclyl; each Rc4, each Rc5, and each Rc7 are independently selected from H, halogen, cyano, ORc13, NRC13RC14, C(O)ORC13, C(O)NRC 13RC14, S(O)2NRC 13RC14, C1-C6 alkyl, halCoa1l-kCy6l, Ci-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-Ci2 cycloalkyl, and 3-12 membered heterocyclyl, two Rc4, two Rc5, or two Rc7, together with the atoms to which they are attached, optionally form partially unsaturated C3-C12 cycloalkyl, partially unsaturated 3-12 membered heterocyclyl, C6-C10 aryl and 5-10 membered heteroaryl; Rc8, Rc9, Rc10 and Rc11 are independently selected from H, halogen, ORc13, NRc13Rc14, C(O)ORc 13, C(O)NRC 13RC14, S(O)2NRC 13RC14, C1-C6 alkyl, hCa1l-oCa6lkyl, hCe1t-eCro6alkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclyl, or
Rc8 and Rc9, and Rc10 and Rc11, together with the atoms to which they are attached, optionally form C3-C12 cycloalkyl and 3-12 membered heterocyclyl;
Rc12 is selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclyl;
Rc13 and Rc14 are independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclyl, or
Rc13 and Rc14 together with the atoms to which they are attached, optionally form 3-12 membered heterocyclyl; me is 0, 1, 2, or 3; nc is 0, 1, 2, or 3; and oc is 0, 1, 2, or 3.
27. A bivalent compound according to any one of claims 14-21, wherein the ligand is a CFTR ligand selected from
Figure imgf000424_0001
wherein. denotes the point of attachment to Linker.
28. A bivalent compound including an AMPK ligand selected from XF137-81 , XF137-82, XF137-83, XF137-84, XF137-85, XF137-86, XF137-87, XF137-88, XF137-89, XF137-90, XF137-91, XF137-92, XF137-93, XF137-94, QC179-047, QC179-048, QC137-049, QC179-050, QC179-051, QC179-052, QC179-053, QC179-054, QC137-055, and examples 367 - 380, or analogs thereof.
29. A bivalent compound including a cGAS ligand selected from ZD178-28, ZD178-29, ZD178-30, ZD178-31, ZD178-32, ZD178-33, ZD178-34, ZD178-35, ZD178-58-1, ZD178-58-2, ZD178-58-3, ZD178-58-4, ZD178-58-5, ZD178-58-6, ZD178-58-7, ZD178-58-8, ZD178-58-9, ZD178-58-10, ZD178-58-11, ZD178-58-12, ZD178-58-13, ZD178-58-14, ZD178-63-1, ZD178- 63-2, ZD178-63-3, ZD178-63-4, ZD178-63-5, ZD178-63-6, ZD178-63-78, ZD178-63-8, ZD178- 63-9, ZD178-63-10, ZD178-63-11, ZD178-63-12, ZD178-63-13, ZD178-63-14, ZD178-63-15, ZD178-69-1, ZD178-69-2, ZD178-69-3, ZD178-69-4, ZD178-69-5, ZD178-69-6, ZD178-69-7, ZD178-69-8, ZD178-69-9, ZD178-69-10, ZD178-69-11, ZD178-69-12, ZD178-69-13, ZD178- 69-14, ZD178-69-15, and examples 381 - 388, or analogs thereof.
30. A bivalent compound including a CFTR ligand selected from QC166-130, QC166-131,
QC166-132, QC166-133, QC166-134, QC166-135, QC166-136, QC166-137, QC166-138,
QC166-139, QC166-140, QC166-141, QC166-142, QC166-143, QC 166- 167, QC166-174,
QC166-175, QC 166- 176, QC166-177, QC 166-178, QC 166- 179, QC166-181, QC166-182,
QC166-183, QC 166- 184, QC166-185, QC179-104, QC179-105, QC 179- 106, QC 179- 107,
QC179-108, QC179-109, QC179-110, QC179-111, QC179-112, QC179-113, QC179-137,
QC179-138, QC179-139, QC179-140, QC179-141, QC179-154, QC179-155, QC179-156,
QC179-157, QC179-158, QC192-153, QC192-154, QC192-155, QC192-156, QC192-157,
QC192-184, QC192-178, QC192-179, QC192-180, QC192-181, QC192-182, or analogs thereof.
31 . The bivalent compound (E)-N-(9-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5- tetrahydro-lH-pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)acetamido)nonyl)-3-(5-(4-(4- (dimethylamino)but-2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)propenamide (ZD 178-29, MS7829).
32. The bivalent compound (E)-Nl-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5- tetrahydro-lH-pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)-Nll-(2-(5-(4-(4- (dimethylamino)but-2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)undecanediamide (ZD178-58-8, MS8588).
33. The bivalent compound (£)-3-(6-(l-(2,2-difluorobenzo[t/][l,3]dioxol-5-yl)cyclopropane- l-carboxamido)-3-methylpyridin-2-yl)-A-(7-((2-(5-(4-(4-(dimethylamino)but-2-enoyl)-2- oxopiperazin- l-yl)thiophen-2-yl)ethyl)amino)-7-oxoheptyl)benzamide (QC 166- 178, MS6178).
34. A pharmaceutical composition, comprising a compound according to any one of claims 1 - 33 and a pharmaceutically acceptable carrier.
35. A pharmaceutical composition according to claim 34, wherein the compound is present in an effective amount.
36. A pharmaceutical composition according to claim 35, wherein the composition is formulated for administration by oral, parenteral intradermal, subcutaneous, topical, rectal or transdermal delivery.
37. A method of treating an AMPK-mediated disease, comprising administering to a subject in need thereof of a bivalent compound according to any one of claims 14 - 23 and 28.
38. A method of treating a cGAS-mediated disease, comprising administering to a subject in need thereof of a bivalent compound according to any one of claims 14 - 21, 24, 25 and 29.
39. A method of treating a CFTR-mediated disease, comprising administering to a subject in need thereof of a bivalent compound according to any one of claims 14 - 21, 26, 27 and 30.
40. The method of any one of claims 37 - 39, wherein the disease is selected from cystic fibrosis, breast cancer, ovarian cancer, prostate cancer, colon cancer, pancreatic cancer, bladder cancer, liver cancer and cervical cancer.
PCT/US2024/025509 2023-04-21 2024-04-19 Otub1 small-molecule binders and otub1-recruiting deubiquitinase-targeting chimeras (dubtacs) WO2024220874A2 (en)

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