WO2024218361A1

WO2024218361A1 - Bispecific cytotoxicity targeting chimeras

Info

Publication number: WO2024218361A1
Application number: PCT/EP2024/060829
Authority: WO
Inventors: Christina Ng Di Marco; Matthew Creed POWELL; Matthew Robert SENDER; Brandon James TURUNEN
Original assignee: Glaxosmithkline Intellectual Property Development Limited
Priority date: 2023-04-21
Filing date: 2024-04-19
Publication date: 2024-10-24

Abstract

The present disclosure relates to heterotrifunctional molecules, referred to as bispecific or dual targeting cytotoxicity targeting chimeras (CyTaCs) or antibody recruiting molecules (ARMs) that are able to simultaneously bind one or two target cell-surface proteins as well as an exogenous antibody protein. The present disclosure also relates to agents capable of binding to a receptor on a surface of a pathogenic cell and inducing the depletion of the pathogenic cell in a subject for use in the treatment of cancer, inflammatory diseases, autoimmune diseases, viral infection, or bacterial infection.

Description

BISPECIFIC CYTOTOXICITY TARGETING CHIMERAS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to United States Provisional Patent Application serial number 63/461,181 , filed April 21, 2023, the contents of which are hereby incorporated by reference in their entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

This application contains a sequence listing which has been submitted electronically in ST.26 format and is hereby incorporated by reference in its entirety (said ST.26 copy, created on April 17, 2024, is named “209277_seqlist.xml” and is 15,181 bytes in size).

FIELD OF THE DISCLOSURE

BACKGROUND

Cell-surface proteins and their ligands play key roles in a range of inflammatory, infectious, and autoimmune diseases as well as tumor initiation, growth and metastasis. Antibody-based therapeutics have promising properties as drug candidates for these indications due to their selectivity for pathogenic cell-surface targets and their ability to direct immune surveillance to target-expressing tissues or cells to induce depletion of the pathogenic cells. Examples of such depletion mechanisms include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement- dependant cytotoxicity (CDC). However, antibody-based therapeutics often suffer from a lack of bioavailability, high cost, thermal instability, and difficult manufacturing due to their size, complexity and peptide based structures. Conversely, small molecule therapeutics often provide affordability, stability, and the convenience of oral dosing, but may suffer from poor selectivity and off-target effects, while also lacking the immune control of therapeutic antibodies. Accordingly, a need exists for improved therapeutic approaches that target pathogenic cells for use in the treatment of disease. Such compositions and related methods are provided in the present disclosure.

SUMMARY

In one aspect, the present disclosure provides a heterotrifunctional molecule referred to as a cytoxicity targeting chimera (CyTaC) or an antibody recruiting molecule (ARM), wherein the ARM comprises moieties that binds one or two target cell-surface proteins on a cell and a moiety that binds an exogenous antibody. In a further aspect, the ARM comprises a trivalent linker that links the target-binding moieties to the antibody-binding moiety. In a further aspect, a target-binding moiety is a C-C chemokine receptor type 2 (CCR2)-binding moiety. In a further aspect, a target-binding moiety is a C-C chemokine receptor type 8 (CCR8)-binding moiety. In a further aspect, a target-binding moiety is a prostate specific membrane antigen (PSMA)-binding moiety. In a further aspect, the exogenous antibody is an anti-cotinine antibody, or antigen-binding fragment thereof.

In a further aspect, the ARM is a compound of Formula (I) defined by:

or a pharmaceutically acceptable salt thereof, wherein:

T¹ and T² are each independently a target binding moiety;

R¹ is C_1-4 alkyl or C_3-6 cycloalkyl;

G is a bond, -CH₂CH₂NH-, -C(O)CH₂CH₂OCH₂CH₂NH-, or -L³-CH₂CH₂NH-;

G¹ and G² are each independently -C(O)CH₂-, -CH₂CH₂NHC(O)CH₂- CH₂CH₂C(O)NH(CH₂CH₂O)₃CH₂CH₂-, or -CH₂CH₂NHC(O)(CH₂CH₂O)₃CH₂CH₂-; L’ and L” are each independently a bond,

each y is an integer of 1 to 9; each w is an integer of 0 to 5;

Y¹ and Y² are each independently a bond or a divalent spacer moiety of one to twelve atoms in length; and

L¹, L² and L³ are each independently a bond or a divalent linker as described herein, wherein at least one of L¹ , L² and L³ is a divalent linker as described herein; wherein each

of a L’ group represents a covalent bond to the Y¹ group of Formula (I), or when Y¹ is a bond, a covalent bond to the T¹ group of Formula (I), and each

of a L’ group represents a covalent bond to the L¹ group of Formula (I), or when L¹ is a bond, a covalent bond to the G¹ group of Formula (I); and wherein each

of a L” group represents a covalent bond to the Y² group of Formula (I), or when Y² is a bond, a covalent bond to the T² group of Formula (I), and each of a L”

group represents a covalent bond to the L² group of Formula (I), or when L² is a bond, a covalent bond to the G² group of Formula (I).

In a further aspect, the ARM is a compound of Formula (I) defined by:

or a pharmaceutically acceptable salt thereof, wherein:

T¹ and T² are each independently a target binding moiety; R¹ is C_1-4 alkyl or C_3-6 cycloalkyl;

G¹ and G² are each independently -C(O)CH₂-, -CH₂CH₂NHC(O)CH₂-, CH₂CH₂C(O)NH(CH₂CH₂O)₃CH₂CH₂-, or -CH₂CH₂NHC(O)(CH₂CH₂O)₃CH₂CH₂-;

L’ and L” are each independently a bond

, or

each y is an integer of 1 to 9; each w is an integer of 0 to 5;

L¹, L² and L³ are each independently a bond or a divalent linker as described herein, wherein at least one of L¹ , L² and L³ is a divalent linker as described herein; wherein each of a L’ group represents a covalent bond to the Y¹ group of Formula (I), or when Y¹ is a bond, a covalent bond to the T¹ group of Formula (I), and each

of a L’ group represents a covalent bond to the L¹ group of Formula (I), or when L¹ is a bond, a covalent bond to the G¹ group of Formula (I); and wherein each of a L” group represents a covalent bond to the Y² group of Formula (I), or when Y² is a bond, a covalent bond to the T² group of Formula (I), and each

of a L” group represents a covalent bond to the L² group of Formula (I), or when L² is a bond, a covalent bond to the G² group of Formula (I).

In one aspect, the present disclosure provides a method of treating and/or preventing a disease or disorder in a patient in need thereof, comprising: administering to the patient a therapeutically effective amount of a compound of Formula (I) as disclosed herein and an anti- cotinine antibody, or antigen-binding fragment thereof.

In one aspect, the present disclosure provides a method of increasing antibody- dependent cell cytotoxicity (ADCC) of target-expressing cells comprising: contacting the cells with an effective amount of the compound of Formula (I) as disclosed herein and an anti- cotinine antibody, or antigen-binding fragment thereof.

In one aspect, the present disclosure provides a method of increasing cell killing of target-expressing cells comprising: contacting the cells with an effective amount of the compound of Formula (I) as disclosed herein and an anti-cotinine antibody, or antigen-binding fragment thereof.

In one aspect, the present disclosure provides a method of depleting target-expressing cells comprising: contacting the cells with an effective amount of a compound of Formula (I) as disclosed herein and an anti-cotinine antibody, or antigen-binding fragment thereof.

In one aspect, the present disclosure provides a compound of Formula (I) as disclosed herein for use in therapy. In a further aspect, the present disclosure provides a combination comprising a compound of Formula (I) as disclosed herein and an anti-cotinine antibody, or antigen-binding fragment thereof, for use in therapy.

In one aspect, the present disclosure provides a combination comprising a compound of Formula (I) as disclosed herein and an anti-cotinine antibody, or antigen-binding fragment thereof, for use in the treatment of a disease or disorder.

In one aspect, the present disclosure provides use of a compound of Formula (I) as disclosed herein in the manufacture of a medicament for the treatment of a disease or disorder. In a further aspect, the present disclosure provides use of a combination comprising a compound of Formula (I) as disclosed herein and an anti-cotinine antibody, or antigen- binding fragment thereof, in the manufacture of a medicament for the treatment of a disease or disorder.

In one aspect, the present disclosure provides a combination comprising a compound of Formula (I) as disclosed herein and an anti-cotinine antibody, or antigen-binding fragment thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 : Schematic representation of cytotoxicity targeting chimeras (CyTaCs) technology compared to current antibody technology.

FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, and FIG. 2F: PK analysis of compounds of Formula (I) in mice as described in Example 12; FIG. 2A shows PK analysis of the compound of Example 1 dosed in the presence of anti-cotinine antibody; FIG. 2B shows PK analysis of the compound of Example 2 dosed in the presence of anti-cotinine antibody; FIG. 2C shows PK analysis of the compound of Example 4 dosed in the presence of anti-cotinine antibody; FIG. 2D shows PK analysis of the compound of Example 7 dosed in the presence of anti- cotinine antibody; FIG. 2E shows PK analysis of the compound of Example 8 dosed in the presence of anti-cotinine antibody; FIG. 2F shows PK analysis of the compound of Example 9 dosed in the presence of anti-cotinine antibody.

DETAILED DESCRIPTION

In one aspect, the present disclosure provides a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

T¹ and T² are each independently a target binding moiety;

R¹ is C_1-4 alkyl or C_3-6 cycloalkyl;

G¹ and G² are each independently -C(O)CH₂-, -CH₂CH₂NHC(O)CH₂- CH₂CH₂C(O)NH(CH₂CH₂O)₃CH₂CH₂-, or -CH₂CH₂NHC(O)(CH₂CH₂O)₃CH₂CH₂-; L' and L'' are each independently a bond,

, or

; each y is an integer of 1 to 9; each w is an integer of 0 to 5;

L¹, L² and L³ are each independently a bond or a divalent linker of Formula (L-a), (L-b), (L-c), (L-d), (L-e), (L-f), (L-g), (L-h), (L-i), (L-j), (L-k), (L-m), (L-n-i), (L-n-ii), (L-n-iii), (L-n-iv), (L-p), (L- q), (L-r), or (L-s), with the proviso that at least one of L¹ , L² and L³ is a divalent linker of Formula (L-a), (L-b), (L-c), (L-d), (L-e), (L-f), (L-g), (L-h), (L-i), (L-j), (L-k), (L-m), (L-n-i), (L-n-ii), (L-n-iii), (L-n-iv), (L-p), (L-q), (L-r), or (L-s); wherein each

of a L’ group represents a covalent bond to the L¹ group of Formula (I), or when L¹ is a bond, a covalent bond to the G¹ group of Formula (I); wherein each

of a L” group represents a covalent bond to the Y² group of Formula (I), or when Y² is a bond, a covalent bond to the T² group of Formula (I), and each

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-a): (L-a), or a stereoisomer thereof,

wherein:

Ring A and Ring B are each independently C_4-6 cycloalkylene;

L^1a is C_3-5 linear alkylene, wherein 1 or 2 methylene units are replaced with -O- or -NR^a-; each R^a is independently hydrogen or C_1-3 alkyl; and L^2a is -O-, -NHC(O)-, or -CH₂-O-; wherein each of a L¹ group represents a covalent bond to the L’ group of Formula (I), or when L’ is a bond, a covalent bond to the Y¹ group of Formula (I), or when both L’ and Y¹ are a bond, a covalent bond to the T¹ group of Formula (I), and each

of a L¹ group represents a covalent bond to the G¹ group of Formula (I); wherein each

of a L² group represents a covalent bond to the L” group of Formula (I), or when L” is a bond, a covalent bond to the Y² group of Formula (I), or when both L” and Y² are a bond, a covalent bond to the T² group of Formula (I), and each

of a L² group represents a covalent bond to the G² group of Formula (I); and wherein each

of a L³ group represents a covalent bond to the N atom of Formula (I), and each of a L³ group represents a covalent bond to the methylene group of the G group of Formula (I). In another embodiment, Ring A and Ring B of Formula (L-a) are each independently

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-a-i):

(L-a-i), or a stereoisomer thereof, wherein:

Ring A is C_4-6 cycloalkylene;

L^1a is C_3-5 linear alkylene, wherein 1 or 2 methylene units are replaced with -O- or -NR^a-; each R^a is independently hydrogen or C_1-3 alkyl; and L^2a is -O-, -NHC(O)-, or -CH₂-O-; wherein each

of a L¹ group represents a covalent bond to the L’ group of Formula (I), or when L’ is a bond, a covalent bond to the Y¹ group of Formula (I), or when both L’ and Y¹ are a bond, a covalent bond to the T¹ group of Formula (I), and each

of a L³ group represents a covalent bond to the N atom of Formula (I), and each of a L³ group represents a covalent bond to the methylene group of the G group of Formula (I).

In another embodiment, Ring A of Formula (L-a-i) is

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-a-ii):

(L-a-ii), or a stereoisomer thereof, wherein:

L^1a is C_3-5 linear alkylene, wherein 1 or 2 methylene units are replaced with -O- or -NR^a-; each R^a is independently hydrogen or C_1-3 alkyl;

L^2a is -O-, -NHC(O)-, or -CH₂-O-; p is 1 or 2; and m is 1 or 2; wherein each

In another embodiment, L^1a of Formula (L-a), (L-a-i), or (L-a-ii) is selected from

wherein: j is 1, 2, 3, or 4; k is 0, 1 , 2, or 3; the sum of j and k is 2, 3, or 4; q is 1 or 2; r is 1 or 2; s is 0 or 1 ; the sum of q, r, and s is 2 or 3;

X¹ and X² are independently -O- or NR^a; and each R^a is independently hydrogen or C_1-3 alkyl; _ wherein

represents a covalent bond to the C(O) group of Formula (L-a), (L-a-i), or (L- a-ii), and

represents a covalent bond to Ring B of Formula (L-a) or to the cyclohexylene group of Formula (L-a-i) or (L-a-ii).

In another embodiment, L^1a of Formula (L-a), (L-a-i), or (L-a-ii) is selected from - (CH₂)₂O-, -(CH₂)₃O-, -(CH₂)₄O-, -(CH₂)₂OCH₂-, -(CH₂)₃OCH₂-, -(CH₂)₂O(CH₂)₂-, -CH₂OCH₂-, - CH₂O(CH₂)₂-, -CH₂O(CH₂)₃-, -CH₂OCH₂O-, or -CH₂OCH₂OCH₂-. In another embodiment, L^1a of Formula (L-a), (L-a-i), or (L-a-ii) is selected from -(CH₂)₂O-, -(CH₂)₃O-, -(CH₂)₂OCH₂-, or - (CH₂)₃OCH₂-. In another embodiment, L^1a of Formula (L-a), (L-a-i), or (L-a-ii) is selected from -(CH₂)₂NR^a-, -(CH₂)₃NR^a-, -(CH₂)₄NR^a-, -(CH₂)₂NR^aCH₂-, -(CH₂)₃NR^aCH₂-, -(CH₂)₂NR^a(CH₂)₂- -CH₂NR^aCH₂-, -CH₂NR^a(CH₂)₂-, -CH₂NR^a(CH₂)₃-, -CH₂NR^aCH₂NR^a-, or -

CH₂NR^aCH₂NR^aCH₂-, wherein each R^a is independently hydrogen or C_1-3 alkyl. In another embodiment, L^{1 a} of Formula (L-a), (L-a-i), or (L-a-ii) is selected from -(CH₂)₂NR^a-, -(CH₂)₃NR^a- , -(CH₂)₂NR^aCH₂-, or -(CH₂)₃NR^aCH₂-, wherein R^a is hydrogen or C_1-3 alkyl. In another embodiment, L^1a of Formula (L-a), (L-a-i), or (L-a-ii) is selected from -(CH₂)₂NH-, -(CH₂)₃NH-, -(CH₂)₄NH-, -(CH₂)₂NHCH₂-, -(CH₂)₃NHCH₂-, -(CH₂)₂NH(CH₂)₂-, -CH₂NHCH₂-, -CH₂NH(CH₂)₂- , -CH₂NH(CH₂)₃-, -CH₂NHCH₂NH-, or -CH₂NHCH₂NHCH₂-. In another embodiment, L^1a of Formula (L-a), (L-a-i), or (L-a-ii) is selected from -(CH₂)₂NH-, -(CH₂)₃NH-, -(CH₂)₂NHCH₂-, or - (CH₂)₃NHCH₂-. In another embodiment, L^1a of Formula (L-a), (L-a-i), or (L-a-ii) is selected from -CH₂OCH₂NR^a-, -CH₂NR^aCH₂O-, -CH₂OCH₂NR^aCH₂-, -CH₂NR^aCH₂OCH₂-, wherein R^a is independently hydrogen or C_1-3 alkyl. In another embodiment, L^1a of Formula (L-a), (L-a-i), or (L-a-ii) is selected from -CH₂OCH₂NH-, -CH₂NHCH₂O-, -CH₂OCH₂NHCH₂-, - CH₂NHCH₂OCH₂-.

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-a-iii):

(L-a-iii), or a stereoisomer thereof,

wherein: p is 1 or 2; m is 1 or 2; and n is 1 , 2, or 3; wherein each

of a L¹ group represents a covalent bond to the G¹ group of Formula (I); wherein each of a L² group represents a covalent bond to the L” group of Formula (I), or when L” is a bond, a covalent bond to the Y² group of Formula (I), or when both L” and Y² are a bond, a covalent bond to the T² group of Formula (I), and each

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-a) selected from the group consisting of:

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-b):

(L-b), or a stereoisomer thereof, wherein:

Ring A is C_4-6 cycloalkylene or C_7-9 bridged bicyclic cycloalkylene;

L^{1 b} is -CH₂-NH-C(O)-, -NHC(O)-, or -C(O)NH-;

L^2b is C_6-12 linear alkylene, wherein 1, 2, 3, or 4 methylene units are replaced with -O-, -NR^{1 b}-

, -C(O)NR^{1 b}-, or -NR^{1 b}C(O)-; or

L^2b is

, wherein n is 1, 2, 3, or 4, and represents a covalent bond to L^{1 b};

and each R^1b is independently hydrogen or C_1-3 alkyl; wherein each of a L¹ group represents a covalent bond to the L’ group of Formula (I),

or when L’ is a bond, a covalent bond to the Y¹ group of Formula (I), or when both L’ and Y¹ are a bond, a covalent bond to the T¹ group of Formula (I), and each

In another embodiment, Ring A of Formula (L-b) is

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-b-i):

(L-b-i), or a stereoisomer thereof, wherein:

L^{1 b} is -CH₂-NH-C(O)-, -NHC(O)-, or -C(O)NH-;

, -C(O)NR^{1 b}-, or -NR^{1 b}C(O)-; or

L^2b is

, wherein n is 1, 2, 3, or 4, and

represents a covalent bond to L^{1 b}; each R^1b is independently hydrogen or C_1-3 alkyl; p is 1 or 2; and m is 1 or 2; wherein each of a L¹ group represents a covalent bond to the L’ group of Formula (I), or when L’ is a bond, a covalent bond to the Y¹ group of Formula (I), or when both L’ and Y¹ are a bond, a covalent bond to the T¹ group of Formula (I), and each

of a L² group represents a covalent bond to the L” group of Formula (I), or when L” is a bond, a covalent bond to the Y² group of Formula (I), or when both L” and Y² are a bond, a covalent bond to the T² group of Formula (I), and each of a L² group

represents a covalent bond to the G² group of Formula (I); and wherein each

of a L³ group represents a covalent bond to the N atom of Formula (I), and each

of a L³ group represents a covalent bond to the methylene group of the G group of Formula (I).

In another embodiment, L^2b of Formula (L-b) or (L-b-i) is selected from or

wherein: j is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; k is 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10; the sum of j and k is 5, 6, 7, 8, 9, 10, or 11; q is 1 , 2, 3, 4, 5, 6, 7, 8, or 9; r is 1 , 2, 3, 4, 5, 6, 7, 8, or 9; s is 0, 1 , 2, 3, 4, 5, 6, 7, or 8; the sum of q, r, and s is 4, 5, 6, 7, 8, 9, or 10; t is 1, 2, 3, 4, 5, 6, or 7; u is 1 , 2, 3, 4, 5, 6, or 7; v is 1 , 2, 3, 4, 5, 6, or 7; w is 0, 1 , 2, 3, 4, 5, or 6; the sum of t, u, v, and w is 3, 4, 5, 6, 7, 8, or 9; a is 1 , 2, 3, 4, or 5; b is 1 , 2, 3, 4, or 5; c is 1 , 2, 3, 4, or 5; d is 1 , 2, 3, 4, or 5; e is 0, 1, 2, 3, or 4; the sum of a, b, c, d, and e is 4, 5, 6, 7, or 8;

X¹ , X², X³, and X⁴ are independently -O-, -NR^{1 b}-, -C(O)NR^{1 b}-, or -NR^{1 b}C(O)-; and each R^1b is independently hydrogen or C_1-3 alkyl; wherein

represents a covalent bond to L^{1 b} of Formula (L-b) or (L-b-i), and

of a

L¹ group, L² group, or L³ group represents a covalent bond to the G¹ group of Formula (I), the G² group of Formula (I), or the methylene group of the G group of Formula (I), respectively.

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-b) selected from the group consisting of:

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-c):

(L-c), or a stereoisomer thereof, wherein:

L^1c is C_2-10 linear alkylene, wherein 1, 2, or 3 methylene units are replaced with -O-, -NH-, - NHC(O)-, or -C(O)NH-;

Ring A is C_4-6 cycloalkylene or C_7-9 bridged bicyclic cycloalkylene; and

L^2c is -O- or a saturated C_2-10 linear alkylene, wherein 1 , 2, or 3 methylene units are replaced with -O-, -NH-, -NHC(O)-, or -C(O)NH-; wherein each

of a L¹ group represents a covalent bond to the G¹ group of Formula (I); wherein each of a L² group represents a covalent bond to the L” group of Formula (I), or when L” is a bond, a covalent bond to the Y² group of Formula (I), or when both L” and Y² are a bond, a covalent bond to the T² group of Formula (I), and each of a L² group

represents a covalent bond to the G² group of Formula (I); and wherein each

In another embodiment, Ring A of Formula (L-c) is

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-c-i):

(L-c-i), or a stereoisomer thereof, wherein:

L^2c is -O- or a saturated C_2-10 linear alkylene, wherein 1 , 2, or 3 methylene units are replaced with -O-, -NH-, -NHC(O)-, or -C(O)NH-; p is 1 or 2; and m is 1 or 2; wherein each of a L¹ group represents a covalent bond to the L’ group of Formula (I),

In another embodiment, L^{1 c} of Formula (L-c) or (L-c-i) is selected from

wherein: j is 1, 2, 3, 4, 5, 6, 7, 8, or 9; k is 0, 1 , 2, 3, 4, 5, 6, 7, or 8; the sum of j and k is 1 , 2, 3, 4, 5, 6, 7, 8, or 9; q is 1 , 2, 3, 4, 5, 6, or 7; r is 1 , 2, 3, 4, 5, 6, or 7; s is 0, 1 , 2, 3, 4, 5, or 6; the sum of q, r, and s is 2, 3, 4, 5, 6, 7, or 8; t is 1, 2, 3, 4, or 5; u is 1 , 2, 3, 4, or 5; v is 1 , 2, 3, 4, or 5; w is 0, 1 , 2, 3, or 4; the sum of t, u, v, and w is 3, 4, 5, 6, or 7; and

X¹ , X² and X³ are independently -O-, -NH-, -NHC(O)-, or -C(O)NH-; _ wherein

represents a covalent bond to the C(O) group of Formula (L-c) or (L-c-i), and

represents a covalent bond to the ring of Formula (L-c) or (L-c-i).

In another embodiment, L^2c of Formula (L-c) or (L-c-i) is selected from

wherein: j is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; k is 0, 1 , 2, 3, 4, 5, 6, 7, 8, or 9; the sum of j and k is 1 , 2, 3, 4, 5, 6, 7, 8, or 9; q is 0, 2, 3, 4, 5, 6, or 7; r is 1 , 2, 3, 4, 5, 6, 7, or 8; s is 0, 1 , 2, 3, 4, 5, 6, or 7; the sum of q, r, and s is 1, 2, 3, 4, 5, 6, 7, or 8; t is 0, 1 , 2, 3, 4, or 5; u is 1 , 2, 3, 4, 5, or 6; v is 1 , 2, 3, 4, 5, or 6; w is 0, 1 , 2, 3, 4, or 5; the sum of t, u, v, and w is 2, 3, 4, 5, 6, or 7; and

X¹ , X² and X³ are independently -O-, -NH-, -NHC(O)-, or -C(O)NH-; wherein

represents a covalent bond to the ring of Formula (L-c) or (L-c-i), and

of a L¹ group, L² group, or L³ group represents a covalent bond to the G¹ group of Formula (I), the G² group of Formula (I), or the methylene group of the G group of Formula (I), respectively.

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-c) selected from the group consisting of:

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-d):

wherein:

L^1d is C_12-31 linear alkylene, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 methylene units are replaced with -NH-, -O-, -C(O)NH-, -NHC(O)-, or -NHC(O)-NH-; wherein each

In another embodiment, L^1d is a C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, C₃₀, or C₃₁ linear alkylene, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 methylene units are replaced with -NH-, -O-, -C(O)NH-, -NHC(O)-, or-NHC(O)-NH-. In another embodiment, L^1d is C₁₂-22 linear alkylene, for example, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, or C₂₂, wherein 1, 2, 3, 4, or 5 methylene units are replaced with -NH-, -O-, -C(O)NH-, - NHC(O)-, or -NHC(O)-NH-.

In another embodiment, L^1d of Formula (L-d) is selected from

wherein: j is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; k is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20; the sum of q andkis 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or21; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19; r is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19; s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; the sum of q, r, and s is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; t is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17; u is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17; v is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17; w is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; the sum oft, u, v, and w is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19; a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; b is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; c is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; d is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; the sum of a, b, c, d, and e is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; f is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13; g is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13; h is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13; i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13; y is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13; z is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; the sum of f, g, h, i, y, and z is 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, or 17; and

X¹ , X², X³, X⁴, and X⁵ are independently -NH-, -O-, -C(O)NH-, -NHC(O)-, or -NHC(O)-NH-; wherein

represents a covalent bond to the C(O) group of Formula (L-d), and

In another embodiment, L^1d of Formula (L-d) is

wherein n is 4, 5, 6, 7, 8, 9, or 10; wherein

represents a covalent bond to the C(O) group of Formula (L-d), and

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-d) selected from the group consisting of:

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-e):

wherein: n is an integer of 3 to 50; wherein each

In another embodiment, n of Formula (L-e) is 3 to 25, 3 to 10, 3 to 8, 3 to 7, 3 to 5, or 3 to 4. In another embodiment, n of Formula (L-e) is 5 to 22, 7 to 15, or 9 to 13. In another embodiment, n of Formula (L-e) is 3, 4, 5, 7, 8, 11 , 22, or 50.

In another embodiment, n of Formula (L-e) is 12 to 50, 15 to 30, 17 to 25, 18 to 24, 18 to 20, 20 to 22, or 22 to 24. In another embodiment, n of Formula (L-e) is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and/or 50. In another embodiment, n of Formula (L-e) is 19 or 23.

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-f):

(L-f), or a stereoisomer thereof, wherein:

L^1f is a bond; C_1-6 linear alkylene, wherein 0, 1 , or 2 methylene units are replaced with -O-, - NH-, or -C(O)-; or -(C_3-6 cycloalkylene)-NHC(O)-; L^2f is a bond, -NHC(O)-, -C(O)NH-, or a C_1-6 linear alkylene, wherein 0, 1, or 2 methylene units are replaced with -O-; and each of Z¹ and Z² is independently N or CH; wherein each of a L¹ group represents a covalent bond to the L’ group of Formula (I),

of a L² group represents a covalent bond to the G² group of Formula (I); and wherein each of a L³ group represents a covalent bond to the N atom of Formula (I), and each of a L³ group represents a covalent bond to the methylene group of the G group of Formula (I).

In another embodiment, L^1f of Formula (L-f) is selected from

wherein: j is 1, 2, 3, 4, or 5; k is 0, 1 , 2, 3, or 4; the sum of j and k is 1 , 2, 3, 4, or 5; q is 1 , 2, or 3; r is 1 , 2, or 3; s is 0, 1 , 2; the sum of q, r, and s is 2, 3, or 4; and

X¹ and X² are independently -O-, -NH-, or -C(O)-; or -(C_3-6 cycloalkylene)-NHC(O)-; wherein

represents a covalent bond to the C(O) group of Formula (L-f), and

represents a covalent bond to the ring of Formula (L-f).

In another embodiment, L^2f of Formula (L-f) is selected from

wherein: j is 1, 2, 3, 4, or 5; k is 0, 1 , 2, 3, or 4; the sum of j and k is 1 , 2, 3, 4, or 5; q is 1 , 2, or 3; r is 1 , 2, or 3; s is 0, 1 , 2; and the sum of q, r, and s is 2, 3, or 4; wherein

represents a covalent bond to the ring of Formula (L-f), and

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-f) selected from the group consisting of:

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-g):

(L-g), wherein:

Ring A is a 5 to 6 membered heteroarylene having 1 or 2 nitrogen ring atoms;

L^1g is a bond, -CH₂-, -NH-, or -O-; and

L^2g is

wherein n is 1 , 2, 3, 4, or 5, and represents a covalent bond to

wherein each of a L¹ group represents a covalent bond to the L’ group of Formula (I),

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-g-i):

(L-g-i), wherein:

L^1g is a bond, -CH₂-, -NH-, or -O-;

L^2g is

wherein n is 1 , 2, 3, 4, or 5, and

represents a covalent bond to Z¹, Z², and Z³ are each independently selected from N or CH, provided that one or two of Z¹, Z², and Z³ is N; wherein each

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-g) selected from the group consisting of:

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-h):

(L-h), or a stereoisomer thereof,

wherein: each Z¹ is independently N or CH;

L^{1 h} is a bond, -C(O)-, -C(O)-NH-, or -NHC(O)-;

L^2h is C_2-10 linear alkylene or

, wherein n is 1, 2, 3, or 4, and

represents a covalent bond to L^{1 h} and

represents a covalent bond to L^3h;

L^3h is a bond, -C(O)CH₂-, -O-(C_3-6 cycloalkylene)-O-, or -C(O)NH(CH₂)₃OCH₂-;

L^4h is a bond, -C(O)-, -CH₂C(O)-, or -C(O)CH₂-; and m is 1 , 2, or 3; wherein each

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-h) selected from the group consisting of:

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-i):

wherein:

L^{1 i} is a bond, C_1-12 linear alkylene, or

, wherein n is 1, 2, 3, 4, or 5, and

represents a covalent bond to L³ⁱ and

represents a covalent bond to NH;

L²ⁱ is a bond, C_1-12 linear alkylene, or

, wherein n is 1 , 2, 3, 4, or 5, and

represents a covalent bond to HN; and L³ⁱ is a bond or -C(O)-; wherein each of a L¹ group represents a covalent bond to the L’ group of Formula (I), or when L’ is a bond, a covalent bond to the Y¹ group of Formula (I), or when both L’ and Y¹ are a bond, a covalent bond to the T¹ group of Formula (I), and each

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-i) selected from the group consisting of:

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-j):

(L-j), or a stereoisomer thereof, wherein:

Z¹ is C, CH, or N; each of Z², Z³, Z⁴ and Z⁵ is independently CH or N, provided that no more than two of Z², Z³, Z⁴ and Z⁵ are N;

L^1j is -NH-, -C(O)NH-, -NHC(O)-, or -O-;

L^2j is C_1-6 linear alkylene or

, wherein n is 1 or 2, and

represents a covalent bond to L^1j; and represents a single bond or a double bond; wherein each of a L¹ group represents a covalent bond to the L’ group of Formula (I),

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-j) selected from the group consisting of:

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-k):

(L-k), or a stereoisomer thereof,

wherein:

Ring A is phenyl or a 5 or 6 membered heteroarylene having 1 or 2 nitrogen ring atoms; each of Z¹ and Z² is independently CH or N;

L^{1 k} is a bond, -C(O)-, -C(O)NH- or -NHC(O)-; and

L^2k is a C₃-8 straight chain alkylene or

, wherein n is 1 , 2, or 3, and

represents a covalent bond to L^{1 k}; wherein each

represents a covalent bond to the G² group of Formula (I); and wherein each

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-k) selected from the group consisting of:

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-m):

(L-m), or a stereoisomer thereof, wherein: Z¹ is CH or N; m is 1 or 2; p is 1 or 2;

0, 1, or 2 hydrogen atoms of

are replaced with F;

|_1 m is a bond, -C(O)-, -C(O)NH-, -NHC(O)-, -S(O)₂NH- or -NHS(O)₂-; and

|_2m is C_3-6 linear alkylene, C_3-6 cycloalkylene, or

, wherein n is 1 or 2, and

represents a covalent bond to L^{1 m}; wherein each

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-m) selected from the group consisting of:

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-n-i):

wherein each

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-n-ii):

wherein each

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-n-iii):

wherein each

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-n-iv):

(L-n-iv) wherein each

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-p):

(L-p), or a stereoisomer thereof, wherein y is an integer of 1 to 9; wherein each

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-q):

(L-q), or a stereoisomer thereof, wherein:

Ring A, Ring B, Ring C, and Ring D are each independently C_4-6 cycloalkylene;

L^1a, L^3a, and L^4a are each independently C_3-5 linear alkylene, wherein 1 or 2 methylene units are replaced with -O- or -NR^a-; each R^a is independently hydrogen or C_1-3 alkyl; and L^2a is -O-, -NHC(O)-, or -CH₂-O-; wherein each

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-q-i):

(L-q-i), or a stereoisomer thereof, wherein: L^1a, L^3a, and L^4a are each independently C_3-5 linear alkylene, wherein 1 or 2 methylene units are replaced with -O- or -NR^a-; each R^a is independently hydrogen or C_1-3 alkyl; and L^2a is -O-, -NHC(O)-, or -CH2-O-; wherein each

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-q-ii): (L-q-ii), or a

stereoisomer thereof, wherein: p is 1 , 2, or 3; m is 1 , 2, or 3; and n is 1 , 2, or 3; wherein each

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-q) having the following structure:

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-r):

wherein n is an integer of 10 to 30; wherein each

of a L¹ group represents a covalent bond to the L’ group of Formula (I), or when L’ is a bond, a covalent bond to the Y¹ group of Formula (I), or when both L’ and Y¹ are a bond, a covalent bond to the T¹ group of Formula (I), and each of a L¹ group

represents a covalent bond to the G¹ group of Formula (I); wherein each

In another embodiment, n of Formula (L-r) is 10 to 20, 10 to 18, 12 to 16, or 13 to 15. In another embodiment, n of Formula (L-r) is 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In another embodiment, n of Formula (L-r) is 14.

In another embodiment, L¹, L² or L³ is a divalent linker of Formula (L-s):

wherein n is an integer of 10 to 30; wherein each

In another embodiment, n of Formula (L-s) is 10 to 20, 10 to 18, 12 to 16, or 13 to 15. In another embodiment, n of Formula (L-s) is 15 to 30, 17 to 28, 18 to 26, 19 to 25, 20 to 24, or 21 to 23. In another embodiment, n of Formula (L-s) is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30. In another embodiment, n of Formula (L-s) is

14 or 22.

In one embodiment of the disclosure, Y¹ and Y² are each independently selected from a bond; -NH-; -( C_1-12 alkylene)-, wherein 1 , 2, or 3 methylene units are replaced with -O-, -NH- , -N(CH₃)-, -C(O)-, -NHC(O)-, -C(O)NH-, -(C_3-6 cycloalkylene)-, -(C_3-6 cycloalkenylene)-, 3- to 10-membered heterocycloalkylene, arylene, or heteroarylene; or -(C_2-12 alkenylene)-, wherein 1 , 2, or 3 methylene units are replaced with -O-, -NH-, -N(CH₃)-, -C(O)-, -NHC(O)-, -C(O)NH- , -(C_3-6 cycloalkylene)-, -(C_3-6 cycloalkenylene)-, 3- to 10-membered heterocycloalkylene, arylene, or heteroarylene.

In another embodiment, Y¹ and Y² are each independently selected from a bond; -NH- ; -(C_1-6 alkylene)-O-; -O-(C_1-6 alkylene)-; -(C_2-6 alkenylene)-O-; -(C_1-6 alkylene)-C(O)-; -(C_2-6 alkenylene)-C(O)-; phenylene; piperidinylene; hydroxypiperidinylene; fluoropiperidinylene; azetidinylene; -C(O)-piperazinylene-; -(C_1-6 alkylene)-oxopiperazinylene-; pyrrolidinylene; 7- to 9-membered bridged bicyclic heterocycloalkylene; -(C_1-6 alkylene)-O-phenylene-; -(C_2-6 alkenylene)-O-piperidinylene; -(C_1-5 alkylene)-NH-, wherein 0, 1, or 2 methylene units are replaced with -O-; -NH-(C_1-5 alkylene)-NH-; -N(CH₃)-(C_1-5 alkylene)-NH-; -NH-(C_1-5 alkylene)- N(CH₃)-; -N(CH₃)-(C_1-5 alkylene)-N(CH₃)-; -(C_3-6 cycloalkylene)-NH-; -C(O)NH-(C_1-5 alkylene)- NH-; -C(O)NH-(C_3-6 cycloalkylene)-NH-; -(C_1-5 alkylene)-O-(C_3-6 cycloalkylene)-NH-;-(C_3-6 cycloalkenylene)-NH-; or

, wherein Y^1a is a bond, -O-, -NH-, -NHC(O)-,

-C(O)NH-, or C_1-3 alkylene; and Y^2a is a bond, -O-, -NH-, -NHC(O)-, -C(O)NH-, or C_1-3 alkylene.

In another embodiment, Y¹ and Y² are each independently selected from the group consisting of:

In another embodiment, Y¹ or Y² is a bond. In another embodiment, Y¹ or Y² is NH. In another embodiment, Y¹ or Y² is:

In another embodiment, R¹ is methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, or t- butyl. In another embodiment, R¹ is methyl. In another embodiment, R¹ is ethyl. In another embodiment, R¹ is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

In another embodiment, y of L’ or L” is independently 2 to 8, 3 to 7, 4 to 7, or 5 to 7. In another embodiment, y of L’ or L” is independently 1 , 2, 3, 4, 5, 6, 7, 8, or 9.

In another embodiment, w of L’ or L” is independently 0 to 4, 0 to 3, 0 to 2, or 1 to 2. In another embodiment, w of L’ or L” is independently 0, 1, 2, 3, 4, or 5.

In another embodiment, L’ and L” are each independently a bond or

wherein : each y is an integer of 1 to 9; and each w is an integer of 0 to 5. In another embodiment, L’ and L” are each independently a bond or

In another embodiment, L’ and L” are each independently a bond.

In another embodiment, T¹ or T² is

(Formula A), wherein R² of Formula A is hydrogen or C_1-4 alkyl; and R³ of Formula A is hydrogen or C_1-4 alkyl. In another embodiment, R² and R³ of Formula A are each independently hydrogen, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, or t-butyl. In another embodiment, R² of Formula A is isopropyl and R³ of Formula A is methyl. In another embodiment, R² of Formula A is t-butyl and R³ of Formula A is hydrogen. In another embodiment, T¹ or T² is

In another embodiment, T¹ orT² is:

In another embodiment, T¹ orT² is

(Formula C).

In another embodiment, T¹ orT² is

In another embodiment, T¹ orT² is

In another embodiment, T¹ orT² is

In another embodiment, T¹ orT² is:

wherein R² and R³ of Formula G1 , Formula G2, Formula G3, and Formula G4 are each independently F or H.

In another embodiment, T¹ or T² is

(Formula H), wherein R² of Formula H is hydrogen or C_1-4 alkyl; and R³ of Formula H is hydrogen or C_1-4 alkyl. In another embodiment, R² and R³ of Formula H are each independently hydrogen, methyl, ethyl, 1 -propyl, 2-propyl, 1-butyl, 2-butyl, or t- butyl. In another embodiment, R² of Formula H is isopropyl and R³ of Formula H is methyl. In another embodiment, R² of Formula H is t-butyl and R³ of Formula H is hydrogen.

In another embodiment, T¹ or T² is

(Formula J), wherein Q is C_1-5 alkylene, wherein 0, 1 , or 2 methylene units are replaced with -O-; and Ar is an optionally substituted 5- to 10-membered aromatic ring or 9- or 10-membered unsaturated fused bicyclic ring.

In another embodiment, Q is -CH₂-, -CH(CH₃)-, -CH₂CH₂CH₂-, -CH(CH₂CH₃)-, or - CH₂CH₂O-. In another embodiment, Q is -CH₂- or -CH(CH₃)-. In another embodiment, Q is - CH(CH₃)-.

In another embodiment, Ar is an optionally substituted 5-, 6-, 7-, 8-, 9-, or 10- membered aromatic ring. In another embodiment, Ar is an optionally substituted 6-membered aromatic ring. In another embodiment, Ar is an optionally substituted 9-membered aromatic ring. In another embodiment, Ar is an optionally substituted 9- or 10-membered unsaturated fused bicyclic ring. In another embodiment, Ar is an optionally substituted 9-membered unsaturated fused bicyclic ring. In another embodiment, Ar is phenyl, pyridinyl, indolyl, indolinyl, dihydrobenzofuranyl, or benzofuranyl, and each Ar is substituted with 0, 1 , or 2 substituent groups. In another embodiment, Ar is phenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-indolyl, 3-indolyl, 4-indolyl, 5- indolyl, 6-indolyl, 7-indolyl, 2-indolinyl, 3-indolinyl, 4-indolinyl, 5-indolinyl, 6-indolinyl, 7- indolinyl, 2-di hydrobenzofuranyl, 3-dihydrobenzofuranyl, 4-dihydrobenzofuranyl, 5- dihydrobenzofuranyl, 6-dihydrobenzofuranyl, 7-dihydrobenzofuranyl, 2-benzofuranyl, 3- benzofuranyl, 4-benzof uranyl, 5-benzofuranyl, 6-benzof uranyl, or 7-benzofuranyl, and each Ar is substituted with 0, 1, or 2 substituent groups.

In another embodiment, the Ar substituent groups are independently selected from C_1- ₃ alkyl, C_1-3 alkoxy, C_1-3 haloalkyl, C_1-3 haloalkoxy, or halo. In another embodiment, the Ar substituent groups are independently selected from methyl, ethyl, methoxy, ethoxy, bromo, chloro, or trifluoromethyl.

In another embodiment, the compound of Formula (I) is selected from a compound as listed in Table 1:

Table 1

Definitions

As used herein and in the claims, the singular forms “a” and “the” include plural reference unless the context clearly dictates otherwise.

As used herein and in the claims , the term “comprising” encompasses “including” or “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional, e.g., X + Y.

The term “consisting essentially of” limits the scope of the feature to the specified materials or steps and those that do not materially affect the basic characteristic(s) of the claimed feature. The term “consisting of” excludes the presence of any additional component(s).

The term “pathogenic cells” includes a cell subset that causes or is capable of causing disease. Examples of pathogenic cells include, but are not limited to, pathogenic immune cells, cancer or tumor cells, and stromal cells. A pathogenic cell can also be a pathogenic agent capable of causing an infection, such as a virus or a bacterial cell.

The term “pathogenic immune cells” includes a particular immune cell subset that causes or is capable of causing disease. These cellular subsets are resident cells or are recruited to particular locations and secrete cytokines, chemokines and other mediators and contribute to the persistence and progression of disease such as cancer in the case of a tumor microenvironment or chronic inflammation of the lung in the case of asthma. Examples of pathogenic immune cells include, but are not limited to myeloid-derived suppressor cells (MDSCs), T regulatory cells (Tregs), neutrophils, macrophages, B regulatory cells (Bregs), CD8 regulatory cells, (CD8regs), and exhausted T cells.

The term “pharmaceutical composition” refers to a formulation of a compound of the invention and a medium generally accepted in the art for the delivery of the biologically active compound to mammals, e.g., humans. Such a medium includes all pharmaceutically acceptable carriers, diluents or excipients therefor.

The terms “effective amount” and “therapeutically effective amount” refer to an amount of a compound, or antibody, or antigen-binding portion thereof, according to the invention, which when administered to a patient in need thereof, is sufficient to effect treatment for disease-states, conditions, or disorders for which the compounds have utility. Such an amount would be sufficient to elicit the biological or medical response of a tissue system, or patient that is sought by a researcher or clinician. The amount of a compound according to the invention which constitutes a therapeutically effective amount will vary depending on such factors as the compound and its biological activity, the composition used for administration, the time of administration, the route of administration, the rate of excretion of the compound, the duration of the treatment, the type of disease-state or disorder being treated and its severity, drugs used in combination with or coincidentally with the compounds of the invention, and the age, body weight, general health, sex and diet of the patient. Such a therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to their own knowledge, the state of the art, and this disclosure.

The term “alkyl” represents a saturated, linear or branched hydrocarbon moiety having the specified number of carbon atoms. The term “C_1-3 alkyl” refers to an unsubstituted alkyl moiety containing 1 , 2 or 3 carbon atoms; exemplary alkyls include methyl, ethyl and propyl.

The term “alkylene” represents a saturated, linear or branched hydrocarbon moiety having the specified number of carbon atoms, with two points of attachment. The two points of attachment can be from the same or different carbon atoms. The term “C_1-3 alkylene” refers to an unsubstituted alkylene moiety containing 1 , 2 or 3 carbon atoms with two points of attachment; exemplary C_1-3 alkylene groups include methylene, ethylene and propylene.

The term “alkenyl” represents an unsaturated, linear or branched hydrocarbon moiety having the specified number of carbon atoms. The term “C_2-6 alkenyl” refers to an unsubstituted alkenyl moiety containing 2, 3, 4, 5, or 6 carbon atoms; exemplary alkenyls include propenyl, butenyl, pentenyl and hexenyl.

The term “alkenylene” represents an unsaturated, linear or branched hydrocarbon moiety having the specified number of carbon atoms, with two points of attachment. The two points of attachment can be from the same or different carbon atoms. The term “C_2-6 alkenylene” refers to an unsubstituted alkenylene moiety containing 2, 3, 4, 5, or 6 carbon atoms with two points of attachment; exemplary C_2-6 alkenylene groups include propenylene, butenylene, pentenylene and hexenylene.

The term “cycloalkyl” represents a saturated cyclic hydrocarbon moiety having the specified number of carbon atoms. The term “C_3-6 cycloalkyl” refers to an unsubstituted cycloalkyl moiety containing 3, 4, 5 or 6 carbon atoms; exemplary cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term “cycloalkylene” represents a saturated cyclic hydrocarbon moiety having the specified number of carbon atoms, with two points of attachment. The two points of attachment can be from the same or different carbon atoms. The term “C_4-6 cycloalkylene” refers to an unsubstituted cycloalkylene moiety containing 4, 5, or 6 carbon atoms with two points of attachment. Exemplary cycloalkylene groups include cyclobutane-1 ,3-diyl, cyclopentane-1 ,3- diyl, cyclohexane-1,3-diyl, or cyclohexane-1,4-diyl.

The term “cycloalkenylene” represents an unsaturated cyclic hydrocarbon moiety having the specified number of carbon atoms, with two points of attachment. The two points of attachment can be from the same or different carbon atoms. The term “C_3-6 cycloalkenylene” refers to an unsubstituted cycloalkenylene moiety containing 3, 4, 5, or 6 carbon atoms with two points of attachment.

The term “heterocycloalkylene” refers to a saturated cyclic hydrocarbon moiety containing 1 or 2 heteroatoms independently selected from oxygen, sulphur or nitrogen atoms, with two points of attachment. The two points of attachment can be from the same or different carbon atoms. The term “3- to 6-membered heterocycloalkylene” refers to a 3- to 6-membered saturated cyclic moiety containing 2, 3, 4 or 5 carbon atoms in addition to 1 or 2 oxygen, sulphur or nitrogen atoms, with two points of attachment. Suitably, the 3- to 6-membered heterocycloalkylene group contains 1 oxygen or nitrogen atom. Suitably such group contains 3 carbon atoms and 1 oxygen or nitrogen atom, such as azetidindiyl or oxetandiyl. Suitably such group contains 4 or 5 carbon atoms and 1 oxygen or nitrogen atom, such as tetrahydrofurandiyl, tetrahydropyrandiyl, pyrrolidindiyl or piperidindiyl.

The term “bridged bicyclic cycloalkylene” refers to a saturated bicyclic hydrocarbon moiety having at least one bridge, with two points of attachment. A “bridge” is an unbranched chain of atoms or an atom or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of the ring system which is bonded to three or more skeletal atoms (excluding hydrogen). The two points of attachment can be from the same or different carbon atoms. The term “C_7-9 bridged bicyclic cycloalkylene” refers to an unsubstituted bridged bicyclic cycloalkylene moiety containing 7, 8, or 9 carbon atoms with two points of attachment.

The term “arylene” refers to a monocyclic or bicyclic ring system wherein at least one ring in the system is aromatic, with two points of attachment. Exemplary arylene groups include phenylene, biphenylene, naphthylene, and anthracylene.

The term “heteroarylene” refers to a monocyclic or bicyclic ring system wherein at least one ring in the system is aromatic, and having, in addition to carbon atoms, from one to five heteroatoms independently selected from oxygen, sulphur or nitrogen atoms, with two points of attachment. The term “5- to 6-membered heteroarylene” refers to a 5- to 6-membered cyclic aromatic moiety containing 2, 3, 4 or 5 carbon atoms in addition to 1 , 2, or 3 heteroatoms independently selected from oxygen, sulphur or nitrogen atoms, with two points of attachment.

The skilled artisan will appreciate that salts, including pharmaceutically acceptable salts, of the compounds according to Formula (I) may be prepared. Indeed, in certain embodiments of the invention, salts including pharmaceutically-acceptable salts of the compounds according to Formula (I) may be preferred over the respective free or unsalted compound. Accordingly, the invention is further directed to salts, including pharmaceutically- acceptable salts, of the compounds according to Formula (I). The invention is further directed to free or unsalted compounds of Formula (I).

The salts, including pharmaceutically acceptable salts, of the compounds of the invention are readily prepared by those of skill in the art.

Representative pharmaceutically acceptable acid addition salts include, but are not limited to, 4-acetamidobenzoate, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate (besylate), benzoate, bisulfate, bitartrate, butyrate, calcium edetate, camphorate, camphorsulfonate (camsylate), caprate (decanoate), caproate (hexanoate), caprylate (octanoate), cinnamate, citrate, cyclamate, digluconate, 2,5-dihydroxybenzoate, disuccinate, dodecylsulfate (estolate), edetate (ethylenediaminetetraacetate), estolate (lauryl sulfate), ethane-1 ,2-disulfonate (edisylate), ethanesulfonate (esylate), formate, fumarate, galactarate (mucate), gentisate (2,5-dihydroxybenzoate), glucoheptonate (gluceptate), gluconate, glucuronate, glutamate, glutarate, glycerophosphorate, glycolate, hexylresorcinate, hippurate, hydrabamine (N,N'-di(dehydroabietyl)-ethylenediamine), hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoate, isobutyrate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, methanesulfonate (mesylate), methylsulfate, mucate, naphthalene-1 ,5-disulfonate (napadisylate), naphthalene-2-sulfonate (napsylate), nicotinate, nitrate, oleate, palmitate, p-aminobenzenesulfonate, p- aminosalicyclate, pamoate (embonate), pantothenate, pectinate, persulfate, phenylacetate, phenylethylbarbiturate, phosphate, polygalacturonate, propionate, p-toluenesulfonate (tosylate), pyroglutamate, pyruvate, salicylate, sebacate, stearate, subacetate, succinate, sulfamate, sulfate, tannate, tartrate, teoclate (8-chlorotheophyllinate), thiocyanate, triethiodide, trifluoroacetate, undecanoate, undecylenate, and valerate.

Representative pharmaceutically acceptable base addition salts include, but are not limited to, aluminium, 2-amino-2-(hydroxymethyl)-1 ,3-propanediol (TRIS, tromethamine), arginine, benethamine (N-benzylphenethylamine), benzathine (N,N'- dibenzylethylenediamine), b/s-(2-hydroxyethyl)amine, bismuth, calcium, chloroprocaine, choline, clemizole (1-p chlorobenzyl-2-pyrrolidine-T-ylmethylbenzimidazole), cyclohexylamine, dibenzylethylenediamine, diethylamine, diethyltriamine, dimethylamine, dimethylethanolamine, dopamine, ethanolamine, ethylenediamine, L-histidine, iron, isoquinoline, lepidine, lithium, lysine, magnesium, meglumine (N-methylglucamine), piperazine, piperidine, potassium, procaine, quinine, quinoline, sodium, strontium, t- butylamine, and zinc.

The compounds according to Formula (I) may contain one or more asymmetric centers (also referred to as a chiral center) and may, therefore, exist as individual enantiomers, diastereomers, or other stereoisomeric forms, or as mixtures thereof. Chiral centers, such as chiral carbon atoms, may be present in a substituent such as an alkyl group. Where the stereochemistry of a chiral center present in a compound of Formula (I), or in any chemical structure illustrated herein, if not specified the structure is intended to encompass all individual stereoisomers and all mixtures thereof. Thus, compounds according to Formula (I) containing one or more chiral centers may be used as racemic mixtures, enantiomerically enriched mixtures, or as enantiomerically pure individual stereoisomers.

A mixture of stereoisomers in which the relative configuration of all of the stereocenters is known may be depicted using the symbol "&" together with an index number (e.g., “&1”). For example, a group of two stereogenic centers labeled with the symbol “&1” represents a mixture of two possible stereoisomers in which the two stereogenic centers have a relative configuration as depicted. Divalent groups are groups having two points of attachment. For all divalent groups, unless otherwise specified, the orientation of the group is implied by the direction in which the formula or structure of the group is written.

Unless defined otherwise, 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. Although any compositions and methods similar or equivalent to those described herein can be used in the practice or testing of the methods of the disclosure, exemplary compositions and methods are described herein. Any of the aspects and embodiments of the disclosure described herein may also be combined. For example, the subject matter of any dependent or independent claim disclosed herein may be multiply combined (e.g., one or more recitations from each dependent claim may be combined into a single claim based on the independent claim on which they depend).

Ranges provided herein include all values within a particular range described and values about an endpoint for a particular range.

Concentrations described herein are determined at ambient temperature and pressure. This may be, for example, the temperature and pressure at room temperature or in a particular portion of a process stream. Preferably, concentrations are determined at a standard state of 25 °C and 1 bar of pressure.

Targets and Target-Binding Moieties

The compounds of Formula (I) as disclosed herein are heterotrifunctional, or dual targeting, synthetic agents designed such that two termini interact with one or two cell surface targets, while the third terminus binds a specific antibody. More specifically, the ARM simultaneously binds the cell surface targets as well as the specific antibody. This multi- component complex directs immune surveillance to one or two target expressing tissue/cells and unites the mechanisms of antibody function with the dose-control of small molecules. This mechanism may include antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), or complement dependant cytotoxicity (CDC), and preferably includes ADCC. The same Fc receptor expressing immune cells that initiate destruction of the ARM/antibody tagged cells also participate in presentation of endogenous antigens for the potential for long term cellular immunity.

The compounds of Formula (I) as disclosed herein include target-binding moieties that are capable of binding one or two target proteins (e.g., a receptor) present on the surface of a cell. A person skilled in the art can select molecules known to bind the target protein for use as the target-binding moieties in the ARM. In one embodiment, the compounds of Formula (I) are designed such that one terminus interacts with a cell surface prostate specific membrane antigen (PSMA) target, a second terminus interacts with a cell surface C-C chemokine receptor type 2 (CCR2) target, and a third terminus interacts with the specific antibody. In another embodiment, the compounds of Formula (I) are designed such that one terminus interacts with a cell surface prostate specific membrane antigen (PSMA) target, a second terminus interacts with a cell surface C-C chemokine receptor type 8 (CCR8) target, and a third terminus interacts with the specific antibody. In another embodiment, the compounds of Formula (I) are designed such that one terminus interacts with a cell surface C-C chemokine receptor type 2 (CCR2) target, a second terminus interacts with a cell surface C-C chemokine receptor type 8 (CCR8) target, and a third terminus interacts with the specific antibody. In another embodiment, the compounds of Formula (I) are designed such that two termini interact with the cell surface PSMA target, and a third terminus interacts with the specific antibody. In another embodiment, the compounds of Formula (I) are designed such that two termini interact with the cell surface CCR2 target, and a third terminus interacts with the specific antibody. In another embodiment, the compounds of Formula (I) are designed such that two termini interact with the cell surface CCR8 target, and a third terminus interacts with the specific antibody.

In one embodiment, the compound of Formula (I) comprises a PSMA-binding moiety and a CCR2-binding moiety. In one embodiment, the compound of Formula (I) comprises a PSMA-binding moiety and a CCR8-binding moiety. In another embodiment, the compound of Formula (I) comprises a CCR2-binding moiety and a CCR8-binding moiety. In another embodiment, the compound of Formula (I) comprises two PSMA-binding moieties. In another embodiment, the compound of Formula (I) comprises two CCR2-binding moieties. In another embodiment, the compound of Formula (I) comprises two CCR8-binding moieties.

In one embodiment, the target of the target binding moiety is a cell surface protein. In a further embodiment, the target of the target binding moiety is a target protein expressed on a pathogenic cell.

In a further embodiment, the pathogenic cell is a pathogenic immune cell, a tumor cell or cancer cell, or a stromal cell (including stromal cells present in a tumor microenvironment).

In a further embodiment, the target of the target binding moiety is present on the surface of a pathogenic agent selected from a virus or a bacterial cell. Examples of a virus expressing cell surface targets include, but are not limited to, influenza. Examples of cell surface targets on influenza virus include, but are not limited to, neuraminidase.

In a further embodiment, the pathogenic immune cells are monocytes, myeloid derived suppressor cells (MDSC), such as monocytic MDSCs (mMDSCs) and polymorphonuclear MDSCs (PMN_MDSCs), T regulatory cells (Tregs), neutrophils (e.g., N2 neutrophils), macrophages (e.g., M2 macrophages), B regulatory cells (Bregs, memory B cells), plasma cells, CD8 cells (e.g., CD8 regulatory cells (CD8regs), memory CD8 cells, effector CD8 cells, naive CD8 Tcells, TEMRA), exhausted T cells, eosinophils, basophils, mast cells, dendritic cells, natural killer (NK) cells, innate lymphoid cells, NK T cells (NKT), or ydT cells.

In a further embodiment, the pathogenic immune cells are myeloid derived suppressor cells (MDSC), such as monocytic MDSCs (mMDSCs) and polymorphonuclear MDSCs (PMN_MDSCs), T regulatory cells (Tregs), neutrophils (e.g., N2 neutrophils), macrophages (e.g., M2 macrophages), B regulatory cells (Bregs), CD8 regulatory cells (CD8regs), or exhausted T cells.

In a further embodiment, the tumor cells or cancer cells are solid tumor cells.

In a further embodiment, the tumor cells or cancer cells are lung cancer cells (e.g., non-small cell lung cancer (NSCLC) cells), hepatocellular carcinoma (HCC) cells, colorectal cancer (CRC) cells, cervical cancer cells (e.g., cervical squamous cell carcinoma (CESC) cells), head and neck cancer cells (e.g., head and neck squamous cell carcinoma (HNSC) cells), pancreatic cancer cells, prostate cancer cells (e.g., metastatic castration-resistant prostate cancer (mCRPC) cells), ovarian cancer cells, endometrial cancer cells, brain cancer cells, endocrine cancer cells, testicular cancer cells, bladder cancer cells, bone cancer cells, esophogeal cancer cells, gastric cancer cells, renal cell cancer cells, melanoma cancer cells, thyroid cancer cells, or breast cancer cells, preferably cells selected from mCRPC cells, breast cancer cells, lung cancer cells, colorectal cancer cells, or renal cell cancer cells.

In a further embodiment, the pathogenic cell is an endothelial cell associated with tumor neovasculature.

In a further embodiment, the stromal cells are cancer associated fibroblasts (CAFs).

In one embodiment, the target of the target binding moiety is selected from a G protein- coupled receptor (GPCR), an enzyme (such as a dehydrogenase, an esterase, a phosphodiesterase, a hydrolase, a lipase, a phosphatase, a kinase, a reductase, or a transferase), a transporter (e.g., an ion channel), a protease, or a receptor. In a further embodiment, the target of the target binding moiety is selected from a GPCR, an enzyme (such as a dehydrogenase, an esterase, a phosphodiesterase, a hydrolase, a lipase, a phosphatase, a kinase, a reductase, or a transferase), a transporter (e..g, an ion channel), a protease, or a receptor, wherein the target is associated with and/or expressed on immune cells (including pathogenic immune cells), tumor cells or cancer cells, or stromal cells (including stromal cells present in a tumor microenvironment).

In a further embodiment, the target of the target binding moiety is selected from 15- hydroxyprostaglandin dehydrogenases, 5-hydroxytryptamine receptors, activated leukocyte cell adhesion molecules, ADAM metallopeptidases, adenosine receptors, adenosine deaminases, adrenoceptor beta, advanced glycosylation end-product specific receptors, membrane alanyl aminopeptidases, alkaline phosphatases, calcium voltage-gated channels, cannabinoid receptors, carcinoembryonic antigen related cell adhesion molecules, C-C motif chemokine receptors, CD14, CD19, CD200 receptors, CD22, CD274, CD276, CD33, CD37, CD38, CD3e, CD4, CD44, CD48, CD70, CD74, CD80, CD99, muscarinic cholinergic receptors, nicotinic cholinergic receptors, coagulation factor II thrombin receptors, colony stimulating factor 2 receptors, complement C5a receptors, C-type lectin domains, C-X-C motif chemokine receptors, cysteinyl leukotriene receptors, cytotoxic T-lymphocyte associated proteins, delta like canonical Notch ligands, dipeptidyl peptidases, ectonucleoside triphosphate diphosphohydrolases, erythropoietin receptors, F11 receptors, formyl peptide receptors, FXYD domain containing ion transport regulators, G protein-coupled bile acid receptors, G protein-coupled receptors, gamma-aminobutyric acid type A receptors, gastric inhibitory polypeptide receptors, glutamate metabotropic receptor, platelet glycoproteins, hepatitis A virus cellular receptors, histamine receptors, hydroxycarboxylic acid receptors, integrins, intercellular adhesion molecules, interleukin receptor accessory proteins, interleukin receptors, killer cell lectin like receptors, KISS1 receptors, leukotriene receptors, lymphocyte activating gene proteins, lymphocyte antigens, mannose receptors, membrane metalloendopeptidases, membrane spanning 4-domains, platelet activating factor receptors, potassium calcium-activated channels, potassium voltage-gated channels, programmed cell death proteins, prostaglandin receptors, prostaglandin synthases, protein tyrosine phosphatases, purinergic receptors, pyrimidinergic receptors, scavenger receptors, selectins, signaling lymphocytic activation molecule (SLAM) proteins, sodium voltage-gated channels, somatostatin receptors, sphingosine-1-phosphate receptors, suppression of tumorigenicity proteins, T cell immunoreceptors, thromboxane receptors, TNF receptors, toll like receptors, transient receptor potential cation channels, triggering receptors expressed on myeloid cells, or V-set immunoregulatory receptors.

In a further embodiment, the target of the target binding moiety is a target as listed in Table 2:

Table 2

In a further embodiment, the target of the target binding moiety is a chemokine receptor (CCR). In a further embodiment, the target of the target binding moiety is selected from CCR1 , CCR2, CCR3, CCR5, or CCR8.

In a further embodiment, the target of the target binding moiety is selected from C-C motif chemokine receptor (CCR) 2 (CCR2), CCR1 , CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, C-X-C motif chemokine receptor 1 (CXCR1), C-X-C motif chemokine receptor 2 (CXCR2), C-X-C motif chemokine receptor 3 (CXCR3), C-X-C motif chemokine receptor 4 (CXCR4), C-X-C motif chemokine receptor 5 (CXCR5), C-X-C motif chemokine receptor 6 (CXCR6), atypical chemokine receptor 3 (ACKR3), integrin αvβ6, fibroblast activation protein-alpha (FAPα), prostate specific membrane antigen (PSMA), folate receptor (folate receptor 1 or folate receptor beta), complement C3a receptor 1 (C3AR1), complement C5a receptor 1 (C5AR1), G protein-coupled receptor (GPR) 65 (GPR65), GRP132, GPR84, GPR183, GPR35, GPR42, cholecystokinin A receptor (CCKAR), leukotriene B4 receptor (LTB4R), somatostatin receptor 2 (SSTR2), free fatty acid receptor 1 (FFAR1), purinergic receptor P2Y2 (P2RY2), prostaglandin D2 receptor (PTGDR), calcitonin receptor (CALCR), CD38, purinergic receptor P2X 7 (P2RX7), integrin subunit alpha V (ITGAV), integrin subunit alpha 5 (ITGA5), integrin subunit beta 1 (ITGB1), integrin subunit beta 6 (ITGB6), integrin subunit beta 3 (ITGB3) prostaglandin D2 receptor 2 (PTGDR2), gastrin releasing peptide receptor (GRPR), MER proto-oncogene tyrosine kinase (MERTK), C-X3-C motif chemokine receptor 1 (CX3CR1), oxidized low density lipoprotein receptor 1 (OLR1), plasminogen activator urokinase receptor (PLAUR), carbonic anhydrase 9 (CA9), carbonic anhydrase 12 (CA12), mas-related G-protein coupled receptor member X2 (MRGPRX2), heat shock protein 90 alpha family class A member 1 (HSP90AA1), dipeptidyl peptidase 4 (DPP4), formyl peptide receptor 2 (FPR2), and succinate receptor 1 (SUCNR1).

In a further embodiment, the target-binding moiety T¹ or T² is a small molecule that binds a target as listed in T able 2. A person skilled in the art can select small molecules known to bind the target protein for use as the target-binding moiety in the ARM. In one embodiment, the target-binding small molecule is modified to include a functional group such as -NH₂ or - COOH to facilitate covalent coupling of the target-binding small molecule to the divalent linker moiety by amide bond formation.

The present disclosure also provides a pharmaceutical composition comprising a compound of Formula (I) as disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent.

Anti-Cotinine Antibodies

The present disclosure provides an antibody, or antigen-binding fragment thereof, that binds to a cotinine moiety. As used herein, the term “anti-cotinine antibody or antigen-binding fragment thereof” refers to an antibody, or antigen binding fragment thereof that binds to a cotinine moiety. Cotinine has the following structure:

As used herein, the term “cotinine moiety” refers to cotinine or an analog of cotinine. Compounds of Formula (I) described herein comprise a cotinine moiety linked via a linker to target-binding moieties, such as a PSMA-binding moiety, a CCR2-binding moiety, and/or a CCR8-binding moiety. In one embodiment, the cotinine moiety has the following structure:

wherein R¹ is C_1-4 alkyl or C_3-6 cycloalkyl. In another embodiment, R¹ is methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, or t-butyl. In another embodiment, R¹ is methyl. In another embodiment, R¹ is ethyl. In another embodiment, R¹ is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

The term “antibody” is used herein in the broadest sense to refer to molecules with an immunoglobulin-like domain (for example IgG, IgM, IgA, IgD or lgE) and includes monoclonal, recombinant, polyclonal, chimeric, human, humanised, multispecific antibodies, including bispecific antibodies, and heteroconjugate antibodies; a single variable domain (e.g. , a domain antibody (DAB)), antigen binding antibody fragments, Fab, F(ab’)2, Fv, disulphide linked Fv, single chain Fv, disulphide-linked scFv, diabodies, TANDABS, etc. and modified versions of any of the foregoing (for a summary of alternative “antibody” formats see Holliger and Hudson, Nature Biotechnology, 2005, 23(9): 1126-1136).

The term, full, whole or intact antibody, used interchangeably herein, refers to a heterotetrameric glycoprotein with an approximate molecular weight of 150,000 daltons. An intact antibody is composed of two identical heavy chains (HCs) and two identical light chains (LCs) linked by covalent disulphide bonds. This H2L2 structure folds to form three functional domains comprising two antigen-binding fragments, known as ‘Fab’ fragments, and a ‘Fc’ crystallisable fragment. The Fab fragment is composed of the variable domain at the amino- terminus, variable heavy (VH) or variable light (VL), and the constant domain at the carboxyl terminus, CH1 (heavy) and CL (light). The Fc fragment is composed of two domains formed by dimerization of paired CH2 and CH3 regions. The Fc may elicit effector functions by binding to receptors on immune cells or by binding C1q, the first component of the classical complement pathway. The five classes of antibodies IgM, IgA, IgG, IgE and IgD are defined by distinct heavy chain amino acid sequences, which are called μ, α, γ, ε and δ respectively, each heavy chain can pair with either a K or A light chain. The majority of antibodies in the serum belong to the IgG class, there are four isotypes of human IgG (lgG1 , lgG2, lgG3 and I gG4), the sequences of which differ mainly in their hinge region.

“CDRs” are defined as the complementarity determining region amino acid sequences of an antibody or antigen binding fragment thereof. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, “CDRs” as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.

Throughout this specification, amino acid residues in variable domain sequences and variable domain regions within full-length antigen binding sequences, e.g. within an antibody heavy chain sequence or antibody light chain sequence, are numbered according to the Kabat numbering convention. Similarly, the terms “CDR”, “CDRL1”, “CDRL2”, “CDRL3”, “CDRH1”, “CDRH2”, “CDRH3” used in the Examples follow the Kabat numbering convention. For further information, see Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987).

It will be apparent to those skilled in the art that there are alternative numbering conventions for amino acid residues in variable domain sequences and full-length antibody sequences. There are also alternative numbering conventions for CDR sequences, for example those set out in Chothia et al., Nature, 1989, 342: 877-883. The structure and protein folding of the antigen binding protein may mean that other residues are considered part of the CDR sequence and would be understood to be so by a skilled person.

Other numbering conventions for CDR sequences available to a skilled person include “AbM” (University of Bath) and “contact” (University College London) methods.

Table 3 below represents one definition using each numbering convention for each CDR or binding unit. It should be noted that some of the CDR definitions may vary depending on the individual publication used.

Table 3

In a further embodiment, the anti-cotinine antibody is humanized. In a further embodiment, the Fc region of the anti-cotinine antibody is modified to increase ADCC activity, ADCP activity, and/or CDC activity, suitable modifications of which are provided below. In a further embodiment, the Fc region of the anti-cotinine antibody is modified to increase ADCC activity.

Fc engineering methods can be applied to modify the functional or pharmacokinetics properties of an antibody. Effector function may be altered by making mutations in the Fc region that increase or decrease binding to C1q or Fcγ receptors and modify CDC or ADCC activity respectively. Modifications to the glycosylation pattern of an antibody can also be made to change the effector function. The in vivo half-life of an antibody can be altered by making mutations that affect binding of the Fc to the FcRn (neonatal Fc receptor). The term “effector function” as used herein refers to one or more of antibody-mediated effects including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-mediated complement activation including complement-dependent cytotoxicity (CDC), complement- dependent cell-mediated phagocytosis (CDCP), antibody dependent complement-mediated cell lysis (ADCML), and Fc-mediated phagocytosis or antibody-dependent cellular phagocytosis (ADCP).

The interaction between the Fc region of an antigen binding protein or antibody and various Fc receptors (FcR), including FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), FcRn, C1q, and type II Fc receptors is believed to mediate the effector functions of the antigen binding protein or antibody. Significant biological effects can be a consequence of effector functionality. Usually, the ability to mediate effector function requires binding of the antigen binding protein or antibody to an antigen and not all antigen binding proteins or antibodies will mediate every effector function.

Effector function can be assessed in a number of ways including, for example, evaluating ADCC effector function of antibody coated to target cells mediated by Natural Killer (NK) cells via FcγRIII, or monocytes/macrophages via FcγRI, or evaluating CDC effector function of antibody coated to target cells mediated by complement cascade via C1q. For example, an antibody, or antigen binding fragment thereof, of the present invention can be assessed for ADCC effector function in a Natural Killer cell assay. Examples of such assays can be found in Shields et al., The Journal of Biological Chemistry, 2001 , 276: 6591 -6604; Chappel et al., The Journal of Biological Chemistry, 1993, 268: 25124-25131 ; Lazar et al., PNAS, 2006, 103: 4005-4010.

Examples of assays to determine CDC function include those described in J Imm Meth, 1995, 184: 29-38.

The effects of mutations on effector functions (e.g., FcRn binding, FcγRs and C1q binding, CDC, ADCML, ADCC, ADCP) can be assessed, e.g., as described in Grevys et al., J Immunol., 2015, 194(11): 5497-5508; Tam et al., Antibodies, 2017, 6(3): 12; or Monnet et al., mAbs, 2014, 6(2): 422-436.

Throughout this specification, amino acid residues in Fc regions, in antibody sequences or full-length antigen binding protein sequences, are numbered according to the EU index numbering convention.

Human lgG1 constant regions containing specific mutations have been shown to enhance binding to Fc receptors. In some cases these mutations have also been shown to enhance effector functions, such as ADCC and CDC, as described below. Antibodies, or antigen binding fragments thereof, of the present invention may include any of the following mutations. Enhanced CDC: Fc engineering can be used to enhance complement-based effector function. For example (with reference to lgG1), K326W/E333S; S267E/H268F/S324T; and lgG1/lgG3 cross subclass can increase C1q binding; E345R (Diebolder et al., Science, 2014, 343: 1260-1293) and E345R/E430G/S440Y results in preformed IgG hexamers (Wang et al., Protein Cell, 2018, 9(1): 63-73).

Enhanced ADCC: Fc engineering can be used to enhance ADCC. For example (with reference to lgG1), F243L/R292P/Y300LA/305I/P396L; S239D/I332E; and

S298A/E333A/K334A increase FcγRllla binding; S239D/I332E/A330L increases FcγRIIIA binding and decreases FcγRllb binding; G236A/S239D/I332E improves binding to FcγRlla, improves the FcYRIIa/FcγRllb binding ratio (activating/inhibitory ratio), and enhances phagocytosis of antibody-coated target cells by macrophages. An asymmetric Fc in which one heavy chain contains L234Y/L235Q/G236W/S239M/H268D/D270E/S298A mutations and D270E/K326D/A330M/K334E in the opposing heavy chain, increases affinity for FcγRllla F158 (a lower-affinity allele) and FcγRllla V158 (a higher-affinity allele) with no increased binding affinity to inhibitory FcγRllb (Mimoto et al., mAbs, 2013, 5(2): 229-236).

Enhanced ADCP: Fc engineering can be used to enhance ADCP. For example (with reference to lgG1), G236A/S239D/I332E increases FcγRlla binding and increases FcγRllla binding (Richards, J. et al., Mol. Cancer Then, 2008, 7: 2517-2527).

Increased co-engagement: Fc engineering can be used to increase co-engagement with FcRs. For example (with reference to lgG1), S267E/L328F increases FcγRllb binding; N325S/L328F increases FcγRlla binding and decreases FcγRllla binding Wang et al., Protein Cell, 2018, 9(1): 63-73).

In a further embodiment, an antibody, or antigen binding fragment thereof, of the present invention may comprise a heavy chain constant region with an altered glycosylation profile, such that the antibody, or antigen binding fragment thereof, has an enhanced effector function, e.g., enhanced ADCC, enhanced CDC, or both enhanced ADCC and CDC. Examples of suitable methodologies to produce an antibody, or antigen binding fragment thereof, with an altered glycosylation profile are described in WO 2003/011878, WO 2006/014679 and EP1229125.

The absence of the a1,6 innermost fucose residues on the Fc glycan moiety on N297 of lgG1 antibodies enhances affinity for FcγRIIIA. As such, afucosylated or low fucosylated monoclonal antibodies may have increased therapeutic efficacy (Shields et al., J Biol Chem., 2002, 277(30): 26733-40 and Monnet et al., mAbs, 2014, 6(2): 422-436).

In one embodiment there is provided an antibody, or antigen binding fragment thereof, comprising a chimeric heavy chain constant region. In an embodiment, the antibody, or antigen binding fragment thereof, comprises an lgG1/lgG3 chimeric heavy chain constant region, such that the antibody, or antigen binding fragment thereof, has an enhanced effector function, for example enhanced ADCC or enhanced CDC, or enhanced ADCC and CDC functions. For example, a chimeric antibody, or antigen binding fragment thereof, of the invention may comprise at least one CH2 domain from lgG3. In one such embodiment, the antibody, or antigen binding fragment thereof, comprises one CH2 domain from lgG3 or both CH2 domains may be from lgG3. In a further embodiment, the chimeric antibody, or antigen binding fragment thereof, comprises an IgG1 CH1 domain, an lgG3 CH2 domain, and an lgG3 CH3 domain. In a further embodiment, the chimeric antibody, or antigen binding fragment thereof, comprises an lgG1 CH1 domain, an lgG3 CH2 domain, and an lgG3 CH3 domain except for position 435 that is histidine.

In a further embodiment, the chimeric antibody, or antigen binding fragment thereof, comprises an lgG1 CH1 domain and at least one CH2 domain from lgG3. In an embodiment, the chimeric antibody, or antigen binding fragment thereof, comprises an lgG1 CH1 domain and the following residues, which correspond to lgG3 residues, in a CH2 domain: 274Q, 276K, 296F, 300F and 339T. In an embodiment, the chimeric antibody, or antigen binding fragment thereof, also comprises 356E, which corresponds to an lgG3 residue, within a CH3 domain. In an embodiment, the antibody, or antigen binding fragment thereof, also comprises one or more of the following residues, which correspond to lgG3 residues within a CH3 domain: 358M, 384S, 392N, 397M, 422I, 435R, and 436F.

Also provided is a method of producing an antibody, or antigen binding fragment thereof, according to the invention comprising the steps of: a) culturing a recombinant host cell comprising an expression vector comprising a nucleic acid sequence encoding a chimeric Fc region having both lgG1 and lgG3 Fc region amino acid residues (e.g. as described above); and b) recovering the antibody, or antigen binding fragment thereof.

Such methods for the production of antibody, or antigen binding fragment thereof, with chimeric heavy chain constant regions can be performed, for example, using the COMPLEGENT technology system available from BioWa, Inc. (Princeton, NJ) and Kyowa Hakko Kirin Co., Ltd. The COMPLEGENT system comprises a recombinant host cell comprising an expression vector in which a nucleic acid sequence encoding a chimeric Fc region having both I gG 1 and lgG3 Fc region amino acid residues is expressed to produce an antibody, or antigen binding fragment thereof, having enhanced CDC activity, i.e. CDC activity is increased relative to an otherwise identical antibody, or antigen binding fragment thereof, lacking such a chimeric Fc region, as described in WO 2007/011041 and US 2007/0148165, each of which are incorporated herein by reference. In an alternative embodiment, CDC activity may be increased by introducing sequence specific mutations into the Fc region of an IgG chain. Those of ordinary skill in the art will also recognize other appropriate systems.

The present invention also provides a method of producing an antibody, or antigen binding fragment thereof, according to the invention comprising the steps of: a) culturing a recombinant host cell comprising an expression vector comprising a nucleic acid encoding the antibody, or antigen binding fragment thereof, optionally wherein the FUT8 gene encoding alpha-1, 6-fucosyltransferase has been inactivated in the recombinant host cell; and b) recovering the antibody, or antigen binding fragment thereof.

Such methods for the production of an antibody, or antigen binding fragment thereof, can be performed, for example, using the POTELLIGENT technology system available from BioWa, Inc. (Princeton, NJ) in which CHOK1SV cells lacking a functional copy of the FUT8 gene produce monoclonal antibodies having enhanced ADCC activity that is increased relative to an identical monoclonal antibody produced in a cell with a functional FUT8 gene as described in US Patent No. 7,214,775, US Patent No. 6,946,292, WO 00/61739 and WO 02/31240, all of which are incorporated herein by reference. Those of ordinary skill in the art will also recognize other appropriate systems.

In one embodiment, the antibody, or antigen binding fragment thereof, is produced in a host cell in which the FUT8 gene has been inactivated. In a further embodiment, the antibody, or antigen binding fragment thereof, is produced in a -/- FUT8 host cell. In a further embodiment, the antibody, or antigen binding fragment thereof, is afucosylated at Asn297 (igGi).

It will be apparent to those skilled in the art that such modifications may not only be used alone but may be used in combination with each other in order to further enhance effector function.

In one such embodiment, there is provided an antibody, or antigen binding fragment thereof, comprising a heavy chain constant region that comprises a both a mutated and chimeric heavy chain constant region, individually described above. For example, an antibody, or antigen binding fragment thereof, comprising at least one CH2 domain from lgG3 and one CH2 domain from lgG1 , and wherein the lgG1 CH2 domain has one or more mutations at positions selected from 239, 332 and 330 (for example the mutations may be selected from S239D, I332E and A330L), such that the antibody, or antigen binding fragment thereof, has enhanced effector function, e.g. enhanced ADCC or enhanced CDC, or enhanced ADCC and enhanced CDC in comparison to an equivalent antibody, or antigen binding fragment thereof, with an lgG1 heavy chain constant region lacking said mutations. In one embodiment, the lgG1 CH2 domain has the mutations S239D and I332E. In another embodiment, the lgG1 CH2 domain has the mutations S239D, A330L, and I332E.

In an alternative embodiment, there is provided an antibody, or antigen binding fragment thereof, comprising both a chimeric heavy chain constant region and an altered glycosylation profile, as individually described above. In an embodiment, the antibody, or antigen binding fragment thereof, comprises an altered glycosylation profile such that the ratio of fucose to mannose is 0.8:3 or less. In one such embodiment, the heavy chain constant region comprises at least one CH2 domain from lgG3 and one CH2 domain from lgG1 and has an altered glycosylation profile such that the ratio of fucose to mannose is 0.8:3 or less, for example wherein the antibody, or antigen binding fragment thereof, is defucosylated. Said antibody, or antigen binding fragment thereof, has an enhanced effector function, e.g. enhanced ADCC or enhanced CDC, or enhanced ADCC and enhanced CDC, in comparison to an equivalent antibody, or antigen binding fragment thereof, with an lgG1 heavy chain constant region lacking said glycosylation profile.

In an alternative embodiment, the antibody, or antigen binding fragment thereof, has at least one lgG3 heavy chain CH2 domain and at least one heavy chain constant domain from lgG1 wherein both IgG CH2 domains are mutated in accordance with the limitations described herein.

In one aspect, there is provided a method of producing an antibody, or antigen binding fragment thereof, according to the invention described herein comprising the steps of: a) culturing a recombinant host cell containing an expression vector comprising a nucleic acid sequence encoding a chimeric Fc domain having both I gG 1 and lgG3 Fc domain amino acid residues (e.g. as described above); and wherein the FUT8 gene encoding alpha- 1 ,6-fucosyltransferase has been inactivated in the recombinant host cell; and b) recovering the antibody, or antigen binding fragment thereof.

Such methods for the production of an antibody, or antigen binding fragment thereof, can be performed, for example, using the ACCRETAMAB technology system available from BioWa, Inc. (Princeton, NJ) that combines the POTELLIGENT and COMPLEGENT technology systems to produce an antibody, or antigen binding fragment thereof, having both enhanced ADCC and CDC activity relative to an otherwise identical monoclonal antibody that lacks a chimeric Fc domain and that is fucosylated.

In another embodiment, there is provided an antibody, or antigen binding fragment thereof, comprising a mutated and chimeric heavy chain constant region wherein said antibody, or antigen binding fragment thereof, has an altered glycosylation profile such that the antibody, or antigen binding fragment thereof, has enhanced effector function, e.g. enhanced ADCC or enhanced CDC, or both enhanced ADCC and CDC. In one embodiment the mutations are selected from positions 239, 332 and 330, e.g. S239D, I332E and A330L. In a further embodiment the heavy chain constant region comprises at least one CH2 domain from lgG3 and one CH1 domain from lgG1. In one embodiment the heavy chain constant region has an altered glycosylation profile such that the ratio of fucose to mannose is 0.8:3 or less, e.g. the antibody, or antigen binding fragment thereof, is defucosylated, such that said antibody, or antigen binding fragment thereof, has an enhanced effector function in comparison with an equivalent non-chimeric antibody, or antigen binding fragment thereof, lacking said mutations and lacking said altered glycosylation profile.

In a further embodiment, the anti-cotinine antibody, or antigen binding fragment thereof, comprises a heavy chain CDR1 having SEQ ID NO: 1 , a heavy chain CDR2 having SEQ ID NO: 2, a heavy chain CDR3 having SEQ ID NO: 3, a light chain CDR1 having SEQ ID NO: 4, a light chain CDR2 having SEQ ID NO: 5, and a light chain CDR3 having SEQ ID NO: 6. In a further embodiment, the anti-cotinine antibody has a heavy chain and a light chain, the heavy chain comprising a CDR1 having SEQ ID NO: 1 , a CDR2 having SEQ ID NO: 2, and a CDR3 having SEQ ID NO: 3, and the light chain comprising a CDR1 having SEQ ID NO: 4, a CDR2 having SEQ ID NO: 5, and a CDR3 having SEQ ID NO: 6. In a further embodiment, the anti-cotinine antibody is of I gG 1 isotype. In a further embodiment, the anti- cotinine antibody is of lgG1 isotype comprising a substitution in an Fc region to increase or enhance ADCC activity. In a further embodiment, the anti-cotinine antibody is of I gG 1 isotype comprising a substitution in an Fc region to increase or enhance ADCC activity, wherein the substitution is S239D/I332E or S239D/I332E/A330L, wherein residue numbering is according to the Ell Index. In a further embodiment, the anti-cotinine antibody is of lgG1 isotype comprising a substitution in an Fc region to increase or enhance ADCC activity, wherein the substitution is S239D/I332E, wherein residue numbering is according to the EU Index.

In a further embodiment, the anti-cotinine antibody, or antigen binding fragment thereof, comprises a heavy chain variable region (VH) having SEQ ID NO: 7 and a light chain variable region (VL) having SEQ ID NO: 8. In a further embodiment, the anti-cotinine antibody has a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region (VH) having SEQ ID NO: 7, and the light chain comprising a light chain variable region (VL) having SEQ ID NO: 8. In a further embodiment, the anti-cotinine antibody is of lgG1 isotype. In a further embodiment, the anti-cotinine antibody is of lgG1 isotype comprising a substitution in an Fc region to increase or enhance ADCC activity. In a further embodiment, the anti- cotinine antibody is of lgG1 isotype comprising a substitution in an Fc region to increase or enhance ADCC activity, wherein the substitution is S239D/I332E or S239D/I332E/A330L, wherein residue numbering is according to the Ell Index. In a further embodiment, the anti- cotinine antibody is of lgG1 isotype comprising a substitution in an Fc region to increase or enhance ADCC activity, wherein the substitution is S239D/I332E, wherein residue numbering is according to the Ell Index.

In a further embodiment, the anti-cotinine antibody has a heavy chain comprising SEQ ID NO: 9 and a light chain comprising SEQ ID NO: 10.

The present disclosure also provides a pharmaceutical composition comprising an anti-cotinine antibody, or antigen binding fragment thereof as disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent.

The present disclosure also provides a combination comprising the compound of Formula (I) as disclosed herein, and an anti-cotinine antibody, or antigen-binding fragment thereof as disclosed herein. The compound of Formula (I) and anti-cotinine antibody, or antigen binding fragment thereof can be present in the same composition or in separate compositions. In one embodiment, a combination comprises a pharmaceutical composition comprising the compound of Formula (I) as disclosed herein and an anti-cotinine antibody, or antigen binding fragment thereof as disclosed herein, and a pharmaceutically acceptable carrier, diluent, or excipient. In another embodiment, a combination comprises a first pharmaceutical composition comprising a compound of Formula (I) as disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient; and a second pharmaceutical composition comprising an anti-cotinine antibody or antigen binding fragment thereof as disclosed herein, and a pharmaceutically acceptable carrier, excipient, or diluent.

Statement of Use

The compounds of Formula (I) and pharmaceutically acceptable salts thereof are capable of simultaneously binding a cell surface-expressed target or targets and an anti- cotinine antibody, or antigen binding fragment thereof to form a multi-component complex for the treatment and/or prevention of diseases or disorders associated with target-expressing cells.

In one embodiment, the present disclosure provides a method of treating and/or preventing a disease or disorder in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and an anti-cotinine antibody, or antigen binding fragment thereof, wherein the disease or disorder is selected from a cancer, an inflammatory disease, an autoimmune disease, a viral infection, or a bacterial infection.

In a further embodiment, the compound and the antibody, or antigen binding fragment thereof, are administered simultaneously. In a further embodiment, the compound and the antibody, or antigen binding fragment thereof, are administered simultaneously from a single composition, including as a fixed-dose composition or by pre-mixing the compound and the antibody, or antigen binding fragment thereof, prior to administration. For example, the compound and the antibody, or antigen binding fragment thereof, can be pre-mixed about 2 seconds to about 30 seconds, about 30 seconds to about 2 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 30 minutes, or about 30 minutes to about 2 hours prior to administration. In a further embodiment, the compound and the antibody, or antigen binding fragment thereof, are administered simultaneously from two separate compositions.

In a further embodiment, the compound and the antibody, or antigen binding fragment thereof, are administered sequentially.

In certain embodiments, the compound and the antibody, or antigen binding fragment thereof, whether administered simultaneously or sequentially, may be administered by the same route or may be administered by different routes. In one embodiment, the compound and the antibody, or antigen binding fragment thereof, are both administered intraveneously or subcutaneously, in the same composition or in separate compositions. In another embodiment, the compound is administered orally and the antibody, or antigen binding fragment thereof, is administered intravenously or subcutaneously.

In a further embodiment, the compound and the antibody, or antigen binding fragment thereof, are administered in a molar ratio of compound to antibody, or antigen binding fragment, thereof of about 2: 1, about 1.8:1 , about 1.6:1 , about 1.5:1 , about 1.4: 1, about 1.3: 1, about 1.2: 1, about 1: 1 , about 1:1.2, about 1 : 1.3, about 1:1.4, about 1 : 1.5, about 1:1.6, about 1 :1.8, about 1 :2, about 2: 1 to about 1.5: 1 , about 1.5: 1 to about 1.2: 1, about 1.2: 1 to about 1 :1, about 1:1 to about 1:1.2, about 1: 1.2 to about 1:1.5, or about 1:1.5 to about 1 :2.

In a further embodiment, the compound and the antibody, or antigen-binding fragment thereof, are present as a combination in a molar ratio of compound to antibody, or antigen- binding fragment thereof, of about 2: 1 , about 1.8:1, about 1.6:1, about 1.5:1 , about 1.4:1, about 1.3:1, about 1.2: 1, about 1:1 , about 1:1.2, about 1: 1.3, about 1 :1.4, about 1:1.5, about 1:1.6, about 1: 1.8, about 1:2, about 2:1 to about 1.5:1, about 1.5:1 to about 1.2: 1 , about 1.2: 1 to about 1:1 , about 1 : 1 to about 1 :1.2, about 1 : 1.2 to about 1 : 1.5, or about 1 : 1.5 to about 1 :2.

In a further embodiment, the compound and the antibody, or antigen-binding fragment thereof, are administered at a dosage of compound of 0.0001 mg/kg to 1 mg/kg and antibody, or antigen-binding fragment thereof, of 0.01 mg/kg to 100 mg/kg. For example, in a further embodiment, the compound is administered at a dosage of about 0.0001 mg/kg to about 0.0002 mg/kg, about 0.0002 mg/kg to about 0.0003 mg/kg, about 0.0003 mg/kg to about 0.0004 mg/kg, about 0.0004 mg/kg to about 0.0005 mg/kg, about 0.0005 mg/kg to about 0.001 mg/kg, about 0.001 mg/kg to about 0.002 mg/kg, about 0.002 mg/kg to about 0.003 mg/kg, about 0.003 mg/kg to about 0.004 mg/kg, about 0.004 mg/kg to about 0.005 mg/kg, about 0.005 mg/kg to about 0.01 mg/kg, about 0.01 mg/kg to about 0.02 mg/kg, about 0.02 mg/kg to about 0.03 mg/kg, about 0.03 mg/kg to about 0.04 mg/kg, about 0.04 mg/kg to about 0.05 mg/kg, about 0.05 mg/kg to about 0.1 mg/kg, about 0.1 mg/kg to about 0.2 mg/kg, about 0.2 mg/kg to about 0.3 mg/kg, about 0.3 mg/kg to about 0.4 mg/kg, about 0.4 mg/kg to about 0.5 mg/kg, and/or about 0.5 mg/kg to about 1 mg/kg, and the antibody, or antigen-binding fragment thereof, is administered at a dosage of about 0.01 mg/kg to about 0.02 mg/kg, about 0.02 mg/kg to about 0.03 mg/kg, about 0.03 mg/kg to about 0.04 mg/kg, about 0.04 mg/kg to about 0.05 mg/kg, about 0.05 mg/kg to about 0.1 mg/kg, about 0.1 mg/kg to about 0.2 mg/kg, about 0.2 mg/kg to about 0.3 mg/kg, about 0.3 mg/kg to about 0.4 mg/kg, about 0.4 mg/kg to about 0.5 mg/kg, about 0.5 mg/kg to about 1 mg/kg, about 1 mg/kg to about 2 mg/kg, about 2 mg/kg to about 3 mg/kg, about 3 mg/kg to about 4 mg/kg, about 4 mg/kg to about 5 mg/kg, about 5 mg/kg to about 10 mg/kg, about 10 mg/kg to about 15 mg/kg, about 15 mg/kg to about 20 mg/kg, about 20 mg/kg to about 25 mg/kg, about 25 mg/kg to about 30 mg/kg, about 30 mg/kg to about 35 mg/kg, about 35 mg/kg to about 40 mg/kg, about 40 mg/kg to about 45 mg/kg, about 45 mg/kg to about 50 mg/kg, about 50 mg/kg to about 60 mg/kg, about 60 mg/kg to about 70 mg/kg, about 70 mg/kg to about 80 mg/kg, about 80 mg/kg to about 90 mg/kg, and/or about 90 mg/kg to about 100 mg/kg.

In a further embodiment, the compound and the antibody, or antigen-binding fragment thereof, are administered at a dosage of compound of 0.007 mg to 70 mg and antibody, or antigen-binding fragment thereof, of 0.7 mg to 7000 mg. For example, in a further embodiment, the compound is administered at a dosage of about 0.007 mg to about 0.01 mg, about 0.01 mg to about 0.02 mg, about 0.02 mg to about 0.03 mg, about 0.03 mg to about 0.04 mg, about 0.04 mg to about 0.05 mg, about 0.05 mg to about 0.1 mg, about 0.1 mg to about 0.2 mg, about 0.2 mg to about 0.3 mg, about 0.3 mg to about 0.4 mg, about 0.4 mg to about 0.5 mg, about 0.5 mg to about 1 mg, about 1 mg to about 2 mg, about 2 mg to about 3 mg, about 3 mg to about 4 mg, about 4 mg to about 5 mg, about 5 mg to about 10 mg, about 10 mg to about 20 mg, about 20 mg to about 30 mg, about 30 mg to about 40 mg, about 40 mg to about 50 mg, about 50 mg to about 60 mg, and/or about 60 mg to about 70 mg, and the antibody, or antigen-binding fragment thereof, is administered at a dosage of about 0.7 mg to about 1 mg, about 1 mg to about 2 mg, about 2 mg to about 3 mg, about 3 mg to about 4 mg, about 4 mg to about 5 mg, about 5 mg to about 10 mg, about 10 mg to about 20 mg, about 20 mg to about 30 mg, about 30 mg to about 40 mg, about 40 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 500 mg, about 500 mg to about 1000 mg, about 1000 mg to about 1500 mg, about 1500 mg to about 2000 mg, about 2000 mg to about 2500 mg, about 2500 mg to about 3000 mg, about 3000 mg to about 3500 mg, about 3500 mg to about 4000 mg, about 4000 mg to about 4500 mg, about 4500 mg to about 5000 mg, about 5000 mg to about 5500 mg, about 5500 mg to about 6000 mg, about 6000 mg to about 6500 mg, and/or about 6500 mg to about 7000 mg. In a further embodiment, the compound and the antibody, or antigen-binding fragment thereof, are administered in a molar ratio and/or dosage as described herein once every week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks for a period of one week to one year, such as a period of one week, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months.

In a further embodiment, the present disclosure provides a therapeutically effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and the antibody, or antigen-binding fragment thereof, for use in therapy. The compound of Formula (I), or a pharmaceutically acceptable salt thereof, and the antibody, or antigen-binding fragment thereof, can be used in treating or preventing a disease or disorder selected from a cancer, an inflammatory disease, an autoimmune disease, a viral infection, or a bacterial infection.

In a further embodiment, the present disclosure provides a therapeutically effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and the antibody, or antigen-binding fragment thereof, for the manufacture of a medicament. The medicament can be used in treating or preventing a disease or disorder selected from a cancer, an inflammatory disease, an autoimmune disease, a viral infection, or a bacterial infection.

In a further embodiment, the disease or disorder is mediated by chemokine receptor 2 (CCR2) and/or is associated with CCR2-positive pathogenic cells. In a further embodiment, CCR2-positive cell types are identified by testing for expression of CCR2 such as by immunohistochemistry or flow cytometry.

In a further embodiment, the disease or disorder is mediated by C-X-C motif chemokine receptor 3 (CXCR3) and/or is associated with CXCR3-positive pathogenic cells. In a further embodiment, CXCR3-positive cell types are identified by testing for expression of CXCR3 such as by immunohistochemistry or flow cytometry.

In a further embodiment, the disease or disorder is mediated by PSMA and/or is associated with PSMA-positive pathogenic cells. In a further embodiment, PSMA-positive cell types are identified by testing for expression of PSMA such as by immunohistochemistry or flow cytometry.

In a further embodiment, the disease or disorder is mediated by integrin aVp6 and/or is associated with integrin aVp6-positive pathogenic cells. In a further embodiment, integrin aVp6-positive cell types are identified by testing for expression of integrin aVp6 such as by immunohistochemistry or flow cytometry. In a further embodiment, the disease or disorder is mediated by folate receptor a (FRα) and/or folate receptor β (FRβ) and/or is associated with FRα- and/or FRβ-positive pathogenic cells. In a further embodiment, FRα- and/or FRβ-positive cell types are identified by testing for expression of FRα and/or FRβ such as by immunohistochemistry or flow cytometry.

In a further embodiment, the disease or disorder is mediated by fibroblast activation protein (FAP) and/or is associated with FAP-positive pathogenic cells. In a further embodiment, FAP-positive cell types are identified by testing for expression of FAP such as by immunohistochemistry or flow cytometry.

In a further embodiment, the disease or disorder is mediated by chemokine receptor 8 (CCR8) and/or is associated with CCR8-positive pathogenic cells. In a further embodiment, CCR8-positive cell types are identified by testing for expression of CCR8 such as by immunohistochemistry or flow cytometry.

In a further embodiment, the disease or disorder is a cancer selected from lung cancer (e.g., non-small cell lung cancer (NSCLC)), hepatocellular carcinoma (HCC), colorectal cancer (CRC), cervical cancer (e.g., cervical squamous cell carcinoma (CESC)), head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSC)), pancreatic cancer, prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), ovarian cancer, endometrial cancer, brain cancer, endocrine cancer, testicular cancer, bladder cancer, bone cancer, esophogeal cancer, gastric cancer, renal cell cancer, melanoma cancer, thyroid cancer, or breast cancer, preferably a cancer selected from mCRPC, breast cancer, lung cancer, colorectal cancer, or renal cell cancer.

In a further embodiment, the disease or disorder is a solid tumor. In a further embodiment, the disease or disorder is a solid tumor selected from lung cancer (e.g., NSCLC), HCC, CRC, cervical cancer (e.g., CESC), head and neck cancer (e.g., HNSC), pancreatic cancer, prostate cancer (e.g., mCRPC), ovarian cancer, endometrial cancer, brain cancer, endocrine cancer, testicular cancer, bladder cancer, bone cancer, esophogeal cancer, gastric cancer, renal cell cancer, melanoma cancer, thyroid cancer, or breast cancer, preferably a solid tumor selected from mCRPC, breast cancer, lung cancer, colorectal cancer, or renal cell cancer.

In a further embodiment, the disease or disorder is a PD-1 relapsed or refractory cancer, such as a PD-1 relapsed or refractory lung cancer (e.g., NSCLC), HCC, CRC, cervical cancer (e.g., CESC), head and neck cancer (e.g., HNSC), pancreatic cancer, prostate cancer (e.g., mCRPC), ovarian cancer, endometrial cancer, brain cancer, endocrine cancer, testicular cancer, bladder cancer, bone cancer, esophogeal cancer, gastric cancer, renal cell cancer, melanoma cancer, thyroid cancer, or breast cancer, preferably a PD-1 relapsed or refractory breast cancer, lung cancer, head and neck cancer, or cervical cancer. In a further embodiment, the disease or disorder is a non-solid cancer. In a further embodiment, the disease or disorder is a leukemia, a lymphoma, or a myeloma.

In a further embodiment, the disease or disorder is a viral infection. In a further embodiment, the viral infection is caused by an influenza virus, a coronavirus (e.g., COVID- 19), or a hepatitis B virus.

In a further embodiment, the disease or disorder is a bacterial infection. In a further embodiment, the bacterial infection is a chronic bacterial infection.

In a further embodiment, the disease is an autoimmune or inflammatory disease selected from vitiligo and type I diabetes.

In one embodiment, the present disclosure provides a method of increasing antibody- dependent cell cytotoxicity (ADCC) of target-expressing cells comprising contacting the cells with an effective amount of the compound of Formula (I), or pharmaceutically acceptable salt thereof, and an anti-cotinine antibody, or antigen-binding fragment thereof, wherein the target- binding moiety of the compound binds the target expressed on the cells.

In one embodiment, the present disclosure provides a method of increasing antibody dependent cellular phagocytosis (ADCP) of target-expressing cells comprising contacting the cells with an effective amount of the compound of Formula (I), or pharmaceutically acceptable salt thereof, and an anti-cotinine antibody, or antigen-binding fragment thereof, wherein the target-binding moiety of the compound binds the target expressed on the cells.

In one embodiment, the present disclosure provides a method of increasing complement dependant cytotoxicity (CDC) of target-expressing cells comprising contacting the cells with an effective amount of the compound of Formula (I), or pharmaceutically acceptable salt thereof, and an anti-cotinine antibody, or antigen-binding fragment thereof, wherein the target-binding moiety of the compound binds the target expressed on the cells.

In one embodiment, the present disclosure provides a method of conditioning a patient for therapy with a chimeric antigen receptor (CAR) T cell therapy or a CAR NK cell therapy, comprising administering to a patient an effective amount of the compound of Formula (I), or pharmaceutically acceptable salt thereof, and an anti-cotinine antibody, or antigen-binding fragment thereof. In some embodiments, the compound of Formula (I), or pharmaceutically acceptable salt thereof, and an anti-cotinine antibody, or antigen-binding fragment thereof, are administered in combination with the CAR-T or CAR-NK cell therapy. A compound of Formula (I), or pharmaceutically acceptable salt thereof, and an anti-cotinine antibody, or antigen- binding fragment thereof, may be administered as a conditioning therapy or combination therapy to improve efficacy in treatment of solid tumor cancers. In other embodiments, a compound of Formula (I), or pharmaceutically acceptable salt thereof, and an anti-cotinine antibody, or antigen-binding fragment thereof, may be administered as a neoadjuvant treatment for other therapies, including but not limited to immunotherapy, surgical resection, radiation, and/or chemotherapy.

In one embodiment, the present disclosure provides method of increasing cell killing of target-expressing cells comprising: contacting the cells with an effective amount of compound of Formula (I), or pharmaceutically acceptable salt thereof, and an anti-cotinine antibody, or antigen-binding fragment thereof, wherein the target-binding moiety of the compound bind the targets expressed on the cells.

In one embodiment, the present disclosure provides a method of depleting target- expressing cells comprising: contacting the cells with an effective amount of a compound of Formula (I), or pharmaceutically acceptable salt thereof, and an anti-cotinine antibody, or antigen-binding fragment thereof, wherein the target-binding moiety of the compound bind the targets expressed on the cells.

In a further embodiment, the target-expressing cells are CCR2-expressing cells. In a further embodiment, the CCR2-expressing cells are myeloid-derived suppressor cells (MDSCs), T regulatory cells (Tregs), neutrophils, macrophages, B regulatory cells (Bregs), CD8 regulatory cells, (CD8regs), exhausted T cells, or cancer-associated fibroblasts (CAFs).

In a further embodiment, the target-expressing cells are CXCR3-expressing cells. In a further embodiment, the CXCR3-expressing cells are activated T cells, autoreactive T cells, T regulatory cells (Tregs), CD4 regulatory T cells (CD4regs), CD8 regulatory T cells, (CD8regs), T helper (Th) T cells, Th1 T cells, natural killer T (NKT) cells, natural killer (NK) cells, dendritic cells, B cells, ybT cells, or tumor cells.

In a further embodiment, the target-expressing cells are PSMA-expressing cells. In a further embodiment, the PSMA-expressing cells are tumor cells.

In a further embodiment, the target-expressing cells are integrin aVp6-expressing cells. In a further embodiment, the integrin aVp6-expressing cells are tumor cells.

In a further embodiment, the target-expressing cells are FRα- and/or FRβ-expressing cells. In a further embodiment, the FRα- and/or FRβ-expressing cells are myeloid derived suppressor cells (MDSCs), macrophages, B cells, or tumor cells.

In a further embodiment, the target-expressing cells are FAP-expressing cells. In a further embodiment, the FAP-expressing cells are cancer-associated fibroblasts (CAFs), macrophages, or tumor cells.

In a further embodiment, the target-expressing cells are CCR8-expressing cells. In a further embodiment, the CCR8-expressing cells are T regulatory cells (Tregs) or tumor cells.

In a further embodiment, the target-expressing cells are pathogenic cells. In a further embodiment, the pathogenic cell is a pathogenic immune cell, a tumor cell or cancer cell, or a stromal cell.

In a further embodiment, the pathogenic immune cells are monocytes, myeloid derived suppressor cells (MDSC), such as monocytic MDSCs (mMDSCs) and polymorphonuclear MDSCs (PMN_MDSCs), T regulatory cells (Tregs), neutrophils (e.g., N2 neutrophils), macrophages (e.g., M2 macrophages), B regulatory cells (Bregs, memory B cells), plasma cells, CD8 cells (e.g., CD8 regulatory cells (CD8regs), memory CD8 cells, effector CD8 cells, naive CD8 Tcells, TEMRA), exhausted T cells, eosinophils, basophils, mast cells, dendritic cells, natural killer (NK) cells, innate lymphoid cells, NK T cells (NKT), or ybT cells.

In a further embodiment, the pathogenic immune cells are myeloid derived suppressor cells (MDSC), such as monocytic MDSCs (mMDSCs) and polymorphonuclear MDSCs (PMN_MDSCs), T regulatory cells (Tregs), neutrophils (e.g., N2 neutrophils), macrophages (e.g., M2 macrophages), B regulatory cells (Bregs), CD8 regulatory cells (CD8regs), exhausted T cells.

In a further embodiment, the tumor cells or cancer cells are solid tumor cells.

Combination Therapies

The compounds of the invention may be employed alone or in combination with other therapeutic agents. Combination therapies according to the present invention thus comprise the administration of at least one compound of Formula (I) or a pharmaceutically acceptable salt thereof, and the use of at least one other pharmaceutically active agent. The compounds of the invention and the other pharmaceutically active agents may be administered together in a single pharmaceutical composition or separately and, when administered separately this may occur simultaneously or sequentially in any order. The amounts of the compounds of the invention and the other pharmaceutically active agents and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect.

It will be appreciated that when the compound of the present invention is administered in combination with one or more other therapeutically active agents normally administered by the inhaled, intravenous, oral, intranasal, ocular topical or other route, that the resultant pharmaceutical composition may be administered by the same route. Alternatively, the individual components of the composition may be administered by different routes.

In one embodiment, the compounds and pharmaceutical composition disclosed herein are used in combination with, or include, one or more additional therapeutic agents. In a further embodiment, the additional therapeutic agent is a checkpoint inhibitor or an immune modulator.

In a further embodiment, the checkpoint inhibitor is selected from a PD-1 inhibitor (e.g., an anti-PD-1 antibody including, but not limited to, pembrolizumab, nivolumab, cemiplimab, or dostarlimab), a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody including, but not limited to, atezolizumab, avelumab, or durvalumab), or a CTLA-4 inhibitor (e.g. , an anti-CTLA-4 antibody including, but not limited to, ipilimumab or tremilumumab).

In a further embodiment, the checkpoint inhibitor is selected from a CD226 axis inhibitor, including but not limited to a TIGIT inhibitor (e.g., an anti-TIGIT antibody), a CD96 inhibitor (e.g., an anti-CD96 antibody), and/or a PVRIG inhibitor (e.g., an anti-PVRIG antibody).

In a further embodiment, the immune modulator is an ICOS agonist (e.g., an anti-ICOS antibody including, but not limited to feladilimab), a PARP inhibitor (e.g., niraparib, olaparib), or a STING agonist.

Pharmaceutical Compositions, Dosages, and Dosage Forms

For the purposes of administration, in certain embodiments, the ARMs described herein are administered as a raw chemical or are formulated as pharmaceutical compositions. Pharmaceutical compositions disclosed herein include an ARM and one or more of: a pharmaceutically acceptable carrier, diluent or excipient. An ARM is present in the composition in an amount which is effective to treat a particular disease, disorder or condition of interest. The activity of the ARM can be determined by one skilled in the art, for example, as described in the biological assays described below. Appropriate concentrations and dosages can be readily determined by one skilled in the art. In certain embodiments, the ARM is present in the pharmaceutical composition in an amount from about 25 mg to about 500 mg. In certain embodiments, the ARM is present in the pharmaceutical composition in an amount of about 0.01 mg to about 300 mg. In certain embodiments, the ARM is present in the pharmaceutical composition in an amount of about 0.01 mg, 0.1 mg, 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg or about 500 mg.

Administration of the compounds of the invention, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, is carried out via any of the accepted modes of administration of agents for serving similar utilities. The pharmaceutical compositions of the invention are prepared by combining a compound of the invention with an appropriate pharmaceutically acceptable carrier, diluent or excipient, and in specific embodiments are formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Exemplary routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral (e.g., intramuscular, subcutaneous, intravenous, or intradermal), sublingual, buccal, rectal, vaginal, and intranasal. Pharmaceutical compositions of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient.

Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound of the invention in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia. College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest in accordance with the teachings described herein.

The pharmaceutical compositions disclosed herein are prepared by methodologies well known in the pharmaceutical art. For example, in certain embodiments, a pharmaceutical composition intended to be administered by injection is prepared by combining a compound of the invention with sterile, distilled water so as to form a solution. In some embodiments, a surfactant is added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the compound of the invention so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.

Traditional antibody therapeutics have several disadvantages that are addressed by the ARMs approach described herein including difficulties in managing adverse events via adjusting dose and dose frequency of administration, challenges in generating antibodies to certain classes of drug targets (e.g., GPCRs, ion channels, and enzymes), and a new cell line for development is required for each new antibody which can be slow and costly. Moreover, different formats of biologies (e.g., bispecifics) can be challenging to manufacture. In contrast, the ARMs approach provides the following advantages: uniting the pharmacology of antibodies with the dose-control of small molecules, dose controlled PK/PD allowing temporal cell depletion, simpler multimerization, and rapid reversal of cell depletion through dosing of the antibody-binding component (e.g., cotinine hapten) which can uncouple therapeutic effects from potential adverse events.

EXAMPLES

The following examples illustrate the invention. These Examples are not intended to limit the scope of the invention, but rather to provide guidance to the skilled artisan to prepare and use the compounds, compositions, and methods of the invention. While particular embodiments of the invention are described, the skilled artisan will appreciate that various changes and modifications can be made. References to preparations carried out in a similar manner to, or by the general method of, other preparations, may encompass variations in routine parameters such as time, temperature, workup conditions, minor changes in reagent amounts etc. Chemical names for all title compounds were generated using ChemDraw Plugin version 16.0.1.13c (90) or ChemDraw desktop version 16.0.1.13 (90). A person of ordinary skill in the art will recognize that compounds of the invention may have alternative names when different naming software is used.

COMPOUND SYNTHESIS

The compounds according to Formula (I) are prepared using conventional organic synthetic methods. A suitable synthetic route is depicted below in the following general reaction schemes. All the starting materials are commercially available or are readily prepared from commercially available starting materials by those of skill in the art. The skilled artisan will appreciate that if a substituent described herein is not compatible with the synthetic methods described herein, the substituent may be protected with a suitable protecting group that is stable to the reaction conditions. The protecting group may be removed at a suitable point in the reaction sequence to provide a desired intermediate or target compound. Suitable protecting groups and the methods for protecting and de-protecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which may be found in T. Greene and P. Wuts, Protecting Groups in Organic Synthesis (4th ed.), John Wiley & Sons, NY (2006). In some instances, a substituent may be specifically selected to be reactive under the reaction conditions used. Under these circumstances, the reaction conditions convert the selected substituent into another substituent that is either useful as an intermediate compound or is a desired substituent in a target compound.

Scheme 1

Intermediate 1 : (2S,3S)-1-Methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid

Commercially available, racemic trans-4-cotininecarboxylic acid (304 g, 1.38 mol) was purified by chiral prep HPLC (61 injections) on Chiralpak 1A 20u 101 x 210 mm at 500 mL/min eluting with 50 % acetonitrile in methanol containing 0.1 % formic acid. The desired fractions were collected and were concentrated at 45 °C. The solid residue was stirred in acetonitrile, was filtered, and was dried under reduced pressure for 18 h to provide the title compound as a 10 white solid (143.6 g, 652 mmol, 94.5 % yield). Analytical chiral HPLC: 95 % ee at ret. time 2.5 min, Chiralpak AD-H 5u 4.6 x 150 mm, methanol with 0.1 % formic acid at 1.0 mL/min; Alpha D = + 58 deg (c=0.3, CH3OH); VCD analysis was used to assign absolute stereochemistry. LCMS m/z 221.1 (M+H)+. ¹ H NMR (400 MHz, DMSO-d₆) δ ppm 2.48 - 2.49 (m, 2 H) 2.53 - 2.61 (m, 1 H) 2.71 - 2.80 (m, 1 H) 3.06 - 3.15 (m, 1 H) 3.34 (br s, 1 H) 4.79 (d, J=6.3 Hz, 1 H) 7.35 15 - 7.57 (m, 1 H) 7.74 (dt, J=7.9, 2.0 Hz, 1 H) 8.54 (d, J=1.8 Hz, 1 H) 8.57 (dd, J=4.7, 1.7 Hz, 1 H) 12.78 (br s, 1 H).

Intermediate 2: (1R,4r)-4-(4-(((1 S,4R)-4-(2-((2S, 3S)-1 -Methyl -5-oxo-2-(pyri di n-3- yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- 20 carboxylic acid, Hydrochloride salt.

Step 1 : methyl (E)-4-(((1r,4r)-4-(2-Dibenzylamino)ethoxy)cyclohexyl)oxy)but-2- enoate.

To a stirred solution of (1 r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexan-1-ol (250 g, 736 mmol) in toluene (2500 mL) were added methyl but-2-ynoate (140 g, 1423 mmol), triphenylphosphine (19.32 g, 73.6 mmol), and acetic acid (16.86 mL, 295 mmol) at RT and the resulting solution was stirred at 115 °C for 16 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to the crude compound. The crude compound was adsorbed on silicagel (500 g, 60-120mesh), and purified by manual column chromatography(1.5 kg , 100-200 mesh) eluted with 15 % EtOAc in pet-ether to afford methyl (E&Z)-4-(((1 r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoate (350 g) as a mixture of E/Z isomers (52.48 % and 21.15 %). To separate both isomers, the compound was adsorbed on silica gel (500 g, 100-200 mesh), and purified by manual column chromatography (1.5 kg , 100-200 mesh) eluted with 15 % EtOAc in pet-ether to afford the title compound (240 g, 463 mmol, 62.9 % yield, 84.45% purity) as a pale yellow liquid. LC-MS m/z 438.3 (M+H)+. Step 2: (E)-4-(((1,4-trans)-4-(2-(Dibenzylamino)ethoxy)cyclohexyl)oxy)but-2- enoic acid

Methyl (E)-4-(((1 ,4-trans)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoate (9.03 g, 20.64 mmol) was dissolved in tetrahydrofuran (THF) (25 mL) and aqueous 5.089 Molar sodium hydroxide (4.87 mL, 24.76 mmol) was added. The homogenous pale-yellow reaction was heated at reflux for 1 hour. Additional 5.089 M sodium hydroxide (1.217 mL, 6.19 mmol) was added and the reaction was refluxed for 50 minutes. The reaction was cooled over 60 minutes and concentrated in vacuo. The residue was azeotroped twice with toluene in order to aid in removal of water. The residue was pumped under high vacuum to afford the title compound (9.9 g, 22.17 mmol, 107% yield, 82% purity, E/Z mixture) as a yellow solid. LC-MS m/z 424.4 (M+H)+.

Step 3: tert-butyl (1R,4r)-4-((E)-4-(((1r,4R)-4-(2- 15 (Dibenzylamino)ethoxy) cyclohexyl)oxy)but-2-enamido)cyclohexane-1 -carboxylate.

(E)-4-(((1 ,4-trans)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoic acid, sodium salt (9.2 g, 20.60 mmol) was suspended in dry DMF (40 ml) with stirring. HATU (8.62 g, 22.66 mmol) was added as a solid and a partially dissolved mixture was observed. The mixture was stirred for 30 minutes to give a partially dissolved greenish solution, tert-butyl (1 ,4-trans)-4- aminocyclohexane-1-carboxylate (4.11 g, 20.60 mmol) was added as a solution in DMF (10 ml) followed by addition of a solution of DIEA (10.80 mL, 61.8 mmol) in DMF (10 ml). An additional 10 ml of DMF was added and the heterogeneous mixture was stirred for 15 hours at room temperature. Additional HATU (1.724 g, 4.53 mmol) was added and the almost homogeneous reaction was stirred for 60 minutes. The cloudy reaction was stirred for an additional 60 minutes. The reaction was diluted with 200 ml of EtOAc and 200 ml of water and stirred for 10 minutes. The resulting homogeneous biphasic mixture was transferred to a separatory funnel and the layers were separated. The aqueous layer was extracted twice more with 150 ml EtOAc and the combined EtOAc layers were washed 4 x with water and 2 x with saturated NaCI in order to remove DMF. The EtOAc extracts were dried over sodium sulfate, filtered, and concentrated in vacuo, and pumped under high vacuum to give an orange syrup which was purified via silica-gel chromatography (Isco Combiflash, 330 gram gold column, 0- 80% EtOAc:heptane over 45 minutes, 150 ml/min flow rate, loaded as a solution in DCM) to give the title compound (4.55 g, 7.52 mmol, 36.5% yield) as a white foamy solid. LC-MS m/z 605.5 (M+H)+.

Step 4: tert-butyl (1R,4r)-4-(4-(((1r,4R)-4-(2-Aminoethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1 -carboxylate

A 500 ml 3-necked RB flask was charged with tert-butyl (1R,4r)-4-((E)-4-(((1r,4R)-4- (2- (dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enamido)cyclohexane-1-carboxylate (6.40 g, 10.58 mmol) and isopropanol (120 mL) and the suspension was stirred until a homogeneous solution was obtained. 10% wet Pd-C (0.640 g, 6.01 mmol) was added and the flask was evacuated and placed under 2 balloons of hydrogen attached to the end necks of the flask. The middle neck was covered with a rubber septum. The reaction was stirred at room temperature for 16 hours. The reaction was degassed and filtered 2 X through celite. The filtrate was concentrated in vacuo and pumped under high vacuum to give a waxy grey solid with a slight odor of isopropanol. The waxy solid was dissolved in DCM and concentrated in vacuo at 54 degrees C for 20 minutes in order to aid in removal of isopropanol. The residue was pumped under high vacuum to afford the title compound (4.44 g, 10.41 mmol, 98% yield) as a waxy grey solid. LC-MS m/z 427.4 (M+H)+. Step 5: tert-butyl (1R,4r)-4-(4-(((1 S,4R)-4-(2-((2S,3S)-1-Methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxylate.

(2S,3S)-1 -Methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (Intermediate 1) (2.287 g, 10.38 mmol) was suspended in 30 ml DCM with stirring in a 500 ml RB flask at room temperature. HATU (4.34 g, 11.42 mmol) was added and the suspension was stirred for 15 minutes. A solution of tert-butyl (1R,4r)-4-(4-(((1r,4R)-4-(2- aminoethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxylate (4.43 g, 10.38 mmol) in DCM (20 ml) was added dropwise via pipet over 15 minutes. After addition was complete, a solution of DIEA (5.44 mL, 31.2 mmol) in DCM (10 ml) was added dropwise over 10 minutes and the resulting homogeneous dark solution was stirred at room temperature for 16 hours. The reaction was concentrated in vacuo in order to remove DCM and DIEA. The residue was dissolved in 100 ml DCM and transferred to a separatory funnel. 20 ml of saturated sodium bicarbonate was added The layers were separated and the DCM layer was washed with saturated NaCI, dried over sodium sulfate, filtered, concentrated in vacuo, and pumped under high vacuum to give an orange syrup which was purified via silica gel chromatography (Isco Combiflash, 0-10% MeOH:DCM over 60 minutes, 330 gram gold column, 150 ml/min flow rate, loaded as a solution in 30 ml DCM) to afford the title compound (4.13 g, 6.57 mmol, 63.2% yield) as a white solid. LC-MS m/z 629.3 (M+H)+.

Step 6: (1R, 4r)-4-(4-((( 1 S,4R)-4-(2-((2S,3S)-1 -Methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3- carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxylic acid, Hydrochloride salt

tert-Butyl (1R,4r)-4-(4-(((1 S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3- carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate (4.13 g, 6.57 mmol) was dissolved in dry 1,4-dioxane (13 ml) with stirring in a 250 ml RB flask. 4 M anhydrous HCI (39 mL, 156 mmol) in 1 ,4-dioxane was added and the mixture was stirred at room temperature. Upon HCI addition, an insoluble oil was observed. The mixture was stirred for 90 minutes at room temperature. The reaction was concentrated in vacuo (at 60 degrees bath temperature) and pumped under high vacuum for 15 hours to afford the title compound (4.187 g, 6.87 mmol, 105% yield) as a white solid. LC-MS m/z 573.4 (M+H)+. 1 H NMR (400 MHz, DMSO-d6) 5 1.09 - 1.37 (m, 10H), 1.58 - 1.70 (m, 2H), 1.73 - 1.91 (m, 7H), 2.03 - 2.16 (m, 3H), 2.43 - 2.49 (m, 1 H), 2.72 (dd, J = 16.9, 9.5 Hz, 1 H), 2.95 - 3.05 (m, 1 H), 3.08 - 3.23 (m, 4H), 3.27 - 3.48 (m, 5H), 3.55 - 3.57 (m, 3H), 4.81 (d, J = 5.9 Hz, 1 H), 7.84 - 7.92 (m, J = 4.9 Hz, 1 H), 8.05 (br t, J = 5.9 Hz, 1 H), 8.19 - 8.28 (m, 1 H), 8.71 - 8.76 (m, 1 H), 8.76 - 8.83 (m, 1 H).

Intermediate 3: Methyl (1R,4r)-4-(4-(((1r,4R)-4-(2- aminoethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate.

Step 1. Sodium (E)-4-(((1 r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2- enoate (Intermediate 3A).

Methyl (E)-4-(((1 r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoate (5.02 g, 11.47 mmol) was dissolved in THF (14.5 mL) and aqueous 5.089M sodium hydroxide (3.38 ml, 17.21 mmol) was added. The unstirred mixture was 2 distinct layers at room temperature. The pale yellow reaction was heated at 80 °C for 18 h, then cooled to RT and concentrated in vacuo. The resultant residue was azeotroped with toluene (2x 50 mL) and dried on high vac to provide the title compound as a stickly yellow solid (6.16 g, 12.3 mmol, theoretical). LCMS m/z 424.2 (M+H)⁺. ¹ H NMR (400 MHz, DMSO-d₆) δ ppm 1.07 - 1.26 (m, 4 H) 1.77 - 1.93 (m, 4 H) 2.53 - 2.58 (m, 2 H) 3.15 - 3.28 (m, 2 H) 3.45 - 3.53 (m, 2 H) 3.56 - 3.65 (m, 4 H) 3.89 - 4.01 (m, 2 H) 5.68 - 5.77 (m, 1 H) 6.09 - 6.26 (m, 1 H) 7.12 - 7.20 (m, 6 H) 7.21 - 7.29 (m, 6 H) 7.30 - 7.39 (m, 8 H).

Step 2. Methyl (1R,4r)-4-((E)-4-(((1r,4R)-4-(2-(dibenzylamino)ethoxy)cyclohexyl) oxy)but-2-enamido)cyclohexane-1-carboxylate (Intermediate 3B)

To a solution of sodium (E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but- 2-enoate (2.25g, 5.05 mmol), methyl (1r,4r)-4-aminocyclohexane-1 -carboxylate hydrochloride (1.39 g, 7.18 mmol), and HATU (2.69 g, 7.07 mmol) in DCM (20 mL) was added DIEA (2.65 ml, 15.2 mmol). The reaction was stirred at RT for 3 nights, then diluted with 10% methanol in DCM (100 mL) and aqueous saturated sodium bicarbonate (50 mL). The layers were separated, and the aqueous layer was back extracted with 10% methanol in DCM (2x 30 mL). The organic fractions were combined, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resultant residue was purified via silica gel chromatography eluting with 0-100% ethyl acetate in heptanes to provide the title compound as an off-white solid (1.94 g, 3.24 mmol, 64.1% yield). LCMS m/z 563.34 (M+H)⁺. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.10 - 1.23 (m, 2H) 1.30 - 1.37 (m, 3H) 1.51 - 1.67 (m, 2 H) 1.91 - 2.15 (m, 10 1-1) 2.21 - 2.32 (m, 1 H) 2.62 - 2.71 (m, 2 H) 3.20 - 3.29 (m, 1 H) 3.29 - 3.39 (m, 1 H) 3.51 - 3.60 (m, 2 H) 3.66 (s, 4 H) 3.69 (s, 3 H) 3.78 - 3.93 (m, 1 H) 4.12 - 4.17 (m, 1 H) 5.37 (br d, J=7.83 Hz, 1 H) 6.01 (dt, J=15.28, 2.14 Hz, 1 H) 6.82 - 6.90 (m, 1 H) 7.20 - 7.27 (m, 2 H) 7.29 - 7.35 (m, 4 H) 7.36 - 7.42 (m, 4 H).

Step 3. Methyl (1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1 -carboxylate (Intermediate 3).

To a solution of methyl (1R,4r)-4-((E)-4-(((1r,4R)-4-(2- (dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enamido)cyclohexane-1-carboxylate (1.94g, 3.44 mmol) under an atmosphere of nitrogen was added Pd-C (10 % wt, 0.366 g, 0.344 mmol). The flask was evacuated, back-filled with a hydrogen gas balloon and stirred at RT overnight for 32 h. The flask was again evaucuated and back-filled with a fresh hydrogen gas balloon. After 22 h, the mixture was filtered through a celite pad, washing with additional methanol (2x 20 mL). The filtrate was concentrated in vacuo and dried on high vac to provide the title compound as an off-white solid (1.19 g, 3.08 mmol, 89% yield). LCMS m/z 563.34 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.06 - 1.26 (m, 4 H) 1.29 - 1.45 (m, 2 H) 1.59 - 1.71 (m, 2 H) 1.74 - 1.82 (m, 2 H) 1.83 - 1.95 (m, 4 H) 2.02 - 2.12 (m, 2 H) 2.19 - 2.31 (m, 1 H) 2.58 - 2.64 (m, 1 H) 3.13 - 3.28 (m, 4 H) 3.29 - 3.38 (m, 8 H) 3.39 - 3.54 (m, 1 H) 3.59 (s, 3 H) 4.04 - 4.15 (m, 1 H) 7.66 (d, J=7.82 Hz, 1 H).

Intermediate 4: Benzyl (1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1 -carboxylate

Step 1 : Methyl 4-(((1r,4r)-4-(2-minoethoxy)cyclohexyl)oxy)butanoate (Intermediate 4A)

To a solution of methyl (E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but- 2-enoate (5.00 g, 11.4 mmol) under an atmosphere of nitrogen gas was added Pd-C (0.608 g, 0.571 mmol). The flask was evacuated and back-filled with a hydrogen gas balloon and stirred at RT for 3 nights. The flask was then evacuated and back-filled nitrogen gas. Additional Pd-C (0.608 g, 0.571 mmol) was added. The flask was again evacuated and back- filled with a fresh hydrogen gas balloon. The reaction was stirred at RT for 24 h, then filtered through a pad of celite. To the filtrate was added Pd-C (0.608 g, 0.571 mmol). The flask was evacuated, back-filled with a fresh hydrogen gas balloon, and stirred at RT for 22 h.

Additional Pd-C (0.608 g, 0.571 mmol was added. The flask was evacuated and back- filled with a fresh hydrogen gas balloon. After another 24 h, additional Pd-C (0.608 g, 0.571 mmol) was added. The flask was again evacuated and back-filled with a hydrogen gas balloon. After stirring 5 h at RT, the mixture was filtered through a pad of celite, washing with additional methanol (2x 20 mL). The filtrate was concentrated in vacuo and dried on high vac to afford the title compound as a yellow semi-solid (5.22 g, 10.1 mmol, 88% yield). LCMS m/z 260.28 (M+H)⁺. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.17 - 1.42 (m, 4 H) 1.84 - 1.93 (m, 2 H) 1.94 - 2.05 (m, 4 H) 2.35 - 2.50 (m, 2 H) 2.84 - 2.93 (m, 2 H) 3.19 - 3.37 (m, 2 H) 3.41 - 3.57 (m, 4 H) 3.69 (s, 3 H). Step 2: Methyl 4-(((1r,4r)-4-(2-((tert-butoxycarbonyl)amino)ethoxy)cyclohexyl) oxy)butanoate (Intermediate 4B)

To a solution of methyl 4-(((1r,4r)-4-(2-minoethoxy)cyclohexyl)oxy)butanoate (1.20 g, 4.63 mmol) in dichloromethane (23 mL) was added di-tert-butyl dicarbonate (1.52 g, 6.94 mmol) and DIPEA (1.616 ml, 9.25 mmol). The mixture was stirred at RT for 19 h, then diiluted with additional dichloromethane (50 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (30 mL) and brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated The resultant residue was purified via silica gel chromatography eluting with 0-10% methanol in dichloromethane to afford the title compound as a yellow oil (1.22 g, 3.00 mmol, 88% yield). LCMS m/z 360.14 (M+H)⁺. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.41 - 1.51 (m, 9 H) 1.85

- 1.93 (m, 3 H) 1.94 - 2.05 (m, 5 H) 2.26 - 2.33 (m, 1 H) 2.42 (t, J=7.34 Hz, 2 H) 3.17 - 3.34 (m, 4 H) 3.36 - 3.60 (m, 6 H) 3.69 (s, 3 H).

Step 3: Sodium 4-(((1r,4r)-4-(2-((tert-butoxycarbonyl)amino)ethoxy) cyclohexyl)oxy)butanoate (1.23 g, 3.35 mmol), benzyl (1 r,4r)-4-aminocyclohexane-1- carboxylate hydrochloride (Intermediate 4C)

To a solution of methyl 4-(((1r,4r)-4-(2-(tert -butoxycarbonyl)amino)ethoxy) cyclohexyl)oxy)butanoate (1.21 g, 3.36 mmol) in THF (13.4 mL) was added aqueous 5.089 Molar sodium hydroxide (0.990 ml, 5.04 mmol). The mixture was stirred at RT for 18 h, then concentrated in vacuo and dried on high vac to provide the title compound as a pale yellow solid (1.234 g, 3.36 mmol, theoretical yield). LCMS m/z 346.18 (M+H)⁺. ¹ H NMR (400 MHz, DMSO-d₆) δ ppm 1.34 - 1.41 (m, 9 H) 1.53 - 1.68 (m, 4 H) 1.77 - 1.93 (m, 6 H) 2.06 - 2.12 (m, 1 H) 2.96 - 3.06 (m, 2 H) 3.29 - 3.41 (m, 7 H). Step 4: Benzyl (1R,4r)-4-(4-(((1r,4R)-4-(2-((tert-butoxycarbonyl)amino)ethoxy) cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate (Intermediate 4D)

To a solution of sodium 4-(((1r,4r)-4-(2-((tert-butoxycarbonyl)amino)ethoxy) cyclohexyl)oxy)butanoate (1.23 g, 3.35 mmol), benzyl (1r,4r)-4-aminocyclohexane-1- carboxylate hydrochloride (0.903 g, 3.35 mmol), and HATU (1.782 g, 4.69 mmol) in DCM (13.4 mL) was added DIPEA (1.75 mL, 10.0 mmol). The mixture was stirred at RT for 3 nights, then diluted with additional DCM (100 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-15% methanol in dichloromethane to afford the title compound as a pale yellow oil. LCMS m/z 561.34 (M+H)⁺. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.42 - 1.53 (m, 9 H) 1.57 - 1.63 (m, 2 H) 1.84 - 1.92 (m, 2 H) 1.96 - 2.03 (m, 4 H) 2.03 - 2.12 (m, 4 H) 2.22 - 2.31 (m, 2 H) 3.16 - 3.25 (m, 4 H) 3.25 - 3.35 (m, 3 H) 3.44 - 3.56 (m, 4 H) 3.66 - 3.84 (m, 5 H) 4.81 - 4.95 (m, 1 H) 5.13 (s, 2 H) 5.58 - 5.65 (m, 1 H) 7.31 - 7.44 (m, 5 H).

Step 5: Benzyl (1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1 -carboxylate (Intermediate 4)

To a solution of benzyl (1R,4r)-4-(4-(((1r,4R)-4-(2-((ted-butoxycarbonyl)amino)ethoxy) cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate

(1.77g, 3.16 mmol) in DCM (12.6 mL) was added TFA (2.43 mL, 31.6 mmol). The mixture was stirred at RT 2 nights. The reaction was then concentrated, azeotroped with toluene (2x), and dried on high vac to afford the title compound as a pale yellow semi-solid (2.98 g, 1.81 mmol, theoretical yield. LCMS m/z 461.33 (M+H)⁺. ¹H NMR (400 MHz, DMSO- δ₆) δ ppm 1.07 - 1.30 (m, 28 H) 1.34 - 1.48 (m, 2 H) 1.59 - 1.73 (m, 2 H) 1.75 - 1.83 (m, 2 H) 1.85 - 1.98 (m, 4 H) 2.03 - 2.11 (m, 1 H) 2.92 - 2.98 (m, 1 H) 3.10 - 3.21 (m, 3 H) 3.30 - 3.37 (m, 2 H) 3.52 - 3.68 (m, 5 H) 5.09 (s, 2 H) 7.29

. 7.44 (m, 4 H) 7.61 - 7.75 (m, 2 H) 8.18 - 8.32 (m, 1 H).

Intermediate 5: (S)-1-((1S,2R,4R)-2-Amino-4-(isopropyl(methyl)amino) cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one, 2Hydrochloric acid salt.

Step 1 : Ethyl (1R,2S,5R)-2-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1-yl)-5- (isopropyl(methyl)amino)cyclohexane-1 -carboxylate

To a mixture of ethyl (1R,2S)-2-((S)-3-((benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1- yl)-5-oxocyclohexane-1-carboxylate (28.0 g, 69.6 mmol) and isopropyl methylamine (12.32 mL, 118 mmol) in dichloromethane (DCM) (270 mL) was added titanium(IV) isopropoxide (30.6 mL, 104 mmol) at rt. The mixture was stirred under an atmosphere of nitrogen at rt for 21 h. 5 % Pt/C (4.07 g, 1.044 mmol) was addd and the mixture was stirred under a balloon atmosphere of hydrogen at rt for 29 h. The hydrogen balloon was refilled after 7h and 16h. The mixture was filtered through Celite® and the catalyst was washed with dichloromethane (DCM). The combined filtrates were concentrated under reduced pressure. The residue was dissolved in dichloromethane (DCM) (80 mL), was placed in an ice bath, and ethyl acetate (250 mL) and Celite® (5 g) were added. The mixture was stirred at rt for4 h and was sonicated at rt for 20 min. The mixture was filtered through Celite® with wet ethyl acetate (4x 70 mL). The combined filtrates were concentrated under reduced pressure and were chased with dichloromethane (DCM) (3x 100 mL) to provide the title compound as a light-brown, oily foam (24.58 g, 77%). LC-MS m/z 460.5 (M+H)⁺.

Step 2: (1R,2S,5R)-2-((S)-3-(((Benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1-yl)- 5-(isopropyl(methyl)amino)cyclohexanecarboxylic acid.

Ethyl (1R,2S,5R)-2-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1-yl)-5- (isopropyl(methyl)amino)cyclohexane-1-carboxylate (24.58 g, 53.5 mmol) was dissolved in toluene (140 mL) and dichloromethane (DCM) (5 mL) and the solution was extracted with 2 N HCI (2x 67 mL). The combined aqueous extracts were placed in a metal insert under an atmosphere of nitrogen and heated at 63 °C for 22 h. The mixture was placed in an ice bath and 10N sodium hydroxide (32 mL) was added. The final temperature of the mixture was 16 °C. The mixture was washed with toluene (150 mL) and the aqueous phase was filtered. An aqueous emulsion (20 mL) was separated and was filtered through Celite®. The combined aqueous phases were cooled in an ice bath, and the pH was adjusted to 6 to 7 with concentrated HCI. The mixture was saturated with NaCI and was extracted with dichloromethane (DCM) (200 mL). Concentrated HCI (0.5 mL) was added and the aqueous phase was extracted with 10% methanol in dichloromethae (DCM) (2x 100 mL). The combined organic extracts were dried over Na₂SO₄, were filtered, and the filtrate was concentrated under reduced pressure to provide the title compound as a beige, foamy solid (14.18 g, 2.74 mmol, 61%). LC-MS m/z 432.4 (M+H)⁺.

Step 3: tert-Butyl ((1R,2S,5R)-2-((S)-3-(((benxyloxy)carbonyl)amino)-2- oxopyrrolidin-1-yl)-5-(isopropyl(methyl)amino)cyclohexyl)carbamate.

(1R,2S,5R)-2-((S)-3-(((Benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1-yl)-5- (isopropyl(methyl)amino)cyclohexanecarboxylic acid (14.18 g, 32.9 mmol) was chased with dichloromethane (DCM) (30 mL) and toluene (3x 330 mL). To a solution of the residue in anhydrous toluene (121 mL) under an atmosphere of nitrogen was added anhydrous tert- butanol (31.0 mL, 329 mmol) and triethylamine (16.0 mL, 115 mmol), and the mixture was heated in a metal insert at 85 °C for 5 min. DPPA (7.79 mL, 36.1 mmol) was added dropwise over 14 min and the mixture was heated under an atmosphere of nitrogen at 85 °C for 2.75 h. The mixture was cooled to rt, ethyl acetate (280 mL) was added, and the internal temp was adjusted to 2 to 3 °C using an ice bath. Saturated NaHCO₃ (280 mL) was added dropwise over a 20 min period and the mixture was stirred at rt for 2 h. The organic phase was washed with brine (40 mL), was dried over Na₂SO₄, was filtered, and the filtrate was concentrated under reduced pressure. The residue was placed under vacuum at rt for 2 days. tert-Butanol (100 mL) was added and the mixture was sonicated at rt for 20 min. 1 N sodium hydroxide (80 mL) was added, and the mixture was stirred at rt for 30 min, and was concentrated under reduced pressure to 80 mL of volume. Dichloromethane (DCM) (200 mL) and water (40 mL) were added, the organic phase was dried over Na₂SO₄, was filtered, and the filtrate was concentrated under reduced pressure. The residue was chased with ethyl acetate (2x 85 mL). The residue was stirred in ethyl acetate (30 mL) and heptane (40 mL), was collected by filtration, and was washed with 1 :10 ethyl acetate/heptane solution to provide the title compound as a white solid (8.03 g, 15.02 mmol, 48.6%). LC-MS m/z 503.5 (M+H)⁺.

Step 4: tert-Butyl ((1R,2S,5R)-2-((S)-3-amino-2-oxopyrrolidin-1-yl)-5- (isopropyl(methyl)amino)cyclohexyl)carbamate

To a solution of tert-butyl ((1R,2S,5R)-2-((S)-3-(((benxyloxy)carbonyl)amino)-2- oxopyrrolidin-1-yl)-5-(isopropyl(methyl)amino)cyclohexyl)carbamate (4.99 g, 9.93 mmol) in ethanol (70 mL) was added 20 wt% Pd(OH)₂/C (0.7 g, 0.993 mmol) and the mixture was stirred under a balloon atmosphere of hydrogen at rt for 23 h. The mixture was filtered through Celite® under an atmosphere of nitrogen, and the catalyst was washed with ethanol (3x 10 mL). The combined filtrates were concentrated under reduced pressure and the residue was placed under vacuum at rt overnight to provide the title compound as a white solid (3.64 g, 9.38 mmol, 99% yield). LC-MS m/z 369.4 (M+H)⁺. Step 5: tert-Butyl ((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamate

To a solution of tert -Butyl ((1R,2S,5R)-2-((S)-3-amino-2-oxopyrrolidin-1-yl)-5-

(isopropyl(methyl)amino)cyclohexyl)carbamate (3.64 g, 9.88 mmol) in ethanol (100 mL) was added commercially-available 4-chloro-6-(trifluoromethyl)quinazoline (2.30 g, 9.88 mmol) and DI PEA (2.77 mL, 15.84 mmol), and the mixture was heated under an atmosphere of nitrogen in a metal insert at 50 °C for 3h. The mixture was concentrated under reduced pressure., dichloromethane (DCM) (150 mL) was added, and the organic phase was washed with water (30 mL). The organic phase was dried over Na₂SO₄, was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by ISCO CombiFlash® Rf (220 g RediSep® Rf Gold column, 120 mL/min) eluting with a gradient of 0 to 75% DCM/DCM in MeOH with 1% NH₄OH and then wash the column with MeOH. The desired fractions were combined and dried under reduced pressure to provide the title compound as a white solid (4.32 g, 7.57 mmol, 77% yield). LC-MS m/z 565.2 (M+H)⁺

Step 6: (S)-1-((1 S,2R,4R)-2-Amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one, 2Hydrochloric acid salt.

To a mixture of tert-Butyl ((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamate (3.18 g, 5.63 mmol) in dichloromethane (DCM) (8 mL) was added HCl (7.04 mL, 28.2 mmol). The mixture was stirred at rt for 3h and was concentrated to dryness to provide the title compound as a white solid (3.5 g, 6.51 mmol, 116% yield). LC-MS m/z 465.3 (M+H)⁺. Intermediate 6: (S)-1-((1S,2R,4R)-2-Amino-4-(tert-butylamino)cyclohexyl)-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one.

Step 1 : Benzyl ((S)-1-((7R,8S)-7-acetamido-1,4-dioxaspiro=[4.5]decan-8-yl)-2- oxo pyrroIidin-3-yI)carbamate.

To a solution of (7R,8S)-8-((S)-3-(((benzyloxy)carbonyl)amino-2-oxopyrrolidin-1-yl)- 1 ,4-dioxaspiro[4.5]decane-7-carboxylic acid (1.5 g, 3.58 mmol) in toluene (15 mL) wa added triethylamine (0.500 L, 3.58 mmol) at rt. The mixture was cooled to -10 °C, isobutyl chloroformate (0.471 mL, 3.58 mmol) was added, and the mixture was stirred at 0 °C to -10 °C for 30 min. A solution of sodiium azide (0.419 g, 6.45 mmol) and tetrabutylammonium bromide (0.058 g, 0.179 mmol) in water (3.00 mL) was added, and the mixture was stirred at 0 °C to -10 °C for 2h. Water (50 mL) and toluene (100 mL) were added and the mixture was stirred for 10 min. The organic phase was dried over molecular sieves (4A), acetic anhydride (0.744 mL, 7.89 mmol) and acetic acid (0.267 mL, 4.66 mmol) were added, and the mixture was stirred at 90 °C for 4h. The mixture was cooled to rt and was concentrated under reduced pressure. The residue was triturated with pentane (20 mL) to provide the title compound as an off-white solid (0.8 g, 1.714 mmol, 47.8% yield). LC-MS m/z 432.2 (M+H)⁺.

Step 2: Benzyl ((S)-1-((1S,2R)-2-acetamido-4-oxocyclohexyl)-2-oxopyrrolidin-3- yl)carbamate.

To a solution of benzyl ((S)-1-((7R,8S)-7-acetamido-1 ,4-dioxaspiro=[4.5]decan-8-yl)- 2-oxopyrrolidin-3-yl)carbamate (800 mg, 1.854 mmol) in acetone (10 mL) was added HCI (5 mL, 5.00 mmol) and the mixture was stirred at 50 °C for 2h. The mixture was cooled to rt and was concentrated. Water (10 mL) was added and the mixture was extracted with dichloromethane (DCM) (2x 50 mL). The combined organic extracts were dried over anhydrous Na₂SO₄, were filtered, and the filtrate was concentrated. The residue was triturated with diethyl ether (10 mL) to provide the title compound as an off-white solid (600 mg, 1.490 mmol, 80% yield). LC-MS m/z 388.2 (M+H)+.

Step 3: Benzyl ((S)-1-((1 S,2R,4R)-2-acetamido-4-(tert-butylamino)cyclohexyl)-2- oxo py rro I i d i n-3-y I )ca rbam ate.

TiCI₂(i-O/Pr)₂ was pre-formed by adding titanium(IV) isopropoxide (0.282 mL, 0.964 mmol) to 1M TiCI₄ in dichloromethane (DCM) (0.964 mL, 0.964 mmol) at 5 - 10 °C and the mixture was stirred for 15 min. The pre-formed TiCI₂(i-O/Pr)₂ was added to a solution of benzyl ((S)-1-((1 S,2R)-2-acetamido-4-oxocyclohexyl)-2-oxopyrrolidin-3-yl)carbamate (600 mg, 1.607 mmol) and tert-butylamine (0.851 mL, 8.03 mmol) in dichloromethane (DCM) (10 mL) at -20 °C. The mixture was warmed to rt and stirred for 2h. Borane-dimethyl sulphide complex (0.153 mL, 1.607 mmol) was added and the mixture was stirred at rt for 16h. Dichloromethane (DCM) (50 mL) and water (50 mL) were added and the mixture was stirred for 10 min. The emulsion was filtered through Celite® and the aqueous phase was extracted with dichloromethane (DCM) (50 mL). 1N HCI (20 mL) was added to the combined organic extracts and the mixture wa stirred for 10 min. Dichloromethane (DCM) (50 mL) was added and the pH was adjusted to 8 to 9 with ammonium hydroxide solution. The organic phase was washed with ammonium chloride solution (14%) (2x 25 mL), was dried over anhydrous Na₂SO₄, was filtered, and the filtrate was evaporated. Purification by column chromatography (neutral alumina column) eluting with 2% methanol in dichloromethane (DCM) provided the title compound as an off- white solid (300 mg, 0.673 mmol, 41.9% yield). LC-MS m/z 445.48 (M+H)⁺. Step 4: N-((1R,2S,5R)-2-((S)-3-Amino-2-oxopyrrolidin-1-yl)-5-(tert-butylamino) cyclohexyl)acetamide.

A mixture of benzyl ((S)-1-((1 S,2R,4R)-2-acetamido-4-(tert-butylamino)cyclohexyl)-2- oxopyrrolidin-3-yl)carbamate (1.00 g, 2.249 mmol) and 10% Pd/C (100 mg, 0.094 mmol) in methanol (10 mL) was stirred under a balloon atmosphere of hydrogen at rt for 2h. The mixture was filtered through Celite®, the catalyst was washed with methanol, and the combined filtrates were concentrated under reduced pressure to provide the title compound as an off- white solid (696.1 mg, 2.242 mmol, 100% yield). LC-MS m/z 311.5 (M+H)⁺.

Step 5: N-(( 1R,2S,5R)-5-(tert-Butylamino)-2-((S)-3-((2-chloro-6-(trifluoromethyl) quinazolin-4-yl)amino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide.

A mixture of N-((1R,2S,5R)-2-((S)-3-amino-2-oxopyrrolidin-1-yl)-5-(tert- butylamino)cyclohexyl)acetamide (683 mg, 2.200 mmol), commercially-available 2,4-dichloro- 6-(trifluoromethyl)quinazoline (587 mg, 2.200 mmol), and DIPEA (0.615 mL, 3.52 mmol) in ethanol (20 mL) was stirred at rt for 2h, and then the mixture was concentrated under reduced pressure. Saturated NaHCO₃ was added and the mixture was extracted with ethyl acetate. The combined organic extracts were washed with saturated NaCI, were dried over Na₂SO₄, were filtered, and the filtrate was concentrated under reduced pressure to provide the title compound as a pale-yellow solid (1.128 g, 2.085 mmol, 95% yield). LC-MS m/z 541.5 (M+H)+. Step 6: N-((1R,2S,5R)-5-(tert-Butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl) quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)acetamide.

To a solution of N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-((2-chloro-6- (trifluoromethyl)quinazolin-4-yl)amino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide (4.61 g, 8.52 mmol) in methanol (150 mL) was added 10% Pd/C (1.360 g, 1.278 mmol) and Cs₂CO₃ (4.16 g, 12.78 mmol). The mixture was stirred under a balloon atmosphere of hydrogen at rt for 3h. The mixture was filtered and the catalyst was washed with methanol and with dichloromethane (DCM). The combined filtrates were concentrated to dryness, the residue was washed with dichloromethane (DCM), and the combined filtrates were concentrated to dryness to provide the title compound as a solid (4.97 g, 9.81 mmol, 115% yield). LC-MS m/z 507.1 (M+H)⁺. Step 7: (S)-1-((1S,2R,4R)-2-Amino-4-(tert-butylamino)cyclohexyl)-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one.

A mixture of N-((1R,2S,5R)-5-(tert-Butylamino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)acetamide (814 mg, 1.607 mmol) and 2M HCl (7.231 mL, 14.46 mmol) was stirred at 50 °C for 6 days. The mixture was basified with saturated NaHCO₃ and was concentrated under reduced pressure to dryness. The residue was suspended in 10% methanol in dichloromethane (DCM), was stirred at rt for 1 h, and was filtered. The solid was washed with 10% methanol in dichloromethane (DCM) and the combined filtrates were concentrated. Purification by ISCO CombiFlash® chromatography (80 g RediSep Rf Gold® column, 60 mL/min) eluting with a gradient of 0 to 15% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM) provided the title compound as a white solid (300 mg, 0.646 mmol, 40.2% yield). LC-MS m/z 465.4 (M+H)⁺.

Intermediate 7: (1S,3R)-3-(((Benzyloxy)carbonyl)amino)-1- isopropylcyclo- pentane-1 -carboxylic acid

To a solution of methyl (1S,3R)-3-((tert-butoxycarbonyl)amino)-1- isopropylcyclopentane-1-carboxylate (3.64 g, 12.75 mmol, J. Med. Chem., 2013, 56(19), 7706) in methanol (75 mL) was added a solution of lithium hydroxide (1.527 g, 63.8 mmol) in water (15 mL), followed by tetrahydrofuran (THF) (7.5 mL). The mixture was refluxed for 24 h and was cooled to rt. 1.0 M HCI (63.8 mL, 63.8 mmol) was added slowly and the mixture was extracted with dichloromethane (DCM) (3 x 150 mL). The combined organic extracts were dried over Na₂SO₄, were filtered, and the filtrate was concentrated under reduced pressure. The residue was dissolved in 2,2,2-trifluoroacetic acid (20 mL, 12.75 mmol). After 1 h, 12 M hydrochloric acid (50.0 mL, 600 mmol) was added, the mixture was heated at 100 °C for 16 h and was concentrated under reduced pressure. The residue was dissolved in a mixture of 1,4- dioxane (50 mL) and 1.0 M sodium hydroxide (50.0 mL, 50.0 mmol), was cooled to 0 °C, and Cbz-CI (2.367 mL, 16.58 mmol) was added dropwise. After stirring at 0 °C for 20 h, the mixture was extracted with ether (100 mL). The aqueous layer was acidified with 1.0 M HCI (50.0 mL, 50.0 mmol) and was extracted with diethyl ether (150 mL). The combined organic extracts were dried over Na₂SO₄, were filtered, and the filtrate was concentrated under reduced pressure to provide the title compound as a brown oil (3.32 g, 10.87 mmol, 85 % yield). LC- MS m/z 306.3 (M+H)⁺. Intermediate 8A: Benzyl (R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6- naphthyridine-6(5H)-carboxylate; and Intermediate 8B: Benzyl (S)-8-amino-3-

(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate.

Step 1 : Benzyl 3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine6(5H)- carboxylate.

A mixture of commercially-available 3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6- naphthyridine, dihydrochloric acid salt (5.0 g, 18.18 mmol) in 2-methyltetrahydrofuran (2- MeTHF) (100 mL) was stirred at rt for 1 min. 1.0 M sodium hydroxide (72.7 mL, 72.7 mmol) was added slowly and the mixture was stirred for 5 min. Cbz-CI (2.72 mL, 19.08 mmol) was added dropwise at rt and the mixture was stirred for 1 h. 2-Methyltetrahydrofuran (2-MeTHF) was removed under reduced pressure and the resulting emulsion stirred until the oil solidified into a yellow solid. The solid was filtered off, was crushed with a spatula into a fine powder, was washed with water, and was air-dried overnight under vacuum filtration to afford the title compound as a yellow solid (6.06 g, 18.02 mmol, 99 % yield). LC-MS m/z 337.3 (M+H)⁺.

Step 2: 6-((Benzyloxy)carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6- naphthyridine 1 -oxide

To a solution of benzyl 3-(trifluoromethyl)-7,8-dihydro-1 ,6-naphthyridine-6(5H)- carboxylate (6.0 g, 17.84 mmol) in dichloromethane (DCM) (10.0 mL) at rt was added methyltrioxorhenium(vii) (0.160 g, 0.642 mmol) followed by dropwise addition of 30 % hydrogen peroxide (3.64 mL, 35.7 mmol) and the mixture was stirred at rt for 20 h. The excess hydrogen peroxide was destroyed by addition of manganese(IV) oxide (1.0 mg, 0.012 mmol) and the mixture was stirred for 30 min. Dichloromethane (DCM) (50 mL) and 1.0 M NaOH (50 mL) were added and the aqueous phase was extracted with dichloromethane (DCM) (50 mL). The combined organic extracts were dried over Na₂SO₄ and were filtered. The filtrate was diluted with toluene (50 mL) and was concentrated under reduced pressure to provide the title compound as a white solid (6.20 g, 17.6 mmol, 99 % yield). LC-MS m/z 353.3 (M+H)⁺.

Step 3: Benzyl 8-bromo-3-(trifluoromethyl)-7,8-dihydro-1,6- naphthyridine-6(5H)- carboxylate.

To a solution of 6-((benzyloxy)carbonyl)-3-(trifluoromethyl)-5,6,7,8- tetrahydro- 1 ,6- naphthyridine 1-oxide (6.20 g, 17.60 mmol) in chloroform (30 mL) were simultaneously added at rt over 7 min the following two solutions dropwise from two separate syringes: a solution of phosphorus oxybromide (10.09 g, 35.2 mmol) in chloroform (10.0 mL) and a solution of triethylamine (1.385 mL, 9.93 mmol) in chloroform (10.0 mL). The mixture was stirred at rt for 45 min, was poured on ice, and was basified with 2.0 M Na₂CO₃. The mixture was extracted with dichloromethane (DCM) (3 x 200 mL). The combined organic extracts were dried over Na₂SO₄, were filtered, and the filtrate was concentrated under reduced pressure. Purification by normal-phase chromatography (330 g silica, 200 mL/min) eluting with a gradient of 0 to 40 % ethyl acetate in heptane provided the title compound as a white crystalline solid (2.83 g, 6.82 mmol, 38.7 % yield). LC-MS m/z 417.1 (M+H)⁺.

Step 4: Benzyl (R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6- naphthyridine- 6(5H)-carboxylate; and Benzyl (S)-8-amino-3-(trifluoromethyl)-7,8- dihydro-1, 6- naphthyridine-6(5H)-carboxylate.

To a solution of benzyl 8-bromo-3-(trifluoromethyl)-7,8-dihydro-1 ,6- naphthyridine- 6(5/-/)-carboxylate (2.70 g, 6.50 mmol) in N,N-dimethylformamide (DMF) (25.0 mL) was added sodium azide (1.268 mg, 19.51 mmol) and the mixture was stirred at rt for 18 h. The flask was purged with nitrogen, freshly degassed tetrahydrofuran (THF), (30.000 mL) and water (6.000 mL) were added followed by 1.0 M trimethylphosphine in toluene (32.5 mL, 32.5 mmol). The mixture was stirred at rt for 20 h and was concentrated under reduced pressure. The residue was partitioned between diethyl ether and water, the organic phase was separated, was dried over Na₂SO₄, and was concentrated under reduced pressure. Preparative chiral resolution using a Lux Amylose 2 column (5 pm, 21 x 250 mm, 20 mL/min) and eluting with 30 % heptane in ethanol containing 0.1% isopropylamine provided the title compound as a white solid (990.1 mg, 2.82 mmol, 43.3 % yield). LC-MS m/z 352.2 (M+H)⁺. Benzyl (S)-8-amino-3- (trifluoromethyl)-7,8-dihydro-1 ,6-naphthyridine-6(5H)-carboxylate was also isolated as a white solid (888.7 mg, 2.53 mmol, 38.9 % yield). LC-MS m/z 352.3 (M+H)⁺. The absolute stereochemistry of both enantiomers was determined by VCD using the following procedure:

Samples were dissolved in CD₃CN (16.8 mM) and placed in a 100 pm pathlength cell with BaF₂ windows. IR and VCD spectra were recorded on a ChirallR2XTM VCD spectrometer (BioTools Inc., Jupiter, FL) equipped with dual PEM accessory, with 4 cm-1 resolution, 6-hour collection for one isomer-E1 and 12-hour collection for the other isomer-E2, and the instrument optimized at 1400 cm-1. A conformational search on the modeled (R)- structure was carried out using MOE at LowMode using MMFF94x forcefield with Born solvation, dielectric constant set at 20 and exterior dielectric constant set at 47. The geometry optimization, frequency, and IR and VCD intensity calculations of the 10 conformers resulted from the conformational search were carried out at the DFT level b3lyp/6-31G(d) serf = (solvent = dimethyl sulfoxide) with Gaussian 16 (Gaussian Inc., Wallingford, CT). The Gaussian output files were converted to VCD and IR spectra using BLAIR. The calculated frequencies were scaled by 0.981 and the IR and VCD intensities were converted to Lorentzian bands with 8-cm-1 half-width for comparison to experimental spectra. The assignment was evaluated by CompareVOA program (BioTools Inc., Jupiter, FL) to generate a confidence level based on current database that includes 88 previous correct assignments for different chiral structures.

Intermediate 9: ((R)-8-Amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin- 6(5H)-yl)((1 S,3R)-1 -isopropyl -3-(((3S,4S)-3-methoxytetrahydro-2H-pyran -4- yl)amino)cyclopentyl)methanone

Step 1 : Benzyl (R)-8-((tert-butoxycarbonyl)amino)-3-(trifluoromethyl)- 7,8- dihydro-1,6-naphthyridine-6(5H)-carboxylate.

To benzyl (R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1 ,6-naphthyridine-6(5/7)- carboxylate (Step 3-4) (4.25 g, 12.10 mmol) was added di-tert-butyl dicarbonate (2.90 g, 13.31 mmol) and water (24.00 mL) and the mixture was stirred for 5 min. Acetone (24.00 mL) was added slowly. The mixture was stirred for 2 h, the precipitate was filtered, was washed with water, and was air-dried to provide the title compound as a pale-yellow solid (4.70 g, 10.41 mmol, 86 % yield). LC-MS m/z 452.1 (M+H)⁺.

Step 2: tert-Butyl (R)-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6- naphthyridin-8- yl)carbamate.

A mixture of benzyl (R)-8-((tert-butoxycarbonyl)amino)-3-(trifluoromethyl)- 7,8-dihydro- 1 ,6-naphthyridine-6(5/-/)-carboxylate (4.60 g, 10.19 mmol) and 20 % palladium hydroxide on carbon (2.216 g, 3.06 mmol) in methanol (100.000 mL) was sealed in a flask and was purged with nitrogen. Hydrogen was bubbled into the mixture and the mixture was stirred for 1.5 h under a balloon atmosphere of hydrogen. The mixture was open to air and was stirred for 10 min. 7.0 M ammonia in methanol (100 mL) was added, the mixture was filtered, and the catalyst was washed with methanol. The combined filtrates were concentrated under reduced pressure. Purification by reverse-phase HPLC chromatography (C18 Aq 275 g Gold column, 125 mL/min) eluting with a gradient of 30 to 60 % acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonia hydroxide (0.075 %) provided the title compound as a pale-yellow foam (2.66 g, 8.38 mmol, 82 % yield). LC-MS m/z 318.1 (M+H)⁺.

Step 3: tert-Butyl ((R)-6-((1 S,3R)-3-(((benzyloxy)carbonyl)amino)-1- isopropylcyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6- naphthyridin-8-yl)carbamate.

To a solution of (1 S,3R)-3-(((benzyloxy)carbonyl)amino)-1- isopropylcyclopentane-1- carboxylic acid (5.12 g, 16.77 mmol) in dichloromethane (DCM) was added DIPEA (9.15 mL, 52.4 mmol) and HATU (5.90 g, 15.51 mmol) and the mixture was stirred for 24 h. The mixture was added to tert-butyl (R)-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8- yl)carbamate (2.66 g, 8.38 mmol), was stirred for 2 min, and the resulting solution was divided into two 20 mL vials. The vials were sealed and were stirred at 80 °C for 22 h. The mixtures were cooled to rt, water was added (1.000 mL each), the vials were sealed, and the mixtures were stirred at 80 °C for 50 min. The two mixtures were cooled to rt, were combined, and water (100 mL) and dichloromethane (DCM) (100 mL) were added. The organic phase was dried over Na₂SO₄, was filtered, and the filtrate was concentrated under reduced pressure. Purification by ISCO CombiFlash® chromatography (330 g silica, 200 mL/min) eluting with a gradient of 0 to 10 % methanol in dichloromethane (DCM) provided the title compound as a white foam (2.95 g, 4.88 mmol, 58.2 % yield). LC-MS m/z 605.2 (M+H)⁺. Step 4: tert-Butyl ((R)-6-((1 S,3R)-3-amino-1-isopropylcyclopentane-1- carbonyl)- 3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8-yl)carbamate.

A mixture of tert-butyl ((R)-6-((1 S,3R)-3-(((benzyloxy)carbonyl)amino)-1- isopropylcyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1 ,6-naphthyridin-8- yl)carbamate (2.95 g, 4.88 mmol) and 20 % palladium hydroxide on carbon (1.028 g, 1.464 mmol) in methanol (50.000 mL) was purged with nitrogen. Hydrogen was bubbled into the mixture and the mixture was stirred for 1 h under a balloon atmosphere of hydrogen. The mixture was opened to air and the mixture was stirred for 15 min. 2.0 M ammonia in methanol (100 mL) was added and the mixture was filtered. The catalyst was washed with methanol and the combined filtrates were concentrated under reduced pressure to provide the title compound as a white foam (2.24 g, 4.76 mmol, 98 % yield). LC-MS m/z 471.4 (M+H)⁺.

Step 5: tert-Butyl ((R)-6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro- 2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro- 1 ,6-naphthyridin-8-yl)carbamate, 2Formic acid salt.

To a solution of the tert-butyl ((R)-6-((1 S,3R)-3-amino-1- isopropylcyclopentane-1- carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1 ,6-naphthyridin-8- yl)carbamate (2.24 g, 4.76 mmol) in isopropyl acetate (50.00 mL) was added 2-propanol (0.917 mL, 11.90 mmol) and triethylamine (0.664 mL, 4.76 mmol). The mixture was cooled to 0 °C, sodium triacetoxyborohydride (2.52 g, 11.90 mmol) was added followed by dropwise addition of a solution of (R)-3-methoxytetrahydro-4/7-pyran-4-one (0.929 g, 7.14 mmol) in isopropyl acetate (50.00 mL). The mixture was stirred at 0 °C for 20 min, was warmed to rt, and was stirred at rt for 2 h. 1.0 M NaOH (200 mL) and isopropyl acetate (200 mL) were added, the organic phase was dried over Na₂SO₄, was filtered, and the filtrate was concentrated under reduced pressure. Purification by reverse-phase HPLC chromatography (C18 275g Gold column; 125 mL/min) eluting with 35 % acetonitrile in water containing formic acid (0.1 %) provided the title compound as a white foam (2.73 g, 4.03 mmol, 85 % yield). The compound was used as an intermediate in the next step. LC-MS m/z 585.2 (M+H)⁺.

Step 6: ((R)-8-Amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)- yl)((1 S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4- yl)amino)cyclopentyl)methanone.

A mixture of tert-butyl ((R)-6-((1 S,3R)-1-isopropyl-3-(((3S,4S)-3- methoxytetrahydro- 2/7-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)- 5,6,7,8-tetrahydro-1 ,6- naphthyridin-8-yl)carbamate, 2formic acid salt (2.73 g, 4.03 mmol) and trifluoroacetic acid (10 mL, 130 mmol) was stirred at rt for 15 min and was concentrated under reduced pressure. Dichloromethane (DCM) (100 mL) and 1.0 M NaOH (100 mL) were added and the aqueous phase was extracted with dichloromethane (DCM) (100 mL). The combined organic extracts were dried over Na₂SO₄, were filtered, and the filtrate were concentrated under reduced pressure to provide the title compound as a white foam (1.77 g, 3.65 mmol, 91 % yield). LC- MS m/z 485.4 (M+H)⁺.

Intermediate 10: di-tert-butyl (((S)-6-((S)-2-amino-3-(naphthalen-2- yl) propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate

Step 1 : Di-tert-butyl (((S)-6-((S)-2-amino-3-(naphthalen-2- yl)propanamido)-1- (tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate

To a solution of (S)-2-((((9/7-fluoren-9-yl)methoxy)carbonyl)amino)-3- (naphthalen-2- yl)propanoic acid (269 mg, 0.615 mmol) and HATU (304 mg, 0.800 mmol) in anhydrous N,N- dimethylformamide (6.2 mL) was added DIPEA (215 μl, 1.23 mmol). After 10 minutes, di-tert- butyl (((S)-6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (300 mg, 0.615 mmol) was added and the reaction was stirred at RT for 23 h. Piperidine (122 μl, 1.23 mmol) was added, and the mixture was stirred at RT for 3 h. The reaction was diluted with EtOAc (50 mL), washed with saturated aqueous sodium bicarbonate (2x 30mL) and brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resultant residue was purified by ISCO CombiFlash® chromatography eluting with a gradient of 0 to 20% ethyl acetate in hexanes to provide the title compound as a yellow solid (261 mg, 381 mmol, 61.9% yield). LC-MS m/z 685.4 (M+H)+.

Intermediate 11 : (S)-5-(6-Methoxy-2-(piperidin-4-yl)quinolin-4-yl)-3-(1-((5- methoxy-6-methylpyridin-2-yl)methyl)piperidin-4-yl)oxazolidin-2-one

Step 1 : tert-Butyl (S)-4-((2-hydroxy-2-(6-methoxyquinolin-4- yl)ethyl)amino)piperidine-1-carboxylate (Intermediate 11 A)

To a solution of (S)-6-methoxy-4-(oxiran-2-yl)quinoline (10.0 g, 49.7 mmol) in Ethanol (150 mL) stirred under nitrogen, was added tert-butyl 4-aminopiperidine-1 -carboxylate (10.95 g, 54.7 mmol) followed by lithium perchlorate (5.82 g, 54.7 mmol) at room temperature. The reaction was heated and stirred at 80°C overnight, then concentrated in vacuo and diluted with water (500 mL). The mixture was extracted with ethyl acetate (4x 500 mL), and the combined organic fractions were washed with brine (1000 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to provide the title compound as an orange liquid (18.0 g, 34.8 mmol, 70.0% yield). LMCS m/z 402.2 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 - 8.69 (m, 1 H) 7.95 - 7.92 (m, 1 H) 7.57 - 7.56 (m, 1 H) 7.41 - 7.38 (m, 2 H) 5.40 - 5.30 (m, 1 H) 3.90 (s, 3 H) 3.83 - 3.78 (m, 4 H) 2.72 - 2.63 (m, 5 H) 1.90 - 1.55 (m 4H) 1.41 - 1.34 (m, 4 H) 1.18 - 1.01 (m 5H).

Step 2: tert-Butyl (S)-4-(5-(6-methoxyquinolin-4-yl)-2-oxooxazolidin-3- yl)piperidine-1-carboxylate (Intermediate 11B)

To a solution of tert-butyl (S)-4-((2-hydroxy-2-(6-methoxyquinolin-4- yl)ethyl)amino)piperidine-1-carboxylate (350 g, 708 mmol) in dichloromethane (5250 mL) stirred under nitrogen, was added DMAP (130 g, 1062 mmol), followed by GDI (172 g, 1062 mmol). The reaction mixture was stirred at room temperature for 16 h, then diluted with water (3000 mL) and extracted with dichloromethane (3x 2000 mL). The combined organic fractions were washed with brine (3000 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant crude black liquid was adsorbed onto silica gel (1500 g, 60- 120 mesh) and purified via silica gel chromatography eluting with 50-100% ethyl acetate in petroleum ether to provide a yellow solid. The yellow solid was adsorbed onto silica gel (700 g, 60-120 mesh) and repurified via silica gel chromatography eluting with 0-5% methanol in dichloromethane to afford the title compound as a pale yellow solid (130 g, 304 mmol, 43.0% yield). Chiral purity 99.87%. LCMS m/z 482.2 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.78 (s, 1 H) 8.02 - 7.99 (m, 1 H) 7.48 - 7.46 (m, 2 H) 7.21 - 7.20 (m, 1 H) 6.38 - 6.34 (m, 1 H) 4.34 - 4.29 (m, 1 H) 4.05 - 3.95 (m, 5 H) 3.78 - 3.72 (m, 1 H) 3.37 - 3.34 (m, 1 H) 2.78 (br s, 2 H) 1.75 - 1.72 (m, 1 H) 1.61 - 1.54 (m, 2 H) 1.45 - 1.37 (m, 10H).

Step 3: (S)-5-(6-Methoxyquinolin-4-yl)-3-(piperidin-4-yl)oxazolidin-2-one hydrochloride (Intermediate 11C)

To a solution of tert-butyl (S)-4-(5-(6-methoxyquinolin-4-yl)-2-oxooxazolidin-3- yl)piperidine-1 -carboxylate (6.00 g, 14.0 mmol) in dichloromethane (24 mL) and Methanol (12 mL) was added 3M HCI in CPME (46.8 ml, 140 mmol). The reaction was stirred at RT for 2 h, and then additional 3M HCI in CPME (10 mL, 30 mmol) was added. After 1.5 h, additional 3M HCI in CPME (6 mL, 18 mmol) was added. After another 1 h, additional 3M HCI in CPME (5 mL, 15 mmol) was added. The milky white reaction mixture was then diluted with methanol and concentrated under a steady stream of nitrogen for 16 h to afford the title compound as a fine white powder (6.25 g, 14.0 mmol, theoretical yield). LCMS m/z 328.3 (M+H)⁺. ¹ H NMR (400 MHz, DMSO-d₆) δ 9.08 - 8.98 (m, 2 H), 8.94 - 8.82 (m, 1 H), 8.30 (d, J = 9.3 Hz, 1 H), 7.79 (d, J = 5.4 Hz, 1 H), 7.75 (dd, J = 9.3, 2.4 Hz, 1 H), 7.44 (d, J = 2.4 Hz, 1 H), 6.56 (dd, J = 9.0, 6.6 Hz, 1 H), 4.40 (t, J = 9.0 Hz, 1 H), 4.01 (s, 3 H), 3.97 - 3.84 (m, 1 H), 3.38 (dd, J = 8.6, 6.6 Hz, 1 H), 3.36 - 3.21 (m, 2 H), 3.06 - 2.91 (m, 2 H), 2.07 - 1.70 (m, 4 H). Step 4: (S)-5-(6-Methoxyquinolin-4-yl)-3-(1-(2,2,2-trifluoroacetyl)piperidin-4- yl)oxazolidin-2-one (Intermediate 11 D)

To a solution of (S)-5-(6-methoxyquinolin-4-yl)-3-(piperidin-4-yl)oxazolidin-2-one hydrochloride (3.00 g, 3.25 mmol) in dichloromethane (55 mL) was added TEA (4.60 mL, 33.0 mmol) and trifluoroacetic anhydride (1.51 mL, 10.7 mmol). The reaction was stirred at RT for 24 h, then concentrated in vacuo. The resultant residue was purified via silica gel chromatography eluting with 0-10% methanol in dichloromethane to afford the title compound as a yellow solid (3.09 g, 6.42 mmol, 78% yield). LCMS m/z 424.22 (M+H)⁺. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.44 - 1.57 (m, 1 H) 1.70 (qd, J=12.72, 4.40 Hz, 1 H) 1.80 - 1.93 (m, 1 H) 2.06 (ddt, J=8.50, 4.22, 2.20, 2.20 Hz, 1 H) 2.84 (q, J=13.86 Hz, 1 H) 3.13 - 3.31 (m, 1 H) 3.41 (td, J=8.56, 6.36 Hz, 1 H) 3.98 (s, 3 H) 4.04 - 4.27 (m, 3 H) 4.55 - 4.78 (m, 1 H) 6.07 - 6.21 (m, 1 H) 6.83 - 6.94 (m, 1 H) 7.47 (dd, J=9.29, 2.45 Hz, 1 H) 7.55 (d, J=4.89 Hz, 1 H) 8.14 (dd, J=9.29, 0.98 Hz, 1 H) 8.84 (d, J=4.40 Hz, 1 H).

Step 5: tert-Butyl (S)-4-(6-methoxy-4-(2-oxo-3-(1-(2,2,2-trifluoroacetyl)piperidin- 4-yl)oxazolidin-5-yl)quinolin-2-yl)piperidine-1 -carboxylate (Intermediate 11 E)

To a solution of (S)-5-(6-methoxyquinolin-4-yl)-3-(1-(2,2,2-trifluoroacetyl)piperidin-4- yl)oxazolidin-2-one (1.50 g, 3.54 mmol), 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (8.12 g, 35.4 mmol) and peroxydisulfuric acid, diammonia salt (4.85 g, 21.3 mmol) in DMSO (35.4 mL) and water (0.059 mL) was bubbled nitrogen gas at RT for 1 h. The mixture was heated at 50 °C for 21 h, then cooled to RT and diluted with water (700 mL). Additional 1 -(tert- butoxycarbonyl)piperidine-4-carboxylic acid (8.12 g, 35.4 mmol) and peroxydisulfuric acid, diammonia salt (4.85 g, 21.3 mmol) were added, and the reaction was heated at 50 °C for 25 h. The mixture was cooled to RT, then diluted with dichloromethane (100 mL) and saturated aqueous sodium bicarbonate (100 mL). The layers were separated, and the aqueous layer was extracted with dichloromethane (100 mL). The organic fractions were combined and washed with saturated aqueous sodium bicarbonate (50 mL) and brine (50 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The resultant residue was adsorbed onto silica gel and purified via silica gel chromatography eluting with 0-5% methanol in dichloromethane to afford the title compound as a yellow solid (0.990 g, 1.13 mmol, 31.8% yield). LCMS m/z 607.27 (M+H)⁺. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.52 (s, 9 H) 1.61 - 1.74 (m, 5 H) 1.83 - 2.02 (m, 2 H) 2.79-2.97 (m, 5H) 3.17-3.44 (m, 3H) 3.97 (s, 3 H) 4.01- 4.23 (m, 3 H) 4.25 - 4.40 (m, 2 H) 6.06 - 6.18 (m, 1 H) 6.83 (d, J=2.45 Hz, 1 H) 7.39 - 7.50 (m, 2 H) 8.00 - 8.12 (m, 1 H).

Step 6: tert-Butyl (S)-4-(6-methoxy-4-(2-oxo-3-(piperidin-4-yl)oxazolidin-5- yl)quinolin-2-yl)piperidine-1 -carboxylate (Intermediate 11 F)

To a RB flask was charged with tert-butyl (S)-4-(6-methoxy-4-(2-oxo-3-(1-(2,2,2- trifluoroacetyl)piperidin-4-yl)oxazolidin-5-yl)quinolin-2-yl)piperidine-1-carboxylate (0.989 g, 1.630 mmol) and potassium carbonate (2.25 g, 16.3 mmol) was added methanol (16.3 mL). The suspension was stirred at RT for 24 h, then poured into a saturated aqueous solution of ammonium chloride. The aqueous layer was extracted with dichloromethane (5x) and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford the title compound as a yellow solid (569 mg, 0.981 mmol, 60.2% yield). LCMS m/z 511.34 (M+H)⁺. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.50 - 1.53 (m, 9 H) 1.55 - 1.67 (m, 2 H) 1.70 - 1.80 (m, 2 H) 1.81 - 2.03 (m, 3 H) 2.68 - 2.97 (m, 5 H) 2.99 - 3.11 (m, 2 H) 3.15 - 3.23 (m, 1 H) 3.24 - 3.31 (m, 1 H) 3.40 - 3.47 (m, 1 H) 3.89 - 4.00 (m, 4 H) 4.18 - 4.27 (m, 1 H) 4.28 - 4.38 (m, 1 H) 6.09 (dd, J=8.80, 6.85 Hz, 1 H) 6.85 (d, J=2.93 Hz, 1 H) 7.39 - 7.48 (m, 2 H) 8.06 (d, J=8.80 Hz, 1 H).

Step 7: tert-Butyl (S)-4-(6-methoxy-4-(3-(1-((5-methoxy-6-methylpyridin-2- yl)methyl)piperidin-4-yl)-2-oxooxazolidin-5-yl)quinolin-2-yl)piperidine-1 -carboxylate (Intermediate 11G)

To a mixture of tert-butyl (S)-4-(6-methoxy-4-(2-oxo-3-(piperidin-4-yl)oxazolidin-5- yl)quinolin-2-yl)piperidine-1 -carboxylate (1.60 g, 3.13 mmol), 5-methoxy-6- methylpicolinaldehyde (568. mg, 3.76 mmol) and sodium triacetoxyborohydride (1.99 g, 9.40 mmol) under nitrogen was added DCE (31 mL) and titanium(IV) isopropoxide (2.05 g, 2.19 mL, 7.21 mmol). The mixture was then heated to 75 °C for 5 h. The reaction was cooled to RT, quenched with saturated aqueous sodium bicarbonate (30 ml), and stirred for 30 minutes at RT. The slurry was filtered through pad of celite to remove solids, then washed with dichloromethane (30 mL). The filtrate was extracted with dichloromethane (3x), and the combined organic fractions were dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0- 10% methanol in dichloromethane to afford the title compound as a yellow solid (882 mg, 1.34 mmol, 42.7% yield). LCMS m/z 646.21 (M+H)⁺. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1 .51 (s, 9 H) 1.58 - 1 .75 (m, 2 H) 1.79 - 1.92 (m, 4 H) 1.93 - 2.00 (m, 2 H) 2.11 - 2.26 (m, 2 H) 2.46 (s, 3 H) 2.83 - 2.97 (m, 3 H) 2.99 - 3.08 (m, 2 H) 3.42 (dd, J=8.31, 6.85 Hz, 1 H) 3.59 (s, 2 H) 3.83 (s, 3 H) 3.85 - 3.90 (m, 1 H) 3.96 (s, 3 H) 4.20 (t, J=8.80 Hz, 1 H) 4.25 - 4.41 (m, 2 H) 6.07 (dd, J=9.05, 7.09 Hz, 1 H) 6.84 (d, J=2.45 Hz, 1 H) 7.01 - 7.07 (m, 1 H) 7.10 - 7.16 (m, 1 H) 7.42 (dd, J=9.29, 2.93 Hz, 1 H) 7.45 (s, 1 H) 8.05 (d, J=9.29 Hz, 1 H). Step 8: (S)-5-(6-Methoxy-2-(piperidin-4-yl)quinolin-4-yl)-3-(1-((5-methoxy-6- methylpyridin-2-yl)methyl)piperidin-4-yl)oxazolidin-2-one (Intermediate 11)

To a solution of tert-butyl (S)-4-(6-methoxy-4-(3-(1-((5-methoxy-6-methylpyridin-2- yl)methyl)piperidin-4-yl)-2-oxooxazolidin-5-yl)quinolin-2-yl)piperidine-1 -carboxylate (880 mg, 1 Eq, 1.36 mmol) in dichloromethane was added 4N HCI in dioxane HO (497 mg, 3.41 mL, 13.6 mmol). The mixture was stirred at RT for 20 h, then concentrated and dried on high vac to afford the title compound as a yellow solid (1.235 g, 2.24 mmol, theoretical yield). LCMS m/z 546.25 (M+H)⁺. ¹ H NMR (400 MHz, DMSO-cfc) δ ppm 1.73 - 1.83 (m, 1 H) 1.89 - 2.01 (m, 1 H) 2.02 - 2.18 (m, 4 H) 2.18 - 2.29 (m, 1 H) 2.39 - 2.46 (m, 2 H) 2.82 - 2.95 (m, 1 H) 2.97 - 3.10 (m, 2 H) 3.11 - 3.27 (m, 2 H) 3.30 - 3.52 (m, 5 H) 3.58 - 3.78 (m, 3 H) 3.87 (s, 3 H) 3.93 - 4.02 (m, 3 H) 4.25 - 4.43 (m, 3 H) 6.40 - 6.49 (m, 1 H) 7.24 - 7.35 (m, 1 H) 7.47 (br s, 1 H) 7.56 - 7.65 (m, 1 H) 8.02 - 8.24 (m, 1 H) 8.78 - 8.92 (m, 1 H) 9.14 - 9.31 (m, 1 H) 10.55 - 10.89 (m, 1 H).

EXAMPLE 1

(((S)-1 -Carboxy-5-((S)-2-((1R,4S)-4-(4-(((1 r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4- (((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl) cycl ohexyl )am i no)-4-oxobutoxy)cyclohexyl )oxy)ethyl )am i no)-2-oxoethyl )-4-(((1 S,4r)-4- (2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy) cyclohexyl)oxy)butanamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexa ne-1-carboxamido)-3-(naphthalen-2-yl)propanamido)pentyl)carbamoyl)-L-glutamic acid

Step 1 : tert-Butyl ((benzyloxy)carbonyl)glycinate

To a solution of benzyl 2-bromoacetate (20g, 87 mmol) and tert-butyl glycinate hydrochloride (16.1 g, 96.0 mmol) in tetrahydrofuran (175 ml) was added DIPEA (35.1 mL, 201 mmol). The reaction was stirred at RT for 44 hours. The mixture was fitlered to remove a white solid, which was washed with ethyl acetate. The organic filtrate was diluted with additional ethyl acetate (200 mL), washed with saturated aqueous sodium bicarbonate (200 mL) and brine (200 ml), dried over sodium sulfate, filtered, and concentrated. The resultant residue was purified by silica gel chromatography eluting with 0-60% ethyl acetate in hexanes to provide the title compound as a yellow oil (13.97 g, 49.0 mmol, 57.3 % yield; 48.0 mmol, 56.1% yield at 98% purity). LC-MS m/z 280.21 (M+H)⁺. ¹H NMR (400 MHz, CHLOROFORM- d) δ ppm 1.48 (s, 9 H) 1.91 (br s, 1 H) 3.38 (s, 2 H) 3.53 (s, 2 H) 5.20 (s, 2 H) 7.32 - 7.43 (m, 5 H).

Step 2: Sodium (E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-

To a solution of methyl (E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but- 2-enoate (5.02 g, 11.5 mmol) was dissolved in tetrahydrofuran (14.6 mL) was added aqueous sodium hydroxide (5.089M, 3.38 mL, 17.2 mmol). The unstirred mixture was 2 distinct layers at room temperature.

The homogenous pale yellow reaction was heated with magnetic stirring at 80 °C for 17 hours. The mixture was concentrated in vacuo, azeotroped with toluene (2 x 50 mL), and dried under high vacuum to provide the title compound as a sticky pale yellow solid (5.33 g, 11.4 mmol, 99.1% yield at 95% purity). LC-MS m/z 424.23 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.11 - 1.26 (m, 4 H) 1.80 - 1.92 (m, 4 H) 2.53 - 2.58 (m, 2 H) 3.16 - 3.19 (m, 1 H) 3.20 - 3.27 (m, 1 H) 3.49 (t, J=Q.11 Hz, 2 H) 3.57 - 3.65 (m, 4 H) 3.95 (dd, J=5.62, 1.71 Hz, 2 H) 5.69 - 5.79 (m, 1 H) 6.13 - 6.30 (m, 1 H) 7.20 - 7.29 (m, 2 H) 7.29 - 7.40 (m, 8 H).

Step 3: Benzyl N-(2-(tert-butoxy)-2-oxoethyl)-N-((E)-4-(((1r,4r)-4-(2- dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoyl)glycinate

To a solution of benzyl (2-(tert-butoxy)-2-oxoethyl)glycinate (500 mg, 1.79 mmol) and sodium (E)-4-(((1 r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoate (797 mg, 1.79 mmol) in dichloromethane (3.58 mL) was added HATU (885 mg, 2.33 mmol), and N- ethyl-N-isopropylpropan-2-amine (468 μl, 2.68 mmol). The reaction was stirred at RT overnight for 19 h. The mixture was diluted with dichloromethane (100 mL) and washed with saturated aqueous sodium bicarbonate (50 mL) and brine (50 mL), dried over sodium sulfate, filtered, and concentrated. The resultant residue was purified by silica gel chromatography eluting with 0-50% ethyl acetate in heptanes to provide the title compound as a yellow oil (961 mg, 1.19 mmol, 66.6% yield at 85% purity). LC-MS m/z 685.29 (M+H)⁺. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.25 - 1.36 (m, 7 H) 1.43 - 1.52 (m, 11 H) 1.81 - 2.04 (m, 4 H) 2.69 (t, J=Q.11 Hz, 2 H) 3.13 - 3.21 (m, 1 H) 3.51 - 3.60 (m, 2 H) 3.65 - 3.72 (m, 4 H) 4.09 - 4.31 (m, 4 H) 5.16 - 5.24 (m, 2 H) 6.82 - 7.07 (m, 1 H) 7.20 - 7.28 (m, 2 H) 7.30 - 7.47 (m, 12 H).

Step 4: N-(2-(Benzyloxy)-2-oxoethyl)-N-((E)-4-(((1r,4r)-4-(2-(dibenzylamino) ethoxy)cyclohexyl)oxy)but-2-enoyl)glycine

To a solution of benzyl N-(2-(tert-butoxy)-2-oxoethyl)-N-((E)-4-(((1 r,4r)-4-(2- (dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoyl)glycinate (960 mg, 1.40 mmol) in Dichloromethane (5.6 mL) was added TFA (2.16 mL, 28.0 mmol). The reaction was stirred at RT for 20 h, then concentrated in vacuo and dried on high vac to provide the title compound as an orange oil (1.57 g, 1.39 mmol, 99 % yield). LC-MS m/z 629.23 (M+H)⁺. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.23 - 1.47 (m, 3 H) 1.48 - 1.58 (m, 1 H) 1.61 - 1.71 (m, 1 H) 1.93 - 2.10 (m, 3 H) 2.70 (dt, J=9.29, 6.36 Hz, 1 H) 2.86 - 3.01 (m, 1 H) 3.25 - 3.43 (m, 3 H) 3.70 - 3.90 (m, 2 H) 4.19 - 4.43 (m, 7 H) 4.51 - 4.64 (m, 2 H) 5.19 - 5.30 (m, 2 H) 6.36 - 6.52 (m, 1 H) 6.92 - 7.09 (m, 1 H) 7.32 - 7.56 (m, 15 H).

Step 5: Methyl (1R,4r)-4-(4-(((1r,4R)-4-((2-((E)-N-(2-(benzyloxy)-2-oxoethyl)-4- (((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enamido)acetamido) methoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate

To a solution of N-(2-(benzyloxy)-2-oxoethyl)-N-((E)-4-(((1 r,4r)-4-(2- (dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoyl)glycine trifluoroacetic acid salt (500 mg, 0.673 mmol) in dichloromethane (6.898 mL) was added HATU (333 mg, 0.875 mmol), and N- ethyl-N-isopropylpropan-2-amine (0.586 mL, 3.37 mmol). tert-Butyl (1R,4r)-4-(4-(((1 r,4R)-4- (2-aminoethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxylate (316 mg, 0.740 mmol) was added, and the reaction was stirred at RT for 3 h. The mixture was diluted with dichloromethane (50 mL), washed with saturated aqueous sodium bicarbonate (20 mL) and brine (20 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The resultant residue was purified by silica gel chormatography eluting with 0-10% methanol in dichloromethane to afford the title compound as a yellow oil (581 mg, 0.429 mmol, 63.7 % yield). LC-MS m/z 1037.42 (M+H)⁺. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.07 - 1.17 (m, 4 H) 1.23 - 1.37 (m, 4 H) 1.39 - 1.54 (m, 9 H) 1.73 (br s, 4 H) 1.83 - 2.19 (m, 10 H) 2.19 - 2.32 (m, 2H) 3.20 (q, J=7.50 Hz, 4H) 3.50 (s, 14 H) 3.74 (dt, J=13.45, 6.48 Hz, 2 H) 4.01 - 4.34 (m, 4 H) 5.32 (s, 2 H) 5.59 - 5.71 (m, 1 H) 6.32 - 6.41 (m, 1 H) 6.97 - 7.09 (m, 1 H) 7.33 - 7.46 (m, 15 H) 8.05 - 8.13 (m, 1 H).

Step 6: Sodium N-(4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy) butanoyl)-N-(2-(((((1R,4r)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4- oxobutoxy)cyclohexyl)oxy)methyl)amino)-2-oxoethyl)glycinate

To a solution of tert-butyl (1R,4r)-4-(4-(((1r,4R)-4-(2-(2-((E)-N-(2-(benzyloxy)-2- oxoethyl)-4-(((1 r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2- enamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate (580 mg, 0.559 mmol) in tetrahydrofuran (5.6 mL) (5591 pl) was added 1 N aqueuous sodium hydroxide. The reaction was stirred at RT for 17 h. The mixture was neutralized with 1 N HCI, concentrated in vacuo, and dried on high vac to provide the title compound as a yellow semi- solid (580 mg, 0.557 mmol, 100% yield). LC-MS 947.44 (M+H)⁺. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.12 - 1.38 (m, 11 H) 1.42 - 1.47 (m, 10 H) 1.89 (br s, 13 H) 2.09 -

2.18 (m, 3 H) 2.21 - 2.30 (m, 3 H) 3.19 (q, J=7.QQ Hz, 7 H) 3.38 - 3.49 (m, 4 H) 3.51 - 3.58 (m,

2 H) 3.58 - 3.67 (m, 2 H) 3.68 - 3.82 (m, 8 H) 5.95 - 6.03 (m, 1 H) 6.28 - 6.44 (m, 1 H) 7.14 -

7.22 (m, 2 H) 7.37 - 7.37 (m, 1 H) 7.37 - 7.49 (m, 8 H).

Step 7: tert-Butyl (1R,4r)-4-(4-(((1r,4R)-4-(2-(2-(4-(((1r,4r)-4-(2-(dibenzylamino) ethoxy)cyclohexyl)oxy)-N-(2-(((((1R,4r)-4-(4-(((1 r,4R)-4-(methoxycarbonyl)cyclohexyl) amino)-4-oxobutoxy)cyclohexyl)oxy)methyl)amino)-2-oxoethyl)butanamido)acetamido )ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate

To a solution of sodium N-(2-((2-(((1R,4r)-4-(4-(((1 r,4R)-4-(tert- butoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-N- ((E)-4-(((1 r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoyl)glycinate (530 mg, 0.547 mmol), methyl (1R,4r)-4-(4-(((1 r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1 -carboxylate (210 mg, 0.547 mmol) and HATU (270 mg, 0.711 mmol) in dichloromethane (22.7 mL) was added DI PEA (0.191 mL, 1.09 mmol). The reaction was stirred at RT for 1h, then diluted with additional dichloromethane (20 mL). The mixture was washed with saturated aqueous sodium bicarbonate (20 mL) and brine (20 mL), dried over sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-10% methanol in dichloromethane to afford the titel compound as a clear film (174 mg, 0.106 mmol, 19.3 % yield). LC-MS m/z 1313.64 (M+H)⁺. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.03 - 1.18 (m, 5 H) 1.21 - 1.33 (m, 11 H) 1.39 - 1.44 (m, 11 H) 1.45 - 1.59 (m, 4 H) 1.79 - 1.87 (m, 4 H) 1.89 - 2.05 (m, 20 H) 2.05 - 2.14 (m, 2 H) 2.17 - 2.26 (m, 6 H) 2.59 - 2.68 (m, 2 H) 3.17 - 3.18 (m, 1 H) 3.17 - 3.32 (m, 5 H) 3.39 - 3.54 (m, 14 H) 3.62 - 3.66 (m, 6 H) 3.67 - 3.78 (m, 2 H) 3.93 - 3.98 (m, 1 H) 4.05 - 4.09 (m, 1 H) 4.09 - 4.13 (m, 1 H) 5.67 - 5.86 (m, 2 H) 6.30 (dt, J=15.04, 1.77 Hz, 1 H) 6.62 (br t, J=5.38 Hz, 1 H) 6.85 - 7.00 (m, 2 H) 7.18 - 7.23 (m, 1 H) 7.25 - 7.32 (m, 4 H) 7.33 - 7.37 (m, 4 H).

Step 8: tert-Butyl (1R,4r)-4-(4-(((1r,4R)-4-(2-(2-(4-(((1r,4r)-4-(2-aminoethoxy) cyclohexyl)oxy)-N-(2-((2-(((1R,4r)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)- 4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)butanamido)acetamido) ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate

To a solution of tert-butyl (1R,4r)-4-(4-(((1r,4R)-4-(2-(2-((E)-4-(((1 r,4r)-4-(2- (dibenzylamino)ethoxy)cyclohexyl)oxy)-N-(2-((2-(((1R,4r)-4-(4-(((1r,4R)-4-(methoxy carbonyl) cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)but-2-enamido) acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxylate (170 mg, 0.129 mmol) in methanol (22.7 mL) under nitrogen was added Pd-C (13.8 mg, 0.013 mmol). The flask was evacuated and back-filled with a helium gas balloon and stirred at RT for 19 h. The flask was evacuated and placed under nitrogen. Additional additional Pd-C (13.8 mg, 0.013 mmol) was added, and then the flask was evacuated and back-filled with new hydrogen gas balloon. The reaction was stirred at RT for 3 nights, then filtered through celite plug, washing with additional methanol (20 mL). The filtrate was concentrated and dried on high vac to provide the crude title compound as a yellow film which was used without further purification or characterization (144 mg, 0.036 mmol, 27.4 % yield). LC-MS m/z 1135.98 (M+H)⁺. Step 9: tert-Butyl (1R,4r)-4-(4-(((1r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4-(((1r,4R)-4- (methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2- oxoethyl)-4-(((1 S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethoxy)cyclohexyl)oxy)butanamido)acetamido)ethoxy)cyclohexyl)oxy)b utanamido)cyclohexane-1 -carboxylate

To a solution of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (50 mg, 0.227 mmol) and HATU (112 mg, 0.295 mmol) in dichloromethane (22.7 mL) was added DIPEA (0.079 ml, 0.454 mmol).

The reaction was stirred at RT for 15 minutes. tert-Butyl (1R,4r)-4-(4-(((1r,4R)-4-(2-(2- (4-(((1r,4r)-4-(2-aminoethoxy)cyclohexyl)oxy)-N-(2-((2-(((1R,4r)-4-(4-(((1r,4R)-4- (methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2- oxoethyl)butanamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxylate (196 mg, 0.173 mmol) was added and the reaction was continued at RT for 3 h. A solution of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (50 mg, 0.227 mmol), HATU (112 mg, 0.295 mmol) and DIPEA (0.079 ml, 0.454 mmol) in 0.5 mL DMF was prepared and added to the above mixture. The reaction was stirred at RT for 3 nights, then diluted with additional dichloromethane (10 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (20 mL) and brine (20 mL), dried over sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-20% methanol in dichloromethane to afford the title compound as a yellow film (88 mg, 0.057 mmol, 28.7% yield). LC-MS m/z 669.80 (M+2H)/2. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.03 - 1.16 (m, 5 H) 1.18 - 1.29 (m, 10 H) 1.35 - 1.38 (m, 4 H) 1.41 (s, 9 H) 1.44 - 1.59 (m, 4H) 1.79 - 1.90 (m, 9H) 1.91 - 2.02 (m, 15 H), 2.05 - 2.14 (m, 1 H) 2.18 - 2.24 (m, 4 H) 2.31 - 2.37 (m, 2 H) 2.62 - 2.67 (m, 3 H) 2.75 - 2.89 (m, 3 H) 3.01 - 3.10 (m, 1 H) 3.13 - 3.31 (m, 7 H) 3.34 - 3.47 (m, 13 H) 3.48 - 3.57 (m, 4 H) 3.60 - 3.76 (m, 6H) 3.90 (s, 1 H), 4.03 (s, 1 H) 4.73 - 4.84 (m, 1 H) 5.70 - 5.88 (m, 2 H) 6.24 (br t, J=4.89 Hz, 1 H) 6.65 (br t, J=5.14 Hz, 1 H) 7.35 (dd, J=7.83, 4.89 Hz, 1 H) 7.56 (dt, J=7.83, 1.96 Hz, 1 H) 8.50 (d, J=1.96 Hz, 1 H) 8.60 (dd, J=4.89, 1.47 Hz, 1 H) 8.93 (br t, J=5.14 Hz, 1 H).

Step 10: (1R, 4r)-4-(4-((( 1 r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4-(((1 r,4R)-4-(methoxy- carbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4- (((1 S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido) ethoxy)cyclohexyl)oxy)butanamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cy clohexane-1 -carboxylic acid

To a solution of tert-butyl (1R,4r)-4-(4-(((1r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4- (((1 r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2- oxoethyl)-4-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido) ethoxy)cyclohexyl)oxy)butanamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexa ne-1 -carboxylate (88 mg, 0.066 mmol) in dichloromethane (2.2 mL) was added TFA (101 μl, 1.316 mmol). The reaction was stirred at RT for 20 h. Additional TFA (101 μl, 1.316 mmol) was added, and the reaction was stirred at RT for 20 h. The mixture was concentrated and dried on high vac to provide the crude title compound as an orange film (130.1 mg, 0.064 mmol, 97% yield). LC-MS m/z 1281.63 (M+H)⁺. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.26

- 1.39 (m, 14 H) 1.41 - 1.49 (m, 10 H) 1.57 - 1.60 (m, 10 H) 1.87 - 2.22 (m, 24 H) 2.29 - 2.51 (m, 4 H) 2.53 - 2.65 (m, 3 H) 2.77 - 2.87 (m, 3 H) 3.15 - 3.24 (m, 2 H) 3.37 - 3.58 (m, 10 H) 3.62 - 3.69 (m, 6 H) 3.72 - 3.82 (m, 6 H) 4.06 - 4.10 (m, 1 H) 4.30 - 4.36 (m, 1 H) 5.30 (br d, J=6.85 Hz, 1 H) 7.57 - 7.73 (m, 2 H) 8.64 - 8.74 (m, 2 H) 8.81 (dd, J=4.65, 1.22 Hz, 1 H) 8.90

- 8.99 (m, 2 H). Step 11 : Methyl (1R,4r)-4-(4-(((1r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4-(((1R,4R)-4- (((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin- 4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy) cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2- (pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido) acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate

To a solution of (1R,4r)-4-(4-(((1r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4-(((1r,4R)-4- (methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)- 4-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy) cyclohexyl)oxy)butanamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxylic acid compound with 2,2,2-trifluoroacetic acid (180 mg, 0.129 mmol) and (S)-1- ((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin- 4-yl)amino)pyrrolidin-2-one hydrochloride (78 mg, 0.155 mmol) in dichloromethane (2.6 mL) was added 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1 , 1,3,3-tetramethylisouronium hexafluorophosphate(V) (63.8 mg, 0.168 mmol), and N-ethyl-N-isopropylpropan-2-amine (112 μl, 0.645 mmol). The reaction was stirred at RT for 1.5 h, then diluted with additional dichloromethane (30 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (20 mL) and brine (20 mL), dried over sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-20% methanol in dichloroethane to afford the title compound as an orange film (182 mg, 0.066 mmol, 51.5% yield). LC-MS m/z 864.95 (M+2H)/2. ¹H N MR (400 MHz, CHLOROFORM-d) δ ppm 1.03 - 1.30 (m, 14 H) 1.34 - 1.41 (m, 2 H) 1.58 (br s, 5 H) 1.61 - 1.72 (m, 3 H) 1.75 - 2.05 (m, 28 H) 2.14 - 2.22 (m, 6 H) 2.26 (br s, 3 H) 2.38 - 2.47 (m, 1 H) 2.60 - 2.65 (m, 4 H) 2.65 - 2.71 (m, 2 H) 2.73 (br s, 5 H) 3.03 - 3.12 (m, 1 H) 3.13 - 3.26 (m, 6 H) 3.33 - 3.43 (m, 20 H) 3.46 - 3.56 (m, 6 H) 3.59 - 3.70 (m, 5 H) 3.89 (s, 2 H) 3.98 - 4.03 (m, 2 H) 4.77 (d, J=6.36 Hz, 1 H) 4.79 - 4.86 (m, 1 H) 5.01 - 5.10 (m, 1 H) 5.82 (br d, J=7.82 Hz, 1 H) 5.85 - 5.92 (m, 1 H) 6.32 (br t, J=4.89 Hz, 1 H) 6.74 (dt, J=14.92, 5.26 Hz, 1 H) 7.33 (dd, J=7.82, 4.89 Hz, 1 H) 7.54 (br d, J=7.82 Hz, 1 H) 7.85 (s, 2 H) 8.45 (br d, J=7.34 Hz, 1 H) 8.48 (d, J=1.96 Hz, 1 H) 8.55 - 8.59 (m, 1 H) 8.61 (d, J=0.98 Hz, 1 H) 8.85 (s, 1 H) 8.91 - 9.01 (m, 1 H) 9.12 - 9.29 (m, 1 H).

Step 12: 4-(4-(((1 r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5- (isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino) pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy) ethyl)amino)-2-oxoethyl)-4-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)acetamido)ethoxy) cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylic acid

To a solution of methyl (1R,4r)-4-(4-(((1r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4- (((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl) amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(((1 S,4r)-4-(2-((2S,3S)-1- methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido) acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate (180 mg, 0.104 mmol) in tetrahydrofuran (2.1 mL) was added 1N aqueous sodium hydroxide (208 μl, 0.208 mmol). The reaction was stirred at RT for 3 nights. The mixture was neutralized with 1 N HCI, concentrated in vacuo, and dried on high vac to afford the crude title compound as a yellow solid (199 mg, 0.083 mmol, 79% yield). LC-MS m/z 857.46 (M+2H)/2. ¹ H NMR (400 MHz, DMSO-d₆) δ 0.77 - 0.92 (m, 1 H) 0.99 - 1.26 (m, 9 H) 1.26 - 1.42 (m, 3H) 1.44 - 1.56 (m, 1 H) 1.58 - 1.75 (m, 4 H) 1.78 - 1.95 (m, 7 H) 1.96 - 2.15 (m, 4 H) 2.18- 2.40 (m, 3 H) 2.39 - 2.58 (m, 12 H) 2.58 - 2.67 (m, 1 H) 2.72 - 3.09 (m, 3 H) 3.13 - 3.47 (m, 13 H) 3.49 - 3.98 (m, 46 H) 3.99 - 4.26 (m, 6 H) 4.84 (br d, J=5.87 Hz, 1 H) 5.38 - 5.52 (m, 1 H) 7.11 - 7.36 (m, 2 H) 7.39 - 7.50 (m, 1 H) 7.64 - 7.78 (m, 1 H) 8.03 - 8.16 (m, 2 H) 8.26 - 8.42 (m, 2 H) 8.73 - 8.91 (m, 2 H) 9.01 (d, J=2.93 Hz, 1 H) 9.41 - 9.63 (m, 1 H) 10.15 - 10.32 (m, 1 H) 11.65 - 12.27 (m, 3 H).

Step 13: (((S)-1 -Carboxy-5-((S)-2-((1R,4S)-4-(4-(((1 r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)- 4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl) cyclohexyl) amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(((1S,4r)-4- (2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy) cyclohexyl)oxy)butanamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexa ne-1 -carboxamido)-3-(naphthalen-2-yl)propanamido)pentyl)carbamoyl)-L-glutamic acid (Example 1)

To a solution of 4-(4-(((1r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4-(((1R,4R)-4- (((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4- yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl) oxy)ethyl)amino)-2-oxoethyl)-4-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)acetamido)ethoxy) cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylic acid (175 mg, 0.102 mmol) in dichloromethane (6.9 mL) was added HATU (58.2 mg, 0.153 mmol), and N-ethyl-N- isopropylpropan-2-amine (0.053 mL, 0.306 mmol). Di-tert-butyl (((S)-6-((S)-2-amino-3- (naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (69.9 mg, 0.102 mmol) was added and the reaction was stirred at RT for 2 h. The mixture was diluted with additional dichloromethane (20 mL), washed with saturated aqueous sodium bicarbonate (15 mL) and brine (15 mL), dried over sodium sulfate, filtered, concentrated in vacuo, and dried on high vac for 1 h. To a solution of the resultant residue in dichloromethane (6.9 mL) was added TFA (0.157 mL, 2.042 mmol). After 19 h, additional TFA (0.157 mL, 2.042 mmol) was added. After 2 h, the mixture was concentrated in vacuo. The resultant residue was purified

The resultant residue was purified via MDAP (XSelect™ CSH C18 5um column, 40 mL/min) eluting with a gradient of 30 to 85 % acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075 %) to provide the title compound as an off-white solid (47.9 mg, 0.020 mmol, 19.3 % yield). LC-MS m/z 1107.30 (M+2H)/2. HPLC: 91.8% pure @ 254 nm. ¹ H NMR (400 MHz, DMSO-d₆) δ ppm 0.93 (br d, J=6.36 Hz, 2 H) 1.01

- 1.07 (m, 3 H) 1.08 - 1.27 (m, 11 H) 1.28 - 1.53 (m, 5 H) 1.54 - 1.68 (m, 7 H) 1.69 - 1.75 (m,

1 H) 1.77 - 1.93 (m, 10 H) 1.96 - 2.08 (m, 4 H) 2.09 - 2.18 (m, 4 H) 2.22 (br t, J=7.09 Hz, 1 H) 2.32 (dt, J=12.47, 6.48 Hz, 2 H) 2.38 - 2.46 (m, 1 H) 2.47 - 2.55 (m, 26 H) 2.61 (br s, 1 H) 2.64

- 2.74 (m, 1 H) 2.89 - 3.06 (m, 3 H) 3.08 - 3.25 (m, 9 H) 3.27 - 3.45 (m, 30 H) 3.47 - 3.58 (m,

2 H) 3.81 - 3.91 (m, 2 H) 3.93 - 4.09 (m, 3 H) 4.44 - 4.60 (m, 2 H) 4.65 (d, J=5.87 Hz, 1 H) 4.93 - 5.04 (m, 1 H) 6.13 - 6.24 (m, 1 H) 6.41 (br d, J=7.82 Hz, 1 H) 7.35 - 7.51 (m, 3 H) 7.60 (br d, J=7.34 Hz, 2 H) 7.65 - 7.73 (m, 2 H) 7.76 - 7.82 (m, 2 H) 7.85 (br d, J=7.34 Hz, 1 H) 7.90 (d, J=8.80 Hz, 1 H) 7.97 - 8.05 (m, 2 H) 8.05 - 8.12 (m, 2 H) 8.25 - 8.31 (m, 1 H) 8.45 - 8.50 (m, 1 H) 8.56 (br d, J=4.40 Hz, 1 H) 8.59 - 8.70 (m, 2 H) 8.75 - 8.84 (m, 1 H) 8.96 (s, 2H).

EXAMPLE 2

(((S)-1 -Carboxy-5-((S)-2-((1R,4S)-4-(4-((( 1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2- (((1R,4R)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl) cycl ohexyl )am i no)-4-oxobutoxy)cyclohexyl )oxy)ethyl )am i no)-2-oxoethy I )-4-(4- (((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxamido) acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxamido)-3- (naphthalen-2-yl)propanamido)pentyl)carbamoyl)-L-glutamic acid compound with 2,2,2-trifluoroacetic acid

Step 1 : tert-Butyl N-(2-methoxy-2-oxoethyl)-N-((1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1- methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1-carbonyl)glycinate

To a solution of (1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylic acid (2.00 g, 3.49 mmol), tert-butyl (2-methoxy-2-oxoethyl)glycinate (0.850 g, 4.18 mmol) and HATU (1.72 g, 4.53 mmol) in Dichloromethane (35 mL) was added N-ethyl-N- isopropylpropan-2-amine (1.21 mL, 6.97 mmol). The reaction was stirred at RT overnight for 22 h, then diluted with saturated aqueous sodium bicarbonate (50 mL). The layers were separated and the organic fraction was washed with brine (50 mL), dried over sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-15% methanol in dichloromethane to provide the title compound as a pale yellow foam (1.79 g, 1.96 mmol, 56.1% yield). ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.12 - 1.33 (m, 7 H) 1.41 - 1.54 (m, 8 H) 1.71 - 1.77 (m, 2 H) 1.80 - 2.02 (m, 8 H) 2.03 - 2.16 (m, 2 H) 2.21 - 2.28 (m, 2 H) 2.34 (ddd, J=11.62, 8.19, 3.67 Hz, 1 H) 2.69 (s, 3 H) 2.74 - 2.93 (m, 3 H) 3.23 (td, J=8.80, 4.40 Hz, 2 H) 3.41 - 3.49 (m, 4 H) 3.65 - 3.86 (m, 4 H) 4.07 (d, J=1.96 Hz, 2 H) 4.17 (s, 2 H) 4.81 (d, J=6.36 Hz, 1 H) 5.51 (br d, J=7.82 Hz, 1 H) 5.78 (br s, 1 H) 7.35 - 7.42 (m, 1 H) 7.54 - 7.65 (m, 1 H) 8.55 (d, J=1.47 Hz, 1 H) 8.65 (dd, J=4.65, 1.71 Hz, 1 H).

Step 2: N-(2-methoxy-2-oxoethyl)-N-((1R,4r)-4-(4-((( 1 S,4R)-4-(2-((2S,3S)-1 - methyl -5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1-carbonyl)glycine

To a solution of tert-butyl N-(2-methoxy-2-oxoethyl)-N-((1R,4r)-4-(4-(((1S,4R)-4-(2- ((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1-carbonyl)glycinate (1.48 g, 1.96 mmol) in anhydrous 1,4-Dioxane (9.78 mL) was added 3M HCI in CPME (9.78 ml, 29.3 mmol). The reaction was stirred at room temperature for 24 h. A gummy white solid was scraped from the sides of the flask. Additional 3M HCI in CPME (3.26 mL, 9.78 mmol) was added, and the mixture was stirred at RT for another 27 h, then concentrated in vacuo and dried on high vac to provide the title compound as a white solid (1.36 g, 1.43 mmol, 73.3% yield). LCMS m/z 702.39 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.07 - 1.25 (m, 5 H) 1.31 - 1.50 (m, 3 H) 1.54 - 1.71 (m, 5 H) 1.72 - 1 .91 (m, 5 H) 2.07 (br t, J=7.58 Hz, 2 H) 2.29 - 2.46 (m, 2 H) 2.66 - 2.77 (m, 2 H) 2.93 - 3.04 (m, 1 H) 3.09 - 3.25 (m, 4 H) 3.27 - 3.51 (m, 5 H) 3.60 - 3.72 (m, 2 H) 3.92 - 4.11 (m, 3 H) 4.20 - 4.39 (m, 3 H) 4.68 - 4.77 (m, 2 H) 7.60 - 7.70 (m, 2 H) 7.90 - 7.98 (m, 1 H) 8.00 - 8.06 (m, 1 H) 8.57 - 8.65 (m, 1 H) 8.66 - 8.72 (m, 1 H).

Step 3: tert-Butyl (1R,4R)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-methoxy-2- oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1 -methyl -5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxamido) acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate

To a solution of N-(2-methoxy-2-oxoethyl)-N-((1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1- methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carbonyl)glycine (250 mg, 0.356 mmol), tert-butyl (1R,4r)-4-(4-(((1 r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1 -carboxylate (182 mg, 0.427 mmol) and HATU (176 mg, 0.463 mmol) in Dichloromethane (23 mL) was added DIPEA (0.124 ml, 0.712 mmol). The reaction was stirred at RT for 2 h, then diluted with additional DCM (20 mL). The mixture was washed with saturated aqueous sodium bicarbonate (20 mL) and brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-20% methanol in dichloromethane to provide the title compound as a clear film (197 mg, 0.149 mmol, 41.8% yield). LCMS m/z 1110.39 (M+H)⁺. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.02 - 1.30 (m, 12 H) 1.38 - 1.41 (m, 9 H) 1.42 - 1 .52 (m, 2 H) 1.54 - 1 .67 (m, 2 H) 1 .70 - 1 .85 (m, 4 H) 1 .87 - 2.03 (m,8 H) 2.15 - 2.26 (m, 4 H) 2.61 (s, 3 H) 2.71 - 2.79 (m, 2 H) 2.81 - 2.90 (m, 1 H) 3.11 - 3.28 (m, 4 H) 3.33 - 3.56 (m, 20 H) 3.61 - 3.72 (m, 3 H) 3.73 - 3.80 (m, 3 H) 4.02 (d, J=13.69 Hz, 3 H) 4.79 (d, J=6.85 Hz, 1 H) 5.85 - 6.07 (m, 2 H) 6.36 - 6.47 (m, 1 H) 7.29 - 7.38 (m, 1 H) 7.59 (dt, J=7.83, 1.96 Hz, 1 H) 8.20 - 8.32 (m, 1 H) 8.49 (d, J=1.96 Hz, 1 H) 8.53 - 8.63 (m, 2 H).

Step 4: N-(2-((2-(((1R,4R)-4-(4-(((1R,4R)-4-(tert-Butoxycarbonyl)cyclohexyl) amino-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-N-((1R,4R)-4-(4-(((1S,4R)-

4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy) cyclohexyl)oxy)butanamido)cyclohexane-1 -carbonyl)glycine

To a solution of tert-butyl (1R,4R)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-methoxy-2- oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate (195 mg, 0.176 mmol) in THF (3.5 mL) was added 1N aqueous sodium hydroxide (351 μl, 0.351 mmol). The reaction was stirred at RT for 1 h, then neutralized with 1 N aqueous HCI. The mixture was concentrated in vacuo and dried on high vac to provide the crude title compound as a white solid (245.4 mg, theoretical) that was used without further purification or characterization.

Step 5: tert-Butyl 4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1r,4R)-4-

(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2- oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1 -methyl -5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxamido) acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate

To a solution of N-(2-((2-(((1R,4R)-4-(4-(((1 r,4R)-4-(tert- butoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-N- ((1R,4R)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carbonyl)glycine (190 mg, 0.173 mmol) and methyl (1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1 -carboxylate (73.3 mg, 0.191 mmol) in DCM (0.9 mL) was added HATU (86 mg, 0.225 mmol) and N-ethyl-N-isopropylpropan-2-amine (60.5 μl, 0.347 mmol). The reaction was stirred at RT for 16 h, then diluted with additional DCM (20 mL). The mixture was washed with saturated aqueous sodium bicarbonate (10 mL) and brine (10 mL), dried over sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chormatography eluting with 0-20% methanol in DCM to afford the title product as a light yellow solid. LCMS m/z 732.13 (M+2H)/2. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.06 - 1.28 (m, 12 H) 1.37 - 1.64 (m, 16 H) 1.76 - 2.06 (m, 28 H) 2.14 - 2.30 (m, 8 H) 2.62 (br s, 3 H) 2.72 - 2.88 (m, 3 H) 3.20 (br s, 6 H) 3.33 - 3.55 (m, 23 H) 3.59 - 3.74 (m, 6 H) 3.86 (br s, 2 H) 4.01 (br s, 2 H) 4.77 (br d, J=5.87 Hz, 1 H) 5.85 (br d, J=6.85 Hz, 3 H) 6.27 (br s, 1 H) 6.56 (br s, 1 H) 7.21 - 7.41 (m, 1 H) 7.55 (br d, J=7.34 Hz, 1 H) 8.37 - 8.78 (m, 2 H) 9.20 (br s, 1 H).

Step 6: (1R, 4r)-4-(4-((( 1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1 r,4R)-4- (Methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)- 2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1 -methyl -5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxamido) acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylic acid

To a solution of tert-butyl 4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4- (((1 r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2- oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxamido)acetamido) ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxylate (120 mg, 0.082 mmol) in DCM (0.4 mL) was added TFA (0.3 mL, 4.1 mmol). The reaction was stirred at RT for 2.5 h, then concentrated in vacuo and dried on high vac to provide the crude title compound as a pale yellow oil (225 mg, 0.081 mmol, 99% yield) that was used without further purification or characterization. LCMS m/z 704.17 (M+H)⁺.

Step 7: Methyl (1R,4r)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4- (((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl) quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4- oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1- methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1-carboxamido)acetamido)ethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1 -carboxylate

To a solution of (1R,4r)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4- (((1 r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2- oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxamido)acetamido) ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxylic acid (125 mg, 0.089 mmol) and N-ethyl-N-isopropylpropan-2-amine (155 μl, 0.889 mmol) in DCM (1.8 mL) was added 2-(3/7-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1 , 1,3,3-tetramethylisouronium hexafluorophosphate(V) (50.7 mg, 0.133 mmol), and (S)-1-((1S,2R,4R)-2-amino-4- (isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2- one hydrochloride (53.4 mg, 0.107 mmol). The reaction was stirred at RT for 3 nights, then diluted with additional DCM (30 mL). The organic mixture was washed with saturated aqueous sodium bicarbonate (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-20% methanol in DCM to provide the title compound as a pale yellow oil (158 mg, 0.083 mmol, 93.1% yield). ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.04 - 1.15 (m, 9 H) 1.23 (br s, 11 H) 1.35 - 1.42 (m, 4 H) 1.44 - 1.54 (m, 3 H) 1.58 - 1.69 (m, 4 H) 1.76 - 1.86 (m, 8 H) 1.88 - 2.04 (m, 10 H) 2.14 - 2.25 (m, 10 H) 2.26 - 2.47 (m, 10 H) 2.64 (s, 3 H) 2.67 - 2.72 (m, 1 H) 2.73 - 2.90 (m, 3 H) 3.09 (q, J=7.34 Hz, 1 H) 3.16 - 3.27 (m, 4 H) 3.35 - 3.53 (m, 33 H) 3.58 - 3.74 (m, 6 H) 3.87 (br s, 2 H) 3.97 - 4.10 (m, 3 H) 4.79 (br d, J=6.36 Hz, 2 H) 5.09 (br d, J=3.91 Hz, 1 H) 5.83 - 5.99 (m, 3 H) 6.34 (br d, J=4.40 Hz, 1 H) 6.59 - 6.73 (m, 1 H) 7.32 - 7.42 (m, 1 H) 7.56 (br d, J=7.82 Hz, 1 H) 7.86 (s, 2 H) 8.50 (br s, 2 H) 8.55 - 8.67 (m, 2 H) 8.83 (br s, 1 H) 9.23 (br s, 1 H). Step 8: 4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-((( 1 R,4R)-4-

(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin- 4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy) cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1 -methyl -5-oxo-2- (pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido) cyclohexane-1 -carboxam id o)acetami do )ethoxy)cy cl o hexyl )oxy)butan amido) cyclohexane-1 -carboxylic acid

To a solution of methyl (1R,4r)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4- (4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl) amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1 S,4R)-4-(2-((2S,3S)-1- methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido) cyclohexane-1-carboxamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxylate (155 mg, 0.084 mmol) in Tetrahydrofuran (1.7 mL) was added 1 N aqueous sodium hydroxide (aq) (167 μl, 0.167 mmol). The reaction was stirred at RT for 18 h, then concentrated in vacuo and dried on high vac to provide the crude title compound as a yellow solid (155 mg, 0.076 mmol, 91% yield). LCMS m/z 919.78 (M+2H)/2. ¹H NMR (400 MHz, METHANOL-d4) δ ppm 0.99 - 1.05 (m, 3 H) 1.12 - 1.18 (m, 3 H) 1.20 - 1.38 (m, 16 H) 1.47 - 1.63 (m, 6 H) 1.66 - 1.76 (m, 3 H) 1.78 - 1.85 (m, 6 H) 1.87 - 1.91 (m, 9H) 1.98 (br d, J=7.34 Hz, 16 H) 2.04 - 2.19 (m, 2 H) 2.20 - 2.25 (m, 6 H) 2.26 - 2.30 (m, 3 H) 2.31 - 2.45 (m, 2 H) 2.48 - 2.60 (m, 1 H) 2.64 - 2.69 (m, 3 H) 2.69 - 2.76 (m, 2 H) 2.80 - 2.90 (m, 1 H) 3.04 - 3.13 (m, 1 H) 3.23 - 3.31 (m, 4 H) 3.32 - 3.38 (m, 6 H) 3.39 - 3.49 (m, 8 H) 3.50 - 3.59 (m, 6 H) 3.61 - 3.69 (m, 4 H) 3.71 - 3.78 (m, 6 H) 3.97 - 4.11 (m, 3 H) 4.18 - 4.28 (m, 2 H) 4.65 (br d, J=2.93 Hz, 1 H) 4.84 (d, J=6.36 Hz, 1 H) 5.26 (t, J=7.83 Hz, 1 H) 7.33 (dd, J=8.31 , 4.40 Hz, 1 H) 7.54 (dd, J=7.83, 4.89 Hz, 1 H) 7.81 (br d, J=7.83 Hz, 1 H) 7.90 (d, J=8.80 Hz, 1 H) 8.05 (dd, J=9.05, 1.71 Hz, 1 H) 8.19 (dd, J=8.31, 1.47 Hz, 1 H) 8.48 - 8.56 (m, 2 H) 8.57 - 8.61 (m, 2 H) 8.81 (s, 1 H).

Step 9: Di-tert-butyl (((S)-1-(tert-butoxy)-6-((S)-2-((1R,4S)-4-(4-(((1R,4R)-4-(2-(2- ((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2- ((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl) carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4- (4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido) ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxamido)acetamido)ethoxy) cyclohexyl)oxy)butanamido)cyclohexane-1-carboxamido)-3-(naphthalen-2- yl)propanamido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate

To a solution of 4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1R,4R)-4- (((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4- yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl) oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1 S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylic acid (130 mg, 71.0 μmol) , di-tert-butyl (((S)-6-((S)-2-amino-3-(naphthalen-2-yl)propanamido)-1- (tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (48.4 mg, 71.0 μmol), HATU (40.3 mg, 106 μmol) in DCM (7 mL) was added DIPEA (27.4 mg, 37.0 μL, 212 μmol) and DMF (0.5 mL). The reaction was stirred at RT for 1 .5 h, then diluted with additional DCM (20 mL). The organic mixture was washed with saturated aqueous sodium bicarbonate (15 mL) and brine (15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated The resultant residue was purified via silica gel chormatography eluting with 0-20% methanol in dichloromethane to provide the title compound as a white solid (104 mg, 42.0 mmol, 58.8% yield). LC-MS m/z 1253.65 (M+2H)/2. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.80 - 1.04 (m, 5 H) 1.04 - 1.18 (m, 9 H) 1.18 - 1.33 (m, 15 H) 1.35 - 1.42 (m, 3 H) 1.44 - 1.47 (m, 16 H) 1.48 - 1.63 (m, 15 H) 1.64 - 1.72 (m, 5 H) 1.89 (br s, 13 H) 1.91 - 2.08 (m, 21 H) 2.12 - 2.27 (m, 11 H) 2.27 - 2.36 (m, 2 H) 2.37 - 2.52 (m, 3 H) 2.67 (s, 3 H) 2.69 - 2.69 (m, 1 H) 2.69 - 2.73 (m, 1 H) 2.86 (br s, 3 H) 2.96 - 3.13 (m, 2 H) 3.18 - 3.32 (m, 7 H) 3.48 (br s, 14 H) 3.50 (br s, 6 H) 3.70 (br s, 2 H) 3.93 (br s, 1 H) 4.00 - 4.14 (m, 3 H) 4.25 (br s, 1 H) 4.53 - 4.68 (m, 1 H) 4.78 - 4.85 (m, 1 H) 4.89 (br d, J=4.40 Hz, 1 H) 5.08 (td, J=7.58, 1.96 Hz, 1 H) 5.51 - 5.64 (m, 1 H) 5.69 (br dd, J=15.65, 7.82 Hz, 2 H) 6.05 (br d, J=2.93 Hz, 1 H) 6.31 - 6.39 (m, 1 H) 6.46 - 6.56 (m 1 H) 7.15 (br s, 1 H) 7.33 - 7.51 (m, 3 H) 7.58 (dt, J=7.83, 1.96 Hz, 2 H) 7.64 (s, 1 H) 7.71 (br d, J=8.31 Hz, 1 H) 7.89 (d, J=1.47 Hz, 2 H) 8.39 - 8.47 (m, 1 H) 8.54 (d, J=1.96 Hz, 1 H) 8.60 -

8.65 (m, 1 H) 8.67 (d, J=2.45 Hz, 1 H) 8.91 (s, 1 H) 9.18 - 9.34 (m, 2 H).

Step 10: (((S)-1 -Carboxy-5-((S)-2-((1R,4S)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2- (((1R,4R)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl) cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(4- (((1 S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido) ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxamido)acetamido) ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxamido)-3-(naphthalen-2- yl)propanamido)pentyl)carbamoyl)-L-glutamic acid compound with 2,2,2-trifluoroacetic acid (Example 2)

To a solution of di-tert-butyl (((S)-1-(tert-butoxy)-6-((S)-2-((1R,4S)-4-(4-(((1R,4R)-4-(2- (2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2- ((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl) cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1 S,4R)-4-(2- ((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl) oxy)butanamido)cyclohexane-1-carboxamido)acetamido)ethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanamido)-1-oxohexan-2- yl)carbamoyl)-L-glutamate (104 mg, 0.042 mmol) in DCM (7 mL) was added TFA (0.272 mL, 3.53 mmol). After 24 h, the mixture was concentrated and the resultant residue was purified via MDAP (XSelect™ CSH C18 5um column, 40 mL/min) eluting with a gradient of 15 to 55 % acetonitrile in water containing trifluoroacetic acid (0.1%) to provide the title compound as a white solid (61.1 mg, 0.021 mmol, 29.4 % yield). LC-MS m/z 1170.25 (M+2H)/2. HPLC: 100% pure @ 254 nm. ¹ H NMR (400 MHz, METHANOL-d4 ) δ ppm 1.40 (br s, 21 H) 1.42 - 1.45 (m,

2 H) 1.47 - 1.64 (m, 5 H) 1.66 - 1.75 (m, 1 H) 1.79 - 1.86 (m, 8 H) 1.87 - 1.91 (m, 2 H) 1.92 -

2.04 (m, 14 H) 2.11 - 2.28 (m, 10 H) 2.35 - 2.50 (m, 4 H) 2.59 - 2.66 (m, 1 H) 2.66 - 2.76 (m,

3 H) 2.80 - 2.83 (m, 2 H) 2.85 - 2.93 (m, 2 H) 3.00 - 3.03 (m, 1 H) 3.05 - 3.17 (m, 3 H) 3.22 -

3.39 (m, 34 H) 3.40 - 3.51 (m, 8 H) 3.51 - 3.60 (m, 5 H) 3.61 - 3.69 (m, 2 H) 3.71 - 3.79 (m, 1

H) 3.80 - 3.94 (m, 2 H) 4.01 (d, J=6.85 Hz, 2 H) 4.16 - 4.25 (m, 3 H) 4.27 - 4.39 (m, 2 H) 4.68

(dd, J=8.56, 6.60 Hz, 1 H) 5.03 (dd, J=6.60, 1.71 Hz, 1 H) 5.61 (br t, J=9.29 Hz, 1 H) 7.35 -

7.51 (m, 3 H) 7.69 (s, 1 H) 7.75 - 7.86 (m, 3 H) 7.94 - 8.06 (m, 3 H) 8.33 (d, J=8.80 Hz, 1 H)

8.40 (br d, J=7.34 Hz, 1 H) 8.78 - 8.86 (m, 2 H) 8.89 (s, 1 H) 9.01 (s, 1 H).

EXAMPLE 3

((( S)-1 -Carboxy-5-((S)-2-(2-((1R,4S)-N-(2-((( 1 R,2S,5R)-5-

(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4- yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1- methyl -5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1-carboxamido)acetamido)-3-(naphthalen-2-yl)propanamido) pentyl)carbamoyl)-L-glutamic acid

Step 1 : di-tert-Butyl (((S)-1-(tert-butoxy)-6-((S)-2-(2-((1R,4S)-N-(2-methoxy-2- oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxamido) acetamido)-3-(naphthalen-2-yl)propanamido)-1-oxohexan-2-yl)carbamoyl) -L- glutamate.

To a solution of N-(2-methoxy-2-oxoethyl)-N-((1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1- methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1-carbonyl)glycine (50 mg, 0.071 mmol), di-tert-butyl (((S)-6-((S)-2- amino-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L- glutamate (48.8 mg, 0.071 mmol) and HATU (32.5 mg, 0.085 mmol) in Dichloromethane (22.7 mL) was added DIPEA (0.025 ml, 0.142 mmol). The reaction was stirred at RT for 19 h, then diluted with additional DCM (20 mL). The mixture was washed with saturated sodium bicarbonate (20 mL) and brine (20 mL), dried over sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-20% methanol in dichloromethane to provide the title compound as and off-white solid (39.5 mg, 0.026 mmol, 40.5% yield). LCMS m/z 685.15 (M+2H)/2. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.58 - 0.73 (m, 1 H) 0.81 - 0.96 (m, 1 H) 1.08 - 1.32 (m, 11 H) 1.33 - 1.48 (m, 27 H) 1.55 - 1.77 (m, 4 H) 1.79 - 2.00 (m, 12 H) 2.02 - 2.13 (m, 2 H) 2.16 - 2.26 (m, 2 H) 2.28 - 2.43 (m, 2 H) 2.67 (s, 3H) 2.76 - 2.88 (m, 3 H) 3.16 - 3.26 (m, 3 H) 3.28 - 3.35 (m, 1 H) 3.38 - 3.52 (m, 7 H) 3.68 - 3.82 (m, 4 H) 3.89 - 4.03 (m, 2 H) 4.09 - 4.24 (m, 2 H) 4.25 - 4.34 (m, 1 H) 4.37 - 4.49 (m, 1 H) 4.77 - 4.86 (m, 1 H) 5.43 - 5.55 (m, 1 H) 5.65 - 5.74 (m, 1 H) 5.77 - 5.94 (m, 2 H) 7.26 - 7.33 (m, 1 H) 7.35 - 7.40 (m, 1 H) 7.42 - 7.50 (m, 2 H) 7.58 (br d, J=7.82 Hz, 1 H) 7.64 - 7.84 (m, 3 H) 8.54 (d, J=1.47 Hz, 1 H) 8.57 - 8.69 (m, 2 H).

Step 2: Sodium N-(2-(((7S,11S,18S)-7,11-bis(tert-butoxycarbonyl)-2,2-dimethyl- 19-(naphthalen-2-yl)-4, 9,17-trioxo-3-oxa-8, 10, 16-tri azanonadecan-18-yl)amino)-2- oxoethyl)-N-((1R,4S)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carbonyl)glycinate.

To a solution of di-tert-butyl (((S)-1-(tert-butoxy)-6-((S)-2-(2-((1R,4S)- N-(2-methoxy-2- oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxamido)acetamido)-3- (naphthalen-2-yl)propanamido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (39 mg, 0.028 mmol) in THF (2.3 mL) was added aqueous 1.0 M sodium hydroxide (42.7 μl, 0.043 mmol). The homogenous pale yellow solution was stirred at RT for 2 nights, then concentrated in vacuo. The resultant residue was azeotroped with toluene (2x) and dried on high vac to provide the title compound as a clear film (38.6 mg, 0.028 mmol, quantitative yield) that was used without further purification or characterization. LCMS m/z 678.11 (M+2H)/2.

Step 3: (((S)-1 -Carboxy-5-((S)-2-(2-((1R,4S)-N-(2-(((1R,2S,5R)-5-(isopropyl

(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1- yl)cyclohexyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin- 3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxamido)acetamido)-3-(naphthalen-2-yl)propanamido)pentyl)carbamoyl)-L- glutamic acid (Example 3).

To a solution of sodium N-(2-(((7S,11S, 18S)-7, 11-bis(tert-butoxycarbonyl)-2,2- dimethyl-19-(naphthalen-2-yl)-4, 9, 17-trioxo-3-oxa-8, 10,16-tri azanonadecan-18-yl)amino)-2- oxoethyl)-N-((1R,4S)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine- 3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carbonyl)glycinate (19 mg, 0.014 mmol) and (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one hydrochloride (6.91 mg, 0.014 mmol) in DCM (69.0 μl) was added HATU (6.82 mg, 0.018 mmol) and DIPEA (7.23 μl, 0.041 mmol). The reaction was stirred at RT for 18 h, then diluted with additional DCM (20 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (10 mL) and brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was redissolved in DCM (2 mL). TFA (106 μl, 1.380 mmol) was added, and the reaction was stirred at RT for 30 h. The reaction was concentrated and dried on high vac. The resultant residue was purified via MDAP (XSelect™ CSH C18 5um column, 40 mL/min) eluting with a gradient of 15 to 55 % acetonitrile in water containing trifluoroacetic acid (0.1%) to provide the title compound as a white solid (11.1 mg, 0.0067 mmol, 48.8% yield). LCMS m/z 817.19 (M+2H)/2. HPLC: 100% pure at 254nm. ¹H NMR (400 MHz, METHANOL-d4) δ ppm 1.22 - 1.31 (m, 5 H) 1.32 - 1.48 (m, 12 H) 1.54 - 1.54 (m, 1 H) 1.54 - 1.67 (m, 2 H) 1.70 - 1.86 (m, 6 H) 1.89 - 2.05 (m, 8 H) 2.08 - 2.33 (m, 10 H) 2.38 - 2.47 (m, 3 H) 2.65 - 2.74 (m, 6 H) 2.76 - 2.84 (m, 3 H) 2.85 - 2.96 (m, 2 H) 3.02 - 3.13 (m, 2 H) 3.14 - 3.22 (m, 2 H) 3.24 - 3.31 (m, 2 H) 3.43 - 3.53 (m, 6 H) 3.64 - 3.78 (m, 4 H) 3.80 - 3.88 (m, 2 H) 3.91 - 4.14 (m, 4 H) 4.16 - 4.36 (m, 7 H) 4.71 - 4.79 (m, 1 H) 5.00 - 5.07 (m, 2 H) 5.59 - 5.69 (m, 1 H) 7.34 - 7.46 (m, 3 H) 7.58 - 7.69 (m, 2 H) 7.72 - 7.81 (m, 2 H) 7.94 - 8.00 (m, 2 H) 8.02 - 8.11 (m, 1 H) 8.31 (br d, J=8.80 Hz, 1 H) 8.37 (br d, J=8.31 Hz, 1 H) 8.74 - 8.84 (m, 3 H) 8.87 - 8.94 (m, 2 H).

EXAMPLE 4 (((S)-1 -Carboxy-5-((S)-2-((1R,4S)-4-(4-(((1 S,4R)-4-(2-(2-((2S,3S)-N-(2-((2-(((1R,4S)- 4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl) carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-1- methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)acetamido) ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxamido)-3-(naphthalen-2- yl)propanamido)pentyl)carbamoyl)-L-glutamic acid

Step 1 : Benzyl (2-(tert-butoxy)-2-oxoethyl)glycinate

To a solution of benzyl 2-bromoacetate (20.0 g, 87.0 mmol) and tert-butyl glycinate hydrochloride (16.1 g, 96.0 mmol) in THF (175 ml) was added DI PEA (35.1 ml, 201 mmol). The reaction was stirred at RT for two nights. A white solid was removed via filtration, washing with ethyl acetate. The organic filtrate was diluted with EtOAc (200 mL) and washed with aqueous sodium bicarbonate (200 mL) and brine (200 mL), washed over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-60% ethyl acetate in hexanes to afford the title compound as a yellow oil. LCMS m/z 280.21 (M+H)⁺. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.48 (s, 9 H) 1.91 (br s, 1 H) 3.38 (s, 2 H) 3.53 (s, 2 H) 5.20 (s, 2 H) 7.32 - 7.43 (m, 5 H).

Step 2: Benzyl N-(2-(tert-butoxy)-2-oxoethyl)-N-((2S,3S)-1-methyl-5-oxo-2- (pyridin-3-yl)pyrrolidine-3-carbonyl)glycinate

To a solution of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (500 mg, 2.270 mmol), benzyl (2-(tert-butoxy)-2-oxoethyl)glycinate (698 mg, 2.497 mmol) and HATU (1122 mg, 2.95 mmol) in Dichloromethane (23 mL) was added DIPEA (0.793 ml, 4.54 mmol). The reaction was stirred at RT 24 h, then diluted with additional DCM (50 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (30 mL) and brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chormatography eluting with 0-10% methanol in dichloromethane to provide the title compound as a yelllow oil (1.20 g, 2.24 mmol, 99% yield. LCMS m/z 482.17 (M+H)⁺. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.35 - 1.47 (m, 9 H) 2.69 - 2.71 (m, 2 H) 2.74 - 2.81 (m, 1 H) 3.07 - 3.15 (m, 1 H) 3.17 - 3.25 (m, 1 H) 3.69 - 3.81 (m, 2 H) 3.81 - 3.96 (m, 1 H) 3.98 - 4.32 (m, 2 H) 4.94 - 5.02 (m, 1 H) 5.07 (s, 1 H) 5.18 (s, 1 H) 7.24 - 7.30 (m, 1 H) 7.31 - 7.44 (m, 5 H) 7.50 - 7.62 (m, 1 H) 8.52 - 8.58 (m, 1 H) 8.64 (dd, J=4.65, 1.71 Hz, 1 H).

Step 3: N-(2-(Benzyloxy)-2-oxoethyl)-N-((2S,3S)-1 -methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carbonyl)glycine

To a solution of benzyl N-(2-(tert-butoxy)-2-oxoethyl)-N-((2S,3S)-1-methyl-5-oxo-2- (pyridin-3-yl)pyrrolidine-3-carbonyl)glycinate (600mg, 1.246 mmol) in DCM (6 mL) was added TFA (1.92 mL, 24.9 mmol). The reaction was stirred at RT 24 h, then concentrated and dried on high vac to provide the title compound as a yellow oil (832 mg, 1.26 mmol, quantitative yield). LCMS m/z 426.13 (M+H)⁺. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 2.78 - 2.85 (m, 3 H) 2.93 - 3.05 (m, 1 H) 3.11 - 3.21 (m, 1 H) 3.29 - 3.44 (m, 1 H) 3.67 - 3.79 (m, 1 H) 3.88 - 4.08 (m, 2 H) 4.10 - 4.20 (m, 1 H) 4.28 - 4.52 (m, 1 H) 5.07 - 5.41 (m, 3 H) 7.25 - 7.31 (m, 1 H) 7.32 - 7.42 (m, 4 H) 7.95 - 8.07 (m, 1 H) 8.31 - 8.38 (m, 1 H) 8.79 - 8.79 (m, 1 H) 8.79 - 8.87 (m, 1 H) 8.91 - 8.96 (m, 1 H).

Step 4: tert-Butyl (1R,4r)-4-(4-(((1 S,4R)-4-(2-(2-((2S,3S)-N-(2-(benzyloxy)-2- oxoethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)acetamido) ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate

To a solution of tert-butyl (1R,4r)-4-(4-(((1r,4R)-4-(2- aminoethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxylate (348 mg, 0.816 mmol) and N-(2-(benzyloxy)-2-oxoethyl)-N-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carbonyl)glycine compound with 2,2,2-trifluoroacetic acid (400 mg, 0.741 mmol) in Dichloromethane (7 mL) was added HATU (367 mg, 0.964 mmol), and N-ethyl-N- isopropylpropan-2-amine (0.387 mL, 2.22 mmol). The reaction was stirred at RT for 20 h, then diluted with additional DCM (50 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-10% methanol in dichloromethane to afford the title compound as a yellow oil (488 mg, 0.556 mmol, 75.0% yield). LCMS m/z 834.39 (M+H)⁺. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.40 - 1.49 (m, 13 H) 1.50 - 1.58 (m, 3 H) 1.83 - 1.92 (m, 2 H) 1.93 - 2.19 (m, 8 H) 2.20 - 2.33 (m, 2 H) 2.63 - 2.75 (m, 4 H) 2.78 - 2.91 (m, 1 H) 3.08 - 3.19 (m, 2 H) 3.22 - 3.32 (m, 2 H) 3.35 - 3.57 (m, 6 H) 3.67 - 3.84 (m, 3 H) 3.89 - 4.26 (m, 3 H) 4.86 - 4.99 (m, 1 H) 5.05 - 5.14 (m, 1 H) 5.50 - 5.61 (m, 1 H) 7.26 - 7.32 (m, 3 H) 7.35 - 7.46 (m, 4 H) 7.49 - 7.61 (m, 1 H) 7.63 - 7.76 (m, 1 H) 8.53 (d, J=1.96 Hz, 1 H) 8.59 - 8.72 (m, 1 H). Step 5: N-(2-((2-(((1R,4S)-4-(4-(((1 r,4R)-4-(tert-butoxycarbonyl)cyclohexyl) amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-N-((2S, 3S)-1 -methyl -5- oxo-2-(pyridin-3-yl)pyrrolidine-3-carbonyl)glycine

To a solution of tert-butyl (1R,4r)-4-(4-(((1 S,4R)-4-(2-(2-((2S,3S)-N-(2-(benzyloxy)-2- oxoethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)acetamido) ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxylate (470 mg, 0.564 mmol) in Methanol (7 mL) under nitrogen was added Pd-C (60.0 mg, 0.056 mmol). The flask was evacuated and backfilled with a hydrogen gas balloon, and stirred at RT for 19 h. The mixture was filtered through a pad of celite, washing with additional methanol. The filtrate was concentrated and dried on high vac to provide the title compound as a clear film (415 mg, 0.463 mmol, 82% yield). LCMS m/z 744.22 (M+H)⁺. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.06 - 1.21 (m, 2 H) 1.21 - 1.33 (m, 4 H) 1.39 - 1.46 (m, 9 H) 1.48 - 1.59 (m, 2 H) 1.80 - 1.90 (m, 2 H) 1.92 - 2.06 (m, 10 H) 2.08 - 2.20 (m, 1 H) 2.25 (td, J=7.09, 2.45 Hz, 2 H) 2.62 - 2.73 (m, 2 H) 2.77 - 2.92 (m, 4 H) 3.11 (q, J=7.34 Hz, 2 H) 3.20 - 3.42 (m, 2 H) 3.49 (s, 4 H) 3.63 - 3.76 (m, 2 H) 4.98 (dd, J=13.45, 6.11 Hz, 1 H) 5.81 (br dd, J=15.65, 7.83 Hz, 1 H) 5.88 - 6.03 (m, 1 H) 7.40 (td, J=8.31, 4.89 Hz, 1 H) 7.58 - 7.67 (m, 1 H) 8.00 - 8.09 (m, 1 H) 8.50 - 8.56 (m, 1 H) 8.61 (td, J=5.62, 1.47 Hz, 1 H) 8.83 - 8.93 (m, 1 H).

Step 6: Benzyl (1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-((2-(((1R,4S)-4-(4- (((1 r,4R)-4-(tert-butoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl) oxy )ethyl)amino)-2-oxoethyl)-1 -methyl -5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxylate

To a solution of benzyl (1R,4r)-4-(4-(((1 r,4R)-4-(2-aminoethoxy)cyclohexyl) oxy)butanamido)cyclohexane-1-carboxylate (180 mg, 0.390 mmol) and N-(2-((2-(((1R,4S)-4- (4-(((1r,4R)-4-(tert-butoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy) ethyl)amino)-2-oxoethyl)-N-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carbonyl)glycine (290 mg, 0.390 mmol) in dichloromethane (7 mL) was added HATU (193 mg, 0.507 mmol), and N-ethyl-N-isopropylpropan-2-amine (0.204 mL, 1.17 mmol). The reaction was stirred at RT for 28 h, then diluted with additional DCM (100 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chormatography eluting with 0-20% methanol in dichloromethane to provide the title compound as a clear film, (93mg, 0.078 mmol, 20.1% yield). LCMS m/z 1186.45 (M+2H)/2. ¹ H NMR (400 MHz, CHLOROFORM-d)v δ ppm 1.05 - 1.19 (m, 4 H) 1.22 - 1.33 (m, 4 H) 1.48 - 1.64 (m, 14 H) 1.79 - 1.88 (m, 4 H) 1.92 - 2.07 (m, 16 H) 2.09 - 2.15 (m, 1 H) 2.18 - 2.33 (m, 5 H) 2.62 - 2.67 (m, 3 H) 2.77 - 2.88 (m, 1 H) 3.05 - 3.19 (m, 4 H) 3.21 - 3.30 (m, 3 H) 3.38 - 3.48 (m, 8 H) 3.49 - 3.57 (m, 2 H) 3.62 - 3.78 (m, 5 H) 3.79 - 3.85 (m, 2 H) 3.86 - 3.91 (m, 1 H) 3.95 - 4.11 (m, 1 H) 4.92 (d, J=6.36 Hz, 1 H) 5.10 (s, 2 H) 5.74 (br d, J=3.91 Hz, 2 H) 6.72 (br d, J=6.85 Hz, 1 H) 7.18 - 7.44 (m, 6 H) 7.56 (br d, J=6.85 Hz, 1 H) 8.49 (s, 1 H) 8.61 (br s, 1 H) 8.78 (br s, 1 H) 11.11 (br s, 1 H).

Step 7: (1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-((2-(((1R,4S)-4-(4-(((1r,4R)-4- ((benzyloxy)carbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl) amino)-2- oxoethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)acetamido) ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylic acid

To a solution of benzyl (1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-((2-(((1R,4S)-4- (4-(((1r,4R)-4-(tert-butoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy) ethyl)amino)-2-oxoethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido) acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxylate (93 mg, 0.078 mmol) in dichloromethane (DCM) (1.6 mL) was added TFA (302 μ 3l,.92 mmol). The reaction was stirred at RT for 30 h, then concentrated in vacuo and dried on high vac to provide the crude title compound as a clear oil (98.0 mg, 0.075 mmol, 95% yield) that was used without further purification or characterization. LCMS m/z 1243.38 (M+H)⁺.

Step 8: di-tert-Butyl (((S)-6-((S)-2-((1R,4S)-4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-((2- (((1R,4S)-4-(4-(((1r,4R)-4-((benzyloxy)carbonyl)cyclohexyl)amino)-4-oxobutyoxy) cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxamido)-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2- yl)carbamoyl)-L-glutamate

To a solution of (1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-((2-(((1R,4S)-4-(4- (((1 r,4R)-4-((benzyloxy)carbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl) amino)-2-oxoethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)acetamido) ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxylic acid compound with 2,2,2- trifluoroacetic acid (98 mg, 0.079 mmol) and di-tert-butyl (((S)-6-((S)-2-amino-3-(naphthalen- 2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (13.5 mg, 0.020 mmol) in dichloromethane (315 pl) was added 2-(3H-[1 ,2,3]triazolo[4,5-b]pyridin-3-yl)-1 , 1 ,3,3- tetramethylisouronium hexafluorophosphate(V) (44.9 mg, 0.118 mmol), and N-ethyl-N- isopropylpropan-2-amine (41.2 μl, 0.236 mmol). The reaction was stirred at RT for 3 nights, Additional N-ethyl-N-isopropylpropan-2-amine (41.2 μl 0, .236 mmol) was added to pH ~10. After another 3 h at RT, additional di-tert-butyl (((S)-6-((S)-2-amino-3-(naphthalen-2- yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (13.5 mg, 0.020 mmol was added. The mixture was stirred another 20 h, and then additional HATU (44.9 mg, 0.118 mmol) was added. After 24 h, DMF (1 mL) and additional di-tert-butyl (((S)-6-((S)-2- amino-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L- glutamate (13.5 mg, 0.020 mmol) were added. The mixture was stirred at RT another 24 h, then diluted with DCM (30 mL), washed with saturated aqueous sodium bicarbonate (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-20% methanol in DCM to provide the title compound as a clear film (69.4 mg, 0.036 mmol, 45.1% yield). LCMS 899.23 (M+H)⁺. ¹H NMR (400 Hz, CHLOROFORM-d) δ ppm 1.22 - 1.38 (m, 2 H) 1.38 - 1.49 (m, 27 H) 1.51 - 1.61 (m, 18 H) 1.82 - 1.93 (m, 8 H) 1.94 - 2.10 (m, 14 H) 2.17 - 2.32 (m, 6 H) 2.34 - 2.47 (m, 2 H) 2.60 - 2.69 (m, 2 H) 2.77 - 2.88 (m, 1 H) 2.93 - 2.97 (m, 1 H) 3.04 - 3.18 (m, 6 H) 3.20 - 3.33 (m, 5 H) 3.40 - 3.55 (m, 10 H) 3.63 - 3.73 (m, 4 H) 3.75 - 3.91 (m, 4 H) 3.98 - 4.08 (m, 1 H) 4.17 - 4.26 (m, 1 H) 4.92 - 4.98 (m, 1 H) 5.10 - 5.15 (m, 2 H) 5.56 - 5.65 (m, 1 H) 5.68 - 5.75 (m, 1 H) 6.50 - 6.60 (m, 1 H) 7.14 - 7.25 (m, 1 H) 7.31 - 7.53 (m, 9 H) 7.55 - 7.68 (m, 3 H) 7.70 - 7.78 (m, 1 H) 8.48 - 8.55 (m, 1 H) 8.62 - 8.68 (m, 1 H)

Step 9: 4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-((2-(((1R,4S)-4-(4-(((1S,4R)-4-

(((7S,11 S,18S)-7,11-bis(tert-butoxycarbonyl)-2,2-dimethyl-19-(naphthalen-2-yl)-4,9,17- trioxo-3-oxa-8,10,16-triazanonadecan-18-yl)carbamoyl)cyclohexyl)amino)-4- oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-1-methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carboxamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido) cyclohexane-1 -carboxylic acid

To a solution of di-tert-butyl (((S)-6-((S)-2-((1R,4S)-4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N- (2-((2-(((1R,4S)-4-(4-(((1r,4R)-4-((benzyloxy)carbonyl)cyclohexyl)amino)-4- oxobutyoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-1-methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carboxamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxamido)-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)- L-glutamate (69 mg, 0.038 mmol) in Methanol (154 pl) under nitrogen gas was added Pd/C (4.09 mg, 3.84 μmol).

The flask was evacuated and back-filled with a hydrogen gas balloon and stirred at RT for 5 h. The flask was evacuated and back-filled with nitrogen gas. Additional Pd/C (4.09 mg, 3.84 μmol) was added. The flask was evacuated and back-filled with a hydroge gas balloon and stirred at RT for 3 nights. The The mixture was filtered through a pad of celite, washing with additional MeOH (10 mL). The filtrate was concentrated and dried on high vac to provide the title compound as a clear oil (65.5 mg, 0.034 mmol, 89% yield). LCMS m/z 854.20 (M+2H)/2. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.10 - 1.19 (m, 4 H) 1.26 - 1.35 (m, 10 H) 1.38 - 1.51 (m, 40 H) 1.83 - 1.93 (m, 5 H) 1.95 - 2.11 (m, 12 H) 2.19 - 2.32 (m, 5 H) 2.33 - 2.45 (m, 3 H) 2.62 - 2.66 (m, 1 H) 2.66 - 2.71 (m, 3 H) 2.80 - 2.89 (m, 2 H) 2.93 - 2.98 (m, 2 H) 2.99 - 3.08 (m, 4 H) 3.09 - 3.18 (m, 3 H) 3.23 - 3.35 (m, 5 H) 3.41 - 3.52 (m, 9 H) 3.53 - 3.65 (m, 5 H) 3.73 - 3.90 (m, 4 H) 3.99 - 4.09 (m, 1 H) 4.18 - 4.39 (m, 2 H) 4.45 - 4.63 (m, 1 H) 4.95 (d, J=6.85 Hz, 1 H) 6.43 - 6.55 (m, 1 H) 7.36 - 7.49 (m, 2 H) 7.59 (br d, J=7.82 Hz, 1 H) 7.63 - 7.69 (m, 1 H) 7.75 (br d, J=7.82 Hz, 1 H) 8.52 (d, J=1.96 Hz, 1 H) 8.64 (dd, J=4.89, 1.47 Hz, 1 H). Step 10: (((S)-1-Carboxy-5-((S)-2-((1R,4S)-4-(4-(((1 S,4R)-4-(2-(2-((2S,3S)-N-(2-((2- (((1R,4S)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl) cyclohexyl)amino)-4-oxobuto xy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-1 -methyl -5- oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)acetamido)ethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanamido)pentyl) carbamoyl)-L-glutamic acid (Example 4)

To a solution of (S)-1-((1 S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3- ((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one hydrochloride (11.7 mg, 0.023 mmoL) and 4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-((2-(((1R,4S)-4-(4-(((1 S,4R)-4- (((7S, 11 S,18S)-7,11-bis(tert-butoxycarbonyl)-2,2-dimethyl-19-(naphthalen-2-yl)-4,9, 17- trioxo-3-oxa-8, 10, 16-triazanonadecan-18-yl)carbamoyl)cyclohexyl)amino)-4- oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-1-methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carboxamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxylic acid (40 mg, 0.023 mmol) in DCM (117 mL) was added HATU (11.6 mg, 0.030 mmol) and DIPEA (12.3 mL, 0.070 mmol). The reaction was stirred at RT for 17 h, then diluted with additional DCM (20 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (10 mL) and brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. To a solution of the resultant residue in DCM (2 mL) was added TFA (90 pl, 1.172 mmol). After 4 h, additional TFA (90 μl, 1.172 mmol) was added, and the mixture was stirred at RT for 21 h, then concentrated in vacuo. The resultant residue was purified via MDAP (XSelect™ CSH C18 5um column, 40 mL/min) eluting with a gradient of 15 to 55 % acetonitrile in water containing trifluoroacetic acid (0.1%) to provide the title compound as a gray solid (13.0 mg, 0.0065 mmol, 27.7% yield). LCMS m/z 993.05 (M+2H)/2. HPLC: >96% pure at 254nm. ¹H NMR (400 MHz, METHANOL-d4) δ ppm 1.10 - 1.24 (m, 4 H) 1.26 - 1.40 (m, 12 H) 1.42 - 1.47 (m, 3 H) 1.48 - 1.74 (m, 7 H) 1.77 - 1.94 (m, 10 H) 1.94 - 2.04 (m, 9 H) 2.10 - 2.29 (m, 10 H) 2.39 - 2.49 (m, 3 H) 2.61 - 2.66 (m, 2 H) 2.67 - 2.74 (m, 5 H) 2.78 - 2.83 (m, 3 H) 2.90 - 3.00 (m, 2 H) 3.04 - 3.18 (m, 3 H) 3.21 - 3.30 (m, 2 H) 3.36 - 3.40 (m, 3 H) 3.40 - 3.60 (m, 13 H) 3.62 - 3.78 (m, 4 H) 3.82 - 3.92 (m, 3 H) 3.98 - 4.15 (m, 5 H) 4.17 - 4.24 (m, 2 H) 4.25 - 4.31 (m, 1 H) 4.31 - 4.37 (m, 2 H) 4.64 - 4.71 (m, 1 H) 5.18 (br d, J=5.87 Hz, 1 H) 5.53 - 5.64 (m, 2 H) 7.38 - 7.42 (m, 1 H) 7.42 - 7.48 (m, 2 H) 7.68 - 7.71 (m, 1 H) 7.76 - 7.84 (m, 3 H) 7.92 - 7.97 (m, 1 H) 7.97 - 8.02 (m, 1 H) 8.30 - 8.35 (m, 2 H) 8.76 (s, 1 H) 8.80 (br d, J=5.38 Hz, 1 H) 8.88 (s, 1 H) 8.98 (s, 1 H).

EXAMPLE 5

(((S)-1 -Carboxy-5-((S)-2-((1R,4S)-4-(4-(((1 S,4R)-4-(2-(2-((2-((2-(((1R,4S)-4-(4- (((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl) carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)(2- ((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethyl) amino)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxamido)-3- (naphthalen-2-yl)propanamido)pentyl)carbamoyl)-L-glutamic acid

Step 1 : (2S,3S)-N-Allyl-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide

To a solution of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (800mg, 3.63 mmol), prop-2-en-1 -amine (0.327 ml, 4.36 mmol), and HATU (1.80 g,4.72 mmol) in dichloromethane (23 mL) was added DIPEA (1.27 ml, 7.27 mmol). The reaction was stirred at RT for 17 h, then diluted with additional dichloromethane (30 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-20% methanol in dichloromethane to afford the title compound as a yellow oil (631 mg, 2.36 mmol, 65.0% yield). ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 2.68 (s, 3 H) 2.75 - 2.94 (m, 3 H) 3.82 - 3.98 (m, 2 H) 4.82 (d, J=6.85 Hz, 1 H) 5.06 - 5.18 (m, 2 H) 5.64 (br s, 1 H) 5.79 (ddt, J=17.12, 10.27, 5.87, 5.87 Hz, 1 H) 7.38 (dd, J=7.58, 5.14 Hz, 1 H) 7.59 (dt, J=7.82, 1.96 Hz, 1 H) 8.54 (d, J=1.96 Hz, 1 H) 8.64 (dd, J=4.65, 1.71 Hz, 1 H).

Step 2: (2S,3S)-1-Methyl-5-oxo-N-(2-oxoethyl)-2-(pyridin-3-yl)pyrrolidine-3- carboxamide.

To a solution of (2S,3S)-N-allyl-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamide (470 mg, 1.81 mmol) in a 3:1 mixture of THF (12mL) and water (4.00 mL) cooled to 0 °C was added osmium tetroxide (3.41 mL, 0.272 mmol). After 15 minutes, sodium periodate (853 mg, 3.99 mmol) was added.

After 1.5 h, the water bath was removed and the mixture was allowed to warm to RT and stir for 1 h. The reaction was then diluted with dichloromethane and stirred at RT for 30 minutes. The mixture was filtered to remove solids, washing with additional dichloromethane (20 mL), followed by 1:1 methanokdichloromethane (2x 20 mL). The combined filtrates were concentrated, and the resultant residue was preabsorbed onto silica gel and purified via silica gel chromatography eluting with 0-15% methanol in dichloromethane to afford the title compound as a clear oil (310 mg, 1.07 mmol, 59.0% yield). LCMS m/z 262.06 (M+H)⁺. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 2.78 - 2.89 (m, 2 H) 3.28 - 3.38 (m, 1 H) 3.48 - 3.55 (m, 3 H) 4.14 - 4.35 (m, 1 H) 4.56 - 4.66 (m, 1 H) 4.75 - 4.85 (m, 1 H) 7.36 - 7.44 (m, 1 H) 7.57 - 7.66 (m, 1 H) 8.52 - 8.58 (m, 1 H) 8.60 - 8.67 (m, 1 H) 9.66 (s, 1 H). Step 3: Benzyl N-(2-(tert-butoxy)-2-oxoethyl)- S N-(2-((2S,3S)-1-methyl-5-oxo-2- (pyridin-3-yl)pyrrolidine-3-carboxamido)ethyl)glycinate.

To a solution of benzyl (2-(tert-butoxy)-2-oxoethyl)glycinate (331 mg, 1.186 mmol) in 1 ,2-dichloroethane (6 mL) was added (2S,3S)-1-methyl-5-oxo-N-(2-oxoethyl)-2-(pyridin-3- yl)pyrrolidine-3-carboxamide (310 mg, 1.186 mmol), and acetic acid (3.40 μl 0, .059 mmol). To this mixture was added sodium triacetoxyborohydride (503 mg, 2.373 mmol). The reaction was stirred at RT for 19 h, then diluted with dichloromethane (50 mL) and washed with saturated aqueous sodium bicarbonate (50 mL) and brine (50 mL), dried over sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-10% methanol in dichloromethane to afford the title compound as a pale yellow oil (343.2 mg, 0.635 mmol, 53.5% yield). LCMS m/z 525.20 (M+H)⁺. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.43 (s, 9 H) 2.69 (s, 3 H) 2.73 - 2.82 (m, 2 H) 2.83 - 2.88 (m, 2 H) 3.24 (q, J=5.22 Hz, 2 H) 3.29 - 3.42 (m, 2 H) 3.44 - 3.58 (m, 3 H) 4.87 (d, J=6.85 Hz, 1 H) 5.10 (s, 2 H) 7.30 - 7.43 (m, 6 H) 7.55 (br t, J=3.91 Hz, 1 H) 7.62 (dt, J=7.83, 1.96 Hz, 1 H) 8.56 (d, J=1.96 Hz, 1 H) 8.59 (dd, J=4.89, 1.47 Hz, 1 H).

Step 4: N-(2-(benzyloxy)-2-oxoethyl)-N-(2-((2S,3S)-1 -methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carboxamido)ethyl)glycine

To a solution of benzyl N-(2-(tert-butoxy)-2-oxoethyl)- S N-(2-((2S,3S)-1-methyl-5-oxo- 2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethyl)glycinate (340 mg, 0.648 mmol) in dichloromethane (3.2 mL) was added TFA (999 μl, 12.96 mmol). The mixture was stirred at RT for 16 h. Additional TFA (999 μl,12.96 mmol) was added, and the reaction was stirred at RT for 5 h. Additional TFA (999 μl,12.96 mmol) was again added, and the mixture was stirred at RT for 17 h. The reaction was concentrated and dried on high vac to afford the title compound as a pale yellow oil (699 mg, 0.648 mmol, theoretical yield). LCMS m/z 469.16 (M+H⁺). ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 2.78 (s, 3 H) 2.89 (dd, J=18.10, 6.85 Hz, 1 H) 3.14 (br dd, J=17.85, 10.03 Hz, 1 H) 3.32 - 3.51 (m, 2 H) 3.62 (br s, 2 H) 3.83 - 3.98 (m, 1 H) 4.06 - 4.17 (m, 2 H) 4.22 - 4.38 (m, 2 H) 5.28 - 5.40 (m, 3 H) 7.35 - 7.53 (m, 5 H) 8.09 (br dd, J=8.07, 5.62 Hz, 1 H) 8.44 (br d, J=7.83 Hz, 1 H) 8.63 - 8.78 (m, 1 H) 8.90 (d, J=5.38 Hz, 1 H) 9.52 (br s, 1 H).

Step 5: tert-Butyl (1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2-(benzyloxy)-2-oxoethyl)(2-

((2S,3S)-1 -ethyl -5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethyl)amino) acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate.

To a solution of N-(2-(benzyloxy)-2-oxoethyl)-N-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin- 3-yl)pyrrolidine-3-carboxamido)ethyl)glycine compound with 2,2,2-trifluoroacetic acid (370 mg, 0.635 mmol) in dichloromethane (7 mL) was added HATU (314 mg, 0.826 mmol), N-ethyl- N-isopropylpropan-2-amine (1.106 mL, 6.35 mmol), and tert-butyl (1R,4r)-4-(4-(((1r,4R)-4-(2- aminoethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate (298 mg, 0.699 mmol). The reaction was stirred at RT for 3 h, then diluted with additional dichloromethane (20 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-20% methanol in dichloromethane to afford the title compound as a yellow oil (385.7 mg, 0.396 mmol, 62.3% yield). LCMS m/z 877.25 (M+H)⁺. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.42 (br s, 9 H) 1.50 - 1.59 (m, 3 H) 1.83 - 1.92 (m, 3 H) 1.92 - 1.98 (m, 4 H) 1.99 - 2.08 (m, 3 H) 2.08 - 2.19 (m, 1 H) 2.26 (t, J=7.34 Hz, 2 H) 2.70 (s, 2 H) 2.76 (t, J=5.62 Hz, 2 H) 2.81 - 2.83 (m, 2 H) 3.14 (q, J=7.34 Hz, 8 H) 3.26 (br d, J=1.47 Hz, 3 H) 3.46 - 3.48 (m, 3 H) 3.67 - 3.77 (m, 8 H) 4.88 (d, J=6.85 Hz, 1 H) 5.13 (s, 2 H) 5.62 - 5.73 (m, 1 H) 7.01 - 7.09 (m, 1 H) 7.16 (br s, 1 H) 7.31 - 7.46 (m, 5 H) 7.61 (dt, J=7.83, 1.96 Hz, 1 H) 8.56 (d, J=2.45 Hz, 1 H) 8.61 (dd, J=4.89, 1.47 Hz, 1 H). Step 6: N-(2-((2-(((1R, 4S)-4-(4-((( 1 r,4R)-4-(tert-Butoxycarbonyl)cyclohexyl) amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-N-(2-((2S,3S)-1 -methyl -5- oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethyl)glycine.

To a solution of tert-butyl (1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2-(benzyloxy)-2-oxoethyl)(2- ((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethyl)amino) acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate (385 mg, 0.439 mmol) in methanol (4.5 mL) under an atmosphere of nitrogen gas was added Pd-C (46.7 mg, 0.044 mmol). The flask was evacuated and backfilled with a hydrogen gas balloon, then stirred at RT for 3 nights. The mixture was filtered through a celite plug, washing with additional methanol (20 mL) The filtrate was concentrated in vacuo and dried on high vac to afford the title compound as a yellow oil. LCMS m/z 787.31 (M+H)⁺. ¹H NMR (400 MHz, CHLOROFORM- d) δ ppm 1.11 - 1.28 m, 4 H) 1.39 - 1.48 (m, 9 H) 1.85 - 2.18 (m, 12 H) 2.26 (br t, J=6.85 Hz, 2 H) 2.71 (br s, 2 H) 2.83 (s, 6 H) 3.15 (q, J=7.34 Hz, 6 H) 3.26 - 3.32 (m, 2 H) 3.33 - 3.38 (m, 2 H) 3.43 - 3.49 (m, 2 H) 3.67 - 3.80 (m, 6 H) 4.90 (d, J=6.36 Hz, 1 H) 5.65 - 5.86 (m, 1 H) 7.41 (br dd, J=7.82, 4.89 Hz, 1 H) 7.64 (br d, J=7.82 Hz, 1 H) 8.56 - 8.63 (m, 1 H) 10.28 - 10.74 (m, 3 H).

Step 7: tert-Butyl ( 1R,4r)-4-(4-((( 1 S,4R)-4-(2-(2-((2-((2-((( 1R,4S)-4-(4-((( 1 r,4R)-4- (methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2- oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethyl) amino)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate.

To a solution of N-(2-((2-(((1R,4S)-4-(4-(((1 r,4R)-4-(tert- butoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-N- (2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethyl)glycine (345 mg, 0.438 mmol) and methyl (1R,4r)-4-(4-(((1r,4R)-4-(2- aminoethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate (185 mg, 0.482 mmol) in dichloromethane (DCM) (2192 pl) was added HATU (217 mg, 0.570 mmol) and N-ethyl-N- isopropylpropan-2-amine (153 μl, 0.877 mmol). The reaction was stirred at RT for 1 h, then diluted with additional dichloromethane (20 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (10 mL) and brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chormatography eluting with 0-20% methanol in dichloromethane to afford the title compound as a tacky yellow solid (417.9 mg, 0.330 mmol, 75% yield). LCMS m/z 1153.40 (M+H +). ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.93 - 1.31 (m, 10 H) 1.32 - 1.65 (m, 14 H) 1.73 - 2.14 (m, 19 H) 2.15 - 2.39 (m, 5 H) 2.53 - 3.03 (m, 8 H) 3.05 - 3.35 (m, 12 H) 3.42 (br s, 10 H) 3.56 - 3.87 (m, 6 H) 3.96 - 4.42 (m, 2 H) 4.86 (br d, J=4.89 Hz, 1 H) 5.87 (br d, J=0.98 Hz, 2 H) 7.04 (br s, 1 H) 7.19 - 7.45 (m, 1 H) 7.51 - 7.78 (m, 2 H) 8.54 (br d, J=17.61 Hz, 2 H).

Step 8: (1R,4r)-4-(4-(((1 S,4R)-4-(2-(2-((2-((2-(((1R,4S)-4-(4-(( ( 1 r,4R)-4-

(Methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2- oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido) ethyl)amino)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylic acid.

To a solution of tert-butyl (1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2-((2-(((1R,4S)-4-(4- (((1 r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2- oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethyl) amino)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate (220 mg, 0.191 mmol) in dichloromethane (1 mL) was added TFA (294 μl, 3.81 mmol). The reaction was stirred at RT for 1.5 h, then concentrated in vacuo and dried on high vac to afford the title compound as a yellow oil (475.5 mg, 0.188 mmol, 99% yield). LCMS m/z 1097.29 (M+H)⁺. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 1.19 - 1.36 (m, 14 H) 1.37 - 1.42 (m, 2 H) 1.44 - 1.58 (m, 4 H) 1.78 - 1.89 (m, 4 H) 1.92 - 2.08 (m, 14 H) 2.20 - 2.38 (m, 6 H) 2.73 (br s, 3 H) 2.77 - 2.85 (m, 1 H) 2.89 - 2.99 (m, 1 H) 3.13 - 3.27 (m, 2 H) 3.28 - 3.39 (m, 4 H) 3.40 - 3.52 (m, 9 H) 3.53 - 3.64 (m, 5 H) 3.67 (s, 3 H) 3.75 (s, 1 H) 4.03 - 4.27 (m, 4 H) 5.30 (d, J=5.87 Hz, 1 H) 7.47 - 7.60 (m, 1 H) 8.13 (dd, J=8.31 , 5.87 Hz, 1 H) 8.43 - 8.51 (m, 1 H) 8.65 (dt, J=8.31, 1.71 Hz, 1 H) 8.76 (dd, J=4.65, 1.22 Hz, 1 H) 8.87 - 8.94 (m, 1 H) 9.04 (d, J=1.47 Hz, 1 H).

Step 9: Methyl (1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2-((2-(((1R,4S)-4-(4-(((1R,4R)-4- (((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin- 4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy) cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carboxamido)ethyl)amino)acetamido)ethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1 -carboxylate.

To a solution of (1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2-((2-(((1R,4S)-4-(4-(((1r,4R)-4- (methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2- oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethyl)amino) acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylic acid (150 mg, 0.137 mmol) and N-ethyl-N-isopropylpropan-2-amine (238 μl, 1.37 mmol) in dichloromethane (3 mL) was added 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1 , 1,3,3-tetramethylisouronium hexafluorophosphate(V) (78.0 mg, 0.205 mmol), and (S)-1-((1S,2R,4R)-2-amino-4- (isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2- one hydrochloride (82.0 mg, 0.164 mmol). The reaction was stirred at RT for 1 .5 h, then diluted with additional dichloromethane (30 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-20% methanol in dichloromethane to afford the title compound as a yellow film (233.6 mg, 0.136 mmol, thoretical yield). LCMS m/z 772.60 (M+2H)/2. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.04 - 1.27 (m, 10 H) 1.34 - 1.43 (m, 9 H) 1.44 - 1.58 (m, 4 H) 1.60 - 1.70 (m, 2 H) 1.77 - 1.85 (m, 4 H) 1.86 - 2.02 (m, 14 H) 2.15 - 2.25 (m, 6 H) 2.37 - 2.49 (m, 1 H) 2.60 - 2.66 (m, 4 H) 2.68 - 2.77 (m, 3 H) 2.77 - 2.89 (m, 3 H) 2.95 (td, J=9.05, 6.85 Hz, 2 H) 3.09 (q, J=7.QQ Hz, 2 H) 3.13 - 3.24 (m, 9 H) 3.26 - 3.35 (m, 4 H) 3.38 - 3.45 (m, 14 H) 3.63 (s, 3 H) 3.69 (dt, J=13.33, 6.79 Hz, 2 H) 3.97 - 4.07 (m, 1 H) 4.75 (br s, 1 H) 4.85 (d, J=6.85 Hz, 1 H) 5.08 (td, J=7.58, 4.40 Hz, 1 H) 5.90 - 6.04 (m, 2 H) 7.16 (dd, J=8.56, 4.16 Hz, 1 H) 7.23 (q, J=5.38 Hz, 2 H) 7.32 (dd, J=7.83, 4.89 Hz, 1 H) 7.57 (dt, J=7.82, 1.96 Hz, 1 H) 7.75 (t, J=4.89 Hz, 1 H) 7.86 (d, J=0.98 Hz, 2 H) 8.12 (dd, J=8.56, 1.22 Hz, 1 H) 8.46 (dd, J=4.16, 1.22 Hz, 1 H) 8.49 (d, J=1.96 Hz, 1 H) 8.54 (dd, J=4.65, 1.71 Hz, 1 H) 8.62 (s, 1 H) 8.82 (s, 1 H).

Step 10: 4-(4-(((1S,4R)-4-(2-(2-((2-((2-(((1R,4S)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5- (lsopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino) pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl) oxy)ethyl)amino)-2-oxoethyl)(2-((2S,3S)-1 -methyl -5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethyl)amino)acetamido)ethoxy)cyclohexyl) oxy)butanamido)cyclohexane-1 -carboxylic acid.

To a solution of methyl (1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2-((2-(((1R,4S)-4-(4-(((1R,4R)- 4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4- yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy) cyclohexyl) oxy)ethyl)amino)-2-oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethyl)amino)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxylate (140 mg, 0.091 mmol) in THF (2 mL) was added 1N aqueous sodium hydroxide (181 μl, 0.181 mmol). The reaction was stirred at RT 21 h, then concentrated and dried on high vac to afford the title compound as a white solid (188.2 mg, 0.091 mmol, theoretical yield). LCMS m/z 765.62 (M+2H)/2. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.84 - 1.00 (m, 4 H) 1.04 - 1.20 (m, 14 H) 1.61 - 1.73 (m, 6 H) 1.75 - 2.02 (m, 22 H) 2.11 - 2.25 (m, 8 H) 2.39 - 2.48 (m, 1 H) 2.52 - 2.72 (m, 7 H) 2.75 - 2.90 (m, 2 H) 2.99 - 3.26 (m, 14 H) 3.28 - 3.45 (m, 13 H) 3.49 - 3.70 (m, 4 H) 3.72 - 3.79 (m, 1 H) 3.98 - 4.09 (m, 1 H) 5.05 - 5.11 (m, 2 H) 6.84 - 7.00 (m, 1 H) 7.58 (br d, J=6.36 Hz, 1 H) 7.81 - 7.90 (m, 2 H) 8.09 - 8.26 (m, 1 H) 8.45 (br d, J=15.16 Hz, 2 H) 8.59 (s, 1 H) 8.86 (br s, 1 H) 9.18 - 9.34 (m, 1 H). Step 11 : (((S)-1 -Carboxy-5-((S)-2-((1R,4S)-4-(4-(((1 S,4R)-4-(2-(2-((2-((2-(((1R,4S)- 4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl) cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)(2-((2S,3S)-1- methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethyl)amino)acetamido) ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxamido)-3-(naphthalen-2- yl)propanamido)pentyl)carbamoyl)-L-glutamic acid (Example 5).

To a solution of 4-(4-(((1S,4R)-4-(2-(2-((2-((2-(((1R,4S)-4-(4-(((1R,4R)-4- (((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4- yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl) oxy)ethyl)amino)-2-oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethyl)amino)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxylic acid (100 mg, 0.065 mmol) in dichloromethane (30 mL) and DMF (3 mL) was added HATU (37.3 mg, 0.098 mmol), N-ethyl-N-isopropylpropan-2-amine (0.034 mL, 0.196 mmol), and di-tert-butyl (((S)-6-((S)-2-amino-3-(naphthalen-2-yl)propanamido)-1-(tert- butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (44.8 mg, 0.065 mmol). The reaction was stirred at RT for 1 h, then diluted with additional DCM (20 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (15 mL) and brine (15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-20% methanol in dichloromethane to afford the intermediate di-tert-butyl (((S)-1-(tert-butoxy)-6-((S)-2-((1R,4S)-4-(4-(((1S,4R)-4-(2-(2-((2-((2- (((1R,4S)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl) amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2- (pyridin-3-yl)pyrrolidine-3-carboxamido)ethyl)amino)acetamido)ethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanamido)-1-oxohexan-2- yl)carbamoyl)-L-glutamate as a white solid (70.5 mg, 0.032 mmol, 49.1 % yield). LCMS m/z 1099.25 (M+2H)/2).

To a solution of the above intermediate (70.5 mg, 0.032 mmol) in dichloromethane (30 mL) was added TFA (0.252 mL, 3.27 mmol). The mixture was stirred at RT for 18 h, then concentrated in vacuo. The resultant residue was purified via MDAP (XSelect™ CSH C18 5um column, 40 mL/min) eluting with a gradient of 15 to 55 % acetonitrile in water containing trifluoroacetic acid (0.1%) to provide the title compound as a white solid (26.0 mg, 0.013 mmol, 19.4% yield). LCMS m/z 1014.75 (M+2H)/2. HPLC: 100% pure at 254nm. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 1.04 - 1.40 (m, 20 H) 1.41 - 1.46 (m, 3 H) 1.48 - 1.74 (m, 6 H) 1.74 - 1.94 (m, 12 H) 1.95 - 2.04 (m, 10 H) 2.08 - 2.28 (m, 11 H) 2.35 - 2.56 (m, 3H) 2.59 - 2.67 (m, 1 H) 2.68 - 2.80 (m, 4 H) 2.80 - 2.85 (m, 3 H) 2.89 - 2.99 (m, 1 H) 3.00 - 3.23 (m, 5 H) 3.23 - 3.31 (m, 3 H) 3.35 - 3.38 (m, 2 H) 3.39 - 3.50 (m, 9 H) 3.52 - 3.61 (m, 6 H) 3.62 - 3.82 (m, 4 H) 3.83 - 3.96 (m, 2 H) 4.00 - 4.15 (m, 4 H) 4.16 - 4.28 (m, 2 H) 4.29 - 4.39 (m, 2 H) 4.68 (dd, J=8.56, 6.60 Hz, 1 H) 5.25 (d, J=5.87 Hz, 1 H) 5.60 - 5.70 (m, 1 H) 7.36 - 7.50 (m, 3 H) 7.69 (s, 1 H) 7.76 - 7.86 (m, 3 H) 8.00 (d, J=8.80 Hz, 1 H) 8.05 (dd, J=8.07, 5.62 Hz, 1 H) 8.32 (dd, J=9.05, 1.71 Hz, 1 H) 8.42 - 8.49 (m, 1 H) 8.54 (br d, J=8.31 Hz, 1 H) 8.85 (d, J=4.40 Hz, 1 H) 8.89 (s, 1 H) 8.99 (s, 1 H) 9.04 (s, 1 H).

EXAMPLE 6

To a solution of 4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1R,4R)-4- (((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4- yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl) oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylic acid (130.0 mg, 70.68 μmol), (S)-1-((1 S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3- ((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one hydrochloride (35.4 mg, 70.7 μmol) and HATU (34.9 mg, 91.9 μmol) in DCM (2.5 mL) was added DIPEA (18.3 mg, 24.6 μL, 141 μmol) The reaction was stirred at RT for 30 h. Additional (S)-1-((1 S,2R,4R)-2-amino-4- (isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2- one hydrochloride (3.54 mg, 7.07 μmol) was added. The reaction was stirred at RT for 19 h, then diluted with additional dichloromethane (20 mL), washed with saturated aqueous sodium bicarbonate (10 mL) and brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via MDAP (XSelect™ CSH C185um column, 40 mL/min) eluting with a gradient of 50 to 99 % acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075 %) to provide the title compound as a white solid (48.32 mg, 0.019 mmol, 27.4 % yield). LCMS m/z 1143.46 (M+2H)/2. HPLC: >91.5% pure at 254nm. ¹ H NMR (400 MHz, DMSO-d₆) δ ppm 0.87 - 0.96 (m, 6 H) 1.00 - 1.06 (m, 6 H) 1.07 - 1.24 (m, 15 H) 1.29 - 1.46 (m, 6 H) 1.52 - 1.72 (m, 14 H) 1.74 - 1.91 (m, 19 H) 1 .94 - 2.09 (m, 10 H) 2.11 - 2.17 (m, 6 H) 2.29 - 2.37 (m, 2 H) 2.39 - 2.47 (m, 2 H) 2.48 - 2.53 (m, 4 H) 2.57 - 2.62 (m, 2 H) 2.65 - 2.75 (m, 2 H) 2.92 - 3.00 (m, 2 H) 3.12 - 3.26 (m, 12 H) 3.28 - 3.46 (m, 28 H) 3.49 - 3.55 (m, 2 H) 3.80 - 3.95 (m, 4 H) 4.07 (br s, 2 H) 4.45 - 4.56 (m, 2 H) 4.65 (d, J=6.36 Hz, 1 H) 4.92 - 5.04 (m, 2 H) 7.41 - 7.47 (m, 1 H) 7.58 - 7.65 (m, 3 H) 7.65 - 7.70 (m, 1 H) 7.87 - 7.90 (m, 1 H) 7.90 - 7.93 (m, 1 H) 7.99 - 8.04 (m, 1 H) 8.05 - 8.10 (m, 2 H) 8.18 - 8.24 (m, 1 H) 8.45 - 8.49 (m, 1 H) 8.54 - 8.57 (m, 1 H) 8.58 - 8.65 (m, 4 H) 8.83 - 8.89 (m, 1 H) 8.94 - 9.02 (m, 4 H).

(((S)-1 -Carboxy-5-((S)-2-((1R,4S)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2- (((1R,4R)-4-(4-(((1R,4R)-4-(((R)-7-((1R,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro- 2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro- 1,7-naphthyridin-8-yl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy) cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1 S,4R)-4-(2-((2S,3S)-1 -m ethyl -5-oxo-2- (pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1-carboxamido)acetamido) ethoxy)cyclo hexyl) oxy)butanamido)cyclohexane-1-carboxamido)-3-(naphthalen-2- yl)propanamido)pentyl)carbamoyl)-L-glutamic acid

Step 1 : (1R,4r)-4-(4-(((1r,4R)-4-(2-(((Benzyloxy)carbonyl)amino)ethoxy) cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylic acid.

To a solution of tert-butyl (1R,4r)-4-(4-(((1r,4R)-4-(2- aminoethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylate (30.0 g, 70.3 mmol) in dichloromethane (100 mL) was added N-ethyl-N-isopropylpropan-2-amine (18.18 g, 141 mmol) followed by dropwise addition of benzyl carbonochloridate (18.00 g, 105 mmol). The reaction was stirred for 16 h, then washed with 1 M HCI and water. The organic solution was concentrated in vacuo. The resultant residue was redissolved in 1:1 DCM:TFA (100 mL) and stirred at RT for 4 h. The reaction was concentrated in vacuo. The resultant residue was purified via silica gel chromatography eluting with 0-40% methanol in dichloromethane to afford the title compound as an off-white solid (25.1 g, 49.7 mmol, 70.7% yield) that was used without further characterization. LCMS m/z 505.2 (M+H)⁺.

Step 2: Di-tert-butyl (((S)-6-((S)-2-((1R,4S)-4-(4-(((1r,4R)-4-(2-(((benzyloxy) carbonyl)amino)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxamido)-3- (naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L- glutamate.

To a solution of (1R,4r)-4-(4-(((1r,4R)-4-(2-(((benzyloxy)carbonyl)amino)ethoxy) cyclohexyl)oxy) butanamido)cyclohexane-1-carboxylic acid (534.9 mg, 1.06 mmol) in dichloromethane was added DIPEA (249 mg, 336 mL, 1.93 mmol), di -tert-butyl (((S)-6-((S)- 2-amino-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L- glutamate (660 mg, 0.964 mmol), and HATU (476 mg, 1.25 mmol). The reactio was stirred at RT for 2 nights, then diluted with dichloromethane (20 mL), washed with saturated aqueous sodium bicarbonate (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-10% methanol in dichloromethane to afford the title compound as an off-white solid (886 mg, 0.650 mmol, 67.5% yield). LCMS m/z = 1172.53 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.05 - 1.36 (m, 13 H) 1.36 - 1.44 (m, 27 H) 1.46 - 1.59 (m, 3 H) 1.59 - 1.72 (m, 6 H) 1.80 - 1.96 (m, 6 H) 2.01 - 2.13 (m, 3 H) 2.15 - 2.31 (m, 2 H) 2.85 - 3.02 (m, 2 H) 3.04 (br s, 6 H) 3.36 - 3.41 (m, 3 H) 3.94 (td, J=8.07, 5.87 Hz, 1 H) 4.00 - 4.09 (m, 1 H) 4.49 - 4.61 (m, 1 H) 5.01 (s, 2 H) 6.27 (dd, J=19.81 , 8.56 Hz, 2 H) 7.20 - 7.27 (m, 1 H) 7.28 - 7.41 (m, 7 H) 7.41 - 7.51 (m, 2 H) 7.56 - 7.62 (m, 1 H) 7.68 (s, 1 H) 7.76 - 7.82 (m, 2 H) 7.83 - 7.88 (m, 1 H) 7.90 - 7.99 (m, 2 H).

Step 3: Di-tert-butyl (((S)-6-((S)-2-((1R,4S)-4-(4-(((1r,4R)-4-(2-aminoethoxy) cyclohexyl)oxy)butanamido)cyclohexane-1-carboxamido)-3-(naphthalen-2- yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate.

To a solution of di-tert-butyl (((S)-6-((S)-2-((1R,4S)-4-(4-(((1r,4R)-4-(2-(((benzyloxy) carbonyl)amino)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxamido)-3- (naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (886 mg, 0.756 mmol) in methanol under an atmosphere of nitrogen gas was added Pd/C (80.5 mg, 10% wt., 0.0756 mmol). The flask was evacuated and back-filled with a hydrogen gas balloon and stirred at RT for 20 h. The mixture was filtered through a celite plug, washing with additional methanol (2x 10 mL), then concentrated in vacuo and dried on high vac to afford the title compound as an off-white solid (759 mg, 0.615 mmol, 81.3% yield). LCMS m/z 1037.72 (M+H)⁺. ¹ H NMR (400 MHz, METHANOL-d₄) δ ppm 1.11 - 1.41 (m, 11 H) 1.43 - 1.53 (m, 27 H) 1.60 - 1.69 (m, 2 H) 1.77 - 1.95 (m, 7 H) 1.96 - 2.08 (m, 6 H) 2.12 - 2.26 (m, 4 H) 2.29 - 2.40 (m, 2 H) 2.82 (t, J=5.38 Hz, 2 H) 3.03 - 3.17 (m, 3 H) 3.22 - 3.29 (m, 1 H) 3.44 - 3.49 (m, 2 H) 3.50 - 3.64 (m, 4 H) 4.08 (dd, J=8.31, 4.89 Hz, 1 H) 4.22 (dd, J=8.56, 5.14 Hz, 1 H) 4.68 (dd, J=8.56, 6.60 Hz, 1 H) 7.38 - 7.42 (m, 1 H) 7.42 - 7.49 (m, 2 H) 7.70 (s, 1 H) 7.77 - 7.87 (m, 3 H).

Step 4: tert-Butyl (2-(((1R,4r)-4-(4-(((1R,4R)-4-(((R)-6-((1 S,3S)-1 -isopropyl-3- (((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3- (trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8-yl)carbamoyl) cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)carbamate.

To a solution of (1R,4r)-4-(4-(((1r,4R)-4-(2-((tert-butoxycarbonyl)amino)ethoxy) cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylic acid (386 mg, 0.819 mmol) in DCM (8 mL) was added HATU (386 mg, 1.01 mmol), DI PEA (504 mg, 0.680 mL, 3.90 mmol), and ((R)-8-amino-3-(trifluoromethyl)-5,8-dihydro-1,7-naphthyridin-7(6/7)-yl)((1R,3R)-1-isopropyl- 3-(((3S,4S)-3-methoxytetrahydro-2/7-pyran-4-yl)amino)cyclopentyl)methanone, 2Hydrochloride (435 mg, 0.780 mmol). The reaction was stirred at RT for 1.5 h, then diluted with additional dichloromethane (20 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The reesultant residue was purified via silica gel chromatography eluting with 0-20% methanol in dichloromethane to afford the title compound as an orange solid (810 mg, 0.622 mmol, 79.8% yield). LCMS m/z 937.63 (M+H)⁺. ¹ H NMR (400 MHz, DMSO-d₆) δ ppm 0.68 - 0.79 (m, 2 H) 0.84 - 0.92 (m, 2 H) 1.09 - 1.28 (m, 15 H) 1.32 - 1.45 (m, 9 H) 1.61 - 1.69 (m, 2 H) 1.74 - 1.93 (m, 6 H) 2.02 - 2.12 (m, 3 H) 2.26 - 2.40 (m, 1 H) 2.53 - 2.58 (m, 3 H) 2.96 - 3.08 (m, 2 H) 3.13 - 3.20 (m, 3 H) 3.33 (br s, 17 H) 3.76 - 3.86 (m, 1 H) 4.04 - 4.17 (m, 2 H) 6.73 (br t, J=5.62 Hz, 1 H) 7.24 (br dd, J=8.56, 4.16 Hz, 1 H) 7.60 - 7.74 (m, 1 H) 8.21 (br d, J=8.31 Hz, 1 H) 8.27 (br s, 1 H) 8.43 (br d, J=3.91 Hz, 1 H) 8.79 - 8.95 (m, 1 H).

Step 5:(1R,4R)-4-(4-(((1r,4R)-4-(2-Aminoethoxy)cyclohexyl)oxy)butanamido)-N- ((R)-6-((1S,3S)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino) cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8- yl)cyclohexane-1 -carboxamide.

To a solution of tert-butyl (2-(((1R,4r)-4-(4-(((1R,4R)-4-(((R)-7-((1R,3R)-1-isopropyl-3- (((3S,4S)-3-methoxytetrahydro-2/7-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3- (trifluoromethyl)-5,6,7,8-tetrahydro-1 ,7-naphthyridin-8-yl)carbamoyl)cyclohexyl)amino)-4- oxobutoxy)cyclohexyl)oxy)ethyl)carbamate (730 mg, 0.779 mmol) in THF was added 4N HCI in dioxane (1.95 mL, 7.79 mmol). The reactio was stirred at RT for 21 h, then concentrated in vacuo and dried on high vac to afford the title compound as an orange solid (652 mg, 0.631 mmol, 81.0% yield). LCMS m/z 837.69 (M+H)⁺.¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.21 - 1.25 (m, 3 H) 1.26 - 1.33 (m, 13 H) 1.63 - 1.69 (m, 2 H) 1.73 - 1.81 (m, 12 H) 1.86 - 1.94 (m, 5 H) 2.03 - 2.12 (m, 3 H) 2.11 - 2.12 (m, 1 H) 2.90 - 2.97 (m, 2 H) 3.08 - 3.19 (m, 2 H) 3.31 - 3.38 (m, 7 H) 3.59 - 3.65 (m, 9 H) 3.81 - 3.88 (m, 1 H) 4.12 - 4.20 (m, 1 H) 7.53 (dd, J=8.31, 4.40 Hz, 1 H) 7.60 - 7.72 (m, 2 H) 8.30 (br s, 1 H) 8.54 (dd, J=8.31, 1.47 Hz, 1 H) 8.77 (dd, J=4.40, 0.98 Hz, 1 H) 8.87 (s, 1 H).

Step 6: Benzyl N-(2-(tert-butoxy)-2-oxoethyl)-N-((1R,4r)-4-(4-(((1 S,4R)-4-(2- ((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy) cyclohexyl)oxy)butanamido)cyclohexane-1 -carbonyl)glycinate.

To a solution of (1R,4r)-4-(4-(((1 S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 -carboxylic acid (1.00 g, 1.75 mol) in dichloromethane was added benzyl (2-(tert-butoxy)-2- oxoethyl)glycinate (585 mg, 2.10 mmol), HATU (863 mg, 2.27 mmol) and DIIPEA (677 mg, 0.912 mL, 5.24 mmol). The reaction was stirred at RT for 6 h. Additional benzyl (2-(tert- butoxy)-2-oxoethyl)glycinate (0.200 g, 0.349 mmol) was added, and the mixture was stirred at RT for an additional 19 h. The reaction was diluted with dichloromethane (20 mL), washed with saturated aqueous sodium bicarbonate (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, fitlered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-20% methanol in dichloromethane to afford the title compound as a tan solid (1.22 g, 1.23 mmol, 70.4% yield). LCMS m/z 834.34 (M+H)⁺. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.47 (d, J=11.25 Hz, 9 H) 1.52 - 1.55 (m, 2 H) 1.64 - 1.82 (m, 9 H) 1.84 - 1.90 (m, 3 H) 1.92 - 1.99 (m, 3 H) 2.00 - 2.13 (m, 2 H) 2.21 - 2.29 (m, 2 H) 2.68 (s, 3 H) 2.76 - 2.84 (m, 2 H) 3.08 - 3.17 (m, 1 H) 3.19 - 3.30 (m, 2 H) 3.38 - 3.48 (m, 4 H) 3.65 - 3.84 (m, 2 H) 4.06 (d, J=6.85 Hz, 2 H) 4.16 - 4.24 (m, 2 H) 4.81 (d, J=6.36 Hz, 1 H) 5.14 - 5.26 (m, 2 H) 5.81 - 5.90 (m, 1 H) 7.31 - 7.43 (m, 6 H) 7.59 (dt, J=7.82, 1.96 Hz, 1 H) 8.55 (d, J=2.45 Hz, 1 H) 8.64 (dd, J=4.89, 1.47 Hz, 1 H). Step 7: N-(2-(benzyloxy)-2-oxoethyl)-N-((1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1- methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1-carbonyl)glycine.

To a solution of benzyl N-(2-(tert-butoxy)-2-oxoethyl)-N-((1R,4r)-4-(4-(((1 S,4R)-4-(2- ((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl) oxy)butanamido)cyclohexane-1-carbonyl)glycinate (1.22 g, 1.47 mmol) in dichloromethane was added 4N HCI in dioxane (1.10 mL, 4.40 mmol). The reaction was stirred at RT for 19 h, and then additional 4N HCI in dioxane (1.10 mL, 4.40 mmol) was added. The mixture was stirred at RT for another 24 h, then concentrated in vacuo and dried on high vac to afford the title compound as an off-white solid (1.52 g, 1.47 mmol, theoretical yield). LCMS m/z 778.36 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.12 (br s, 5 H) 1.26 - 1.42 (m, 4 H) 1.57 - 1.70 (m, 3 H) 1.71 - 1.79 (m, 2 H) 1.81 - 1.90 (m, 3 H) 2.04 - 2.12 (m, 2 H) 2.52 - 2.56 (m, 3 H) 2.65 - 2.78 (m, 1 H) 2.98 - 3.07 (m, 1 H) 3.12 - 3.27 (m, 4 H) 3.31 - 3.45 (m, 4 H) 3.57 (s, 3 H) 3.92 - 4.47 (m, 4 H) 4.83 (d, J=6.36 Hz, 1 H) 5.06 - 5.23 (m, 2 H) 5.76 (s, 1 H) 7.31 - 7.43 (m, 4 H) 7.66 (br dd, J=7.58, 3.67 Hz, 1 H) 7.97 (dd, J=7.58, 5.62 Hz, 1 H) 8.06 (t, J=5.62 Hz, 1 H) 8.34 (br d, J=7.82 Hz, 1 H) 8.83 (d, J=1.47 Hz, 1 H) 8.86 (d, J=5.38 Hz, 1 H).

Step 8:vDi-tert-butyl (((S)-6-((S)-2-(( 1R,4S)-4-(4-((( 1R,4R)-4-(2-(2-((1 R,4R)-N-{2- (benzyloxy)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxamido)-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2- yl)carbamoyl)-L-glutamate.

To a solution of N-(2-(benzyloxy)-2-oxoethyl)-N-((1R,4r)-4-(4-(((1 S,4R)-4-(2-((2S,3S)- 1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy) butanamido)cyclohexane-1-carbonyl)glycine (225 mg, 0.289 mmol) in dichloromethane (6 mL) was added DIPEA (0.252 mL, 0.289 mmol), di-tert-butyl (((S)-6-((S)-2-((1R,4S)-4-(4- (((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxamido)-3- (naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (300 mg, 0.289 mmol) and HATU (143 mg, 0.376 mmol). The reaction was stirred at RT for 19 h, then diluted with additional dichloromethane (20 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-20% methanol in dichloromethane to afford the title compound as an off-white solid (316 mg, 0.135 mmol, 46.8% yield). LCMS m/z 899.31 (M+2H)/2. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.93 - 1.22 (m, 9 H) 1.23 - 1.32 (m, 7 H) 1.57 - 1.66 (m, 27 H) 1.67 - 1.75 (m, 3 H) 1.78 - 2.01 (m, 15 H) 2.03 - 2.13 (m, 3 H) 2.14 - 2.35 (m, 6 H) 2.38 - 2.47 (m, 2 H) 2.69 (s, 3 H) 2.77 - 2.89 (m, 3 H) 2.93 - 3.03 (m, 1 H) 3.05 - 3.15 (m, 1 H) 3.19 - 3.34 (m, 5 H) 3.38 - 3.59 (m, 20 H) 3.62 - 3.70 (m, 1 H) 3.74 - 3.85 (m, 2 H) 4.03 - 4.15 (m, 3 H) 4.18 - 4.30 (m, 2 H) 4.49 - 4.64 (m, 2 H) 4.82 (br dd, J=4.40, 2.45 Hz, 2 H) 5.17 - 5.24 (m, 2 H) 5.43 - 5.51 (m, 1 H) 5.55 - 5.63 (m, 1 H) 5.84 - 5.92 (m, 1 H) 7.18 - 7.26 (m, 1 H) 7.34 - 7.41 (m, 4 H) 7.42 - 7.51 (m, 3 H) 7.56 - 7.61 (m, 1 H) 7.66 (s, 2 H) 7.75 (br d, J=7.34 Hz, 1 H) 8.16 - 8.24 (m, 1 H) 8.56 (d, J=1.96 Hz, 1 H) 8.64 (dd, J=4.65, 1.71 Hz, 1 H).

Step 9: N-(2-((2-(((1R,4R)-4-(4-(((1 S,4R)-4-(((7S,11S,18S)-7,11-Bis(tert- butoxycarbonyl)-2,2-dimethyl-19-(naphthalen-2-yl)-4,9, 17-trioxo-3-oxa-8, 10, 16- tri azanonadecan-18-yl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy) ethyl)amino)-2-oxoethyl)-N-((1R,4R)-4-(4-(((1 S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2- (pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido) cyclohexane-1-carbonyl)glycine.

To a solution of di-tert-butyl (((S)-6-((S)-2-((1R,4S)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)- N-(2-(benzyloxy)-2-oxoethyl)-4-(4-(((1 S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxamido)-3- (naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate in methanol (3.5 mL) under an atmosphere of nitrogen gas was added Pd/C (18.7 mg, 10% wt., 0.018 mmol). The flask was evacuated and back-filled with a hydrogen gas balloon, then stirred at RT for 18 h. The mixture was filtered through a celite pad, washing with additional methanol (2x 10 mL). The filtrate was concentrated in vacuo and dried on high vac to afford the title compound as a pink solid (185 mg, 0.084 mmol, 48.1% yield). LCMS m/z 854.33 (M+2H)/2. ¹ H NMR (400 MHz, DMSO-d₆) δ ppm 1.01 - 1.24 (m, 16 H) 1.36 - 1.44 (m, 26 H) 1.46 - 1.57 (m, 3 H) 1.66 (dt, J=13.57, 6.66 Hz, 10 H) 1.74 - 1.89 (m, 12 H) 2.01 - 2.10 (m, 5 H) 2.22 (br s, 2 H) 2.29 - 2.37 (m, 1 H) 2.39 - 2.46 (m, 1 H) 2.64 - 2.75 (m, 2 H) 2.87 - 3.01 (m, 4 H) 3.02 - 3.28 (m, 16 H) 3.38 - 3.44 (m, 3 H) 3.80 - 3.86 (m, 1 H) 3.88 - 3.98 (m, 3 H) 4.01 - 4.13 (m, 5 H) 4.48 - 4.59 (m, 2 H) 4.66 (d, J=6.36 Hz, 1 H) 6.20 - 6.34 (m, 3 H) 7.35 - 7.52 (m, 5 H) 7.59 (br d, J=7.82 Hz, 1 H) 7.63 - 7.69 (m, 3 H) 7.77 - 7.82 (m, 2 H) 7.84 - 7.89 (m, 1 H) 7.91 - 7.98 (m, 2 H) 8.00 - 8.06 (m, 1 H) 8.47 (d, J=2.45 Hz, 1 H) 8.56 (dd, J=4.40, 1.47 Hz, 1 H).

Step 10: (((S)-1 -Carboxy-5-((S)-2-((1R,4S)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2- (((1R,4R)-4-(4-(((1R,4R)-4-(((R)-7-((1R,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro- 2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro- 1,7-naphthyridin-8-yl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy) ethyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3- yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxamido)-3-(naphthalen-2-yl)propanamido)pentyl)carbamoyl)-L-glutamic acid (Example 7).

To a solution of N-(2-((2-(((1R,4R)-4-(4-(((1 S,4R)-4-(((7S,11 S,18S)-7,11-bis(tert- butoxycarbonyl)-2,2-dimethyl-19-(naphthalen-2-yl)-4, 9, 17-trioxo-3-oxa-8, 10,16- triazanonadecan-18-yl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy) ethyl)amino)-2-oxoethyl)-N-((1R,4R)-4-(4-(((1 S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin- 3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carbonyl)glycine (80.0 mg, 0.047 mmol) in dichlormethane was added HATU (23.2 mg, 0.061 mmol, DI PEA (0.041 mL, 0.23 mol), and (1R,4R)-4-(4-(((1r,4R)-4-(2- aminoethoxy)cyclohexyl)oxy)butanamido)-N-((R)-7-((1R,3R)-1-isopropyl-3-(((3S,4S)-3- methoxytetrahydro-2/7-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)- 5,6,7,8-tetrahydro-1 ,7-naphthyridin-8-yl)cyclohexane-1-carboxamide, 2Hydrochloride (46.9 mg, 0.052 mmol). The reaction was stirred at RT for 2.5 h, then diluted with additional dichloromethane (20 mL), washed with saturated aqueous sodium bicarbonate (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. To a solution of the resultant residue in dichloromethane (0.5 mL) was added TFA (0.36 mL, 4.7 mmol). The mixture was stirred at RT for 19 h, then concentrated in vacuo. The resultant residue was purified via MDAP (XSelect™ CSH C18 5um column, 40 mL/min) eluting with a gradient of 15 to 55 % acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075 %) to provide the title compound as an off-white solid (28.3 mg, 0.011 mmol, 24.3% yield). LCMS m/z 786.87 (M+2H)/2. HPLC: 100% pure at 254nm. ¹ H NMR (400 MHz, DMSO-d₆) δ ppm 0.68 - 0.75 (m, 2 H) 0.83 - 0.88 (m, 2 H) 1.05 - 1.27 (m, 24 H) 1.28 - 1.35 (m, 6 H) 1.40 - 1.51 (m, 8 H) 1.61 - 1.71 (m, 14 H) 1.74 - 1.91 (m, 24 H) 2.01 - 2.11 (m, 12 H) 2.54 - 2.56 (m, 6 H) 2.65 - 2.74 (m, 6 H) 3.38 (br s, 21 H) 3.80 - 4.03 (m, 14 H) 4.04 - 4.15 (m, 5 H) 4.49 - 4.58 (m, 1 H) 4.61 - 4.70 (m, 1 H) 4.75 - 4.86 (m, 1 H) 4.88 - 5.04 (m, 1 H) 6.13 - 6.22 (m, 1 H) 6.35 - 6.42 (m, 1 H) 7.36 - 7.52 (m, 6 H) 7.55 - 7.71 (m, 7 H) 7.76 - 7.81 (m, 2 H) 7.83 - 7.89 (m, 1 H) 7.90 - 8.08 (m, 3 H) 8.09 - 8.29 (m, 3 H) 8.43 - 8.52 (m, 1 H) 8.52 - 8.58 (m, 1 H) 8.76 - 8.91 (m, 2 H).

EXAMPLE 8

(((S)-1 -Carboxy-5-((S)-2-((1R,4S)-4-(4-(((1R,4R)-4-(2-(2-((1 R,4R)-N-(2-((2-

(((1R,4R)-4-(4-((( 1 S,4R)-4-(4-(6-methoxy-4-((S)-3-(1 -((5-methoxy-6-methylpyridin-2- yl)methyl)piperidin-4-yl)-2-oxooxazolidin-5-yl)quinolin-2-yl)piperidine-1- carbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4- (4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 - carboxamido)-3-(naphthalen-2-yl)propanamido)pentyl)carbamoyl)-L-glutamic acid

Example 8 was prepared in an analogous fashion to Example 7, substituting Intermediate 11 for ((R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1 ,6-naphthyridin-6(5H)- yl)((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl) methanone, to afford the title compound as an off-white solid (26.1 mg, 0.011 mmol, 19.2% yield). LCMS m/z 1294.31 (M+2H)/2. HPLC: 100% pure at 254nm. ¹H NMR (400 MHz, DMSO- d₆) δ ppm 1.02 - 1.27 (m, 18 H) 1.28 - 1.38 (m, 4 H) 1.60 - 1.74 (m, 13 H) 1.76 (br s, 16 H) 1.99 - 2.12 (m, 8 H) 2.13 - 2.18 (m, 1 H) 2.31 (s, 3 H) 2.33 - 2.38 (m, 1 H) 2.38 - 2.47 (m, 2 H) 2.55 (s, 5 H) 2.65 - 2.74 (m, 2 H) 2.77 - 2.92 (m, 3 H) 2.94 - 3.05 (m, 4 H) 3.07 - 3.26 (m, 18 H) 3.26 - 3.43 (m, 31 H) 3.44 - 3.49 (m, 4 H) 3.52 - 3.61 (m, 2 H) 3.78 (s, 3 H) 3.87 (br s, 2 H) 3.93 (s, 3 H) 3.96 - 4.04 (m, 2 H) 4.05 - 4.14 (m, 3 H) 4.27 - 4.36 (m, 1 H) 4.49 - 4.60 (m, 2 H) 4.65 (d, J=5.87 Hz, 1 H) 6.13 - 6.23 (m, 1 H) 6.28 - 6.35 (m, 1 H) 6.41 (br d, J=7.83 Hz, 1 H) 6.57 - 6.69 (m, 1 H) 7.13 - 7.22 (m, 2 H) 7.27 (d, J=8.80 Hz, 1 H) 7.34 - 7.50 (m, 6 H) 7.57 - 7.71 (m, 5 H) 7.76 - 7.82 (m, 2 H) 7.82 - 7.88 (m, 1 H) 7.93 (d, J=9.29 Hz, 1 H) 7.97 - 8.06 (m, 2 H) 8.07 - 8.14 (m, 1 H) 8.22 (quin, J=5.26 Hz, 1 H) 8.47 (d, J=1.96 Hz, 1 H) 8.56 (dd, J=4.65, 1.71 Hz, 1 H) 8.80 - 8.92 (m, 1 H).

EXAMPLE 9

(((S)-1 -Carboxy-5-((S)-2-((1R,4S)-4-(4-(((1R,4R)-4-(2-(2-((1 R,4R)-N-(2-((2- (((1R,4R)-4-(4-(((1 S,4R)-4-(4-(6-methoxy-4-((S)-3-(1 -(4-methoxy-3- methylbenzyl)piperidin-4-yl)-2-oxooxazolidin-5-yl)quinolin-2-yl)piperidine-1- carbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4- (4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1- carboxamido)acetamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1 - carboxamido)-3-(naphthalen-2-yl)propanamido)pentyl)carbamoyl)-/_-glutamic acid

Example 9 was prepared in an analogous fashion to Example 8, substituting 4- methoxy-3-methylbenzaldehyde for 5-methoxy-6-methylpicolinaldehyde to afford the title compound as an off-white solid (22.1 mg, 0.0090 mmol, 15.8% yield). LCMS m/z 1294.29 (M+2H)/2. HPLC: 96% pure at 254nm. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.01 - 1.28 (m, 18 H) 1.29 - 1.37 (m, 3 H) 1.49 - 1.60 (m, 4 H) 1.61 - 1.74 (m, 12 H) 1.75 - 1.92 (m, 15 H) 1.92 - 2.01 (m, 3 H) 2.01 - 2.09 (m, 5 H) 2.10 - 2.13 (m, 3 H) 2.31 - 2.36 (m, 1 H) 2.39 - 2.46 (m, 1 H) 2.49 - 2.50 (m, 2 H) 2.58 (br s, 3 H) 2.65 - 2.75 (m, 2 H) 2.76 - 2.82 (m, 1 H) 2.84 - 2.90 (m, 1 H) 2.91 - 3.05 (m, 5 H) 3.08 - 3.27 (m, 21 H) 3.28 - 3.47 (m, 32 H) 3.48 - 3.60 (m, 4 H) 3.72 - 3.77 (m, 3 H) 3.84 - 3.89 (m, 2 H) 3.91 - 3.95 (m, 3 H) 3.95 - 4.04 (m, 2 H) 4.05 - 4.14 (m, 3 H) 4.31 (br t, J=8.80 Hz, 1 H) 4.49 - 4.61 (m, 2 H) 4.65 (d, J=5.87 Hz, 1 H) 6.13 - 6.24 (m, 1 H) 6.27 - 6.36 (m, 1 H) 6.41 (br d, J=8.31 Hz, 1 H) 6.53 - 6.71 (m, 2 H) 6.84 (d, J=8.80 Hz, 1 H) 6.99 - 7.08 (m, 2 H) 7.18 (d, J=2.93 Hz, 1 H) 7.36 - 7.51 (m, 5 H) 7.55 - 7.76 (m, 5 H) 7.76 - 7.82 (m, 2 H) 7.82 - 7.88 (m, 1 H) 7.92 (d, J=9.29 Hz, 1 H) 7.97 - 8.06 (m, 2 H) 8.09 (br d, J=7.34 Hz, 1 H) 8.22 (br t, J=5.62 Hz, 1 H) 8.47 (d, J=1.96 Hz, 1 H) 8.56 (dd, J=4.65, 1.71 Hz, 1 H) 8.81 - 8.91 (m, 1 H).

EXAMPLE 10

Step 1 : tert-Butyl (S)-4-(6-methoxy-4-(3-(1-(4-methoxy-3-methylbenzyl)piperidin- 4-yl)-2-oxooxazolidin-5-yl)quinolin-2-yl)piperidine-1 -carboxylate

To a solution of (S)-5-(6-methoxyquinolin-4-yl)-3-(1-(2,2,2-trifluoroacetyl)piperidin-4- yl)oxazolidin-2-one (569 mg, 1.11 mmol) in DCE (11 mL) was added 4-methoxy-3- methylbenzaldehyde (201 mg, 1.34 mmol) and acetic acid (3.19 μl, 0.056 mmol).

Sodium triacetoxyhydroborate (472 mg, 2.23 mmol) was added, and the reaction was stirred at RT for 23 h. The reaction was diluted with saturated aqueous sodium bicarbonate (50 mL) and extracted with dichloromethane (3x 30 mL). The combined organic fractions were dried over sodium sulfate, filtered, and concentrated. The resultant residue was purified via silica gel chromatography eluting with 0-10% methanol in dichloromethane to afford the titlec compound as a pale yellow solid (512 mg, 0.779 mmol, 69.9% yield). LCMS m/z 645.42 (M+H)⁺. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.51 (s, 9 H) 1.60 - 1.69 (m, 2 H) 1.80 - 1.92 (m, 4 H) 1.93 - 2.02 (m, 2 H) 2.03 - 2.17 (m, 2 H) 2.19 - 2.23 (m, 3 H) 2.79 - 3.09 (m, 5 H) 3.37 - 3.44 (m, 1 H) 3.45 - 3.48 (m, 2 H) 3.79 - 3.89 (m, 4 H) 3.95 (s, 3 H) 4.16 - 4.24 (m, 1 H) 4.25 - 4.39 (m, 2 H) 6.07 (dd, J=8.80, 6.85 Hz, 1 H) 6.72 - 6.79 (m, 1 H) 6.83 (d, J=2.93 Hz, 1 H) 7.01 - 7.09 (m, 2 H) 7.38 - 7.46 (m, 2 H) 8.05 (d, J=9.29 Hz, 1 H).

Step 2: tert-Butyl (S)-22-(4-(6-methoxy-4-(3-(1-(4-methoxy-3-methyl benzyl) piperidin-4-yl)-2-oxooxazolidin-5-yl)quinolin-2-yl)piperidin-1-yl)-22-oxo-4,7,10,13,16,19- hexaoxadocosanoate.

To a solution of tert-butyl (S)-4-(6-methoxy-4-(3-(1-(4-methoxy-3- methylbenzyl)piperidin-4-yl)-2-oxooxazolidin-5-yl)quinolin-2-yl)piperidine-1 -carboxylate (53 mg, 0.082 mmol) in dichloromethane (2 mL) was added 4N HCI/Dioxane (0.205 mL, 0.822 mmol). The mixture was stirred at RT for 2 h, then concentrated in vacuo. To a solution of the resultant oil in DMF (2 mL) was added DIPEA (0.187 mL, 1.07 mmol), 2,2-dimethyl-4-oxo- 3,7,10,13,16,19,22-heptaoxapentacosan-25-oic acid (36.0 mg, 0.082 mmol), and HATU (34.4 mg, 0.090 mmol). The reaction was stirred at RT for 1 h, then purified via MDAP (XSelect™ CSH C18 5um column, 40 mL/min) eluting with a gradient of 15 to 55 % acetonitrile in water containing TFA (0.1 %) to provide the title compound as a clear oil (66.0 mg, 0.066 mmol, 81% yield). LCMS m/z 965.7 (M+H)⁺. ¹H NMR (CHLOROFORM-d, 400MHz): δ ppm 8.49 (d, J=9.3 Hz, 1 H) 7.83 (d, J=6.4 Hz, 1 H) 7.72 (d, J=7.8 Hz, 1 H) 7.15 - 7.25 (m, 1 H) 7.02 (s, 1 H) 6.87 (d, J=8.3 Hz, 1 H) 6.26 (br. s., 1 H) 4.89 (d, J=12.0 Hz, 1 H) 4.46 - 4.60 (m, 1 H) 3.98-4.25 (m, 5 H) 3.53 - 3.92 (m, 29 H) 3.46 (br. s., 1 H) 3.32 (t,J=12.6 Hz, 1 H) 2.70-2.94 (m, 3 H) 2.65 (t, J=5.9 Hz, 1 H) 2.52 (dt, J=8.6, 6.6 Hz, 3 H) 2.05-2.30 (m, 4 H) 1.94 (d, J=10.8 Hz, 2 H) 1.47 (d, J=3.2 Hz, 9 H). Step 3: (S)-22-(4-(6-Methoxy-4-(3-(1-(4-methoxy-3-methylbenzyl)piperdin-4-yl)-2- oxooxazolidin-5-yl)quinolin-2-yl)piperidin-1-yl)-22-oxo-4,7,10,13,16,19- hexaoxadocosanoic acid.

To a solution of tert-butyl (S)-22-(4-(6-methoxy-4-(3-(1-(4-methoxy-3- methylbenzyl)piperidin-4-yl)-2-oxooxazolidin-5-yl)quinolin-2-yl)piperidin-1-yl)-22-oxo- 4,7,10,13,16,19-hexaoxadocosanoate (250 mg, 0.259 mmol) in dichloromethane was added 4N HCI in dioxane (648 μl, 2.59 mmol). The solution was stirred at RT for 3 h, then concentrated in vacuo to provide the title compound as a tan oil (244 mg, 0.263 mmol, theoretical yield). LCMS, m/z 909.7 (M+H)⁺. ¹ H NMR (METHANOL-d₄, OOM Hz): δ ppm 8.19

(d, J=9.3 Hz, 1 H) 7.87 (s, 1 H) 7.78 (br. s., 1 H) 7.26 - 7.39 (m, 3 H) 7.01 (d, J=8.3 Hz, 1 H) 6.53 (br. s., 1 H) 5.51 (s, 3 H) 4.50 (t, J=9.3 Hz, 1 H) 4.31 (br. s., 1 H) 4.23 (s, 2 H) 4.06 (s, 3 H) 3.99 (d, J=7.Q Hz, 1 H) 3.87 (s, 3 H) 3.78 - 3.85 (m, 2 H) 3.49-3.78 (m, 44 H) 3.06 - 3.18 (m, 3 H) 2.77 - 2.91 (m, 2 H) 2.61 - 2.74 (m, 1 H) 2.47 - 2.60 (m, 5 H) 2.23 (s, 3 H) 1.78 - 2.21 (m, 9 H).

Step 4: tert-Butyl (21-(((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-21-oxo- 3,6,9, 12, 15, 18-hexaoxahenicosyl)carbamate.

To a solution of 2,2-dimethyl-4-oxo-3,8, 11,14, 17,20,23-heptaoxa-5-azahexacosan-26- oic acid (68.3 mg, 0.151 mmol) in DMF (1 mL) was added HATU (63.0 mg, 0.166 mmol) and DIEA (0.066 ml, 0.377 mmol). The mixture was stirred at for 10 min, and then (S)- 1-((1S,2R,4R)-2-amino-4-(tert-butylamino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4- yl)amino)pyrrolidin-2-one (70 mg, 0.151 mmol) was added. The reaction was strired at RT overnight, and then purified via MDAP (XSelect™ CSH C18 5um column, 40 mL/min) eluting with a gradient of 15 to 55 % acetonitrile in water containing TFA (0.1 %) to provide the title compound as a clear oil (134 mg, 0.128 mmol, 85% yield). LCMS m/z 900.7 (M+H)⁺. 1 H NMR (CHLOROFORM-d, 400MHz):δ ppm 10.04 - 11.88 (m, 5 H) 8.57 - 9.10 (m, 3 H) 7.90 - 8.29 (m, 3 H) 7.14 (br. s., 1 H), 5.53 (d, J=7.Q Hz, 1 H) 4.15 - 4.49 (m, 2 H) 3.04 - 4.04 (m, 33 H) 1.78 - 2.76 (m, 11 H) 1.45 (d, J=7.8 Hz, 18 H).

Step 5: 1-Amino-N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-

(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)-3,6,9,12,15,18- hexaoxahenicosan-21 -amide, Hydrochloride.

To a solution of tert-butyl (21-(((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-21-oxo-

3,6,9, 12, 15, 18-hexaoxahenicosyl)carbamate (134 mg, 0.149 mmol) in dichloromethane (2 mL) was added 4N HCI in dioxane (0.372 mL, 1.489 mmol). The mixture was stirred at RT for 2 h, then concentrated in vacuo to afford the title compound as a light tan oil (109 mg, 0.129 mmol, 87% yield). LCMS mz/ 3,6,9, 12,15, 18-hexaoxahenicosan-21 -amide, Hydrochloride (109 mg, 0.129 mmol, 87 % yield) as a light tan oil. LCMS m/z 800.5 (M+H)⁺. ¹H NMR (METHANOL-d₄, 400MHz): δ ppm 9.30 (s, 1 H) 8.95 (s, 1 H), 8.36 (dd, J=8.8, 1.5 Hz, 1 H), 8.06 (d, J=8.8 Hz, 1 H), 5.70 (t, J=9.5 Hz, 1 H) 4.46 (br. s., 1 H) 4.29 (dt, J=12.5, 4.5 Hz, 1 H) 4.01 - 4.11 (m, 1 H) 3.57-3.89 (m, 37 H) 3.54 (s, 4 H) 3.11 - 3.21 (m, 2 H) 2.56 - 2.74 (m, 4 H) 1.95 - 2.26 (m, 6 H) 1.43 - 1.56 (m, 9 H).

Step 6: tert-Butyl (2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)(34-(((1R,2S,5R)-5-

(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1- yl)cyclohexyl)amino)-12,34-dioxo-3,6,9,16,19,22,25,28,31 -nonaoxa-13- azatetratri aconty I )ca rbam ate.

To a solution of 1-amino-N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)-3,6,9, 12,15,18- hexaoxahenicosan-21 -amide (83 mg, 0.104 mmol) and 1-azido-12-(tert-butoxycarbonyl)- 3,6,9,15, 18,21-hexaoxa-12-azatetracosan-24-oic acid (56.9 mg, 0.109 mmol) in DMF (1 mL) was added HATU (43.4 mg, 0.114 mmol) and DIPEA (0.054 ml, 0.31 mmol). The solution was stirred at RT for 1 h, then purified via MDAP (XSelect™ CSH C18 5um column, 40 mL/min) eluting with a gradient of 15 to 55 % acetonitrile in water containing TFA (0.1 %) to provide the title compound as a clear oil (117 mg, 0.088 mmol, 85% yield). LCMS m/z 653.1 (M+H)⁺. ¹ H NMR (CHLOROFORM-d, 400MHz): δ ppm 10.44 (d, J=6.1 Hz, 1 H) 8.74 - 8.99 (m, 2 H) 8.05 - 8.21 (m, 2 H) 7.16 - 7.33 (m, 1 H) 5.53 (d, J=8.1 Hz, 1 H) 4.38 (br. s., 1 H) 4.15 (br. s., 1 H) 3.86 (t, J=8.7 Hz, 1 H) 3.33-3.78 (m, 39 H) 1.97 - 2.70 (m, 7 H) 1.85 (br. s., 1 H) 1.38 - 1.54 ppm (m, 11 H).

Step 7: 1 -Azido-N-(21 -(((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-

(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-21-oxo- 3,6,9, 12, 15, 18-hexaoxahenicosyl)-3,6,9, 15, 18,21 -hexaoxa-12-azatetracosan-24-am ide, Hydrochloride.

To a solution of tert-butyl (2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)(34- (((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4- yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-12,34-dioxo-3,6,9,16, 19,22,25,28,31-nonaoxa-13- azatetratriacontyl)carbamate (109 mg, 0.084 mmol) in dichloromethane (2 mL) was added 4N HCI in dioxane (0.209 mL, 0.836 mmol). The solution was stirred at RT for 2 h, then concentrated in vacuo to provide the title compound as a light tan oil (122 mg, 0.087 mmol, theoretical yield). LCMS m/z 603.1 (M+H)⁺. 1 H NMR (CHLOROFORM-d, 400MHz): δ ppm 11.03 (d, J=6.4 Hz, 1 H) 9.42 (br. s., 1 H) 9.10 (d, J=10.3 Hz, 1 H) 8.97 (br. s., 1 H) 8.79 (br. s., 1 H) 8.50 - 8.73 (m, 3 H) 8.37 (d, J=8.3 Hz, 1 H) 8.10 (d, J=8.8 Hz, 1 H) 7.71 (br. s., 1 H) 5.68 (br. s., 1 H) 4.34 - 4.56 (m, 2 H) 4.22 (br. s., 1 H) 3.17 - 3.94 (m, 86 H) 2.35 - 2.84 (m, 9 H) 1.85 - 2.34 (m, 5 H) 1.46 - 1.68 (m, 10 H). Step 8: 3-(2-((2S,3S)-1 -Methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido) ethoxy)propanoic acid.

To a solution of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (2.02 g, 9.17 mmol) and (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (2.02 g, 9.17 mmol) in acetonitrile (92 mL) was added N-methylmorpholine (3.00 mL, 27.3 mmol). The mixture was stirred at RT for 16.5 h, then concentrated in vacuo. The resultant residue was purified via reverse phase preparative chromatography (EZ Prep Isco, C18 Aq 275g Gold, 0-25% gradient, Acetonitrile with 0.1% Formic acid/Water with 0.1% Formic acid, 150 mL/min flow rate, 30 min overall run time) to afford the title compound as an off-white solid (3.2 g, 9.54 mmol, 104 % yield) that was used without further purification or characterization. LCMS m/z 336.2 (M+H)⁺.

Step 9: (2S,3S)-N-(7-(2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl)-41-

(((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl) amino)pyrrolidin-1-yl)cyclohexyl)amino)-6,19,41-trioxo-3,10,13,16,23,26,29,32,35,38- decaoxa-7,20-diazahentetracontyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamide.

To a solution of 3-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethoxy)propanoic acid (29.2 mg, 0.087 mmol) in DMF (1 mL) was added HATU (34.7 mg, 0.091 mmol), 1-azido-N-(21-(((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6- (trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-21-oxo- 3,6,9,12, 15, 18-hexaoxahenicosyl)-3,6,9,15, 18,21-hexaoxa-12-azatetracosan-24-amide (100 mg, 0.083 mmol), and DIPEA (0.051 mL, 0.291 mmol). The reaction was stirred at RT until completion by LCMS, then purified via MDAP (XSelect™ CSH C18 5um column, 40 mL/min) eluting with a gradient of 15 to 55 % acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075 %) to afford the title compound as a clear film (87 mg, 0.055 mmol, 67% yield). LCMS m/z 761.7 (M+H)⁺. ¹ H NMR (METHANOL-d₄, 400MHz): δ ppm 8.86 (s, 1 H) 8.56 - 8.64 (m, 2 H) 8.53 (d, J=1.7 Hz, 1 H) 8.07 (dd, J=8.8, 2.0 Hz, 1 H) 7.91 (d, J=8.8 Hz, 1 H) 7.78 - 7.84 (m, 1 H) 7.54 (dd, J=7.9, 5.0 Hz, 1 H) 5.32 (t, J=7.9 Hz, 1 H), 4.84 (d, J=6.6 Hz, 1 H) 4.65 (br s, 1 H) 3.97 - 4.08 (m, 1 H) 3.81 - 3.90 (m, 1 H) 3.44 - 3.80 (m, 57 H) 3.35 - 3.41 (m, 16 H) 3.03 - 3.14 (m, 1 H) 2.80 - 2.91 (m, 1 H) 2.62 - 2.77 (m, 6 H) 2.40 - 2.59 (m, 5 H) 2.18 - 2.33 (m, 1 H) 1.82 - 2.03 (m, 4 H) 1.65 - 1.80 (m, 2 H) 1.20 (s, 9 H).

Step 10: (2S,3S)-N-(7-(2-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)ethyl)-41-

(((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4- yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-6,19,41-trioxo-3,10,13,16,23,26,29,32,35,38- decaoxa-7,20-diazahentetracontyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamide.

To a solution of 3-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3- carboxamido)ethoxy)propanoic acid (29.2 mg, 0.087 mmol) in DMF (1 mL) was added HATU (34.7 mg, 0.091 mmol), (2S,3S)-N-(7-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-41- (((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino) pyrrolidin-1-yl)cyclohexyl)amino)-6,19,41-trioxo-3, 10, 13,16,23,26,29,32,35,38-decaoxa-7,20- diazahentetracontyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide (89 mg, 0.058 mmol), and DIEA (0.051 mL, 0.291 mmol). The solution was stirred at RT until completion of the reaction as monitored by LCMS, then purified via MDAP (XSelect™ CSH C185um column, 40 mL/min) eluting with a gradient of 15 to 55 % acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075 %) to afford the title compound as a clear film (87 mg, 0.055 mmol, 67% yield). LCMS m/z 761.7 (M+H)⁺. ¹H NMR (METHANOL- d₄, 400MHz): δ ppm 8.86 (s, 1 H) 8.56 - 8.64 (m, 2H), 8.53 (d, J=1.7 Hz, 1 H) 8.07 (dd, J=8.8, 2.0 Hz, 1 H) 7.91 (d, J=8.8 Hz, 1 H) 7.78 - 7.84 (m, 1 H) 7.54 (dd, J=7.9, 5.0 Hz, 1 H) 5.32 (t, J=7.9 Hz, 1 H) 4.84 (d, J=Q.Q Hz, 1 H) 4.65 (br s, 1 H) 2.97 - 4.08 (m, 1 H) 3.81 - 3.90 (m, 1 H) 3.44 - 3.80 (m, 57 H) 3.35 - 3.41 (m, 16 H) 3.03 - 3.14 (m, 1 H) 2.80 - 2.91 (m, 1 H) 2.62 - 2.77 (m, 6 H) 2.40 - 2.59 (m, 5 H) 2.18 - 2.33 (m, 1 H) 1.82 - 2.03 (m, 4 H) 1.65 - 1.80 (m, 2 H) 1.20 (s, 9 H).

Step 11 : (2R,3R)-N-(7-(34-(((1R,2S,5R)-5-(tert-Butylamino)-2-((S)-2-oxo-3-((6-

(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-12,34-dioxo- 3,6,9,16,19,22,25,28,31 -nonaoxa-13-azatetratriacontyl)-41-(4-(6-methoxy-4-((S)-3-(1-(4- methoxy-3-methylbenzyl)piperidin-4-yl)-2-oxooxazolidin-5-yl)quinolin-2-yl)piperidin-1- yl)-6,20,41 -trioxo-3, 10,13,16,23,26,29,32,35,38-decaoxa-7,19-diazahentetracontyl)-1 - methyl -5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide (Example 10).

To a solution of (S)-22-(4-(6-methoxy-4-(3-(1-(4-methoxy-3-methylbenzyl)piperidin-4- yl)-2-oxooxazolidin-5-yl)quinolin-2-yl)piperidin-1-yl)-22-oxo-4,7, 10, 13,16,19- hexaoxadocosanoic acid (30 mg, 0.033 mmol) in DMF (1 mL) was added HATU (13.2 mg, 0.035 mmol), followed by DIPEA (0.050 mL, 0.286 mmol). The solution was stirred at RT for 5 minutes, and then (2S,3S)-N-(7-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-41- (((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4- yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-6, 19,41-trioxo-3,10, 13, 16,23,26,29,32,35,38- decaoxa-7,20-diazahentetracontyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide (49.4 mg, 0.033 mmol) The reaction was stirred at RT for 1 h, then quenched with 3 drops of deionized water. The crude mixture was purified via MDAP (XSelect™ CSH C18 5um column, 40 mL/min) eluting with a gradient of 15 to 55 % acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075 %) to afford the title compound as a clear oil (31 mg, 0.012 mmol, 37% yield). LCMS m/z 679.3 (M+H)⁺. HPLC: 97.0% pure at 254nm. ¹ H NMR (DMSO-d₆, 400MHz): δ ppm 10.01 (br. s., 1 H) 9.01 (br. s., 1 H) 8.52 - 8.83 (m, 3 H) 8.47 (s, 1 H) 8.02 - 8.14 (m, 2 H) 7.84 - 7.99 (m, 4 H) 7.67 (d, J=7.8 Hz, 1 H) 7.34 -

7.53 (m, 3 H) 7.18 (br s, 1 H) 6.97 - 7.10 (m, 2 H) 6.83 (d, J=8.3 Hz, 1 H) 6.31 (t, J=7.8 Hz, 1 H) 5.00 (t, J=6.4 Hz, 1 H) 4.65 (d, J=5.9 Hz, 1 H), 4.44 - 4.60 (m, 2 H) 4.31 (t, J=9.0 Hz, 1 H)

3.54 - 4.21 (m, 34 H) 3.04 - 3.41 (m, 113 H) 2.54 - 2.98 (m, 15 H) 2.24 - 2.43 (m, 8 H) 2.07 - 2.21 (m, 4 H) 1.44 - 2.02 (m, 18 H) 1.10 (s, 9 H).

BIOLOGICAL ASSAYS

Example Compounds 1-10 which are compounds of Formula (I) having one or more binding moieties targeting PSMA, CCR2, and/or CCR8 were tested in various biological assays as described in more detail below.

EXAMPLE 11 : Antibody Dependent Cellular Cytotoxicity Reporter Assay

An antibody dependent cellular cytotoxicity (ADCC) reporter assay was conducted using the following four assay components: (i) ARM compound of Formula (I) targeting PSMA, CCR2, and/or CCR8 (concentrations ranging from 1 pM to 10 pM); (ii) anti-cotinine antibody having a heavy chain sequence of SEQ ID NO: 11 and a light chain sequence of SEQ ID NO: 12 (rabbit variable region with human IgG 1 Fc domain containing DE mutation (S239D/I332E)) (concentrations ranging from 0.01 μg / mL - 200 pg I mL); (iii) Target cells: LNCaP cells (PSMA-positive) (typically 1000-20,000 cells per well), hCCR2 CHOK1 cells (hCCR2+), mCCR2 CHOK1 cells (mCCR2+), or hCCR8 CHOK1 cells (CCR8+); and (iv) Reporter cells — Jurkat cells engineered to express FcγRllla with a reporter gene luciferase under the control of the NFAT promoter (typically 3000-75,000 cells per well). Reagents were combined in final volume of 20 μL in 384 - well tissue culture treated plate. All four assay components were incubated together for about 12-18 hours. Thereafter, BioGio Detection reagent (Promega) was added to the wells to lyse the cells and provide a substrate for the luciferase reporter protein.

Luminescence signal was measured on a microplate reader capable of measuring luminescence and signal background was calculated by dividing the signal of a test well by the signal obtained when no compound of formula (I) was included in the assay. EC50 calculations were done using Graphpad Prism Software, specifically a nonlinear regression curve fit ( Y = Bottom + ( Top - Bottom ) / ( 1 + 10 ^ ( ( Log EC50 - X ) * HillSlope ) ) ).

ARMs compounds of Formula (I) were tested for ADCC activity in the above assay in one or more experimental runs and the results are shown in Table 4 below. Potency of the compounds of Formula (I) is reported as a pEC₅₀ values. The pEC₅₀ value is the negative log of the EC₅₀ value, wherein the EC₅₀ value is half maximal effective concentration measured in molar (M). For compounds tested in more than one experimental run, the pEC₅₀ value is reported as an average.

Table 4: Results for ADCC Reporter Assay (Example 11)

¹ inactive= pEC₅₀ <5; ²N/A = not tested EXAMPLE 12: PK Analysis

Mice (C57BL6) were dosed intravenously with a PBS solution containing a compound of Formula (I) of Examples 1, 2, 4, 7, 8 and 9. Peripheral blood from IV dosed mice was analyzed to determine PK properties of the ARM compounds of Formula (I).

Formulations preparation: On the day of experiment, stock solution of the compound of formula (I) was removed from storage at -20°C and thawed at room temperature. Anti- cotinine antibody having a heavy chain sequence of SEQ ID NO: 13 and a light chain sequence of SEQ ID NO: 14 (rabbit variable region sequence with mouse lgG2a Fc domain), if required was removed from storage at -80°C and thawed at room temperature. Antibody vials were immediately transferred into wet ice after thawing. Compounds of formula (I) were further diluted in DMSO as per experimental requirements.

Formulation composition: The formulation composition was Saline: DMSO: PBS. Saline was added based on the quantity required and then stock solution of the compound of formula (I) prepared in DMSO, followed by addition of antibody in PBS. Formulations were incubated at room temperature for 30 minutes before administration to the mouse. DMSO was used at 1 to 2 % (v/v) in the final formulation.

Administration to Animal: Solution formulation of antibody and compound of formula (I) was injected (bolus injection) to the restrained mouse in the right/left lateral tail vein.

Collection of Blood for PK: Blood was collected at various time points, typically ranging from 0.033hrs to 72hours following administration (50 μL/time point) through retro-orbital bleeding under mild isoflurane anesthesia.

Terminal bleeding at end of experiment (72hr): Approximately 250 μL of blood in K2EDTA tube and approximately 250 μL of blood in SST (serum separation tube) was collected from each mouse through retro-orbital bleeding under deep isoflurane anesthesia. After bleeding, each mouse was sacrificed by cervical dislocation. The blood distribution at termination was determined as follows: 50 μL of K2EDTA blood was transferred to another tube for PK.

Blood drug concentration analysis: Drug concentration in blood samples was determined by an LC-MS/MS-based bioanalytical method developed at Syngene. Samples were analyzed on Q-Trap, API-5500 LC-MS/MS system coupled with Exion UHPLC system from SCIEX, USA operated in multiple reaction monitoring mode employing electrospray ionization technique in positive polarity. Analyte and internal standard peaks were resolved on Synergi Polar, 75 X 2.0 mm, 4 μ column using mobile phase 10 mM Ammonium acetate in Milli-Q water as phase A and 0.1 % Formic acid in acetonitrile as Phase B. Gradient elution was performed with initial composition 95 % Phase A at 0.0 min, holding it for 0.2 minutes, ramping to 5 % by 1 .0 minute, keeping the same for next 0.5 minutes and coming back to 95 % by 1.6 minutes. The total run time was 2 minutes.

Working dilutions for calibration curve and quality control standards were prepared by serially diluting 20 mg/mL stock solution with DMSO. Spiked concentrations for calibration curve in the whole blood ranged from 1 ng/mL to 1000 ng/mL. The working solution of internal standard (Verapamil, 25 ng/mL) was prepared in acetonitrile. 10 μL of the study sample and calibration curve, quality control, and blank whole blood samples were aliquoted in 96 deep well plates for processing. 10 μL of Milli-Q water was added to all the samples and briefly vortexed to initiate complete hemolysis. 10 μL of 20 mM dithiothreitol (DTT) was added to all the samples and incubated for 30 minutes at 37°C. The addition of DTT enhanced the recovery of ARM compounds of formula (I) from the biological matrix. 300 μL of working internal standard solution was added to all samples except total blank and wash samples, where 300 μL of acetonitrile was added. All the samples were vortex mixed for 5 minutes, followed by centrifugation at 4000 rpm for 10 minutes at 4 °C. Supernatants were transferred to the loading plate and injected 3 μL to LC-MS/MS system for analysis. The results are shown in FIGs. 2A- 2F. The results demonstrate that dosing the ARM compound of Formula (I) in the presence of anti-cotinine antibody extends the half-life of the compound of Formula (I).

SEQUENCE LISTINGS

Heavy chain CDR1 amino acid sequence

SEQ ID NO: 1

Heavy chain CDR2 amino acid sequence

SEQ ID NO: 2

Heavy chain CDR3 amino acid sequence

SEQ ID NO: 3

Light chain CDR1 amino acid sequence

SEQ ID NO: 4

Light chain CDR2 amino acid sequence

SEQ ID NO: 5

Light chain CDR3 amino acid sequence

SEQ ID NO: 6

Variable heavy chain amino acid sequence

SEQ ID NO: 7

Variable light chain amino acid sequence

SEQ ID NO: 8

Heavy chain amino acid sequence

SEQ ID NO: 9

Light chain amino acid sequence

SEQ ID NO: 10

Heavy chain amino acid sequence

SEQ ID NO: 11

Light chain amino acid sequence

SEQ ID NO: 12

Heavy chain amino acid sequence

SEQ ID NO: 13

Light chain amino acid sequence

SEQ ID NO: 14

Claims

1. A compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

T¹ and T² are each independently a target binding moiety;

R¹ is C_1-4 alkyl or C_3-6 cycloalkyl;

G¹ and G² are each independently -C(O)CH₂-, -CH₂CH₂NHC(O)CH₂- CH₂CH₂C(O)NH(CH₂CH₂O)₃CH₂CH₂-, or -CH₂CH₂NHC(O)(CH₂CH₂O)₃CH₂CH₂-; bond,

each y is an integer of 1 to 9; each w is an integer of 0 to 5;

L¹, L² and L³ are each independently a bond or a divalent linker of Formula (L- a), (L-c), or (L-e), with the proviso that at least one of L¹, L² and L³ is a divalent linker of Formula (L-a), (L-c), or (L-e): (L-a), or a stereoisomer thereof,

wherein:

Ring A and Ring B are each independently C_4-6 cycloalkylene;

L^{1 a} is C_3-5 linear alkylene, wherein 1 or 2 methylene units are replaced with -O- or -NR^a-; each R^a is independently hydrogen or C_1-3 alkyl; and

L^2a is -O-, -NHC(O)-, or -CH₂-O-;

(L-c), or a stereoisomer thereof, wherein:

L^{1 c} is C_2-10 linear alkylene, wherein 1, 2, or 3 methylene units are replaced with -O-, -NH-, -NHC(O)-, or -C(O)NH-;

Ring A is C_4-6 cycloalkylene or C_7-9 bridged bicyclic cycloalkylene; and

L^2c is -O- or a saturated C_2-10 linear alkylene, wherein 1, 2, or 3 methylene units are replaced with -O-, -NH-, -NHC(O)-, or - C(O)NH-;

(L-e), wherein n is an integer of 3 to 50; wherein each

of a L” group represents a covalent bond to the L² group of Formula (I), or when L² is a bond, a covalent bond to the G² group of Formula (I); wherein each

of a L³ group represents a covalent bond to the methylene group of the G group of Formula (I); and

Y¹ and Y² are each independently a bond or a divalent spacer moiety of one to twelve atoms in length.

2. The compound of claim 1 , or a pharmaceutically acceptable salt thereof, wherein R¹ is -CH₃.

3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein

L’ and L” are each independently a bond or

4. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein L¹ , L² or L³ is a divalent linker of Formula (L-a-i):

(L-a-i), or a stereoisomer thereof, wherein Ring A, L^1a, L^2a,

are as defined for Formula (L-a).

5. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein L¹ , L² or L³ is a divalent linker of Formula (L-a-ii):

(L-a-ii), or a stereoisomer thereof, wherein L^1a, L^2a,

are as defined for Formula (L-a); p is 1 or 2; and m is 1 or 2.

6. The compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein L¹ , L² or L³ is a divalent linker of Formula (L-a-iii):

(L-a-iii), or a stereoisomer thereof, wherein p is 1 or 2; m is 1 or 2; n is 1 , 2, or 3; and

are as defined for Formula (L-a).

7. The compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein L¹, L² or L³ is a divalent linker of Formula (L-a) selected from the group consisting of:

8. The compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein L¹, L² or L³ is a divalent linker of Formula (L-c-i):

(L-c-i) , or a stereoisomer thereof, wherein L^1c, L^2c,

, and

are as defined for Formula (L-c); p is 1 or 2; and m is 1 or 2.

9. The compound of any one of claims 1 to 3, wherein L¹, L² or L³ is a divalent linker of Formula (L-c) selected from the group consisting of:

10. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein Y¹ and Y² are each independently selected from a bond; -NH-; - (C_1-12 alkylene)-, wherein 1 , 2, or 3 methylene units are replaced with -O-, -NH-, N(CH₃)-, -C(O)-, -NHC(O)-, -C(O)NH-, -(C_3-6 cycloalkylene)-, -(C_3-6 cycloalkenylene)-, 3- to 10-membered heterocycloalkylene, arylene, or heteroarylene; or -(C_2-12 alkenylene)-, wherein 1 , 2, or 3 methylene units are replaced with -O-, -NH-, N(CH₃)-, -C(O)-, NHC(O)-, -C(O)NH-, -(C_3-6 cycloalkylene)-, -(C_3-6 cycloalkenylene)-, 3- to 10- membered heterocycloalkylene, arylene, or heteroarylene.

11. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein Y¹ and Y² are each independently selected from a bond; -NH-; - (C_1-6 alkylene)-O-; -O-(C_1-6 alkylene)-; -(C_2-6 alkenylene)-O-; -(C_1-6 alkylene)-C(O)-; - (C_2-6 alkenylene)-C(O)-; phenylene; piperidinylene; hydroxypiperidinylene; fluoropiperidinylene; azetidinylene; -C(O)-piperazinylene-; -(C_1-6 alkylene)- oxopiperazinylene-; pyrrolidinylene; 7- to 9-membered bridged bicyclic heterocycloalkylene; -(C_1-6 alkylene)-O-phenylene-; -(C_2-6 alkenylene)-O- piperidinylene; -(C_1-5 alkylene)-NH-, wherein 0, 1 , or 2 methylene units are replaced with -O-; -NH-(C_1-5 alkylene)-NH-; -N(CH₃)-(C_1-5 alkylene)-NH-; NH-(C_1-5 alkylene)- N(CH₃)-; -N(CH₃)-(C_1-5 alkylene)-N(CH₃)-; -(C_3-6 cycloalkylene)-NH-; -C(O)NH-(C_1-5 alkylene)-NH-; -C(O)NH-(C_3-6 cycloalkylene)-NH-; -(C_1-5 alkylene)-O-(C_3-6 cycloalkylene)-NH-; -(C_3-6 cycloalkenylene)-NH-; or

, wherein

Y^1a is a bond, -O-, -NH-, -NHC(O)-, -C(O)NH-, or C_1-3 alkylene; and Y^2a is a bond, -O-, -NH-, -NHC(O)-, -C(O)NH-, or C_1-3 alkylene.

12. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein Y¹ and Y² are each independently selected from the group consisting

13. The compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein Y¹ and Y² are each independently a bond,

14. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein T¹ or T² is:

wherein R² is hydrogen or C_1-4 alkyl; and R³ is hydrogen or C_1-4 alkyl.

15. The compound of claim 14, wherein T¹ or T² is:

16. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein T¹ or T² is selected from the group consisting of:

17. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein T¹ or T² is:

18. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein T¹ or T² is:

19. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein T¹ or T² is:

20. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein T¹ or T² is:

21. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein T¹ or T² is selected from the group consisting of:

R² and R³ are each independently F or H.

22. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein T¹ or T² is:

R² is hydrogen or C_1-4 alkyl; and

R³ is hydrogen or C_1-4 alkyl.

23. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein T¹ or T² is:

Q is C_1-5 alkylene, wherein 0, 1, or 2 methylene units are replaced with -O-; and

Ar is an optionally substituted 5- to 10-membered aromatic ring or 9- or 10-membered unsaturated fused bicyclic ring.

24. The compound of claim 1, wherein the compound is selected from a compound as listed in Table 1.

25. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein the target of the target binding moiety T¹ or T² is selected from G protein-coupled receptor (GPCRs), enzymes, ion channels, proteases, and receptors.

26. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein the target of the target binding moiety T¹ or T² is present on a surface of a pathogenic immune cell, a tumor cell or cancer cell, or a stromal cell.

27. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein the target of the target binding moiety T¹ or T² is present on the surface of a pathogenic agent selected from a virus or a bacterial cell.

28. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein the target of the target binding moiety T¹ or T² is present on a surface of monocytic myeloid-derived suppressor cells (mMDSCs), T regulatory cells (Tregs), neutrophils, macrophages, B regulatory cells (Bregs), CD8 regulatory cells (CD8regs), exhausted T cells, polymorphonuclear myeloid derived suppressor cells (PMN- MDSCs), or cancer-associated fibroblasts (CAFs).

29. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein the target of the target binding moiety T¹ orT² is a chemokine receptor (OCR).

30. The compound of any one of claims 1 to 13, wherein the target of the target binding moiety T¹ or T² is selected from CCR1 , CCR2, CCR3, CCR5, or CCR8.

31. The compound of any one of claims 1 to 13, wherein the target of the target binding moiety T¹ orT² is selected from C-C motif chemokine receptor (CCR) 2 (CCR2), CCR1 , CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, C-X-C motif chemokine receptor 1 (CXCR1), C-X-C motif chemokine receptor 2 (CXCR2), C-X-C motif chemokine receptor 3 (CXCR3), C-X-C motif chemokine receptor 4 (CXCR4), C-X-C motif chemokine receptor 5 (CXCR5), C-X-C motif chemokine receptor 6 (CXCR6), atypical chemokine receptor 3 (ACKR3), integrin avp6, fibroblast activation protein- alpha (FAPa), prostate specific membrane antigen (PSMA), folate receptor (folate receptor 1 or folate receptor beta), complement C3a receptor 1 (C3AR1), complement C5a receptor 1 (C5AR1), G protein-coupled receptor (GPR) 65 (GPR65), GRP132, GPR84, GPR183, GPR35, GPR42, cholecystokinin A receptor (CCKAR), leukotriene B4 receptor (LTB4R), somatostatin receptor 2 (SSTR2), free fatty acid receptor 1 (FFAR1), purinergic receptor P2Y2 (P2RY2), prostaglandin D2 receptor (PTGDR), calcitonin receptor (CALCR), CD38, purinergic receptor P2X 7 (P2RX7), integrin subunit alpha V (ITGAV), integrin subunit alpha 5 (ITGA5), integrin subunit beta 1 (ITGB1), integrin subunit beta 6 (ITGB6), integrin subunit beta 3 (ITGB3) prostaglandin D2 receptor 2 (PTGDR2), gastrin releasing peptide receptor (GRPR), MER proto- oncogene tyrosine kinase (MERTK), C-X3-C motif chemokine receptor 1 (CX3CR1), oxidized low density lipoprotein receptor 1 (OLR1), plasminogen activator urokinase receptor (PLAUR), carbonic anhydrase 9 (CA9), carbonic anhydrase 12 (CA12), mas- related G-protein coupled receptor member X2 (MRGPRX2), heat shock protein 90 alpha family class A member 1 (HSP90AA1), dipeptidyl peptidase 4 (DPP4), formyl peptide receptor 2 (FPR2), and succinate receptor 1 (SUCNR1).

32. A method of treating and/or preventing a disease or disorder in a patient in need thereof, the method comprising: administering to the patient a therapeutically effective amount of the compound of any one of claims 1 to 31 and an anti-cotinine antibody, or antigen-binding fragment thereof, wherein the disease or disorder is selected from a cancer, an inflammatory disease, an autoimmune disease, a viral infection, or a bacterial infection.

33. The method of claim 32, wherein the disease or disorder is mediated by chemokine receptor 2 (CCR2) and/or is associated with CCR2-positive pathogenic cells.

34. The method of claim 32, wherein the disease or disorder is mediated by C-X-C motif chemokine receptor 3 (CXCR3) and/or is associated with CXCR3-positive pathogenic cells.

35. The method of claim 32, wherein the disease or disorder is mediated by PSMA and/or is associated with PSMA-positive pathogenic cells.

36. The method of claim 32, wherein the disease or disorder is mediated by integrin aVp6 and/or is associated with integrin aVp6-positive pathogenic cells.

37. The method of claim 32, wherein the disease or disorder is mediated by folate receptor a (FRα) and/or folate receptor β (FRβ) and/or is associated with FRα- and/or FRβ- positive pathogenic cells.

38. The method of claim 32, wherein the disease or disorder is mediated by fibroblast activation protein (FAP) and/or is associated with AAP-positive pathogenic cells.

39. The method of claim 32, wherein the disease or disorder is mediated by chemokine receptor 8 (CCR8) and/or is associated with CCR8-positive pathogenic cells.

40. The method of any one of claims 32 to 39, wherein the disease is a cancer that is a solid tumor.

41. The method of any one of claims 32 to 40, wherein the cancer is selected from lung cancer (e.g., non-small cell lung cancer (NSCLC)), hepatocellular carcinoma (HCC), colorectal cancer (CRC), cervical cancer (e.g., cervical squamous cell carcinoma (CESC)), head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSC)), pancreatic cancer, prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), ovarian cancer, endometrial cancer, brain cancer, endocrine cancer, testicular cancer, bladder cancer, bone cancer, esophogeal cancer, gastric cancer, renal cell cancer, melanoma cancer, thyroid cancer, or breast cancer.

42. The method of claim 32 or 34, wherein the disease is an autoimmune or inflammatory disease selected from vitiligo and type I diabetes.

43. The method of any one of claims 32 to 42, wherein the compound the antibody, or antigen-binding fragment thereof are administered simultaneously.

44. The method of any one of claims 32 to 42, wherein the compound and the antibody, or antigen-binding fragment thereof are administered sequentially.

45. A method of increasing antibody-dependent cell cytotoxicity (ADCC) of target- expressing cells, the method comprising: contacting the cells with an effective amount of the compound of any one of claims 1 to 31 and an anti-cotinine antibody, or antigen- binding fragment thereof, wherein the target-binding moiety of the compound binds the target expressed on the cells.

46. A method of depleting target-expressing cells, the method comprising: contacting the cells with an effective amount of the compound of any one of claims 1 to 31 and an anti-cotinine antibody, or antigen-binding fragment thereof, wherein the target-binding moiety of the compound binds the target expressed on the cells.

47. The method of claim 45 or 46, wherein the target-expressing cells are myeloid-derived suppressor cells (MDSCs), T regulatory cells (Tregs), neutrophils, macrophages, B regulatory cells (Bregs), CD8 regulatory cells (CD8regs), exhausted T cells, or cancer- associated fibroblasts (CAFs).

48. The method of any one of claims 45 to 47, wherein the target-expressing cells are CCR2-expressing cells.

49. The method of claim 45 or 46, wherein the target-expressing cells are CXCR3- expressing cells.

50. The method of claim 45 or 46, wherein the target-expressing cells are PSMA- expressing cells.

51. The method of claim 45 or 46, wherein the target-expressing cells are integrin aVp6- expressing cells.

52. The method of claim 45 or 46, wherein the target-expressing cells are FRα- and/or FRβ-expressing cells.

53. The method of claim 45 or 46, wherein the target-expressing cells are FAP-expressing cells.

54. The method of claim 45 or 46, wherein the target-expressing cells are CCR8- expressing cells.

55. The method of any one of claims 32 to 54, wherein the anti-cotinine antibody has a heavy chain and a light chain, the heavy chain comprising a CDR1 having SEQ ID NO: 1, a CDR2 having SEQ ID NO: 2, and a CDR3 having SEQ ID NO: 3, and the light chain comprising a CDR1 having SEQ ID NO: 4, a CDR2 having SEQ ID NO: 5, and a CDR3 having SEQ ID NO: 6.

56. The method of any one of claims 32 to 55, wherein the anti-cotinine antibody has a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region (VH) having SEQ ID NO: 7, and the light chain comprising a light chain variable region (VL) having SEQ ID NO: 8.

57. The method of any one of claims 32 to 56, wherein the anti-cotinine antibody is of I gG 1 isotype comprising a substitution in an Fc region to increase ADCC activity.

58. The method of claim 57, wherein the substitution in the Fc region is S239D/I332E, wherein residue numbering is according to the Ell Index.

59. The method of any one of claims 32 to 58, wherein the anti-cotinine antibody has a heavy chain comprising SEQ ID NO: 9 and a light chain comprising SEQ ID NO: 10.

60. A combination comprising the compound of any one of claims 1 to 31 and an anti- cotinine antibody, or antigen-binding fragment thereof.

61. The combination of claim 60, wherein the anti-cotinine antibody has a heavy chain and a light chain, the heavy chain comprising a CDR1 having SEQ ID NO: 1 , a CDR2 having SEQ ID NO: 2, and a CDR3 having SEQ ID NO: 3, and the light chain comprising a CDR1 having SEQ ID NO: 4, a CDR2 having SEQ ID NO: 5, and a CDR3 having SEQ ID NO: 6.

62. The combination of claim 60 or 61 , wherein the anti-cotinine antibody has a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region (VH) having SEQ ID NO: 7, and the light chain comprising a light chain variable region (VL) having SEQ ID NO: 8.

63. The combination of any one of claims 60 to 62, wherein the anti-cotinine antibody is of I gG 1 isotype comprising a substitution in an Fc region to increase ADCC activity.

64. The combination of claim 63, wherein the substitution in the Fc region is S239D/I332E, wherein residue numbering is according to the Ell Index.

65. The combination of any one of claims 60 to 64, wherein the anti-cotinine antibody has a heavy chain comprising SEQ ID NO: 9 and a light chain comprising SEQ ID NO: 10.