WO2022237626A1

WO2022237626A1 - Substituted spiro derivatives

Info

Publication number: WO2022237626A1
Application number: PCT/CN2022/091065
Authority: WO
Inventors: Olivier Alexis Georges Querolle; Xuedong Dai; Wei Cai; Johannes Wilhelmus J. Thuring; Lichao FANG; Ming Li; Lianzhu LIU; Yingtao LIU; Luoheng QIN; Jianping Wu; Yanping Xu; Patrick René Angibaud; Hélène France Solange Colombel; Isabelle Noëlle Constance PILATTE; Virginie Sophie Poncelet; Carsten Sven KRAMER; Vineet PANDE
Original assignee: Janssen Pharmaceutica Nv; Johnson & Johnson (China) Investment Ltd.
Priority date: 2021-05-08
Filing date: 2022-05-06
Publication date: 2022-11-17
Also published as: JP2024518434A; KR20240006542A; CN117321049A; EP4334310A1; BR112023023154A2; CA3215379A1; AU2022272692A1; MX2023013174A

Abstract

The present invention relates to pharmaceutical agents useful for therapy and/or prophylaxis in a mammal, pharmaceutical composition comprising such compounds, and their use as menin/MLL protein/protein interaction inhibitors, useful for treating diseases such as cancer, myelodysplastic syndrome (MDS) and diabetes.

Description

SUBSTITUTED SPIRO DERIVATIVES

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Chromosomal rearrangements affecting the mixed lineage leukemia gene (MLL; MLL1; KMT2A) result in aggressive acute leukemias across all age groups and still represent mostly incurable diseases emphasizing the urgent need for novel therapeutic approaches. Acute leukemias harboring these chromosomal translocations of MLL represent as lymphoid, myeloid or biphenotypic disease and constitute 5 to 10%of acute leukemias in adults and approximately 70%in infants (Marschalek, Br J Haematol 2011. 152 (2) , 141-54; Tomizawa et al., Pediatr Blood Cancer 2007. 49 (2) , 127-32) .

MLL is a histone methyltransferase that methylates histone H3 on lysine 4 (H3K4) and functions in multiprotein complexes. Use of inducible loss-of-function alleles of Mll1 demonstrated that Mll1 plays an essential role in sustaining hematopoietic stem cells (HSCs) and developing B cells although its histone methyltransferase activity is dispensable for hematopoiesis (Mishra et al., Cell Rep 2014. 7 (4) , 1239-47) .

Fusion of MLL with more than 60 different partners has been reported to date and has been associated with leukemia formation/progression (Meyer et al., Leukemia 2013. 27, 2165–2176) . Interestingly, the SET (Su (var) 3–9, enhancer of zeste, and trithorax) domain of MLL is not retained in chimeric proteins but is replaced by the fusion partner (Thiel et al., Bioessays 2012. 34, 771-80) . Recruitment of chromatin modifying enzymes like Dot1L and/or the pTEFb complex by the fusion partner leads to enhanced transcription and transcriptional elongation of MLL target genes including HOXA genes (e.g. HOXA9) and the HOX cofactor MEIS1 as the most prominent ones. Aberrant expression of these genes in turn blocks hematopoietic differentiation and enhances proliferation.

Menin which is encoded by the Multiple Endocrine Neoplasia type 1 (MEN1) gene is expressed ubiquitously and is predominantly localized in the nucleus. It has been shown to interact with numerous proteins and is, therefore, involved in a variety of cellular processes. The best understood function of menin is its role as an oncogenic cofactor of MLL fusion proteins. Menin interacts with two motifs within the N-terminal fragment of MLL that is retained in all fusion proteins, MBM1 (menin-binding motif 1) and MBM2 (Thiel et al., Bioessays 2012. 34, 771-80) . Menin/MLL interaction leads to the formation of a new interaction surface for lens epithelium-derived growth factor (LEDGF) . Although MLL directly binds to LEDGF, menin is obligatory for the stable interaction between MLL and LEDGF and the gene specific chromatin recruitment of the MLL complex via the PWWP domain of LEDGF (Cermakova et al., Cancer Res 2014. 15, 5139-51; Yokoyama & Cleary, Cancer Cell 2008. 8, 36-46) . Furthermore, numerous genetic studies have shown that menin is strictly required for oncogenic transformation by MLL fusion proteins suggesting the menin/MLL interaction as an attractive therapeutic target. For example, conditional deletion of Men1 prevents leukomogenesis in bone marrow progenitor cells ectopically expressing MLL fusions (Chen et al., Proc Natl Acad Sci 2006. 103, 1018-23) . Similarly, genetic disruption of menin/MLL fusion interaction by loss-of-function mutations abrogates the oncogenic properties of the MLL fusion proteins, blocks the development of leukemia in vivo and releases the differentiation block of MLL-transformed leukemic blasts. These studies also showed that menin is required for the maintenance of HOX gene expression by MLL fusion proteins (Yokoyama et al., Cell 2005. 123, 207-18) . In addition, small molecule inhibitors of menin/MLL interaction have been developed suggesting druggability of this protein/protein interaction and have also demonstrated efficacy in preclinical models of AML (Borkin et al., Cancer Cell 2015. 27, 589-602; Cierpicki and Grembecka, Future Med Chem 2014. 6, 447-462) . Together with the observation that menin is not a requisite cofactor of MLL1 during normal hematopoiesis (Li et al., Blood 2013. 122, 2039-2046) , these data validate the disruption of menin/MLL interaction as a promising new therapeutic approach for the treatment of MLL rearranged leukemia and other cancers with an active HOX/MEIS1 gene signature. For example, an internal partial tandem duplication (PTD) within the 5’region of the MLL gene represents another major aberration that is found predominantly in de novo and secondary AML as well as myeloid dysplasia syndromes. Although the molecular mechanism and the biological function of MLL-PTD is not well understood, new therapeutic targeting strategies affecting the menin/MLL interaction might also prove effective in the treatment of MLL-PTD-related leukemias. Furthermore, castration-resistant prostate cancer has been shown to be dependent on the menin/MLL interaction (Malik et al., Nat Med 2015. 21, 344-52) .

MLL protein is also known as Histone-lysine N-methyltransferase 2A (KMT2A) protein in the scientific field (UniProt Accession # Q03164) .

Several references describe inhibitors targeting the menin-MLL interaction: WO2011029054, J Med Chem 2016, 59, 892-913 describe the preparation of thienopyrimidine and benzodiazepine derivatives; WO2014164543 describes thienopyrimidine and thienopyridine derivatives; Nature Chemical Biology March 2012, 8, 277-284 and Ren, J.; et al. Bioorg Med Chem Lett (2016) , 26 (18) , 4472-4476 describe thienopyrimidine derivatives; J Med Chem 2014, 57, 1543-1556 describes hydroxy-and aminomethylpiperidine derivatives; Future Med Chem 2014, 6, 447-462 reviews small molecule and peptidomimetic compounds; WO2016195776 describes furo [2, 3-d] pyrimidine, 9H-purine, [1, 3] oxazolo [5, 4-d] pyrimidine, [1, 3] oxazolo [4, 5-d] pyrimidine, [1, 3] thiazolo [5, 4-d] pyrimidine, thieno [2, 3-b] pyridine and thieno [2, 3-d] pyrimidine derivatives; WO2016197027 describes 5, 6, 7, 8-tetrahydropyrido [3, 4-d] pyrimidine, 5, 6, 7, 8-tetrahydropyrido] 4, 3-d] pyrimidine, pyrido [2, 3-d] pyrimidine and quinoline derivatives; and WO2016040330 describes thienopyrimidine and thienopyridine compounds. WO2017192543 describes piperidines as Menin inhibitors. WO2017112768, WO2017207387, WO2017214367, WO2018053267 and WO2018024602 describe inhibitors of the menin-MLL interaction. WO2017161002 and WO2017161028 describe inhibitors of menin-MLL. WO2018050686, WO2018050684 and WO2018109088 describe inhibitors of the menin-MLL interaction. WO2018226976 describes methods and compositions for inhibiting the interaction of menin with MLL proteins. WO2019060365 describes substituted inhibitors of menin-MLL. Krivtsov et al., Cancer Cell 2019. No. 6 Vol. 36, 660-673 describes a menin-MLL inhibitor.

WO2020069027 discloses inhibitors of Menin. WO2018175746 discloses methods for treating hematological malignancies and ewing's sarcoma. WO2020045334 discloses azabicyclic derivative used in pharmaceutical compositions. WO2019120209 discloses substituted heterocyclic compounds as menin/MLL protein/protein interaction inhibitors. CN111297863 discloses use of menin-mixed lineage leukemia (MLL) inhibitors. WO2021121327 describes substituted straight chain spiro derivatives and their use as menin/MLL protein/protein interaction inhibitors.

DESCRIPTION OF THE INVENTION

The present invention concerns novel compounds of Formula (I) ,

and the tautomers and the stereoisomeric forms thereof, wherein

R ^1a represents -C (=O) -NR ^xaR ^xb; or

R ^xa and R ^xb are each independently selected from the group consisting of hydrogen; C _3-6cycloalkyl; C _1-4alkyl; C _1-4alkyl substituted with 1, 2 or 3 halo atoms; and C _1-4alkyl substituted with one -OH, -OC _1-4alkyl, or NR ^11cR ^11d;

R ^1b represents F or Cl;

Y ¹ represents -CR ^5aR ^5b-, -O-, -S-, or -NR ^5c-;

R ² is selected from the group consisting of hydrogen, halo, C _1-4alkyl, -O-C _1-4alkyl, and -NR ^7aR ^7b;

U ¹ and U ² each independently represent N or CH;

n1, n2, n3 and n4 are each independently selected from 1 and 2;

X ¹ represents CH, and X ² represents N;

R ⁴ represents C _1-5alkyl;

R ^5a, R ^5b, R ^5c, R ^7a, and R ^7b, are each independently selected from the group consisting of hydrogen, C _1-4alkyl and C _3-6cycloalkyl;

R ³ is selected from the group consisting of Het ¹, Het ², Cy ², and -C _1-6alkyl-NR ^xcR ^xd;

R ^xc represents Cy ¹; Het ⁵; -C _1-6alkyl-Cy ¹; -C _1-6alkyl-Het ³; -C _1-6alkyl-Het ⁴;

or -C _1-6alkyl-phenyl;

R ^xd represents hydrogen; C _1-4alkyl; or C _1-4alkyl substituted with one, two or three substituents selected from the group consisting of halo, -OH, -O-C _1-4alkyl, and cyano; or R ^xc and R ^xd are taken together to form together with the N-atom to which they are attached a 4-to 7-membered monocyclic fully saturated heterocyclyl containing one N-atom and optionally one additional heteroatom selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted with one, two or three substituents selected from the group consisting of halo, -OH, -O-C _1- ₄alkyl, and cyano;

Het ¹ represents a monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; or a bicyclic C-linked 6-to 11-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted on one nitrogen with a substituent selected from the group consisting of R ⁶ and -C (=O) -R ⁸; and wherein said heterocyclyl is optionally substituted on one or two carbon atoms with in total one, two, three or four substituents each independently selected from the group consisting of halo, R ⁶, Het ^6a, Het ^6b, C _1-4alkyl, oxo, -NR ^9aR ^9b and -OH;

Het ² represents C-linked pyrazolyl or triazolyl; which is substituted on one nitrogen atom with R ^6a;

R ⁶ is selected from the group consisting of Het ³; -C (=O) -NH-R ⁸;

C _1-6alkyl optionally substituted with one or two substituents each independently selected from the group consisting of Het ³, Het ⁴, Het ^6a, Het ^6b, Cy ¹, -CN, -OH, -O-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl-C _3-6cycloalkyl, -C (=O) -OH, -NR ^11aR ^11b and -NH-S (=O) ₂-C _1-4alkyl; and C _3-6cycloalkyl optionally substituted by one or two substituents each independently selected from the group consisting of -CN, -OH, -O-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl, -NH-S (=O) ₂-C _1-4alkyl, and C _1-4alkyl optionally substituted with one substituent selected from the group consisting of OH, -O-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl and -NH-S (=O) ₂-C _1-4alkyl;

R ^6a represents C _1-6alkyl substituted with one substituent selected from the group consisting of -NR ^11aR ^11b, Het ^3a, and Het ^6a;

R ⁸ represents C _1-6alkyl optionally substituted with one, two or three substituents each independently selected from -OH, halo, cyano, -NR ^11aR ^11b, Het ^3a, and Het ^6a;

Het ³ and Het ⁵ each independently represent a monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one, two or three heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; or a bicyclic C-linked 6-to 11-membered fully saturated heterocyclyl containing one, two or three heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted on one carbon atom with C _1-4alkyl, halo, -OH, -NR ^11aR ^11b, or oxo; and wherein said heterocyclyl is optionally substituted on one nitrogen atom with C _1-4alkyl;

Het ^3a and Het ^5a each independently represent a monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom, and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; or a bicyclic C-linked 6-to 11-membered fully saturated heterocyclyl containing one N-atom, and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂;

wherein said heterocyclyl is optionally substituted on one carbon atom with C _1-4alkyl, halo, -OH, -NR ^11aR ^11b, or oxo; and wherein said heterocyclyl is optionally substituted on one nitrogen atom with C _1-4alkyl;

Het ⁴ and Het ⁷ each independently represent a monocyclic C-linked 5-or 6-membered aromatic ring containing one, two, three or four heteroatoms each independently selected from O, S, and N; wherein said 5-membered aromatic ring is optionally substituted on one nitrogen atom with C _1-4alkyl; and wherein said 5-or 6-membered aromatic ring is optionally substituted on one carbon atom with -OH;

Het ^6a and Het ⁸ each independently represent a monocyclic N-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted on one or two carbon atoms with in total one, two, three or four substituents each independently selected from the group consisting of halo, -OH, oxo, - (C=O) -NR ^10aR ^10b, -O-C _3-6cycloalkyl, -S (=O) ₂-C _1-4alkyl, cyano, C _1-4alkyl, -C _1-4alkyl-OH, -O-C _1-4alkyl, -O- (C=O) -NR ^10aR ^10b, and -O- (C=O) -C _1-4alkyl; and wherein said heterocyclyl is optionally substituted on one nitrogen with a substituent selected from the group consisting of -C (=O) -C _1-4alkyl and - (C=O) -NR ^10aR ^10b;

Het ^8a each independently represent a monocyclic N-linked 4-to 7-membered fully saturated heterocyclyl containing two N-atoms and optionally one additional heteroatom selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted on one or two carbon atoms with in total one, two, three or four substituents each independently selected from the group consisting of halo, -OH, oxo, - (C=O) -NR ^10aR ^10b, -O-C _3-6cycloalkyl, -S (=O) ₂-C _1-4alkyl, cyano, C _1-4alkyl, C _1-4alkyl-OH, -O-C _1-4alkyl, -O- (C=O) -NR ^10aR ^10b, and -O- (C=O) -C _1-4alkyl; and wherein said heterocyclyl is optionally substituted on one nitrogen with a substituent selected from the group consisting of -C (=O) -C _1-4alkyl and - (C=O) -NR ^10aR ^10b;

Het ^6b represents a bicyclic N-linked 6-to 11-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted on one or two carbon atoms with in total one or two substituents each independently selected from the group consisting of C _1- ₄alkyl, -OH, oxo, - (C=O) -NR ^10aR ^10b, -NH-C (=O) -C _1-4alkyl, -NH-C (=O) -Cy ³, and -O-C _1-4alkyl; and wherein said heterocyclyl is optionally substituted on one nitrogen with a substituent selected from the group consisting of -C (=O) -C _1-4alkyl, -C (=O) -Cy ³, - (C=O) -C _1-4alkyl-OH, -C (=O) -C _1-4alkyl-O-C _1-4alkyl, -C (=O) -C _1-4alkyl-NR ^11aR ^11b, and C _1-4alkyl;

Cy ¹ represents C _3-6cycloalkyl optionally substituted with one, two or three substituents selected from the group consisting of -OH, -NH-C (=O) -C _1-4alkyl, C _1-4alkyl, -NH-S (=O) ₂-C _1-4alkyl, -S (=O) ₂-C _1-4alkyl, and -O-C _1-4alkyl;

Cy ² represents C _3-7cycloalkyl substituted with one or two substituents each independently selected from the group consisting of -NR ^9aR ^9b; Het ^6a; Het ^6b; and C _1-6alkyl substituted with one or two substituents each independently selected from the group consisting of Het ^3a, Het ^6a, Het ^6b, and -NR ^9aR ^9b; and said C _3-7cycloalkyl is optionally substituted with one or two additional substituents each independently selected from the group consisting of halo, R ⁶, C _1-4alkyl, and -OH;

Cy ³ represents C _3-7cycloalkyl; wherein said C _3-7cycloalkyl is optionally substituted with one, two or three halo substituents;

R ^9a and R ^9b are each independently selected from the group consisting of hydrogen; C _1-4alkyl; C _3-6cycloalkyl; Het ⁵; -C _1-4alkyl-R ¹⁶; -C (=O) -C _1-4alkyl-Het ^3a; -C (=O) -R ¹⁴; C _3-6cycloalkyl substituted with one, two or three substituents selected from the group consisting of halo, -OH, -O-C _1-4alkyl, -NR ^11aR ^11b, and cyano; and C _1-4alkyl substituted with one, two or three substituents selected from the group consisting of halo, -OH, -O-C _1-4alkyl, -NR ^11aR ^11b, and cyano;

R ^11a, R ^11b, R ^13a, R ^13b, R ^15a, R ^15b, R ^17a, and R ^17b are each independently selected from the group consisting of hydrogen and C _1-4alkyl;

R ^11c and R ^11d are each independently selected from the group consisting of hydrogen, C _1-6alkyl, and -C (=O) -C _1-4alkyl;

R ¹⁴ represents Het ^5a; Het ^8a; or C _1-4alkyl substituted with one, two or three substituents selected from the group consisting of -NR ^13aR ^13b and Het ^8a;

R ¹⁶ represents -C (=O) -NR ^17aR ^17b, -S (=O) ₂-C _1-4alkyl, Het ⁵, Het ⁷, or Het ⁸;

and the pharmaceutically acceptable salts and the solvates thereof.

The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula (I) , a pharmaceutically acceptable salt, or a solvate thereof, and a pharmaceutically acceptable carrier or excipient.

Additionally, the invention relates to a compound of Formula (I) , a pharmaceutically acceptable salt, or a solvate thereof, for use as a medicament, and to a compound of Formula (I) , a pharmaceutically acceptable salt, or a solvate thereof, for use in the treatment or in the prevention of cancer, myelodysplastic syndrome (MDS) and diabetes.

In a particular embodiment, the invention relates to a compound of Formula (I) , a pharmaceutically acceptable salt, or a solvate thereof, for use in the treatment or in the prevention of cancer.

In a specific embodiment said cancer is selected from leukemias, myeloma or a solid tumor cancer (e.g. prostate cancer, lung cancer, breast cancer, pancreatic cancer, colon cancer, liver cancer, melanoma and glioblastoma, etc. ) . In some embodiments, the leukemias include acute leukemias, chronic leukemias, myeloid leukemias, myelogeneous leukemias, lymphoblastic leukemias, lymphocytic leukemias, Acute myelogeneous leukemias (AML) , Chronic myelogenous leukemias (CML) , Acute lymphoblastic leukemias (ALL) , Chronic lymphocytic leukemias (CLL) , T cell prolymphocytic leukemias (T-PLL) , Large granular lymphocytic leukemia, Hairy cell leukemia (HCL) , MLL-rearranged leukemias, MLL-PTD leukemias, MLL amplified leukemias, MLL-positive leukemias, leukemias exhibiting HOX/MEIS1 gene expression signatures etc.

The invention also relates to the use of a compound of Formula (I) , a pharmaceutically acceptable salt, or a solvate thereof, in combination with an additional pharmaceutical agent for use in the treatment or prevention of cancer, myelodysplastic syndrome (MDS) and diabetes.

Furthermore, the invention relates to a process for preparing a pharmaceutical composition according to the invention, characterized in that a pharmaceutically acceptable carrier is intimately mixed with a therapeutically effective amount of a compound of Formula (I) , a pharmaceutically acceptable salt, or a solvate thereof.

The invention also relates to a product comprising a compound of Formula (I) , a pharmaceutically acceptable salt, or a solvate thereof, and an additional pharmaceutical agent, as a combined preparation for simultaneous, separate or sequential use in the treatment or prevention of cancer, myelodysplastic syndrome (MDS) and diabetes.

Additionally, the invention relates to a method of treating or preventing a cell proliferative disease in a warm-blooded animal which comprises administering to the said animal an effective amount of a compound of Formula (I) , a pharmaceutically acceptable salt, or a solvate thereof, as defined herein, or a pharmaceutical composition or combination as defined herein.

DETAILED DESCRIPTION OF THE INVENTION

The term ‘halo’ or ‘halogen’ as used herein represents fluoro, chloro, bromo and iodo.

The prefix ‘C _x-y’ (where x and y are integers) as used herein refers to the number of carbon atoms in a given group. Thus, a C _1-6alkyl group contains from 1 to 6 carbon atoms, and so on.

The term ‘C _1-4alkyl’ as used herein as a group or part of a group represents a straight or branched chain saturated hydrocarbon radical having from 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl and the like.

Similar, the term ‘C _1-6alkyl’ as used herein as a group or part of a group represents a straight or branched chain saturated hydrocarbon radical having from 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl, n-pentyl, n-hexyl and the like.

The term ‘C _3-6cycloalkyl’ as used herein as a group or part of a group defines a saturated, cyclic hydrocarbon radical having from 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The term ‘C _3-7cycloalkyl’ as used herein as a group or part of a group defines a saturated, cyclic hydrocarbon radical having from 3 to 7 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

It will be clear for the skilled person that S (=O) ₂ or SO ₂ represents a sulfonyl moiety.

It will be clear for the skilled person that CO or C (=O) represents a carbonyl moiety.

It will be clear for the skilled person that a group such as -CRR-represents

An example of such a group is -CR ^5aR ^5b-.

It will be clear for the skilled person that a group such as -NR-represents

An example of such a group is -NR ^5c-.

The term ‘monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom, and optionally one or two additional heteroatoms each independently selected from O, S, and N’, defines a fully saturated, cyclic hydrocarbon radical having from 4 to 7 ring members and containing 1 nitrogen atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, such as for example C-linked azetidinyl, C-linked pyrrolidinyl, C-linked morpholinyl and C-linked piperidinyl. The term ‘monocyclic N-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N’, is defined similar but is attached to the remainder of the molecule of formula (I) via a nitrogen atom. Examples are N-linked azetidinyl, N-linked pyrrolidinyl, N-linked morpholinyl, N-linked thiomorpholinyl, N-linked piperazinyl, N-linked 1, 4-diazepanyl, and N-linked piperidinyl. Two R groups taken together to form together with the N-atom to which they are attached a 4-to 7-membered monocyclic fully saturated heterocyclyl containing one N-atom and optionally one additional heteroatom selected from O, S, and N, are defined similar.

The term ‘monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one, two or three heteroatoms each independently selected from O, S, and N’, defines a fully saturated, cyclic hydrocarbon radical having from 4 to 7 ring members and containing one, two or three heteroatoms each independently selected from O, S, and N, such as for example C-linked azetidinyl, C-linked pyrrolidinyl, C-linked morpholinyl, C-linked tetrahydrofuranyl, C-linked thiolanyl, C-linked oxetanyl, C-linked thietanyl, C-linked tetrahydropyranyl, and C-linked piperidinyl. The term ‘monocyclic N-linked 4-to 7-membered fully saturated heterocyclyl containing two N-atoms and optionally one additional heteroatom selected from O, S, and N’, defines a fully saturated, cyclic hydrocarbon radical having from 4 to 7 ring members and containing 2 nitrogen atoms and optionally one additional heteroatom selected from O, S, and N, such as for example N-linked piperazinyl, and N-linked 1, 4-diazepanyl.

For clarity, the 4-to 7-membered fully or partially saturated heterocyclyls have from 4 to 7 ring members including the heteroatoms.

Non-limiting examples of ‘monocyclic 5-or 6-membered aromatic rings containing one or two nitrogen atoms and optionally a carbonyl moiety’, include, but are not limited to pyrazolyl, imidazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1H-1, 2, 4-triazolyl, 4H-1, 2, 4-triazolyl, 1, 2, 4-triazinyl, 1, 2-dihydro-2-oxo-5-pyrimidinyl, 1, 2-dihydro-2-oxo-6-pyridinyl, 1, 2-dihydro-2-oxo-4-pyridinyl, and 1, 6-dihydro-6-oxo-3-pyridazinyl.

Non-limiting examples of ‘monocyclic C-linked 5-or 6-membered aromatic rings containing one, two or three heteroatoms each independently selected from O, S, and N’, include, but are not limited to C-linked pyrazolyl, C-linked imidazolyl, C-linked pyridinyl, C-linked triazolyl, C-linked pyridazinyl, C-linked pyrimidinyl, C-linked oxazolyl, C-linked furanyl, C-linked isothiazolyl, or C-linked pyrazinyl.

Within the context of this invention, bicyclic C-linked 6-to 11-membered fully saturated heterocyclyl groups, include fused, spiro and bridged bicycles.

Within the context of this invention, bicyclic N-linked 6-to 11-membered fully saturated heterocyclyl groups, include fused, spiro and bridged bicycles.

Fused bicyclic groups are two cycles that share two atoms and the bond between these atoms.

Spiro bicyclic groups are two cycles that are joined at a single atom.

Bridged bicyclic groups are two cycles that share more than two atoms.

Examples of bicyclic C-linked 6-to 11-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, include, but are not limited to

and the like.

Examples of bicyclic C-linked 6-to 11-membered fully saturated heterocyclyl containing one, two or three heteroatoms each independently selected from O, S, and N, include, but are not limited to

and the like.

Examples of bicyclic N-linked 6-to 11-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, include, but are not limited to

and the like.

Whenever substituents are represented by chemical structure, such as for example

‘----’ represents the bond of attachment to the remainder of the molecule of Formula (I) .

When any variable occurs more than one time in any constituent, each definition is independent.

When any variable occurs more than one time in any formula (e.g. Formula (I) ) , each definition is independent.

In this context, it will also be clear that a term like “optionally substituted with one, two or three substituents selected from the group consisting of” is equivalent to “optionally substituted with one, two or three substituents each independently selected from the group consisting of” .

In general, whenever the term ‘substituted’ is used in the present invention, it is meant, unless otherwise indicated or clear from the context, to indicate that one or more hydrogens, in particular from 1 to 4 hydrogens, more in particular from 1 to 3 hydrogens, preferably 1 or 2 hydrogens, more preferably 1 hydrogen, on the atom or radical indicated in the expression using ‘substituted’ are replaced with a selection from the indicated group, provided that the normal valency is not exceeded, and that the substitution results in a chemically stable compound, i.e. a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture. In a particular embodiment, when the number of substituents is not explicitly specified, the number of substituents is one.

Combinations of substituents and/or variables are permissible only if such combinations result in chemically stable compounds. ‘Stable compound’ is meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture.

The skilled person will understand that the term ‘optionally substituted’ means that the atom or radical indicated in the expression using ‘optionally substituted’ may or may not be substituted (this means substituted or unsubstituted respectively) .

When two or more substituents are present on a moiety they may, where possible and unless otherwise indicated or clear from the context, replace hydrogens on the same atom or they may replace hydrogen atoms on different atoms in the moiety.

Within the context of this invention ‘saturated’ means ‘fully saturated’ , if not otherwise specified.

Unless otherwise specified or clear from the context, aromatic rings and heterocyclyl goups, can be attached to the remainder of the molecule of Formula (I) through any available ring carbon atom (C-linked) or nitrogen atom (N-linked) .

Unless otherwise specified or clear from the context, aromatic rings and heterocyclyl goups, may optionally be substituted, where possible, on carbon and/or nitrogen atoms according to the embodiments.

The term “subject” as used herein, refers to an animal, preferably a mammal (e.g. cat, dog, primate or human) , more preferably a human, who is or has been the object of treatment, observation or experiment.

The term “therapeutically effective amount” as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medicinal doctor or other clinician, which includes alleviation or reversal of the symptoms of the disease or disorder being treated.

The term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.

The term “treatment” , as used herein, is intended to refer to all processes wherein there may be a slowing, interrupting, arresting or stopping of the progression of a disease, but does not necessarily indicate a total elimination of all symptoms.

The term “compound (s) of the (present) invention” or “compound (s) according to the (present) invention” as used herein, is meant to include the compounds of Formula (I) and the pharmaceutically acceptable salts, and the solvates thereof.

As used herein, any chemical formula with bonds shown only as solid lines and not as solid wedged or hashed wedged bonds, or otherwise indicated as having a particular configuration (e.g. R, S) around one or more atoms, contemplates each possible stereoisomer, or mixture of two or more stereoisomers.

Hereinbefore and hereinafter, the term “compound (s) of Formula (I) ” is meant to include the tautomers thereof and the stereoisomeric forms thereof.

The terms “stereoisomers” , “stereoisomeric forms” or “stereochemically isomeric forms” hereinbefore or hereinafter are used interchangeably.

The invention includes all stereoisomers of the compounds of the invention either as a pure stereoisomer or as a mixture of two or more stereoisomers.

Enantiomers are stereoisomers that are non-superimposable mirror images of each other. A 1: 1 mixture of a pair of enantiomers is a racemate or racemic mixture.

Atropisomers (or atropoisomers) are stereoisomers which have a particular spatial configuration, resulting from a restricted rotation about a single bond, due to large steric hindrance. All atropisomeric forms of the compounds of Formula (I) are intended to be included within the scope of the present invention.

Diastereomers (or diastereoisomers) are stereoisomers that are not enantiomers, i.e. they are not related as mirror images. If a compound contains a double bond, the substituents may be in the E or the Z configuration.

Substituents on bivalent cyclic saturated or partially saturated radicals may have either the cis-or trans-configuration; for example if a compound contains a disubstituted cycloalkyl group, the substituents may be in the cis or trans configuration.

Therefore, the invention includes enantiomers, atropisomers, diastereomers, racemates, E isomers, Z isomers, cis isomers, trans isomers and mixtures thereof, whenever chemically possible.

The meaning of all those terms, i.e. enantiomers, atropisomers, diastereomers, racemates, E isomers, Z isomers, cis isomers, trans isomers and mixtures thereof are known to the skilled person.

The absolute configuration is specified according to the Cahn-Ingold-Prelog system. The configuration at an asymmetric atom is specified by either R or S. Resolved stereoisomers whose absolute configuration is not known can be designated by (+) or

(-) depending on the direction in which they rotate plane polarized light. For instance, resolved enantiomers whose absolute configuration is not known can be designated by (+) or

(-) depending on the direction in which they rotate plane polarized light.

When a specific stereoisomer is identified, this means that said stereoisomer is substantially free, i.e. associated with less than 50%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, in particular less than 2%and most preferably less than 1%, of the other stereoisomers. Thus, when a compound of Formula (I) is for instance specified as (R) , this means that the compound is substantially free of the (S) isomer; when a compound of Formula (I) is for instance specified as E, this means that the compound is substantially free of the Z isomer; when a compound of Formula (I) is for instance specified as cis, this means that the compound is substantially free of the trans isomer.

Some of the compounds according to Formula (I) may also exist in their tautomeric form.

Such forms in so far as they may exist, although not explicitly indicated in the above Formula (I) are intended to be included within the scope of the present invention.

For example

is equivalent to

It follows that a single compound may exist in both stereoisomeric and tautomeric form.

Pharmaceutically acceptable salts include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form with one or more equivalents of an appropriate base or acid, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration) . Salts may also be prepared by exchanging a counter-ion of a compound of the invention in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

The pharmaceutically acceptable salts as mentioned hereinabove or hereinafter are meant to comprise the therapeutically active non-toxic acid and base salt forms which the compounds of Formula (I) and solvates thereof, are able to form.

Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic) , malonic, succinic (i.e. butanedioic acid) , maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids. Conversely said salt forms can be converted by treatment with an appropriate base into the free base form.

The compounds of Formula (I) and solvates thereof containing an acidic proton may also be converted into their non-toxic metal or amine salt forms by treatment with appropriate organic and inorganic bases.

Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g. the lithium, sodium, potassium, cesium, magnesium, calcium salts and the like, salts with organic bases, e.g. primary, secondary and tertiary aliphatic and aromatic amines such as methylamine, ethylamine, propylamine, isopropylamine, the four butylamine isomers, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, quinuclidine, pyridine, quinoline and isoquinoline; the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like. Conversely the salt form can be converted by treatment with acid into the free acid form.

The term solvate comprises the solvent addition forms as well as the salts thereof, which the compounds of Formula (I) are able to form. Examples of such solvent addition forms are e.g. hydrates, alcoholates and the like.

The compounds of the invention as prepared in the processes described below may be synthesized in the form of mixtures of enantiomers, in particular racemic mixtures of enantiomers, that can be separated from one another following art-known resolution procedures. A manner of separating the enantiomeric forms of the compounds of Formula (I) , and pharmaceutically acceptable salts, and solvates thereof, involves liquid chromatography using a chiral stationary phase. Said pure stereochemically isomeric forms may also be derived from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction occurs stereospecifically. Preferably if a specific stereoisomer is desired, said compound would be synthesized by stereospecific methods of preparation. These methods will advantageously employ enantiomerically pure starting materials.

The term "enantiomerically pure" as used herein means that the product contains at least 80%by weight of one enantiomer and 20%by weight or less of the other enantiomer. Preferably the product contains at least 90%by weight of one enantiomer and 10%by weight or less of the other enantiomer. In the most preferred embodiment the term "enantiomerically pure" means that the composition contains at least 99%by weight of one enantiomer and 1%or less of the other enantiomer.

The present invention also embraces isotopically-labeled compounds of the present invention which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature (or the most abundant one found in nature) .

All isotopes and isotopic mixtures of any particular atom or element as specified herein are contemplated within the scope of the compounds of the invention, either naturally occurring or synthetically produced, either with natural abundance or in an isotopically enriched form. Exemplary isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine and iodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C , ¹³N, ¹⁵O, ¹⁷O, ¹⁸O, ³²P, ³³P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²²I, ¹²³I, ¹²⁵I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br and ⁸²Br. Preferably, the isotope is selected from the group of ²H, ³H, ¹¹C and ¹⁸F. More preferably, the isotope is ²H. In particular, deuterated compounds are intended to be included within the scope of the present invention.

Certain isotopically-labeled compounds of the present invention (e.g., those labeled with ³H and ¹⁴C) may be useful for example in substrate tissue distribution assays. Tritiated ( ³H) and carbon-l4 ( ¹⁴C) isotopes are useful for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e., ²H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Positron emitting isotopes such as ¹⁵O, ¹³N, ¹¹C and ¹⁸F are useful for positron emission tomography (PET) studies. PET imaging in cancer finds utility in helping locate and identify tumours, stage the disease and determine suitable treatment. Human cancer cells overexpress many receptors or proteins that are potential disease-specific molecular targets. Radiolabelled tracers that bind with high affinity and specificity to such receptors or proteins on tumour cells have great potential for diagnostic imaging and targeted radionuclide therapy (Charron, Carlie L. et al. Tetrahedron Lett. 2016, 57 (37) , 4119-4127) . Additionally, target-specific PET radiotracers may be used as biomarkers to examine and evaluate pathology, by for example, measuring target expression and treatment response (Austin R. et al. Cancer Letters (2016) , doi: 10.1016/j. canlet. 2016.05.008) .

The present invention relates in particular to compounds of Formula (I) as defined herein, and the tautomers and the stereoisomeric forms thereof, wherein

R ^1a represents -C (=O) -NR ^xaR ^xb; or

R ^xa and R ^xb are each independently selected from the group consisting of hydrogen; C _3-6cycloalkyl; C _1-4alkyl; and C _1-4alkyl substituted with 1, 2 or 3 halo atoms;

R ^1b represents F or Cl;

Y ¹ represents -CR ^5aR ^5b-, -O-, -S-, or -NR ^5c-;

U ¹ and U ² each independently represent N or CH;

n1, n2, n3 and n4 are each independently selected from 1 and 2;

X ¹ represents CH, and X ² represents N;

R ⁴ represents C _1-5alkyl;

or -C _1-6alkyl-phenyl;

R ^xd represents hydrogen; C _1-4alkyl; or C _1-4alkyl substituted with one, two or three substituents selected from the group consisting of halo, -OH, -O-C _1-4alkyl, and cyano;

or R ^xc and R ^xd are taken together to form together with the N-atom to which they are attached a 4-to 7-membered monocyclic fully saturated heterocyclyl containing one N-atom and optionally one additional heteroatom selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted with one, two or three substituents selected from the group consisting of halo, -OH, -O-C _1-4alkyl, and cyano;

Het ¹ represents a monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; or a bicyclic C-linked 6-to 11-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂;

wherein said heterocyclyl is optionally substituted on one nitrogen with a substituent selected from the group consisting of R ⁶ and -C (=O) -R ⁸; and wherein said heterocyclyl is optionally substituted on one or two carbon atoms with in total one, two, three or four substituents each independently selected from the group consisting of halo, R ⁶, Het ^6a, Het ^6b, C _1-4alkyl, oxo, -NR ^9aR ^9b and -OH;

R ⁶ is selected from the group consisting of Het ³; -C (=O) -NH-R ⁸;

C _1-6alkyl optionally substituted with one or two substituents each independently selected from the group consisting of Het ³, Het ⁴, Het ^6a, Het ^6b, Cy ¹, -CN, -OH, -O-C _1-4alkyl, -C (=O) -NH-C _1- ₄alkyl, -C (=O) -NH-C _1-4alkyl-C _3-6cycloalkyl, -C (=O) -OH, -NR ^11aR ^11b and -NH-S (=O) ₂-C _1- ₄alkyl; and

C _3-6cycloalkyl optionally substituted by one or two substituents each independently selected from the group consisting of -CN, -OH, -O-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl, -NH-S (=O) ₂-C _1-4alkyl, and C _1-4alkyl optionally substituted with one substituent selected from the group consisting of OH, -O-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl and -NH-S (=O) ₂-C _1-4alkyl;

R ⁸ represents C _1-6alkyl substituted with one substituent selected from the group consisting of -OH, -NR ^11aR ^11b, Het ^3a, and Het ^6a;

Het ³ and Het ⁵ each independently represent a monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one, two or three heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; or a bicyclic C-linked 6-to 11-membered fully saturated heterocyclyl containing one, two or three heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂;

Het ⁴ and Het ⁷ each independently represent a monocyclic C-linked 5-or 6-membered aromatic ring containing one, two or three heteroatoms each independently selected from O, S, and N; wherein said 5-membered aromatic ring is optionally substituted on one nitrogen atom with C _1-4alkyl; and wherein said 5-or 6-membered aromatic ring is optionally substituted on one carbon atom with -OH;

Het ^6b represents a bicyclic N-linked 6-to 11-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted on one carbon atom with a substituent selected from the group consisting of -OH, oxo, - (C=O) -NR ^10aR ^10b, -NH-C (=O) -C _1-4alkyl, -NH-C (=O) -Cy ³, and -O-C _1-4alkyl; and wherein said heterocyclyl is optionally substituted on one nitrogen with a substituent selected from the group consisting of -C (=O) -C _1-4alkyl, -C (=O) -Cy ³, and C _1-4alkyl;

R ^9a and R ^9b are each independently selected from the group consisting of hydrogen; C _1-4alkyl; C _3-6cycloalkyl; Het ⁵; -C _1-4alkyl-R ¹⁶; -C (=O) -C _1-4alkyl-Het ^3a; -C (=O) -R ¹⁴; C _3-6cycloalkyl substituted with one, two or three substituents selected from the group consisting of halo, -OH, -O-C _1-4alkyl, and cyano; and C _1-4alkyl substituted with one, two or three substituents selected from the group consisting of halo, -OH, -O-C _1-4alkyl, and cyano;

R ^10a, R ^10b, R ^11a, R ^11b, R ^13a, R ^13b, R ^15a, R ^15b, R ^17a, and R ^17b are each independently selected from the group consisting of hydrogen and C _1-4alkyl;

and the pharmaceutically acceptable salts and the solvates thereof.

R ^1a represents -C (=O) -NR ^xaR ^xb;

R ^xa and R ^xb are each independently selected from the group consisting of C _3-6cycloalkyl; C _1-4alkyl; and C _1-4alkyl substituted with 1, 2 or 3 halo atoms;

R ^1b represents F;

Y ¹ represents -O-;

R ² represents hydrogen;

U ¹ and U ² each independently represent N or CH;

n1, n2, n3 and n4 are each independently selected from 1 and 2;

X ¹ represents CH, and X ² represents N;

R ⁴ represents C _1-5alkyl;

R ³ is selected from the group consisting of Het ¹ and Cy ²;

Het ¹ represents a monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom;

wherein said heterocyclyl is optionally substituted on one nitrogen with a substituent selected from the group consisting of R ⁶ and -C (=O) -R ⁸; and wherein said heterocyclyl is optionally substituted on one or two carbon atoms with in total one, two, three or four halo substituents;

R ⁶ is selected from the group consisting of Het ³;

C _1-6alkyl optionally substituted with one or two substituents each independently selected from the group consisting of Het ³, Het ⁴, Het ^6a, Cy ¹, -OH, -O-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl-C _3-6cycloalkyl, and -NH-S (=O) ₂-C _1-4alkyl;

R ⁸ represents C _1-6alkyl substituted with one substituent selected from the group consisting of - OH and -NR ^11aR ^11b;

Het ³ and Het ⁵ each independently represent a monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one, two or three heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted on one carbon atom with -OH or oxo;

Het ⁴ represents a monocyclic C-linked 5-or 6-membered aromatic ring containing one, two or three heteroatoms each independently selected from O, S, and N; wherein said 5-or 6-membered aromatic ring is optionally substituted on one carbon atom with -OH;

Het ^6a represents a monocyclic N-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted on one or two carbon atoms with in total one, two, three or four substituents each independently selected from the group consisting of oxo, -S (=O) ₂-C _1-4alkyl, and -O-C _1-4alkyl; and wherein said heterocyclyl is optionally substituted on one nitrogen with -C (=O) -C _1-4alkyl;

Het ^6b represents a bicyclic N-linked 6-to 11-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted on one carbon atom with - (C=O) -NR ^10aR ^10b; and wherein said heterocyclyl is optionally substituted on one nitrogen with -C (=O) -C _1-4alkyl;

Cy ¹ represents C _3-6cycloalkyl optionally substituted with one, two or three substituents selected from the group consisting of -OH, -NH-C (=O) -C _1-4alkyl, C _1-4alkyl, -NH-S (=O) ₂-C _1-4alkyl, and -O-C _1-4alkyl;

Cy ² represents C _3-7cycloalkyl substituted with one or two substituents each independently selected from the group consisting of -NR ^9aR ^9b; Het ^6a; and Het ^6b;

R ^9a and R ^9b are each independently selected from the group consisting of hydrogen; C _1-4alkyl; C _3-6cycloalkyl; Het ⁵; -C _1-4alkyl-R ¹⁶; and C _1-4alkyl substituted with one, two or three -O-C _1-4alkyl substituents;

R ^10a, R ^10b, R ^11a, and R ^11b represent C _1-4alkyl;

R ¹⁶ represents Het ⁵;

and the pharmaceutically acceptable salts and the solvates thereof.

R ^1a represents -C (=O) -NR ^xaR ^xb;

R ^xa and R ^xb represent C _1-4alkyl;

R ^1b represents F;

Y ¹ represents -O-;

R ² represents hydrogen;

U ¹ and U ² each independently represent N or CH;

n1, n2, n3 and n4 are each independently selected from 1 and 2;

X ¹ represents CH, and X ² represents N;

R ⁴ represents isopropyl;

R ³ is selected from the group consisting of Het ¹ and Cy ²;

wherein said heterocyclyl is optionally substituted on one nitrogen with a substituent selected from the group consisting of R ⁶;

R ⁶ represents C _1-6alkyl substituted with one Het ³;

Het ³ represents a monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one, two or three heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂;

Het ^6a represents a monocyclic N-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom; wherein said heterocyclyl is optionally substituted on one carbon atom with one -O-C _1-4alkyl;

Het ^6b represents a bicyclic N-linked 6-to 11-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O and N; wherein said heterocyclyl is optionally substituted on one carbon atom with - (C=O) -NR ^10aR ^10b; and wherein said heterocyclyl is optionally substituted on one nitrogen with -C (=O) -C _1-4alkyl;

Cy ² represents C _3-7cycloalkyl substituted with one substituents selected from the group consisting of Het ^6a and Het ^6b;

R ^10a and R ^10b represent C _1-4alkyl;

and the pharmaceutically acceptable salts and the solvates thereof.

In an embodiment, the present invention relates to those compounds of Formula (I) and the pharmaceutically acceptable salts, and the solvates thereof, or any subgroup thereof as mentioned in any of the other embodiments, wherein

R ^1a represents -C (=O) -NR ^xaR ^xb.

R ^1b represents F.

R ² represents hydrogen.

In an embodiment, the present invention relates to those compounds of Formula (I) and the pharmaceutically acceptable salts, and the solvates thereof, or any subgroup thereof as mentioned in any of the other embodiments, wherein n1 is 1, n2 is 2, n3 is 1, and n4 is 1.

In an embodiment, the present invention relates to those compounds of Formula (I) and the pharmaceutically acceptable salts, and the solvates thereof, or any subgroup thereof as mentioned in any of the other embodiments, wherein U ¹ represents N.

In an embodiment, the present invention relates to those compounds of Formula (I) and the pharmaceutically acceptable salts, and the solvates thereof, or any subgroup thereof as mentioned in any of the other embodiments, wherein U ¹ represents N, and U ² represents N.

In an embodiment, the present invention relates to those compounds of Formula (I) and the pharmaceutically acceptable salts, and the solvates thereof, or any subgroup thereof as mentioned in any of the other embodiments, wherein U ¹ represents CH, and U ² represents N.

Y ¹ represents -O-.

Y ¹ represents -O-; and

U ² represents N.

Y ¹ represents -O-; and

U ¹ represents N.

Y ¹ represents -O-;

U ² represents N;

R ^1b represents F; and

R ² represents hydrogen.

Y ¹ represents -O-;

U ¹ represents N;

R ^1b represents F; and

R ² represents hydrogen.

Y ¹ represents -O-;

U ¹ represents N;

R ^1b represents F;

R ² represents hydrogen; and

R ⁴ represents isopropyl.

R ³ represents Het ¹ or Cy ².

R ³ represents Cy ².

R ³ represents Het ¹.

wherein said heterocyclyl is optionally substituted on one nitrogen with R ⁶; and wherein said heterocyclyl is optionally substituted on one or two carbon atoms with in total one, two, three or four substituents each independently selected from the group consisting of halo, R ⁶, Het ^6a, Het ^6b, C _1-4alkyl, oxo, -NR ^9aR ^9b and -OH.

R ³ represents Het ¹;

R ⁶ is selected from the group consisting of Het ³; -C (=O) -NH-R ⁸;

C _3-6cycloalkyl substituted by one or two substituents each independently selected from the group consisting of -CN, -OH, -O-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl, -NH-S (=O) ₂-C _1-4alkyl, and C _1-4alkyl optionally substituted with one substituent selected from the group consisting of OH, -O-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl and -NH-S (=O) ₂-C _1-4alkyl.

R ⁶ is selected from the group consisting of Het ³; -C (=O) -NH-R ⁸;

C _1-6alkyl substituted with one or two substituents each independently selected from the group consisting of Het ³, Het ⁴, Het ^6a, Het ^6b, Cy ¹, -CN, -OH, -O-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl-C _3-6cycloalkyl, -C (=O) -OH, -NR ^11aR ^11b and -NH-S (=O) ₂-C _1-4alkyl; and C _3-6cycloalkyl substituted by one or two substituents each independently selected from the group consisting of -CN, -OH, -O-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl, -NH-S (=O) ₂-C _1-4alkyl, and C _1-4alkyl optionally substituted with one substituent selected from the group consisting of OH, -O-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl and -NH-S (=O) ₂-C _1-4alkyl.

R ⁴ represents

R ³ represents Het ¹;

wherein said heterocyclyl is substituted on one nitrogen with R ⁶; and wherein said heterocyclyl is optionally substituted on one or two carbon atoms with in total one, two, three or four halo substituents;

R ⁶ is selected from the group consisting of C _1-6alkyl optionally substituted with one or two substituents each independently selected from the group consisting of Het ³, Het ⁴, Het ^6a, and Cy ¹.

U ¹ represents N;

R ³ represents Het ¹;

U ¹ represents N;

Y ¹ represents -O-;

R ^1b represents F;

R ² represents hydrogen;

R ⁴ represents isopropyl;

R ³ represents Het ¹;

wherein said heterocyclyl is substituted on one nitrogen with R ⁶;

R ⁶ represents C _1-6alkyl substituted with one Het ³.

U ¹ represents N;

R ³ represents Het ¹;

wherein said heterocyclyl is substituted on one nitrogen with R ⁶;

R ⁶ represents C _1-6alkyl substituted with one Het ³.

U ¹ represents N;

Y ¹ represents -O-;

R ^1b represents F;

R ² represents hydrogen;

R ⁴ represents isopropyl;

R ³ represents Het ¹;

wherein said heterocyclyl is substituted on one nitrogen with R ⁶;

R ⁶ represents C _1-6alkyl substituted with one Het ³.

In an embodiment, the present invention relates to those compounds of Formula (I) and the pharmaceutically acceptable salts, and the solvates thereof, or any subgroup thereof as mentioned in any of the other embodiments, wherein the compounds of Formula (I) are restricted to compounds of Formula (I-y) :

wherein R ³ is as defined for the compounds of Formula (I) or any subgroup thereof as mentioned in any of the other embodiments.

wherein R ³ represents Het ¹.

wherein R ³ represents Cy ².

In an embodiment, the present invention relates to those compounds of Formula (I) and the pharmaceutically acceptable salts, and the solvates thereof, or any subgroup thereof as mentioned in any of the other embodiments, wherein the compounds of Formula (I) are restricted to compounds of Formula (I-z) :

wherein R ³ represents Het ¹.

wherein R ³ represents Cy ².

In an embodiment, the present invention relates to a subgroup of Formula (I) as defined in the general reaction schemes.

In an embodiment the compound of Formula (I) is selected from the group consisting of any of the exemplified compounds, tautomers and stereoisomeric forms thereof, and the free bases, any pharmaceutically acceptable salts, and the solvates thereof.

All possible combinations of the above indicated embodiments are considered to be embraced within the scope of the invention.

METHODS FOR THE PREPARATION OF COMPOUNDS OF FORMULA (I)

In this section, as in all other sections unless the context indicates otherwise, references to Formula (I) also include all other sub-groups and examples thereof as defined herein. The general preparation of some typical examples of the compounds of Formula (I) is described hereunder and in the specific examples, and are generally prepared from starting materials which are either commercially available or prepared by standard synthetic processes commonly used by those skilled in the art of organic chemistry. The following schemes are only meant to represent examples of the invention and are in no way meant to be a limit of the invention. Alternatively, compounds of the present invention may also be prepared by analogous reaction protocols as described in the general schemes below, combined with standard synthetic processes commonly used by those skilled in the art.

The skilled person will realize that in the reactions described in the Schemes, although this is not always explicitly shown, it may be necessary to protect reactive functional groups (for example hydroxy, amino, or carboxy groups) where these are desired in the final product, to avoid their unwanted participation in the reactions. In general, conventional protecting groups can be used in accordance with standard practice. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.

The skilled person will realize that in the reactions described in the Schemes, it may be advisable or necessary to perform the reaction under an inert atmosphere, such as for example under N ₂-gas atmosphere.

It will be apparent for the skilled person that it may be necessary to cool the reaction mixture before reaction work-up (refers to the series of manipulations required to isolate and purify the product (s) of a chemical reaction such as for example quenching, column chromatography, extraction) .

The skilled person will realize that heating the reaction mixture under stirring may enhance the reaction outcome. In some reactions microwave heating may be used instead of conventional heating to shorten the overall reaction time.

The skilled person will realize that another sequence of the chemical reactions shown in the Schemes below, may also result in the desired compound of Formula (I) .

The skilled person will realize that intermediates and final compounds shown in the Schemes below may be further functionalized according to methods well-known by the person skilled in the art. The intermediates and compounds described herein can be isolated in free form or as a salt, or a solvate thereof. The intermediates and compounds described herein may be synthesized in the form of mixtures of tautomers and stereoisomeric forms that can be separated from one another following art-known resolution procedures.

SCHEME 1

In general, compounds of Formula (I) wherein Y ¹ is limited to Y ^1a being -O-or -NR ^5c-, hereby named compounds of Formula (Ia) , (Ib) , (Ic) , (Id) , (Ie) , can be prepared according to the following reaction Scheme 1. In Scheme 1, W ¹ represents fluoro, chloro, bromo or iodo; all other variables are defined according to the scope of the present invention.

In Scheme 1, the following reaction conditions apply:

Step 1: at a suitable temperature such as ranged from room temperature to 90 ℃, in the presence of a suitable base such as for example diisopropylethylamine or triethylamine or sodium carbonate, in a suitable solvent such as for example acetonitrile or dimethylformamide or dichloromethane;

Step 2: at a suitable temperature range from room temperature to 130℃, in presence of a suitable base such as for example cesium carbonate, in a suitable solvent such as for example dimethylformamide or 1-methyl-2-pyrrolidinone;

Alternatively, at a suitable temperature such as for example room temperature, in the presence of a suitable deprotonating agent such as for example sodium hydride, in a suitable solvent such as for example dimethylsulfoxide;

Alternatively, at a suitable temperature such as room temperature, in the presence a suitable base such as 1, 8-Diazabicyclo [5.4.0] undec-7-ene (DBU) , in a suitable solvent such as for example tetrahydrofuran;

Step 3: at a suitable temperature such as room temperature, in the presence of a suitable catalyst such as palladium on charcoal (Pd/C) , in a suitable solvent such as methanol, under H ₂ pressure such as for example from 1 to 3 bar, optionally in the presence of a base such as triethylamine;

Alternatively, at a suitable temperature such as room temperature, in the presence of a suitable catalyst such as for example 1, 1'-Bis (diphenylphosphino) ferrocene-palladium (II) dichloride dichloromethane complex, a suitable reducing agent such sodium borohydride, a suitable base such as for example N, N, N', N'-tetramethylethylenediamine, in a suitable solvent such as for example tetrahydrofuran;

Step 4: at a suitable temperature range from 100 to 130℃, in presence of a suitable base such as for example cesium carbonate, in a suitable solvent such as for example dimethylformamide or 1-methyl-2-pyrrolidinone;

Step 5: at a suitable temperature range from 100 to 130℃, in presence of a suitable base such as for example cesium carbonate, in a suitable solvent such as for example dimethylformamide or 1-methyl-2-pyrrolidinone;

alternatively, at a suitable temperature ranged from 80 to 100℃, in presence of a suitable catalyst such as palladium acetate (Pd (OAc) ₂) , in presence of a suitable ligand such as for example 2, 2′-bis (diphenylphosphino) -1, 1′-binaphthyl, in presence of a suitable base such as cesium carbonate, in a suitable solvent such as for example dioxane;

Step 6: at a suitable temperature from room temperature to 60℃, in presence of a suitable catalyst such as palladium acetate (Pd (OAc) ₂) or Tris (dibenzylideneacetone) dipalladium (0) (Pd ₂dba ₃) , in presence or not of a suitable ligand such as for example triphenylphosphine, in a suitable solvent such as for example dioxane;

SCHEME 2

In general, compounds of Formula (I) wherein Y ¹ is limited to -CH ₂-, and R ² is limited to W ¹, hereby named compounds of Formula (If) , can be prepared according to the following reaction Scheme 2. In Scheme 2, all other variables are defined according to the scope of the present invention.

In Scheme 2, the following reaction conditions apply:

Step 1: at a suitable temperature ranged from 60℃ to 100℃, in presence of a suitable catalyst such as palladium acetate (Pd (OAc) ₂) or Tris (dibenzylideneacetone) dipalladium (0) (Pd ₂ (dba) ₃) or Tetrakis (triphenylphosphine) palladium (0) , in a suitable solvent such as for example tetrahydrofuran or dioxane.

The skilled person will realize that starting from compound (If) , analogous chemistry as reported in steps 3, 4, 5 and 6 in scheme 1 could be performed.

SCHEME 3

In general, compounds of Formula (I) wherein Y ¹ is limited to -CR ^5aR ^5b-and R ² is limited to W ¹, hereby named compounds of Formula (Ig) , can be prepared according to the following reaction Scheme 3. In Scheme 3 at least one of R ^5a and R ^5b is other than hydrogen. All other variables are defined according to the scope of the present invention.

In Scheme 3, the following reaction condition apply:

Step 1: at a suitable temperature ranged from 80℃ to 200℃, in presence of a suitable catalyst such as palladium acetate (Pd (OAc) ₂) , in the presence of a suitable ligand such as for example triphenylphosphine or tricyclohexylphosphine, in a suitable solvent such as for example dioxane, preferably in sealed conditions, optionally under microwave irradiation.

The skilled person will realize that starting from compound (Ig) , analogous chemistry as reported in steps 3, 4, 5 and 6 in scheme 1 could be performed.

SCHEME 4

In general, compounds of Formula (I) hereby named compounds of Formula (Ib) can be alternatively prepared according to the following reaction Scheme 4. In Scheme 4, PG ¹ represents a suitable protecting group, such as for example tert-butyloxycarbonyl and LG ¹ is a leaving group such as for example chloro, bromo, iodo or tosylate or mesylate; all other variables are defined as listed before or according to the scope of the present invention.

In Scheme 4, the following reaction conditions apply:

Step 3: at a suitable temperature such as room temperature, in the presence of a suitable catalyst such as palladium on charcoal (Pd/C) , in a suitable solvent such as methanol, under H ₂ pressure such as for example from 1 to 3 bar;

Alternatively, at a suitable temperature such as room temperature, in the presence of a suitable catalyst such as for example 1, 1'-Bis (diphenylphosphino) ferrocene-palladium (II) dichloride dichloromethane complex, a suitable reducing agent such sodium borohydride , a suitable base such as for example N, N, N', N'-tetramethylethylenediamine, in a suitable solvent such as for example tetrahydrofuran;

Step 4: when PG ¹ is tert-butyloxycarbonyl, at a suitable temperature range such as for example from 0 ℃ to room temperature, in the presence of suitable cleavage conditions, such as for example an acid such as HCl or trifluoroacetic acid in a suitable solvent such as acetonitrile or DCM or methanol (MeOH) ;

Step 5: represents all type of reactions, such as for examples reductive amination, nucleophilic substitution, leading to final examples (Ib) ;

The skilled person will realize that starting from intermediate XI, analogous chemistry as reported in steps 3, 4, 5 and 6 in scheme 1 could be performed.

SCHEME 5

In general, compounds of Formula (I) wherein U is limited to N and Y ¹ is limited to Y ^1b being O, hereby named compounds of Formula (Iba) can be prepared according to the following reaction Scheme 5. In Scheme 5, PG ¹ represents a suitable protecting group, such as for example tert-butyloxycarbonyl and W ² a leaving group such as for example chloro, tosylate or mesylate; all other variables are defined according to the scope of the present invention.

In Scheme 5, the following reaction conditions apply:

Step 1: at a suitable temperature such as room temperature, in the presence of a suitable base such as for example potassium carbonate, in a suitable solvent such as for example dimethylformamide;

Step 2: at a suitable temperature such as room temperature, in presence of a suitable base such as lithium hydroxyde, in a suitable solvent such as for example a mixture of tetrahydrofuran, ethanol and water;

Step 3: at a suitable temperature such as room temperature, in the presence of a dibromoisocyanurate, in a suitable solvent such as dichloroethane;

Step 4: when W ² is chloro, at a suitable temperature range such as room temperature, in the presence of a chlorinating reagent such as oxalyl chlorine, in the presence of a catalytic amount of dimethylformamide, in the presence of a suitable base such as triethylamine, in a suitable solvent such as dichloromethane;

When W ² is a trifluoroethoxy, at a suitable temperature such as 65℃, in the presence of 2, 2, 2-trifluoroethanol as solvent or not, suitable activating agents such as 1, 3-dibromo-1, 3, 5-triazinane-2, 4, 6-trione, in the presence of molecular sieve;

Step 5: At a suitable temperature such as room temperature, in the presence of a suitable base such as for example triethylamine or 1, 8-Diazabicyclo [5.4.0] undec-7-ene (DBU) , in a suitable solvent such as for example dichloromethane or acetonitrile;

Step 6: At a suitable temperature such as room temperature, in the presence of a suitable base such as for example triethylamine or 1, 8-Diazabicyclo [5.4.0] undec-7-ene (DBU) , in a suitable solvent such as for example dichloromethane or acetonitrile;

Step 7: when PG ¹ is tert-butyloxycarbonyl, at a suitable temperature range such as for example from 0 ℃ to room temperature, in the presence of suitable cleavage conditions, such as for example an acid such as HCl or trifluoroacetic acid in a suitable solvent such as acetonitrile or DCM or methanol (MeOH) ;

Step 8: represents all type of reactions, such as for examples reductive amination, nucleophilic substitution leading to final examples (Iba) .

SCHEME 6

In general, intermediates of formula IIIa can be prepared according to the following reaction Scheme 5. In Scheme 5, PG ² represents a suitable protecting group, such as for example benzyloxycarbonyl; all other variables are defined according to the scope of the present invention or as defined in the previous schemes.

Step 1: at a suitable temperature such as room temperature, in the presence of benzyl chloroformate, in the presence of a suitable base such as as for example triethymaine, in a suitable solvent such as for example dichloromethane;

Step 2: when PG ¹ is tert-butyloxycarbonyl, at a suitable temperature range such as for example from 0 ℃ to room temperature, in the presence of suitable cleavage conditions, such as for example an acid such as HCl or trifluoroacetic acid in a suitable solvent such as acetonitrile or DCM or methanol (MeOH) ;

Step 3: represents all type of reactions, such as for examples reductive amination, nucleophilic substitution leading to intermediate IIIa.

SCHEME 7

In general, intermediates of formula XXVI can be prepared according to the following reaction Scheme 7. Variables are defined according to the scope of the present invention or as defined in the previous schemes.

Step 1: at a suitable temperature such as 120℃, in the presence of a suitable base such as for example cesium carbonate, in a suitable solvent such as for example dimethylacetamide;

Step 2: at a suitable temperature such as 0℃ to room temperature, in the presence of a suitable oxidative agent such as for example urea hydrogen peroxide, in the presence of a suitable reagent such as trifluoroacetic anhydride, in a suitable solvent such as for example tetrahydrofuran;

Step 3: at a suitable temperature such as 0℃ to room temperature, in the presence of a suitable chlorinated agent such as for example phosphoryl chloride, in the presence of a suitable base such as diisopropylethylamine, in a suitable solvent such as for example ethyl acetate;

It will be clear for someone skilled in the art that starting from intermediate XXVI, similar chemistry as reported in Scheme 4 starting from intermediate II could be performed.

SCHEME 8

In general, intermediates of formula XXVIII can be prepared according to the following reaction Scheme 8. Variables are defined according to the scope of the present invention or as defined in the previous schemes.

It will be clear for someone skilled in the art that starting from intermediate XXVIII, similar chemistry as reported in Scheme 1 (ie steps 3, 4, 5 and 6) could be applied to functionalize first position 2. Then, from the obtained intermediate, similar chemistry as reported in scheme 2 and 3 could be applied to functionalize position 5 with intermediates IV, VIII and VIIa.

It will be appreciated that where appropriate functional groups exist, compounds of various formulae or any intermediates used in their preparation may be further derivatized by one or more standard synthetic methods employing condensation, substitution, oxidation, reduction, or cleavage reactions. Particular substitution approaches include conventional alkylation, arylation, heteroarylation, acylation, sulfonylation, halogenation, nitration, formylation and coupling procedures.

The compounds of Formula (I) may be synthesized in the form of racemic mixtures of enantiomers which can be separated from one another following art-known resolution procedures. The racemic compounds of Formula (I) containing a basic nitrogen atom may be converted into the corresponding diastereomeric salt forms by reaction with a suitable chiral acid. Said diastereomeric salt forms are subsequently separated, for example, by selective or fractional crystallization and the enantiomers are liberated therefrom by alkali. An alternative manner of separating the enantiomeric forms of the compounds of Formula (I) involves liquid chromatography using a chiral stationary phase. Said pure stereochemically isomeric forms may also be derived from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction occurs stereospecifically.

In the preparation of compounds of the present invention, protection of remote functionality (e.g., primary or secondary amine) of intermediates may be necessary. The need for such protection will vary depending on the nature of the remote functionality and the conditions of the preparation methods. Suitable amino-protecting groups (NH-Pg) include acetyl, trifluoroacetyl, t-butoxycarbonyl (Boc) , benzyloxycarbonyl (CBz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc) . The need for such protection is readily determined by one skilled in the art. For a general description of protecting groups and their use, see T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 4th ed., Wiley, Hoboken, New Jersey, 2007.

PHARMACOLOGY

It has been found that the compounds of the present invention block the interaction of menin with MLL proteins and oncogenic MLL fusion proteins. Therefore the compounds according to the present invention and the pharmaceutical compositions comprising such compounds may be useful for the treatment or prevention, in particular treatment, of diseases such as cancer, myelodysplastic syndrome (MDS) and diabetes.

In particular, the compounds according to the present invention and the pharmaceutical compositions thereof may be useful in the treatment or prevention of cancer. According to one embodiment, cancers that may benefit from a treatment with menin/MLL inhibitors of the invention comprise leukemias, myeloma or a solid tumor cancer (e.g. prostate cancer, lung cancer, breast cancer, pancreatic cancer, colon cancer, liver cancer, melanoma and glioblastoma, etc. ) . In some embodiments, the leukemias include acute leukemias, chronic leukemias, myeloid leukemias, myelogeneous leukemias, lymphoblastic leukemias, lymphocytic leukemias, Acute myelogeneous leukemias (AML) , Chronic myelogenous leukemias (CML) , Acute lymphoblastic leukemias (ALL) , Chronic lymphocytic leukemias (CLL) , T cell prolymphocytic leukemias (T-PLL) , Large granular lymphocytic leukemia, Hairy cell leukemia (HCL) , MLL-rearranged leukemias, MLL-PTD leukemias, MLL amplified leukemias, MLL-positive leukemias, leukemias exphibiting HOX/MEIS1 gene expression signatures etc.

Hence, the invention relates to compounds of Formula (I) , the tautomers and the stereoisomeric forms thereof, and the pharmaceutically acceptable salts, and the solvates thereof, for use as a medicament.

The invention also relates to the use of a compound of Formula (I) , a tautomer or a stereoisomeric form thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a pharmaceutical composition according to the invention, for the manufacture of a medicament.

The present invention also relates to a compound of Formula (I) , a tautomer or a stereoisomeric form thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a pharmaceutical composition according to the invention, for use in the treatment, prevention, amelioration, control or reduction of the risk of disorders associated with the interaction of menin with MLL proteins and oncogenic MLL fusion proteins in a mammal, including a human, the treatment or prevention of which is affected or facilitated by blocking the interaction of menin with MLL proteins and oncogenic MLL fusion proteins.

Also, the present invention relates to the use of a compound of Formula (I) , a tautomer or a stereoisomeric form thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a pharmaceutical composition according to the invention, for the manufacture of a medicament for treating, preventing, ameliorating, controlling or reducing the risk of disorders associated with the interaction of menin with MLL proteins and oncogenic MLL fusion proteins in a mammal, including a human, the treatment or prevention of which is affected or facilitated by blocking the interaction of menin with MLL proteins and oncogenic MLL fusion proteins.

The invention also relates to a compound of Formula (I) , a tautomer or a stereoisomeric form thereof, or a pharmaceutically acceptable salt, or a solvate thereof, for use in the treatment or prevention of any one of the diseases mentioned hereinbefore.

The invention also relates to a compound of Formula (I) , a tautomer or a stereoisomeric form thereof, or a pharmaceutically acceptable salt, or a solvate thereof, for use in treating or preventing any one of the diseases mentioned hereinbefore.

The invention also relates to the use of a compound of Formula (I) , a tautomer or a stereoisomeric form thereof, or a pharmaceutically acceptable salt, or a solvate thereof, for the manufacture of a medicament for the treatment or prevention of any one of the disease conditions mentioned hereinbefore.

The compounds of the present invention can be administered to mammals, preferably humans, for the treatment or prevention of any one of the diseases mentioned hereinbefore.

In view of the utility of the compounds of Formula (I) , the tautomers and the stereoisomeric forms thereof, and the pharmaceutically acceptable salts, and the solvates thereof, there is provided a method of treating warm-blooded animals, including humans, suffering from any one of the diseases mentioned hereinbefore.

Said method comprises the administration, i.e. the systemic or topical administration, of a therapeutically effective amount of a compound of Formula (I) , a tautomer or a stereoisomeric form thereof, or a pharmaceutically acceptable salt, or a solvate thereof, to warm-blooded animals, including humans.

Therefore, the invention also relates to a method for the treatment or prevention of any one of the diseases mentioned hereinbefore comprising administering a therapeutically effective amount of compound according to the invention to a patient in need thereof.

One skilled in the art will recognize that a therapeutically effective amount of the compounds of the present invention is the amount sufficient to have therapeutic activity and that this amount varies inter alias, depending on the type of disease, the concentration of the compound in the therapeutic formulation, and the condition of the patient. An effective therapeutic daily amount would be from about 0.005 mg/kg to 100 mg/kg. The amount of a compound according to the present invention, also referred to herein as the active ingredient, which is required to achieve a therapeutically effect may vary on case-by-case basis, for example with the particular compound, the route of administration, the age and condition of the recipient, and the particular disorder or disease being treated. A method of treatment may also include administering the active ingredient on a regimen of between one and four intakes per day. In these methods of treatment the compounds according to the invention are preferably formulated prior to administration.

The present invention also provides compositions for preventing or treating the disorders referred to herein. Said compositions comprising a therapeutically effective amount of a compound of Formula (I) , a tautomer or a stereoisomeric form thereof, or a pharmaceutically acceptable salt, or a solvate thereof, and a pharmaceutically acceptable carrier or diluent.

While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical composition. Accordingly, the present invention further provides a pharmaceutical composition comprising a compound according to the present invention, together with a pharmaceutically acceptable carrier or diluent. The carrier or diluent must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof.

The pharmaceutical compositions may be prepared by any methods well known in the art of pharmacy, for example, using methods such as those described in Gennaro et al. Remington’s Pharmaceutical Sciences (18 ^th ed., Mack Publishing Company, 1990, see especially Part 8: Pharmaceutical preparations and their Manufacture) .

The compounds of the present invention may be administered alone or in combination with one or more additional therapeutic agents. Combination therapy includes administration of a single pharmaceutical dosage formulation which contains a compound according to the present invention and one or more additional therapeutic agents, as well as administration of the compound according to the present invention and each additional therapeutic agent in its own separate pharmaceutical dosage formulation.

Therefore, an embodiment of the present invention relates to a product containing as first active ingredient a compound according to the invention and as further active ingredient one or more anticancer agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of patients suffering from cancer.

The one or more other medicinal agents and the compound according to the present invention may be administered simultaneously (e.g. in separate or unitary compositions) or sequentially in either order. In the latter case, the two or more compounds will be administered within a period and in an amount and manner that is sufficient to ensure that an advantageous or synergistic effect is achieved. It will be appreciated that the preferred method and order of administration and the respective dosage amounts and regimes for each component of the combination will depend on the particular other medicinal agent and compound of the present invention being administered, their route of administration, the particular condition, in particular tumour, being treated and the particular host being treated.

The following examples further illustrate the present invention.

EXAMPLES

Several methods for preparing the compounds of this invention are illustrated in the following examples. Unless otherwise noted, all starting materials were obtained from commercial suppliers and used without further purification, or alternatively can be synthesized by a skilled person by using well-known methods.

As understood by a person skilled in the art, compounds synthesized using the protocols as indicated may exist as a solvate e.g. hydrate, and/or contain residual solvent or minor impurities. Compounds isolated as a salt form, may be integer stoichiometric i.e. mono-or di-salts, or of intermediate stoichiometry. When an intermediate or compound in the experimental part below is indicated as ‘HCl salt’ without indication of the number of equivalents of HCl, this means that the number of equivalents of HCl was not determined.

The stereochemical configuration for centers in some compounds may be designated “R” or “S” when the mixture (s) was separated; for some compounds, the stereochemical configuration at indicated centers has been designated as “*R” or “*S” when the absolute stereochemistry is undetermined (even if the bonds are drawn stereo specifically) although the compound itself has been isolated as a single stereoisomer and is enantiomerically pure.

For example, it will be clear that Compound 3

is

The paragraphs above about stereochemical configurations, also apply to intermediates.

A skilled person will realize that, even where not mentioned explicitly in the experimental protocols below, typically after a column chromatography purification, the desired fractions were collected and the solvent was evaporated.

In case no stereochemistry is indicated, this means it is a mixture of stereoisomers, unless otherwise is indicated or is clear from the context.

When a stereocenter is indicated with ‘RS’ this means that a racemic mixture was obtained at the indicated centre, unless otherwise indicated.

A skilled person will understand that when Intermediates or Compounds are reported in Tables, the synthetic methodology from the indicated starting material to desired Intermediate/Compound might go over one or more reaction steps.

When two enantiomers, diastereomers or isomers are present in the same cell of one of the tables below, a skilled person will understand that these Intermediates or Compounds were separated from each other by using a suitable chromatographic method e.g. SFC or reversed phase separation.

Preparation of intermediates

For intermediates that were used in a next reaction step as a crude or as a partially purified intermediate, in some cases no mol amounts are mentioned for such intermediate in the next reaction step or alternatively estimated mol amounts or theoretical mol amounts for such intermediate in the next reaction step are indicated in the reaction protocols described below.

Hereinafter, the terms : ‘ACN' or ‘MeCN’ means acetonitrile, ‘DCM’ means dichloromethane, ‘DIPEA or DIEA’ means N, N-diisopropylethylamine, ‘h’ means hours (s) , ‘min’ means minute (s) , ‘DMF’ means N, N-dimethylformamide, ‘TEA’ or ‘Et ₃N’ means triethyl amine, ‘EtOAc’ or ‘EA’ means ethyl acetate, ‘THF’ means tetrahydrofuran; ‘HPLC’ means High-performance Liquid Chromatography, ‘Prep-HPLC’ means preparative HPLC; ‘MeOH’ means methanol, ‘NMR’ means Nuclear Magnetic Resonance, ‘rt or ‘RT’ means room temperature, ‘SFC’ means supercritical fluid chromatography, ‘q.s. ’ means quantum satis, ‘DMSO’ means dimethylsulfoxide, ‘Pd/C’ or “Pd/C (10%) ” means palladium on carbon, ‘atm’ means atmosphere, ‘ee’ means enantiomeric excess, ‘PE’ means petroleum ether, ‘NaBH (OAc) ₃’ means sodium triacetoxyborohydride, ‘TFA’ means trifluoroacetic acid, ‘DCE’ means dichloroethane, and ‘DMA’ means N, N-dimethylacetamide; “IPA” means isopropyl alcohol; “iPrNH ₂” means isopropylamine; NH ₄OH means ammonium hydroxide; “Pd (OH) ₂ means palladium hydroxide; DBU means 1, 8-diazabicyclo [5.4.0] undec-7-ene; Cbz” means benzoylcarbonyl; NaBH ₃CN means sodium cyanoborohydride; NaBH ₄ means sodium borohydride; tlc means thin-layer chromatography ; FCC means Flash Column Chromatography ; HATU means 1- [Bis (dimethylamino) methylene] -1H-1, 2, 3-triazolo [4, 5- b] pyridinium 3-oxid hexafluorophosphate, N- [ (Dimethylamino) -1H-1, 2, 3-triazolo- [4, 5-b] pyridin-1-ylmethylene] -N-methylmethanaminium hexafluorophosphate N-oxide; EDCI means N- (3-Dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride; ‘HOBT’ or ‘HOBt’ means 1-Hydroxybenzotriazole hydrate; TMEDA means N, N, N′, N′-Tetramethylethylenediamine; Pd (dppf) Cl ₂. DCM means [1, 1′-Bis (diphenylphosphino) ferrocene] dichloropalladium (II) , complex with dichloromethane; “Ni (acac) ₂” means Nickel (II) acetylacetonate; “Zn” means Zinc; “MS” means molecular sieve; “Boc ₂O” means di-tert-butyl decarbonate; “Ar” means argon; “FA” means formic acid; “CC” means column chromatography; “T ₃P” means propyl phosphonic anhydride.

A. Preparation of the intermediates

Example A1

Preparation of intermediate 1

A mixture of 2, 6-diazaspiro [3.3] heptane-2-carboxylic acid, phenylmethyl ester (1.084 g, 4.667 mmol) , tert-butyl 3-isobutyrylazetidine-1-carboxylate (1.3 g, 5.6 mmol) , sodium cyanoborohydride (1.5 g, 23.33 mmol) and acetic acid (267 μL, 4.67 mmol) in methanol (50 mL) was stirred at 50℃ overnight. The mixture was gathered with another reaction performed on 100 mg of 2, 6-diazaspiro [3.3] heptane-2-carboxylic acid, phenylmethyl ester and poured onto 10%aqueous solution of K ₂CO ₃. The resulting mixture was extracted with DCM. The organic layer was decanted, washed with water, dried over MgSO ₄, filtered and evaporated to dryness. The residue was purified by chromatography over silica gel (irregular SiOH, 40g; mobile phase: gradient from 0%NH ₄OH, 0%MeOH, 100%DCM to 0.3%NH ₄OH, 3%MeOH, 97%DCM) . The pure fractions were collected and evaporated to dryness. The resulting residue was purified a second time by chromatography over silica gel (irregular SiOH, 40g; mobile phase: gradient from 40%EtOAc, 60%heptane to 60%EtOAc, 40%heptane) . The pure fractions were collected and evaporated to dryness yielding 1.58 g of intermediate 1 (70%yield) .

Preparation of intermediate 2

A mixture of intermediate 1 (500 mg; 1.127 mmol) and TFA (1.5 mL) in DCM (5 mL) was stirred at room temperature overnight. The reaction mixture was diluted with ACN and evaporated to dryness (twice) . The residue was dissolved in DCM and basified with 15%aqueous solution of NH ₄OH. The organic layer was washed again with 15%aqueous solution of NH ₄OH, then, with water, filtered over

and evaporated to dryness yielding 330 mg of intermediate 2 (85%) which was directly engaged in the next step without any further purification.

Preparation of intermediate 3

Acetic acid (55 μL; 0.96 mmol) was added at room temperature to a solution of intermediate 2 (330 mg; 0.96 mmol) and oxetane-3-carbaldehyde (132 μL; 1.92 mmol) in THF (12 mL) . The mixture was stirred at rt for overnight then NaBH (OAc) ₃ (611 mg; 2.88 mmol) was added portionwise. The mixture was stirred at rt for 3 hours. The reaction mixture was partitioned between aqueous 10%K ₂CO ₃ and EtOAc. The layers were separated and the aqueous layer was extracted once with DCM. The organic layers were mixed, dried over MgSO ₄ and evaporated to dryness. The residue was purified by chromatography over silica gel (irregular SiOH, 10g+24g; mobile phase: gradient from 0.5%NH ₄OH, 5%MeOH, 95%DCM to 1%NH ₄OH, 10%MeOH, 90%DCM) . The pure fractions were collected and evaporated to dryness yielding 264 mg of intermediate 3 (66%yield) .

Preparation of intermediate 4

A mixture of intermediate 3 (264 mg; 0.638 mmol) and Pd/C (10%) (68 mg; 0.0638 mmol) in ethanol (10 mL) was hydrogenated under 3 bars of H ₂ for 2 hours. Pd/C (10%) was removed by filtration over

and the solvent was evaporated to dryness yielding 173 mg of intermediate 4 (97%yield) .

Example A2

Preparation of intermediate 5

In a round bottom flask, 2, 6-diazaspiro [3.4] octane-6-carboxylic acid, phenylmethyl ester (500 mg; 2.03 mmol) , tert-butyl 3-isobutyrylazetidine-1-carboxylate (553.7 mg; 2.43 mmol) , sodium cyanoborohydride (382.7 mg; 6.09 mmol) and acetic acid (0.116 mL; 2.03 mmol) were diluted in MeOH. Then, the reaction mixture was heated overnight at 50℃ and cooled down to room temperature. Carefully, a saturated solution of NaHCO ₃ was added until pH > 9. The resulting mixture was extracted with DCM. The organic layer was decanted, washed with water, dried over MgSO ₄, filtered and evaporated to dryness. The residue was purified by chromatography over silica gel (irregular SiOH, 40g; mobile phase: gradient from 0%NH ₄OH, 0%MeOH, 100%DCM to 0.3%NH ₄OH, 3%MeOH, 97%DCM) . The pure fractions were collected and evaporated to dryness to afford 700 mg of intermediate 5 (75%yield) .

Preparation of intermediate 6

In a round bottom flask, at 0℃, TFA (2.34 mL, 30.59 mmol) was added to intermediate 5 (700 mg, 1.53 mmol) in DCM (33.7 mL) . Then, the reaction was warmed to room temperature and the reaction mixture was stirred overnight at room temperature. The residue was dissolved in 4 mL of water. Then, the solution was basified with a solution of NaOH 1M (12 mL) until pH=8-9. After stirring for 10 min at room temperature, the resulting mixture was extracted with dichloromethane (3 x 30 mL) . The combined organic layers were washed with brine (1 x 50 mL) , dried over MgSO ₄, filtered and evaporated till dryness to give 482 mg of intermediate 6 which was directly engaged in the next step without any further treatment.

Preparation of intermediate 7

Acetic acid (119 μL; 2.07 mmol) was added at room temperature to a solution of intermediate 6 (482 mg; 1.34 mmol) and oxetane-3-carbaldehyde (188 μL; 2.72 mmol) in THF (20 mL) . The mixture was stirred at rt for 4h then NaBH (OAc) ₃ (870 mg; 4.1 mmol) was added portionwise. The mixture was stirred at room temperature for 2 hours. The reaction mixture was poured into ice water, basified with an aqueous solution of K ₂CO ₃ 10%and EtOAc was added. The organic layer was separated, washed with brine, dried over MgSO ₄, filtered and evaporated till dryness to give 438 mg of an intermediate residue. The residue (438 mg) was purified by silica gel chromatography (Stationary phase: irregular SiOH 15-40μm 24g Mobile phase: Gradient from 97%DCM, 3%MeOH (+10%NH ₄OH) to 90%DCM, 10%MeOH (+10%NH ₄OH) ) . The fractions containing the product were mixed and concentrated to give 127 mg of intermediate 7 (22%yield) .

Preparation of intermediate 8

A mixture of intermediate 7 (63 mg; 0.147 mmol) , Pd (OH) ₂ (21 mg; 0.174 mmol) in MeOH (3 mL) and THF (0.5 mL) was hydrogenated under atmospheric pressure overnight. The catalyst was removed by filtration through a pad of

washed with MeOH and the filtrate was evaporated to give 35 mg of intermediate 8 (81%yield) .

Example A3

Preparation of intermediate 9

To the mixture of 5-fluoro-2-methoxybenzoic acid (8.00 g, 47.0 mmol) and N-ethylpropan-2-amine (8.19 g, 94.0 mmol) in dry DCM (150 mL) cooled at 0 ℃, were slowly added HATU (21.5 g, 56.5 mmol) and DIEA (9.10 g, 70.4 mmol) in portions. The resulting mixture was slowly warmed to RT and stirred for 8 h. The organic layer was washed with water (20 mL x 3) and dried over anhydrous Na ₂SO ₄. After filtration, the solvent was removed under reduced pressure and the crude product was purified by FCC (EtOAc/PE = 0%to 20%of EtOAc) to afford intermediate 9 (12.0 g, 96%yield) as a white solid.

The following intermediate was synthesized by an analogous method as described above for the preparation of intermediate 9

Preparation of intermediate 11 (Method A)

To the solution of intermediate 9 (12.0 g, 50.1 mmol) in dry DCM (100 mL) cooled at -78 ℃was slowly added BBr ₃ (14.4 mL, 152 mmol) and the resulting mixture was slowly warmed to RT and stirred for 8 h. The mixture was cooled to -78 ℃ again and MeOH (5 mL) was added dropwise to quench the reaction. The resulting mixture was slowly warmed to RT and the pH value was adjusted to about 8 by adding a saturated solution of NaHCO ₃. The aqueous layer was extracted by DCM (50 mL x 3) and the combined organic layers were dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure to give the crude product which was purified by FCC (EtOAc/PE = 0%to 20%of EtOAc) to afford intermediate 11 (9.0 g, 78%yield) as a white solid.

Alternative preparation of intermediate 11 (Method B)

A solution of 5-fluorosalicylic acid (30.0 g, 192.2 mmol) in thionyl chloride (200 mL) was stirred for 5 hours at 80 ℃. Then, the resulting mixture was concentrated under reduced pressure to give the acyl chloride. To a stirred solution of N-ethylpropan-2-amine (33.5 g, 384.3 mmol) and triethylamine (58.3 g, 576.5 mmol) in dichloromethane (200 mL) was added a solution of acyl chloride in dichloromethane (100 mL) dropwise at 0 ℃. After stirring overnight at room temperature, the resulting mixture was concentrated under reduced pressure. The crude product was dissolved in methanol (300 mL) . Then, a solution of sodium hydroxide (20 g) in water (100 mL) was added. After stirring for 1 hour at room temperature, the resulting mixture was diluted with water (100 mL) and concentrated under reduced pressure to remove the excess methanol, adjusted to pH value 4 and extracted with ethyl acetate (2 x 150 mL) . The combined organic layer was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with (EA/PE, 16.3: 83.7) to afford 29.4 g (66%yield) of intermediate 11 as an off-white solid.

The following intermediate was synthesized by an analogous method as described above for intermediate 11 (Method A)

The following intermediate was synthesized by an analogous method as described above for intermediate 11 (Method B)

Example A4

Preparation of intermediate 13

To the solution of 3, 5, 6-trichloro-1, 2, 4-triazine (10.0 g, 54.2 mmol) and TEA (15.2 mL, 109 mmol) in DCM (100 mL) cooled at 0 ℃ was added tert-butyl 2, 6-diazaspiro [3.4] octane-2-carboxylate (9.21 g, 43.4 mmol) and the mixture was warmed to RT and stirred for 1 h. The mixture was diluted with water (20 mL) and extracted with DCM (30 mL x 3) . The combined organic layers were washed with brine, dried over Na ₂SO ₄, filtered and concentrated under reduced pressure to give the crude product which was purified by FCC on silica gel (Mobile phase A: PE; Mobile phase B: EtOAc, eluent from 0-25%Mobile phase B) to afford intermediate 13 (12.0 g, 58%yield) as a yellow solid.

Preparation of intermediate 14

The mixture of intermediate 13 (12.0 g, 33.3 mmol) , intermediate 11 (7.5 g, 33.3 mmol) and DBU (6.1 g, 40.1 mmol) in THF (120 mL) was stirred at 25 ℃ for 8 h. The mixture was diluted with water (30 mL) and extracted with DCM (30 mL x 3) . The combined organic layers were washed with brine, dried over Na ₂SO ₄, filtered and concentrated under reduced pressure to give the crude product which was purified by FCC on silica gel (Mobile phase A: PE; Mobile phase B: EtOAc, eluent from 0-25%Mobile phase B) to afford intermediate 14 (14.0 g, 73%yield) as a green solid.

The following intermediates was synthesized by an analogous method as described above for intermediate 14

Preparation of intermediate 16

Method A:

To the mixture intermediate 14 (20 g, 36.4 mmol) , NaBH ₄ (2.48 g, 65.7 mmol) and TMEDA (8.54 g, 73.5 mmol) in THF (500 mL) was added Pd (dppf) Cl ₂·DCM (1.70 g, 2.08 mmol) under N ₂ atmosphere. After addition, the reaction mixture was stirred at 25 ℃ for 14 h. The reaction mixture was filtered, and the filtrate was concentrated, the residue was purified by FCC on silica gel (eluent with EtOAc) to afford intermediate 16 (15 g, 74%yield) as brown solid.

Method B:

To the solution of intermediate 14 (22.0 g, 40.1 mmol) , TEA (15 mL) in MeOH (100 mL) was added Pd/C (wet, 5.0 g, 10%) The resulting mixture was stirred under H ₂ atmosphere (30 psi) at 25 ℃ for 8hr. The reaction mixture was filtered through a celite

and the filtrate was concentrated in vacuo to afford intermediate 16 (25.0 g, crude) , which was used directly in next step without further purification.

The following intermediate was synthesized by an analogous method described above for intermediate 16

Preparation of intermediate 18

To the solution of intermediate 16 (300 mg, 0.583 mmol) in DCM (5 mL) was added TFA (0.5 mL, 6.4 mmol) and the resulting mixture was stirred at RT for 3 h. Then, 10%NaOH (5 mL) solution was slowly added into the mixture to adjust the pH value to about 12 and the resulting mixture was extracted with DCM (10 mL x 3) . The combined organic layers were dried over anhydrous Na ₂SO ₄, filtered, and concentrated in vacuo to afford intermediate 18 (220 mg, 90%yield) as a white solid.

The following intermediate was synthesized by an analogous method described above for intermediate 18

Example A5

Preparation of intermediate 20:

To a solution of cis-3- [ [ (1, 1-dimethylethoxy) carbonyl] amino] -cyclobutanecarboxylic acid (10.0 g, 46.5 mmol) in DMF (100 mL) was added HOBt (8.15 g, 60.3 mmol) , EDCI (11.6 g, 60.5 mmol) and DIEA (30.0 mL, 182 mmol, 0.782 g/mL) at 0 ℃. Then, N, O-dimethylhydroxylamine hydrochloride (5.90 g, 60.5 mmol) was added at 0 ℃. The mixture was stirred at room temperature for 16 hours. The mixture was diluted with ethyl acetate (500 mL) . The mixture was washed with 1 M HCl (150 mL) , saturated NaHCO ₃ (100 mL x 2) and brine (300 mL x 3) , dried over Na ₂SO ₄, filtered and concentrated under reduced pressure to give intermediate 20 (11.0 g, crude) as a white solid, which was used in the next step without further purification.

Preparation of intermediate 21:

To a solution of intermediate 20 (11.0 g, 6.97 mmol) in THF (100 mL) was added isopropylmagnesium chloride (64.0 mL, 128 mmol, 2M in THF) dropwise at 0 ℃ under N ₂ atmosphere. The mixture was stirred at room temperature for 12 hours under N ₂ atmosphere. The mixture was quenched with saturated NH ₄Cl (100 mL) . The mixture was filtered through a pad of

and the filtrate was concentrated under reduced pressure. The mixture was extracted with ethyl acetate (200 mL x 2) . The combined organic layers were washed with brine (200 mL x 2) , dried over Na ₂SO ₄, filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography over silica gel (eluent: petroleum ether: ethyl acetate from 1: 0 to 5: 1, to yield intermediate 21 (6.30 g) as a white solid.

Example A6

Preparation of intermediate 22:

Under N ₂, at rt, to a mixture of intermediate18 (1 g, 2.41 mmol) , intermediate 21 (873 mg, 3.62 mmol) , acetic acid (276 μL, 4.83 mmol) in MeOH (50 mL) was added NaBH ₃CN (455 mg, 7.24 mmol) . Then, the reaction was heated at 50℃ overnight. The reaction mixture was cooled to rt, poured into ice water, basified with a saturated solution of NaHCO ₃ and DCM was added. The organic layer was separated, washed with brine, dried over MgSO ₄, filtered and evaporated till dryness. The crude was purified by silica gel chromatography (Stationary phase: irregular SiOH 15-40μm 40g, Mobile phase: Gradient from 0%NH ₄OH, 100%DCM, 0%MeOH to 0.1%NH ₄OH, 95%DCM, 5%MeOH) . The fraction containing the product were mixed and concentrated to afford 1.37g (89%yield) of intermediate 22.

Example A7

Preparation of intermediate 23:

To a solution of 3, 3-dimethoxycyclobutanecarboxylic acid (12.0 g, 75 mmol) in DCM (145 mL) was added T ₃P (100 mL, 168 mmol, 50%in EtOAc) and DIEA (64 mL, 372 mmol) at 0℃. Then N, O-dimethylhydroxylamine hydrochloride (8.8 g, 89.5 mmol) was added at 0℃. The mixture was stirred at room temperature for 16 hours. The mixture was poured onto a saturated solution NaHCO ₃ and EtOAc was added. The organic layer was separated, washed with brine, dried over MgSO ₄, filtered and concentrated under reduced pressure to give intermediate 23 (16.0 g, crude) which was used in the next step without further purification.

Preparation of intermediate 24:

The reaction was performed twice on 15.7 g of intermediate 23 and respective reaction media were mixed for the work-up and purification. To a solution of intermediate 23 (15.7 g, 77.7 mmol) in THF (420 mL) was added isopropylmagnesium chloride (178.5 mL, 232 mmol, 2M in THF) dropwise at 0 ℃ under N ₂ atmosphere. The reaction mixtures were stirred at room temperature for 12 hours under N ₂ atmosphere and then, poured onto ice-water and a 10%aqueous solution of NH ₄Cl. The mixture obtained was combined with the mixture obtained from the second reaction, and the combined mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO ₄, filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography over silica gel (mobile phase: Heptane: EtOAc 9: 1) . The pure fractions were collected and evaporated to dryness yielding 22 g (76%yield) of intermediate 24 as a colourless oil.

Example A8

Preparation of intermediate 25

A mixture of intermediate 18 (10 g, 24.13 mmol) , intermediate 24 (4.94 g, 26.54 mmol) and acetic acid (1.5 mL, 26.54 mmol) in MeOH (80 mL) was stirred at room temperature for 20 min. Then, NaBH ₃CN (1.82 g, 28.95 mmol) was added and the mixture was stirred at 50 ℃overnight. The reaction solution was poured into ice water and extracted with DCM. The organic layer was washed with water and brine, then dried over Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (Mobile phase A: PE; Mobile phase B: EtOAc, eluent from 0-100%EtOAc) to give 9.21 g (64%yield) of intermediate 25 as a light yellow solid.

Preparation of intermediate 26

and intermediate 27

Intermediate 25 (9.2g) was purified via chiral SFC (Stationary phase: C+HIRALPAK AD-H 5μm 250*21.2mm, Mobile phase: 83%CO ₂, 17%mixture of EtOH/ACN 80/20 v/v (+0.3%iPrNH ₂) ) . The fractions containing the products were mixed and concentrated to afford 4.07g (44%yield) of intermediate 26 and 4.06 g (44%yield) of intermediate 27 and 273 mg of a residual fraction of intermediate 25.

Method A for intermediate 27:

To a solution of intermediate 35 (2.24 g, 3.618 mmol) in methanol (45 mL) was added palladium on activated carbon (10%palladium) (635 mg, 0.597 mmol) . Then, the mixture was stirred at room temperature for 5 hours under the hydrogen. The mixture was diluted with methanol, filtered through a pad of

and the filtrate was evaporated under reduced pressure. The residue was dissolved with ethyl acetate, washed with sodium hydroxide solution (1M in water) and brine. The organic layer was dried over anhydrous sodium sulfate, evaporated under reduced pressure to give 1.4 g (62%yield) of intermediate 27 as a yellow solid.

Method B for intermediate 27:

A mixture of intermediate 35 (1.44 g; 2.33 mmol) and TMEDA (0.54 mL; 3.63 mmol) in dry THF (55 mL) was degassed by N ₂ bubbling. Then, Pd (dppf) Cl ₂. DCM (216 mg; 0.26 mmol) and sodium borohydride (144 mg; 3.81 mmol) were added. The reaction mixture was stirred at 50℃ overnight in a sealed glassware. The solution was cooled, poured out into cooled water. EtOAc was added and the mixture was filtered through a

The product was extracted with EtOAc and the organic layer was dried over MgSO ₄, filtered and evaporated to dryness. The crude residue (1.7g) was purified by silica gel chromatography (Stationary phase: irregular SiOH 40 μm 40 g, Mobile phase: Gradient from 100%DCM, 0%MeOH (+10%NH4OH) to 95%DCM, 5%MeOH (+10%NH4OH) ) . The fractions containing the product were mixed and concentrated to afford 2 fractions of intermediate 27 (680 mg, 50%yield, 96%purity by LCMS and 360 mg; 26%yield, 91%purity by LCMS)

Preparation of intermediate 28:

A solution of intermediate 26 (2 g, 3.42 mmol) and TFA (2.9 mL; 37.9 mmol) in DCM (29 mL) was stirred at rt overnight. ACN was then added and the solution was evaporated to dryness. The residue was then dissolved in EtOAc and iced water, basified with NH ₄OH. The layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were dried over MgSO ₄, filtered and evaporated to give 1.80 g (98%yield) of intermediate 28.

Preparation of intermediate 29:

Intermediate 27 (1.87 g, 3.20 mmol) in TFA (2.7 mL) and DCM (27 mL) was stirred at rt overnight. The solution was evaporated to dryness. The residue was then dissolved in DCM and iced water, basified with a 30%aqueous NH ₄OH solution. The aqueous layer was extracted with DCM. The combined organic layers were dried over MgSO ₄, filtered and evaporated to give 1.35 g (78%yield) of intermediate 29 as a pale yellow solid.

Alternative preparation of intermediate 29:

To a solution of intermediate 27 (1.40 g, 2.25 mmol) in acetone (30 mL) and water (14 mL) was added p-Toluenesulfonic acid (1.94 g, 11.276 mmol) . The reaction solution was stirred at 65 degrees for 5 hours. The resulting mixture was quenched with water and ethyl acetate. The combined organic layers were washed with water and brine, dried over anhydrous sodium sulfate. The solid was filtered off. The residue was concentrated under reduce pressure to give 1.01 g (78%) of intermediate 29 as a yellow solid.

Preparation of intermediate 29a:

Intermediate 29a was prepared accordingly to intermediate 28 starting from intermediate 25

Example A9

Preparation of intermediate 30:

To a stirring solution of 2, 6-diazaspiro [3.4] octane-6-carboxylic acid, phenylmethyl ester (15 g, 60.9 mmol) in methanol (300 mL) was added intermediate 24 (13.61 g, 73.08 mmol) and acetic acid (4.02 g, 66.99 mmol) . After stirring for 0.5 hour at room temperature, sodium cyanoborohydride was added (7.65 g, 121.8 mmol) . After stirring overnight at 50℃, the reaction mixture was quenched with a potassium carbonate solution (10%in water) and extracted with ethyl acetate. The combined organic layers were washed with brine and dried over anhydrous sodium sulfate. The solid was filtered off. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (Mobile phase A: PE; Mobile phase B: EtOAc, eluent from 0-50%EtOAc) to give 17.8 g (69%yield) of intermediate 30 as a light yellow oil.

Preparation of intermediate 31:

and intermediate 32:

170 g of intermediate 30 was purified by SFC with the following conditions: Column: CHIRALPAK IG, 5*25cm, 10um; Mobile Phase A: CO ₂, Mobile Phase B: EtOH: ACN: DCM=1: 1: 1; Flow rate: 150 mL/min; Gradient: 40%B; 220 nm; retention time 1 = 4.45 min; retention time 2 = 5.88 min; Injection Volumn: 3.8 ml; Number of Runs: 237 to give two fractions. Fraction A: 67.0 g (>99%purity by LCMS, 39%yield, retention time 2: 5.88 min) of intermediate 31 as a light-yellow oil. Fraction B: 65 g (99%purity, 38%yield, retention time 1: 4.45 min) of intermediate 32 as a light-yellow oil.

Preparation of intermediate 33

To a solution of intermediate 31 (15 g, 36.01 mmol) in methanol (300 mL) was added palladium on activated carbon (10%palladium) (8g, 7.517 mmol) . Then the mixture was stirred at room temperature for 5 hours under the hydrogen (2-3 atm. ) . The mixture was diluted with methanol and filtered through a pad of

The filtrate was evaporated under reduced pressure to give 9.5 g of desired product as a yellow oil which was directly used in the next step without any further modifications.

Preparation of intermediate 34

To a solution of 3, 5, 6-trichloro-1, 2, 4-triazine (9.4 g, 50.99 mmol) in dichloromethane (100 mL) were added the mixture of intermediate 33 (12.0 g, 42.49 mmol) and triethylamine (12 mL, 84.98 mmol) in dichloromethane (150 mL) under nitrogen at 0 ℃. After stirring for 3 hours at room temperature under nitrogen, the mixture was quenched with water and extracted with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate. The solid was filtered off. The filtrate was concentrated under reduced pressure to give 17.3 g (83%yield, 88%purity by LCMS) of intermediate 34 as a yellow solid.

Preparation of intermediate 35:

A solution of intermediate 34 (1.6g; 3.72 mmol) , intermediate 11 (1g; 4.44 mmol) and DBU (2.7 mL; 18.45 mmol) in THF (150 mL) was stirred at rt for 72 hours. The solution was poured into cooled water and the product was extracted with EtOAc. The organic layer was dried over MgSO ₄, filtered and evaporated to dryness. The crude (3g) was purified by silica gel chromatography (Stationary phase: irregular bare silica 80g, Mobile phase: 63%Heptane, 2%MeOH (+10%NH ₄OH) , 35%EtOAc) . The fraction containing the product were mixed and concentrated to afford 1.48g (64%yield) of intermediate 35.

Alternative preparation of intermediate 35:

To a solution of intermediate 34 (3.00 g, 6.971 mmol) and intermediate 11 (1.88 g, 8.365 mmol) in tetrahydrofuran (60 mL) was added tetramethylguanidine (1.37 g, 11.85 mmol) . The reaction solution was stirred for 2 days at room temperature. The resulting mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were washed with sodium hydroxide (0.5 M/L) , water and brine, dried over anhydrous sodium sulfate. The solid was filtered off. The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography column (ethyl acetate/hexane 2: 1) to give 2.60 g (59%yield) of intermediate 35 as a yellow solid.

Preparation of intermediate 82:

To a mixture of intermediate 34 (10.0 g, 23.27 mmol) and intermediate 81 (5.89 g, 27.887 mmol) in THF (250 mL) was added tetramethylguanidine (7.3 mL, 58.09 mmol) . After stirring at room temperature for 48 hours, the reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were washed with water and brine and dried over Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel column chromatography (Mobile phase A: PE; Mobile phase B: EtOAc, eluent from 0-93%EtOAc) to give 7.5 g (49%yield) of intermediate 82 as a yellow solid.

Preparation of intermediate 83

To a mixture of intermediate 82 (7.0 g, 11.57 mmol) in tetrahydrofuran (140 mL) were added 1, 1'-bis (diphenylphosphino) ferrocene-palladium (II) dichloride dichloromethane complex (472 mg, 0.58 mmol) , sodium borohydride (744 mg, 19.67 mmol) and N, N, N', N'-tetramethylethylenediamine (2.9 mL, 19.67 mmol) . After stirring at room temperature overnight under the N ₂ atmosphere, the reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were washed with water and brine and dried over Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel column chromatography (MeOH/DCM, 0%MeOH to 9%MeOH) to give 4.8 g (54%yield, 85.1%purity based on LC/MS) of intermediate 83 as a brown solid.

Preparation of intermediate 84:

To a mixture of intermediate 83 (4.8 g, 8.32 mmol) in acetone (100 mL) and water (50 mL) was added p-toluenesulfonic acid (7.17 g, 41.62 mmol) . After stirring at 65 ℃ overnight, the reaction mixture was quenched with saturated sodium bicarbonate solution and extracted with dichloromethane. The combined organic layers were washed with water and brine, dried over Na ₂SO ₄, filtered and concentrated under reduced pressure to give 3.1 g (45%yield, 62.8%purity based on LC/MS) of intermediate 84 as a brown solid.

Example A10

Preparation of intermediate 36:

To a stirring solution of 1- (tert-butoxycarbonyl) azetidine-3-carboxylic acid (30.0 g, 149.09 mmol) , 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (42.9 g, 223.64 mmol) and N, O-dimethylhydroxylamine (21.8 g, 223.64 mmol) in DCM (500 mL) were added N, N-diisopropylethylamine (61.7 mL, 372.73 mmol) and 4-dimethylaminopyridine (3.6 g, 29.82 mmol) . After stirring overnight at room temperature, the reaction solution was diluted with DCM (500 mL) and washed with water, 10%of citric acid aqueous solution, water and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give 24.0 g of intermediate 36 as a light-yellow oil.

Preparation of intermediate 37:

To a stirred solution of intermediate 36 (26.5 g, 108.5 mmol) in tetrahydrofuran (250 mL) was added isopropylmagnesium chloride (271 mL, 542.0 mmol, 2M in THF) at 0℃. After stirring overnight at room temperature, the reaction mixture was quenched with brine (300 mL) at 0℃and extracted with ethyl acetate (3 x 500 mL) . The combined organic layer was washed with water and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE/EA, 8: 2) to give 19.5 g of intermediate 37 (87%purity, 68%yield) as a light yellow oil.

Preparation of intermediate 39:

To a stirred mixture of tert-butyl 2, 6-diazaspiro [3.4] octane-2-carboxylate (25.5 g, 120.12 mmol) in tetrahydrofuran (250 mL) and potassium carbonate (36.52 g, 264.262 mmol) in water (250 mL) was added benzyl chloroformate (20.3 mL, 144.143 mmol) at 0℃. After stirring overnight at room temperature, the reaction mixture was extracted with ethyl acetate (3 x 300 mL) . The combined organic layer was washed with water and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue purified by silica gel column chromatography (PE/EA, 6: 4) to give 39.10 g of intermediate 39 (99%purity, 93%yield) as a light-yellow oil.

Preparation of intermediate 40:

To a solution of intermediate 39 (55.5 g, 160.2 mmol) in DCM (550 mL) was added TFA (110 mL) . After stirring for 2 hours at room temperature, the reaction solution was concentrated. The residue was dissolved in water (300 mL) . The resulting aqueous solution was basified to pH=8 with a saturated solution of NaHCO ₃ and extracted with DCM/MeOH (10: 1, 4 x 500 mL) . The combined organic layer was washed with brine (2 x 300 mL) , dried over anhydrous Na ₂SO ₄ and filtered. The filtrate was concentrated under reduced pressure to give 45.3 g (74%yield) of intermediate 40 as TFA salt and as a light brown solid.

Preparation of intermediate 41:

To a stirred solution of intermediate 40 (5.00 g, 13.88 mmol) in methanol (50 mL) was intermediate 37 (3.79 g, 16.65 mmol) . After stirring for 0.5 hour at room temperature, sodium cyanoborohydride (4.36 g, 69.38 mmol) was added. The resulting mixture was stirring overnight at 50 ℃. Additional intermediate 37 (1.58 g, 6.94 mmol) and sodium cyanoborohydride (2.62 g, 41.63 mmol) were added. After stirring for 6 hours at 50℃, additional sodium cyanoborohydride (1.31 g, 20.814 mmol) was added. After stirring overnight at 50℃, the reaction mixture was quenched with saturated sodium bicarbonate solution (100 mL) and extracted with ethyl acetate (3 x 300 mL) . The combined organic layer was washed with water, brine and dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with (PE/EA, 7: 3) to give 4.5 g (67%yield) of intermediate 41 as a light yellow oil

Preparation of intermediate 42:

To a stirred solution of intermediate 41 (3.60 g, 7.87 mmol) in ethanol (40 mL) was added palladium on activated carbon 10%Pd (800 mg) . After stirring under a hydrogen stream (2-3 atm) at room temperature for 2 hours, the reaction mixture was filtered through a pad of

which was washed with ethanol and DCM. The filtrate was concentrated under reduced pressure to give 2.5 g of intermediate 42 as a grey oil.

Preparation of intermediate 43:

To a stirred solution of 3, 4, 6-trichloropyridazine (700 mg, 2.164 mmol) in N, N-dimethylformamide (15 ml) was added intermediate 42 (397 mg, 2.164 mmol) and triethylamine (0.9 mL, 6.492 mmol) . After stirring 3h at temperature, the reaction mixture was quenched with water (30 mL) and extracted with ethyl acetate (3 x 50 mL) . The combined organic layer was washed with water, brine and dried over anhydrous sodium sulfate and filtered off. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with (PE: EA = 55: 45) to give 900 mg (84%yield) of intermediate 43 as a white solid.

Preparation of intermediate 44:

To a stirring solution of intermediate 43 (800 mg, 1.701 mmol) in N, N-dimethylacetamide (15 mL) were added intermediate 11 (383 mg, 1.70 mmol) and cesium carbonate (1.66 g, 5.10 mmol) . After stirring for 3h at 130℃, the reaction mixture was cooled to room temperature, quenched with water (100 mL) and extracted with EA (3 x 80 mL) . The combined organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with (PE: EA = 55: 45) to give 800 mg (70%yield) of intermediate 44 as a white solid.

Preparation of intermediate 45:

To a stirred solution of intermediate 44 (750 mg, 1.138 mmol) in ethyl acetate (15 mL) was added palladium on activated carbon 10%Pd (800 mg) . After stirring under a hydrogen stream (2-3 atm) at room temperature overnight, the reaction mixture was filtered through a pad of

which was washed with ethyl acetate and ethanol. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with (DCM: MeOH = 6: 4) to give 303 mg of intermediate 45 (42%yield) as an off-white solid.

Preparation of intermediate 50:

A mixture of intermediate 18 (740 mg, 1.78 mmol) , intermediate 37 (487 mg, 2.1 mmol) , NaBH ₃CN (337 mg, 5.4 mmol) and acetic acid (102 μL, 1.78 mmol) in MeOH (15 mL) was stirred at 50℃ overnight. The reaction mixture was mixed with another reaction performed on 220 mg of intermediate 18. The resulting reaction mixture was poured into ice water, basified with a saturated solution of NaHCO ₃ and DCM was added. The organic layer was separated, washed with brine, dried over MgSO ₄, filtered and evaporated till dryness. The residue was purified by silica gel chromatography (Stationary phase: irregular SiOH 15-40μm 24g MERCK, Mobile phase: Gradient from 99%DCM, 1%MeOH (+10%NH ₄OH) to 95%DCM, 5%MeOH (+10%NH ₄OH) ) . The fractions containing the product were mixed and concentrated to afford 1.04g (93%yield) of intermediate 50.

Preparation of intermediate 51:

and intermediate 52:

Intermediate 50 (1.04g) was purified by chiral SFC (Stationary phase: CHIRALPAK IC 5μm 250*30mm, Mobile phase: 50%CO ₂, 50%EtOH (0.3%iPrNH ₂) ) . The fractions containing the products were mixed and concentrated to afford 411 mg (37%yield) of intermediate 51 and 427 mg (38%yield) of intermediate 52.

Alternative preparation of intermediate 50:

Under N ₂ flow, intermediate 18 (854 mg; 1.13 mmol) and intermediate 37 (385 mg; 1.7 mmol) in THF (15 mL) were stirred at rt for 24h. Then, sodium triacetoxyborohydride (718 mg; 3.39 mmol) was added portionwise. The mixture was stirred at room temperature for 24h. The solution was poured out into cooled water, basified with a solution of NaOH 3N and EtOAc was added. The organic layer was separated, dried over MgSO ₄, filtered and evaporated to dryness. The residue was purified by silica gel chromatography (Stationary phase: irregular SiOH 15-40μm 12g, Mobile phase: Gradient from 99%DCM, 1%MeOH (+10%NH ₄OH) to 95%DCM, 5%MeOH (+10%NH ₄OH) ) . The fractions containing the product were mixed and concentrated to afford 200mg (28%yield) of intermediate 50.

Example A12

Preparation of intermediate 53

Phosphorus oxychloride (9.42 g, 61.4 mmol) was added dropwise to a 0 ℃ (ice/water) solution consisting of 4-chloropyridazin-3-ol (2.00 g, 38.3 mmol) and ACN (20 mL) . Then, the reaction mixture was heated and stirred at 80 ℃ for 3 hours before cooling to RT. The reaction mixture was slowly poured into water (50 mL) and adjusted to pH=8 by the saturated solution of sodium bicarbonate. The mixture was extracted with DCM (50 mL x 3) . The combined organic layers were dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure to give the crude product which was purified by FCC (silica gel, Mobile Phase A: PE; Mobile Phase B: EtOAc, eluent with 0-25%EtOAc) to give the intermediate 53 (2.00 g, 88%yield) as a yellow solid.

Preparation of intermediate 54:

A stir bar, intermediate 53 (500 mg, 3.36 mmol) , tert-butyl 2, 6-diazaspiro [3.4] octane-2-carboxylate (712 mg, 3.35 mmol) , triethylamine (1.02 g, 10.1 mmol) and dry DCM (10 mL) were added to a 40 mL glass bottle before the resultant mixture was stirred at 25 ℃ for 8 h. The mixture was diluted into DCM (20 mL) and washed with water (10 mL x 3) . The organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure to give the crude which was purified by FCC (silica gel, Mobile Phase A: PE; Mobile Phase B: EtOAc, eluent with 0-100%EtOAc) to give the intermediate 54 (500 mg, 42%yield) as a yellow solid.

Preparation of intermediate 55

A stir bar, intermediate 11 (346 mg, 1.54 mmol) , intermediate 54 (500 mg, 1.54 mmol) , cesium carbonate (1.51 g, 4.63 mmol) and dry N, N-dimethylformamide (10 mL) were added to a 50 mL round-bottomed flask before the resultant mixture was heated and stirred at 130 ℃ for 8 h. The mixture was cooled to room temperature and concentrated under reduced pressure to give a residue. The residue was suspended into dichloromethane (20 mL) and washed with water (10 mL x 3) . The organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure to give a crude which was purified by FCC (silica gel, Mobile Phase A: EtOAc; Mobile Phase B: MeOH, eluent with 0-10%MeOH) to give the intermediate 55 (700 mg, 80%yield) as a yellow solid.

Preparation of intermediate 56:

A stir bar, intermediate 55 (700 mg, 1.36 mmol) , trifluoroacetic acid (4 mL) and dry dichloromethane (2 mL) were added to a 25 mL round-bottomed flask before the mixture was stirred at 25 ℃ for 40 min. The mixture was concentrated under reduced pressure to give a residue. The residue was diluted into dichloromethane (20 mL) and pH wasadjusted to pH =12 by a solution of sodium hydroxide (3 M, 8 mL) . The aqueous layer was extracted with dichloromethane (10 mL x 2) . The combined organic layers were dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure to give intermediate 56 (600 mg, crude) as a yellow oil.

Preparation of intermediate 57:

HATU (99.5 g, 262 mmol) was added in portions to a 0 ℃ (ice/water) mixture consisting of 1- (tert-butoxycarbonyl) piperidine-4-carboxylic acid (50.0 g, 218 mmol) , N, O-dimethylhydroxylamine hydrochloride (23.4 g, 240 mmol) , Et ₃N (90.9 mL, 654 mmol) , and dichloromethane (500 mL) . The reaction mixture was stirred at room-temperature for 12 hours. The reaction mixture was concentrated to dryness under reduced pressure. The residue was diluted with water (1500 mL) and extracted with dichloromethane (500 mL x 3) . The combined organic extracts were dried over anhydrous Na ₂SO ₄, filtered, and concentrated to dryness under reduced pressure to afford the crude product, which was purified by FCC (silica gel, Mobile Phase A: PE; Mobile Phase B: EtOAc, eluent with 0-50%EtOAc) to afford the intermediate 57 (54 g, yield: 82%) as a yellow oil.

Preparation of intermediate 58:

Intermediate 57 (54.0 g, 198 mmol) and THF (500 mL) were added into a 1 L three-necked round-bottomed flask. i-PrMgCl (198 mL, 397 mmol, 2 M in THF) was added dropwise into the mixture at 0 ℃ (ice/water) under N ₂. The mixture was stirred with warming to room temperature for 10 hours before pouring into water (2000 mL) and extracted with EtOAc (1000 mL x 3) . The organic phase was washed with brine, dried over Na ₂SO ₄, filtered and concentrated under reduced pressure to give the crude which was purified by flash column chromatography on silica gel (silica gel, Mobile Phase A: PE; Mobile Phase B: EtOAc, eluent with 0-35%EtOAc) to give the intermediate 58 (19.2 g, 34%yield) as a yellow oil.

Preparation of intermediate 59:

A stir bar, intermediate 58 (278 mg, 1.09 mmol) , intermediate 56 (300 mg, 0.726 mmol) , zinc chloride (200 mg, 1.47 mmol) and dry methanol (6 mL) were added to a 40 mL glass bottle before the mixture was heated and stirred at 45 ℃ for 4 h. Then, sodium cyanotrihydroborate (91.2 mg, 1.45 mmol) was added to the mixture. The resultant mixture was stirred at 45 ℃ for another 40 h. The mixture was diluted into dichloromethane (40 mL) and washed with water (10 mL x 3) . The organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure to give the crude which was purified by FCC (silica gel, Mobile Phase A: EtOAc; Mobile Phase B: MeOH, eluent with 0-10%MeOH) to give intermediate 59 (150 mg, 29%yield) as a yellow solid.

Example A12

Preparation of intermediate 60:

Intermediate 18 (120 mg, 0.29 mmol) , intermediate 58 (150 mg, 0.585 mmol) and ZnCl ₂ (80 mg, 0.59 mmol) were added to a 25 mL round bottomed flask and the resulting mixture was dissolved in MeOH (5 mL) . The mixture was heated and stirred at 80 ℃ for 4 hours. Sodium cyanoborohydride (37 mg, 0.59 mmol) was added to the mixture. Then, the mixture was stirred at 80 ℃ for 16 hours. Then, additional intermediate 58 (150 mg, 0.585 mmol) , ZnCl ₂ (80 mg, 0.59 mmol) , and NaBH ₃CN (37 mg, 0.59 mmol) were added into the above solution. Then, the mixture was stirred at 80 ℃ for 6 hours. The reaction mixture was concentrated to dryness under reduced pressure to give the crude product which was purified by preparative HPLC using a Boston Green ODS 150 mm x 30 mm x 5 μm column (eluent: 25%to 55% (v/v) CH ₃CN and H ₂O with 0.04%NH ₃H ₂O+10mM NH ₄HCO ₃) to afford pure intermediate 60 which was suspended in water (10 mL) . The mixture was frozen using dry ice/acetone, and then lyophilized to dryness to afford the intermediate 60 (60 mg) as a white solid.

Preparation of intermediate 60a

and intermediate 60b:

Intermediate 60 (375 mg, 0.57 mmol) was purified by supercritical fluid chromatography (Separation condition: DAICEL CHIRALPAK IG (250 mm x 30 mm x 10 um) ; Mobile phase: A: Supercritical CO ₂, B: 0.1%NH ₃H ₂O IPA, A: B =45: 55 at 80 mL/min; Column Temp: 38 ; Nozzle Pressure: 100Bar; Nozzle Temp: 60; Evaporator Temp: 20; Trimmer Temp: 25 ; Wavelength: 220nm) . The pure fractions were collected, and the volatiles were removed under vacuum. The resulting product was lyophilized to dryness to remove the solvent residue completely. Desired product intermediate 60a (15 mg, 4%yield) and intermediate 60b (19 mg, 5%yield) were obtained as white solid.

Preparation of intermediate 61:

EDCI (34.0 g, 177 mmol) was added to a solution consisting of (R) -1- (tert-butoxycarbonyl) pyrrolidine-3-carboxylic acid (25.0 g, 116 mmol) , HOBT (24.0 g, 178 mmol) , DIPEA (102.5 mL, 586.9 mmol) and DMF (250 mL) at 0 ℃. The reaction mixture was stirred at for 5 min. N, O-dimethylhydroxylamine (12.5 g, 128 mmol) was added the reaction mixture. The reaction mixture was stirred at room-temperature for 10 h before cooling to room temperature. The mixture was poured into water (1000 mL) and extracted with ethyl acetate (400 mL x 3) . The organic phase was washed with 5%aqueous citric acid solution (400 mL x 3) , sat. NaHCO ₃ (400 mL x 2) , brine (400 mL x 2) , dried over Na ₂SO ₄, filtered and concentrated under reduced pressure to give crude intermediate 61 (26 g, 82%yield) as a colourless oil.

Preparation of intermediate 62:

i-PrMgCl (101 mL, 202 mmol, 2 M, in THF) was added dropwise to a 0 ℃ (ice/water) solution of intermediate 61 (26.0 g, 101 mmol) and THF (250 mL) . The reaction mixture was stirred at room-temperature for 10 hours. The mixture was quenched with a saturated solution of NH ₄Cl (500 mL) and extracted with ethyl acetate (500 mL x 3) . The combined organic layers were dried over anhydrous Na ₂SO ₄, filtered, and concentrated in vacuum to give the product which was purified by FCC (silica gel, Mobile Phase A: PE; Mobile Phase B: EtOAc, eluent with 0-50%EtOAc) to afford the intermediate 62 (15.0 g, 56%yield ) as a yellow oil.

Preparation of intermediate 63:

and intermediate 64:

To a solution of intermediate 18 (300 mg, 0.724 mmol) and intermediate 62 (524 mg, 2.17 mmol) in 15 mL of MeOH was added ZnCl ₂ (395 mg, 2.90 mmol) . After addition, the reaction mixture was stirred at 75 ℃ for 3 hours, then NaBH ₃CN (182 mg, 2.90 mmol) was added into the reaction and the mixture was stirred at the same temperature for 4 hours. Additional intermediate 62 (300 mg) was added and the mixture was stirred at 75 ℃ for 16 hours. The reaction mixture was concentrated in vacuum and the residue was purified by preparative HPLC (Column Welch Xtimate C18 150 x 25mm x 5um, Mobile Phase A: water (0.04%NH ₃H ₂O+10mM NH ₄HCO ₃) , Mobile Phase B: acetonitrile, Flow rate: 30 mL/min, gradient condition from 61%B to 81%B) . The pure fractions were collected, and the solvent was evaporated under vacuum. The aqueous layers were lyophilized to afford intermediate 63 (75.0 mg, 16%yield) as a white solid and intermediate 64 (88 mg, 18%yield) as a white solid.

Preparation of intermediate 65:

(S) -1- (tert-Butoxycarbonyl) pyrrolidine-3-carboxylic acid (15.0 g, 69.7 mmol) , EDCI (20.039 g, 104.53 mmol) , HOBT (14.125 g, 104.53 mmol) and DIEA (45.034 g, 348.44 mmol) were added to DMF (100 mL) at 10 ℃. After 5 min N, O-dimethylhydroxylamine hydrochloride (7.477 g, 76.66 mmol) was added to the mixture. The mixture was stirred at 40 ℃ for 10 h before poured into water (400 mL) and extracted with ethyl acetate (300 mL x 3) . The organic phase was washed with 5%aqueous citric acid solution (3 x 300 mL) , sat. NaHCO ₃ (2 x 300 mL) , brine (2 x 300 mL) , dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure to give the intermediate 65 (12.41 g, 74%) as a yellow oil.

Preparation of intermediate 66:

The intermediate 65 (12.4 g, 48.0 mmol) and THF (20 mL) were added into a 250 mL round-bottomed flask. Isopropylmagnesium chloride (49 mL, 98 mmol, 2 M in THF) was added dropwise into the mixture at 0 ℃ (ice/water) under N ₂. The mixture was stirred with warming to room temperature for 10 h. The mixture was quenched with a saturated aqueous solution of NH ₄Cl (100 mL) and extracted with EtOAc (200 mL x 3) . The combined organic extracts were dried over anhydrous Na ₂SO ₄, filtered, and concentrated to dryness under reduced pressure to give the crude product which was purified by FCC (silica gel, Mobile Phase A: PE; Mobile Phase B: EtOAc, eluent with 0-35%EtOAc) to afford the intermediate 66 (7.6 g, 65%) as a light yellow oil.

Preparation of intermediate 67:

and intermediate 68:

To a solution of intermediate 18 (300 mg, 0.724 mmol) and intermediate 66 (524 mg, 2.17 mmol) in 15 mL of MeOH was added ZnCl ₂ (395 mg, 2.90 mmol) . After addition, the reaction mixture was stirred at 75 ℃ for 3 hours. Then, NaBH ₃CN (182 mg, 2.90 mmol) was added into the reaction mixture and the mixture was stirred at the same temperature for 4 hours. Additional intermediate 67 (300 mg) was added and the mixture was stirred at 75 ℃ for 16 hours. The reaction mixture was cooled to 25 ℃ and concentrated in vacuum. The residue was purified by preparative HPLC (Column Welch Xtimate C18 150 x 25mm x 5um, Mobile Phase A: water (0.04%NH ₃H ₂O+10mM NH ₄HCO ₃) , Mobile Phase B: acetonitrile, Flow rate: 30 mL/min, gradient condition from 61%B to 81%B) . The pure fractions were collected and the solvent was evaporated under vacuum. The aqueous layers were lyophilized to afford the intermediate 67 (100 mg, 21%yield) as a white solid and the intermediate 68 (105 mg, 22%yield) as a white solid.

Preparation of intermediate 70:

A mixture of 2- [ (4-chloro-5-pyrimidinyl) oxy] -N-ethyl-5-fluoro-N- (1-methylethyl) -benzamide (4.5 g, 13.322 mmol) and intermediate 42 (4.31 g, 13.322 mmol) in acetonitrile (100 mL) was added sodium carbonate (5.65 g, 53.29 mmol) at room temperature. After stirring for 2 hours at 90℃, the resulting mixture was cooled to room temperature and filtered through a pad of

The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with (DCM/MeOH, 96.7: 3.3) to afford 6.7g (78%yield) of intermediate 70.

Preparation of intermediate 71:

and intermediate 72

Intermediate 70 (6.7g) was purified by via chiral SFC (Stationary phase: CHIRACEL OJ-H 5μm 250*30mm, Mobile phase: 94%CO ₂, 6%MeOH (0.3%iPrNH ₂) ) . The fraction containing the product were mixed and concentrated to afford 3.18g (47%yield) of intermediate 71 and 3.16g (47%yield) of intermediate 72.

Preparation of intermediate 73:

EDCI (3.12 g, 13.7 mmol) was added to a solution of 1- (tert-butoxycarbonyl) -3-fluoroazetidine-3-carboxylic acid (2.00 g, 9.12 mmol) , DIEA (6.5 mL, 36.7 mmol) , N, O-dimethylhydroxylamine hydrochloride (1.78 g, 18.2 mmol) , and HOBT (1.85 g, 13.7 mmol) in acetonitrile (20 mL) and the reaction mixture was allowed to stirred at 25℃ under N ₂ for 2 h. The mixture was quenched with water (50 mL) and extracted with EtOAc (100 mL x 3) . The EtOAc layer was dried over Na ₂SO ₄, filtered and evaporated to give a residue, which was purified by FCC (silica gel, from PE: EA=100: 0 to 60: 40) to give intermediate 73 (1.5 g, 63%yield) as a light yellow oil.

Preparation of intermediate 74:

Under N ₂ at 5℃, i-PrMgCl 2M in THF (10 mL, 20 mmol) was added to a solution of intermediate 73 (3.00 g, 11.4 mmol) in THF (30 mL) dropwise. The solution was stirred at 5℃for 30 min, allowed to slowly rise to 20℃ and stirred for 12h. The reaction mixture was poured out into a mixture of ice water and a saturated aqueous NH ₄Cl solution and extracted with EtOAc (200 mL x 2) . The organic layer was decanted, dried over Na ₂SO ₄, filtered and evaporated to dryness. The resulting crude was purified by FCC (silica gel, from PE: EA=100: 0 to 70: 30) to yield intermediate 74 (1.6 g, 51 %yield) as a colourless oil.

Preparation of intermediate 75:

To a solution of bicyclo [1.1.1] pentane-1-carboxylic acid (1.00 g, 8.92 mmol) , tert-butyl 4-bromopiperidine-1-carboxylate (4.71 g, 17.8 mmol) , 2, 2'-bipyridine (696 mg, 4.46 mmol) , Ni (acac) ₂ (916 mg, 3.57 mmol) , MgCl ₂ (2.55 g, 26.8 mmol) , Zn (4.00 g, 61.2 mmol) ,

MS (10.0 g) and DIEA (4.5 mL, 27.2 mmol) in THF/DMF (100 mL/30 mL) was added Boc ₂O (7.79 g, 35.7 mmol) under an Ar atmosphere at 30℃. After addition, the reaction mixture was stirred at 30 ℃ for 60 hours. The reaction mixture was poured into 150 mL of water and extracted with EtOAc (150 mL x 2) . The combined extracts were washed with brine (200 mL) , dried over Na ₂SO ₄, filtered and concentrated in vacuum. The resulting residue was purified by column chromatography (silica gel, eluent from PE/EtOAc = 100: 0 to 85: 15) to afford intermediate 75 (580 mg, purity 60%based on LCMS, 14%yield) as a colourless oil.

Preparation of intermediate 76:

To a solution of intermediate 75 (580 mg, 60%purity, 1.25 mmol) , intermediate 18 (568 mg, 1.37 mmol) and AcOH (449 mg, 7.47 mmol) in 50 mL of MeOH was added NaBH ₃CN (470 mg, 7.47 mmol) . After addition, the reaction mixture was stirred at 60℃ for 16 hours. The reaction mixture was concentrated under vacuum and the residue was diluted with 100 mL of water and extracted with EtOAc (100 mL x 2) . The combined extracts were concentrated in vacuum and the resulting residue was purified by preparative HPLC (Column Phenomenex Gemini NX-C18 (75*30mm*3um) , Mobile Phase A: water (0.2%FA) , Mobile Phase B: acetonitrile, Flow rate: 30 mL/min, gradient condition from 25%B to 55%B) . The pure fractions were collected and lyophilized to afford intermediate 76 (310 mg, 37%yield) as a white solid.

Preparation of intermediate 76:

LiCl (565.2 mg, 13.333 mmol) was dried under high vacuum by heating with a heat gun and allowed afterwards to cool to room temperature. Then, Mg turnings (324 mg, 13.333 mmol) and THF (11.1 mL, 1 M, 11.1 mmol) were added. The reaction mixture was cooled to 0 ℃ and bromocyclobutane (1.5 g, 11.1 mmol) was then added. The reaction mixture was stirred at room temperature for 2 hrs. In this time a grey solution was formed. The THF solution of cyclobutylmagnesium bromide. LiCl or intermediate 76 (approx. 1 M) was used directly in the following reaction.

Preparation of intermediate 77:

In flask, intermediate 57 (1.01 g, 3.704 mmol) was dissolved in dry THF (10 mL) . The solution was cooled in an ice bath and treated with a solution of freshly prepared intermediate 76 (11.1mL, approx. 1 M, 11.1 mmol) dropwise at this temperature. The reaction mixture was stirred over night and was allowed to come to room temperature. Then saturated ammonium chloride solution was added, and the water phase was extracted with ethyl acetate for three times. After the organic phase was dried with magnesium sulfate and filtered, the organic phase was evaporated. The crude product (953 mg) was purified with flash CC (silica gel, 15%EA in n-heptane) to give 833 mg (28%yield) of intermediate 77 as a colorless oil.

Preparation of intermediate 78:

intermediate 79:

and intermediate 80:

To a solution of intermediate 18 (90.0 mg, 0.217 mmol) , 2 drops of acetic acid and intermediate 77 (145.1 mg, 0.543 mmol) in methanol (4 mL) was added sodium cyanoborohydride (54.6 mg, 0.869 mmol) . After stirring at 60 ℃ overnight, the solvent was removed under vacuum. Then, the reaction was quenched with a saturated solution of sodium carbonate and extracted with ethyl acetate. The combined organic layers were washed with water, brine and dried over anhydrous magnesium sulfate. The solid was filtered off. The filtrate was concentrated under reduced pressure. The crude product (200 mg) was obtained as a colorless oil and purified by preparative CC (12 g silica gel, eluent from 2.5 to 5%MeOH in DCM) to give intermediate 78 (106 mg, 73%yield) as a white solid. A enantiomeric separation was performed via Preparative SFC (Stationary phase: Chiralpak Daicel IG 20 x 250 mm, Mobile phase: CO, EtOH + 0.4 iPrNH ₂) yielding 224 mg of intermediate 79 and 261 mg intermediate 80 containing 5%of intermediate 79 .

Preparation of intermediate 85:

To a mixture of 4-chloro-3-iodopyridine (2.00 g, 8.35 mmol) in DMF (30 mL) was added tert-butyl 2, 6-diazaspiro [3.4] octane-2-carboxylate (1.95 g, 9.19 mmol) and Cs ₂CO ₃ (8.2 g, 25.2 mmol) . The resultant mixture was stirred at 110 ℃ overnight. The reaction was cooled to room temperature and diluted by water (100 mL) , extracted with ethyl acetate (40 mL x 3) . The combined organic layers were washed with brine (20 mL x 3) , dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduce pressure to give the crude which was purified by FCC (100%petroleum ether to petroleum ether: ethyl acetate = 1: 1) to give intermediate 85 (1.7 g, purity 100%, yield 49%) as a white solid.

Preparation of intermediate 86:

To a mixture of intermediate 11 (2.72 g, 12.1 mmol) in N-methyl-2-pyrrolidone (20 mL) was added intermediate 85 (1.70 g, 4.09 mmol) and Cs ₂CO ₃ (4.00 g, 12.3 mmol) . The mixture was replaced with argon. Then CuCl (255 mg, 2.58 mmol) and 2, 2, 6, 6-tetramethyl-3, 5-heptanedione (0.4 mL, 1.91 mmol) was added under the protection of argon. The resultant mixture was stirred at 140 ℃ overnight under argon atmosphere. The mixture was cooled to room temperature and diluted by water (100 mL) , extracted with ethyl acetate (40 mL x 3) . The combined organic layers was washed by brine (20 mL x 3) , dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduce pressure to give the crude, which was purified by FCC (100%DCM to DCM: MeOH = 10: 1) to afford intermediate 86 (780 mg, purity 89.93%, yield 33%) as a brown solid.

Preparation of intermediate 87:

To a mixture of intermediate 86 (200 mg, 0.390 mmol) in DCM (2 mL) was added TFA (0.5 mL) at room temperature. The mixture was stirred at room temperature for 0.5 h. The reaction mixture was evaporated under reduce pressure. The residue was diluted by 2 M NaOH (5 mL) , extracted with DCM (10 mL x3) . The combined organic layers was dried over by anhydrous Na ₂SO ₄, filtered and concentrated under reduce pressure to give intermediate 87 (160 mg, 99%yield) as a yellow solid, which was used in next step without further purification.

Preparation of intermediate 88:

To a mixture of intermediate 87 (160 mg, 0.388 mmol) in MeOH (4 mL) was added intermediate 21 (187 mg, 0.775 mmol) and AcOH (47 mg, 0.783 mmol) . The mixture was stirred at 70 ℃ for 1 h. Then the mixture was cooled to room temperature and NaBH ₃CN (48 mg, 0.764 mmol) was added to the mixture. The resultant mixture was stirred at 70 ℃ for another 1 h. The reaction mixture was cooled to room temperature and evaporated to remove solvent. The residue was diluted by saturated NaHCO ₃ aqueous solution (10 mL) , extracted with dichloromethane (10 mL x 3) . The combined organic layers were washed with brine (5 mL) , dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure to give the crude, which was purified by FCC (100%petroleum ether to 100%ethyl acetate; TLC: ethyl acetate, Rf = 0.1) to give intermediate 88 (100 mg, purity 99%, yield 40%) as a yellow solid.

Preparation of intermediate 89:

To a solution of intermediate 19 (800 mg, 1.475 mmol) in MeOH (10 mL) was added intermediate 37 (838 mg, 3.686 mmol) . The mixture was stirring at room temperature for 1 h. The mixture was added NaBHCN (556 mg, 8.848 mmol) at 0℃. The mixture was stirring at room temperature for overnight. The mixture was quenched with Sodium bicarbonate solution, extracted with EA, washed with water and brine, dried over Na ₂SO ₄, filtered and evaporated under reduced pressure. The residue was purified by flash chromatography (silica gel, eluent from 100%DCM to 10%MeOH in DCM) to give 300 mg of intermediate 89 as a yellow oil.

B. Preparation of compounds

Preparation of compound 1:

A mixture of 2- [ (4-chloro-5-pyrimidinyl) oxy] -N-ethyl-5-fluoro-N- (1-methylethyl) -benzamide (174 mg; 0.516 mmol) , intermediate 4 (173 mg; 0.62 mmol) and sodium carbonate (218 mg; 2.064 mmol) in ACN (20 mL) was refluxed (90℃) for 2 hours. The reaction mixture was cooled to RT, poured onto iced water and extracted with DCM. The organic layer was decanted, washed with water, filtered over

and evaporated to dryness. The residue was purified by chromatography over silica gel (irregular SiOH, 24g; mobile phase: gradient from 0%NH ₄OH, 0%MeOH, 100%DCM to 1%NH ₄OH, 10%MeOH, 90%DCM) . The pure fractions were collected and evaporated to dryness yielding 150 mg of compound 1 (50%yield) .

Preparation of compound 2

A mixture of 2- [ (4-chloro-5-pyrimidinyl) oxy] -N-ethyl-5-fluoro-N- (1-methylethyl) -benzamide (33.5 mg; 0.099 mmol) , intermediate 8 (35 mg; 0.119 mmol) and sodium carbonate (42 mg; 0.398 mmol) in ACN (3.7 mL) was refluxed (90℃) for 2h. A similar work up and purification than the one used to isolate compound 1 was applied and led to compound 2.

Preparation of compound 3:

and compound 4:

Compound 2 (361 mg) was purified via chiral SFC (Stationary phase: CHIRALPAK AD-H 5μm 250*30mm, Mobile phase: 88%CO ₂, 12%EtOH (0.3%iPrNH ₂) ) . The fractions containing the product were mixed and concentrated to afford 156 mg of a fraction A which was taken up with Et ₂O and evaporated till dryness to give 150 mg of compound 3 and 156 mg of fraction B which was taken up with Et ₂O and evaporated till dryness to give compound 4.

Alternative preparation of compound 4:

The reaction was performed twice on (1.3g; 2.48 mmol) of compound 76.

Under N ₂ flow, NaBH (OAc) ₃ (1.56 g; 7.43 mmol) was added dropwise to a solution of compound 76 (2.6 g; 5 mmol) , oxetane-3-carbaldehyde (0.37 mL; 5.35 mmol) in THF (100 mL) . Then, the reaction mixture was stirred at RT for 2h. Both reactions (performed on 1.3g of compound 76) were mixed with another reaction performed on 700 mg of compound 76 and the resulting mixture was poured into ice water, basified with an aqueous solution of K ₂CO ₃ 10%and EtOAc was added. The organic layer was separated, washed with brine, dried over MgSO ₄, filtered and evaporated till dryness to give 3.93 g of crude compound 4 which was mixed with additional 392 mg of crude compound 4. The resulting crude was purified by silica gel chromatography (Stationary phase: irregular SiOH 15-40μm 80g MERCK, Mobile phase: Gradient from 97%DCM, 3%MeOH (+10%NH4OH) to 90%DCM, 10%MeOH (+10%NH4OH) ) . The fractions containing the product were mixed and concentrated to afford 2.5 g of compound 4 (white product) and 1.2 g of impure (71.6%purity evaluated by LC/MC) compound 4

Analogous reaction protocols as reported for compounds 1 and 2 could be used for the preparation of compounds listed in the table below starting from the appropriate starting materials.

Preparation of compound 22:

At 0℃, TFA (3.2mL; 42.2mmol) was added to intermediate 22 (1.35g; 2.11mmol) in DCM (32 mL) . Then, the reaction was warmed to room temperature and stirred for 15h at room temperature.

The reaction mixture was concentrated, and the residue was dissolved in 40 mL of water. The solution was basified with a solution of NaOH 1M until pH=8-9. After stirring for 10 min at room temperature, the resulting mixture was extracted with DCM. The combined organic layers were washed with brine and dried over MgSO ₄, filtered and evaporated till dryness to give 0.84g (74%) of compound 22.

Preparation of compound 32:

A solution of intermediate 45 (100 mg; 0.16 mmol) and TFA (0.25 mL; 3.27 mmol) in DCM (2.5 mL) was stirred at rt overnight. TFA was eliminated by evaporation. The residue was taken up with water, basified with an aqueous solution of NH ₄OH. The organic layer was extracted with DCM, dried over MgSO ₄ and evaporated to dryness to give 84 mg of compound 32 (quantitative) .

Preparation of compound 33:

At 0℃, TFA (0.49 mL; 6.4 mmol) was added to a solution of intermediate 50 (200 mg; 0.32 mmol) in DCM (7 mL) . The reaction mixture was stirred overnight at RT. The residue was evaporated till dryness. The residue (420 mg) was purified by silica gel chromatography (Stationary phase: irregular SiOH 15-40μm 12g, Mobile phase: Gradient from 90%DCM, 10%MeOH (+10%NH ₄OH) to 85%DCM, 15%MeOH (+10%NH ₄OH) ) . The fraction containing the product were mixed and concentrated to afford 144 mg (85%yield) of compound 33.

Preparation of compound 34:

At 0℃, TFA (0.42 mL; 5.5 mmol) was added to a solution of intermediate 51 (173 mg; 0.28 mmol) in DCM (6 mL) . The reaction mixture was stirred overnight at RT. The solvent was evaporated. The residue was dissolved in water. Then, the solution was basified with a solution of NaOH 1M until pH=9-10. After stirring for 10 min at room temperature, the resulting mixture was extracted with dichloromethane (3x) . The combined organic layer was washed with brine and dried over MgSO ₄, filtered and evaporated till dryness to give 150 mg (quantitative) of compound 34.

Preparation of compound 35:

Compound 35 was prepared accordingly to compound 34 starting from intermediate 52.

Preparation of compound 36:

A stir bar, intermediate 59 (130 mg, 0.199 mmol) , trifluoroacetic acid (2 mL) and dry dichloromethane (1 mL) were added to a 25 mL round-bottomed flask before the resultant mixture was stirred at 25 ℃ for 1 h. The mixture was concentrated under reduced pressure to give e compound 36 (130.0 mg, crude) as colourless oil which was used to the next step directly without further purification.

Preparation of compound 37:

Intermediate 60 (40 mg, 0.061 mmol) , 1, 4-dioxane (0.5 mL) and HCl/1, 4-dioxane (0.2 mL, 4 M) were added to a 10 mL round-bottomed flask. The reaction mixture was stirred at room-temperature for 12 hours. The reaction mixture was concentrated to dryness under reduced pressure to afford the title compound which was dissolved in H ₂O (10 mL) . The resultant solution was basified with solid NaHCO ₃ to pH = 8 and extracted with ethyl acetate (10 mL x 3) . The combined organic extracts were dried over anhydrous Na ₂SO ₄, filtered, and concentrated to dryness under reduced pressure. The residue was suspended in water (10 mL) . The mixture was frozen using dry ice/acetone and then lyophilized to afford compound 37 (30 mg, crude) , as a white solid which was used in the next step without further purification.

Preparation of compound 78:

HCl/dioxane (200 uL, 0.400 mmol, 2M) was added to a solution consisting of intermediate 60a (15 mg, 0.023 mmol) in dioxane (1 mL) . The reaction mixture was stirred at r. t. for 2 hr. White solid was precipitated. The solvent was removed by syringe and the white solid was concentrated to dryness under reduced pressure to afford the title compound which was suspended in water (10 mL) and frozen using dry ice/ethanol, and then lyophilized to dryness to afford the compound 78 (6.47 mg, 47%yield) as a white solid.

Preparation of compound 79:

HCl/dioxane (200 uL, 0.400 mmol) was added to a solution consisting of intermediate 60b (19 mg, 0.029 mmol) and dioxane (1 mL) . The reaction mixture was stirred at r. t. for 2 h. White solid was precipitated. The solvent was removed by syringe and the white solid was concentrated to dryness under reduced pressure to afford the title compound which was suspended in water (10 mL) and frozen using dry ice/ethanol, and then lyophilized to dryness to afford compound 79 (7.32 mg, 42%yield) as a white solid.

Preparation of compound 45:

HCl/dioxane (150 uL, 0.300 mmol, 2 M) was added to a solution consisting of intermediate 63 (20 mg, 0.031 mmol) and dioxane (1 mL) . The reaction mixture was stirred at room-temperature for 4 hours. The white solid was precipitated. The solvent (dioxane) was removed by syringe and the white solid was concentrated to dryness under reduced pressure to afford the title compound which was suspended in water (10 mL) . The mixture was frozen using dry ice/ethanol and then lyophilized to dryness to afford the compound 45 (3.17 mg, 17%yield) as a white solid.

Preparation of compound 46

HCl/dioxane (150 uL, 0.300 mmol, 2 M) was added to a solution consisting of intermediate 64 (19 mg, 0.030 mmol) and dioxane (1 mL) . The reaction mixture was stirred at room-temperature for 4 hours. The white solid was precipitated. The solvent (dioxane) was removed by syringe and the white solid was concentrated to dryness under reduced pressure to afford the title compound which was suspended in water (10 mL) . The mixture frozen using dry ice/ethanol and then lyophilized to dryness to afford the compound 46 (8.01 mg, 46%yield) as a white solid.

Preparation of compound 49:

HCl/1, 4-dioxane (0.3 mL) was added to the mixture consisting of intermediate 67 (25 mg, 0.039 mmol) and 1, 4-dioxane (1 mL) at 0 ℃. The resultant mixture was stirred at room-temperature for 1 h. The reaction mixture was concentrated to dryness under reduced pressure to afford the crude product which was purified by preparative HPLC using a YMC-Triart Prep C18 250 x 50 mm x 10 μm column (eluent: 45%to 75% (v/v) CH ₃CN with 0.04%NH ₃H ₂O+10mM NH ₄HCO ₃) to afford pure product. The product was suspended in water (10 mL) . The mixture was frozen using dry ice/ethanol, and then lyophilized to dryness to afford the compound 49 (3.88 mg, 89%. purity based on LC/MS, 16%yield) as a white solid.

Alternative procedure for the preparation of compound 49:

A stir bar, intermediate 67 (70.0 mg, 0.109 mmol) and hydrochloric acid/dioxane (2 mL, 8.0 mmol, 4 M in dioxane) were added to a 10 mL round-bottomed flask before the mixture was stirred at 25 ℃ for 1 h. The mixture was concentrated under reduced pressure to give 70 mg of crude compound 49 (HCl salt) as a white solid which was used in the next step without further purification.

Preparation of compound 50:

To a solution of intermediate 68 (40.0 mg, 0.063 mmol) in 1 mL of dioxane was added HCl/dioxane (3 mL) . After addition, the reaction mixture was stirred at 10 ℃ for 45 minutes. The reaction mixture was concentrated in vacuum and the residue was purified by preparative HPLC (Column Boston Prime C18 150 x 30mm x 5um, Mobile Phase A: water (0.04%NH ₃H ₂O+10mM NH ₄HCO ₃) , Mobile Phase B: acetonitrile, Flow rate: 25 mL/min, gradient condition from 45%B to 75%) . The pure fractions were collected, and the solvent was evaporated under vacuum. The aqueous layer was lyophilized to afford compound 50 (12 mg, 34%yield) as white solid.

Preparation of compound 76:

In a round bottom flask, at 0℃, TFA (12.7mL; 166 mmol) was added to intermediate 71 (5.18 g; 8.29 mmol) in DCM (175 mL) . Then, the reaction was warmed to room temperature and the reaction mixture was stirred overnight at room temperature. The residue was dissolved in 20 mL of water. Then, the solution was basified with a solution of NaOH 1M (70 mL) until pH=8-9. After stirring for 10 min at room temperature the resulting mixture was extracted with dichloromethane (5 x 100 mL) . The combined organic layers were washed with brine (1 x 150 mL) and dried over MgSO ₄, filtered and evaporated till dryness to give 4 g (92%yield) of compound 76.

The compounds listed in the table below were prepared following analogous reaction protocols as reported for the preparation of compounds 76 starting from the corresponding starting materials.

Preparation of compound 80:

To a solution of intermediate 76 (380 mg, 0.561 mmol) in 3 mL of DCM was added TFA (6 mL) . After addition, the reaction mixture was stirred at 27℃ for 1 hour. The reaction mixture was concentrated in vacuum to afford compound 80 (350 mg, crude, TFA salt) which was used in next step without any purification.

Preparation of compound 112:

In a vial intermediate 80 (208 mg, 0.312 mmol) was dissolved in DCM (3.00 mL, 46.9 mmol) and cooled to 0 ℃. The mixture was treated with TFA (0.478 mL, 6.25 mmol) , then the cooling bath was removed. After stirring overnight sat. sodium carbonate solution was added as well as DCM. The water phase was further basified with 1 N aqueous NaOH solution to pH 13. The water phase was extracted multiple times with DCM and then with ethyl acetate. The collected organic solvents were dried with MgSO ₄, filtered, then the solvents were removed to yield compound 112 (150 mg, 85%yield) as a white solid.

Preparation of compound 113:

In a vial intermediate 79 (224 mg, 0.336 mmol) was dissolved in DCM (3.23 mL, 50.5 mmol) and cooled to 0 ℃. The mixture was treated with TFA (0.515 mL, 6.73 mmol) . The cooling bath was removed. After stirring overnight saturated sodium carbonate solution was added as well as DCM. The water phase was further basified with 1 N aqueous NaOH solution to pH 13. The water phase was extracted multiple times with DCM and ethyl acetate. The collected organic solvents were dried with MgSO4, filtered, then the solvents were removed to yield crude compound 113 (239 mg) . 35 mg of crude compound 113 were used and purified via preparative HPLC (Stationary phase: RP XBridge Prep C18 OBD-10μm, 30x150mm, Mobile phase: 0.25%NH ₄HCO ₃ solution in water, CH ₃CN) yielding 16 mg of compound 113 as white solid.

Preparation of compound 54:

compound 55:

and compound 56

In a sealed tube, NaBH ₃CN (45.8 mg, 0.729 mmol) was added to a mixture of intermediate 29 (157 mg, 0.291 mmol) , (S) -3-methoxypyrrolidine (88.4 mg, 0.874 mmol) and acetic acid (16.7 μL, 0.291 mmol) in methanol (4 mL) . The reaction mixture was stirred at 60 ℃ for 18 h. A saturated aqueous solution of NaHCO ₃ and EtOAc were added. The layers were separated. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO ₄, filtered, concentrated and purified by silica gel chromatography (irregular SiOH 40 μm, 12 g, liquid loading (DCM) , mobile phase gradient: from DCM/ (MeOH/aq. NH ₃: 9/1) : 99/1 to 90/10) . The fractions containing product were evaporated to give 164 mg of compound 54 which was purified by reverse phase (Stationary phase: YMC-actus Triart C18 10 μm 30*150 mm, Mobile phase: Gradient: (aq. NH ₄HCO ₃ 0.2%, pH=9.5) / (MeCN/MeOH: 1/1) : from 40/60 to 10/90) . The fractions containing products were evaporated, solubilized in MeCN, extended with water and freeze-dried to give 81 mg (45%yield) of compound 55 as a white fluffy solid and 22 mg (12%yield) of compound 56 as a white fluffy solid.

Preparation of compound 57:

and compound 58:

NaBH ₃CN (47 mg; 0.75 mmol) was added to a mixture of intermediate 29 (200 mg; 0.37 mmol) , (cis) -hexahydro-1H-furo [3, 4-c] pyrrole (0.13 mL; 1.12 mmol) and AcOH (21 μL; 0.37 mmol) in THF (10 mL) and the reaction mixture was stirred at 60℃ for 18 h. The reaction mixture was cooled to RT, poured onto a 10%aqueous solution of K ₂CO ₃ and EtOAc. The organic layer was decanted, separated, dried over MgSO ₄, filtered and evaporated to dryness g. The crude (340 mg) was purified by silica gel chromatography (Stationary phase: irregular bare silica 12g, Mobile phase: Gradient from 99%DCM, 1%MeOH (+10%NH ₄OH) to 90%DCM, 10%MeOH (+10%NH ₄OH) ) . The fractions containing the product were mixed and concentrated to afford an intermediate fraction (220mg) which was purified via reverse phase (Stationary phase: YMC-actus Triart C18 15 μm 35*220 mm, Mobile phase: Gradient from 40% (aq. NH ₄HCO ₃ 0.2%pH=9.5) /MeCN/MeOH to 40/30/30 to 20/40/40) . yielding 135 mg of compound 57 which were freeze dried with acetonitrile/water 20/80 to give 120 mg (55 %yield) of compound 57 as a white powder and 40 mg of compound 58 which were freeze-dried with acetonitrile/water 20/80 to give 38 mg (16%yield) of compound 58 as a white powder.

The compounds listed in the table below were prepared following the same procedures as reported for the preparation of compounds 54, 55 and 56 starting from the corresponding starting materials.

Preparation of compound 131:

and compound 132:

To a solution of intermediate 29 (120 mg, 0.22 mmol) in methanol (2 ml) was added the cis- N, N-dimethyl-3-azabicyclo [3.1.0] hexane-6-carboxamide (41 mg, 0.27 mmol) . After stirring for 20 minutes at room temperature, sodium cyanoborohydride (28 mg, 0.47 mmol) was added to the mixture. After stirring at 50℃ overnight, the resulting mixture was quenched with a saturated sodium bicarbonate solution and extracted with dichloromethane. The combined organic layers were washed with water and brine and dried over anhydrous sodium sulfate. The solid was filtered off. The filtrate was concentrated under reduced pressure and the resulting residue was purified by preparative HPLC with the following conditions (Column: XSelect CSH Prep C18 OBD Column, 5um, 19*150mm ; Mobile Phase A: Water (10mmol/L NH ₄HCO ₃) , Mobile Phase B: ACN; Flow rate: 25 mL/min; Gradient: 30%B to 60%B in 7 min; 220 nm; retention time 1: 6.35 min; retention time 2: 6.90 min. Mixed of the pure fractions and lyophilization afforded 46.5 mg (30.6%yield, retention time 1: 6.35 min) of compound 131 as white solid and 3 mg (1.9%yield, retention time 2: 6.90 min) of compound 132 as white solid.

Preparation of compound 120:

and compound 121:

To a solution of intermediate 84 (2.5 g, 2.99 mmol, 62.8%purity) in MeOH (50 mL) was added cis-hexahydro-1H-furo [3, 4-c] pyrrole hydrochloride (1.07 g, 7.15 mmol) . After stirring at room temperature for 30 minutes, NaBH ₃CN (599 mg, 9.53 mmol) was added to the reaction mixture. The resulting reaction mixture was stirred at 50 ℃ overnight and quenched with saturated sodium bicarbonate solution and extracted with dichloromethane. The combined organic layers were washed with water and brine, dried over Na ₂SO ₄, filtered and concentrated under reduced pressure to give 800 mg of crude product as a yellow solid. The crude product was purified by preparative-HPLC (YMC-Actus Triart C18, 30 mm X 150 mm, 5um; Mobile Phase A: Water (10 mmol/L NH ₄HCO ₃+0.1%NH ₃. H ₂O) , Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 40%B to 60%B in 7 min; 254 nm; RT1: 6.95 min; RT2: 8.27 min) . The fractions containing the products were mixed. The solvents were concentrated and both compounds were freeze dried to afford to give 102.6 mg of compound 120 as a white solid and 23.1 mg of compound 121 as a white solid.

CONVERSION

Preparation of compound 23:

compound 24:

and compound 25:

At room temperature, NaBH (OAc) ₃ (144 mg; 0.68 mmol) was added to a solution of compound 22 (240 mg; 0.445 mmol) and tetrahydro-4H-pyran-4-one (48 μL; 0.53 mmol) in dichloroethane (6 mL) . The mixture was stirred at rt overnight. The solution was cooled and poured into cooled water, basified with K ₂CO ₃ powder and the product was extracted with DCM. The organic layer was dried over MgSO ₄, filtered and evaporated to dryness giving 380 mg of compound 23

Separation of the enantiomers (380 mg of compound 23) was performed via chiral SFC (Stationary phase: Chiralpak IG 5μm 250*20mm, Mobile phase: 50%CO ₂, 50%EtOH (0.3%iPrNH ₂) ) . The fractions containing the products were mixed, concentrated and freeze-dried with a mixture of acetonitrile/water (20/80) to afford 90 mf (32%yield) of compound 24 as a white powder and 98 mg (35%yield) of compound 25 as a white powder.

The compounds listed below were prepared by using analogous reaction protocols as reported for compound 23 starting from the respective starting materials

Preparation of compound 38:

Acetic acid (16 μL; 0.28 mmol) was added at room temperature to a solution of compound 32 (88 mg; 0.17 mmol) and oxetane-3-carbaldehyde (24 μL; 0.35 mmol) in THF (3 mL) . The mixture was stirred at rt for 4h. Then, NaBH (OAc) ₃ (107 mg; 0.51 mmol) was added portionwise. The mixture was stirred at room temperature for 2 hours and then, poured into ice water, basified with a 10%aqueous solution of K ₂CO ₃. EtOAc was added. The organic layer was separated, washed with brine, dried over MgSO ₄, filtered and evaporated till dryness. The resulting residue (98 mg) was purified by silica gel chromatography (Stationary phase: irregular SiOH 40 μm 4 g, Mobile phase: Gradient from 100%DCM to 80%DCM, 20%MeOH (+10%NH ₄OH) ) . The fractions containing the product were mixed and concentrated to give 60 mg of compound 38 which was further purified via reverse phase (Stationary phase: YMC-actus Triart C18 10μm 30*150mm, Mobile phase: Gradient from 40% NH ₄HCO ₃ 0.2%, 60%MeOH to 20% NH ₄HCO ₃ 0.2%, 80%MeOH) . The fractions containing the product were mixed and concentrated to give 26 mg of compound 38 which was freeze-dried with acetonitrile/water (20/80) to give 24 mg (25%yield) of compound 38 as a white powder.

Preparation of compound 39:

Acetic acid (24 μL; 0.42 mmol) was added at room temperature to a solution of compound 33 (144 mg; 0.27 mmol) and oxetane-3-carbaldehyde (38 μL; 0.55 mmol) in THF (4 mL) . The mixture was stirred at rt overnight then NaBH (OAc) ₃ (177 mg; 0.83 mmol) was added portionwise. The mixture was stirred at room temperature for 24 hours. The mixture was poured into iced water. The aqueous layer was basified with K ₂CO ₃ powder and the mixture was extracted with EtOAc (x2) . The organic layers were combined, dried over MgSO ₄ and evaporated till dryness. The residue (137 mg) was purified by silica gel chromatography (Stationary phase: irregular SiOH 15-40μm 12g, Mobile phase: Gradient from 97%DCM, 3%MeOH (+10%NH ₄OH) to 90%DCM, 10%MeOH (+10%NH ₄OH) ) . The fractions containing the product were mixed and concentrated to give 90 mg of an intermediate impure fraction which was further purified by reverse phase (Stationary phase: YMC-actus Triart C18 10μm 30*150mm, Mobile phase: Gradient from 65% NH ₄HCO ₃ 0.2%, 35%ACN to 25% NH ₄HCO ₃ 0.2%, 75%ACN) . The fraction containing the product were mixed and concentrated and the resulting residue (44 mg) was freeze-dried with Acetonitrile/water (20/80) to give 42 mg (26%yield) of compound 39.

Preparation of compound 40:

NaBH (OAc) ₃ (91 mg; 0.43 mmol) was added dropwise to a solution of compound 34 (150 mg; 0.28 mmol) and oxetane-3-carbaldehyde (21μL; 0.3 mmol) in THF (6 mL) . The reaction mixture was stirred at RT for 1.5 h. The reaction mixture was poured into ice water, basified with a solution of K ₂CO ₃ 10%and EtOAc was added. The organic layer was separated, washed with brine, dried over MgSO4, filtered and evaporated till dryness. The residue (143 mg) was purified by silica gel chromatography (Stationary phase: irregular SiOH 15-40μm 12g, Mobile phase: Gradient from 97%DCM, 3%MeOH (+10%NH ₄OH) to 85%DCM, 15%MeOH (+10%NH ₄OH) ) . The fractions containing the product were mixed, concentrated and the resulting residue (54 mg) was freeze-dried with acetonitrile/water (20/80) to give 50 mg (29%yield) of compound 40.

Preparation of compound 41:

NaBH (OAc) ₃ (82 mg; 0.39 mmol) was added dropwise to a solution of compound 35 (135 mg; 0.25 mmol) and oxetane-3-carbaldehyde (19μL; 0.27 mmol) in THF (5 mL) . The reaction mixture was stirred at RT for 1.5 h. The reaction mixture was poured into ice water, basified with a solution of K ₂CO ₃ 10%and EtOAc was added. The organic layer was separated, washed with brine, dried over MgSO ₄, filtered and evaporated till dryness. The residue (130 mg) was purified by silica gel chromatography (Stationary phase: irregular SiOH 15-40μm 12g, Mobile phase: Gradient from 95%DCM, 5%MeOH (+10%NH ₄OH) to 92%DCM, 8%MeOH (+10%NH ₄OH) ) . The fractions containing the product were mixed and concentrated to afford a fraction of 84 mg which was taken up with Et ₂O and evaporated till dryness to give 70 mg (45%yield) of compound 41.

Preparation of compound 42:

A stir bar, compound 37 (130 mg, 0.195 mmol) , oxetane-3-carbaldehyde (16.8 mg, 0.195 mmol) , triethylamine (98.7 mg, 0.975 mmol) and dry dichloromethane (4 mL) were added to a 8 mL glass bottle before the mixture was stirred at 25 ℃ for 1 h. Then, sodium cyanotrihydroborate (36.7 mg, 0.584 mmol) was added to the mixture. The resultant mixture was stirred at 25 ℃ for another 1 h. The mixture was diluted into dichloromethane (40 mL) and washed with water (20 mL x 3) . The organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure to give the crude which was purified by preparative HPLC (Column: Phenomenex Gemini-NX 150*30mm*5um, Mobile Phase A: water (0.04%NH ₃H ₂O+10mMwNH ₄HCO ₃) , Mobile Phase B: acetonitrile, Flow rate: 30 mL/min, gradient condition from 43%B to 71%B) . The pure fractions were collected, and the solvent was evaporated under vacuum to give a residue. The residue was partitioned between acetonitrile (2 mL) and water (10 mL) . The solution was lyophilized to dryness to give compound 42 (24.34 mg, 19%yield) as a white powder.

Preparation of compound 43:

and compound 44:

NaBH (OAc) ₃ (120 mg, 0.566 mmol) was added in portions to a 0 ℃ (ice/water) solution consisting of compound 37 (100 mg, crude) , oxetane-3-carbaldehyde (30.0 mg, 0.348 mmol) , Et ₃N (100 uL, 0.719 mmol) and dichloromethane (5 mL) . The resultant mixture was stirred at room-temperature of 1.5 hours. The reaction mixture was concentrated under reduced pressure to give the crude product, which was purified by preparative HPLC using a Welch Xtimate C18 150*25mm*5um (eluent: 38%to 68% (v/v) CH ₃CN and H ₂O with 0.04%NH ₃H ₂O+10mM NH ₄HCO ₃) to afford pure product which was suspended in water (10 mL) . The mixture frozen using dry ice/acetone, and then lyophilized to dryness to afford a white solid (80 mg) which was further purified by SFC (DAICEL CHIRALPAK AD-H (250mm*30mm, 5um) , isocratic elution: i-PrOH (containing 0.1%of 25%aq. NH ₃) : supercritical CO ₂, 30%: 70%to 30%: 70% (v/v) ) . The pure fractions were collected, and the volatiles were removed under reduced pressure. The product was suspended in water (10 mL) . The mixture was frozen using dry ice/acetone and then, lyophilized to dryness to afford compound 43 (37.00 mg, 41%yield) as a white solid and compound 44 (33.96 mg, 38%yield) as a light yellow solid.

The compounds listed in the table below were prepared following analogous reaction protocols as reported for the preparation of compounds 39 or 42 starting from the corresponding starting materials.

Preparation of compound 47:

To a solution of compound 45 (70.0 mg, 0.130 mmol) and oxetane-3-carbaldehyde (50 mg, 0.581 mmol) in DCM (5 mL) was added TEA (80.0 mg, 0.791 mmol) . The mixture was stirred at rt for 10 mins, then NaBH ₃CN (100 mg, 1.59 mmol) was added. The reaction mixture was stirred at rt for 1 hour and then concentrated to give a residue. The residue was purified by preparative HPLC (Column: YMC-Triart Prep C18 250 x 50mm x 10um, Mobile Phase A: water (0.04%NH ₃H ₂O+10mM NH ₄HCO ₃) , Mobile Phase B: acetonitrile, Flow rate: 25 mL/min, gradient condition: from 45%B to 75%B) to afford compound 47 (20.0 mg, 24%yield) as white solid.

Preparation of compound 48:

To a solution of compound 46 (75.0 mg, crude) , oxetane-3-carbaldehyde (35.9 mg, 0.417 mmol) and TEA (70.3 mg, 0.695 mmol) in 5 mL of DCM was added NaBH ₃CN. After addition, the reaction mixture was stirred at 10 ℃ for 2 hours. The reaction mixture was concentrated in vacuum and the residue was purified by prep-HPLC (Column Boston Prime C18 150 x 30mm x 5um, Mobile Phase A: water (0.04%NH ₃H ₂O+10mM NH ₄HCO ₃, Mobile Phase B: acetonitrile, Flow rate: 25 mL/min, gradient condition from 45%B to 75%) . The pure fractions were collected and lyophilized to afford the compound 48 (7.0 mg) as white powder.

Preparation of compound 51:

A stir bar, the compound 49 hydrochloride (70.0 mg, 0.121 mmol) , oxetane-3-carbaldehyde (15.7 mg, 0.182 mmol) , sodium cyanoborohydride (15.3 mg, 0.243 mmol) , triethylamine (61.5 mg, 0.608 mmol) and dry dichloromethane (2 mL) were added to a 10 mL round-bottomed flask before the resultant mixture was stirred at 25 ℃ for 1 h. The mixture was concentrated under reduced pressure to give the crude which was purified by preparative HPLC (Column: Boston Prime C18 150*30mm*5um, Mobile Phase A: water (0.04%NH ₃H ₂O 10mM NH ₄HCO ₃) , Mobile Phase B: acetonitrile, Flow rate: 25 mL/min, gradient condition from 40%B to 70%) . The pure fractions were collected, and the solvent was evaporated under vacuum to give a residue. The residue was partitioned between acetonitrile (2 mL) and water (10 mL) . The solution was lyophilized to dryness to give the compound 51 (14.3 mg, 19%yield) as a white powder.

Preparation of compound 52:

To a solution of the compound 50 (50.0 mg, 0.093 mmol) , oxetane-2-carbaldehyde (23.9 mg, 0.278 mmol) and TEA (18.8 mg, 0.185 mmol) in 5 mL of DCM was added NaBH ₃CN (29.1 mg, 0.463 mmol) . After addition, the reaction mixture was stirred at 10 ℃ for 1 hour. The reaction mixture was concentrated in vacuum and the residue was purified by preparative HPLC (Column Boston Prime C18 150 x 30mm x 5um, Mobile Phase A: water (0.04%NH ₃H ₂O+10mM NH ₄HCO ₃) , Mobile Phase B: acetonitrile, Flow rate: 25 mL/min, gradient condition from 45%B to 75%B) . The pure fractions were collected and lyophilized to afford the compound 52 (8.38 mg, 1 15%yield) as white solid.

Preparation of compound 53:

A stir bar, compound 36 (130 mg, 0.195 mmol) , oxetane-3-carbaldehyde (16.8 mg, 0.195 mmol) , triethylamine (98.7 mg, 0.975 mmol) and dry dichloromethane (4 mL) were added to a 8 mL glass bottle and the mixture was stirred at 25 ℃ for 1 h. Then, sodium cyanoborohydride (36.7 mg, 0.584 mmol) was added to the mixture which was stirred at 25 ℃ for another 1 h. The mixture was diluted into dichloromethane (40 mL) and washed with water (20 mL x 3) . The organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure to give the crude which was purified by preparative HPLC (Column: Phenomenex Gemini-NX 150*30mm*5um, Mobile Phase A: water (0.04%NH ₃H ₂O+10mM NH ₄HCO ₃) , Mobile Phase B: acetonitrile, Flow rate: 30 mL/min, gradient condition from 43%B to 71%) . The pure fractions were collected, and the solvent was evaporated under vacuum to give a residue which was partitioned between acetonitrile (2 mL) and water (10 mL) . The solution was lyophilized to dryness to give compound 53 (24.3 mg, 19. yield) as a white powder.

Preparation of compound 71:

Sodium triacetoxyborohydrate (50 mg; 0.24 mmol) was added at room temperature to a solution of compound 35 (60 mg; 0.11 mmol) and dihydro-3 (2H) -furanone (18 μL; 0.23 mmol) in dichloroethane (8 mL) . The mixture was stirred at rt for 2.5 h. The solution was poured into cooled water, basified with K ₂CO ₃ powder and the product was extracted with DCM. The organic layer was dried over MgSO ₄, filtered and evaporated to dryness. The crude (79 mg) was purified via Reverse phase (Stationary phase: YMC-actus Triart C18 10μm 30*150mm, Mobile phase: Gradient from 65%NH ₄HCO ₃ 0.2%, 35%ACN to 35%NH ₄HCO ₃ 0.2%, 65%ACN) . The fractions containing the product were mixed and concentrated to afford 48 mg of an intermediate fraction which was freeze-dried with acetonitrile/water (20/80) to give 40 mg (59 %yield) of compound 71 as a white powder and a mixture of two diastereoisomers.

The compounds listed in the table below were prepared accordingly to compound 71 started from the corresponding intermediates.

Preparation of compound 81:

To a solution of compound 80 (350 mg, TFA salt, 0.506 mmol) , oxetane-2-carbaldehyde (200 mg, 2.32 mmol) and TEA (500 mg, 4.94 mmol) in 50 mL of DCM was added NaBH ₃CN (200 mg, 3.18 mmol) . After addition, the reaction mixture was stirred at 28℃ for 3 hours. The reaction mixture was filtered and the filtrate was concentrated in vacuum and the residue was purified by preparative-HPLC (Column Boston Prime C18 150*30mm*5um, Mobile Phase A: water (0.04%NH ₃H ₂O+10mM NH ₄HCO ₃) , Mobile Phase B: acetonitrile, Flow rate: 25 mL/min, gradient condition from 50%B to 80%B) . The pure fractions were collected and lyophilized to afford compound 81 (170 mg, 45%yield) as white solid.

Preparation of compound 82:

and compound 83:

170 mg of compound 81 were separated by Supercritical Fluid Chromatography (separation condition: DAICEL CHIRALPAK AS-H (250mm*30mm, 5um; Mobile phase: A: Supercritical CO ₂, B: 0.1%NH ₃H ₂O-ETOH, A: B =55: 45 at 80 mL/min) to afford impure compound 82 (60 mg, 91.5%purity based on LCMS) and impure compound 83 (60 mg, 94.5%purity based on LCMS) both as white solid. The compound 82 (60 mg, 91.5%purity based on LCMS) was further purified by preparative HPLC (Column Boston Prime C18 150*30mm*5um, Mobile Phase A: water (0.04%NH ₃H ₂O+10mM NH ₄HCO ₃, Mobile Phase B: acetonitrile, Flow rate: 30 mL/min, gradient condition from 50%B to 80%B) to afford compound 82 (40.0 mg, 27%yield) as white solid. The compound 83 (60 mg, 94.5%purity based on LCMS) was further purified by preparative HPLC (Column Boston Prime C18 150*30mm*5um, Mobile Phase A: water (0.04%NH ₃H ₂O+10mM NH ₄HCO ₃, Mobile Phase B: acetonitrile, Flow rate: 30 mL/min, gradient condition from 50%B to 80%B) to afford compound 83 as white solid.

The compounds listed in the table below were prepared following analogous reaction protocols as reported for the preparation of compounds 81, 82 and 83 starting from the corresponding starting materials. Someone skilled in the art, will realize that in some cases, additional deprotection steps might be required to get the final compounds.

Preparation of compound 114:

In a flask compound 113 (59 mg, 0.104 mmol) was dissolved in methanol (1.27 mL, 31.3 mmol) and treated with oxetane-3-carbaldehyde (35.9 mg, 0.417 mmol) , sodium cyanoborohydride (32.8 mg, 0.521 mmol) and 2 drops of AcOH. The mixture was stirred over night at 60 ℃. The solvent was evaporated and then, a saturated sodium carbonate solution was added along with DCM. Then, the water phase was further basified with 1 N aqueous NaOH solution until pH 13. The water phase was extracted multiple times with DCM and ethyl acetate. Drying with magnesium sulfate, filtration and evaporation of solvents afforded the crude material that was purified. The purification was performed via preparative HPLC (Stationary phase: RP XBridge Prep C18 OBD-10μm, 30x150mm, Mobile phase: 0.25%NH ₄HCO ₃ solution in water, CH ₃CN) yielding 42 mg (63%yield) of compound 114 as a white solid.

Preparation of compound 115:

In a flask, compound 112 (50 mg, 0.0884 mmol) was dissolved in MeOH (1.07 mL, 26.5 mmol) and treated with formaldehyde, 37%aqueous. solution (0.132 mL, 1.77 mmol) , 2 drops of HOAc, and, then with sodium cyanoborohydride (27.8 mg, 0.442 mmol) . The mixture was heated over 2 h at 60 ℃. The solvent was evaporated. Then, a saturated. sodium carbonate solution was added along with DCM. The water phase was basified with 1 N aqueous NaOH solution to pH 13. The water phase was extracted multiple times with DCM and ethyl acetate. Drying with magnesium sulfate, filtration and evaporation of solvents afforded crude product (60 mg) . A purification was performed via preparative HPLC (Stationary phase: RP XBridge Prep C18 OBD-10μm, 30x150mm, Mobile phase: 0.25%NH ₄HCO ₃ solution in water, CH ₃CN) affording of compound 115 as a white solid.

The compounds listed below were prepared following an analogous reaction protocol as reported for the preparation of compounds 114 and 115

Preparation of compound 116:

In a flask, compound 112 (45 mg, 0.0795 mmol) was dissolved in dry DMF (1.23 mL, 15.9 mmol) and treated with DIPEA (0.0411 mL, 0.239 mmol) and bromomethoxyethane (12.2 mg, 0.0875 mmol) . The reaction was stirred for 3 h at 80 ℃. A saturated sodium carbonate solution was added along with DCM. Then, the water phase was basified with 1 N aqueous NaOH solution to pH 13. The water phase was extracted multiple times with DCM and ethyl acetate. Drying with magnesium sulfate, filtration and evaporation of solvents afforded the crude product. A purification was performed via preparative HPLC (Stationary phase: RP XBridge Prep C18 OBD-10μm, 30x150mm, Mobile phase: 0.25%NH ₄HCO ₃ solution in water, CH ₃CN) affording 19 mg (yield 38%) of compound 116 as a white solid.

Preparation of compound 119:

To a solution of compound 37 (200 mg, 0.36 mmol) in ACN (5 mL) were added 2-bromo-N, 2-dimethylpropanamide (98 mg, 0.54 mmol) and K ₂CO ₃ (250 mg, 1.81 mmol) . After stirring at 70 ℃ overnight, the reaction mixture was quenched with a saturated solution sodium bicarbonate and extracted with ethyl acetate. The combined organic layers were washed with water and brine and dried over Na ₂SO ₄, filtered and evaporated under reduced pressure. The residue was purified by preparative-TLC (MeOH/DCM, 1: 10) . The obtained crude product (200 mg;white solid) was purified by preparative -HPLC (Column: XBridge Shield RP18 OBD Column, 19*250mm, 10um; Mobile Phase A: Water (10 mmol/L NH ₄HCO ₃) , Mobile Phase B: ACN; Flow rate: 25 mL/min; Gradient: 55%B to 65%B in 7 min; 254/220 nm; RT: 5.93 min) . The fractions containing the product were mixed and concentrated to afford 40.4 mg (16%yield) of compound 119 as a white solid.

Preparation of compound 133:

To a mixture of intermediate 88 (100 mg, 0.157 mmol) in DCM (3 mL) was added TFA (1 mL) at room temperature. The mixture was stirred at room temperature for 0.5 h. The reaction mixture was evaporated under reduce pressure. The residue was diluted by 2M NaOH (5 mL) , extracted with DCM (5 mL x 5) . The combined organic layers was dried over anhydrous Na ₂SO ₄, filtered, and concentrated to dryness under reduce pressure to afford compound 133 (84 mg, 99.6%yield) as a yellow solid.

Preparation of compound 134:

To a mixture of compound 133 (84 mg, 0.156 mmol) in MeOH (2 mL) was added formaldehyde (257 mg, 3.17 mmol, 37%in water) and acetic acid (20 mg, 0.333 mmol) . The mixture was stirred at room temperature for 30 minutes. Then NaBH ₃CN (20 mg, 0.318 mmol) was added to the mixture and the resultant mixture was stirred at room temperature for 1 h. The reaction mixture was evaporated to remove solvent. The residue was diluted by 2M NaOH (5 mL) , extracted with DCM (10 mL x 3) . The combined organic layers were dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure to give the crude, which was purified by preparative HPLC (Column: Welch Xtimate C18 150*30mm*5μm, Mobile Phase A: water (0.05%NH ₃H ₂O+10mM NH ₄HCO ₃) , Mobile Phase B: acetonitrile, Flow rate: 30 mL/min, gradient condition from 55%B to 85%) to afford two fractions. The pure desired fraction were collected and the volatile solvent was removed by evaporation. The aqueous residue was lyophilized to afford compound 134 (40 mg, 99.52%purity, yield 45%) as white powder. The impure desired fraction were collected and the volatile solvent was removed by evaporation. The aqueous residue was lyophilized to afford compound 134 (12 mg, yield 14%, ～95%purity by NMR) as white powder.

Preparation of compound 135:

and compound 136

Compound 134 (40 mg, 0.071 mmol) was purified by SFC (column: DAICEL CHIRALCEL OD-H (250mm*30mm, 5um) , Mobile phase: A: Supercritical CO ₂, B: 0.1%NH ₃H ₂O IPA; Isocratic: A: B = 75: 25; Flow rate: 80 mL/min) to afford two fractions. The pure fractions of first peak were collected and the volatile solvent was evaporated under vacuum. The residue was treated with H ₂O (3 mL) and CH ₃CN (1 mL) . The mixture was lyophilized to give compound 135 (11 mg, 98.17%purity, yield 27%) as white powder. The pure fractions of second peak were collected and the volatile solvent was evaporated under vacuum. The residue was treated with H ₂O (3 mL) and CH ₃CN (1 mL) . The mixture was lyophilized to give compound 136 (10 mg, 96.28 purity, yield 24%) as a white powder.

Preparation of compound 137:

To a solution of compound 76 (200 mg, crude) and 3- (dimethylamino) propanoic acid hydrochloride (49.0 mg, 0.32 mmol) in DCM (10 mL) was added HATU (121 mg, 0.32 mmol) and DIEA (0.21 mL, 1.26 mmol) . The mixture was stirred for 16 hours at rt. 20 mL DCM and 20 mL H ₂O was added to the mixture solution. The mixture was extracted with DCM (30 mL x 2) , the combined extracts were washed with brine (30 mL) and dried over Na ₂SO ₄, the mixture was filtered and the filtrate was concentrated in vacuum. The residue was purified by pre-HPLC (Column: YMC-Triart Prep C18 250*50mm*10um, Mobile Phase A: water (0.04%NH ₃H ₂O+10mM NH ₄HCO ₃) , Mobile Phase B: acetonitrile, Flow rate: 25 mL/min, gradient condition from 45%B to 75%) . The pure fractions were collected and the solvent was lyophilized to give the title compound compound 137 (20 mg, 96.7%purity, 12%yield) as a light yellow solid.

Preparation of compound 138:

To a solution of intermediate 89 (150 mg, 0.23 mmol) in dichloromethane (5.0 mL) were added Trifluoroacetic acid (1.7 mL) at 0 ℃. The resulting mixture was stirred at room temperature for 3 hours. The resulting mixture was concentrated under reduced pressure to give 150 mg of compound 138 (97.5%purity, as trifluoroacetate) as a colorless oil.

Preparation of compound 139:

To a mixture of compound 138 (150 mg, 0.241 mmol, purity 86.63%) , glycolic acid (22 mg, 0.289 mmol) and N, N-diisopropylethylamine (0.12 mL, 0.722 mmol) in N, N-dimethylformamide (2 mL) was added HATU (110 mg, 0.289 mmol) in portions at 0℃ and stirred for 2 hours at room temperature. The reaction mixture was quenched by the addition of water (5 mL) and extracted with ethyl acetate (4 x 5 mL) . The combined organic layers were washed with water (3 x 20 mL) , brine (20 mL) and dried over anhydrous sodium sulfate. Filtration, concentration and the residue was pirified by reverse flash chromatography with the following conditions: Column: SunFire C18 OBD Prep Column, 19 mm X 250 mm; Mobile Phase A: Water (0.1%NH ₄HCO ₃) , Mobile Phase B: ACN; Flow rate: 20 mL/min; Gradient: 15%B to 40%B in 11 min; 254/220 nm; Rt: 9.12 min to afford 46.9 mg of compound 139 as a white solid.

LCMS (Liquid chromatography/Mass spectrometry)

General procedure

The High-Performance Liquid Chromatography (HPLC) measurement was performed using a LC pump, a diode-array (DAD) or a UV detector and a column as specified in the respective methods. If necessary, additional detectors were included (see table of methods below) .

Flow from the column was brought to the Mass Spectrometer (MS) which was configured with an atmospheric pressure ion source. It is within the knowledge of the skilled person to set the tune parameters (e.g. scanning range, dwell time…) in order to obtain ions allowing the identification of the compound’s nominal monoisotopic molecular weight (MW) . Data acquisition was performed with appropriate software.

Compounds are described by their experimental retention times (R _t) and ions. If not specified differently in the table of data, the reported molecular ion corresponds to the [M+H] ⁺ (protonated molecule) and/or [M-H] ^- (deprotonated molecule) . In case the compound was not directly ionizable the type of adduct is specified (i.e. [M+NH ₄] ⁺, [M+HCOO] ^-, etc…) . For molecules with multiple isotopic patterns (Br, Cl.. ) , the reported value is the one obtained for the lowest isotope mass. All results were obtained with experimental uncertainties that are commonly associated with the method used.

Hereinafter, “SQD” means Single Quadrupole Detector, “RT” room temperature, “BEH” bridged ethylsiloxane/silica hybrid, “HSS” High Strength Silica, “DAD” Diode Array Detector.

Table 1a: LCMS Method codes (Flow expressed in mL/min; column temperature (T) in ℃; Run time in minutes) . “TFA” means trifluoroacetic acid

Table 1b: LCMS and melting point data. Co. No. means compound number; R _t means retention time in min.

SFC-Methods

General procedure for SFC methods

The SFC measurement was performed using an Analytical Supercritical fluid chromatography (SFC) system composed by a binary pump for delivering carbon dioxide (CO ₂) and modifier, an autosampler, a column oven, a diode array detector equipped with a high-pressure flow cell standing up to 400 bars. If configured with a Mass Spectrometer (MS) the flow from the column was brought to the (MS) . It is within the knowledge of the skilled person to set the tune parameters (e.g. scanning range, dwell time…) in order to obtain ions allowing the identification of the compound’s nominal monoisotopic molecular weight (MW) . Data acquisition was performed with appropriate software.

Table 2a. Analytical SFC Methods (Flow expressed in mL/min; column temperature (T) in ℃; Run time in minutes, Backpressure (BPR) , “DEA” means diethylamine.

Table 2b. SFC data.

OPTICAL ROTATION (OR)

Optical Rotation is measured with a polarimeter 341 Perkin Elmer. The polarized light is passed through a sample with a path length of 1 decimeter and a sample concentration of 0.2 to 0.4 gram per 100 milliliters. 2 to 4 mg of the product in vial are weight, then dissolved with 1 to 1.2 ml of spectroscopy solvent (DMF for example) . The cell is filled with the solution and put into the polarimeter at a temperature of 20 ℃. The OR is read with 0.004° of precision.

Calculation of the concentration: weight in gram x 100/volume in ml

[α] _d ²⁰ : (read rotation x 100) / (1.000 dm x concentration) .

^dis sodium D line (589 nanometer) .

Table 3. OR data: wavelength: 589 nm (specicied if different) ; solvent: DMF (specicied if different) ; temperature: 20 ℃; ‘conc’ means concentration (g/100 mL) ; ‘OR’ means optical rotation.

Co. No.	OR (°)	Conc.
3	-15.14	0.284
4	+8.3	0.265
7	-10.38	0.26
8	+12.6	0.262
24	+15.72	0.318
25	-16.59	0.416
27	+14.22	0.228
29	-18.04	0.316
30	-16.67	0.228
40	-12.5	0.26
41	+13.67	0.256
55	+12.75	0.251
56	-4.43	0.271
62	+10.4	0.25

NMR

Some NMR experiments were carried out using a Bruker Avance 500 spectrometer equipped with a Bruker 5mm BBFO probe head with z gradients and operating at 500 MHz for the proton and 125 MHz for carbon. Chemical shifts (d) are reported in parts per million (ppm) . J values are expressed in Hz.

NMR experiments were carried out using a Bruker Avance III 400 spectrometer, using internal deuterium lock and equipped with reverse double-resonance ( ¹H, ¹³C, SEI) probe head with z gradients and operating at 400 MHz for the proton and 100MHz for carbon. Chemical shifts (d) are reported in parts per million (ppm) . J values are expressed in Hz.

Some NMR experiments were carried out using a Bruker Avance III 400 spectrometer at ambient temperature (298.6 K) , using internal deuterium lock and equipped with BBO 400MHz S1 5 mm probe head with z gradients and operating at 400 MHz for the proton and 100MHz for carbon. Chemical shifts (d) are reported in parts per million (ppm) . J values are expressed in Hz.

Some NMR experiments were carried out using a Varian 400-MR spectrometer at ambient temperature (298.6 K) , using internal deuterium lock and equipped with Varian 400 4NUC PFG probe head with z gradients and operating at 400 MHz for the proton and 100MHz for carbon. Chemical shifts (d) are reported in parts per million (ppm) . J values are expressed in Hz.

Some NMR experiments were carried out using a Varian 400-VNMRS spectrometer at ambient temperature (298.6 K) , using internal deuterium lock and equipped with Varian 400 ASW PFG probe head with z gradients and operating at 400 MHz for the proton and 100MHz for carbon. Chemical shifts (d) are reported in parts per million (ppm) . J values are expressed in Hz.

Compound 4

Major rotamer (75%)

¹H NMR (400 MHz, DMSO-d ₆) δ ppm 8.30 (s, 1H) , 7.69 -7.81 (m, 1H) , 7.15 -7.38 (m, 2H) , 6.88 -7.02 (m, 1H) , 4.55 (dd, J=7.7, 5.9 Hz, 2H) , 4.20 (t, J=5.5 Hz, 2H) , 3.73 -3.84 (m, 1H) , 3.37 -3.70 (m, 5H) , 3.12 -3.27 (m, 3H) , 3.05 (br d, J=6.2 Hz, 1H) , 2.92 -3.02 (m, 3H) , 2.86 (dt, J=14.1, 7.1 Hz, 1H) , 2.73 (br t, J=7.2 Hz, 1H) , 2.60 (br dd, J=8.7, 6.4 Hz, 1H) , 2.54 (br d, J=7.6 Hz, 2H) , 2.23 -2.36 (m, 1H) , 2.12 (br d, J=9.4 Hz, 1H) , 1.95 (br t, J=7.0 Hz, 2H) , 1.45 -1.59 (m, 1H) , 1.21 (br d, J=3.4 Hz, 2H) , 0.98 -1.15 (m, 7H) , 0.72 -0.80 (m, 6H)

Minor rotamer (25%)

¹H NMR (400 MHz, DMSO-d ₆) δ ppm 8.28 (s, 1H) , 7.69 -7.81 (m, 1H) , 7.15 -7.38 (m, 2H) , 6.88 -7.02 (m, 1H) , 4.55 (dd, J=7.7, 5.9 Hz, 2H) , 4.41 (dt, J=13.8, 6.8 Hz, 1H) , 4.20 (t, J=5.5 Hz, 2H) , 3.37 -3.70 (m, 5H) , 3.12 -3.27 (m, 3H) , 3.05 (br d, J=6.2 Hz, 1H) , 2.92 -3.02 (m, 3H) , 2.86 (dt, J=14.1, 7.1 Hz, 1H) , 2.73 (br t, J=7.2 Hz, 1H) , 2.60 (br dd, J=8.7, 6.4 Hz, 1H) , 2.54 (br d, J=7.6 Hz, 2H) , 2.23 -2.36 (m, 1H) , 2.12 (br d, J=9.4 Hz, 1H) , 1.95 (br t, J=7.0 Hz, 2H) , 1.45 -1.59 (m, 1H) , 1.21 (br d, J=3.4 Hz, 2H) , 0.98 -1.15 (m, 7H) , 0.72 -0.80 (m, 6H)

Compound 38

¹H NMR (500 MHz, DMSO-d ₆) δ ppm 8.4 (d, J=5.7 Hz, 1H) , 7.2 -7.4 (m, 3H) , 6.5 (br d, J=5.7 Hz, 1H) , 4.6 (dd, J=7.7, 5.8 Hz, 2H) , 4.2 (td, J=6.0, 2.2 Hz, 2H) , 3.3 -3.8 (m, 7H) , 3.0 -3.1 (m, 5H) , 2.9 (dt, J=14.3, 6.9 Hz, 1H) , 2.7 -2.8 (m, 1H) , 2.5 -2.6 (m, 3H) , 2.3 -2.4 (m, 1H) , 2.2 (dd, J=9.5, 1.9 Hz, 1H) , 2.0 (br t, J=6.8 Hz, 2H) , 1.5 -1.6 (m, 1H) , 1.0 (br d, J=6.3 Hz, 4H) , 0.9 -1.0 (m, 4H) , 0.8 (dd, J=12.9, 6.9 Hz, 6H) , 0.6 (br s, 2H)

Compound 41

¹H NMR (500 MHz, DMSO-d ₆) δ ppm 8.48 (s, 1H) , 7.22 -7.52 (m, 3H) , 4.56 (br t, J=6.8 Hz, 2H) , 4.17 -4.30 (m, 2H) , 3.87 -4.13 (m, 2H) , 3.49 -3.72 (m, 3H) , 3.30 -3.41 (m, 1H) , 2.97 -3.20 (m, 6H) , 2.88 (dt, J=14.3, 6.9 Hz, 1H) , 2.77 (br d, J=1.3 Hz, 1H) , 2.55 -2.69 (m, 2H) , 2.30 -2.37 (m, 1H) , 2.17 (br d, J=7.6 Hz, 1H) , 1.96 -2.10 (m, 2H) , 1.51 -1.66 (m, 1H) , 0.92 -1.15 (m, 8H) , 0.65 -0.84 (m, 9 H)

Compound 42

¹H NMR (400 MHz, CDCl ₃) δ ppm 8.43 -8.36 (m, 1H) , 7.23 -7.16 (m, 1H) , 7.13 -7.06 (m, 1H) , 7.05 -6.98 (m, 1H) , 6.33 -6.28 (m, 1H) , 4.83 -4.76 (m, 2H) , 4.44 -4.34 (m, 2H) , 4.01 -3.89 (m, 1H) , 3.75 -3.56 (m, 4H) , 3.55 -3.47 (m, 1H) , 3.27 -3.05 (m, 6H) , 2.82 -2.72 (m, 2H) , 2.66 (d, J = 7.2 Hz, 2H) , 2.14 -2.06 (m, 2H) , 1.93 -1.72 (m, 4H) , 1.50 -0.98 (m, 12H) , 0.93 -0.84 (m, 6H) , 0.71 (d, J = 6.4 Hz, 2H) .

Compound 43

¹H NMR (400 MHz, CDCl ₃) δ ppm 8.50 (s, 1H) , 7.26 -7.19 (m, 1H) , 7.18 -7.09 (m, 1H) , 7.08 -6.97 (m, 1H) , 4.89 -4.73 (m, 2H) , 4.49 -4.34 (m, 2H) , 4.33 -4.01 (m, 2H) , 4.00 -3.83 (m, 1H) , 3.81 -3.60 (m, 2H) , 3.59 -3.38 (m, 1H) , 3.31 -3.03 (m, 6H) , 2.86 -2.61 (m, 4H) , 2.21 -2.07 (m, 2H) , 1.96 -1.71 (m, 5H) , 1.56 -1.33 (m, 5H) , 1.15 -1.05 (m, 6H) , 0.93 -0.84 (m, 6H) , 0.82 -0.72 (m, 2H)

Compound 44

¹H NMR (400 MHz, CDCl ₃) δ ppm 8.53 -8.44 (m, 1H) , 7.27 -7.18 (m, 1H) , 7.18 -7.09 (m, 1H) , 7.08 -6.97 (m, 1H) , 4.81 (t, J = 6.8 Hz, 2H) , 4.51 -4.35 (m, 2H) , 4.30 -3.84 (m, 3H) , 3.79 -3.55 (m, 2H) , 3.54 -3.41 (m, 1H) , 3.38 -3.06 (m, 6H) , 3.01 -2.62 (m, 4H) , 2.21 -2.11 (m, 2H) , 2.11 -1.64 (m, 5H) , 1.64 -1.29 (m, 5H) , 1.14 -1.03 (m, 6H) , 0.95 -0.85 (m, 6H) , 0.82 -0.70 (m, 2H) .

Compound 45

¹H NMR (400 MHz, CD ₃OD) δ ppm 8.87 -8.69 (m, 1H) , 7.67 -7.42 (m, 1H) , 7.39 -7.18 (m, 2H) , 4.66 -4.15 (m, 6H) , 4.12 -3.76 (m, 2H) , 3.74 -3.53 (m, 4H) , 3.52 -3.31 (m, 3H) , 3.26 -3.15 (m, 1H) , 3.08 -2.94 (m, 1H) , 2.79 -2.23 (m, 3H) , 2.21 -2.09 (m, 1H) , 2.00 -1.85 (m, 1H) , 1.31 -0.86 (m, 15H)

Compound 46

¹H NMR (400 MHz, CD ₃OD) δ ppm 8.95 -8.76 (m, 1H) , 7.74 -7.41 (m, 1H) , 7.39 -7.22 (m, 2H) , 4.69 -4.09 (m, 6H) , 4.06 -3.78 (m, 2H) , 3.76 -3.50 (m, 4H) , 3.50 -3.32 (m, 3H) , 3.26 -3.10 (m, 2H) , 2.80 -2.27 (m, 3H) , 2.25 -2.09 (m, 1H) , 1.93 -1.73 (m, 1H) , 1.33 -0.96 (m, 15H) .

Compound 47

¹H NMR (400 MHz, CD ₃OD) δ ppm 8.50 (s, 1H) , 7.19-7.25 (m, 1H) , 7.13 (s, 1H) , 6.98-7.07 (m, 1H) , 4.78 (t, J = 6.8 Hz, 2H) , 4.37-4.45 (m, 2H) , 3.95-4.25 (m, 2H) , 3.82-3.93 (m, 1H) , 3.73 (s, 1H) , 3.44 -3.68 (m, 2H) , 3.20 (d, J = 9.2 Hz, 6H) , 2.62-2.82 (m, 4H) , 2.27 (d, J = 6.4 Hz, 2H) , 2.06-2.19 (m, 3H) , 2.03 (s, 1H) , 1.89 (s, 1H) , 1.67-1.81 (m, 2H) , 1.01-1.22 (m, 7H) , 0.72-0.91 (m, 8H) .

Compound 48

¹H NMR (400 MHz, CDCl ₃) δ ppm 8.50 (br. s, 1H) , 7.18-7.25 (m, 1H) , 7.08-7.17 (m, 1H) , 6.94-7.07 (m, 1H) , 4.69-4.87 (m, 2H) , 4.37-4.49 (m, 2H) , 3.97-4.34 (m, 2H) , 3.40-3.92 (m, 4H) , 3.03-3.32 (m, 6H) , 2.52-2.85 (m, 4H) , 2.19-2.45 (m, 3H) , 2.03-2.17 (m, 3H) , 1.83-1.95 (m, 1H) , 1.70-1.79 (m, 1H) , 1.48-1.58 (m, 1H) , 1.00-1.33 (m, 7H) , 0.68-0.96 (m, 8H) .

Compound 49

¹H NMR (400 MHz, CDCl ₃) δ ppm 8.48 (s, 1H) , 7.52 -7.40 (m, 1H) , 7.39 -7.31 (m, 2H) , 4.30 -3.86 (m, 2H) , 3.67 -3.59 (m, 2H) , 3.27 -2.97 (m, 9H) , 2.94 -2.75 (m, 2H) , 2.24 -1.82 (m, 5H) , 1.77 -1.62 (m, 2H) , 1.11 -0.57 (m, 16H)

Compound 50

¹H NMR (400 MHz, DMSO-d ₆) δ ppm 8.48 (s, 1H) , 7.40-7.48 (m, 1H) , 7.29-7.39 (m, 2H) , 3.47-4.31 (m, 9H) , 3.24-3.45 (m, 3H) , 2.92-3.12 (m, 3H) , 1.93-2.22 (m, 4H) , 1.50-1.92 (m, 3H) , 0.91-1.14 (m, 7H) , 0.88 (d, J = 6.80 Hz, 3H) , 0.83 (d, J = 6.40 Hz, 3H) , 0.61-0.79 (m, 2H) .

Compound 51

¹H NMR (400 MHz, CDCl ₃) δ ppm 8.50 (s, 1H) , 7.26 -7.23 (m, 1H) , 7.17 -7.10 (m, 1H) , 7.07 -6.99 (m, 1H) , 4.84 -4.75 (m, 2H) , 4.46 -4.38 (m, 2H) , 4.34 -4.26 (m, 0.2H) , 4.22 -3.99 (m, 2H) , 3.92 -3.82 (m, 0.8H) , 3.80 -3.71 (m, 1H) , 3.68 -3.60 (m, 1H) , 3.56 -3.45 (m, 1H) , 3.30 -3.14 (m, 5H) , 3.13 -3.04 (m, 1H) , 2.82 -2.63 (m, 4H) , 2.34 -2.23 (m, 2H) , 2.16 -2.10 (m, 2H) , 2.06 -2.02 (m, 1H) , 1.97 -1.85 (m, 1H) , 1.80 -1.67 (m, 3H) , 1.12 -1.02 (m, 6H) , 0.91 -0.72 (m, 9H) .

Compound 52

¹H NMR (400 MHz, CDCl ₃) δ ppm 8.50 (s, 1H) , 7.19-7.25 (m, 1H) , 7.08-7.18 (m, 1H) , 6.96-7.08 (m, 1H) , 4.71-4.89 (m, 2H) , 4.38-4.48 (m, 2H) , 3.44-4.34 (m, 6H) , 3.01-3.32 (m, 6H) , 2.50-2.89 (m, 3H) , 2.19-2.47 (m, 3H) , 2.04-2.18 (m, 3H) , 1.83-1.95 (m, 1H) , 1.70-1.77 (m, 1H) , 1.62-1.70 (m, 1H) , 1.50-1.60 (m, 1H) , 1.01-1.27 (m, 7H) , 0.72-0.92 (m, 8H) .

Compound 78

¹H NMR CD ₃OD (Varian-400 MHz) : 9.00 -8.78 (m, 1H) , 7.69 -7.44 (m, 1H) , 7.41 -7.19 (m, 2H) , 4.56 -4.13 (m, 6H) , 4.05 -3.78 (m, 2H) , 3.58 (s, 1H) , 3.49 -3.32 (m, 5H) , 3.13 -2.97 (m, 2H) , 2.72 -2.51 (m, 1H) , 2.49 -2.31 (m, 1H) , 2.23 -1.91 (m, 4H) , 1.89 -1.65 (m, 2H) , 1.29 -0.93 (m, 15H) .

Compound 79

¹H NMR CD ₃OD (Varian-400 MHz) : 8.97 -8.81 (m, 1H) , 7.72 -7.43 (m, 1H) , 7.40 -7.15 (m, 2H) , 4.61 -4.15 (m, 6H) , 4.08 -3.70 (m, 2H) , 3.58 (s, 1H) , 3.50 -3.34 (m, 5H) , 3.14 -2.94 (m, 2H) , 2.73 -2.52 (m, 1H) , 2.50 -2.30 (m, 1H) , 2.22 -1.89 (m, 4H) , 1.89 -1.64 (m, 2H) , 1.29 -1.02 (m, 15H)

Compound 82:

¹H NMR (400 MHz, CDCl ₃) : 8.55 -8.41 (m, 1H) , 7.25 -7.18 (m, 1H) , 7.17 -7.07 (m, 1H) , 7.06 -6.95 (m, 1H) , 4.78 (t, J = 6.8 Hz, 2H) , 4.39 (t, J = 6.0 Hz, 2H) , 4.31 -3.95 (m, 2H) , 3.93 -3.81 (m, 1H) , 3.78 -3.42 (m, 3H) , 3.39 -2.99 (m, 5H) , 2.85 -2.57 (m, 4H) , 2.49 -2.34 (m, 1H) , 2.21 -2.00 (m, 3H) , 1.91 -1.70 (m, 13H) , 1.49 -1.38 (m, 1H) , 1.35 -1.22 (m, 1H) , 1.18 -0.69 (m, 8H) .

Compound 83:

¹H NMR (400 MHz, CDCl ₃) : 8.54 -8.39 (m, 1H) , 7.25 -7.19 (m, 1H) , 7.13 (s, 1H) , 7.06 -6.97 (m, 1H) , 4.78 (t, J = 6.8 Hz, 2H) , 4.39 (t, J = 6.2 Hz, 2H) , 4.32 -3.95 (m, 2H) , 3.93 -3.00 (m, 8H) , 2.87 -2.57 (m, 4H) , 2.40 (s, 1H) , 2.24 -1.98 (m, 3H) , 1.91 -1.68 (m, 14H) , 1.50 -1.38 (m, 1H) , 1.36 -1.22 (m, 1H) , 1.18 -0.67 (m, 8H) .

PHARMACOLOGICAL PART

1) Menin/MLL homogenous time-resolved fluorescence (HTRF) assay

To an untreated, white 384-well microtiter plate was added 40 nL 200X test compound in DMSO and 4 μL 2X terbium chelate-labeled menin (vide infra for preparation) in assay buffer (40 mM Tris·HCl, pH 7.5, 50 mM NaCl, 1 mM DTT (dithiothreitol) and 0.05%Pluronic F-127) . After incubation of test compound and terbium chelate-labeled menin for 30 min at ambient temperature, 4 μL 2X FITC-MBM1 peptide (FITC-β-alanine-SARWRFPARPGT-NH ₂) ( “FITC” means fluorescein isothiocyanate) in assay buffer was added, the microtiter plate centrifuged at 1000 rpm for 1 min and the assay mixtures incubated for 15 min at ambient temperature. The relative amount of menin·FITC-MBM1 complex present in an assay mixture is determined by measuring the homogenous time-resolved fluorescence (HTRF) of the terbium/FITC donor /acceptor fluorphore pair using an EnVision microplate reader (ex. 337 nm/terbium em. 490 nm/FITC em. 520 nm) at ambient temperature. The degree of fluorescence resonance energy transfer (the HTRF value) is expressed as the ratio of the fluorescence emission intensities of the FITC and terbium fluorophores (F ^em 520 nm/F ^em 490 nm) . The final concentrations of reagents in the binding assay are 200 pM terbium chelate-labeled menin, 75 nM FITC-MBM1 peptide and 0.5%DMSO in assay buffer. Dose-response titrations of test compounds are conducted using an 11 point, four-fold serial dilution scheme, starting typically at 10 μM.

Compound potencies were determined by first calculating %inhibition at each compound concentration according to equation 1:

%inhibition = ( (HC -LC) - (HTRF ^compound -LC) ) / (HC -LC) ) *100 (Eqn 1)

Where LC and HC are the HTRF values of the assay in the presence or absence of a saturating concentration of a compound that competes with FITC-MBM1 for binding to menin, and HTRF ^compound is the measured HTRF value in the presence of the test compound. HC and LC HTRF values represent an average of at least 10 replicates per plate. For each test compound, %inhibition values were plotted vs. the logarithm of the test compound concentration, and the IC ₅₀ value derived from fitting these data to equation 2:

%inhibition = Bottom + (Top-Bottom) / (1+10^ ( (logIC ₅₀-log [cmpd] ) *h) ) (Eqn 2)

Where Bottom and Top are the lower and upper asymptotes of the dose-response curve, respectively, IC ₅₀ is the concentration of compound that yields 50%inhibition of signal and h is the Hill coefficient.

Preparation of Terbium cryptate labeling of Menin: Menin (a. a1-610-6xhis tag, 2.3 mg/mL in 20mM Hepes (2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethane sulfonic acid) , 80 mM NaCl, 5mM DTT (Dithiothreitol) , pH 7.5) was labeled with terbium cryptate as follows. 200 μg of Menin was buffer exchanged into 1x Hepes buffer. 6.67 μM Menin was incubated with 8-fold molar excess NHS (N-hydroxysuccinimide) -terbium cryptate for 40 minutes at room temperature. Half of the labeled protein was purified away from free label by running the reaction over a NAP5 column with elution buffer (0.1M Hepes, pH 7 + 0.1%BSA (bovine serum albumin) ) . The other half was eluted with 0.1M phosphate buffered saline (PBS) , pH7.400 μl of eluent was collected for each, aliquoted and frozen at -80℃. The final concentration of terbium-labeled Menin protein was 115 μg/mL in Hepes buffer and 85 μg/mL in PBS buffer, respectively.

MENIN Protein Sequence (SEQ ID NO: 1) :

2a) Proliferation assay

The anti-proliferative effect of menin/MLL protein/protein interaction inhibitor test compounds was assessed in human leukemia cell lines. The cell line MOLM14 harbors a MLL translocation and expresses the MLL fusion protein MLL-AF9, respectively, as well as the wildtype protein from the second allele. OCI-AML3 cells that carry the NPM1c gene mutation were also tested. MLL rearranged cell lines (e.g. MOLM14) and NPM1c mutated cell lines exhibit stem cell-like HOXA/MEIS1 gene expression signatures. KO-52 was used as a control cell line containing two MLL (KMT2A) wildtype alleles in order to exclude compounds that display general cytotoxic effects.

MOLM14 cells were cultured in RPMI-1640 (Sigma Aldrich) supplemented with 10%heat-inactivated fetal bovine serum (HyClone) , 2 mM L-glutamine (Sigma Aldrich) and 50μg/ml gentamycin (Gibco) . KO-52 and OCI-AML3 cell lines were propagated in alpha-MEM (Sigma Aldrich) supplemented with 20%heat-inactivated fetal bovine serum (HyClone) , 2 mM L-glutamine (Sigma Aldrich) and 50μg/ml gentamycin (Gibco) . Cells were kept at 0.3 –2.5 million cells per ml during culturing and passage numbers did not exceed 20.

In order to assess the anti-proliferative effects, 200 MOLM14 cells, 200 OCI-AML3 cells or 300 KO-52 cells were seeded in 200μl media per well in 96-well round bottom, ultra-low attachment plates (Costar, catalogue number 7007) . Cell seeding numbers were chosen based on growth curves to ensure linear growth throughout the experiment. Test compounds were added at different concentrations and the DMSO content was normalized to 0.3%. Cells were incubated for 8 days at 37℃ and 5%CO ₂. Spheroid like growth was measured in real-time by live-cell imaging (IncuCyteZOOM, Essenbio, 4x objective) acquiring images at day 8. Confluence (%) as a measure of spheroid size was determined using an integrated analysis tool.

In order to determine the effect of the test compounds over time, the confluence in each well as a measure of spheroid size, was calculated. Confluence of the highest dose of a reference compound was used as baseline for the LC (Low control) and the confluence of DMSO treated cells was used as 0%cytotoxicity (High Control, HC) .

Absolute IC ₅₀ values were calculated as percent change in confluence as follows:

LC = Low Control: cells treated with e.g. 1 μM of the cytotoxic agent staurosporin, or e.g. cells treated with a high concentration of an alternative reference compound

HC = High Control: Mean confluence (%) (DMSO treated cells)

%Effect = 100 - (100* (Sample-LC) / (HC-LC) )

GraphPad Prism (version 7.00) was used to calculate the IC ₅₀. Dose-response equation was used for the plot of %Effect vs Log10 compound concentration with a variable slope and fixing the maximum to 100%and the minimum to 0%.

2b) MEIS1 mRNA expression assay

MEIS1 mRNA expression upon treatment of compound was examined by Quantigene Singleplex assay (Thermo Fisher Scientific) . This technology allows for direct quantification of mRNA targets using probes hybridizing to defined target sequences of interest and the signal is detected using a Multimode plate reader Envision (PerkinElmer) . The MOLM14 cell line was used for this experiment. Cells were plated in 96-well plates at 3, 750 cells/well in the presence of increasing concentrations of compounds. After incubation of 48 hours with compounds, cells were lysed in lysis buffer and incubated for 45 minutes at 55℃. Cell lysates were mixed with human MEIS1 specific capture probe or human RPL28 (Ribosomal Protein L28) specific probe as a normalization control, as well as blocking probes. Cell lysates were then transferred to the custom assay hybridization plate (Thermo Fisher Scientific) and incubated for 18 to 22 hours at 55℃. Subsequently, plates were washed to remove unbound materials followed by sequential addition of preamplifiers, amplifiers, and label probe. Signals (= gene counts) were measured with a Multimode plate reader Envision. IC ₅₀s were calculated by dose-response modelling using appropriate software. For all non-housekeeper genes response equal counts corrected for background and relative expression. For each sample, each test gene signal (background subtracted) was divided by the normalization gene signal (RPL28: background subtracted) . Fold changes were calculated by dividing the normalized values for the treated samples by the normalized values for the DMSO treated sample. Fold changes of each target gene were used for the calculation of IC ₅₀s.

Table 3. Biological data –HTRF assay, proliferation assay, and MEIS1 mRNA expression assay

Claims

A compound of Formula (I)

or a tautomer or a stereoisomeric form thereof, wherein

R ^1a represents -C (=O) -NR ^xaR ^xb; or

R ^xa and R ^xb are each independently selected from the group consisting of hydrogen; C _3-6cycloalkyl; C _1-4alkyl; C _1-4alkyl substituted with 1, 2 or 3 halo atoms; and C _1-4alkyl substituted with one -OH, -OC _1-4alkyl, or NR ^11cR ^11d;

R ^1b represents F or Cl;

Y ¹ represents -CR ^5aR ^5b-, -O-, -S-, or -NR ^5c-;

R ² is selected from the group consisting of hydrogen, halo, C _1-4alkyl, -O-C _1-4alkyl, and -NR ^7aR ^7b;

U ¹ and U ² each independently represent N or CH;

n1, n2, n3 and n4 are each independently selected from 1 and 2;

X ¹ represents CH, and X ² represents N;

R ⁴ represents C _1-5alkyl;

R ^5a, R ^5b, R ^5c, R ^7a, and R ^7b, are each independently selected from the group consisting of hydrogen, C _1-4alkyl and C _3-6cycloalkyl;

R ³ is selected from the group consisting of Het ¹, Het ², Cy ², and -C _1-6alkyl-NR ^xcR ^xd;

R ^xc represents Cy ¹; Het ⁵; -C _1-6alkyl-Cy ¹; -C _1-6alkyl-Het ³; -C _1-6alkyl-Het ⁴;

or -C _1-6alkyl-phenyl;

R ^xd represents hydrogen; C _1-4alkyl; or C _1-4alkyl substituted with one, two or three substituents selected from the group consisting of halo, -OH, -O-C _1-4alkyl, and cyano;

or R ^xc and R ^xd are taken together to form together with the N-atom to which they are attached a 4-to 7-membered monocyclic fully saturated heterocyclyl containing one N-atom and optionally one additional heteroatom selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted with one, two or three substituents selected from the group consisting of halo, -OH, -O-C _1-4alkyl, and cyano;

Het ¹ represents a monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; or a bicyclic C-linked 6-to 11-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂;

wherein said heterocyclyl is optionally substituted on one nitrogen with a substituent selected from the group consisting of R ⁶ and -C (=O) -R ⁸; and wherein said heterocyclyl is optionally substituted on one or two carbon atoms with in total one, two, three or four substituents each independently selected from the group consisting of halo, R ⁶, Het ^6a, Het ^6b, C _1-4alkyl, oxo, -NR ^9aR ^9b and -OH;

Het ² represents C-linked pyrazolyl or triazolyl; which is substituted on one nitrogen atom with R ^6a;

R ⁶ is selected from the group consisting of

Het ³; -C (=O) -NH-R ⁸;

C _1-6alkyl optionally substituted with one or two substituents each independently selected from the group consisting of Het ³, Het ⁴, Het ^6a, Het ^6b, Cy ¹, -CN, -OH,

-O-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl-C _3-6cycloalkyl, -C (=O) -OH, -NR ^11aR ^11b and -NH-S (=O) ₂-C _1-4alkyl; and

C _3-6cycloalkyl optionally substituted by one or two substituents each independently selected from the group consisting of -CN, -OH, -O-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl,

-NH-S (=O) ₂-C _1-4alkyl, and C _1-4alkyl optionally substituted with one substituent selected from the group consisting of OH, -O-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl and

-NH-S (=O) ₂-C _1-4alkyl;

R ^6a represents C _1-6alkyl substituted with one substituent selected from the group consisting of -NR ^11aR ^11b, Het ^3a, and Het ^6a;

R ⁸ represents C _1-6alkyl optionally substituted with one, two or three substituents each independently selected from -OH, halo, cyano, -NR ^11aR ^11b, Het ^3a, and Het ^6a;

Het ³ and Het ⁵ each independently represent a monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one, two or three heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; or a bicyclic C-linked 6-to 11-membered fully saturated heterocyclyl containing one, two or three heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂;

wherein said heterocyclyl is optionally substituted on one carbon atom with C _1-4alkyl, halo, -OH, -NR ^11aR ^11b, or oxo; and wherein said heterocyclyl is optionally substituted on one nitrogen atom with C _1-4alkyl;

Het ^3a and Het ^5a each independently represent a monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom, and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; or a bicyclic C-linked 6-to 11-membered fully saturated heterocyclyl containing one N-atom, and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂;

wherein said heterocyclyl is optionally substituted on one carbon atom with C _1-4alkyl, halo, -OH, -NR ^11aR ^11b, or oxo; and wherein said heterocyclyl is optionally substituted on one nitrogen atom with C _1-4alkyl;

Het ⁴ and Het ⁷ each independently represent a monocyclic C-linked 5-or 6-membered aromatic ring containing one, two, three or four heteroatoms each independently selected from O, S, and N; wherein said 5-membered aromatic ring is optionally substituted on one nitrogen atom with C _1-4alkyl; and wherein said 5-or 6-membered aromatic ring is optionally substituted on one carbon atom with -OH;

Het ^6a and Het ⁸ each independently represent a monocyclic N-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted on one or two carbon atoms with in total one, two, three or four substituents each independently selected from the group consisting of halo, -OH, oxo, - (C=O) -NR ^10aR ^10b, -O-C _3-6cycloalkyl, -S (=O) ₂-C _1-4alkyl, cyano, C _1-4alkyl, -C _1-4alkyl-OH,

-O-C _1-4alkyl, -O- (C=O) -NR ^10aR ^10b, and

-O- (C=O) -C _1-4alkyl; and wherein said heterocyclyl is optionally substituted on one nitrogen with a substituent selected from the group consisting of -C (=O) -C _1-4alkyl and

- (C=O) -NR ^10aR ^10b;

Het ^8a each independently represent a monocyclic N-linked 4-to 7-membered fully saturated heterocyclyl containing two N-atoms and optionally one additional heteroatom selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted on one or two carbon atoms with in total one, two, three or four substituents each independently selected from the group consisting of halo, -OH, oxo, - (C=O) -NR ^10aR ^10b,

-O-C _3-6cycloalkyl, -S (=O) ₂-C _1-4alkyl, cyano, C _1-4alkyl, C _1-4alkyl-OH, -O-C _1-4alkyl,

-O- (C=O) -NR ^10aR ^10b, and -O- (C=O) -C _1-4alkyl; and wherein said heterocyclyl is optionally substituted on one nitrogen with a substituent selected from the group consisting of -C (=O) -C _1-4alkyl and - (C=O) -NR ^10aR ^10b;

Het ^6b represents a bicyclic N-linked 6-to 11-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted on one or two carbon atoms with in total one or two substituents each independently selected from the group consisting of C _1- ₄alkyl, -OH, oxo, - (C=O) -NR ^10aR ^10b,

-NH-C (=O) -C _1-4alkyl, -NH-C (=O) -Cy ³, and -O-C _1-4alkyl; and wherein said heterocyclyl is optionally substituted on one nitrogen with a substituent selected from the group consisting of -C (=O) -C _1-4alkyl, -C (=O) -Cy ³, - (C=O) -C _1-4alkyl-OH, -C (=O) -C _1-4alkyl-O-C _1-4alkyl, -C (=O) -C _1-4alkyl-NR ^11aR ^11b, and C _1-4alkyl;

Cy ¹ represents C _3-6cycloalkyl optionally substituted with one, two or three substituents selected from the group consisting of -OH, -NH-C (=O) -C _1-4alkyl, C _1-4alkyl, -NH-S (=O) ₂-C _1-4alkyl, -S (=O) ₂-C _1-4alkyl, and -O-C _1-4alkyl;

Cy ² represents C _3-7cycloalkyl substituted with one or two substituents each independently selected from the group consisting of -NR ^9aR ^9b; Het ^6a; Het ^6b; and C _1-6alkyl substituted with one or two substituents each independently selected from the group consisting of Het ^3a, Het ^6a, Het ^6b, and -NR ^9aR ^9b; and said C _3-7cycloalkyl is optionally substituted with one or two additional substituents each independently selected from the group consisting of halo, R ⁶, C _1-4alkyl, and -OH;

Cy ³ represents C _3-7cycloalkyl; wherein said C _3-7cycloalkyl is optionally substituted with one, two or three halo substituents;

R ^9a and R ^9b are each independently selected from the group consisting of hydrogen;

C _1-4alkyl; C _3-6cycloalkyl; Het ⁵; -C _1-4alkyl-R ¹⁶; -C (=O) -C _1-4alkyl-Het ^3a; -C (=O) -R ¹⁴;

C _3-6cycloalkyl substituted with one, two or three substituents selected from the group consisting of halo, -OH, -O-C _1-4alkyl, -NR ^11aR ^11b, and cyano; and

C _1-4alkyl substituted with one, two or three substituents selected from the group consisting of halo, -OH, -O-C _1-4alkyl, -NR ^11aR ^11b, and cyano;

R ^11a, R ^11b, R ^13a, R ^13b, R ^15a, R ^15b, R ^17a, and R ^17b are each independently selected from the group consisting of hydrogen and C _1-4alkyl;

R ^11c and R ^11d are each independently selected from the group consisting of hydrogen, C _1-6alkyl, and -C (=O) -C _1-4alkyl;

R ¹⁴ represents Het ^5a; Het ^8a; or C _1-4alkyl substituted with one, two or three substituents selected from the group consisting of -NR ^13aR ^13b and Het ^8a;

R ¹⁶ represents -C (=O) -NR ^17aR ^17b, -S (=O) ₂-C _1-4alkyl, Het ⁵, Het ⁷, or Het ⁸;

or a pharmaceutically acceptable salt or a solvate thereof.
The compound according to claim 1, wherein

R ^1a represents -C (=O) -NR ^xaR ^xb;

R ^xa and R ^xb are each independently selected from the group consisting of

C _3-6cycloalkyl; C _1-4alkyl; and C _1-4alkyl substituted with 1, 2 or 3 halo atoms;

R ^1b represents F;

Y ¹ represents -O-;

R ² represents hydrogen;

R ⁴ represents C _1-5alkyl;

R ³ is selected from the group consisting of Het ¹ and Cy ²;

Het ¹ represents a monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom;

wherein said heterocyclyl is optionally substituted on one nitrogen with a substituent selected from the group consisting of R ⁶ and -C (=O) -R ⁸; and wherein said heterocyclyl is optionally substituted on one or two carbon atoms with in total one, two, three or four halo substituents;

R ⁶ is selected from the group consisting of Het ³;

C _1-6alkyl optionally substituted with one or two substituents each independently selected from the group consisting of Het ³, Het ⁴, Het ^6a, Cy ¹, -OH, -O-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl, -C (=O) -NH-C _1-4alkyl-C _3-6cycloalkyl, and -NH-S (=O) ₂-C _1-4alkyl;

R ⁸ represents C _1-6alkyl substituted with one substituent selected from the group consisting of -OH and -NR ^11aR ^11b;

Het ³ and Het ⁵ each independently represent a monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one, two or three heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂;

wherein said heterocyclyl is optionally substituted on one carbon atom with -OH or oxo;

Het ⁴ represents a monocyclic C-linked 5-or 6-membered aromatic ring containing one, two or three heteroatoms each independently selected from O, S, and N; wherein said 5-or 6-membered aromatic ring is optionally substituted on one carbon atom with -OH;

Het ^6a represents a monocyclic N-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted on one or two carbon atoms with in total one, two, three or four substituents each independently selected from the group consisting of oxo, -S (=O) ₂-C _1-4alkyl, and -O-C _1-4alkyl; and wherein said heterocyclyl is optionally substituted on one nitrogen with

-C (=O) -C _1-4alkyl;

Het ^6b represents a bicyclic N-linked 6-to 11-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂; wherein said heterocyclyl is optionally substituted on one carbon atom with - (C=O) -NR ^10aR ^10b; and wherein said heterocyclyl is optionally substituted on one nitrogen with -C (=O) -C _1-4alkyl;

Cy ¹ represents C _3-6cycloalkyl optionally substituted with one, two or three substituents selected from the group consisting of -OH, -NH-C (=O) -C _1-4alkyl, C _1-4alkyl, -NH-S (=O) ₂-C _1-4alkyl, and -O-C _1-4alkyl;

Cy ² represents C _3-7cycloalkyl substituted with one or two substituents each independently selected from the group consisting of -NR ^9aR ^9b; Het ^6a; and Het ^6b;

R ^9a and R ^9b are each independently selected from the group consisting of hydrogen;

C _1-4alkyl; C _3-6cycloalkyl; Het ⁵; -C _1-4alkyl-R ¹⁶; and

C _1-4alkyl substituted with one, two or three -O-C _1-4alkyl substituents;

R ^10a, R ^10b, R ^11a, and R ^11b represent C _1-4alkyl;

R ¹⁶ represents Het ⁵.
The compound according to claim 1, wherein

R ^1a represents -C (=O) -NR ^xaR ^xb;

R ^xa and R ^xb represent C _1-4alkyl;

R ^1b represents F;

Y ¹ represents -O-;

R ² represents hydrogen;

R ⁴ represents isopropyl;

R ³ is selected from the group consisting of Het ¹ and Cy ²;

Het ¹ represents a monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom;

wherein said heterocyclyl is optionally substituted on one nitrogen with a substituent selected from the group consisting of R ⁶;

R ⁶ represents C _1-6alkyl substituted with one Het ³;

Het ³ represents a monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one, two or three heteroatoms each independently selected from O, S, and N, wherein said S-atom might be substituted to form S (=O) or S (=O) ₂;

Het ^6a represents a monocyclic N-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom; wherein said heterocyclyl is optionally substituted on one carbon atom with one -O-C _1-4alkyl;

Het ^6b represents a bicyclic N-linked 6-to 11-membered fully saturated heterocyclyl containing one N-atom and optionally one or two additional heteroatoms each independently selected from O and N; wherein said heterocyclyl is optionally substituted on one carbon atom with - (C=O) -NR ^10aR ^10b; and wherein said heterocyclyl is optionally substituted on one nitrogen with -C (=O) -C _1-4alkyl;

Cy ² represents C _3-7cycloalkyl substituted with one substituents selected from the group consisting of Het ^6a and Het ^6b;

R ^10a and R ^10b represent C _1-4alkyl.
The compound according to claim 1, wherein R ³ represents Het ¹.
The compound according to claim 1, wherein n1 is 1, n2 is 2, n3 is 1, and n4 is 1.
The compound according to claim 1, wherein U ¹ represents N.
The compound according to claim 1, wherein

R ³ represents Het ¹;

Het ¹ represents a monocyclic C-linked 4-to 7-membered fully saturated heterocyclyl containing one N-atom;

wherein said heterocyclyl is substituted on one nitrogen with R ⁶; and wherein said heterocyclyl is optionally substituted on one or two carbon atoms with in total one, two, three or four halo substituents;

R ⁶ is selected from the group consisting of C _1-6alkyl optionally substituted with one or two substituents each independently selected from the group consisting of Het ³, Het ⁴, Het ^6a, and Cy ¹.
A pharmaceutical composition comprising a compound as claimed in any one of claims 1 to 7 and a pharmaceutically acceptable carrier or diluent.
A process for preparing a pharmaceutical composition as defined in claim 8 comprising mixing a pharmaceutically acceptable carrier with a therapeutically effective amount of a compound according to any one of claims 1 to 7.
A compound as claimed in any one of claims 1 to 7 or a pharmaceutical composition as claimed in claim 8 for use as a medicament.
A compound as claimed in any one of claims 1 to 7 or a pharmaceutical composition as claimed in claim 8 for use in the prevention or treatment of cancer, myelodysplastic syndrome (MDS) and diabetes.
The compound or a pharmaceutical composition for use according to claim 11, wherein cancer is selected from leukemias, myeloma or a solid tumor cancer such as prostate cancer, lung cancer, breast cancer, pancreatic cancer, colon cancer, liver cancer, melanoma and glioblastoma.
The compound or a pharmaceutical composition for use according to claim 12, wherein the leukemia is selected from acute leukemias, chronic leukemias, myeloid leukemias, myelogeneous leukemias, lymphoblastic leukemias, lymphocytic leukemias, Acute myelogeneous leukemias (AML) , Chronic myelogenous leukemias (CML) , Acute lymphoblastic leukemias (ALL) , Chronic lymphocytic leukemias (CLL) , T cell prolymphocytic leukemias (T-PLL) , Large granular lymphocytic leukemia, Hairy cell leukemia (HCL) , MLL-rearranged leukemias, MLL-PTD leukemias, MLL amplified leukemias, MLL-positive leukemias, and leukemias exhibiting HOX/MEIS1 gene expression signatures.
A method of treating or preventing a disorder selected from cancer, myelodysplastic syndrome (MDS) and diabetes comprising administering to a subject in need thereof, a therapeutically effective amount of a compound as claimed in any one of claims 1 to 7 or a pharmaceutical composition as claimed in claim 8.
The method according to claim 13 wherein the disorder is cancer.