Nitrogen-containing five-membered heterocyclic derivatives as checkpoint kinase 1 inhibitors and uses thereof

Abstract

The present disclosure relates generally to nitrogen-containing five-membered heterocyclic derivatives, particularly more particularly to CHK-1 enzymes, for use in the treatment of conditions associated with checkpoint kinase (CHK), methods of use, compositions, and methods of synthesis thereof.

Description

Technical Field

[0001] The present invention generally relates to nitrogen-containing five-membered heterocyclic derivatives having checkpoint kinase-1 (CHK-1) ​​inhibitory activity, and to the use of such compounds in the treatment of proliferative disorders such as cancer, pulmonary arterial hypertension (PAH) and idiopathic pulmonary fibrosis (IPF). The present invention also provides methods for synthesizing the compounds, methods for using the compounds, pharmaceutical compositions containing the compounds and methods for using the same. Background Art

[0002] A variety of cancer chemotherapeutics act through DNA damage pathways to induce DNA damage, thereby causing tumor growth inhibition. However, these chemotherapeutics cause cell cycle arrest by inducing checkpoints at the S phase or G2 / M boundary. G2 arrest gives cells time to repair damaged DNA before entering mitosis. Checkpoint kinase-1 (CHK-1) ​​and unrelated serine / threonine kinase checkpoint kinase-2 (CHK-2) play a central role in arresting the cell cycle at the G2-M boundary (O'Connell et al., 1997). CHK-1 and / or CHK-2 induce the checkpoint by phosphorylating CDC25 phosphatase, inhibiting the removal of inactivating phosphates on cyclin-dependent kinases (CDKs) (Karlsson-Rosenthal et al., 2006; Zheng et al., 1998). Another overlapping pathway mediated by p53 also triggers cycle arrest in response to DNA damage. However, p53 is inactivated by mutation in many cancers, resulting in partial defects in its ability to initiate DNA repair responses. If CHK-1 activity is also inhibited in p53-negative cancers, all abilities to arrest and repair DNA in response to DNA damage are abolished, and this leads to mitotic catastrophe and enhances the effects of DNA damaging agents (Bunch and Eastman, 1996; Konarias et al., 2001; Tenzer and Pruschy, 2003).

[0003] Thus, CHK-1 inhibition represents a novel therapeutic strategy to enhance the lethality of DNA-damaging chemotherapeutics in p53 pathway-deficient cancers (Ma et al., 2012). Abrogation of the remaining intact checkpoint should result in increased tumor cell death. CHK-1 inhibitors have demonstrated potentiated effects of cytotoxic chemotherapeutics both in vitro and in a range of preclinical models of human cancer, including gemcitabine, irinotecan, cytarabine, and cisplatin (Qiu et al., 2018). This “synthetic lethality” approach should enhance the therapeutic activity of chemotherapeutics without increasing systemic toxicity, as normal cells should remain protected by their functional p53 pathways. Thus, CHK-1 inhibitors have the potential to be used in combination with a broad range of cytotoxic chemotherapeutics to treat a variety of human cancers.

[0004] Furthermore, excessive and sustained proliferation, resistance to apoptosis through finely regulated cell cycle and DNA repair machinery are a few of the pathogenic mechanisms of cancer, pulmonary arterial hypertension (PAH) and idiopathic pulmonary fibrosis (IPF). PAH is a devastating disease with progressive vascular remodeling of the distal pulmonary arteries, leading to concurrent increases in pulmonary artery pressure, perivascular inflammation, fibrotic changes, right ventricular hypertrophy and death (Bourgeois et al., 2019). It is a complication of idiopathic pulmonary fibrosis and affects its survival, functional status and progression, yet currently no treatment is available except lung transplantation. In addition to genetic susceptibility, many epigenetic factors such as oxidative stress and generation of reactive oxygen species lead to DNA damage in pulmonary artery smooth muscle cells (PASMCs) and alter cellular functions similar to cancer cells (Ranchoux et al., 2016). Since DNA repair machinery has been successfully targeted to characterize the underlying molecular mechanisms of cancer, and a wide range of chemotherapeutic agents are being explored that act through DNA damage pathways to cause tumor growth inhibition, a similar approach could be adopted for PAH and IPF (Sharma and Aldred, 2020; Wu et al., 2022). In cancer, these chemotherapeutic agents cause cell cycle arrest by inducing checkpoints at the S phase or the G2 / M boundary, where the G2 arrest allows cells to repair damaged DNA before entering mitosis. Checkpoint kinase-1 (CHK-1) ​​and checkpoint kinase-2 (CHK-2) are serine / threonine kinases that are key components of the DNA damage response and crucial regulators of DNA repair and cell cycle progression. They are upregulated in cancer cells and play a central role in arresting the cell cycle at the G2-M boundary to promote DNA repair (O'Connell et al., 1997). CHK-1 and / or CHK-2 induce this checkpoint by phosphorylating CDC25 phosphatase, inhibiting the removal of inactivating phosphates on cyclin-dependent kinases (CDKs) (Zheng et al., 1998). In PAH, proliferating PAH-PASMCs show increased levels of γ-H2AX and pRPA32, markers of DNA damage / replication stress, and also exhibit enhanced expression and activation of CHK1. Furthermore, pharmacological inhibition of CHK1 reduces vascular remodeling and improves hemodynamic parameters in clinically relevant rat models, suggesting that CHK1 inhibition may also be an attractive therapeutic option for PAH (Bourgeois et al., 2019).

[0005] Various attempts have been made to develop CHK-1 kinase inhibitors. For example, US10000481B2 (Vernalis) discloses 1H-pyrrolo[2,3-B]pyridine derivative compounds as CHK-1 kinase inhibitors. US10010547B2 (Cascadian Therapeutics) discloses pyrazolaminopyrazine derivatives as kinase inhibitors. WO / 2018 / 086546A1 (Zhejiang University) discloses 2-polysubstituted aromatic ring pyrimidine derivatives as CHK-1 inhibitors. Some small molecule inhibitors of CHK-1 (presatinib / LY2606368, LY2603618, and SRA737) are currently being used in combination with gemcitabine, pemetrexed, fludarabine, cytarabine, and cisplatin for phase I / II clinical evaluation. These CHK-1 kinase inhibitors are not nitrogen-containing five-membered heterocyclic derivatives.

[0006] The main features of pulmonary hypertension (PH) in IPF patients (IPF-PH) are excessive proliferation of fibroblasts and pulmonary artery (PA) smooth muscle cells (PASMCs) and resistance to apoptosis, which leads to abnormal accumulation of extracellular matrix and extensive vascular remodeling within the parenchyma. It can be hypothesized that upregulated and activated CHK1 / 2 contribute to fibrosis and vascular lesions in IPF-PH. This is associated with γH2AX, a sensitive molecular marker of DNA damage, which in turn is associated with PAH remodeling and fibrosis scores (Sharma and Aldred, 2020). Increased DNA repair in IPF is associated with significant upregulation of CHK1 and CHK2 in the lungs and distal PAs of IPF patients, and it is predominantly localized within PASMCs and fibrotic lesions. Some drugs targeting proliferation could be targeted for PAH prevention, and since CHK1 activation in PAH-PASMCs is known to be a decisive event in the initiation of pulmonary vascular remodeling in PAH, CHK1 could be a potential therapeutic target for PAH and IPF (Bourgeois et al., 2019; Satoh et al., 2018; Wu et al., 2022).

[0007] The activation of ATR-CHK1 signaling in PAH-PASMCs and the significant therapeutic effects observed by inhibiting this axis in animal models that mimic PAH suggest that CHK1 may represent a new therapeutic avenue for patients with PAH. Inhibition of CHK1 signaling blocks or reverses pulmonary vascular remodeling, a key pathological feature of PAH for which currently approved therapies have limited efficacy. In addition, the therapeutic effects observed in cancer by inhibiting CHK1 also highlight the continued need for the development of novel CHK-1 inhibitors with pharmacokinetic and pharmacodynamic properties that make them suitable for use as pharmaceutical agents. It is therefore an object of the present invention to provide such agents and methods of treatment. Summary of the invention

[0008] In one aspect, the present invention provides a nitrogen-containing five-membered heterocyclic compound of formula (I):

[0009]

[0010] or a pharmaceutically acceptable salt thereof, wherein R1, R2, R4, R6, R7 and X are as described in detail herein.

[0011] In one aspect, the compound of Formula (I) or a pharmaceutically acceptable salt thereof is a compound of Table 1 or a pharmaceutically acceptable salt thereof, as described in detail herein.

[0012] In another aspect, the present invention provides a method for treating a disease or condition associated with the CHK kinase enzyme, more specifically the CHK-1 kinase enzyme, in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0013] In another aspect, the present invention provides a method of treating cancer in an individual in need thereof, wherein the method comprises administering to the individual an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0014] In another aspect, the present invention provides a method of treating idiopathic pulmonary fibrosis (IPF) in an individual in need thereof, wherein the method comprises administering to the individual an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0015] In another aspect, the present invention provides a method of treating Type I pulmonary arterial hypertension (PAH) in an individual in need thereof, wherein the method comprises administering to the individual an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0016] In another aspect, the present invention provides a method for treating a disease or condition associated with the CHK kinase enzyme, more specifically the CHK-1 kinase enzyme, in an individual in need thereof, wherein the method comprises administering to the individual an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof in combination with another therapeutic agent.

[0017] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0018] In another aspect, the present invention provides a method for treating a disease or condition associated with the CHK kinase enzyme (or more specifically, the CHK-1 kinase enzyme) in an individual in need thereof, wherein the method comprises administering to the individual an effective amount of a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0019] In another aspect, the present invention provides methods for preparing the compounds and intermediates thereof disclosed in the present invention.

[0020] In another aspect, the present invention provides a kit comprising the compound or a pharmaceutically acceptable salt thereof. DETAILED DESCRIPTION

[0021] definition

[0022] "Alkyl" refers to and includes saturated straight and branched chain monovalent hydrocarbon structures and combinations thereof having the specified number of carbon atoms (i.e., C1-C 10 A specific alkyl group is an alkyl group having 1 to 20 carbon atoms ("C1-C 20 More specific alkyl groups are alkyl groups having 1 to 8 carbon atoms ("C1-C8 alkyl"), 3 to 8 carbon atoms ("C3-C8 alkyl"), 1 to 6 carbon atoms ("C1-C6 alkyl"), 1 to 5 carbon atoms ("C1-C5 alkyl"), or 1 to 4 carbon atoms ("C1-C4 alkyl"). Examples of alkyl groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,3-dimethylbutyl, or 2,2-dimethylbutyl, and their homologs and isomers.

[0023] As used herein, "alkylene" refers to the same residue as an alkyl group, but with a divalence. Specific alkylene groups are alkylene groups having 1 to 6 carbon atoms ("C1-C6 alkylene"), 1 to 5 carbon atoms ("C1-C5 alkylene"), 1 to 4 carbon atoms ("C1-C4 alkylene"), or 1 to 3 carbon atoms ("C1-C3 alkylene"). Examples of alkylene groups include, but are not limited to, groups such as methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-).

[0024] "Cycloalkyl" refers to and includes cyclic monovalent hydrocarbon structures which may be fully saturated, monounsaturated or polyunsaturated, but which are non-aromatic, having the specified number of carbon atoms (e.g., C1-C 10"Cycloalkyl" refers to one to ten carbon atoms. Cycloalkyl groups may consist of one ring (e.g., cyclohexyl) or multiple rings (e.g., adamantyl), but do not include aryl groups. Cycloalkyl groups containing more than one ring may be fused, spirocyclic, or bridged, or a combination thereof. Preferred cycloalkyl groups are cyclic hydrocarbons having 3 to 13 ring carbon atoms. More preferred cycloalkyl groups are cyclic hydrocarbons having 3 to 8 ring carbon atoms ("C3-C8 cycloalkyl"). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, norbornyl, and the like.

[0025] "Heterocycle" or "heterocyclic radical" refers to a saturated or unsaturated non-aromatic group having 3 to 8 ring carbon atoms and 1 to 2 ring heteroatoms (such as nitrogen or oxygen), and the nitrogen atom is optionally substituted. The heterocyclic radical group can be a monocyclic, bicyclic or spirocyclic 4 to 10-membered heterocyclic radical. The heterocyclic ring containing more than one ring can be fused, spirocyclic or bridged, or any combination thereof. In a fused ring system, one or more of the fused rings can be an aryl or heteroaryl group. Examples of heterocyclic radical groups include, but are not limited to, aziridine, azetidine, oxetane, morpholinyl, azepanyl, tetrahydropyranyl, dihydropyranyl, piperidinyl, piperazinyl, pyrrolidinyl, tetrahydrofuranyl, azaspiro [3.3] heptyl, etc.

[0026] "Ring" is partially or completely unsaturated and substituted or unsubstituted. So-called rings include monocyclic rings, interlocking rings, spiral rings, parallel rings or bridged rings. The number of atoms on the ring is usually defined as the number of elements of the ring. For example, "8-membered ring" means 8 atoms arranged in a circle. Unless otherwise specified, the ring optionally contains 1 to 3 heteroatoms.

[0027] "CHK" refers to checkpoint kinase, which includes CHK-1 and CHK-2. CHK herein specifically refers to CHK-1.

[0028] "Optionally substituted" unless otherwise indicated, refers to a group that may be unsubstituted or substituted with one or more (e.g., 1, 2, or 3) of the substituents listed for the group, wherein the substituents may be the same or different. In one embodiment, the optionally substituted group has one substituent. In another embodiment, the optionally substituted group has two substituents. In another embodiment, the optionally substituted group has three substituents.

[0029] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation other than the active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0030] As used herein, "treatment" or "treatment" is a method for obtaining beneficial or desired results (including clinical results). For example, beneficial or desired results include, but are not limited to, one or more of the following: alleviating symptoms caused by the disease, improving the quality of life of people with the disease, reducing the dose of other drugs required to treat the disease, delaying the progression of the disease, and / or prolonging the survival of individuals. With regard to cancer or other undesirable cell proliferation (idiopathic pulmonary fibrosis (IPF)), beneficial or desired results include shrinking tumors (reducing tumor size); reducing the growth rate of tumors (e.g., inhibiting tumor growth); reducing the number of cancer cells; inhibiting, delaying or slowing down and preferably preventing the infiltration of cancer cells to peripheral organs to a certain extent; inhibiting (slowing down and preferably preventing to a certain extent) tumor metastasis; inhibiting tumor growth; preventing or delaying tumor occurrence and / or recurrence; and / or alleviating one or more symptoms associated with cancer to a certain extent. In some embodiments, beneficial or desired results include preventing or delaying the occurrence and / or recurrence of, for example, undesirable cell proliferation (idiopathic pulmonary fibrosis (IPF)).

[0031] As used herein, "delaying the development of a disease" refers to postponing, hindering, slowing, delaying, stabilizing and / or postponing the development of a disease (e.g., cancer, pulmonary arterial hypertension (PAH), and idiopathic pulmonary fibrosis (IPF)). Such a delay may be of varying lengths of time, depending on the history of the disease and / or the individual being treated. As will be clear to one skilled in the art, a sufficient or significant delay may actually encompass prevention, i.e., the individual will not develop the disease. For example, the development of an advanced cancer, such as metastasis, may be delayed.

[0032] As used herein, an "effective dose" or "effective amount" of a compound or its salt or pharmaceutical composition is an amount sufficient to produce a beneficial or desired result. For preventive purposes, beneficial or desired results include, for example, elimination or reduction of risk, reduction of severity of the disease, or delay of disease onset, including results of biochemical, histological and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that occur during disease development. For therapeutic purposes, beneficial or desired results include improvement, alleviation, mitigation, delay or reduction of one or more symptoms caused by the disease, improvement of the quality of life of people with the disease, reduction of the dose of other drugs required to treat the disease, enhancement of the effect of another drug (e.g., by targeting), delay of disease progression, and / or extension of survival. With respect to cancer or other undesirable cell proliferation, an effective amount includes an amount sufficient to cause tumor shrinkage and / or reduce tumor growth rate (e.g., inhibit tumor growth) or prevent or delay other undesirable cell proliferation (idiopathic pulmonary fibrosis (IPF)). With respect to pulmonary hypertension (PAH), an effective amount includes an amount sufficient to prevent or delay the development of pulmonary hypertension (PAH). In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay occurrence and / or recurrence. An effective amount can be administered in one or more administrations. In the case of cancer, an effective amount of a drug or composition can: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow down and preferably prevent cancer cells from infiltrating peripheral organs to a certain extent; (iv) inhibit (i.e., slow down and preferably prevent to a certain extent) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor occurrence and / or recurrence; and / or (vii) alleviate one or more symptoms associated with cancer to a certain extent.

[0033] As used herein, the term "subject" is a mammal, including humans. Individuals include, but are not limited to, humans, cattle, horses, cats, dogs, rodents, or primates. In some embodiments, the individual is a human. The individual (e.g., a human) may have an advanced disease or a less severe disease, such as a low tumor burden. In some embodiments, the individual is in the early stages of a proliferative disease (e.g., cancer or idiopathic pulmonary fibrosis (IPF)). In some embodiments, the individual is in the late stages of a proliferative disease (e.g., advanced cancer).

[0034] References herein to "about" a value or a parameter include (and describe) embodiments for that value or parameter itself. For example, descriptions referring to "about X" include descriptions of "X".

[0035] It should be understood that the aspects and variations described herein also include “consisting of” and / or “consisting essentially of” aspects and variations.

[0036] Compound

[0037] The present invention provides a nitrogen-containing five-membered heterocyclic compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0038]

[0039] in,

[0040] X is selected from O or NH;

[0041] R1 is selected from C1-C6 alkyl, C1-C6 alkylene, C3-C8 cycloalkyl or absent;

[0042] R2 is selected from H, -NH2, unsaturated or saturated monocyclic, bicyclic or spirocyclic 4 to 10 membered heterocyclic group, wherein the heterocyclic group contains 1-2 independently selected from N (R3) n and O heteroatoms;

[0043] R4, R6 and R7 are independently -A-R5 or absent;

[0044] A is selected from:

[0045] (i) key;

[0046] (ii) CH2;

[0047] (iii) a saturated chain of 2 to 10 chain members in length containing at least one carbon atom chain member, at least one heteroatom chain member selected from nitrogen and oxygen, and optionally one or more further carbon atom chain members and / or heteroatom chain members selected from nitrogen, oxygen, sulfur, sulfinyl and sulfonyl; the saturated chain is optionally substituted with one or more heteroatoms selected from ═O, C 1-4 Substitution with hydrocarbyl and fluorine substituents, wherein two hydrocarbyl substituents on the same carbon atom may optionally be linked to form a ring of three to five ring members;

[0048] R5 is selected from:

[0049] (i) H,

[0050] (ii) CH2,

[0051] (iii) a saturated or unsaturated monocyclic 5-6 membered heterocyclic group, wherein the heterocyclic group contains 1-3 independently selected from N(R3) n , O or S heteroatom; optionally each R3 is selected from H or C1-C3 alkyl; n is 0, 1 or 2;

[0052] Or R4 and R6 together with the carbon to which they are attached form a 5, 6, 7 or 8 membered ring, wherein the ring is partially or fully unsaturated and is substituted or unsubstituted.

[0053] In some embodiments, the nitrogen-containing five-membered heterocyclic derivative of formula (I) is

[0054]

[0055] or a pharmaceutically acceptable salt thereof,

[0056] in,

[0057] R1 is selected from C1-C6 alkyl, C1-C6 alkylene, C3-C8 cycloalkyl or absent;

[0058] R2 is selected from H, -NH2, unsaturated or saturated monocyclic, bicyclic or spirocyclic 4 to 10 membered heterocyclic group, wherein the heterocyclic group contains 1-2 independently selected from N (R3) n and O heteroatoms;

[0059] R4 is -A-R5;

[0060] A is selected from:

[0061] (i) key;

[0062] (ii) CH2;

[0063] (iii) a saturated chain of 2 to 10 chain members in length containing at least one carbon atom chain member, at least one heteroatom chain member selected from nitrogen and oxygen, and optionally one or more further carbon atom chain members and / or heteroatom chain members selected from nitrogen, oxygen, sulfur, sulfinyl and sulfonyl; the saturated chain is optionally substituted with one or more heteroatoms selected from ═O, C 1-4 Substitution with hydrocarbyl and fluorine substituents, wherein two hydrocarbyl substituents on the same carbon atom may optionally be linked to form a ring of three to five ring members;

[0064] R5 is selected from:

[0065] (i) H,

[0066] (ii) a saturated or unsaturated monocyclic 5-6 membered heterocyclic group, wherein the heterocyclic group contains 1-3 independently selected from N(R3) n , O or S heteroatom; optionally each R3 is selected from H or C1-C3 alkyl; n is 0, 1 or 2.

[0067] In some embodiments, the nitrogen-containing five-membered heterocyclic derivative of formula (II) is selected from:

[0068]

[0069]

[0070] In some embodiments, the compound of formula (II) or a pharmaceutically acceptable salt thereof, wherein:

[0071] R4 is -A-R5;

[0072] A is selected from:

[0073] (i) a key; or

[0074] (ii) a saturated chain of 2 to 4 chain members in length containing at least one carbon atom chain member, at least one heteroatom chain member selected from nitrogen, and optionally one or more further carbon atom chain members and / or heteroatom chain members selected from nitrogen, oxygen, sulfur, sulfinyl and sulfonyl; the saturated chain is optionally substituted with one or more heteroatoms selected from ═O, C 1-4 Substitution with hydrocarbyl and fluorine substituents, wherein two hydrocarbyl substituents on the same carbon atom may optionally be linked to form a ring of three to five ring members;

[0075] R5 is selected from:

[0076] (i) H, or

[0077] (ii) a saturated or unsaturated monocyclic 6-membered heterocyclic group, wherein the heterocyclic group contains 1-2 independently selected from N(R3) n wherein each R3 is selected from H or C1-C3 alkyl, and n is 0, 1 or 2.

[0078] In some embodiments, the compound of formula (II) or a pharmaceutically acceptable salt thereof, wherein:

[0079] R4 is -A-R5;

[0080] A is selected from:

[0081] (i) a key; or

[0082] (ii) –(CH2) 1-4 、-NH-(CH2) 1-3 ;

[0083] R5 is selected from:

[0084] (i) H, or

[0085] (ii)

[0086] In some embodiments, the nitrogen-containing five-membered heterocyclic derivative of formula (I) is

[0087]

[0088] in,

[0089] R1 is selected from C1-C6 alkyl, C1-C6 alkylene, C3-C8 cycloalkyl or absent;

[0090] R2 is selected from H, -NH2, unsaturated or saturated monocyclic, bicyclic or spirocyclic 4 to 10 membered heterocyclic group, wherein the heterocyclic group contains 1-2 independently selected from N (R3) n and O heteroatoms;

[0091] R4, R6 and R7 are independently -A-R5;

[0092] A is selected from:

[0093] (i) key;

[0094] (ii) CH2;

[0095] (iii) a saturated chain of 2 to 10 chain members in length containing at least one carbon atom chain member, at least one heteroatom chain member selected from nitrogen and oxygen, and optionally one or more further carbon atom chain members and / or heteroatom chain members selected from nitrogen, oxygen, sulfur, sulfinyl and sulfonyl; the saturated chain is optionally substituted with one or more heteroatoms selected from ═O, C 1-4 Substitution with hydrocarbyl and fluorine substituents, wherein two hydrocarbyl substituents on the same carbon atom may optionally be linked to form a ring of three to five ring members;

[0096] R5 is selected from:

[0097] (i) H,

[0098] (ii) CH2,

[0099] (iii) a saturated or unsaturated monocyclic 5-6 membered heterocyclic group, wherein the heterocyclic group contains 1-3 independently selected from N(R3) n , O or S heteroatom; optionally each R3 is selected from H or C1-C3 alkyl; n is 0, 1 or 2;

[0100] Or R4 and R6 together with the carbon to which they are attached form a 5, 6, 7 or 8 membered ring, wherein the ring is partially or fully unsaturated and is substituted or unsubstituted.

[0101] In some embodiments, the nitrogen-containing five-membered heterocyclic derivative of formula (III) is

[0102]

[0103] wherein R8 is -A-R5;

[0104] A is selected from:

[0105] (i) key;

[0106] (ii) CH2;

[0107] (iii) a saturated chain of 2 to 10 chain members in length containing at least one carbon atom chain member, at least one heteroatom chain member selected from nitrogen and oxygen, and optionally one or more further carbon atom chain members and / or heteroatom chain members selected from nitrogen, oxygen, sulfur, sulfinyl and sulfonyl; the saturated chain is optionally substituted with one or more heteroatoms selected from ═O, C 1-4 Substitution with hydrocarbyl and fluorine substituents, wherein two hydrocarbyl substituents on the same carbon atom may optionally be linked to form a ring of three to five ring members;

[0108] R5 is selected from:

[0109] (i) H,

[0110] (ii) CH2,

[0111] (iii) a saturated or unsaturated monocyclic 5-6 membered heterocyclic group, wherein the heterocyclic group contains 1-3 independently selected from N(R3) n , O or S heteroatom; optionally each R3 is selected from H or C1-C3 alkyl; n is 0, 1 or 2.

[0112] In some embodiments, the nitrogen-containing five-membered heterocyclic derivative of formula (IV) is

[0113]

[0114] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0115] R1 is selected from C1-C3 alkyl, C1-C4 alkylene, C3-C6 cycloalkyl or absent;

[0116] R2 is selected from H, -NH2, unsaturated or saturated monocyclic or spirocyclic 4 to 6 membered heterocyclic group, wherein the heterocyclic group contains 1-2 independently selected from N (R3) n and O heteroatoms.

[0117] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R1 is selected from methyl, ethyl, n-propyl, isopropyl, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or absent.

[0118] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R2 is selected from H, -NH2,

[0119] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R3 is selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,3-dimethylbutyl or 2,2-dimethylbutyl.

[0120] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound is selected from Table 1.

[0121] Table 1: Compounds

[0122]

[0123]

[0124]

[0125]

[0126] Also provided are salts of the compounds mentioned herein, such as pharmaceutically acceptable salts.The present invention also includes any or all stereochemical forms of the compounds, including any enantiomeric or diastereomeric forms, as well as any tautomeric or other forms.

[0127] Compounds as described in detail herein can be in purified form on the one hand, and compositions comprising purified form compounds are described in detail herein. Compositions comprising compounds or salts thereof as described in detail herein, such as compositions of substantially pure compounds are provided. In some embodiments, compositions containing compounds or salts thereof as described in detail herein are in substantially pure form. Unless otherwise stated, "substantially pure" refers to compositions containing no more than 75% impurities, wherein impurities represent compounds other than compounds containing most of the composition or its salt. In some embodiments, compositions of substantially pure compounds or their salts are provided, wherein the compositions contain no more than 25%, 20%, 15%, 10% or 5% impurities. In some embodiments, compositions of substantially pure compounds or their salts are provided, wherein the compositions contain or do not exceed 3%, 2%, 1% or 0.5% impurities.

[0128] Representative compounds of formula (I) are listed in Table 1. It should be understood that the individual enantiomers and diastereomers are included in the general compound structures shown in Table 1. Examples of specific synthetic methods for preparing Compound Nos. 1.1 to 1.22 and 2.1 to 2.7 in Table 1 are provided herein.

[0129] In some embodiments, provided herein are compounds described in Table 1, or salts, polymorphs, solvates, enantiomers, stereoisomers or tautomers thereof, and uses thereof.

[0130] The embodiments and variations described herein apply to compounds of any formula detailed herein, as applicable.

[0131] Representative examples of compounds detailed herein are described herein, including intermediates and final compounds according to the present disclosure. It should be understood that in one aspect, any compound can be used in the methods detailed herein, including, if applicable, intermediate compounds that can be isolated and applied to individuals.

[0132] The compounds described herein may exist as salts, even if no salts are described, and it is understood that the present disclosure covers all salts and solvates of the compounds described herein, as well as non-salt and non-solvate forms of the compounds, as well as those skilled in the art. In some embodiments, the salts of the compounds provided herein are pharmaceutically acceptable salts. In some embodiments, the pharmaceutically acceptable salts are formate.

[0133] When any compound described herein may exist in tautomeric forms, even though only one or a few tautomeric forms may be explicitly described, each and every tautomeric form is intended to be described. The specifically described tautomeric form may or may not be the predominant form in solution or when used according to the methods described herein.

[0134] The present disclosure also includes any or all stereochemical forms of the compounds, including any enantiomeric or diastereomeric forms. The structure or name is intended to cover all possible stereoisomers of the compounds described. The present invention also covers all forms of the compounds, such as crystalline or non-crystalline forms of the compounds. The present invention is also intended to cover compositions comprising the compounds of the present invention, such as compositions of substantially pure compounds, including their specific stereochemical forms, or compositions comprising mixtures of the compounds of the present invention in any ratio, including two or more stereochemical forms, such as racemic or non-racemic mixtures.

[0135] The present invention is also intended to include isotopically labeled and / or isotopically enriched forms of the compounds described herein. The compounds herein may contain unnatural proportions of atomic isotopes at one or more of the atoms that make up such compounds. In some embodiments, the compounds are isotopically labeled, such as isotopically labeled compounds of Formula (I) or its variants described herein, wherein a portion of one or more atoms are replaced by isotopes of the same element. Exemplary isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, chlorine, for example 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15O. 17 O. 32 P. 35 S. 18 F. 36 Cl. Certain isotope-labeled compounds (e.g. 3 H and 14 C) can be used to study the tissue distribution of compounds or substrates. 2 H)) may offer certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and may therefore be preferred in certain circumstances.

[0136] Isotopically labeled compounds of the invention can generally be prepared by standard methods and techniques known to those skilled in the art or by procedures analogous to those described in the accompanying examples by substituting an appropriate isotopically labeled reagent for the corresponding non-labeled reagent.

[0137] The present invention also includes any or all metabolites of any of the compounds described. Metabolites may include any chemical species generated by the biotransformation of any of the compounds described, such as intermediates and products of compound metabolism, such as would be generated in vivo after administration to a human.

[0138] Provided are articles of manufacture comprising a compound described herein, or a salt or solvate thereof, in a suitable container. The container may be a vial, a can, an ampoule, a prefilled syringe, an intravenous bag, etc.

[0139] Preferably, the compounds detailed herein are orally bioavailable. However, the compounds may also be formulated for parenteral (eg, intravenous) administration.

[0140] One or more compounds described herein can be used to prepare a medicament by combining the compound or compounds as active ingredients with a pharmacologically acceptable carrier known in the art. Depending on the therapeutic form of the medicament, the carrier can have various forms. In one variation, the manufacture of the medicament is used in any method disclosed herein, for example, for treating cancer.

[0141] General synthetic scheme

[0142] The compounds of the present invention can be prepared by a number of methods as generally described below and more specifically in the Examples below (e.g., the schemes provided in the Examples below). In the following process descriptions, when symbols are used in the described formulae, they should be understood to represent the above-mentioned groups associated with the formulae herein.

[0143] When it is necessary to obtain a specific enantiomer of a compound, this can be achieved from a corresponding enantiomeric mixture using any suitable conventional procedure for separating or resolving enantiomers. Therefore, for example, diastereomeric derivatives can be produced by reacting an enantiomeric mixture (e.g., racemate) with an appropriate chiral compound. The diastereomer can then be separated by any convenient means (e.g., by crystallization) and the desired enantiomer can be recovered. In another method of resolution, a chiral high performance liquid chromatography can be used to separate the racemate. Alternatively, if desired, a specific enantiomer can be obtained by using an appropriate chiral intermediate in one of the methods.

[0144] When it is desired to obtain a specific isomer of a compound or to otherwise purify a reaction product, chromatography, recrystallization and other conventional separation procedures may also be employed on the intermediates or final products.

[0145] Pharmaceutical compositions and preparations

[0146] The present disclosure covers pharmaceutical compositions of any compound detailed herein. Thus, the present disclosure includes pharmaceutical compositions comprising a compound as detailed herein or a salt thereof and a pharmaceutically acceptable carrier or excipient. In one aspect, a pharmaceutically acceptable salt is an acid addition salt, such as a salt formed with an inorganic acid or an organic acid. In some embodiments, a pharmaceutically acceptable salt is a formate.

[0147] The pharmaceutical composition may be in a form suitable for oral, buccal, parenteral, nasal, topical or rectal administration, or in a form suitable for administration by inhalation.

[0148] Compounds as described in detail herein can be in purified form on the one hand, and compositions comprising compounds in purified form are described in detail herein. Compositions comprising compounds or salts thereof as described in detail herein are provided, e.g., compositions of substantially pure compounds. In some embodiments, compositions containing compounds or salts thereof as described in detail herein are in substantially pure form.

[0149] In one variation, the compounds herein are synthetic compounds prepared for administration to an individual. In another variation, compositions containing the compounds in substantially pure form are provided. In another variation, the disclosure covers pharmaceutical compositions comprising the compounds detailed herein and a pharmaceutically acceptable carrier. In another variation, methods of administering the compounds are provided. The purified forms, pharmaceutical compositions, and methods of administering the compounds are suitable for any compound or form thereof detailed herein.

[0150] The compounds or salts thereof described in detail herein can be formulated for any available delivery route, including oral, mucosal (e.g., nasal, sublingual, vaginal, buccal or rectal), parenteral (e.g., intramuscular, subcutaneous or intravenous), topical or transdermal delivery forms. The compounds or salts thereof can be formulated with a suitable carrier to provide a delivery form, including but not limited to tablets, caplets, capsules (e.g., hard gelatin capsules or soft elastic gelatin capsules), cachets, lozenges, lozenges, gums, dispersions, suppositories, ointments, cataplasms (mud compresses), pastes, powders, dressings, creams, solutions, patches, aerosols (e.g., nasal sprays or inhalers), gels, suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions or water-in-oil liquid emulsions), solutions and elixirs.

[0151] Compositions comprising the compounds provided herein are also described. In one variation, the composition comprises the compound or its salt and a pharmaceutically acceptable carrier or excipient. In another variation, a composition of a substantially pure compound is provided.

[0152] How to use

[0153] The compounds and compositions described in detail herein, such as pharmaceutical compositions containing a compound of any formula provided herein or a salt thereof and a pharmaceutically acceptable carrier or excipient, can be used in methods of administration and treatment as provided herein. The compounds and compositions can also be used in in vitro methods, such as in vitro methods of administering a compound or composition to a cell for screening purposes and / or for performing quality control assays.

[0154] Provided herein is a method for treating an individual disease, comprising administering to the individual an effective amount of a compound of the present invention (collectively referred to as a compound of formula (I) or any embodiment, variant or aspect thereof or a compound of the present invention or a compound described or described herein) or a pharmaceutically acceptable salt thereof. Also provided herein is a method for treating an individual proliferative disease, comprising administering to the individual an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof. Also provided herein is a method for treating an individual cancer, pulmonary arterial hypertension (PAH) or idiopathic pulmonary fibrosis (IPF), comprising administering to the individual an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is administered to the individual according to the dosage and / or administration method described herein.

[0155] Another aspect of the present invention relates to a method for treating a disease or condition associated with a checkpoint kinase. The method involves administering an effective amount of the compositions and compounds of the present invention to a patient in need of treatment for a disease or condition associated with a checkpoint kinase.

[0156] Another aspect of the present invention relates to a method of inhibiting checkpoint kinases. The method involves administering an effective amount of a composition or compound of the present invention (collectively referred to as a compound of formula (I)) to a patient in need thereof.

[0157] Use of the compounds of the present invention for the preparation of a medicament for the treatment of proliferative diseases such as cancer, pulmonary arterial hypertension (PAH) and idiopathic pulmonary fibrosis (IPF).

[0158] The use of the compound of the present invention for preparing a medicament for treating cancer, wherein the cancer is selected from cancer, such as bladder cancer, breast cancer, colon cancer, kidney cancer, epidermal cancer, liver cancer, lung cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer, gastrointestinal cancer or skin cancer; hematopoietic system tumors such as leukemia, B cell lymphoma, T cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma or Burkett lymphoma; myeloid hematopoietic system tumors, such as acute and chronic myeloid leukemia, myelodysplasia Syndrome or promyelocytic leukemia; p53 negative or mutated tumors; MYC oncogene-driven cancers, such as B-cell lymphoma, leukemia, neuroblastoma, breast cancer or lung cancer; thyroid follicular carcinoma; mesenchymal tumors, such as fibrosarcoma or rhabdomyosarcoma; central or peripheral nervous system tumors, such as astrocytoma, neuroblastoma, glioma or schwannoma; melanoma; seminoma; teratocarcinoma; osteosarcoma; xeroderma pigmentosum; keratotic squamous cell tumor; thyroid follicular carcinoma; Ewing's sarcoma or Kaposi's sarcoma. The compounds of the present invention (collectively referred to as compounds of formula (I)) are used for the preparation of a medicament for the treatment of cancer, wherein the cancer is a cancer characterized by a defect in a DNA repair mechanism or a cell cycle defect.

[0159] The compounds of the present invention (collectively referred to as compounds of formula (I)) are used for the preparation of a medicament for the treatment of idiopathic pulmonary fibrosis (IPF). IPF is a chronic scarring lung disease characterized by a gradual and irreversible decline in lung function. It is closely related to concurrent pulmonary hypertension, in which there is abnormal proliferation of arterial cells, vascular remodeling, inflammation, and differentiation of fibroblasts into myofibroblasts. The goals of IPF treatment are to manage symptoms, delay disease progression, prevent acute exacerbations, and prolong survival.

[0160] The compounds of the present invention are used for the preparation of a medicament for the treatment of pulmonary arterial hypertension (PAH). Pulmonary arterial hypertension (PAH) is a debilitating disease associated with progressive vascular remodeling of the distal pulmonary artery, which can lead to elevated pulmonary artery pressure, right ventricular hypertrophy and death. Despite showing high levels of DNA damage that would normally compromise their viability, pulmonary artery smooth muscle cells (PASMCs) from PAH patients exhibit a cancer-like pro-proliferation and anti-apoptotic phenotype, which leads to vascular lumen occlusion. CHK1 expression is significantly increased in isolated PASMCs and distal PAs from PAH patients.

[0161] Using pharmacological and molecular loss-of-function approaches, it has been described that CHK1 promotes PAH-PASMC proliferation and resistance to apoptosis. In vivo, pharmacological inhibition of CHK1 significantly reduced vascular remodeling and improved hemodynamic parameters in an experimental rat model of PAH.

[0162] The compounds disclosed herein may be administered in an amount effective to treat or prevent a disorder and / or prevent the development of a disorder in a subject.

[0163] The present invention can be further understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting.

[0164] Example

[0165] Example-1: Synthesis of 5-(5-(2-(morpholin-2-ylmethoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.1)

[0166]

[0167] Step-1: Synthesis of 2-(3-aminoisoxazol-5-yl)phenol: To a solution of 5-(2-methoxyphenyl)isoxazol-3-amine (synthesized as described in WO2020 / 002587A1) (1.4 g, 7.36 mmol, 1.0 equiv) in DCM (10 mL) was added boron tribromide (2.1 mL, 22.08 mmol, 3.0 equiv) at 0°C. The resulting reaction mixture was allowed to reach room temperature and stirred for 10 hours. Product formation was confirmed by TLC and LCMS. After completion of the reaction, the reaction mixture was added dropwise to ice water (30 mL). The resulting product was filtered and dried under vacuum to obtain 2-(3-aminoisoxazol-5-yl)phenol (980 mg). LCMS: 177.3 [M+1] +

[0168] Step-2: Synthesis of tert-butyl 2-((2-(3-aminoisoxazol-5-yl)phenoxy)methyl)morpholine-4-carboxylate: To a solution of 2-(3-aminoisoxazol-5-yl)phenol (0.426 g, 2.42 mmol, 1.0 equiv) in DMF (6 mL) were added tert-butyl 2-(tosyloxymethyl)morpholine-4-carboxylate (synthesized as shown in WO2014151616A1) (988 mg, 2.66 mmol, 1.1 equiv) and Cs2CO3 (1.1 g, 3.39 mmol, 1.4 equiv). The resulting reaction mixture was added.

[0169] The mixture was stirred at 90°C for 16 hours. The product formation was confirmed by TLC and LCMS. After the reaction was completed, the reaction mixture was quenched with ice water (30 mL) and washed with ethyl acetate (2×20

[0170] The collected organic layer was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired product (420 mg). LCMS: 376.3 [M+1] +

[0171] Step-3: Synthesis of tert-butyl 2-((2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)methyl)morpholine-4-carboxylate: A solution of tert-butyl 2-((2-(3-aminoisoxazol-5-yl)phenoxy)methyl)morpholine-4-carboxylate (300 mg, 0.8 mmol, 1.0 eq.) and 5-chloropyrazine-2-carbonitrile (134 mg, 0.96 mmol, 1.2 eq.) in toluene (9 mL) was purged with nitrogen.

[0172] 5 minutes, then Cs2CO3 (780 mg, 2.4 mmol, 3.0 equiv) was added. The resulting reaction mixture was purged again for 5 minutes, then Xantphos (28 mg, 0.048 mmol,

[0173] 0.06 equiv.) and Pd2dba3 (58 mg, 0.064 mmol, 0.08 equiv.), and the reaction mixture was subjected to microwave treatment at 130 ° C for 1 hour. The product formation was confirmed by TLC and LCMS. After the reaction was completed, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (3×15 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12 g The crude product was purified by flash chromatography on a column and eluted in an ethyl acetate-hexane solvent system to obtain the desired compound (120 mg). LCMS: 479.2 [M+1] +

[0174] Step-4: Synthesis of 5-(5-(2-(morpholin-2-ylmethoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 2-((2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)methyl)morpholine-4-carboxylate (100 mg, 0.209 mmol, 1.0 eq.) in DCM (5 mL) was added TFA (0.4 mL) at 0°C and kept stirring for 2 hours. The reaction mixture was then concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound\ (18 mg, formate salt). LCMS: 379.4 [M+1] + UPLC:97.99@220nm99.22@254nm. 1 H NMR (400 MHz, DMSO-d6, after adding D2O) δ8.73 (s, 1H), 8.55 (s, 1H), 8.32 (s, 1H, formate), 7.84 (d, J = 7.7 Hz, 1H), 7.48 (t, J = 7.9 Hz, 1H), 7.30 (s, 1H), 7.20-7.08 (m, 2H), 4.22-4.09 (m, 2H), 3.97-3.89 (m, 2H), 3.67 (t, J = 11.8 Hz, 1H), 3.17 (t, J = 11.9 Hz, 1H), 3.01 (d, J = 12.8 Hz, 1H), 2.85 (q, J = 11.4 Hz, 2H).

[0175] Example-2: Synthesis of 5-(5-(2-(piperidin-4-ylmethoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.2)

[0176]

[0177] Step-1: Synthesis of 2-(3-aminoisoxazol-5-yl)phenol: Same as described in compound 1.1

[0178] Step-2: Synthesis of tert-butyl 4-((2-(3-aminoisoxazol-5-yl)phenoxy)methyl)piperidine-1-carboxylate: To a solution of 2-(3-aminoisoxazol-5-yl)phenol (476 mg, 2.7 mmol, 1.0 eq.) in DMF (8 mL) was added tert-butyl 4-(tosyloxymethyl)piperidine-1-carboxylate (synthesized as shown in WO2020028724A1) (998 mg, 2.7 mmol, 1.0 eq.) and Cs2CO3 (1.14 g, 3.5 mmol, 1.3 eq.). The resulting reaction mixture was stirred at 90 ° C for 16 hours. Product formation was confirmed by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with ice water (30 mL) and extracted with ethyl acetate (2×20 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12g The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired product (420 mg). LCMS: 374.3 [M+1] +

[0179] Step 3: Synthesis of tert-butyl 4-((2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)methyl)piperidine-1-carboxylate: A solution of tert-butyl 4-((2-(3-aminoisoxazol-5-yl)phenoxy)methyl)piperidine-1-carboxylate (150 mg, 0.40 mmol, 1.0 eq) and 5-chloropyrazine-2-carbonitrile (67 mg, 0.48 mmol, 1.2 eq) in toluene (5 mL) was purged with nitrogen for 5 min, and then CsCO (393 mg, 1.20 mmol, 3.0 eq) was added. The resulting reaction mixture was purged again for 5 min, and then Xantphos (14 mg, 0.024 mmol, 0.06 eq) and Pddba (29 mg, 0.032 mmol, 0.08 eq) were added, and the reaction mixture was microwaved at 140 °C for 45 min. The product formation was confirmed by TLC and LCMS. After the reaction was completed, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (3 × 15 mL). The collected organic layer was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12 g The crude product was purified by flash chromatography on a column and eluted in ethyl acetate: hexane solvent system to obtain the desired compound (60 mg). LCMS: 477.3 [M+1] +

[0180] Step-4: Synthesis of 5-(5-(2-(piperidin-4-ylmethoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 4-((2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)methyl)piperidine-1-carboxylate (60 mg, 0.126 mmol, 1.0 eq.) in DCM (5 mL) was added TFA (0.4 mL) at 0°C and kept stirring for 2 hours. The reaction mixture was then concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound (10 mg, formate salt). LCMS: 377.3 [M+1] + UPLC:96.32@220nm 96.41@254nm; 1 H NMR (400 MHz, methanol-d4) δ 8.65 (d, J = 11.6 Hz, 2H), 8.55 (s, 1H, formate), 7.90 (dd, J = 8.0, 1.8 Hz, 1H), 7.53-7.45 (m, 1H), 7.26 (s, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 4.11 (d, J = 6.4 Hz, 2H), 3.46 (d, J = 12.0 Hz, 2H), 3.07 (td, J = 12.9, 2.9 Hz, 2H), 2.32 (s, 1H), 2.19 (d, J = 14.3 Hz, 2H), 1.69-1.56 (m, 2H).

[0181] Example-3: Synthesis of 5-(5-(2-(piperidin-3-ylmethoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.3)

[0182]

[0183] Step-1: Synthesis of 2-(3-aminoisoxazol-5-yl)phenol: Same as described in compound 1.1

[0184] Step-2: Synthesis of tert-butyl 3-((2-(3-aminoisoxazol-5-yl)phenoxy)methyl)piperidine-1-carboxylate: To a solution of 2-(3-aminoisoxazol-5-yl)phenol (286 mg, 1.62 mmol, 1.0 eq.) in DMF (8 mL) were added tert-butyl 3-(tosyloxymethyl)piperidine-1-carboxylate (synthesized as shown in J. Med. Chem. 2020, 63(19), 11054-84) (600 mg, 1.62 mmol, 1.0 eq.) and Cs2CO3 (688 mg, 2.11 mmol, 1.3 eq.). The resulting reaction mixture was stirred at 90 °C for 16 h. Product formation was confirmed by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with ice water (30 mL) and extracted with ethyl acetate (2×20 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired product (240 mg). LCMS: 374.3 [M+1] +

[0185] Step-3: Synthesis of tert-butyl 3-((2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)methyl)piperidine-1-carboxylate: A solution of tert-butyl 3-((2-(3-aminoisoxazol-5-yl)phenoxy)methyl)piperidine-1-carboxylate (150 mg, 0.40 mmol, 1.0 equiv) and 5-chloropyrazine-2-carbonitrile (67 mg, 0.48 mmol, 1.2 equiv) in toluene (5 mL) was purged with nitrogen for 5 min and then Cs2CO3 (393 mg, 1.20 mmol, 3.0 equiv) was added. The resulting reaction mixture was purged again for 5 minutes, then Xantphos (14 mg, 0.024 mmol, 0.06 equiv) and Pd2dba3 (29 mg, 0.032 mmol, 0.08 equiv) were added, and the reaction mixture was subjected to microwave treatment at 130 ° C for 45 minutes. The product formation was confirmed by TLC and LCMS. After the reaction was completed, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (3 × 15 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12g The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired compound. LCMS: 477.3 [M+1] +

[0186] Step-4: Synthesis of 5-(5-(2-(piperidin-3-ylmethoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 3-((2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)methyl)piperidine-1-carboxylate (60 mg, 0.126 mmol, 1.0 eq.) in DCM (5 mL) was added TFA (0.4 mL) at 0°C and kept stirring for 2 hours. The reaction mixture was then concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound (15 mg, formate salt). LCMS: 377.3 [M+1] + UPLC:99.31@220nm 99.57@254nm; 1 H NMR (400 MHz, methanol-d4) δ 8.64 (d, J = 8.8 Hz, 2H), 8.55 (s, 1H, formate), 7.91-7.84 (m, 1H), 7.49 (t, J = 7.7 Hz, 1H), 7.26 (s, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.13 (t, J = 7.6 Hz, 1H), 4.62 (s, 1H), 4.15 (qd, J = 9.6,6.1Hz,2H),3.54(d,J=12.5Hz,1H),2.93(ddt,J=13.4,10.0,4.5Hz,2H),2.38(s,1H),2. 13(d,J=18.8Hz,1H),2.07-1.94(m,1H),1.81(t,J=13.2Hz,1H),1.56(q,J=15.2,13.9Hz,1H).

[0187] Example-4: Synthesis of 5-(5-(2-(azetidin-3-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.4)

[0188]

[0189] Step-1: Synthesis of 2-(3-aminoisoxazol-5-yl)phenol: Same as described in compound 1.1

[0190] Step-2: Synthesis of tert-butyl 3-(2-(3-aminoisoxazol-5-yl)phenoxy)azetidine-1-carboxylate: To a solution of 2-(3-aminoisoxazol-5-yl)phenol (310 mg, 1.76 mmol, 1.0 eq.) in DMF (5 mL) were added tert-butyl 3-iodoazetidine-1-carboxylate (498 mg, 1.76 mmol, 1.0 eq.) and Cs2CO3 (803 mg, 2.46 mmol, 1.4 eq.). The resulting reaction mixture was stirred at 90 ° C for 16 hours. Product formation was confirmed by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with ice water (30 mL) and extracted with ethyl acetate (2×20 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12 g The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired product (210 mg). LCMS: 332.3 [M+1] +

[0191] Step-3: Synthesis of tert-butyl 3-(2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)azetidine-1-carboxylate: A solution of tert-butyl 3-(2-(3-aminoisoxazol-5-yl)phenoxy)azetidine-1-carboxylate (200 mg, 0.604 mmol, 1.0 equiv) and 5-chloropyrazine-2-carbonitrile (101 mg, 0.725 mmol, 1.2 equiv) in toluene (5 mL) was purged with N2 for 5 min and then Cs2CO3 (590 mg, 1.81 mmol, 3.0 equiv) was added. The resulting reaction mixture was purged again for 5 minutes, then Xantphos (21 mg, 0.036 mmol, 0.06 equiv) and Pd2dba3 (44 mg, 0.048 mmol, 0.08 equiv) were added, and the reaction mixture was subjected to microwave treatment at 140 ° C for 45 minutes. The product formation was confirmed by TLC and LCMS. After the reaction was completed, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (3 × 15 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12g The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired compound (70 mg). LCMS: 435.3 [M+1] +

[0192] Step-4: Synthesis of 5-(5-(2-(azetidin-3-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 3-(2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)azetidine-1-carboxylate (70 mg, 0.161 mmol, 1.0 eq.) in DCM (4.0 mL) was added TFA (0.4 mL) at 0°C and kept stirring for 2 hours. The product formation was confirmed by TLC and LCMS. The reaction mixture was then concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound (40 mg, trifluoroacetate). LCMS: 335.3 [M+1] + 99.73@220nm99.93@254nm. 1 H NMR (400MHz, DMSO-d6, after adding D2O) δ8.80 (s, 1H), 8.60 (s, 1H), 7.91 (dd, J = 7.7, 1.7Hz, 1H), 7.50 (t, J = 7.7Hz, 1H), 7.45 (s, 1H), 7.22 (t,J=7.6Hz,1H),6.91(d,J=8.3Hz,1H),5.26(h,J=6.0,5.3Hz,1H),4.56(dd,J=12.3,6.6Hz,2H),4.10(dd,J=12.6,4.6Hz,2H).

[0193] Example-5: Synthesis of 5-(5-(2-(3-aminopropoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.5)

[0194]

[0195] Step-1: Synthesis of 2-(3-aminoisoxazol-5-yl)phenol: Same as described in compound 1.1

[0196] Step-2: Synthesis of tert-butyl 3-(2-(3-aminoisoxazol-5-yl)phenoxy)propylcarbamate: To a solution of 2-(3-aminoisoxazol-5-yl)phenol (270 mg, 1.53 mmol, 1.0 equiv.) in DMF (5 mL) were added 3-(tert-butoxycarbonylamino)propyl 4-methylbenzenesulfonate (synthesized as shown in WO2020251971A1) (505 mg, 1.53 mmol, 1.0 equiv.) and Cs2CO3 (699 mg, 2.15 mmol, 1.4 equiv.). The resulting reaction mixture was stirred at 90 ° C for 16 hours. Product formation was confirmed by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with ice water (30 mL) and extracted with ethyl acetate (2×20 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12 g The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired product (180 mg). LCMS: 334.3 [M+1] +

[0197] Step-3: Synthesis of tert-butyl 3-(2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)propylcarbamate: A solution of tert-butyl 3-(2-(3-aminoisoxazol-5-yl)phenoxy)propylcarbamate (150 mg, 0.45 mmol, 1.0 eq.) and 5-chloropyrazine-2-carbonitrile (75 mg, 0.54 mmol, 1.2 eq.) in toluene (3 mL) was purged with N2 for 5 minutes and then Cs2CO3 (439 mg, 1.35 mmol, 3.0 eq.) was added. The resulting reaction mixture was purged again for 5 minutes and then Xantphos (16 mg, 0.027 mmol, 0.06 eq.) and Pd2dba3 (33 mg, 0.036 mmol, 0.08 eq.) were added and the reaction mixture was subjected to microwave treatment at 140°C for 45 minutes. The product formation was confirmed by TLC and LCMS. After the reaction was completed, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (3 × 15 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12 g The crude product was purified by flash chromatography on a column and eluted in ethyl acetate: hexane solvent system to obtain the desired compound (50 mg). LCMS: 437.3 [M+1] +

[0198] Step-4: Synthesis of 5-(5-(2-(3-aminopropoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 3-(2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)propylcarbamate (50 mg, 0.11 mmol, 1.0 eq.) in DCM (3.0 mL) was added TFA (0.3 mL) at 0°C and kept stirring for 2 hours. The formation of the product was confirmed by TLC and LCMS. The reaction mixture was then concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound (17 mg, formate salt). LCMS: 337.3 [M+1] + ; 96.86@220nm 98.44@254nm. 1 H NMR (400 MHz, methanol-d4) δ 8.79 (s, 1H), 8.60 (s, 1H), 8.41 (s, 1H, formate), 7.87 (d, J = 7.6 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.33 (s, 1H), 7.22 (d, J = 8.5 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 4.24 (t, J = 6.1 Hz, 2H), 3.07 (t, J = 7.3 Hz, 2H), 2.14 (p, J = 6.5 Hz, 2H).

[0199] Example-6: Synthesis of 5-(5-(2-(4-aminobutyloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.6)

[0200]

[0201] Step-1: Synthesis of 2-(3-aminoisoxazol-5-yl)phenol: Same as described in compound 1.1

[0202] Step-2: Synthesis of tert-butyl 4-(2-(3-aminoisoxazol-5-yl)phenoxy)butylcarbamate: To a solution of 2-(3-aminoisoxazol-5-yl)phenol (205 mg, 1.16 mmol, 1.0 equiv.) in DMF (3 mL), 4-(tert-butoxycarbonylamino)butyl 4-methylbenzenesulfonate (synthesized as shown in WO2021155321A2) (400 mg, 1.16 mmol, 1.0 equiv.) and Cs2CO3 (531 mg, 1.63 mmol, 1.4 equiv.) were added. The resulting reaction mixture was stirred at 90 ° C for 16 hours. Product formation was confirmed by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with ice water (30 mL) and extracted with ethyl acetate (2×20 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12g The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired product (150 mg). LCMS: 348.3 [M+1] +

[0203] Step-3: Synthesis of tert-butyl 4-(2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)butylcarbamate: A solution of tert-butyl 4-(2-(3-aminoisoxazol-5-yl)phenoxy)butylcarbamate (140 mg, 0.403 mmol, 1.0 equiv) and 5-chloropyrazine-2-carbonitrile (67 mg, 0.483 mmol, 1.2 equiv) in toluene (5 mL) was purged with N2 for 5 minutes and then Cs2CO3 (393 mg, 1.21 mmol, 3.0 equiv) was added. The resulting reaction mixture was purged again for 5 minutes and then Xantphos (14.0 mg, 0.024 mmol, 0.06 equiv) and Pd2dba3 (30 mg, 0.032 mmol, 0.08 equiv) were added and the reaction mixture was subjected to microwave treatment at 140°C for 45 minutes. The product formation was confirmed by TLC and LCMS. After the reaction was completed, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (3 × 15 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12 g The crude product was purified by flash chromatography on a column and eluted in ethyl acetate: hexane solvent system to obtain the desired compound (90 mg). LCMS: 451.4 [M+1] +

[0204] Step-4: Synthesis of 5-(5-(2-(4-aminobutoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 4-(2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)butylcarbamate (90 mg, 0.20 mmol, 1.0 eq.) in DCM (5.0 mL) was added TFA (0.3 mL) at 0°C and kept stirring for 2 hours. The formation of the product was confirmed by TLC and LCMS. The reaction mixture was then concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound (29 mg, formate salt). LCMS: 351.4 [M+1] + 95.84@220nm. 1 H NMR (400MHz, DMSO-d6) δ8.75 (s, 1H), 8.62 (s, 1H), 8.41 (s, 1H, formate), 7.86 (d, J = 8.1Hz, 1H), 7.50 (t, J = 7.8Hz, 1H), 7.32 (s, 1H), 7.22 (d,J=8.5Hz,1H),7.12(t,J=7.6Hz,1H),4.17(t,J=6.1Hz,2H),2.86(t,J=7.5Hz,2H),1.91(p,J=6.6Hz,2H),1.77(p,J=7.5Hz,2H).

[0205] Example-7: Synthesis of 5-(5-(2-(2-aminoethoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.7)

[0206]

[0207] Step-1: Synthesis of 2-(3-aminoisoxazol-5-yl)phenol: Same as described in compound 1.1

[0208] Step 2: Synthesis of tert-butyl 2-(2-(3-aminoisoxazol-5-yl)phenoxy)ethylcarbamate: To a solution of 2-(3-aminoisoxazol-5-yl)phenol (250 mg, 1.42 mmol, 1.0 equiv.) in DMF (3 mL), tert-butyl 2-bromoethylcarbamate (318 mg, 1.42 mmol, 1.0 equiv.) and Cs2CO3 (601 mg, 1.84 mmol, 1.3 equiv.) were added. The resulting reaction mixture was stirred at 90 ° C for 16 hours. Product formation was confirmed by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with ice water (30 mL) and extracted with ethyl acetate (2×20 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12 g The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired product (210 mg). LCMS: 320.4 [M+1] +

[0209] Step-3: Synthesis of tert-butyl 2-(2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)ethylcarbamate: A solution of tert-butyl 2-(2-(3-aminoisoxazol-5-yl)phenoxy)ethylcarbamate (200 mg, 0.626 mmol, 1.0 eq.) and 5-chloropyrazine-2-carbonitrile (105 mg, 0.75 mmol, 1.2 eq.) in toluene (5 mL) was purged with N2 for 5 minutes and then Cs2CO3 (612 mg, 1.88 mmol, 3.0 eq.) was added. The resulting reaction mixture was purged again for 5 minutes and then Xantphos (21 mg, 0.037 mmol, 0.06 eq.) and Pd2dba3 (45 mg, 0.050 mmol, 0.08 eq.) were added and the reaction mixture was subjected to microwave treatment at 140°C for 45 minutes. The product formation was confirmed by TLC and LCMS. After the reaction was completed, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (3 × 15 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12 g The crude product was purified by combined flash chromatography by eluting in ethyl acetate: hexane solvent system to obtain the desired compound (70 mg). LCMS: 423.3 [M+1] +

[0210] Step-4: Synthesis of 5-(5-(2-(2-aminoethoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 2-(2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)ethylcarbamate (70 mg, 0.17 mmol, 1.0 eq.) in DCM (5.0 mL) was added TFA (0.4 mL) at 0°C and kept stirring for 2 hours. The formation of the product was confirmed by TLC and LCMS. The reaction mixture was then concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound (20 mg, formate salt). LCMS: 323.3 [M+1] + ; 99.96@220nm 99.97@254nm. 1H NMR (400 MHz, DMSO-d6, after adding D2O) δ8.78 (s, 1H), 8.60 (s, 1H, formate), 8.26 (s, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.52 (t, J = 7.9 Hz, 1H), 7.36 (s, 1H), 7.24 (d, J = 8.5 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 4.31 (t, J = 5.4 Hz, 2H), 3.29 (t, J = 5.3 Hz, 2H).

[0211] Example-8: Synthesis of 5-(5-(2-((4-methylmorpholin-2-yl)methoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.8)

[0212]

[0213] Step-1: Synthesis of 5-(5-(2-((4-methylmorpholin-2-yl)methoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a stirred solution of 5-(5-(2-(morpholin-2-ylmethoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (70 mg, 0.185 mmol, 1 eq.) in dichloroethane were added acetic acid (0.1 mL) and formaldehyde (37% solution) (0.1 mL, 1.11 mmol, 6 eq.). The resulting reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then cooled to 0°C and sodium cyanoborohydride (58 mg, 0.925 mmol, 5 eq.) was added thereto. The reaction mixture was stirred at room temperature for 3 hours. The product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with saturated sodium bicarbonate (2×5 mL), the organic layer was separated and concentrated in vacuo to afford the crude product. The crude product was purified by R-HPLC to obtain the desired compound (8 mg, formate salt). LCMS: 393.5[M+1] + 97.91@220nm; 99.10@254nm. 1H NMR(400MHz,DMSO-d6)δ11.32(br.s.,1H,NH),8.83-8.79(m,1H),8.63(s,1H),8.1 5(s,1H, formate CH),7.88(dd,J=7.87,1.19Hz,1H),7.53-7.47(m,1H),7.39(s,1H),7. 23(d,J=8.58Hz,1H),7.13(t,J=7.63Hz,1H),4.16(d,J=5.25Hz,2H),3.99-3.91(m ,2H),3.87(d,J=11.44Hz,2H),3.66-3.55(m,1H),2.18(s,3H),2.06-1.89(m,2H).

[0214] Example-9: Synthesis of 5-(3-(2-methoxy-4-(1,2,3,6-tetrahydropyridin-4-yl)phenyl)isoxazol-5-ylamino)pyrazine-2-carbonitrile (Compound 1.9)

[0215]

[0216] Step-1: Synthesis of methyl 4-bromo-2-methoxybenzoate: The synthesis was carried out as shown in WO2006044775A2.

[0217] Step-2: Synthesis of 3-(4-bromo-2-methoxyphenyl)-3-oxopropionitrile: To a solution of methyl 4-bromo-2-methoxybenzoate (5.0 g, 20.4 mmol, 1.0 equiv.) in THF (50 mL) was added dropwise a 1.0 M solution of LiHMDS in THF (30.6 mL, 30.6 mmol, 1.5 equiv.) at -78°C. The resulting reaction mixture was stirred at the same temperature for 2 hours. Acetonitrile (15 mL) was then added dropwise to the reaction mixture and stirred for 1 hour. The product formation was confirmed by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with a saturated ammonium chloride solution (100 mL) and extracted with ethyl acetate (3×50 mL), the organic layer was separated, and washed with saturated brine (50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain the desired compound (4.5 g). LCMS: 254.3 [M+1] +

[0218] Step-3: Synthesis of 3-(4-bromo-2-methoxyphenyl)isoxazol-5-amine (1b) and 5-(4-bromo-2-methoxyphenyl)isoxazol-3-amine (1a): To a solution of 3-(4-bromo-2-methoxyphenyl)-3-oxopropionitrile (2.0 g, 7.87 mmol, 1.0 eq.) in EtOH:H2O (20 mL:8 mL) were added hydroxylamine hydrochloride (602 mg, 8.66 mmol, 1.1 eq.) and sodium hydroxide (346 mg, 8.66 mmol, 1.1 eq.) and stirred at 80°C for 16 hours. The progress of the reaction was monitored by TLC and LCMS. After completion of the reaction, it was quenched with saturated sodium bicarbonate solution (50 mL) and extracted with ethyl acetate (3×50 mL), the organic layer was separated and washed with saturated brine (50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired compounds 1a (1.2 g) and 1b (0.65 g). LCMS: 1a: 269.2 [M] + ; 271.2[M+2] + ; 1b:269.3[M] + ; 271.3[M+2] +

[0219] Step-4: Synthesis of tert-butyl 4-(4-(5-aminoisoxazol-3-yl)-3-methoxyphenyl)-5,6-dihydropyridine-1(2H)-carboxylate: A solution of 3-(4-bromo-2-methoxyphenyl)isoxazol-5-amine (1.2 g, 4.46 mmol, 1.0 equiv.) and tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate (1.52 g, 4.91 mmol, 1.1 equiv.) in dioxane:H2O (10 mL:2.0 mL) was purged with N2 for 5 min. K2CO3 (1.54 g, 11.1 mmol, 2.5 eq) was added to the reaction mixture and purged again for 5 minutes, then tetrakis(triphenylphosphine)palladium(0) (515 mg, 0.445 mmol, 0.1 eq) was added and the reaction mixture was stirred at 110 °C for 16 hours. The product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with ice water (50 mL) and extracted with ethyl acetate (3 × 30 mL). The organic layer was washed with saturated brine (30 mL) and dried over Na2SO4, then concentrated under reduced pressure to give a crude product. By using 14 g The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired compound (500 mg). LCMS: 372.4 [M+1] +

[0220] Step-5: Synthesis of tert-butyl 4-(4-(5-((5-cyanopyrazin-2-ylamino)isoxazol-3-yl)-3-methoxyphenyl)-5,6-dihydropyridine-1(2H)-carboxylate: A solution of tert-butyl 4-(4-(5-aminoisoxazol-3-yl)-3-methoxyphenyl)-5,6-dihydropyridine-1(2H)-carboxylate (230 mg, 0.619 mmol, 1.0 eq.) and 5-chloropyrazine-2-carbonitrile (104 mg, 0.743 mmol, 1.2 eq.) in DMF (5.0 mL) was purged with N2 for 5 min, and then Cs2CO3 (605 mg, 1.86 mmol, 4 mmol) was added. l, 3.0 equiv.). The resulting reaction mixture was purged again for 5 minutes, then Xantphos (7.1 mg, 0.012 mmol, 0.02 equiv.) and Pd2dba3 (28 mg, 0.031 mmol, 0.05 equiv.) were added, and the reaction mixture was microwaved at 140 ° C for 45 minutes. Product formation was confirmed by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (3×15 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12 g The crude product was purified by flash chromatography on a column and eluted in ethyl acetate: hexane solvent system to obtain the desired compound (120 mg). LCMS: 475.3 [M+1] +

[0221] Step-6: Synthesis of 5-(3-(2-methoxy-4-(1,2,3,6-tetrahydropyridin-4-yl)phenyl)isoxazol-5-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 4-(4-(5-((5-cyanopyrazin-2-ylamino)isoxazol-3-yl)-3-methoxyphenyl)-5,6-dihydropyridine-1(2H)-carboxylate (120 mg, 0.253 mmol, 1 eq.) in DCM (3 mL) was added TFA (0.096 mL, 1.26 mmol, 5 eq.) at 0°C and kept stirring for 6 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound (15 mg, trifluoroacetate). LCMS: 375.5 [M+1] + 97.1%@220nm,98.88@254nm. 1HNMR (400 MHz, after adding D2O to DMSO-d6): δ8.80 (d, J = 1.4 Hz, 1H), 8.39 (d, J = 1.4 Hz, 1H), 7.72 (d, J = 7.9 Hz, 1H), 7.21-7.14 (m, 2H), 6.83 (s, 1H), 6.33 (s, 1H), 3.90 (s, 3H), 3.87 (s, 2H), 3.33 (t, J = 6.1 Hz, 2H), 2.72 (s, 2H).

[0222] Example-10: Synthesis of 5-(5-(2-(1-methylazetidin-3-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.10)

[0223]

[0224] Step-1: Synthesis of 5-(5-(2-(1-methylazetidin-3-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a stirred solution of 5-(5-(2-(azetidin-3-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (70 mg, 0.209 mmol, 1 eq.) in dichloroethane were added acetic acid (0.1 mL) and formaldehyde (37% solution) (0.1 mL, 1.05 mmol, 5 eq.). The resulting reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then cooled to 0°C and sodium cyanoborohydride (66 mg, 1.05 mmol, 5 eq.) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 3 hours. Product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with saturated sodium bicarbonate (2×5 mL), the organic layer was separated and concentrated in vacuo to afford the crude product. The crude product was purified by R-HPLC to give the desired compound (10 mg, formate salt). LCMS: 349.5 [M+1] + 99.39@220nm,99.27@254nm. 1 H NMR (400 MHz, DMSO-d6 after adding D2O) δ8.79 (s, 1H), 8.65 (s, 1H), 8.17 (s, 1H, formate CH), 7.89 (d, J = 7.9 Hz, 1H), 7.47 (t, J = 7.8 Hz, 1H), 7.39 (s, 1H), 7.16 (t, J = 7.6 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 5.02 (p, J = 5.5 Hz, 1H), 4.00 (dd, J = 9.1, 5.9 Hz, 2H), 3.36 (dd, J = 9.2, 4.9 Hz, 2H), 2.46 (s, 3H).

[0225] Example-11: Synthesis of 5-(5-(2-methoxyphenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.11)

[0226]

[0227] Synthesis of 5-(5-(2-methoxyphenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: A solution of 5-(2-methoxyphenyl)isoxazol-3-amine (200 mg, 1.05 mmol, 1.0 equiv) and 5-chloropyrazine-2-carbonitrile (147 mg, 1.05 mmol, 1.0 equiv) in toluene (5 mL) was purged with nitrogen for 5 minutes, and then Cs2CO3 (1.03 mg, 3.15 mmol, 3.0 equiv) was added. The resulting reaction mixture was purged again for 5 minutes, and then Xantphos (12.0 mg, 0.021 mmol, 0.02 equiv) and Pd2dba3 (19 mg, 0.021 mmol, 0.02 equiv) were added, and the reaction mixture was microwaved at 100 ° C for 45 minutes. Product formation was confirmed by TLC and LCMS. After the reaction was completed, the reaction mixture was quenched with ice water (10 mL) and extracted with ethyl acetate (2 × 20 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. The crude product was ground with ether (2 × 5 mL) to obtain the desired compound (12 mg) without any further purification. LCMS: 294.3 [M + 1] + 97.31@220nm 98.2@254nm. 1 H NMR (400 MHz, DMSO-d6, after adding D2O) δ8.75 (s, 1H), 8.62 (s, 1H), 7.84 (d, J = 7.7 Hz, 1H), 7.51 (t, J = 7.9 Hz, 1H), 7.27–7.17 (m, 2H), 7.12 (t, J = 7.5 Hz, 1H), 3.93 (s, 3H).

[0228] Example-12: Synthesis of 5-(5-(2-(1-(4-methylpentan-2-yl)azetidin-3-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.12)

[0229]

[0230] Step-1: Synthesis of 5-(5-(2-(1-(4-methylpentan-2-yl)azetidin-3-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a stirred solution of 5-(5-(2-(azetidin-3-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (70 mg, 0.209 mmol, 1 eq.) in dichloroethane were added acetic acid (0.1 mL) and 4-methylpentan-2-one (105 mg, 1.05 mmol, 5 eq.). The resulting reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then cooled to 0°C and sodium cyanoborohydride (66 mg, 1.05 mmol, 5 eq.) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 3 hours. Product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with saturated sodium bicarbonate (2×5 mL), the organic layer was separated and concentrated in vacuo to provide a crude product. The crude product was purified by R-HPLC to give the desired compound (23 mg, formate salt). LCMS: 419.2 [M+1] + 96.82@220nm 97.95@254nm. 1 H NMR (400 MHz, DMSO-d6 after adding D2O) δ8.73 (s, 1H), 8.66 (s, 1H), 8.19 (s, 1H, formate CH), 7.88 (dd, J = 7.7, 1.7 Hz, 1H), 7.53-7.42 (m, 1H), 7.35 (s, 1H), 7.15 (t, J = 7.6 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 4.96 (p, J = 5.4 Hz, 1H), 3.91 (q, J = 7.9 Hz, 2H),3.20(q,J=6.5Hz,2H),2.43(q,J=5.3,4.0Hz,1H),1.57(dq,J=11.8,5.8Hz,1H),1.21(ddd,J=13.2,9.8,3.6 Hz,1H),1.02(ddd,J=13.6,9.7,4.3Hz,1H),0.90(d,J=5.9Hz,3H),0.87(d,J=6.6Hz,3H),0.81(d,J=6.4Hz,3H).

[0231] Example-13: Synthesis of 5-(5-(2-(1-isopropylazetidin-3-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.13)

[0232]

[0233] Step-1: Synthesis of 5-(5-(2-(1-isopropylazetidin-3-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a stirred solution of 5-(5-(2-(azetidin-3-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (70 mg, 0.209 mmol, 1 eq.) in dichloroethane were added acetic acid (0.1 mL) and acetone (63 mg, 1.05 mmol, 5 eq.). The resulting reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then cooled to 0 °C and sodium cyanoborohydride (66 mg, 1.05 mmol, 5 eq.) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 3 hours. The product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with saturated sodium bicarbonate (2×5 mL), the organic layer was separated and concentrated in vacuo to afford the crude product. The crude product was purified by R-HPLC to obtain the desired compound (7 mg, formate salt). LCMS: 377.3[M+1] + 98.99@220nm 99.41@254nm. 1 H NMR (400 MHz, DMSO-d6 after adding D2O) δ8.74 (s, 1H), 8.67 (s, 1H), 8.21 (s, 1H, formate CH), 7.87 (d, J = 7.7 Hz, 1H), 7.47 (t, J = 8.0 Hz, 1H), 7.31 (s, 1H), 7.15 (t, J = 7.5 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 4.97 (q, J = 5.6 Hz, 1H), 3.94 (t, J = 7.2 Hz, 2H), 3.27 (dd, J = 9.2, 5.0 Hz, 2H), 2.58 (d, J = 6.4 Hz, 1H), 0.94 (d, J = 6.2 Hz, 6H).

[0234] Example-14: Synthesis of 5-(5-(2-(pyrrolidin-3-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.14)

[0235]

[0236] Step-1: Synthesis of 2-(3-aminoisoxazol-5-yl)phenol: Same as described in compound 1.1

[0237] Step-2: Synthesis of tert-butyl 3-(2-(3-aminoisoxazol-5-yl)phenoxy)pyrrolidine-1-carboxylate: To a solution of 2-(3-aminoisoxazol-5-yl)phenol (300 mg, 1.7 mmol, 1.0 eq.) in DMF was added tert-butyl 3-(tosyloxy)pyrrolidine-1-carboxylate (synthesized as described in J. Med. Chem, 2020, 63(19), 11054-84) (698 mg, 2.04 mmol, 1.2 eq.) and Cs2CO3 (1.11 g, 3.41 mmol, 2 eq.). The resulting reaction mixture was stirred at 90 °C for 16 h. Product formation was confirmed by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with ice water (30 mL) and extracted with ethyl acetate (2×20 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired compound (200 mg) as a colorless liquid. LCMS: 346.3 [M+1] +

[0238] Step-3: Synthesis of tert-butyl 3-(2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)pyrrolidine-1-carboxylate: A solution of tert-butyl 3-(2-(3-aminoisoxazol-5-yl)phenoxy)pyrrolidine-1-carboxylate (200 mg, 0.579 mmol, 1.0 equiv) and 5-chloropyrazine-2-carbonitrile (97 mg, 0.695 mmol, 1.2 equiv) in toluene (3.0 mL) was purged with N2 for 5 min and then Cs2CO3 (566 mg, 1.74 mmol, 3.0 equiv) was added. The resulting reaction mixture was purged again for 5 minutes, then Xantphos (6.7 mg, 0.012 mmol, 0.02 equiv) and Pd2dba3 (26 mg, 0.029 mmol, 0.05 equiv) were added, and the reaction mixture was subjected to microwave treatment at 140 ° C for 45 minutes. The product formation was confirmed by TLC and LCMS. After the reaction was completed, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (3×15 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 4g The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired compound (50 mg). LCMS: 449.2 [M+1] +

[0239] Step-4: Synthesis of 5-(5-(2-(pyrrolidin-3-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 3-(2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)pyrrolidine-1-carboxylate (50 mg, 0.111 mmol, 1 eq.) in DCM (4 mL) was added TFA (0.1 mL) at 0°C and kept stirring for 6 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was treated with 5 mL of ethyl acetate, resulting in the precipitation of the product (8 mg, trifluoroacetate). The product was filtered and characterized by NMR, MS to confirm the formation of the desired compound. LCMS: 349.3 [M+1] + UPLC:97.13@220nm 97.97@254nm; 1 H NMR (400 MHz, methanol-d4) δ 8.65 (d, J = 1.5 Hz, 1H), 8.60 (s, 1H), 7.93 (dd, J = 7.7, 1.7 Hz, 1H), 7.55-7.49 (m, 1H), 7.33 (s, 1H), 7.23-7.16 (m, 2H), 5.40 (s, 1H), 3.68-3.56 (m, 4H), 2.53-2.39 (m, 2H).

[0240] Example-15: Synthesis of 5-(5-(2-((1s,4s)-4-aminocyclohexyloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.15)

[0241]

[0242] Step-1: Synthesis of 2-(3-aminoisoxazol-5-yl)phenol: Same as described in compound 1.1

[0243] Step-2: Synthesis of tert-butyl (1s, 4s)-4-(2-(3-aminoisoxazol-5-yl)phenoxy)cyclohexylcarbamate: To a solution of 2-(3-aminoisoxazol-5-yl)phenol (0.476 g, 2.70 mmol, 1.0 equiv) in DMF (10 mL) was added (1r, 4r)-4-(tert-butoxycarbonylamino)cyclohexyl 4-methylbenzenesulfonate (synthesized as described in WO2021080015A1) (0.998 g, 2.70 mmol, 1.0 equiv) and Cs2CO3 (1.76 g, 5.40 mmol, 2.0 equiv). The resulting reaction mixture was stirred at 90 ° C for 16 hours. Product formation was confirmed by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with ice water (100 mL) and extracted with ethyl acetate (2×50 mL). The collected organic layer was washed with saturated brine (50 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired product (320 mg). LCMS: 374.3 [M+1] +

[0244] Step-3: Synthesis of tert-butyl (1s,4s)-4-(2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)cyclohexylcarbamate: A solution of tert-butyl (1s,4s)-4-(2-(3-aminoisoxazol-5-yl)phenoxy)cyclohexylcarbamate (150 mg, 0.40 mmol, 1.0 equiv) and 5-chloropyrazine-2-carbonitrile (56 mg, 0.40 mmol, 1.0 equiv) in toluene (5 mL) was purged with N2 for 5 min and then Cs2CO3 (392 mg, 1.20 mmol, 3.0 equiv) was added. The resulting reaction mixture was purged again for 5 minutes, then Xantphos (14 mg, 0.024 mmol, 0.06 equiv) and Pd2dba3 (29 mg, 0.032 mmol, 0.08 equiv) were added, and the reaction mixture was subjected to microwave treatment at 110 ° C for 45 minutes. The product formation was confirmed by TLC and LCMS. After the reaction was completed, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (3 × 15 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12g The crude product was purified by flash chromatography on a column and eluted in ethyl acetate: hexane solvent system to obtain the desired compound (70 mg). LCMS: 477.2 [M+1] +

[0245] Step-4: Synthesis of 5-(5-(2-((1s,4s)-4-aminocyclohexyloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl (1s,4s)-4-(2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)cyclohexylcarbamate (70 mg, 0.14 mmol, 1.0 eq.) in DCM (5.0 mL) was added TFA (0.4 mL) at 0°C and kept stirring for 2 hours. The formation of the product was confirmed by TLC and LCMS. The reaction mixture was then concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound (20 mg, formate salt). LCMS: 377.3 [M+1] + 99.99@220nm; 99.94@254nm. 1 H NMR (400 MHz, DMSO-d6, after adding D2O) δ8.67 (s, 1H), 8.54 (s, 1H), 8.39 (s, 1H, formate CH), 7.87 (d, J = 7.6 Hz, 1H), 7.51-7.42 (m, 2H), 7.21 (d, J = 8.5 Hz, 1H), 7.09 (t, J = 7.6 Hz, 1H), 4.81 (s, 1H), 3.12 (dt, J = 13.1, 6.2 Hz, 1H), 2.12 (d, J = 12.6 Hz, 2H), 1.86 (dd, J = 10.1, 4.7 Hz, 2H), 1.72 (h, J = 12.6, 11.5 Hz, 4H).

[0246] Example-16: Synthesis of 5-(5-(2-((1-methylpiperidin-3-yl)methoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.16)

[0247]

[0248] Step-1: Synthesis of 5-(5-(2-((1-methylpiperidin-3-yl)methoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a stirred solution of 5-(5-(2-(piperidin-3-ylmethoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (70 mg, 0.185 mmol, 1 eq.) in dichloroethane (5 mL) were added acetic acid (0.1 mL) and formaldehyde (37% solution) (0.075 ml, 0.929 mmol, 5 eq.). The resulting reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then cooled to 0°C and sodium cyanoborohydride (58 mg, 0.929 mmol, 5 eq.) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 3 hours. The product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with saturated sodium bicarbonate (2×5 mL), the organic layer was separated and concentrated in vacuo to afford the crude product. The crude product was purified by R-HPLC to give the desired compound (10 mg, trifluoroacetate salt). LCMS: 391.5 [M+1] + 99.59@220nm; 99.78@254nm. 1 H NMR (400MHz, methanol-d4) δ8.65(d,J=15.0Hz,2H),7.86(d,J=7.7Hz,1H),7.50(t,J=7.9Hz,1H),7.27-7.06(m,3H),4.16(p,J=9.9Hz,2H),3.71-3 .49(m,2H),2.95(dd,J=12.5,7.8Hz,2H),2.91(s,3H),2.44(s,1H),2.13(t,J=15.8Hz,2H),1.92-1.78(m,1H),1.51(tt,J=13.9,7.0Hz,1H).

[0249] Example-17: Synthesis of 5-(5-(2-(azetidin-3-ylmethoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.17)

[0250]

[0251] Step-1: Synthesis of 2-(3-aminoisoxazol-5-yl)phenol: Same as described in compound 1.1

[0252] Step-2: Synthesis of tert-butyl 3-((2-(3-aminoisoxazol-5-yl)phenoxy)methyl)azetidine-1-carboxylate: To a solution of 2-(3-aminoisoxazol-5-yl)phenol (1.0 g, 5.68 mmol, 1.0 eq.) in DMF (25 mL) was added tert-butyl 3-(tosyloxymethyl)azetidine-1-carboxylate (synthesized as described in EP2676965A1) (2.33 g, 6.81 mmol, 1.2 eq.) and Cs2CO3 (3.7 g, 11.35 mmol, 2.0 eq.). The resulting reaction mixture was stirred at 90°C for 16 hours. Product formation was confirmed by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with ice water (30 mL) and extracted with ethyl acetate (2×20 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash chromatography using ethyl acetate: hexane solvent system to obtain the desired product (800 mg). LCMS: 346.4 [M+1] +

[0253] Step-3: Synthesis of tert-butyl 3-((2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)methyl)azetidine-1-carboxylate: To a solution of tert-butyl 3-((2-(3-aminoisoxazol-5-yl)phenoxy)methyl)azetidine-1-carboxylate (0.5 g, 1.45 mmol, 1.0 eq.) in THF (10.0 mL) was added sodium hydride (60% dispersion in mineral oil) (0.173 g, 4.34 mmol, 3.0 eq.) portionwise at 0°C for a period of 10 minutes. The reaction mixture was then stirred at 0°C for 30 minutes. To the reaction mixture was added 5-chloropyrazine-2-carbonitrile (0.241 g, 1.74 mmol, 1.2 eq.) and it was stirred at room temperature for 16 hours. The product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with ice water (30 mL) and extracted with ethyl acetate (2×50 mL). The combined organic layer was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired product (260 mg). LCMS: 449.1 [M+1] +

[0254] Step-4: Synthesis of 5-(5-(2-(azetidin-3-ylmethoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 3-((2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)methyl)azetidine-1-carboxylate (260 mg, 0.580 mmol, 1.0 eq.) in DCM (10.0 mL) was added TFA (0.5 mL) at 0°C and kept stirring for 2 hours. The product formation was confirmed by TLC and LCMS. The reaction mixture was then concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound (210 mg, trifluoroacetate). LCMS: 349.5 [M+1] + 98.73@220nm; 99.64@254nm. 1 H NMR (400 MHz, DMSO-d6, after adding D2O) δ8.80 (s, 1H), 8.54 (s, 1H), 7.87 (d, J = 7.7 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.23 (d, J = 8.5 Hz, 1H), 7.18 (d, J = 12.8 Hz, 2H), 4.34 (d, J = 7.3 Hz, 2H), 4.15 (t, J = 9.8 Hz, 2H), 3.94 (t, J = 9.3 Hz, 2H), 3.48-3.28 (m, 1H)

[0255] Example-18: Synthesis of 5-(5-(2-(1-sec-butylazetidin-3-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.18)

[0256]

[0257] Step-1: Synthesis of 5-(5-(2-(1-sec-butylazetidin-3-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a stirred solution of 5-(5-(2-(azetidin-3-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (70 mg, 0.209 mmol, 1 eq.) in dichloroethane were added acetic acid (0.1 mL) and butan-2-one (75 mg, 1.05 mmol, 5 eq.). The resulting reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then cooled to 0°C and sodium cyanoborohydride (66 mg, 1.05 mmol, 5 eq.) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 3 hours. Product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with saturated sodium bicarbonate (2×5 mL), the organic layer was separated and concentrated in vacuo to afford the crude product. The crude product was purified by R-HPLC to give the desired compound (10 mg, trifluoroacetate salt). LCMS: 391.5 [M+1] + 99.84@220nm 99.63@254nm. 1 H NMR (400 MHz, DMSO-d6 after adding D2O) δ8.74 (s, 1H), 8.63 (s, 1H), 7.90 (d, J = 7.7 Hz, 1H), 7.55-

[0258] 7.47(m,1H),7.33(s,1H),7.22(t,J=7.5Hz,1H),6.92(s,1H),5.26-5.14(m,1H),4.64(s,2H),4.22(dd,J=12.5,4.5Hz ,2H),3.28(s,1H),1.66(d,J=12.2Hz,1H),1.32(dt,J=14.5,7.6Hz,1H),1.13(d,J=6.3Hz,3H),0.87(t,J=7.3Hz,3H).

[0259] Example-19: Synthesis of 5-(5-(2-methoxy-4-(1,2,3,6-tetrahydropyridin-4-yl)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.19)

[0260]

[0261] Step 1-3: Same as shown in the synthesis of compound 1.9

[0262] Step-4: Synthesis of tert-butyl 4-(4-(3-aminoisoxazol-5-yl)-3-methoxyphenyl)-5,6-dihydropyridine-1(2H)-carboxylate: A solution of 5-(4-bromo-2-methoxyphenyl)isoxazol-3-amine (1.2 g, 4.46 mmol, 1.0 equiv.) and tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate (1.52 g, 4.91 mmol, 1.1 equiv.) in dioxane:water (10 mL:2.0 mL) was purged with N2 for 5 min. K2CO3 (1.54 g, 11.1 mmol, 2.5 eq) was added to the reaction mixture and purged again for 5 minutes, then tetrakis(triphenylphosphine)palladium(0) (515 mg, 0.445 mmol, 0.1 eq) was added and the reaction mixture was stirred at 110 °C for 16 hours. The product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with ice water (50 mL) and extracted with ethyl acetate (3 × 30 mL). The organic layer was washed with saturated brine (30 mL) and dried over Na2SO4, then concentrated under reduced pressure to give a crude product. By using 24 g The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired compound (500 mg). LCMS: 372.4 [M+1] +

[0263] Step-5: Synthesis of tert-butyl 4-(4-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)-3-methoxyphenyl)-5,6-dihydropyridine-1(2H)-carboxylate: To a solution of tert-butyl 4-(4-(3-aminoisoxazol-5-yl)-3-methoxyphenyl)-5,6-dihydropyridine-1(2H)-carboxylate (250 mg, 0.673 mmol, 1.0 eq.) in THF (5.0 mL) was added sodium hydride (60% dispersion in mineral oil) (134 mg, 3.37 mmol, 5.0 eq.) portionwise at 0°C over a period of 10 minutes. To the reaction mixture was added 5-chloropyrazine-2-carbonitrile (122 mg, 0.875 mmol, 1.3 eq.) and it was stirred at room temperature for 16 hours. The product formation was confirmed by TLC and LCMS. After the reaction was completed, the reaction mixture was quenched with ice water (10 mL) and extracted with ethyl acetate (2×20 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12 g The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired compound (120 mg). LCMS: 475.3 [M+1] +

[0264] Step-6: Synthesis of 5-(5-(2-methoxy-4-(1,2,3,6-tetrahydropyridin-4-yl)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 4-(4-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)-3-methoxyphenyl)-5,6-dihydropyridine-1(2H)-carboxylate (120 mg, 0.253 mmol, 1 eq.) in dioxane (3.0 mL) was added 4.0 M HCl in dioxane (3.0 mL) at 0°C and stirred for 6 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was triturated with diethyl ether (2×5 mL) to obtain the desired compound (20 mg, hydrochloride salt). LCMS: 375.5 [M+1] + 93.97@220nm; 93.79@254nm 1 H NMR (400 MHz, DMSO-d6, after adding D2O) δ8.77 (s, 1H), 8.63 (s, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.23 (t, J = 6.3 Hz, 3H), 6.38 (s, 1H), 3.99 (s, 3H), 3.78 (s, 2H), 3.33 (t, J = 6.0 Hz, 2H), 2.73 (s, 2H).

[0265] Example-20: Synthesis of 5-(5-(2-(3-aminocyclobutyloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.20)

[0266]

[0267] Step-1: Synthesis of 2-(3-aminoisoxazol-5-yl)phenol: Same as described in compound 1.1

[0268] Step-2: Synthesis of tert-butyl 3-(2-(3-aminoisoxazol-5-yl)phenoxy)cyclobutylcarbamate: To a solution of 2-(3-aminoisoxazol-5-yl)phenol (300 mg, 1.7 mmol, 1.0 equiv) in DMF (5 mL) was added 3-(tert-butoxycarbonylamino)cyclobutyl 4-methylbenzenesulfonate (synthesized as described in WO2018234978A1) (698 mg, 2.04 mmol, 1.2 equiv) and Cs2CO3 (1.11 g, 3.41 mmol, 2.0 equiv). The resulting reaction mixture was stirred at 90 ° C for 16 hours. Product formation was confirmed by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with ice water (30 mL) and extracted with ethyl acetate (2×20 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12g The crude product was purified by flash chromatography on a column in ethyl acetate: hexane solvent system to obtain the desired product as a colorless liquid (200 mg). LCMS: 346.3 [M+1] +

[0269] Step-3: Synthesis of tert-butyl 3-(2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)cyclobutylcarbamate: To a solution of tert-butyl (3-(2-(3-aminoisoxazol-5-yl)phenoxy)cyclobutyl)carbamate (200 mg, 0.579 mmol, 1.0 eq.) in THF (10.0 mL) was added sodium hydride (60% dispersion in mineral oil) (115 mg, 2.9 mmol, 5.0 eq.) portionwise at 0°C for a period of 10 minutes. The reaction mixture was stirred at 0°C for 30 minutes and then 5-chloropyrazine-2-carbonitrile (97 mg, 0.695 mmol, 1.2 eq.) was added. The reaction mixture was stirred at room temperature for 16 hours. The product formation was confirmed by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with ice water (30 mL) and extracted with ethyl acetate (3×50 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, then concentrated under reduced pressure to obtain a crude product, which was used as is in the next step (100 mg). LCMS: 449.3 [M+1] +

[0270] Step-4: Synthesis of 5-(5-(2-(3-aminocyclobutyloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 3-(2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)cyclobutylcarbamate (100 mg, 0.223 mmol, 1.0 eq.) in DCM (4.0 mL) at 0°C, TFA (0.1 mL) was added and stirring was maintained for 6 hours. The formation of the product was confirmed by TLC and LCMS. The reaction mixture was then concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound (10 mg, trifluoroacetate). LCMS: 349.5 [M+1] + 97.3@220nm 98.04@254nm. 1H NMR (400 MHz, DMSO-d6, after adding D2O) δ 8.76 (d, J = 8.1 Hz, 1H), 8.62 (d, J = 5.3 Hz, 1H), 7.89 (d, J = 7.4 Hz, 1H), 7.50 (t, J = 7.8 Hz, 1H), 7.43 (d, J = 6.1 Hz, 1H), 7.19-7.11 (m, 1H), 7.00 (dd, J = 29.2, 8.3 Hz, 1H), 5.12 (s, 1H), 3.91 (dd, J = 14.2, 7.1 Hz, 1H), 2.98 (s, 1H), 2.75-2.65 (m, 2H), 2.58-2.55 (m, 1H).

[0271] Example-21: Synthesis of 5-(5-(2-(2-azaspiro[3.3]hept-6-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.21)

[0272]

[0273] Step-1: Synthesis of 2-(3-aminoisoxazol-5-yl)phenol: Same as described in compound 1.1

[0274] Step-2: Synthesis of tert-butyl 6-(2-(3-aminoisoxazol-5-yl)phenoxy)-2-azaspiro[3.3]heptane-2-carboxylate: To a solution of 2-(3-aminoisoxazol-5-yl)phenol (240 mg, 1.36 mmol, 1.0 eq.) in DMF (5 mL) was added tert-butyl 6-(tosyloxy)-2-azaspiro[3.3]heptane-2-carboxylate (synthesized as described in WO2021124222A1) (500 mg, 1.36 mmol, 1.0 eq.) and Cs2CO3 (577 mg, 1.77 mmol, 1.3 eq.). The resulting reaction mixture was stirred at 90 ° C for 16 hours. Product formation was confirmed by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with ice water (30 mL) and extracted with ethyl acetate (2×20 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash chromatography in ethyl acetate: hexane solvent system to obtain the desired product (210 mg). LCMS: 372.3 [M+1] +

[0275] Step-3: Synthesis of tert-butyl 6-(2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)-2-azaspiro[3.3]heptane-2-carboxylate: A solution of tert-butyl 6-(2-(3-aminoisoxazol-5-yl)phenoxy)-2-azaspiro[3.3]heptane-2-carboxylate (200 mg, 0.538 mmol, 1.0 equiv) and 5-chloropyrazine-2-carbonitrile (90 mg, 0.646 mmol, 1.2 equiv) in toluene (5 mL) was purged with N2 for 5 min and then Cs2CO3 (525 mg, 1.61 mmol, 3.0 equiv) was added. The resulting reaction mixture was purged again for 5 minutes, then Xantphos (18 mg, 0.032 mmol, 0.06 equiv) and Pd2dba3 (39 mg, 0.043 mmol, 0.08 equiv) were added, and the reaction mixture was subjected to microwave treatment at 110 ° C for 45 minutes. The product formation was confirmed by TLC and LCMS. After the reaction was completed, the reaction mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (3 × 15 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. By using 12g The crude product was purified by combined flash chromatography on a column and eluted in ethyl acetate: hexane solvent system to obtain the desired compound (70 mg). LCMS: 475.3 [M+1] +

[0276] Step-4: Synthesis of 5-(5-(2-(2-azaspiro[3.3]hept-6-yloxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 6-(2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)-2-azaspiro[3.3]hept-2-carboxylate (70 mg, 0.147 mmol, 1.0 eq.) in DCM (4.0 mL) was added TFA (0.4 mL) at 0 °C and kept stirring for 2 hours. The formation of product was confirmed by TLC and LCMS. The reaction mixture was then concentrated under reduced pressure to obtain the crude product. The crude product was purified by R-HPLC to obtain the desired compound (10 mg, trifluoroacetate). LCMS: 375.5[M+1] + 99.66@220nm; 99.72@254nm. 1H NMR (400MHz, DMSO-d6, after adding D2O) δ8.79(s,1H),8.60(s,1H),7.87(d,J=7.8Hz,1H),7.47(t,J=7.8Hz,1H),7.36(s,1H),7.13(t,J=7 .6Hz,1H),6.99(d,J=8.3Hz,1H),4.81(q,J=6.6Hz,1H),4.03(d,J=18.9Hz,4H),2.89(dd,J=12.7,6.4Hz,2H),2.44-2.31(m,2H).

[0277] Example-22: Synthesis of 5-(3-(2-methoxy-4-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)phenyl)isoxazol-5-ylamino)pyrazine-2-carbonitrile (Compound 1.22)

[0278]

[0279] Step-1: Synthesis of 5-(3-(2-methoxy-4-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)phenyl)isoxazol-5-ylamino)pyrazine-2-carbonitrile: To a stirred solution of 5-(3-(2-methoxy-4-(1,2,3,6-tetrahydropyridin-4-yl)phenyl)isoxazol-5-ylamino)pyrazine-2-carbonitrile (80 mg, 0.213 mmol, 1 eq.) in dichloroethane (4 mL) were added acetic acid (0.1 mL) and formaldehyde (37% solution) (0.032 mL, 1.07 mmol, 5 eq.). The resulting reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then cooled to 0°C and sodium cyanoborohydride (33 mg, 0.534 mmol, 2.5 eq.) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 5 hours. The product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with saturated sodium bicarbonate (2×5 mL), and the organic layer was separated and concentrated in vacuo to provide the crude product. The crude product was purified by R-HPLC to give the desired compound (4 mg, formate salt). LCMS: 389.5 [M+1] + 97.23@220nm; 1 H NMR (400 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.34 (s, 1H), 8.23 ​​(s, 1H, formate CH), 7.70 (d, J = 8.1 Hz, 1H), 7.21-7.10 (m, 2H), 6.81 (s, 1H), 6.31 (s, 1H), 3.89 (s, 3H), 3.41 (s, 2H), 2.96 (s, 2H), 2.64 (s, 2H), 2.54 (s, 3H).

[0280] Example-23: Synthesis of 5-(5-(2-(pyrrolidin-3-ylmethoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile (Compound 1.23)

[0281]

[0282] Step-1: Synthesis of 2-(3-aminoisoxazol-5-yl)phenol: Same as described in compound 1.1

[0283] Step-2: Synthesis of tert-butyl 3-((2-(3-aminoisoxazol-5-yl)phenoxy)methyl)pyrrolidine-1-carboxylate: To a solution of 2-(3-aminoisoxazol-5-yl)phenol (300 mg, 1.70 mmol, 1.0 eq.) in DMF were added tert-butyl 3-(tosyloxymethyl)pyrrolidine-1-carboxylate (synthesized as described in J. Med. Chem, 2020, 63(19), 11054-84) (726.1 mg, 2.04 mmol, 1.2 eq.) and Cs2CO3 (1.1 g, 3.40 mmol, 2.0 eq.). The resulting reaction mixture was stirred at 90 °C for 16 h. Product formation was confirmed by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with ice water (30 mL) and extracted with ethyl acetate (2×20 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash chromatography using ethyl acetate: hexane solvent system to obtain the desired compound (300 mg) as a colorless liquid. LCMS: 360.3 [M+1] +

[0284] Step-3: Synthesis of tert-butyl 3-((2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)methyl)pyrrolidine-1-carboxylate: To a solution of tert-butyl 3-((2-(3-aminoisoxazol-5-yl)phenoxy)methyl)pyrrolidine-1-carboxylate (300 mg, 0.835 mmol, 1.0 eq.) in THF (5 mL) was added sodium hydride (60% dispersion in mineral oil) (167 mg, 4.17 mmol, 5.0 eq.) portionwise at 0°C for a period of 10 minutes. The reaction mixture was then stirred at 0°C for 30 minutes followed by the addition of 5-chloropyrazine-2-carbonitrile (232.1 mg, 1.67 mmol, 2.0 eq.) and stirred at room temperature for 16 hours. The product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with ice water (30 mL) and extracted with ethyl acetate (2×50 mL). The combined organic layers were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product (350 mg) as a pale yellow solid, which was used as such in the next step. LCMS: 463.2 [M+1] +

[0285] Step-4: Synthesis of 5-(5-(2-(pyrrolidin-3-ylmethoxy)phenyl)isoxazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 3-((2-(3-(5-cyanopyrazin-2-ylamino)isoxazol-5-yl)phenoxy)methyl)pyrrolidine-1-carboxylate (200 mg, 0.431 mmol, 1.0 eq.) in DCM (5 mL) was added TFA (0.5 mL) at 0°C and kept stirring for 6 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound (50 mg, trifluoroacetate). LCMS: 363.5 [M+1] + 97.53@220nm 97.76@254nm. 1 H NMR (400MHz, methanol-d4) δ8.64(s,1H),8.59(s,1H),7.89(dd,J=7.9,1.8Hz,1H),7.53-7.46 (m,1H),7.30(s,1H),7.21(d,J=8.5Hz,1H),7.14(t,J=7.6Hz,1H),4.28(dd,J=9.4,6.2 Hz,1H),4.22-4.14(m,1H),3.63(dd,J=11.9,7.9Hz,1H),3.50-3.41(m,1H),3.41-3.36 (m,1H),3.29-3.23(m,1H),3.06(q,J=7.5Hz,1H),2.48-2.34(m,1H),2.06-1.93(m,1H).

[0286] Example-24: Synthesis of 5-(5-(3-(piperidin-4-ylmethoxy)naphthalen-2-yl)-1H-pyrazol-3-ylamino)pyrazine-2-carbonitrile (Compound 2.1)

[0287]

[0288] Step-1: Synthesis of methyl 3-hydroxy-2-naphthoate: Synthesized as described in WO2000009169A1.

[0289] Step-2: Synthesis of tert-butyl 4-((3-(methoxycarbonyl)naphthalen-2-yloxy)methyl)piperidine-1-carboxylate: To a solution of tert-butyl 4-(tosyloxymethyl)piperidine-1-carboxylate (2.0 g, 5.41 mmol, 1.0 eq.) in DMF (40 mL) was added Cs2CO3 (4.41 g, 13.53 mmol, 2.5 eq.), followed by addition of methyl 3-hydroxy-2-naphthoate (1.42 g, 7.04 mmol, 1.3 eq.) and the reaction mixture was kept stirring at 90 °C overnight. The progress of the reaction was monitored by TLC and LCMS. The reaction mixture was quenched by the addition of ice water (50 mL) and extracted with ethyl acetate (3×25 mL). The collected organic layer was washed with saturated brine (25 mL), dried over Na2SO4, and then concentrated under reduced pressure to obtain a crude product. The product was purified by using 24 g of 4-nitropropane-1-ol. The crude product was purified by flash chromatography on a column and eluted in a 30% ethyl acetate: hexane solvent system to obtain the desired compound (1.4 g). LCMS: 300.3 [M+H-100] + ; Remove Boc peak

[0290] Step-3: Synthesis of tert-butyl 4-((3-(2-cyanoacetyl)naphthalene-2-yloxy)methyl)piperidine-1-carboxylate: To a solution of acetonitrile (10 mL) in THF (8 mL) was added LiHMDS (1M in THF) (10.5 mL, 10.51 mmol, 3.0 eq) at -78 °C and stirred at the same temperature for 2 hours. Then tert-butyl 4-((3-(methoxycarbonyl)naphthalene-2-yloxy)methyl)piperidine-1-carboxylate (1.4 g, 3.5 mmol, 1.0 eq) was added and stirred for 2 h. Product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (3×15 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over Na2SO4, and then concentrated under reduced pressure to obtain a crude product. By using 12 g The crude product was purified by flash chromatography on a column and eluted in 30% ethyl acetate: hexane solvent system to obtain the desired compound (800 mg). LCMS: 353.3 [M+H-56] +

[0291] Step-4: Synthesis of tert-butyl 4-((3-(3-amino-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)piperidine-1-carboxylate: To a solution of tert-butyl 4-((3-(2-cyanoacetyl)naphthalen-2-yloxy)methyl)piperidine-1-carboxylate (500 mg, 1.22 mmol, 1.0 eq.) in EtOH (5.0 mL) was added hydrazine hydrate (0.3 mL, 6.12 mmol, 5.0 eq.), followed by dropwise addition of acetic acid (5 drops), and the resulting reaction mixture was kept stirring at 90 °C overnight. The product formation was confirmed by TLC and LCMS. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The product was purified by using 12 g of HPLC purifying the product. The crude product was purified by flash chromatography on a column and eluted in 2% MeOH / DCM solvent system to obtain the desired compound (300 mg). LCMS: 423.3 [M+H] +

[0292] Step-5: Synthesis of tert-butyl 4-((3-(3-(5-cyanopyrazin-2-ylamino)-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)piperidine-1-carboxylate: To a solution of tert-butyl 4-((3-(3-amino-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)piperidine-1-carboxylate (300 mg, 0.710 mmol, 1.0 eq.) in EtOH (5.0 mL) was added DIPEA (0.3 mL, 1.78 mmol, 2.5 eq.) followed by 5-bromopyrazine-2-carbonitrile (261 mg, 1.42 mmol, 2.0 eq.) and the reaction mixture was stirred at 90 °C overnight. The product formation was confirmed by TLC and LCMS. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The product was purified by using 12 g of crude product. The crude product was purified by flash chromatography on a column and eluted in 2% MeOH / DCM solvent system to obtain the desired compound (150 mg). LCMS: 426.3 [M+H-Boc] +

[0293] Step-6: Synthesis of 5-(5-(3-(piperidin-4-ylmethoxy)naphthalen-2-yl)-1H-pyrazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 4-((3-(3-(5-cyanopyrazin-2-ylamino)-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)piperidine-1-carboxylate (150 mg, 0.285 mmol, 1.0 eq.) in DCM (5.0 mL) was added TFA (113 mg, 0.998 mmol, 3.5 eq.) at 0°C and kept stirring for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound (15 mg, formate salt). LCMS: 426.3 [M+1] + ; 99.85@220nm,99.88@254nm. 1 H NMR (400MHz, DMSO-d6) δ8.63(s,1H),8.50(s,1H),8.38(s,1H),8.21(s,1H),7.85(dd,J=15.1,8.2Hz,2H),7.48(d,J=8.6Hz,2H),7.40(t,J=7.5Hz,1 H),7.13(s,1H),4.09(d,J=6.8Hz,2H),3.29(d,J=12.1Hz,2H),2.89(t,J= 12.3Hz, 2H), 2.23 (s, 1H), 2.03 (d, J = 13.1Hz, 2H), 1.49 (d, J = 14.1Hz, 2H).

[0294] Example-25: Synthesis of 5-(5-(3-(azetidin-3-ylmethoxy)naphthalen-2-yl)-1H-pyrazol-3-ylamino)pyrazine-2-carbonitrile (Compound 2.2)

[0295]

[0296] Step-1: Synthesis of methyl 3-hydroxy-2-naphthoate: Synthesized as described in WO2000009169A1

[0297] Step-2: Synthesis of tert-butyl 3-((3-(methoxycarbonyl)naphthalen-2-yloxy)methyl)azetidine-1-carboxylate: To a solution of tert-butyl 3-(tosyloxymethyl)azetidine-1-carboxylate (2.0 g, 5.86 mmol, 1.0 eq.) in DMF (40 mL) was added Cs2CO3 (4.77 g, 14.64 mmol, 2.5 eq.), followed by the addition of methyl 3-hydroxy-2-naphthoate (1.54 g, 7.62 mmol, 1.3 eq.) and the reaction mixture was stirred at 90 °C overnight. The progress of the reaction was monitored by TLC and LCMS. The reaction mixture was quenched with ice water (50 mL) and extracted with ethyl acetate (3×25 mL). The collected organic layer was washed with saturated brine (25 mL) and dried over Na2SO4, and then concentrated under reduced pressure to obtain a crude product. The product was obtained by using 24 g of 4-naphthoic acid. The crude product was purified by flash chromatography on a column and eluted in a 20% ethyl acetate: hexane solvent system to obtain the desired compound (1.8 g). LCMS: 372.4 [M+H] +

[0298] Step-3: Synthesis of tert-butyl 3-((3-(2-cyanoacetyl)naphthalene-2-yloxy)methyl)azetidine-1-carboxylate: To a solution of ACN (10 mL) in THF (8.0 mL) was added LiHMDS (1M in THF) (14.54 mL, 14.54 mmol, 3.0 eq) at -78 °C and stirred at the same temperature for 2 hours. Then tert-butyl 3-((3-(methoxycarbonyl)naphthalene-2-yloxy)methyl)azetidine-1-carboxylate (1.8 g, 4.85 mmol, 1.0 eq) was added and stirred for 2 hours. Product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (3×15 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over Na2SO4, and then concentrated under reduced pressure to give a crude product. By using 12 g The crude product was purified by flash chromatography on a column and eluted in a 30% ethyl acetate / hexane solvent system to obtain the desired compound (1 g). LCMS: 381.3 [M+H] +

[0299] Step-4: Synthesis of tert-butyl 3-((3-(3-amino-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)azetidine-1-carboxylate: To a solution of tert-butyl 3-((3-(2-cyanoacetyl)naphthalen-2-yloxy)methyl)azetidine-1-carboxylate (500 mg, 1.31 mmol, 1.0 eq.) in EtOH (5.0 mL) was added hydrazine hydrate (0.3 mL, 6.57 mmol, 5.0 eq.), followed by dropwise addition of acetic acid (5 drops), and the reaction mixture was stirred at 90 °C overnight. Product formation was confirmed by TLC and LCMS. The reaction mixture was concentrated under reduced pressure to obtain a crude product. By using 12 g of The crude product was purified by flash chromatography on a column and eluted in 2% MeOH / DCM solvent system to obtain the desired compound (300 mg). LCMS: 395.4 [M+H] +

[0300] Step-5: Synthesis of tert-butyl 3-((3-(3-(5-cyanopyrazin-2-ylamino)-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)azetidine-1-carboxylate: To a solution of tert-butyl 3-((3-(3-amino-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)azetidine-1-carboxylate (150 mg, 0.380 mmol, 1.0 eq.) in EtOH (5 mL) was added DIPEA (0.14 mL, 0.76 mmol, 2 eq.) followed by 5-bromopyrazine-2-carbonitrile (140 mg, 0.76 mmol, 2.0 eq.) and the reaction mixture was stirred at 90 °C overnight. The product formation was confirmed by TLC and LCMS. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The product was purified by using 12 g of crude product. The crude product was purified by flash chromatography using a 2% MeOH / DCM solvent system to give the desired compound (100 mg). LCMS: 442.3 [(M+1)-56] +

[0301] Step-6: Synthesis of 5-(5-(3-(azetidin-3-ylmethoxy)naphthalen-2-yl)-1H-pyrazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 3-((3-(3-((5-cyanopyrazin-2-ylamino)-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)azetidine-1-carboxylate (100 mg, 0.201 mmol, 1.0 eq.) in DCM (5.0 mL) was added TFA (80 mg, 0.704 mmol, 3.5 eq.) at 0°C and kept stirring for 2 h. Then the reaction mixture was concentrated under reduced pressure to get the crude product. The crude product was purified by R-HPLC to get the desired compound (15 mg, formate salt). LCMS: 398.3 [M+H]+ 99.89@220nm,99.85@254nm 1 H NMR (400MHz, DMSO-d6) δ8.71(s,1H),8.51(s,1H),8.35(s,1H),8.31(s,1H),7.92(d,J=8.3Hz,1H),7.84(d,J=8.2Hz,1H),7.52(d,J=3.1Hz, 1H),7.49(d,J=7.8Hz,1H),7.40(t,J=7.4Hz,1H),7.17(s,1H),4.40(d,J=4.7Hz,2H),4.05(t,J=8.7Hz,2H),3.60(s,2H),3.21–3.16(m,1H).

[0302] Example-26: Synthesis of 5-(5-(3-(morpholin-2-ylmethoxy)naphthalen-2-yl)-1H-pyrazol-3-ylamino)pyrazine-2-carbonitrile (Compound 2.3)

[0303]

[0304] Step-1: Synthesis of methyl 3-hydroxy-2-naphthoate: Synthesized as described in WO2000009169A1

[0305] Step-2: Synthesis of tert-butyl 2-((3-(methoxycarbonyl)naphthalen-2-yloxy)methyl)morpholine-4-carboxylate: To a solution of tert-butyl 2-(tosyloxy)methyl)morpholine-4-carboxylate (1.5 g, 4.04 mmol, 1.0 eq.) in DMF (40 mL) was added CsCO (3.2 g, 10.10 mmol, 2.5 eq.) followed by methyl 3-hydroxy-2-naphthoate (1.06 g, 5.25 mmol, 1.3 eq.) and the reaction mixture was stirred at 90 °C overnight. The progress of the reaction was monitored by TLC and LCMS. The reaction mixture was quenched with ice water (50 mL) and extracted with ethyl acetate (3 x 25 mL). The collected organic layer was washed with saturated brine (25 mL) and dried over NaSO, then concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash chromatography using 20% ​​ethyl acetate / hexane solvent system to give the desired compound (1.3 g). LCMS: 302.4 [M+H-100]

[0306] Step-3: Synthesis of tert-butyl 2-((3-(2-cyanoacetyl)naphthalene-2-yloxy)methyl)morpholine-4-carboxylate: To a solution of ACN (10 mL) in THF (8.0 mL) was added LiHMDS (1M in THF) (8.97 mL, 8.97 mmol, 3.0 eq) at -78 °C and stirred at the same temperature for 2 hours. Then tert-butyl 2-((3-(methoxycarbonyl)naphthalene-2-yloxy)methyl)morpholine-4-carboxylate (1.2 g, 2.99 mmol, 1.0 eq) was added and stirred for 2 h. Product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (3×15 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over Na2SO4, and then concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash chromatography using 30% ethyl acetate / hexane solvent system to give the desired compound (800 mg). LCMS: 311.3 [M+H-100] +

[0307] Step-4: Synthesis of tert-butyl 2-((3-(3-amino-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)morpholine-4-carboxylate: To a solution of tert-butyl 2-((3-(2-cyanoacetyl)naphthalen-2-yloxy)methyl)morpholine-4-carboxylate (500 mg, 1.22 mmol, 1.0 eq.) in EtOH (5.0 mL) was added hydrazine hydrate (0.3 mL, 6.09 mmol, 5.0 eq.), followed by acetic acid (5 drops) and the reaction mixture was stirred at 90 °C overnight. The product formation was confirmed by TLC and LCMS. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash chromatography using 2% MeOH / DCM solvent system to obtain the desired compound (400 mg). LCMS: 325.7 [M+H-100] +

[0308] Step-5: Synthesis of tert-butyl 2-((3-(3-(5-cyanopyrazin-2-ylamino)-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)morpholine-4-carboxylate: To a solution of tert-butyl 2-((3-(3-amino-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)morpholine-4-carboxylate (200 mg, 0.471 mmol, 1.0 equiv) in EtOH (5.0 mL) was added DIPEA (0.21 mL, 1.18 mmol, 2.5 equiv) followed by 5-bromopyrazine-2-carbonitrile (172 mg, 0.942 mmol, 2.0 equiv) and the reaction mixture was stirred at 90 °C overnight. The product formation was confirmed by TLC and LCMS. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude product was then purified by flash chromatography by using 2% MeOH / DCM solvent system to obtain the desired compound (150 mg). LCMS: 528.3[(M+1)] +

[0309] Step-6: Synthesis of 5-((5-(3-(morpholin-2-ylmethoxy)naphthalen-2-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carbonitrile: To a solution of tert-butyl 2-((3-(3-((5-cyanopyrazin-2-yl)amino)-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)morpholine-4-carboxylate (100 mg, 0.190 mmol, 1.0 eq.) in DCM (5.0 mL) was added TFA (75 mg, 0.663 mmol, 3.5 eq.) at 0°C and kept stirring for 2 hours. Then the reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude product was purified by R-HPLC to obtain the desired compound (15 mg, formate salt). LCMS: 428.3 [M+H] + 99.25@220nm,99.23@254nm. 1 H NMR(400MHz,DMSO-d6)δ8.68(s,1H),8.46(s,1H),8.28(s,1H),8.23(s,1H),7.88(d,J =8.1Hz,1H),7.82(d,J=8.2Hz,1H),7.52–7.46(m,2H),7.40(t,J=7.5Hz,1H),7.18(s, 1H), 4.23 (qd, J = 10.4, 4.5Hz, 2H), 4.04 (s, 1H), 3.94 (d, J = 12.0Hz, 1H), 3.64 (d, J = 11. 5Hz, 1H), 3.15 (d, J = 12.2Hz, 1H), 2.93 (d, J = 12.6Hz, 1H), 2.81 (q, J = 13.3, 12.8Hz, 2H).

[0310] Example-27: Synthesis of 5-(5-(3-(pyrrolidin-3-ylmethoxy)naphthalen-2-yl)-1H-pyrazol-3-ylamino)pyrazine-2-carbonitrile (Compound 2.4)

[0311]

[0312] Step-1: Synthesis of methyl 3-hydroxy-2-naphthoate: Synthesized as described in WO2000009169A1

[0313] Step-2: Synthesis of tert-butyl 3-((3-(methoxycarbonyl)naphthalen-2-yloxy)methyl)pyrrolidine-1-carboxylate: To a solution of methyl 3-hydroxy-2-naphthoate (1.37 g, 6.75 mmol, 1.2 eq.) in DMF (30 mL) was added Cs2CO3 (2.75 g, 8.44 mmol, 1.5 eq.) followed by addition of tert-butyl 3-(tosyloxymethyl)pyrrolidine-1-carboxylate (2.0 g, 5.63 mmol, 1.0 eq.) and the reaction mixture was kept stirring at 90 °C overnight. The progress of the reaction was monitored by TLC and LCMS. The reaction mixture was quenched by the addition of ice water (50 mL) and extracted with ethyl acetate (3×25 mL). The collected organic layer was washed with saturated brine (25 mL), dried over Na2SO4, and then concentrated under reduced pressure to obtain a crude product. The product was purified by using 24 g of The crude product was purified by flash chromatography on a column and eluted in a 30% ethyl acetate: hexane solvent system to obtain the desired compound (1.62 g). LCMS: 286.3 [M+H-100] + ; Remove Boc peak

[0314] Step-3: Synthesis of tert-butyl 3-((3-(2-cyanoacetyl)naphthalene-2-yloxy)methyl)pyrrolidine-1-carboxylate: To a solution of acetonitrile (12 mL) in THF (20 mL) was added LiHMDS (1M in THF) (14.7 mL, 14.7 mmol, 3.5 eq) at -78°C and stirred at the same temperature for 2 hours. Then tert-butyl 3-((3-(methoxycarbonyl)naphthalene-2-yloxy)methyl)pyrrolidine-1-carboxylate (1.62 g, 4.20 mmol, 1.0 eq) was added and stirred for 2 hours. Product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (3×15 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over Na2SO4, and then concentrated under reduced pressure to obtain a crude product. By using 12 g The crude product was purified by flash chromatography on a column and eluted in a 30% ethyl acetate: hexane solvent system to obtain the desired compound (620 mg). LCMS: 295.3 [M+H-100] +

[0315] Step-4: Synthesis of tert-butyl 3-((3-(3-amino-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)pyrrolidine-1-carboxylate: To a solution of tert-butyl 3-((3-(2-cyanoacetyl)naphthalen-2-yloxy)methyl)pyrrolidine-1-carboxylate (620 mg, 1.57 mmol, 1.0 eq.) in EtOH (12 mL) was added hydrazine hydrate (1.18 g, 23.5 mmol, 15.0 eq.), followed by addition of acetic acid (5 drops) dropwise and the resulting reaction mixture was kept stirring at 90 °C overnight. The formation of the product was confirmed by TLC and LCMS. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The product was purified by using 12 g of The crude product was purified by flash chromatography on a column and eluted in 2% MeOH / DCM solvent system to obtain the desired compound (300 mg). LCMS: 409.1 [M+H] +

[0316] Step-5: Synthesis of tert-butyl 3-((3-(3-(5-cyanopyrazin-2-ylamino)-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)pyrrolidine-1-carboxylate: To a solution of tert-butyl 3-(((3-(3-amino-1H-pyrazol-5-yl)naphthalen-2-yl)oxy)methyl)pyrrolidine-1-carboxylate (150 mg, 0.367 mmol, 1.0 eq.) in EtOH (5.0 mL) was added DIPEA (0.17 mL, 0.918 mmol, 2.5 eq.) followed by 5-bromopyrazine-2-carbonitrile (135 mg, 0.734 mmol, 2.0 eq.) and the reaction mixture was stirred at 90 °C overnight. The product formation was confirmed by TLC and LCMS. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The product was purified by using 12 g of crude product. The crude product was purified by flash chromatography on a column and eluted in 2% MeOH / DCM solvent system to obtain the desired compound (70 mg). LCMS: 512.3 [M+H] +

[0317] Step-6: Synthesis of 5-(5-(3-(pyrrolidin-3-ylmethoxy)naphthalen-2-yl)-1H-pyrazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 3-((3-(3-(5-cyanopyrazin-2-ylamino)-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)pyrrolidine-1-carboxylate (70 mg, 0.137 mmol, 1.0 eq.) in DCM (5.0 mL) was added TFA (54.6 mg, 0.479 mmol, 3.5 eq.) at 0°C, and the reaction mixture was stirred for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound (12 mg, formate salt). LCMS: 412.3 [M+1] + ; 99.89@220nm,99.86@254nm. 1 H NMR(400MHz,DMSO-d6)δ8.66(s,1H),8.47(s,1H),8.37(s,1H),8.24(s,1H),7.85 (dd,J=15.8,8.1Hz,2H),7.57–7.46(m,2H),7.41(t,J=7.4Hz,1H),7.17(s,1H),4. 26(dd,J=9.4,6.2Hz,1H),4.16(t,J=8.7Hz,1H),3.46–3.36(m,1H),3.28–3.04(m, 3H),2.93–2.86(m,1H),2.21(dq,J=14.1,7.3Hz,1H),1.81(dq,J=14.8,7.6Hz,1H)

[0318] Example-28: Synthesis of 5-(5-(3-(piperidin-3-ylmethoxy)naphthalen-2-yl)-1H-pyrazol-3-ylamino)pyrazine-2-carbonitrile (Compound 2.5)

[0319]

[0320] Step-1: Synthesis of methyl 3-hydroxy-2-naphthoate: Synthesized as described in WO2000009169A1.

[0321] Step-2: Synthesis of tert-butyl 3-((3-(methoxycarbonyl)naphthalen-2-yloxy)methyl)piperidine-1-carboxylate: To a solution of tert-butyl 3-(tosyloxymethyl)piperidine-1-carboxylate (2.0 g, 5.41 mmol, 1.0 eq.) in DMF (40 mL) was added Cs2CO3 (4.41 g, 13.53 mmol, 2.5 eq.), followed by addition of methyl 3-hydroxy-2-naphthoate (1.42 g, 7.04 mmol, 1.3 eq.) and the reaction mixture was kept stirring at 90 °C overnight. The progress of the reaction was monitored by TLC and LCMS. The reaction mixture was quenched by the addition of ice water (50 mL) and extracted with ethyl acetate (3×25 mL). The collected organic layer was washed with saturated brine (25 mL), dried over Na2SO4, and then concentrated under reduced pressure to obtain a crude product. The product was purified by using 24 g of HPLC purifying the product. The crude product was purified by flash chromatography on a column and eluted in a 30% ethyl acetate: hexane solvent system to obtain the desired compound (1.3 g). LCMS: 300.3 [M+H-100] + ; Remove Boc peak

[0322] Step-3: Synthesis of tert-butyl 3-((3-(2-cyanoacetyl)naphthalene-2-yloxy)methyl)piperidine-1-carboxylate: To a solution of acetonitrile (7.0 mL) in THF (15 mL) was added LiHMDS (1M in THF) (8.7 mL, 8.7 mmol, 3.5 eq) at -78 °C and stirred at the same temperature for 2 hours. Tert-butyl 3-((3-(methoxycarbonyl)naphthalene-2-yloxy)methyl)piperidine-1-carboxylate (1.0 g, 2.50 mmol, 1.0 eq) was then added and the reaction mixture was stirred for 2 hours. Product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (3×15 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over Na2SO4, and then concentrated under reduced pressure to give a crude product. By using 12 g The crude product was purified by flash chromatography on a column and eluted in a 30% ethyl acetate: hexane solvent system to obtain the desired compound (550 mg). LCMS: 309.4 [M+H-Boc] +

[0323] Step-4: Synthesis of tert-butyl 3-((3-(3-amino-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)piperidine-1-carboxylate: To a solution of tert-butyl 3-((3-(2-cyanoacetyl)naphthalen-2-yloxy)methyl)piperidine-1-carboxylate (550 mg, 1.35 mmol, 1.0 eq.) in EtOH (5.0 mL) was added hydrazine hydrate (1.01 mL, 20.2 mmol, 15.0 eq.), followed by dropwise addition of acetic acid (0.1 mL), and the resulting reaction mixture was kept stirring at 90 °C overnight. The product formation was confirmed by TLC and LCMS. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The product was purified by using 12 g of crude oil. The crude product was purified by flash chromatography on a column and eluted in 2% MeOH / DCM solvent system to obtain the desired compound (250 mg). LCMS: 423.7 [M+H] +

[0324] Step-5: Synthesis of tert-butyl 3-((3-(3-(5-cyanopyrazin-2-ylamino)-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)piperidine-1-carboxylate: To a solution of tert-butyl 3-((3-(3-amino-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)piperidine-1-carboxylate (150 mg, 0.355 mmol, 1.0 eq.) in EtOH (5.0 mL) was added DIPEA (0.19 mL, 1.07 mmol, 3.0 eq.) followed by 5-bromopyrazine-2-carbonitrile (78.3 mg, 0.426 mmol, 1.2 eq.) and added to the reaction mixture and kept stirring at 90 °C for 6 hours. The product formation was confirmed by TLC and LCMS. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The product was purified by using 12 g of The crude product was purified by flash chromatography on a column and eluted in a 2% MeOH / DCM solvent system to obtain the desired compound (110 mg). LCMS: 526.3 [M+H] +

[0325] Step-6: Synthesis of 5-(5-(3-(piperidin-3-ylmethoxy)naphthalen-2-yl)-1H-pyrazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 3-((3-(3-(5-cyanopyrazin-2-ylamino)-1H-pyrazol-5-yl)naphthalen-2-yloxy)methyl)piperidine-1-carboxylate (110 mg, 0.209 mmol, 1.0 eq.) in DCM (5.0 mL) was added TFA (83.5 mg, 0.733 mmol, 3.5 eq.) at 0°C and kept stirring for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound (15 mg). LCMS: 426.3 [M+H]+ ;UPLC:99.86@220nm,99.90@254nm. 1 HNMR (400MHz, methanol-d4) δ8.56(s,1H),8.53(s,1H),8.48(s,1H),8.10(s,1H),7.86(d,J=8.1Hz,1H),7.8 2(d,J=8.2Hz,1H),7.54–7.45(m,1H),7.45(s,1H),7.40(t,J=7.3Hz,1H),7.04(s,1H),4.27(dd,J=9.8 ,5.0Hz,1H),4.17(dd,J=9.7,6.9Hz,1H),3.58–3.49(m,1H),3.38(s,1H),2.93(td,J=12.9,12.3,8.7 Hz,2H),2.44(s,1H),2.09(d,J=14.0Hz,1H),2.03–1.97(m,1H),1.87–1.71(m,1H),1.65–1.51(m,1H).

[0326] Example-29: Synthesis of 5-((5-(3-(2-aminoethoxy)naphthalen-2-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carbonitrile (Compound 2.6)

[0327]

[0328] Step-1: Synthesis of methyl 3-hydroxy-2-naphthoate: Synthesized as described in WO2000009169A1

[0329] Step-2: Synthesis of methyl 3-(2-(tert-butoxycarbonylamino)ethoxy)-2-naphthoate: To a solution of methyl 3-hydroxy-2-naphthoate (2.0 g, 9.89 mmol, 1.0 eq.) in DMF (40 mL) was added Cs2CO3 (4.83 g, 14.84 mmol, 1.5 eq.) followed by addition of tert-butyl (2-bromoethyl)carbamate (2.66 g, 11.87 mmol, 1.2 eq.) and the reaction mixture was kept stirring at 90 °C overnight. The progress of the reaction was monitored by TLC and LCMS. The reaction mixture was quenched by the addition of ice water (50 mL) and extracted with ethyl acetate (3×25 mL). The collected organic layer was washed with saturated brine (25 mL), dried over Na2SO4, and then concentrated under reduced pressure to obtain a crude product. The product was purified by using 24 g of HPLC purifying the product. The crude product was purified by flash chromatography on a column and eluted in a 30% ethyl acetate: hexane solvent system to obtain the desired compound (620 mg). LCMS: 246.3 [M+H-100] + ; Remove Boc peak

[0330] Step-3: Synthesis of tert-butyl (2-((3-(2-cyanoacetyl)naphthalen-2-yl)oxy)ethyl)carbamate: To a solution of acetonitrile (10 mL) in THF (8 mL) was added LiHMDS (1M in THF) (6.28 mL, 6.28 mmol, 3.5 eq) at -78 °C and stirred at the same temperature for 2 hours. Then 3-(2-(tert-butoxycarbonyl)aminoethoxy)-2-naphthoic acid methyl ester (620 mg, 1.8 mmol, 1.0 eq) was added and the reaction mixture was stirred for 2 hours. Product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (3×15 mL). The collected organic layer was washed with saturated brine (20 mL), dried over Na2SO4, and then concentrated under reduced pressure to obtain a crude product. By using 12 g The crude product was purified by flash chromatography on a column and eluted in a 30% ethyl acetate: hexane solvent system to obtain the desired compound (320 mg). LCMS: 255.3 [M+H-100] +

[0331] Step-4: Synthesis of tert-butyl 2-(3-(3-amino-1H-pyrazol-5-yl)naphthalen-2-yloxy)ethylcarbamate: To a solution of tert-butyl 2-(3-(2-cyanoacetyl)naphthalen-2-yloxy)ethylcarbamate (300 mg, 0.847 mmol, 1.0 eq.) in EtOH (5.0 mL) was added hydrazine hydrate (635 mg, 12.71 mmol, 15.0 eq.), followed by dropwise addition of acetic acid (5 drops), and the resulting reaction mixture was kept stirring at 90 °C overnight. The formation of the product was confirmed by TLC and LCMS. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The product was purified by using 12 g of HPLC. The crude product was purified by flash chromatography on a column and eluted in a 2% MeOH / DCM solvent system to obtain the desired compound (60 mg). LCMS: 369.4 [M+H] +

[0332] Step-5: Synthesis of tert-butyl 2-(3-(3-(5-cyanopyrazin-2-yl)amino)-1H-pyrazol-5-yl)naphthalen-2-yloxy)ethylcarbamate: To a solution of tert-butyl 2-(3-(3-amino-1H-pyrazol-5-yl)naphthalen-2-yloxy)ethylcarbamate (60 mg, 0.163 mmol, 1.0 eq.) in EtOH (5.0 mL) was added DIPEA (0.07 mL, 0.407 mmol, 2.5 eq.) followed by 5-bromopyrazine-2-carbonitrile (59.9 mg, 0.326 mmol, 2.0 eq.) and the reaction mixture was stirred at 90 °C overnight. The product formation was confirmed by TLC and LCMS. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The product was purified by using 12 g of HPLC purifying the product. The crude product was purified by flash chromatography on a column and eluted in a 2% MeOH / DCM solvent system to obtain the desired compound (40 mg). LCMS: 472.3 [M+H] +

[0333] Step-6: Synthesis of 5-(5-(3-(2-aminoethoxy)naphthalen-2-yl)-1H-pyrazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 2-(3-(3-(5-cyanopyrazin-2-ylamino)-1H-pyrazol-5-yl)naphthalen-2-yloxy)ethylcarbamate (40 mg, 0.084 mmol, 1.0 eq.) in DCM (4.0 mL) was added TFA (34 mg, 0.297 mmol, 3.5 eq.) at 0°C and kept stirring for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound (4 mg, formate salt). LCMS: 372.3 [M+H] + ;UPLC:99.03@220nm,98.98@254nm. 1 H NMR (400MHz, DMSO-d6) δ8.67(s,1H),8.52(s,1H),8.32(s,1H),8.24(s,1H),7.91(d,J=8.2Hz,1H),7. 85(d,J=8.1Hz,1H),7.58–7.47(m,2H),7.41(t,J=7.5Hz,1H),7.12(s,1H),4.35(s,2H),3.27(s,2H).

[0334] Example-30: Synthesis of 5-(5-(3-(3-aminopropoxy)naphthalen-2-yl)-1H-pyrazol-3-ylamino)pyrazine-2-carbonitrile (Compound 2.7)

[0335]

[0336] Step-1: Synthesis of methyl 3-hydroxy-2-naphthoate: Synthesized as described in WO2000009169A1.

[0337] Step-2: Synthesis of methyl 3-(3-(tert-butoxycarbonylamino)propoxy)-2-naphthoate: To a solution of methyl 3-hydroxy-2-naphthoate (2.5 g, 12.36 mmol, 1.0 eq.) in DMF (40 mL) was added Cs2CO3 (10.0 g, 30.9 mmol, 2.5 eq.), followed by addition of tert-butyl 3-bromopropylcarbamate (3.83 g, 16.07 mmol, 1.3 eq.), and the reaction mixture was kept stirring at 90 °C overnight. The progress of the reaction was monitored by TLC and LCMS. The reaction mixture was quenched by the addition of ice water (50 mL) and extracted with ethyl acetate (3×30 mL). The collected organic layer was washed with saturated brine (50 mL), dried over Na2SO4, and then concentrated under reduced pressure to obtain a crude product. By using 24 g The crude product was purified by flash chromatography on a column and eluted in a 30% ethyl acetate: hexane solvent system to obtain the desired compound (2.0 g). LCMS: 260.4 [M+H-100] + ; Remove Boc peak

[0338] Step-3: Synthesis of tert-butyl (3-(3-(2-cyanoacetyl)naphthalen-2-yloxy)propyl)carbamate: To a solution of acetonitrile (10.0 mL) in THF (30 mL) was added LiHMDS (1M in THF) (16.7 mL, 16.7 mmol, 3.0 equiv) at -78°C and stirred at the same temperature for 2 hours. Then 3-(3-(tert-butoxycarbonylamino)propoxy)-2-naphthoic acid methyl ester (2.0 g, 5.56 mmol, 1.0 equiv) was added, and the reaction mixture was stirred for 2 hours. Product formation was confirmed by TLC and LCMS. The reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (3×25 mL). The collected organic layer was washed with saturated brine (20 mL) and dried over Na2SO4, and then concentrated under reduced pressure to obtain a crude product. By using 12 g The crude product was purified by flash chromatography on a column and eluted in 30% ethyl acetate: hexane solvent system to obtain the desired compound (1.1 g). 1H NMR (400MHz, DMSO-d6) δ8.27(s,1H),7.95(d,J=8.2Hz,1H),7.84(d,J=8.4Hz,1H),7.56(t,J=7.5Hz,1H),7.45(s,1H),7.41(t,J= 7.5Hz, 1H), 6.90 (t, J = 5.7Hz, 1H), 4.14 (t, J = 6.0Hz, 2H), 3.86 (s, 2H), 3.16 (q, J = 6.5Hz, 2H), 1.90 (p, J = 6.4Hz, 2H), 1.37 (s, 9H).

[0339] Step-4: Synthesis of tert-butyl 3-(3-(3-amino-1H-pyrazol-5-yl)naphthalen-2-yloxy)propylcarbamate: To a solution of tert-butyl 3-(3-(2-cyanoacetyl)naphthalen-2-yloxy)propylcarbamate (500 mg, 1.36 mmol, 1.0 eq.) in EtOH (5.0 mL) was added hydrazine hydrate (1.02 mL, 20.36 mmol, 15.0 eq.), followed by dropwise addition of acetic acid (0.1 mL), and the resulting reaction mixture was kept stirring at 90 °C overnight. The product formation was confirmed by TLC and LCMS. The reaction mixture was concentrated under reduced pressure to obtain a crude product. By using 12 g of The crude product was purified by flash chromatography on a column and eluted in a 2% MeOH / DCM solvent system to obtain the desired compound (130 mg). LCMS: 383.4 [M+H] +

[0340] Step-5: Synthesis of tert-butyl 3-(3-(3-(5-cyanopyrazin-2-ylamino)-1H-pyrazol-5-yl)naphthalen-2-yloxy)propylcarbamate: To a solution of tert-butyl 3-(3-(3-amino-1H-pyrazol-5-yl)naphthalen-2-yloxy)propylcarbamate (130 mg, 0.339 mmol, 1.0 eq.) in EtOH (5.0 mL) was added DIPEA (0.15 mL, 0.849 mmol, 2.5 eq.) followed by 4-bromo-1-naphthalenecarbonitrile (125 mg, 0.670 mmol, 2.0 eq.) and the reaction mixture was added and kept stirring at 90 °C for 6 hours. The product formation was confirmed by TLC and LCMS. The reaction mixture was concentrated under reduced pressure to obtain a crude product. By using 12 g The crude product was purified by flash chromatography on a column and eluted in a 2% MeOH / DCM solvent system to obtain the desired compound (80 mg). LCMS: 486.3 [M+H] +

[0341] Step-6: Synthesis of 5-(5-(3-(3-aminopropoxy)naphthalen-2-yl)-1H-pyrazol-3-ylamino)pyrazine-2-carbonitrile: To a solution of tert-butyl 3-(3-(3-(5-cyanopyrazin-2-ylamino)-1H-pyrazol-5-yl)naphthalen-2-yloxy)propylcarbamate (80 mg, 0.164 mmol, 1.0 eq.) in DCM (5.0 mL) was added TFA (65.8 mg, 0.577 mmol, 3.5 eq.) at 0°C and kept stirring for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by R-HPLC to obtain the desired compound (15 mg). LCMS: 386.3 [M+H] + ;UPLC:99.73@220NM;99.63@254NM; 1 HNMR(400MHz,DMSO-d6)δ8.67(s,1H),8.47(s,1H),8.39(s,1H),8.22(s,1H),7.88(d,J=8.3Hz,1H),7.83(d,J=8.2Hz,1H),7 .49(d,J=7.2Hz,2H),7.40(t,J=7.5Hz,1H),7.14(s,1H),4.29(t,J=5.9Hz,2H),3.06(t,J=7.1Hz,2H),2.15(p,J=6.5Hz,2H).

[0342] Biological Examples

[0343] Example-31: Determination of % inhibition of CHK-1 kinase

[0344] The inhibition of CHK-1 kinase or the inhibition rate at a specific concentration was determined by TR-FRET assay. Kinase reactions were performed in a low-volume 384-well plate (lot number: Corning 4511) in a volume of 20 μL. The concentration of the substrate fluorescein-CREB peptide in the assay was maintained at 0.2 μM, and the kinase reaction buffer consisted of 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. Serially diluted compounds (3-fold) in DMSO (0.5% in the final reaction) were incubated with a mixture of CHK-1 kinase (6 nM; lot number PR3959B, ThermoFisher), fluorescein-CREB peptide (0.2 μM; lot number PV3508, ThermoFisher), ATP (15 μM; lot number A1852, Sigma) and kinase reaction buffer. After 1 hour of kinase reaction at room temperature, 10 μL of detection mixture (8 nM Tb-CREB [phosphorylated at Serine 133] antibody, Lot No. PV3542, ThermoFisher) and 20 mM EDTA in TR-FRET buffer, Lot No. PV3574, ThermoFisher) were used in a plate reader (Synergy TM TR-FRET on the plate was read by using the PCR amplification system (NEO; Biotek).

[0345] The resulting TR-FRET emission ratio was used to calculate the inhibition ratio by normalizing it to the control using the following formula:

[0346] [% inhibition = 1 - {(% phosphorylation 样品 / % Phosphorylation 对照(0％抑制)}×100]

[0347] The results of % inhibition of CHK1 enzyme at 30 nM and 300 nM are shown in Table B1.

[0348]

[0349]

[0350] CHK1 IC 50 The results are shown in Table-B2

[0351] Table-B2

[0352] Compound No. <![CDATA[CHK1 IC 50 (nM)]]> Compound 1.1 67.7 Compound 1.2 13.3 Compound 1.3 7.77 Compound 1.4 86.4 Compound 1.5 7.82 Compound 1.6 12.3 Compound 1.8 76.6 Compound 1.9 >10000 Compound 2.1 2.46 Compound 2.2 4.14 Compound 2.3 8.15 Compound 2.4 2.34 Compound 2.5 4.36 Compound 2.6 15.7 Compound 2.7 0.933

[0353] Example-32: Microsome Stability Assay

[0354] The incubation mixture contained 0.2 mg / mL microsomes, 1 mmol NADPH, and 1 μM of the test compound in 100 mmol potassium phosphate buffer at pH 7.4. The mixture was incubated at 37°C for 5, 15, and 30 minutes. Aliquots of the mixture were taken at 0, 5, 15, and 30 minutes and quenched with chilled acetonitrile: methanol (50:50) containing an internal standard. After the incubation was complete, the samples were centrifuged at 4,000 rpm at 4°C. The supernatant was analyzed by LC-MS / MS, and the half-life and intrinsic clearance (Clint) were calculated.

[0355] The results of the microsomal stability assay are provided in Table-B3.

[0356] Table-B3

[0357]

[0358]

[0359] HLM: human liver microsomes; MLM: mouse liver microsomes; RLM: rat liver microsomes

[0360] Example-33: Kinetic Solubility Determination

[0361] In 96-well V-bottom dilution plates (#3363, Corning), the test samples were serially diluted in DMSO to a concentration range from 10mM to 0.078mM. From each well, 1 μL of the test sample was transferred to a 96-well flat-bottom transparent plate (#655101, Greiner) containing 99 μL of pH-7.4 PBS, which gave a final concentration range of 100 μM to 0.78 μM for the test drug. The samples were incubated at 37°C for 1 hour, and then the light scattering at 625nm was measured using a laser-based microplate turbidimeter (Nephlostar Plus, BMG Labtech). The turbidimeter was then used as a reading to calculate the solubility concentration (μM) by segmented regression.

[0362] The results of the kinetic solubility assay are provided in Table-B4.

[0363] Table-B4

[0364]

[0365]

[0366] Example-34: Caco-2 cell permeability assay

[0367] After Caco-2 cells are thawed, they are cultured in a culture medium containing 10% FBS, 1% sodium pyruvate, 1% NEAA and 1% Glutamax MEM, and then placed in an incubator containing 5% CO2 at 37°C, and then the culture medium is replaced every 2-3 days. When the confluence reaches 80%, subculture is performed. 5mL of 1M HEPES is added to 500mL of HBSS for the preparation of transport buffer (pH 7.4), and the final concentration of HEPES is 10mM. Before the transport study (days 21-28), the culture medium is removed from the cell culture plate, the cell culture plate is washed twice with preheated HBSS buffer (10mM HEPES, pH 7.4), and 100 μL HBSS buffer is added to each well. After incubation at 37°C for 30 minutes, the Teer value is measured by a cell resistance meter (Millicell-ERS2) to confirm the integrity of the monolayer cells. Discard the HBSS buffer, transfer the upper plate containing the cell monolayer membrane to the corresponding receiving plate, add the test compound solution or buffer to the top (A side) and the bottom (B side), respectively, place the cell plate in the corresponding 96-well receiving plate, and add the prepared positive control compound, test compound or HBSS buffer (containing 0.5% BSA) to the A side or B side of the filter membrane of the Transwell cell plate according to the following grouping, and incubate the cell plate at 37°C. When incubated for 5 minutes, take 8 μL of sample solution from the A side and the B side respectively, and add 72 μL of transport buffer (diluted 10 times) as the T0 of the initial solution. After incubation for 120 minutes, collect the samples at both ends of the filter membrane of the Transwell cell plate; take 80 μL of sample solution from the receiving side as the sample of the receiving side after transport, and take 8 μL of sample solution from the application side and mix it with 72 μL of transport buffer, and use the mixed solution as the sample of the application side after transport. After collecting the bilateral samples, gently tap, discard the remaining solution in the cell plate, wash the cell plate with transport buffer; and add 100 μL of acetonitrile solution containing internal standard working solution to each well to lyse the cells; after mixing evenly by blowing up and down, take 80 μL of cell lysate from each well as the post-cell lysis sample. After collecting all samples in the transport system (T0 samples from the receiving side and the application side), add 160 μL of IS / ACN solution to each well, add 80 μL of IS / ACN solution to the collected lysate samples, and then seal them. Shake for 10 minutes and store the sample plate at -20 ° C before LC-MS / MS analysis.

[0368] The following equations were used to calculate the apparent permeability coefficient (Papp, cm / s), efflux ratio (ER), solution recovery (% recovery) and total recovery (% total recovery).

[0369]

[0370] in,

[0371] V 接收体 is the volume of the receiving end solution (0.25 mL from A to B and 0.1 mL from B to A);

[0372] V 供体 is the volume of the solution at the application end (A to B is 0.1 mL and B to A is 0.25 mL);

[0373] V 裂解物 is the volume of cell lysate (0.1 mL);

[0374] The area is the relative surface area of ​​the cell monolayer (0.0804 cm 2 );

[0375] Time is the incubation time (7200 s);

[0376] [drug] 接收体 is the drug concentration of the receiving end or control compound (nM);

[0377] [drug] 供体 is the concentration of the drug or control compound at the administration end (nM);

[0378] [drug] 裂解物 is the concentration of drug or control compound in cell lysate (nM);

[0379] [drug] 初始 is the initial concentration of the test or control compound (nM).

[0380] Table-B5

[0381]

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: in, X is selected from O or NH; R1 is selected from C1-C6 alkyl, C1-C6 alkylene, C3-C8 cycloalkyl or absent; R2 is selected from H, -NH2, unsaturated or saturated monocyclic, bicyclic or spirocyclic 4 to 10 membered heterocyclic group, wherein the heterocyclic group contains 1-2 independently selected from N (R3) n and O heteroatoms; R4, R6 and R7 are independently -A-R5 or absent; A is selected from: (i) key; (ii) CH2; (iii) a saturated chain of 2 to 10 chain members in length containing at least one carbon atom chain member, at least one heteroatom chain member selected from nitrogen and oxygen and optionally one or more further carbon atom chain members and / or heteroatom chain members selected from nitrogen, oxygen, sulfur, sulfinyl and sulfonyl; the saturated chain is optionally substituted by one or more heteroatoms selected from ═O, C 1-4 Substitution with hydrocarbyl and fluorine substituents, wherein two hydrocarbyl substituents on the same carbon atom may optionally be linked to form a ring of three to five ring members; R5 is selected from: (i) H, (ii) CH2, (iii) a saturated or unsaturated monocyclic 5-6 membered heterocyclic group, wherein the heterocyclic group contains 1-3 independently selected from N(R3) n , O or S heteroatom; optionally each R3 is selected from H or C1-C3 alkyl; n is 0, 1 or 2; Or R4 and R6 together with the carbon to which they are attached form a 5, 6, 7 or 8 membered ring, wherein the ring is partially or fully unsaturated and is substituted or unsubstituted.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein formula (I) is in, R1 is selected from C1-C6 alkyl, C1-C6 alkylene, C3-C8 cycloalkyl or absent; R2 is selected from H, -NH2, unsaturated or saturated monocyclic, bicyclic or spirocyclic 4 to 10 membered heterocyclic group, wherein the heterocyclic group contains 1-2 independently selected from N (R3) n and O heteroatoms; R4 is -A-R5; A is selected from: (i) key; (ii) CH2; (iii) a saturated chain of 2 to 10 chain members in length containing at least one carbon atom chain member, at least one heteroatom chain member selected from nitrogen and oxygen and optionally one or more further carbon atom chain members and / or heteroatom chain members selected from nitrogen, oxygen, sulfur, sulfinyl and sulfonyl; the saturated chain is optionally substituted by one or more heteroatoms selected from ═O, C 1-4 Substitution with hydrocarbyl and fluorine substituents, wherein two hydrocarbyl substituents on the same carbon atom may optionally be linked to form a ring of three to five ring members; R5 is selected from: (i) H, (ii) a saturated or unsaturated monocyclic 5-6 membered heterocyclic group, wherein the heterocyclic group contains 1-3 independently selected from N(R3) n , O or S heteroatom; optionally each R3 is selected from H or C1-C3 alkyl; n is 0, 1 or 2.

3. The compound according to claim 2, wherein formula (II) is selected from:

4. The compound according to claim 2 or 3, wherein R4 is -A-R5; A is selected from: (i) a key; or (ii) a saturated chain of 2 to 4 chain members in length containing at least one carbon atom chain member, at least one heteroatom chain member selected from nitrogen and optionally one or more further carbon atom chain members and / or heteroatom chain members selected from nitrogen, oxygen, sulfur, sulfinyl and sulfonyl; the saturated chain is optionally substituted by one or more heteroatoms selected from ═O, C 1-4 Substitution with hydrocarbyl and fluorine substituents, wherein two hydrocarbyl substituents on the same carbon atom may optionally be linked to form a ring of three to five ring members; R5 is selected from: (i) H, or (ii) a saturated or unsaturated monocyclic 6-membered heterocyclic group, wherein the heterocyclic group contains 1-2 independently selected from N(R3) n optionally each R3 is selected from H or C1-C3 alkyl; n is 0, 1 or 2.

5. A compound according to any one of claims 2 to 4, wherein R4 is -A-R5; A is selected from: (i) a key; or (ii)–(CH2) 1-4 、-NH-(CH2) 1-3 ; R5 is selected from: (i) H, or (ii) 6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein formula (I) is in, R1 is selected from C1-C6 alkyl, C1-C6 alkylene, C3-C8 cycloalkyl or absent; R2 is selected from H, -NH2, unsaturated or saturated monocyclic, bicyclic or spirocyclic 4 to 10 membered heterocyclic group, wherein the heterocyclic group contains 1-2 independently selected from N (R3) n and O heteroatoms; R4, R6 and R7 are independently -A-R5; A is selected from: (i) key; (ii) CH2; (iii) a saturated chain of 2 to 10 chain members in length containing at least one carbon atom chain member, at least one heteroatom chain member selected from nitrogen and oxygen and optionally one or more further carbon atom chain members and / or heteroatom chain members selected from nitrogen, oxygen, sulfur, sulfinyl and sulfonyl; the saturated chain is optionally substituted by one or more heteroatoms selected from ═O, C 1-4 Substitution with hydrocarbyl and fluorine substituents, wherein two hydrocarbyl substituents on the same carbon atom may optionally be linked to form a ring of three to five ring members; R5 is selected from: (i) H, (ii) CH2, (iii) a saturated or unsaturated monocyclic 5-6 membered heterocyclic group, wherein the heterocyclic group contains 1-3 independently selected from N(R3) n , O or S heteroatom; optionally each R3 is selected from H or C1-C3 alkyl; n is 0, 1 or 2; Or R4 and R6 together with the carbon to which they are attached form a 5, 6, 7 or 8 membered ring, wherein the ring is partially or fully unsaturated and is substituted or unsubstituted.

7. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein formula (III) is wherein R8 is -A-R5; A is selected from: (i) key; (ii) CH2; (iii) a saturated chain of 2 to 10 chain members in length containing at least one carbon atom chain member, at least one heteroatom chain member selected from nitrogen and oxygen and optionally one or more further carbon atom chain members and / or heteroatom chain members selected from nitrogen, oxygen, sulfur, sulfinyl and sulfonyl; the saturated chain is optionally substituted by one or more heteroatoms selected from ═O, C 1-4 Substitution with hydrocarbyl and fluorine substituents, wherein two hydrocarbyl substituents on the same carbon atom may optionally be linked to form a ring of three to five ring members; R5 is selected from: (i) H, (ii) CH2, (iii) a saturated or unsaturated monocyclic 5-6 membered heterocyclic group, wherein the heterocyclic group contains 1-3 independently selected from N(R3) n , O or S heteroatom; optionally each R3 is selected from H or C1-C3 alkyl; n is 0, 1 or 2.

8. The compound according to claim 7 or a pharmaceutically acceptable salt thereof, wherein formula (IV) is 9. The compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, wherein R1 is selected from C1-C3 alkyl, C1-C4 alkylene, C3-C6 cycloalkyl or absent; R2 is selected from H, -NH2, unsaturated or saturated monocyclic or spirocyclic 4 to 6 membered heterocyclic group, wherein the heterocyclic group contains 1-2 independently selected from N (R3) n and O heteroatoms.

10. The compound according to any one of claims 1 to 9, wherein R1 is selected from methyl, ethyl, n-propyl, isopropyl, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or absent.

11. A compound according to any one of claims 1 to 10, wherein R2 is selected from H, -NH2, 12. A compound according to any one of claims 1 to 11, wherein R3 is selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,3-dimethylbutyl or 2,2-dimethylbutyl.

13. The compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

14. A compound according to any one of claims 1 to 13 for use in the manufacture of a medicament for treating a disorder associated with the checkpoint kinase-1 (CHK-1) ​​biological pathway.

15. A method of treating a disease associated with checkpoint kinase-1 (CHK-1) ​​in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a compound according to any one of claims 1 to 13.

16. A method of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a compound according to any one of claims 1 to 13.

17. A method of inhibiting checkpoint kinase-1 (CHK-1) ​​in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a compound according to any one of claims 1 to 13.

18. A method of treating idiopathic pulmonary fibrosis (IPF) in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a compound according to any one of claims 1 to 13.

19. A method of treating pulmonary arterial hypertension (PAH) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 13.

20. A method of treating cancer, IPF or PAH in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a compound according to any one of claims 1 to 13 in combination with another therapeutic agent.

21. A pharmaceutical composition comprising a compound according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

22. A method of treating a disease associated with checkpoint kinase-1 (CHK-1) ​​in an individual in need thereof, comprising administering to the individual the pharmaceutical composition of claim 21.

23. Use of a compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a disease mediated by checkpoint kinase-1 (CHK-1).

24. A kit comprising the compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof.

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