Selective inhibitor as well as preparation method and application thereof

Abstract

The invention provides an RET selective inhibitor and a preparation method and application thereof. The present invention provides compounds of formula (I) and pharmaceutically acceptable salts, solvates, polymorphs or isomers thereof, pharmaceutical compositions comprising these compounds, and the use of such compounds in the treatment of RET-related diseases

Description

Technical Field

[0001] The present invention relates to compounds that selectively inhibit RET protein activity, and also relates to methods for preparing these compounds and uses of their pharmaceutical compositions. Background Art

[0002] RET is a proto-oncogene located on chromosome 10. The RET protein encoded by the RET gene is a receptor tyrosine kinase (RTK) present on the cell membrane. It belongs to the cadherin superfamily and is essential for many physiological functions such as early embryonic development, nervous system development, kidney morphogenesis, spermatogenesis and hematopoiesis (Nat. Rev. Cancer 2014, 14(3): 173-86.).

[0003] Abnormalities of the ret gene, including activating point mutations and gene fusions, can lead to abnormal dimerization of RET or promote ligand-independent activation, and are carcinogenic drivers of various solid tumors. Point mutations of the ret gene, such as V804M, M918T, C620R / S, C634R / W / Y, etc., can promote abnormal dimerization of RET or enhance its ability to bind to ATP, and are common in multiple endocrine tumors and 50% of patients with medullary thyroid cancer. Chromosomal rearrangements of the ret gene can cause the kinase domain of the ret gene to fuse with different genes such as kif5b, ccdc6, and ncoa4, leading to high expression of RET or ligand-independent dimerization activation, becoming a driving factor for malignant tumor proliferation (Transl Lung Cancer Res. 2015; 4(2): 156-164). RET gene fusion is present in 5%-40% of papillary thyroid carcinoma and 1-2% of non-small cell lung cancer patients; as well as a variety of solid tumors, including prostate cancer and breast cancer. RET fusion-positive NSCLC patients tend to be younger (≤ 60 years old), have little or no smoking history, have a more advanced disease stage, and have a higher risk of brain metastasis (Nat Rev Clin Oncol, 2018. 15(3): p. 151-167).

[0004] NSCLC is a common malignancy with a high incidence. According to statistics, the number of new cases of RET fusion-positive NSCLC is about 10,000 per year, which is roughly the same as the incidence of chronic myeloid leukemia. In 2012, RET fusion-positive was first identified as a driver of NSCLC (Nat Med, 2012, 18(3): p.375-7.), at that time, only a few multikinase inhibitors (MKIs) with RET inhibitory activity were available, including: Cabozantinib, Vandetanib, Lenvatinib, Sorafenib, Alectinib, etc. Preliminary data showed that MKIs had limited clinical benefits for RET fusion-positive NSCLC, with an objective response rate (ORR) of 16-53% (depending on the specific MKI and patient population), and a median progression-free survival (PFS) of only 3.4-7.3 months (Oncologist, 2023, 28(5): p.402-413.), and were accompanied by toxic side effects due to their significant inhibitory effects on other kinases (such as vascular endothelial growth factor receptor 2 (VEGFR2)). Off-target toxicity led to dose reductions in 23-73% of patients and drug discontinuation in 8-24% of patients (Nat Rev Clin Oncol. 2018, 15(3): 151-167). In addition, although anti-PD1 / PD-L1-directed immune checkpoint inhibitors (ICIs) are widely used to treat NSCLC patients, the effect of ICIs on RET fusion-positive NSCLC patients, including those with positive PD-L1 expression, is poor (ORR is only 0%-20%, and the median PFS is 2.2-3.4 months) (J Hematol Oncol, 2020, 13(1): p. 37). Therefore, the development of highly selective RET inhibitors has attracted the research interest of many pharmaceutical companies, and the development of specific RET inhibitors has become an emerging therapeutic approach to improve the treatment of RET-driven cancers.

[0005] Recently, according to the results of the LIBRETTO-001 (NCT03157128) and ARROW studies (NCT03037385), two selective RET inhibitors, selpercatinib and pralsetinib, have rapid and durable clinical efficacy in advanced RET-altered solid tumors, with fewer treatment-related adverse events. These two drugs have been approved by the FDA for the treatment of adult patients with RET fusion-positive NSCLC and adult and pediatric patients with RET mutation or RET fusion-positive thyroid cancer, and have become first-line treatments for these patients (Journal of Clinical Oncology, 2023, 41(2): p.385-394). Pralsetinib is currently approved in China for the treatment of locally advanced or metastatic RET fusion-positive NSCLC after platinum chemotherapy (https: / / www.cstonepharma.com / en / html / news / 2566.html).

[0006] Despite the encouraging efficacy of highly selective RET tyrosine kinase inhibitors (RET-TKIs), experience with targeted therapy for NSCLC has shown that drug resistance is still an inevitable and important problem in the use of RET-TKIs to treat RET fusion-positive NSCLC, and the ultimate acquired resistance will limit the duration of benefit of highly selective RET inhibitors. In 2020, it was first reported that RET G810 solvent-front mutations occurred in patients with RET fusion-positive NSCLC after treatment with selpercatinib, and G810R / S / C existed simultaneously in multiple tissues of the patients, leading to drug resistance and disease progression (J Thorac Oncol. 2020 April; 15(4): 541-549). Subsequently, in 2021, RET G810 mutations were also reported in patients with RET fusion-positive NSCLC after treatment with pralsetinib. L730 roof mutation leads to pralsetinib resistance (Precision Oncology (2021) 5: 48). Currently, the reported RET acquired resistance mutation sites include solvent front mutation (RETG810), roof mutation (RET L730), gatekeeper mutation (RET V804), hinge mutation (RETY806) and RETV804M / G810S double mutation, etc. These mutations can interfere with the binding of inhibitors to the RET kinase domain and show cross-resistance to selpercatinib and pralsetinib (Ann Oncol. 2021 February; 32(2): 261-268.); Through retrospective analysis, 13% of patients developed acquired resistance to RET mutations, which is a high incidence rate. These patients have shorter duration of remission (DoR) and progression-free survival (PFS). It is foreseeable that as more and more patients are treated with RET selective inhibitors, the increase in the number of resistant patients will also become a major problem. In the future, there is a huge clinical need to develop a new generation of RET inhibitors targeting RET resistance mutations to effectively overcome the resistance problem of these patients. Summary of the invention

[0007] The present invention provides a RET selective inhibitor, which is a compound represented by the general formula (I) or a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof. The present invention also provides a series of compounds represented by the general formula (I) and pharmaceutically acceptable salts, solvates, polymorphs or isomers thereof, pharmaceutical compositions containing these compounds, and methods for treating diseases using such compounds.

[0008] In one aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof:

[0009]

[0010] in,

[0011] L is -NH-(CO)-CR 3 R 4 -(CO)-NH-, -NH-(CO)-NH-, -CR 3 R 4 -(CO)-NH-, or -(CO)-NH-,

[0012] R 1 Selected from 5-6 membered heteroaryl, 3-12 membered heterocyclic group, -OC 1-6 Alkyl and -SC 1-6 The alkyl group, the heteroaryl group and the heterocyclic group may each be optionally substituted by halogen, C 1-6 Alkyl, -CN, -OH, -NR 6 R 7 、-OC 1-6 Alkyl, or -SC 1-6 Alkyl substituted, the alkyl group may be optionally substituted with halogen, -CN, -OH, -NR 6 R 7 、-OC 1-6 Alkyl, or -SC 1-6 Alkyl substitution,

[0013] R 2 Selected from -CN, -C≡C-(C 1-6 alkyl), 5-6 membered heteroaryl and 3-12 membered heterocyclic group, each of which may be optionally substituted by halogen, C 1-6 Alkyl, -CN, -OH, -NR 6 R 7 、-OC 1-6 Alkyl, or -SC 1-6 Alkyl substitution,

[0014] X, X 1 , X 2 , X 3 and X 4 Each independently selected from N and CR 8 ,

[0015] R 8 Each is independently selected from H, halogen, -CN, -OH, -NR 6 R 7 , C 1-6 Alkyl, -OC 1-6 Alkyl and -SC 1-6 alkyl,

[0016] A is phenyl, 5-6 membered heteroaryl, 3-8 membered heterocyclic group, or 3-8 membered cycloalkyl, and the phenyl, heteroaryl, cycloalkyl and heterocyclic group may be optionally replaced by R 5 replace,

[0017] R 5 Selected from halogen, -CN, -OH, -NR 6 R 7 , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -(CH 2 ) 0-1 -(3-8 membered cycloalkyl), -(CH 2 ) 0-1 -(3-8 membered heterocyclic group), -(CH 2 ) 0-1 -(6-10 membered aryl), -(CH 2 ) 0-1 -(5-12 membered heteroaryl), -OC 1-6 Alkyl, or -SC 1-6 Alkyl substituted, the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl may be optionally substituted with halogen, -CN, -OH, -NR 6 R 7 , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OC 1-6 Alkyl, or -SC 1-6 Alkyl substitution,

[0018] R 3 and R 4 Each independently selected from H and C 1-6 Alkyl, or R 3 and R 4 Connected together to form a 3-6 membered carbon ring,

[0019] R 6 and R 7 Each independently selected from H and C 1-6 alkyl,

[0020] R 9 H, -NH 2 , or -NH-C 1-6 alkyl.

[0021] In some embodiments, L is -CR 3 R 4 -(CO)-NH-, R 3 and R 4 As defined above.

[0022] In some embodiments, R 2 is selected from 5-6 membered heteroaryl and 3-12 membered heterocyclic group, wherein the heteroaryl and heterocyclic group may be each optionally substituted by halogen, C 1-6 Alkyl, -CN, -OH, -NR 6 R 7 、-OC 1-6 Alkyl, or -SC 1-6 Alkyl substitution, R 6 and R 7 As defined above.

[0023] In some embodiments, R 9 H or -NH 2 , preferably -NH 2 .

[0024] In some embodiments of the present invention, the present invention provides the following compounds:

[0025]

[0026]

[0027] Or a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof.

[0028] Another aspect of the present invention also relates to a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof, and a pharmaceutically acceptable carrier;

[0029] In another aspect, the present invention provides a method for treating a disease associated with RET, the method comprising administering to a subject an effective amount of a compound of the present invention or a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof, or the above composition; in some embodiments, the disease associated with RET is lung cancer, papillary thyroid cancer, medullary thyroid cancer, differentiated thyroid cancer, recurrent thyroid cancer, refractory differentiated thyroid cancer, multiple endocrine neoplasia type 2A or 2B (MEN2A or MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer, colorectal cancer, papillary renal cell carcinoma, gastrointestinal mucosal gangliocytoma and cervical cancer, preferably RET fusion lung cancer or medullary thyroid cancer, more preferably small cell lung cancer, non-small cell lung cancer, bronchiolar lung cancer or lung adenocarcinoma;

[0030] In some embodiments of the present invention, the subject involved in the present invention is a mammal including a human;

[0031] On the other hand, the present invention provides a use of a compound of the present invention or a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof, or the above composition in the preparation of a medicament for treating a disease associated with RET; in some embodiments, the disease associated with RET is lung cancer, papillary thyroid cancer, medullary thyroid cancer, differentiated thyroid cancer, recurrent thyroid cancer, refractory differentiated thyroid cancer, multiple endocrine neoplasia type 2A or 2B (MEN2A or MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer, colorectal cancer, papillary renal cell carcinoma, gastrointestinal mucosal gangliocytoma, or cervical cancer, preferably RET fusion lung cancer or medullary thyroid cancer, more preferably small cell lung cancer, non-small cell lung cancer, or bronchiolar lung cancer or lung adenocarcinoma. DETAILED DESCRIPTION OF THE INVENTION

[0033] Exemplary embodiments utilizing the principles of the present invention are set forth in the following detailed description of the invention. Features and advantages of the present invention may be better understood by reference to the following summary of the invention.

[0034] It should be understood that the scope of protection of various aspects of the present invention is determined by the claims, and the methods and structures within the scope of these claims and their equivalent methods and structures are within the scope of the claims.

[0035] Unless otherwise defined, all technical terms herein have the same meaning as commonly understood by those skilled in the art to which the subject matter of the claims belongs. Unless otherwise indicated, all patents, patent applications, and public materials cited herein are incorporated herein by reference in their entirety.

[0036] It should be understood that the above brief description and the following detailed description are exemplary and explanatory, rather than limiting on any subject matter of the present invention. Unless otherwise specifically stated, the singular form also includes the plural form. Unless otherwise specified, the use of "or" and "or" means "and / or". In addition, the use of the term "including" and other forms, such as "including", "containing" and "containing" are not limiting.

[0037] Some chemical terms

[0038] The term "optional", "optional" or "optionally" means that the event or situation described subsequently may or may not occur, and the description includes the occurrence of the event or situation and the non-occurrence of the event or situation. For example, "optionally substituted alkyl" means "unsubstituted alkyl" or "substituted alkyl". Also, the optionally substituted group can be unsubstituted (for example: -CH 2 CH 3 ), completely replaced (for example: -CF 2 CF 3), monosubstituted (e.g. -CH 2 CH 2 F) or any level between mono- and fully substituted (e.g. -CH 2 CHF 2 , -CF 2 CH 3 ,-CFHCHF 2 It will be understood by those skilled in the art that for any group containing one or more substituents, no substitution or substitution pattern will be introduced that is sterically impossible to exist and / or cannot be synthesized.

[0039] Unless otherwise indicated, conventional methods within the skill of the art are used, such as mass spectrometry, nuclear magnetic resonance, high performance liquid chromatography, infrared and ultraviolet / visible spectroscopy and pharmacological methods. Unless specifically defined, the relevant terms and experimental procedures and techniques in analytical chemistry, organic synthetic chemistry, and pharmaceutical and medicinal chemistry herein are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and treatment of patients. For example, the manufacturer's instructions for the use of the kit can be used, or the reaction and purification can be carried out in a manner known in the art or in the description of the present invention. The above techniques and methods can usually be implemented according to the descriptions in the multiple general and more specific references cited and discussed in this specification, according to conventional methods well known in the art. In this specification, groups and substituents thereof can be selected by those skilled in the art to provide stable structural moieties and compounds.

[0040] When substituents are described by conventional chemical formulas written from left to right, the substituents also include chemically equivalent substituents that would result if the formula were written from right to left. For example, -CH 2 O- is equivalent to -OCH 2 -.

[0041] As used herein, the terms "group" and "chemical group" refer to a specific part or functional group of a molecule. A chemical group is often considered to be a chemical entity embedded in or attached to a molecule.

[0042] Some of the chemical groups named herein may be abbreviated to indicate the total number of carbon atoms. For example, C 1 -C 6 Alkyl describes an alkyl group, as defined below, having a total of 1 to 6 carbon atoms. The total number of carbon atoms indicated in the shorthand notation does not include carbon atoms in possible substituents.

[0043] The terms "halogen", "halo" or "halide" refer to bromine, chlorine, fluorine or iodine.

[0044] As used herein, the terms "aromatic", "aromatic ring", "aromatic", "aromatic", "aromatic ring" refer to a planar ring or ring moiety having a delocalized electron conjugated system containing 4n+2 electrons, where n is an integer. Aromatic rings may be formed by 5, 6, 7, 8, 9 or more atoms. Aromatic compounds may be optionally substituted and may be monocyclic or polycyclic with fused rings. The term aromatic compound includes all carbon rings (such as benzene rings) and rings containing one or more heteroatoms (such as pyridine).

[0045] The term "heteroatom" or "hetero" as used herein, alone or as part of another component, refers to atoms other than carbon and hydrogen. Heteroatoms are independently selected from oxygen, nitrogen, sulfur, phosphorus, silicon, selenium and tin, but are not limited to these atoms. In embodiments where two or more heteroatoms are present, the two or more heteroatoms may be the same as each other, or some or all of the two or more heteroatoms may be different from each other.

[0046] The terms "fused" or "fused ring", as used herein, alone or in combination, refer to a cyclic structure in which two or more rings share one or more bonds.

[0047] The term "spiro" or "spirocycle", as used herein, alone or in combination, refers to a cyclic structure in which two or more rings share one or more atoms.

[0048] The term "alkyl" as used herein alone or as part of another component (e.g., monoalkylamino) refers to an optionally substituted straight chain or optionally substituted branched monovalent saturated hydrocarbon having 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms, connected to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, n-octyl, n-nonyl, n-decyl, etc.

[0049] The term "alkenyl" as used herein, alone or in combination, refers to an optionally substituted straight chain or optionally substituted branched monovalent hydrocarbon radical having one or more C=C double bonds and having 2 to about 10 carbon atoms, more preferably 2 to about 6 carbon atoms. The double bonds in these groups may be in the cis or trans configuration and are understood to include both isomers. Examples include, but are not limited to, vinyl (CH=CH 2 ), 1-propenyl (CH 2 CH=CH 2 ), isopropenyl (C(CH 3 )=CH 2 ), butenyl and 1,3-butadienyl, etc. When a numeric range appears for alkenyl as defined herein, for example, "C 2 -C 6 "Alkenyl" or "C 2-6"Alkenyl" refers to an alkenyl group which may be composed of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms, and the alkenyl group herein also encompasses the case where no numerical range is specified.

[0050] The term "alkynyl" as used herein, alone or in combination, refers to an optionally substituted straight or branched monovalent hydrocarbon radical having one or more C≡C triple bonds and having 2 to about 10 carbon atoms, more preferably 2 to about 6 carbon atoms. Examples include, but are not limited to, ethynyl, 2-propynyl, 2-butynyl, and 1,3-butadiynyl. When a numerical range appears for alkynyl as defined herein, for example, "C 2 -C 6 Alkynyl" or "C 2-6 "Alkynyl" refers to an alkynyl group which may be composed of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms. The alkynyl group herein also encompasses the case where no numerical range is specified.

[0051] The term "aryl" refers to an all-carbon monocyclic or fused ring having a completely conjugated π electron system, having 6-14 carbon atoms, preferably 6-12 carbon atoms, and most preferably 6 carbon atoms. Aryl groups may be unsubstituted or substituted with one or more substituents, examples of which include, but are not limited to, alkyl, alkyloxy, aryl, aralkyl, amino, halogen, hydroxy, sulfonyl, sulfinyl, phosphoryl, and heteroalicyclic groups. Non-limiting examples of unsubstituted aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl.

[0052] The term "heteroaryl" refers to a monocyclic or fused ring of 5-12 ring atoms, having 5, 6, 7, 8, 9, 10, 11 or 12 ring atoms, of which 1, 2, 3 or 4 ring atoms are selected from N, O, S, and the remaining ring atoms are C, and have a completely conjugated π-electron system. Heteroaryl can be unsubstituted or substituted, and the substituents include but are not limited to alkyl, alkyloxy, aryl, aralkyl, amino, halogen, hydroxyl, cyano, nitro, carbonyl and heteroalicyclic groups. Non-limiting examples of unsubstituted heteroaryl include but are not limited to pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, quinolyl, isoquinolyl, tetrazolyl, triazinyl.

[0053] The term "cycloalkyl" as used herein alone or as part of other components refers to a stable monovalent non-aromatic monocyclic or polycyclic hydrocarbon group, containing only carbon atoms and hydrogen atoms, which may include fused rings, spiro rings or bridged ring systems, containing 3-15 ring carbon atoms, preferably 3-10 ring carbon atoms, more preferably 3-8 ring carbon atoms, which may be saturated or unsaturated, and connected to the rest of the molecule by a single bond. Non-limiting examples of "cycloalkyl" include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, etc.

[0054] The term "carbocycle" refers to a structure formed by a covalently closed ring of carbon, which may be saturated or partially unsaturated. The carbocycle may be formed by 3, 4, 5, 6, 7, 8, 9 or more atoms. The difference between the term carbocycle and heterocycle is that the ring skeleton of the heterocycle contains at least one atom different from carbon. The "carbocycle" herein may be a monocyclic ring or a polycyclic ring, and the polycyclic carbocycle includes a spirocyclic ring, a fused ring and a bridged ring. The carbocycle may be arbitrarily substituted. The "carbocycle" herein preferably contains about 5 to about 20 or 5 to 10 or 5-8 or 5-6 skeleton ring atoms.

[0055] The terms "heterocyclyl", "heterocycloalkyl", and "heterocycle" as used herein alone or as part of other components refer to stable 3-18-membered monovalent non-aromatic rings, including 2-12 carbon atoms, 1-6 heteroatoms selected from nitrogen, oxygen and sulfur. Unless otherwise specified, the heterocyclyl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may contain fused rings, spirocyclic or bridged ring systems, the nitrogen, carbon or sulfur on the heterocyclyl can be optionally oxidized, the nitrogen atom can be optionally quaternized, and the heterocyclyl can be partially or fully saturated. The heterocyclyl can be connected to the rest of the molecule through a single bond through a carbon atom or a heteroatom on the ring. The heterocyclyl containing fused rings can contain one or more aromatic rings or heteroaromatic rings, as long as the atoms on the non-aromatic rings that connect to the rest of the molecule are connected. For the purpose of the present application, the heterocyclic group is preferably a stable 4-11 membered monovalent non-aromatic monocyclic or bicyclic ring containing 1-3 heteroatoms selected from nitrogen, oxygen and sulfur, and more preferably a stable 4-8 membered monovalent non-aromatic monocyclic ring containing 1-3 heteroatoms selected from nitrogen, oxygen and sulfur. Non-limiting examples of heterocyclic groups include azepanyl, azetidinyl, decahydroisoquinolinyl, dihydrofuranyl, dihydroindolinyl, dioxolanyl, 1,1-dioxo-thiomorpholinyl, imidazolidinyl, imidazolinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazinyl, piperazinyl, piperidinyl, 4-piperidonyl, pyranyl, pyrazolidinyl, pyrrolidinyl, quinolizinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydropyranyl, and the like.

[0056] The term "polymorph" or "polymorphism" as used herein refers to compounds of the present invention having multiple crystal lattice forms. Some compounds of the present invention may have more than one crystal form, and the present invention encompasses all polymorphic forms or mixtures thereof.

[0057] Intermediate compounds of the compounds of the present invention and polymorphs thereof are also within the scope of the present invention.

[0058] Unless otherwise specified, compounds of the present invention containing olefinic double bonds include E and Z isomers.

[0059] It should be understood that the compounds of the present invention may contain asymmetric centers. These asymmetric centers may independently be in R or S configuration. Some of the compounds of the present invention may also exhibit cis-trans isomerism, which is obvious to those skilled in the art. It should be understood that the compounds of the present invention include their individual geometric isomers and stereoisomers and their mixtures, including racemic mixtures. These isomers can be separated from their mixtures by implementing or modifying known methods, such as chromatography techniques and recrystallization techniques, or they can be prepared separately from the appropriate isomers of their intermediates.

[0060] The term "pharmaceutically acceptable salt" used herein includes both acid-addition salts and base-addition salts.

[0061] "Pharmaceutically acceptable salts of added acids" refer to those salts that retain the biological effectiveness and properties of the free base of the compound, are not biologically or otherwise undesirable, and are formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., or organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, capric acid, caproic acid, carbonic acid, cinnamic acid, citric acid, etc. "Pharmaceutically acceptable base-added salts" refer to those salts that retain the biological effectiveness and properties of the free acid of the compound, are not biologically or otherwise undesirable. These salts are prepared by reacting the free acid with an inorganic base or an organic base. Salts formed by reacting with an inorganic base include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, etc. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and manganese salts.

[0062] Organic bases for forming salts include, but are not limited to, primary amines, secondary amines, tertiary amines, cyclic amines, and the like, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, ethanolamine, dicyclohexylamine, ethylenediamine, purine, piperazine, piperidine, choline, and caffeine. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0063] Crystallization often produces solvates of the compounds of the invention. The term "solvate" as used herein refers to a complex formed by the combination of one or more molecules of the compounds of the invention and one or more solvent molecules.

[0064] The solvent may be water, in which case the solvate is a hydrate. It may also be an organic solvent. Therefore, the compounds of the present invention may exist as hydrates, including monohydrates, dihydrates, hemihydrates, trihydrates, tetrahydrates, etc., and corresponding solvated forms. The compounds of the present invention may be true solvates, but in other cases, the compounds of the present invention may only accidentally retain water or a mixture of water and some other solvents. The compounds of the present invention may react in a solvent or precipitate or crystallize in a solvent. Solvates of the compounds of the present invention are also included in the scope of the present invention.

[0065] The term "pharmaceutical composition" as used herein refers to a preparation containing a mixture of the compound of the present invention and a medium generally accepted in the art for delivering biologically active compounds to mammals (such as humans). Such a medium includes all pharmaceutically acceptable carriers.

[0066] The term "acceptable" with respect to a formulation, composition or ingredient, as used herein, means having no persistent detrimental effect on the general health of the subject being treated.

[0067] The term "pharmaceutically acceptable" as used herein refers to a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compounds of the present invention and is relatively non-toxic, that is, the substance can be administered to a subject without causing adverse biological reactions or interacting in an adverse manner with any components contained in the composition.

[0068] "Pharmaceutically acceptable carriers" include, but are not limited to, adjuvants, carriers, excipients, auxiliary agents, deodorants, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants and wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that have been approved by relevant government administrative departments for use in humans and domesticated animals.

[0069] As used herein, the terms "subject", "patient", "subject" or "individual" refer to individuals suffering from a disease, disorder or condition, etc., including mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates (e.g., chimpanzees and other apes and monkeys); livestock, such as cattle, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs and cats; laboratory animals, including rodents, such as rats, mice and guinea pigs, etc. Examples of non-human mammals include, but are not limited to, birds and fish, etc. In one embodiment of the methods and compositions provided herein, the mammal is a human.

[0070] The term "treatment" as used herein refers to the treatment of a disease or condition in a mammal, especially a human, including

[0071] (i) preventing a mammal from developing a disease or condition, particularly a mammal that has been previously exposed to the disease or condition but has not yet been diagnosed with the disease or condition;

[0072] (ii) inhibiting the disease or condition, i.e., controlling its development;

[0073] (iii) alleviate the disease or condition, i.e., cause the disease or condition to regress;

[0074] (iv) Alleviate the symptoms caused by a disease or condition.

[0075] As used herein, the terms "disease" and "disorder" may be used interchangeably or may have different meanings because certain specific diseases or disorders do not yet have a known causative agent (so the cause of the disease is still unclear), and therefore cannot be considered a disease but can only be viewed as an unwanted condition or syndrome, the syndrome of which more or less specific symptoms have been confirmed by clinical researchers.

[0076] As used herein, the terms "effective amount", "therapeutically effective amount" or "pharmaceutically effective amount" refer to an amount of at least one agent or compound sufficient to relieve to some extent one or more symptoms of the disease or condition being treated after administration. The result can be a reduction and / or alleviation of signs, symptoms or causes, or any other desired change in a biological system. For example, an "effective amount" for treatment is the amount of a composition comprising a compound disclosed herein required to provide a clinically significant relief of the condition. Techniques such as dose escalation trials can be used to determine the effective amount appropriate for any individual case.

[0077] As used herein, the terms "administering", "administering", "administering" and the like refer to methods capable of delivering a compound or composition to a desired site for biological action. These methods include, but are not limited to, oral routes, intraduodenal routes, parenteral injections (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical administration, and rectal administration. In a preferred embodiment, the compounds and compositions discussed herein are administered orally.

[0078] Preparation of the compounds of the present invention

[0079] The following reaction scheme shows the method for preparing the compounds of the present invention.

[0080] It should be understood that in the following description, combinations of substituent groups and / or variables of the formula are allowed only if stable compounds are formed.

[0081] Those skilled in the art will also appreciate that in the processes described below, the functional groups of the intermediate compounds may need to be protected by suitable protecting groups. These functional groups include hydroxyl, amino, thiol and carboxyl. Suitable hydroxyl protecting groups include trialkylsilyl or diarylalkylsilyl (e.g. tert-butylmethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, etc. Suitable amino, amidine and guanidine protecting groups include tert-butyloxycarbonyl, benzyloxycarbonyl, etc. Suitable protecting groups for thiol include -C(O)-R" (R" represents alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl, etc. Suitable carboxyl protecting groups include alkyl, aryl or arylalkyl esters. Protecting groups can be added or removed by standard techniques known to those skilled in the art. Example

[0082] The following non-limiting examples are merely illustrative and do not limit the present invention in any way.

[0083] Unless otherwise stated, temperatures are in degrees Celsius. Reagents were purchased from commercial suppliers such as Sinopharm Chemical Reagents Beijing Co., Ltd., Alfa Aesar, or Beijing J&K Technology Co., Ltd. and were used directly without further purification unless otherwise stated.

[0084] Unless otherwise stated, the following reactions were performed at room temperature, in anhydrous solvents, under a positive pressure of nitrogen or argon, or using a drying tube; the reaction flasks were fitted with rubber septa to facilitate the addition of substrates and reagents via syringe; and the glassware was oven-dried and / or heat-dried.

[0085] Unless otherwise specified, column chromatography purification used 200-300 mesh silica gel from Qingdao Ocean Chemical Plant; preparative thin layer chromatography used thin layer chromatography silica gel precast plates (HSGF254) produced by Yantai Institute of Chemical Industry; MS was determined using a ThermoLCQ Fleet (ESI) liquid chromatography-mass spectrometer; optical rotation was determined using an SGW-3 automatic polarimeter, Shanghai Shenguang Instrument Co., Ltd.

[0086] NMR data( 1 H NMR) was performed using a Varian instrument at 400 MHz. The solvent used for the NMR data was CDCl 3 , CD 3 OD, D 2 O, DMSO-d6, etc., based on tetramethylsilane (0.00ppm) or residual solvent (CDCl 3 :7.26ppm;CD 3 OD: 3.31ppm; D 2O: 4.79 ppm; d6-DMSO: 2.50 ppm). When peak shape diversity is indicated, the following abbreviations represent different peak shapes: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), dd (double of doublets), dt (double of triplets). If coupling constants are given, they are in Hertz (Hz).

[0087] Abbreviations:

[0088] <![CDATA[Pd 2 (dba) 3 ]]> Tris(dibenzylideneacetone)dipalladium (±)BINAP (±)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl <![CDATA[Pd(dppf)Cl 2 ]]> 1,1'-Bis(diphenylphosphino)ferrocenepalladium(II) chloride HATU 2-(7-Azabenzotriazole)-N,N,N′,N′-tetramethyluronium hexafluorophosphate KK Potassium acetate <![CDATA[Pd(PPh 3 ) 4 ]]> Tetrakis(triphenylphosphine)palladium <![CDATA[Cs 2 CO 3 ]]> Cesium carbonate

[0089] Example 1: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(4-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[11,5-a]pyridin-3-yl)phenyl)acetamide

[0090]

[0091] Step 1: 6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-4-carbonitrile

[0092] Under nitrogen protection, 4-bromo-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine (2.77 g), zinc cyanide (2.35 g) and tetrakis(triphenylphosphine)palladium (1.1 g) were added to DMF (100 mL), heated to 120°C and stirred overnight. The mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 1:1 (V:V)) to give 6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-4-carbonitrile (1.4 g).

[0093] Step 2: 3-Bromo-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-4-carbonitrile

[0094] At room temperature, 6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-4-carbonitrile (223 mg) was dissolved in DMF (5 mL), N-bromosuccinimide (180 mg) was slowly added, and the mixture was stirred for 2 hours. The reaction solution was poured into ice water and stirred continuously. Solids precipitated and were filtered. The filter cake was washed with methyl tert-butyl ether and dried in vacuo to obtain the target product (195 mg).

[0095] Step 3: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(4-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0096] Under nitrogen protection, 3-bromo-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-4-carbonitrile (30 mg), N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetamide (39 mg), tetrakistriphenylphosphine palladium (11 mg) and potassium carbonate (28 mg) were added to dioxane and water (20 mL, volume ratio 5:1) in sequence, heated to 80°C and stirred overnight. After cooling to room temperature, the reaction solution was poured into water (50 mL), then extracted with ethyl acetate, the extract was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography (developing solvent: dichloromethane: methanol = 20:1 (V:V)) to obtain the target compound (29 mg).

[0097] MS m / z[LC-MS]: 480.60[M+1]. 1 H NMR (400MHz, DMSO-d6) δ11.22 (s, 1H), 9.40 (d, J = 1.2Hz, 1H), 8.35 (s, 2H), 8.27 (s, 1H), 8.10 (s, 1H) , 7.51 (d, J=8.4Hz, 2H), 7.38 (d, J=8.0Hz, 2H), 6.58 (s, 1H), 3.87 (s, 3H), 3.71 (s, 2H), 1.26 (s, 9H).

[0098] Example 2: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(4-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-2,2-difluoroacetamide

[0099]

[0100] Step 1: 2-(4-bromophenyl)-N-(5-(tert-butyl)isoxazol-3-yl)-2,2-difluoroacetamide

[0101] At room temperature, 2-(4-bromophenyl)-2,2-difluoroacetic acid (250 mg), 5-(tert-butyl)isoxazol-3-amine (140 mg) and HATU (400 mg) were dissolved in DMF (10 mL), DIEA (260 mg) was added, and the mixture was stirred for 12 hours. The reaction solution was poured into ice water and stirred continuously. Solids precipitated, which were filtered and dried in vacuo to obtain the target product (285 mg).

[0102] Step 2: N-(5-(tert-butyl)isoxazol-3-yl)-2,2-difluoro-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetamide

[0103] Under nitrogen protection, 2-(4-bromophenyl)-N-(5-(tert-butyl)isoxazol-3-yl)-2,2-difluoroacetamide (186 mg), biboric acid pinacol ester (254 mg), [1,1′-bis(diphenylphosphino)ferrocene] palladium dichloride (37 mg) and potassium acetate (100 mg) were added to dry dioxane (20 mL), heated to 90° C. and stirred overnight. Cooled to room temperature, filtered, the filter cake was washed with dichloromethane, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was directly used in the next step reaction.

[0104] Step 3: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(4-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-2,2-difluoroacetamide

[0105] Under nitrogen protection, 3-bromo-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-4-carbonitrile (30 mg), N-(5-(tert-butyl)isoxazol-3-yl)-2,2-difluoro-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetamide (42 mg), tetrakistriphenylphosphine palladium (11 mg) and potassium carbonate (28 mg) were added to dioxane and water (20 mL, volume ratio 5:1) in sequence, heated to 80°C and stirred overnight. After cooling to room temperature, the reaction solution was poured into water (50 mL), then extracted with ethyl acetate, the extract was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography (developing solvent: dichloromethane: methanol = 20:1 (V:V)) to obtain the target compound (25 mg).

[0106] MS m / z[LC-MS]: 516.20[M+1].

[0107] Example 3: N-(5-(tert-butyl)isoxazol-3-yl)-2-(5-(4-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl)pyridin-2-yl)acetamide

[0108]

[0109] The target compound (20 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 2.

[0110] MS m / z[LC-MS]:481.23[M+1]. 1 H NMR (400 MHz, CDCl 3)δ9.03 (s, 1H), 8.75 (s, 1H), 8.74 (d, J=1.2Hz, 1H), 8.46 (s, 2H), 8.04 (s, 1H), 7.81 (s, 1H), 7.70 (s, 1H), 7.25 (d, J=1.2Hz, 1H), 7.06 (d, J=8.0Hz, 2H), 6.88 (d, J=8.0Hz, 2H), 6.73 (s, 1H), 3.99 (s, 3H), 3.73 (s, 2H), 1.34 (s, 9H).

[0111] Example 4: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrimidin-5-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0112]

[0113] The target compound (19 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 1.

[0114] MS m / z [LC-MS]: 533.24 [M+1]. 1 H NMR (400 MHz, CDCl 3 )δ10.53 (s, 1H), 8.82 (d, J = 1.2Hz, 1H), 8.78 (d, J = 2.0Hz, 1H), 8.13 (s, 1H), 7.88 (dd, J = 8.0Hz, 2.0Hz, 1H), 7.77-7.80 (m, 2H), 7.69 (s, 1H), 7.40 (d, J=8.0Hz, 1H), 6.67 (s, 1H), 4.01 (s, 3H), 3.97 (s, 2H), 1.33 (s, 9H).

[0115] Example 5: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrimidin-5-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0116]

[0117] Step 1: 2-methyl-4-(6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl)but-3-yn-2-ol

[0118] Under nitrogen protection, 4-bromo-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine (27 mg), 2-methylbut-3-yn-2-ol (16 mg), bis(triphenylphosphine)palladium(II) dichloride (7 mg), cuprous iodide (4 mg) and triethylamine (100 mg) were added to dioxane (10 mL) in sequence, heated to 100°C and stirred overnight. The mixture was cooled to room temperature, filtered, and the filter cake was washed with dichloromethane. The filtrate was concentrated under reduced pressure and the residue was purified by thin layer chromatography (developing solvent: dichloromethane: methanol = 20:1 (V:V)) to obtain the target compound (21 mg).

[0119] Steps 2 and 3 were carried out according to the synthesis method in Example 1 using the corresponding starting materials to obtain the target compound (9 mg). MS m / z [LC-MS]: 537.26 [MM+1].

[0120] Example 6: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-morpholinopyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0121]

[0122] Step 1: 4-(6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl)morpholine

[0123] Under nitrogen protection, 4-bromo-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine (27 mg), morpholine (17 mg), tris(dibenzylideneacetone)dipalladium (9 mg), (±)-2,2′-bis(diphenylphosphino)-1,1′-binaphthalene (13 mg) and cesium carbonate (660 mg) were added to toluene (5 mL) in sequence, heated to 110°C and stirred overnight. The mixture was cooled to room temperature, filtered, the filter cake was washed with dichloromethane, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 1:1 (V:V)) to obtain the target compound (21 mg).

[0124] Steps 2 and 3 were prepared by using the corresponding raw materials in accordance with the synthesis method in Example 1 to obtain the target compound (19 mg). MS m / z [LC-MS]: 540.26 [M+1].

[0125] Example 7: 2-(4-(4,6-bis(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-N-(5-(tert-butyl)isoxazol-3-yl)acetamide

[0126]

[0127] The target compound (30 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 1.

[0128] MS m / z [LC-MS]: 535.27 [M+1].

[0129] Example 8: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(4-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl)2-fluorophenyl)acetamide

[0130]

[0131] The target compound (22 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 2.

[0132] MS m / z[LC-MS]:498.21[M+1]. 1 H NMR (400 MHz, CDCl 3 )δ8.80(d, J=1.2Hz, 1H), 8.12(s, 1H), 7.94(s, 1H), 7.77-7.78(m, 2H), 7.68(s, 1H), 7.42-7.45(m , 1H), 7.34-7.36(m, 1H), 7.27-7.31(m, 1H), 6.69(s, 1H), 4.01(s, 3H), 3.83(s, 2H), 1.33(s, 9H).

[0133] Example 9: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyridin-3-yl)pyrazolo[1,5-a]pyridin-3-yl]phenyl)acetamide

[0134]

[0135] The target compound (17 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 1.

[0136] MS m / z [LC-MS]: 532.25 [M+1]. 1H NMR (400MHz, DMSO-d6) δ11.08 (s, 1H), 9.11 (d, J = 1.2Hz, 1H), 8.43 (d, J = 2.0Hz, 1H), 8.34 (s, 1H), 8.26 (dd, J = 8.8Hz, 1.6Hz, 1H), 8.10 (s, 1H), 8.0 8(s, 1H), 7.53(d, J=1.2Hz, 1H), 7.37-7.40(m, 1H), 6.93-6.98(m, 3H), 6. 79 (d, J=8.4Hz, 2H), 6.58 (s, 1H), 3.67 (s, 3H), 3.53 (s, 2H), 1.27 (s, 9H).

[0137] Example 10: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-phenylpyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0138]

[0139] The target compound (27 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 1.

[0140] MS m / z [LC-MS]: 531.26 [M+1].

[0141] Example 11: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(6-(1-dimethyl-1H-pyrazol-4-yl)-4-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0142]

[0143] The target compound (21 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 1.

[0144] MS m / z [LC-MS]: 532.27 [M+1].

[0145] Example 12: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0146]

[0147] Step 1: 6-(1-methyl-1H-pyrazol-4-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[1,5-a]pyridine

[0148] Under nitrogen protection, 4-bromo-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine (277 mg), biboronic acid pinacol ester (510 mg), [1,1′-bis(diphenylphosphino)ferrocene] palladium dichloride (75 mg) and potassium acetate (200 mg) were added to dry dioxane (20 mL), heated to 90° C. and stirred overnight. The mixture was cooled to room temperature, filtered, the filter cake was washed with dichloromethane, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was directly used in the next step reaction.

[0149] Steps 2, 3, and 4 were prepared by referring to the synthesis method in Example 1 using the corresponding raw materials to obtain the target compound (18 mg). MS m / z [LC-MS]: 533.25 [M+1]. 1 H NMR (400MHz, DMSO-d6) δ11.09 (s, 1H), 9.19 (d, J = 1.2Hz, 1H), 8.54 (d, J = 1.2Hz, 1H), 8.34 (s, 1H), 8.32 (d, J = 2.4Hz, 1H), 8.23-8.24 (m, 1H), 8.1 3 (s, 1H), 8.08 (s, 1H), 7.72 (d, J = 1.6Hz, 1H), 6.97 (d, J = 8.0Hz, 2H), 6.7 6(d, J=8.0Hz, 2H), 6.57(s, 1H), 3.86(s, 3H), 3.54(s, 2H), 1.26(s, 9H).

[0150] Example 13: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(4-(-4-cyanophenyl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0151]

[0152] The target compound (20 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 1.

[0153] MS m / z [LC-MS]: 556.26 [M+1].

[0154] Example 14: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(4-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0155]

[0156] The target compound (30 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 1.

[0157] MS m / z [LC-MS]: 599.27 [M+1].

[0158] Example 15: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(thiophen-3-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0159]

[0160] The target compound (15 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 1.

[0161] MS m / z [LC-MS]: 537.22 [M+1].

[0162] Example 16: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(4-(6-cyanopyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl]phenyl)acetamide

[0163]

[0164] The target compound (14 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 1.

[0165] MS m / z [LC-MS]: 557.25 [M+1]. 1 H NMR (400MHz, DMSO-d6) δ11.09 (s, 1H), 9.19 (d, J = 1.2Hz, 1H), 8.54 (d, J = 1.2Hz, 1H), 8.34 (s, 1H), 8.32 (d, J = 2.4Hz, 1H), 8.23-8.24 (m, 1H), 8.1 3 (s, 1H), 8.08 (s, 1H), 7.72 (d, J = 1.6Hz, 1H), 6.97 (d, J = 8.0Hz, 2H), 6.7 6(d, J=8.0Hz, 2H), 6.57(s, 1H), 3.86(s, 3H), 3.54(s, 2H), 1.26(s, 9H).

[0166] Example 17: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(trifluoromethyl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-3-yl]phenyl)acetamide

[0167]

[0168] The target compound (30 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 1.

[0169] MS m / z[LC-MS]: 600.24[M+1].

[0170] Example 18: 2-(4-(4-(6-aminopyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl]phenyl)-N-(5-(tert-butyl)isoxazol-3-yl)acetamide

[0171]

[0172] The target compound (12 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 1.

[0173] MS m / z [LC-MS]: 547.25 [M+1]. 1 H NMR (400MHz, DMSO-d6) δ11.11 (s, 1H), 9.00 (d, J = 1.2Hz, 1H), 8.31 (s, 1H), 8.06 (s, 1H), 8.05 (s, 1H), 7.78 (d, J = 2.4Hz, 1H), 7.39 (s, 1H), 7.09-7.16 (m, 1H), 7.04 (d, J=8.0Hz, 2H), 6.90 (d, J=8.0Hz, 2H), 6.55 (s, 1H), 5.95-6.40 (m, 3H), 3.86 (s, 3H), 3.58 (s, 2H), 1.25 (s, 9H).

[0174] Example 19: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0175]

[0176] The target compound (12 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 12.

[0177] MS m / z [LC-MS]: 533.25 [M+1].

[0178] Example 20: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-3-yl]phenyl)acetamide

[0179]

[0180] The target compound (15 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 1.

[0181] MS m / z [LC-MS]: 616.33 [M+1].

[0182] Example 21: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(6-(2-hydroxy-2-methylpropoxy)-4-(pyrazin-2-yl)pyrazolo[1,5-alpyridin-3-yl)phenyl)acetamide

[0183]

[0184] The target compound (16 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 1.

[0185] MS m / z [LC-MS]: 541.26 [M+1]. 1 H NMR (400MHz, DMSO-d6) δ11.09 (s, 1H), 8.59 (d, J=2.0Hz, 1H), 8.45 (s, 1H), 8.30 (d, J=2.4Hz, 1H), 8.22-8.24 (m, 1H), 8.03 (s, 1H), 7.28 (d, J=1.6Hz, 1H), 6.95 (d, J=8.0Hz, 2H), 6.72 (d, J=8.0Hz, 2H), 6.57 (s, 1H), 4.69 (s, 1H), 3.86 (s, 2H), 3.53 (s, 2H), 1.26 (s, 9H), 1.22 (s, 6H).

[0186] Example 22: 5-(tert-butyl)-N-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)benzyl)isoxazole-3-carboxamide

[0187]

[0188] The target compound (18 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 1.

[0189] MS m / z [LC-MS]: 533.24 [M+1].

[0190] Example 23: 2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-N-(4-(((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)acetamide

[0191]

[0192] The target compound (12 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 2.

[0193] MS m / z [LC-MS]: 666.30 [M+1]. 1 H NMR (400MHz, DMSO-d6) δ10.50 (s, 1H), 9.93-10.09 (brs, 1H), 9.18 (s, 1H), 8.53 (s, 1H), 8.34 (s, 1H), 8.28 (s, 1H), 8.25 ( s, 1H), 8.13 (s, 1H), 8.08 (s, 1H), 8.04 (s, 1H), 7.84 (d, J=8.8Hz, 1H), 7.72 (s, 1H), 7.64 (d, J=8.8Hz, 1H), 7.00 (d, J=7.6H z , 2H), 6.77 (d, J=7.6Hz, 2H), 3.86 (s, 3H), 3.60 (s, 2H), 3.55 (s, 2H), 2.67-3.05 (m, 9H), 2.32-2.44 (m, 2H).

[0194] Example 24: N-(4-(1,1,1,3,3-hexafluoro-2-hydroxypropyl-2-yl)phenyl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4.(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0195]

[0196] The target compound (18 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 23.

[0197] MS m / z[LC-MS]: 652.20[M+1]. 1H NMR (400MHz, DMSO-d6) δ10.32 (s, 1H), 9.18 (d, J = 1.2Hz, 1H), 8.59 (s, 1H), 8.54 (d, J = 1.2Hz, 1H), 8.34 (s, 1H), 7.25 (d, J = 2.4Hz, 1H), 8.21-8.22 (m , 1H), 8.13 (s, 1H), 8.08 (s, 1H), 7.71-7.74 (m, 3H), 7.60 (d, J=8.4Hz, 2H) , 7.00 (d, J=8.0Hz, 2H), 6.76 (d, J=8.0Hz, 2H), 3.86 (s, 3H), 3.54 (s, 2H).

[0198] Example 25: N-(6-methoxypyridin-3-yl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl]phenyl)acetamide

[0199]

[0200] The target compound (10 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 23.

[0201] MS m / z[LC-MS]:517.23[M+1].

[0202] Example 26: 2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-N-(5-methylisoxazol-3-yl)acetamide

[0203]

[0204] The target compound (9 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 23.

[0205] MS m / z[LC-MS]: 491.20[M+1].

[0206] Example 27: N-(4-cyano-3-(trifluoromethyl)phenyl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0207]

[0208] The target compound (19 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 23.

[0209] MS m / z[LC-MS]:579.19[M+1].

[0210] Example 28: 2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-N-(5-(trifluoromethyl)pyridin-3-yl

[0211]

[0212] The target compound (19 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 23.

[0213] MS m / z[LC-MS]:555.20[M+1]. 1 H NMR (400MHz, DMSO-d6) δ10.71 (s, 1H), 9.19 (d, J=1.2Hz, 1H), 8.95 (s, 1H), 8.65 (s, 1H), 8.52 (s, 1H), 8.49 (s, 1H), 8.34 (s, 1H), 8 .27-8.31 (m, 2H), 8.14 (s, 1H), 8.08 (s, 1H), 7.72 (s, 1H), 7.01 (d, J=8.0Hz, 2H), 6.78 (d, J=8.0Hz, 2H), 3.86 (s, 3H), 3.61 (s, 2H).

[0214] Example 29: N-cyclopropyl-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0215]

[0216] The target compound (29 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 23.

[0217] MS m / z [LC-MS]: 450.22 [M+1].

[0218] Example 30: N-(4-chloro-3-(trifluoromethyl)phenyl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0219]

[0220] The target compound (24 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 23.

[0221] MS m / z [LC-MS]: 588.16 [M+1]. 1 H NMR (400MHz, DMSO-d6) δ10.54(s, 1H), 9.18(s, 1H), 8.53(s, 1H), 8.34(s, 1H), 8.28(s, 1H), 8.25(s, 1H), 8.18(s, 1H), 8.14(s, 1H), 8.08( s, 1H), 7.86 (d, J=8.4Hz, 1H), 7.72 (s, 1H), 7.66 (d, J=8.4Hz, 1H), 6.99 (d, J=8.0Hz, 2H), 6.77 (d, J=8.0Hz, 2H), 3.86 (s, 3H), 3.56 (s, 2H).

[0222] Example 31: 2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-N-(2-(trifluoromethyl)pyridin-4-yl)acetamide

[0223]

[0224] The target compound (24 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 23.

[0225] MS m / z[LC-MS]:555.20[M+1]. 1 H NMR (400MHz, DMSO-d6) δ10.86 (s, 1H), 9.19 (d, J = 1.6Hz, 1H), 8.61 (d, J = 5.6Hz, 1H), 8.53 (d, J=1.2Hz, 1H), 8.34 (s, 1H), 8.29 (d, J=2.4Hz, 1H), 8.25-8.26 (m, 1H), 8.14 ( s, 1H), 8.11 (d, J = 2.0Hz, 1H), 8.08 (s, 1H), 7.78 (dd, J = 5.6Hz, 2.0Hz, 1H), 7.72 (d, J =1.6Hz, 1H), 6.99 (d, J = 8.0Hz, 2H), 6.78 (d, J = 8.0Hz, 2H), 3.86 (s, 3H), 3.62 (s, 2H).

[0226] Example 32: N-(3-(tert-butyl)isoxazol-5-yl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0227]

[0228] The target compound (20 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 23.

[0229] MS m / z [LC-MS]: 533.26 [M+1]. 1 H NMR (400MHz, DMSO-d6) δ11.70 (s, 1H), 9.18 (d, J = 1.6Hz, 1H), 9.19 (d, J = 1.6Hz, 1H), 8.54 (d, J = 1.6Hz, 1H), 8.34 (s, 1H), 8.29 (d, J = 2.8Hz, 1H), 8.21 -8.22 (m, 1H), 8.13 (s, 1H), 7.72 (d, J = 1.6Hz, 1H), 6.96 (d, J = 8.0Hz, 2H), 6 .76(d, J=8.0Hz, 2H), 6.21(s, 1H), 3.86(s, 3H), 3.57(s, 2H), 1.23(s, 9H).

[0230] Example 33: N-(5-(tert-butyl)thiazol-2-yl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0231]

[0232] The target compound (22 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 23.

[0233] MS m / z [LC-MS]: 549.23 [M+1]. 1 H NMR (400MHz, DMSO-d6) δ12.08 (s, 1H), 9.18 (d, J = 1.2Hz, 1H), 8.55 (d, J = 1.2Hz, 1H), 8.34 (s, 1H), 8.27 (d, J = 2.4Hz, 1H), 8.18-8.19 (m, 1H), 8.1 3 (s, 1H), 8.08 (s, 1H), 7.72 (d, J=1.2Hz, 1H), 7.16 (s, 1H), 6.97 (d, J=8. 0Hz, 2H), 6.76 (d, J=8.0Hz, 2H), 3.86 (s, 3H), 3.61 (s, 2H), 1.30 (s, 9H).

[0234] Example 34: N-(3,3-difluorocyclobutyl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0235]

[0236] The target compound (14 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 23.

[0237] MS m / z [LC-MS]: 549.23 [M+1].

[0238] Example 35: 2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-N-(5-(trifluoromethyl)isoxazol-3-yl)acetamide

[0239]

[0240] The target compound (26 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 23.

[0241] MS m / z[LC-MS]: 545.17[M+1].

[0242] Example 36: N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(6-(1-methyl-1H-pyrazin-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0243]

[0244] The target compound (16 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 23.

[0245] MS m / z [LC-MS]: 516.23 [M+1]. 1 H NMR (400MHz, DMSO-d6) δ12.03 (s, 1H), 10.36 (s, 1H), 9.18 (d, J=1.2Hz, 1H), 8.56 (d, J= 1.6Hz, 1H), 8.34 (s, 1H), 8.29 (d, J=2.8Hz, 1H), 8.17-8.18 (m, 1H), 8.12 (s, 1H), 8.08 ( s, 1H), 7.71 (d, J=1.2Hz, 1H), 6.96 (d, J=8.0Hz, 2H), 6.73 (d, J=8.0Hz, 2H), 6.14 (s, 1H ), 3.86(s, 3H), 3.45(s, 2H), 1.79-1.86(m, 1H), 0.85-0.89(m, 2H), 0.60-0.64(m, 2H).

[0246] Example 37: 2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-N-(3-phenylisoxazol-5-yl)acetamide

[0247]

[0248] The target compound (16 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 23.

[0249] MS m / z [LC-MS]:553.21[M+1]. 1 H NMR (400MHz, DMSO-d6) δ11.89 (s, 1H), 9.19 (d, J = 1.2Hz, 1H), 8.55 (s, 1H), 8.34(s, 1H), 8.33(d, =2.4Hz, 1H), 8.23-8.24(m, 1H), 8.14(s, 1H), 8.08(s, 1H), 7.82-7.85 (m, 2H), 7.72 (d, J=1.2Hz, 1H), 7.45-7.50 (m, 3H), 7.00 (d, J =8.0Hz, 2H), 6.78 (d, J=8.0Hz, 2H), 6.73 (s, 1H), 3.86 (s, 3H), 3.63 (s, 2H).

[0250] Example 38: 2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)acetamide

[0251]

[0252] The target compound (16 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 23.

[0253] MS m / z [LC-MS]: 598.30 [M+1].

[0254] Example 39: N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0255]

[0256] The target compound (17 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 23.

[0257] MS m / z [LC-MS]: 613.31 [M+1]. 1 H NMR (400MHz, DMSO-d6) δ10.60 (s, 1H), 9.18 (d, J = 1.2Hz, 1H), 8.54 (d, J = 1.2Hz, 1H), 8.34 (s, 1H), 8.26 (d, J=2.4Hz, 1H), 8.17-8.21 (m, 2H), 8.13 (s, 1H), 8.08 (s, 1H), 8.02 (d, J=8.4Hz, 1 H), 7.71 (d, J=1.2Hz, 1H), 7.66 (dd, J=8.8Hz, 2.0Hz, 1H), 7.00 (d, J=8.0Hz, 2H), 6.75 (d, J=8 .0Hz, 2H), 3.86 (s, 3H), 3.58 (s, 2H), 3.41 (s, 2H), 2.08-2.54 (m, 10H), 0.94 (t, J=7.2Hz, 3H).

[0258] Example 40: 2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-N-(pyridin-4-yl)acetamide

[0259]

[0260] The target compound (7 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 23.

[0261] MS m / z[LC-MS]: 487.20[M+1].

[0262] Example 41: 2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-N-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)acetamide

[0263]

[0264] The target compound (15 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 23.

[0265] MS m / z[LC-MS]:587.22[M+1]. 1H NMR (400MHz, DMSO-d6) δ11.27 (s, 1H), 9.18 (s, 1H), 8.53 (s, 1H), 8.34 (s, 1H), 8.30 (d, J=2.4Hz, 1H), 8.21-8.24 (m, 1H), 8.13 (s, 1H), 8.08 (s, 1H), 7.72 (s, 1H), 6.97 (d, J=8.0Hz, 2H), 6.95 (s, 1H), 6.76 (d, J=8.0Hz, 2H), 3.86 (s, 3H), 3.57 (s, 2H), 1.53 (s, 6H).

[0266] Example 42: 2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-N-(3-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-5-yl)acetamide

[0267]

[0268] The target compound (10 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 23.

[0269] MS m / z[LC-MS]:587.22[M+1]. 1 H NMR (400MHz, DMSO-d6) δ11.92 (s, 1H), 9.18 (s, 1H), 8.53 (d, J = 1.2Hz, 1H), 8.33 (s, 1H), 8.28 (d, J = 2.4Hz, 1H), 8.21-8.22 (m, 1H), 8.13 (s, 1H), 8.07 (s, 1H), 7.71 (d, J=1.6Hz, 1H), 6.97 (d, J=8.0Hz, 2H), 6.76 (d, J=8.0Hz, 2H), 6.34 (s, 1H), 3.86 (s, 3H), 3.59 (s, 2H), 1.48 (s, 6H).

[0270] Example 43: 2-(4-(6-methoxy-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-N-(3-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-5-yl)acetamide

[0271]

[0272] The target compound (19 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 1.

[0273] MS m / z [LC-MS]: 537.19 [M+1].

[0274] Example 44: N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(6-(1-(difluoromethyl)-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0275]

[0276] The target compound (12 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 1.

[0277] MS m / z [LC-MS]: 569.23 [M+1].

[0278] Example 45: N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-2-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0279]

[0280] The target compound (9 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 2.

[0281] MS m / z[LC-MS]:680.31[M+1].

[0282] Example 46: N-(5-(tert-butyl)isoxazol-3-yl)-2-(2-fluoro-4-(6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)acetamide

[0283]

[0284] The target compound (12 mg) was obtained by using the corresponding raw materials according to the synthesis method in Example 2.

[0285] MS m / z[LC-MS]:473.21[M+1].

[0286] Example 47: 1-(4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-3-(4-(((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)urea

[0287]

[0288] The target compound (11 mg) was obtained using the intermediate in Example 12 and the corresponding boronate ester.

[0289] MS m / z[LC-MS]: 667.30[M+1].

[0290] Example 48: 1-(4-(6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-3-(4-(((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)urea

[0291]

[0292] The target compound (22 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 2.

[0293] MS m / z[LC-MS]:589.27[M+1].

[0294] Example 49: 2-(4-(6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)acetamide

[0295]

[0296] The target compound (5 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 2.

[0297] MS m / z [LC-MS]: 602.29 [M+1].

[0298] Example 50: 1-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-3-(4-(6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)urea

[0299]

[0300] The target compound (13 mg) was obtained by using the corresponding starting materials according to the synthetic method in Reference Example 2.

[0301] MS m / z [LC-MS]: 603.28 [M+1].

[0302] Example 51: 1-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(6-(1-methyl-1H-pyrazol-4-yl)-4-(pyrazin-2-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)urea

[0303]

[0304] The target compound (11 mg) was obtained using the intermediate in Example 12 and the corresponding boronate ester.

[0305] MS m / z[LC-MS]: 699.31[M+1].

[0306] Biological activity test

[0307] RET compound biological activity test method

[0308] 1. Effects of Compounds on RET WT In vitro enzyme activity assay

[0309] The compounds in this patent have an effect on RET WT IC of enzyme activity inhibition 50 The value was determined by homogeneous time-resolved fluorescence (HTRF) method. The compound was diluted 5 times with 100% DMSO starting from 0.2 mM (7 concentrations in total), and 2 μL of the compound was added to 48 μL of reaction buffer (50 mM HEPES pH 7.5, 0.1 mM Na 3 VO 4 , 5mM MgCl 2 , 1 mM DTT, 0.001% Tween 20 and 100 μg / ml BSA) for dilution and mixing. Take 2.5 μL and add it to a 384-well plate (OptiPlate-384, purchased from PerkinElmer), and then add 5 μL of GST-RET WT (658-1114aa, final concentration of 0.5nM), centrifuged and mixed, and then added 2.5μL of ATP (final concentration 25μM) and TK Peptide Substrate mixture (final concentration 1μM, purchased from Cisbio) to start the reaction, and the total reaction volume was 10μL. The 384-well plate was placed in an incubator at 23°C for 2 hours, and then 5μL of TK Antibody (purchased from Cisbio) and 5μL of Streptavidin-XL665 (purchased from Cisbio) were added to stop the reaction. After incubation in the incubator for another 1 hour, the fluorescence value was read on Envision (purchased from PerkinElmer) (320nm excitation, detection of 665nm and 620nm emission light, the ratio of the two is the enzyme activity signal). Each compound was measured at 7 concentrations of RET WT The enzymatic activity signal was calculated using GraphPad Prism software to obtain the IC 50 value.

[0310] 2. Effects of Compounds on RET V804M In vitro enzyme activity assay

[0311] The compounds in this patent have an effect on RET V804M IC of enzyme activity inhibition 50 The value was determined by homogeneous time-resolved fluorescence (HTRF) method. The compound was diluted 5 times with 100% DMSO starting from 0.2 mM (7 concentrations in total), and 2 μL of the compound was added to 48 μL of reaction buffer (50 mM HEPES pH 7.5, 0.1 mM Na 3 VO 4 , 5mM MgCl 2 , 1mM DTT, 0.001% Tween 20, 20nM SEB (purchased from Cisbio) and 100μg / ml BSA) for dilution and mixing. Take 2.5μL and add it to a 384-well plate (OpriPlate-384, purchased from PerkinElmer), and then add 5μL of GST-RET V804M (658-1114aa, final concentration of 0.02nM), centrifuged and mixed, and then added 2.5μL of ATP (final concentration of 1.5μM) and TK Peptide Substrate mixture (final concentration of 1μM, purchased from Cisbio) to start the reaction, and the total reaction volume was 10μL. The 384-well plate was placed in an incubator at 23℃ for 1 hour, and then 5μL of TK Antibody (purchased from Cisbio) and 5μL of Streptavidin-XL665 (purchased from Cisbio) were added to stop the reaction. After incubation in the incubator for another 1 hour, the fluorescence value was read on Envision (purchased from PerkinElmer) (320nm excitation, detection of 665nm and 620nm emission light, the ratio of the two is the enzyme activity signal). Each compound was measured at 7 concentrations of RET V804M The enzymatic activity signal was calculated using GraphPad Prism software to obtain the IC 50 value.

[0312] 3. Effects of Compounds on RET G810R In vitro enzyme activity assay

[0313] The RET (658-1114aa) gene was cloned and connected to the pFASTBac plasmid. The primers RET G810RF: GGAGTACGCCAAATACcggTCCCTGCGGGGCTT and RET G810RR: AAGCCCCGCAGGGAccgGTATTTGGCGTACTCC were used to perform site-directed mutagenesis to introduce the RET protein 810 G mutation to R. The cells were expressed in insect cells Sf9. The cells were collected and resuspended in Lysis Buffer (50mM Tris-HCl, pH 7.5, 150mM NaCl, 0.1mM EDTA, 0.5% Triton X-100, 20% Glycerol, 1mM DTT, 1*protease inhibitor), and lysed by ultrasonication. The lysate was passed through a GSTrap HP (GE Healthcare) affinity column and washed with wash buffer (50mM Tris-HCl, pH 7.5, 150mM NaCl, 0.1mM EDTA, 20% Glycerol, 1 mM DTI) was used to wash away impurities, and then GST-RET was eluted with Elution Buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 20% Glycerol, 20 mM Glutathione, 1 mM DTT) G810R The protein was concentrated, aliquoted, and quickly frozen in liquid nitrogen and stored at -80°C.

[0314] 4. In vitro enzymatic activity assay of compounds on VEGFR2

[0315] The compounds in this patent inhibit the enzymatic activity of VEGFR2 by IC 50 The value was determined by homogeneous time-resolved fluorescence (HTRF) method. The compound was diluted 5 times with 100% DMSO starting from 1 mM (7 concentrations in total), and 2 μL of the compound was added to 48 μL of reaction buffer (50 mM HEPES pH 7.5, 0.1 mM Na 3 VO 4 , 5mM MgCl 2 , 1 mM MnCl 2, 1mM DTT, 0.001% Tween 20 and 100μg / ml BSA) for dilution and mixing. Take 2.5μL and add it to a 384-well plate (OptiPlate-384, purchased from PerkinElmer), then add 5μL of GST-VEGFR2 (final concentration of 0.1nM), centrifuge and mix, then add 2.5μL of ATP (final concentration of 2.5μM) and TKPeptide Substrate mixture (final concentration of 1μM, purchased from Cisbio) to start the reaction, the total reaction volume is 10μL. Place the 384-well plate in an incubator at 23℃ for 1.5 hours, then add 5μL of TK Antibody (purchased from Cisbio), 5 μL of Streptavidin-XL665 (purchased from Cisbio) was used to stop the reaction. After incubation in the incubator for another hour, the fluorescence value was read on Envision (purchased from PerkinElmer) (excitation at 320 nm, detection of emission light at 665 nm and 620 nm, the ratio of the two is the activity signal of the enzyme). The enzymatic activity signal of VEGFR2 was measured at 7 concentrations for each compound, and the data were calculated using GraphPad Prism software to obtain the IC value of the compound. 50 value.

[0316] 5. Determination of compound proliferation activity in TT cells

[0317] Human thyroid cancer cell line TT cells were cultured in Ham's F-12K medium supplemented with 15% fetal bovine serum (FBS, purchased from Biological Industries, BI) and 1% penicillin / streptomycin (P / S, purchased from Life Techonology) at 37°C and 5% CO. 2 .One day before the compound test, TT cells were plated in a 96-well plate (#3917, purchased from Corning) at a concentration of 5000 cells / 195 μL / well. 24 hours later, the compound was diluted 3 times from 10 mM with 100% DMSO (a total of 10 concentrations), and then 2 μL of the compound was added to 48 μL of Ham's F-12K medium for dilution. 5 μL of the diluted compound was added to the plated cell suspension at each concentration, and the compound and cells were co-incubated in a cell culture incubator for 120 h (5 days). After the culture medium was aspirated, 25 μL of Cell-TiterGlo (G7570, purchased from Promega) reagent was added and incubated again for 5-10 minutes. The fluorescence value was then read on Envision, and the data were calculated using GraphPad Prism software to obtain the IC value of the compound's inhibition of cell proliferation. 50 value.

[0318] 6. Compounds in KIF5B-RET / HEK293T V804M Cell proliferation activity assay

[0319] The RET fusion cell line KIF5B-RET / HEK293T cells constructed by lentiviral infection were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS, purchased from Biological Industries, BI) and 1% penicillin / streptomycin (P / S, purchased from Life Techonology) at 37°C and 5% CO 2 .One day before the compound detection, KIF5B-RET / HEK293T cells were plated in a 96-well plate (#3917, purchased from Corning) at a concentration of 2000 cells / 195 μL / well. After 24 hours, the compound was diluted 3 times with 100% DMSO starting from 10 mM (a total of 10 concentrations), and then 2 μL of the compound was added to 48 μL of DMEM medium for dilution. 5 μL of each concentration of the diluted compound was added to the plated cell suspension, and the compound and cells were co-incubated in a cell culture incubator for 72 h (3 days). After the culture medium was aspirated, 25 μL of Cell-Titer Glo (G7570, purchased from Promega) reagent was added and incubated again for 5-10 minutes. The fluorescence value was then read on Envision, and the data were calculated using GraphPad Prism software to obtain the IC value of the compound's inhibition of cell proliferation. 50 value.

[0320] 7. Compounds in BaF3-KIF5B-RET G810R Cell proliferation activity assay

[0321] The KIF5B-RET (K15; R12) fusion gene was constructed into the plasmid pCDHL, and the RET protein was mutated from G to R at position 810 by site-directed mutagenesis. The gene was co-transfected with pSPAX2 and pMD2G (4:2:1) plasmids into 293T cells, and lentiviral particles were packaged and the lentiviral suspension was collected 48 hours after transfection. After infecting BaF3 cells for 48 hours, puromycin (purchased from Solebol) was added at a final concentration of 1 μg / ml for screening. One week later, IL-3-independent BaF3 cells were screened for monoclonal clones to construct BaF3-KIF5B-RET. G810R Cell lines.

[0322] BaF3-KIF5B-RET G810RThe cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, purchased from Biological Industries, BI) and 1% penicillin / streptomycin (P / S, purchased from Life Science) at 37°C and 5% CO. 2 One day before compound testing, BaF3-KIF5B-RET G810R The cells were plated in a 96-well plate (#3917, purchased from Corning) at a concentration of 1000 cells / 195 μL / well. After 24 hours, the compound was diluted 3 times from 10 mM with 100% DMSO (a total of 10 concentrations), and then 2 μL of the compound was added to 48 μL of DMEM medium for dilution. 5 μL of the diluted compound was added to the plated cell suspension at each concentration, and the compound and cells were co-incubated in a cell culture incubator for 72 hours (3 days), and 40 μL of Cell-Titer Glo (G7570, purchased from Promega) reagent was added and incubated again for 5-10 minutes. The fluorescence value was read on Envision, and the data was calculated using GraphPadPrism software to obtain the IC value of the compound's inhibition of cell proliferation. 50 value.

[0323] Protein activity and cytological data

[0324]

[0325] “-” means not tested

[0326] 8. Pharmacokinetic data of the compound:

[0327] Male SD rats were obtained from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The rats were divided into groups, 3 in each group, and the suspension of the sample to be tested (5me / kg, suspension was MC 0.5%) was orally administered. The animals were fasted overnight before the experiment, and the fasting time was from 10 hours before administration to 4 hours after administration. Blood was collected at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration. After anesthesia with isoflurane using a small animal anesthesia machine, 0.3 mL of whole blood was collected through the retinal venous plexus and placed in a heparin anticoagulation tube. The sample was centrifuged at 4°C and 4000 rpm for 5 minutes, and the plasma was transferred to a centrifuge tube and stored at -80°C until analysis. The sample in the plasma was extracted using protein precipitation method, and the extract was analyzed by LC / MS.

[0328]

[0329] Industrial Applicability

[0330] The present invention provides a RET selective inhibitor and its preparation and use. The present invention also provides a series of compounds represented by general formula (I) and pharmaceutically acceptable salts, solvates, polymorphs or isomers thereof, pharmaceutical compositions comprising these compounds, and methods for treating diseases with such compounds. The RET selective inhibitor provided by the present invention has high activity, strong drug resistance, and small clinical side effects, can effectively overcome the problem of drug resistance in tumor treatment, and has good economic value and application prospects.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof: in, L is -NH-(CO)-CR3R4-(CO)-NH-, -NH-(CO)-NH-, -CR3R4-(CO)-NH-, or -(CO)-NH-, R1 is selected from 5-6 membered heteroaryl, 3-12 membered heterocyclic group, -OC 1-6 Alkyl and -SC 1-6 The alkyl group, the heteroaryl group and the heterocyclic group may each be optionally substituted by halogen, C 1-6 Alkyl, -CN, -OH, -NR 6 R 7 、-OC 1-6 Alkyl, or -SC 1-6 Alkyl substituted, the alkyl group may be optionally substituted with halogen, -CN, -OH, -NR 6 R 7 、-OC 1-6 Alkyl, or -SC 1-6 Alkyl substitution, R2 is selected from -CN, -C≡C-(C 1-6 alkyl), 5-6 membered heteroaryl and 3-12 membered heterocyclic group, each of which may be optionally substituted by halogen, C 1-6 Alkyl, -CN, -OH, -NR 6 R 7 、-OC 1-6 Alkyl, or -SC 1-6 Alkyl substitution, X, X1, X2, X3 and X4 are each independently selected from N and CR8, R8 are each independently selected from H, halogen, -CN, -OH, -NR 6 R 7 , C 1-6 Alkyl, -OC 1-6 Alkyl and -SC 1-6 alkyl, A is phenyl, 5-6 membered heteroaryl, 3-8 membered heterocyclyl, or 3-8 membered cycloalkyl, and the phenyl, heteroaryl, cycloalkyl and heterocyclyl may be optionally substituted by R5, R5 is selected from halogen, -CN, -OH, -NR 6 R 7 , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -(CH2) 0-1 -(3-8 membered cycloalkyl), -(CH2) 0-1 -(3-8 membered heterocyclic group), -(CH2) 0-1 -(6-10 membered aryl), -(CH2) 0-1 -(5-12 membered heteroaryl), -OC 1-6 Alkyl, or -SC 1-6 Alkyl substituted, the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl may be optionally substituted with halogen, -CN, -OH, -NR 6 R 7 , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OC 1-6 Alkyl, or -SC 1-6 Alkyl substitution, R3 and R4 are each independently selected from H and C 1-6 Alkyl, or R3 and R4 are linked together to form a 3-6 membered carbon ring, R 6 and R 7 Each independently selected from H and C 1-6 alkyl, R9 is H, -NH2, or -NH-C 1-6 alkyl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof, wherein L is -CR3R4-(CO)-NH-, and R3 and R4 are as defined in claim 1.

3. The compound according to claim 1 or a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof, wherein R2 is selected from a 5-6 membered heteroaryl and a 3-12 membered heterocyclic group, wherein the heteroaryl and heterocyclic group can each be optionally substituted by halogen, C 1-6 Alkyl, -CN, -OH, -NR 6 R 7 、-OC 1-6 Alkyl, or -SC 1-6 Alkyl substituted, R6 and R7 are as defined in claim 1.

4. The compound according to claim 1 or a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof, wherein R9 is H or -NH 2。 5. The following compound or its pharmaceutically acceptable salt, solvate, polymorph or isomer:

6. A pharmaceutical composition comprising the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof, and a pharmaceutically acceptable carrier.

7. Use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof, or the composition according to claim 6 in the preparation of a medicament for treating a disease associated with RET.

8. The use according to claim 7, wherein the RET-related disease is lung cancer, papillary thyroid cancer, medullary thyroid cancer, differentiated thyroid cancer, recurrent thyroid cancer, multiple endocrine neoplasia type 2A or 2B, pheochromocytoma, parathyroid hyperplasia, breast cancer, colorectal cancer, papillary renal cell carcinoma, gastrointestinal mucosal ganglioneuroma, or cervical cancer.

9. The use according to claim 7, wherein the RET-related disease is RET fusion lung cancer or medullary thyroid cancer.

10. The use according to claim 7, wherein the RET-related disease is small cell lung cancer, non-small cell lung cancer, or bronchiolar lung cancer or lung adenocarcinoma.

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