A pyrazolo[1,5-a]pyridine derivative and its preparation method and application

Abstract

The present invention discloses a novel pyrazolo[1,5-a]pyridine derivative, a preparation method thereof and medical application thereof. More specifically, the pyrazolo[1,5-a]pyridine derivative of the present invention can be used as a drug for treating proliferative diseases and other diseases related to abnormal expression of TRK or various kinases such as RET, FGHR, PDGFR, and VEGFR. Its structure is shown in the general formula (I), wherein L 1 , L 2 , L 3 , R 1 , R 2 and R 3 Definitions see the manual:

Description

Technical Field

[0001] The present invention relates to a novel pyrazolo[1,5-a]pyridine derivative, a preparation method and application thereof. Specifically, the present invention relates to a novel pyrazolo[1,5-a]pyridine derivative having the ability to inhibit the growth of a tumor cell line with high kinase expression or a tumor cell line with corresponding kinase mutation and a preparation method thereof. In particular, these compounds can be used as drugs for treating proliferative disorders and other diseases associated with abnormal expression of any one or more kinases such as TRK, RET, FGHR, PDGFR, VEGFR, etc. In addition, the present invention relates to drugs containing these compounds and the application of these compounds in the preparation of drugs. Background Art

[0002] Tumor is one of the major diseases that threaten human health, and society has a great demand for tumor treatment. With the development of tumor molecular biology and tumor pharmacology, chemotherapy and immunotherapy for tumors have also made great progress. And in various tumor treatments, small molecule targeted drug therapy for tumors is playing an increasingly important role.

[0003] [NTRK / TRK (Tropomyosin receptor kinase)] is a neurotrophic factor tyrosine kinase receptor. The TRK family mainly includes three members, NTRK1 / TRKa, NTRK2 / TRKb and NTRK3 / TRKc. The complete TRK kinase consists of three parts: the extracellular region, the transmembrane region and the intracellular region. After the extracellular region of the TRK kinase binds to the corresponding ligand, it can cause the kinase conformation to change and form a dimer. The intracellular region of the TRK kinase undergoes autophosphorylation to activate its own kinase activity, and then further activates the downstream signal transduction pathways (such as MAPK, AKT, PKC, etc.), producing corresponding biological functions; among them, NGF (nerve growth factor) binds to TRKa, BDNF (derived neurotrophic factor) binds to TRKb, and NT3 (neurotrophic factor 3) binds to TRKc.

[0004] A large number of studies have shown that the activation of the TRK signal transduction pathway is also strongly correlated with the occurrence and development of tumors. Activated TRK signaling proteins have been found in neuroblastoma, prostate cancer, breast cancer, etc. In recent years, the discovery of a variety of TRK fusion proteins has further demonstrated their biological function in promoting tumorigenesis (Sharan K Bagal, Mark Andrews, Bruce M. Bechle, Jianwei Bian, James Bilsland, David C Blakemore, John Braganza, Peter J. Bungay, Matthew S. Corbett, Ciarán N Cronin, Jingrong Jean Cui, Rebecca Dias, Neil J Flanagan, Samantha E Greasley, Rachel Grimley, Kim James, Eric Johnson, Linda Kitching, Michelle L Kraus, Indrawan McAlpine, Asako Nagata, Sacha Ninkovic, Kiyoyuki Omoto, Stephanie Scales, Sarah E. Skerratt, Jianmin Sun, Michelle Tran-Dubé, Gareth J. Waldron, Fen Wang, and Joseph S Warmus. Discovery of Potent, Selective and Peripherally Restricted Pan-Trk Kinase Inhibitors for the Treatment of Pain.J.Med.Chem.,Just Accepted Manuscript·DOI:10.1021 / acs.jmedchem.8b00633·Publication Date(Web):26Jun 2018).

[0005] In recent years, TRK fusion protein has become an effective anti-cancer target and research hotspot. For example, TRK kinase inhibitors represented by Larotrectinib (1) LISA JARVIS. Bayer, Loxo to develop TRK inhibitors. C&EN, 20 November 2017, 11; 2) (WO2010048314), Entrectinib (WO2009013126A1), etc. have entered clinical application. More TRK kinase inhibitors are already in various clinical stages. WO2012116217, WO2010033941, JP2018044010 A, MX2017007748 A, US2017057948A1, etc. disclose TRK kinase inhibitors with different mother nuclei. In view of the importance of TRK physiological functions, TRK inhibitors with stronger activity and wider adaptability to gene fusion have attracted widespread interest. In particular, there is still an unmet need in society for TRK inhibitors that can inhibit not only TRK a, b and c, but also their mutant forms (e.g., G595R, G667C, A608D, F589L, G623R).

[0006]

[0007] On the other hand, rearranged in transfection (RET) is a nerve growth factor receptor tyrosine kinase; abnormal RET kinase activity is associated with many tumors. Therefore, RET is also a highly valued anti-tumor target.

[0008] RET is a neuronal growth factor receptor tyrosine kinase. RET kinase knockout mice lack enteric neurons and have other neurological abnormalities, suggesting that a functional RET kinase protein product is required for development. Studies of Hirschsprung's disease patient populations have shown a high frequency of familial and sporadic functional RET mutations. Aberrant RET kinase activity is associated with multiple endocrine tumors (MEN 2A and 2B), familial medullary thyroid tumor (FMTC), papillary thyroid carcinoma (PTC), and Hirschsprung's disease (HSCR) (Maria Grazia Borrello, Elena Ardini, Laura D Locati, Angela Greco, Lisa Licitra & Marco A Pierotti (2013). RET inhibition: implications in cancer therapy. Expert Opinion on Therapeutic Targets, 17:4, 403-419). MEN 2A is a cancer syndrome caused by mutations in the extracellular cysteine-rich region of RET, which leads to dimerization of disulfide bonds, resulting in continuous activation of tyrosine kinase activity (Samuel A. Wells, Jr., Furio Pacini, Bruce G. Robinson, and Massimo Santoro. Multiple Endocrine Neoplasia Type 2 and Familial Medullary Thyroid Carcinoma: An Update. J Clin Endocrinol Metab 98: 3149–3164, 2013). Individuals with this mutation may develop medullary thyroid tumors (MTC), thyroid hyperplasia, and pheochromocytoma. MEN 2B is similar to MEN 2A, but without thyroid hyperplasia, and also causes various mucosal ganglion cysts of the lips, tongue, and intestines. RET is believed to be involved in the tumor initiation of PTC during chromosome remake. PTC includes 80% of thyroid tumors (Viglietto, G. et al., Oncogene, 1995, 11: 1207).

[0009] These facts all indicate an ideal treatment for tumors related to persistent RET activation. The research on RET inhibitors has received widespread attention and has made rapid progress. Among them, RET inhibitors represented by Pralsetinib (WO2017011776A1ArrayLoxo 292) and Selpercatinib (WO2017079140A1 Blu667) have been approved by the FDA for marketing and have been used to treat various related fusion tumors regardless of tumor type and have been successful. This has inspired various other development attempts. ((1)Lucille Lopez-Delisle,Cécile Pierre-Eugène,Caroline Louis-Brennetot,Didier Surdez,Virginie Raynal,Sylvain Baulande,Valentina Boeva,Sandrine Grossetête-Lalami,Valérie Combaret,Michel Peuchmaur6,Olivier Delattre,Isabelle Janoueix-Lerosey.Activated ALK signals through the ERK–ETV5–RET pathway to drive neuroblastoma oncogenesis.Oncogene(2018)37:1417–1429.(2)WO 2014 / 141187A1.RET Kinase Inhibitors May Treat Cancer andGastrointestinal Disorders.(3)Minsoo Song.Progress in Discovery of KIF5B-RETKinase Inhibitors for the Treatment of Non-Small-Cell Lung Cancer.Miniperspective.J.Med.Chem.2015,58,3672-3681.)

[0010]

[0011] As mentioned above, the research and development of kinase inhibitors represented by TRK and RET have achieved great success in the treatment of fusion tumors, bringing good news to the majority of patients. However, the proportion of tumors with TRK and RET as the core and fusion with other genes is still small (Alexander Drilon, Zishuo I. Hu, Gillianne G. Y. Lai & Daniel SWTan Targeting RET-driven cancers: lessons from evolving preclinical and clinical landscapes. Nature Reviews Clinical Oncology volume 15, 2018, 151–167). Obviously, if TRK, RET, or even one or more kinases such as FGHR, PDGFR, and VEGFR can be effectively inhibited at the same time, it will be an effective choice. On the other hand, drug resistance is always the main problem for almost all kinase targets. Therefore, there is still a huge social demand for more multi-target kinase inhibitors in this field. Summary of the invention

[0012] The purpose of the present invention is to disclose a novel pyrazolo[1,5-a]pyridine derivative and its preparation method and application. This type of compound can be used for the treatment of tumors, endocrine disorders, immune system diseases, genetic diseases and neurodegenerative diseases.

[0013] In order to achieve the above-mentioned purpose, the inventors have conducted long-term research and found that the compound represented by the following general formula (I) or its pharmaceutically acceptable salt has excellent effect of inhibiting kinase activity, especially inhibiting the activity of any one or more kinases such as TRK, RET, FGHR, PDGFR, VEGFR, etc., and can be used as a drug for treating proliferative diseases and other diseases related to abnormal expression of various kinases.

[0014] In order to achieve the above object, the technical solution of the present invention is as follows:

[0015] A pyrazolo[1,5-a]pyridine derivative is a compound represented by the general formula (I), and an optical isomer thereof or a pharmaceutically acceptable salt thereof:

[0016]

[0017] In the above general formula (I),

[0018] R 1Selected from: hydrogen atom, carboxyl, nitro, amino, cyano, acyl, alkyl, alkoxy, alkenyl, alkynyl, halogen, isotope, haloalkenyl, heteroalkyl, arylalkyl, cycloalkyl, aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkenyl, alkoxyalkyl, alkenyloxy, alkynyloxy, alkylamino, aminoalkyl, alkylaminocarbonyl, sulfonyl, alkylsulfonyl, alkylsulfinyl, or aminosulfonyl; any of the above groups can be independently substituted by one or more substituents, including but not limited to halogen, isotope, amino, carboxyl, phenyl, benzyl, phenyloxy, =O, -CF 3 , haloalkyl, alkyl, heteroalkyl, alkenyl, alkynyl, hydroxy, hydroxyalkyl, alkoxy, and alkoxyalkyl;

[0019] L 1 Selected from: covalent bond, -CH 2 -CH 2 -, -CH=CH-, -C≡C-, -NH-, Any of the above groups may be independently substituted by one or more substituents, including but not limited to halogen, isotope, =O, -CF 3 , alkyl, haloalkyl, hydroxy, alkoxy, and alkoxyalkyl;

[0020] L 2 Selected from: covalent bond, alkylene, heteroalkylene, carbonyl, -C(O)-NH-, -alkyl-C(O)-NH-, -alkyl-NH-C(O)-, -alkyl-O-; any of the above groups may be independently substituted by one or more substituents, including hydrogen atoms, isotopes, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl;

[0021] L 3 Selected from: covalent bond, alkylene, heteroalkylene, carbonyl, -O-, -NH-, -C(O)-NH-, -NH-C(O)-, -alkyl-C(O)-NH-, -alkyl-NH-C(O)-, -alkyl-NH-, -alkyl-O-; any of the above groups may be independently substituted by one or more substituents, including hydrogen atoms, isotopes, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl;

[0022] R 2Selected from: hydrogen atom, alkyl, heteroalkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, aryl, heteroaryl, heteroarylalkyl, heterocycloalkyl, alkyl ether, heteroalkyl ether, arylalkyl ether, cycloalkyl ether, aryl ether, heteroaryl ether, heteroaryl alkyl ether, heterocycloalkyl ether, carboxyl, carboxylalkylaminocarbonyl, cycloalkylaminocarbonyl, heteroarylaminocarbonyl, heteroarylaminocarbonyl; any of the above groups may be independently unsubstituted or may be substituted by one or more substituents, and these substituents include but are not limited to halogen, isotope, amino, carboxyl, phenyl, benzyl, phenyloxy, =O, -CF 3 , haloalkyl, alkyl, alkenyl, alkynyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, heteroalkyl, arylalkyl, cycloalkyl, aryl, heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkenyl, alkoxyalkyl, alkenyloxy, alkynyloxy, alkylamino, aminoalkyl, alkylaminocarbonyl, sulfonyl, alkylsulfonyl, alkylsulfinyl, or aminosulfonyl.

[0023] R 3 Selected from: hydrogen atom, alkyl, heteroalkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, aryl, heteroaryl, heteroarylalkyl, heterocycloalkyl, alkyl ether, heteroalkyl ether, arylalkyl ether, cycloalkyl ether, aryl ether, heteroaryl ether, heteroarylalkyl ether, heterocycloalkyl ether, alkylamino, heteroalkylamino, arylalkylamino, cycloalkylamino, arylamino, heteroarylamino, heteroarylalkylamino, heterocycloalkylamino, carboxyl, carboxylalkylaminocarbonyl, cycloalkylaminocarbonyl, heteroarylaminocarbonyl, heteroarylaminocarbonyl; any of the above groups may be independently unsubstituted or may be substituted by one or more substituents, and these substituents are not limited to halogen, isotope, amino, carboxyl, phenyl, benzyl, phenyloxy, =O, -CF 3 , haloalkyl, alkyl, alkenyl, alkynyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, heteroalkyl, arylalkyl, cycloalkyl, aryl, heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkenyl, alkoxyalkyl, alkenyloxy, alkynyloxy, alkylamino, aminoalkyl, alkylaminocarbonyl, sulfonyl, alkylsulfonyl, alkylsulfinyl, or aminosulfonyl.

[0024] In certain embodiments, in Formula (I), R 1 is a hydrogen atom, a carboxyl group, a nitro group, an amino group, a cyano group, an acyl group, an alkyl group, an alkoxy group, an alkenyl group, an alkynyl group, a halogen group, an isotope group, a haloalkenyl group, a heteroalkyl group, a heteroalkyl group, an aryl group, or a heteroaryl group; in the above groups, each of them may be unsubstituted or substituted by one or more substituents, and these substituents include: halogen group, isotope group, =O, -CF 3 , alkyl, alkenyl, alkynyl, carboxyl, hydroxyl, hydroxyalkyl, alkoxy, alkoxyalkyl, -C≡N.

[0025] In other embodiments, in the general formula (I), L 3 Selected from O-CH 2 -R 4 ; where R 4 Selected from

[0026] In certain embodiments, in Formula (I), R 3 Selected from C1-C5 alkyl, heterocyclyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl; any of the above groups independently may be unsubstituted or may be substituted by one or more substituents, and these substituents include but are not limited to halogen, isotope, amino, alkyl.

[0027] In certain other embodiments, in the general formula (I), L 1 Selected from: -CH 2 -CH 2 -, -CH=CH-, -C≡C-.

[0028] In certain embodiments, in Formula (I), L 2 Selected from: alkylene, heteroalkylene, carbonyl, -C(O)-NH-, -alkyl-C(O)-NH-, -alkyl-NH-C(O)-.

[0029] In certain other embodiments, in the general formula (I), L 2 Selected from: pyridine, substituted pyridine, pyrimidine, substituted pyrimidine, substituted benzene ring, -CO-NH-, -C(O)-, -CH 2 -NH-, -C(CH 2 )-NH-, -CO-N(OH)-; the alkyl group is a C1-C5 straight chain or branched chain alkyl group; the substituent of the substituted pyridine is a C1-C5 straight chain or branched chain alkyl group; the substituent of the substituted pyrimidine is a C1-C5 straight chain or branched chain alkyl group, or an amino group.

[0030] In certain embodiments, in Formula (I), R 2 Selected from: C1-C8 alkyl, Amino, Benzene ring, substituted benzene ring, methylpyridine, CO-NH-R 7 , Pyrimidine, substituted pyrimidine, Pyridine, substituted pyridine,

[0031] -CH2 -CN,

[0032] -NH-OH,

[0033]

[0034] R 5 Selected from hydrogen,

[0035] R 6 Selected from hydrogen, Benzene ring, substituted benzene ring, Pyrimidine;

[0036] The substituted benzene ring contains 1-3 substituents, and the substituents of the substituted benzene ring are selected from amino, C1-C5 alkyl, halogen, trifluoromethyl, trichloromethyl, tribromomethyl, trifluoroethyl, trifluoropropyl, Methyl ether, ethyl ether, propyl ether, trifluoromethyl ether, trifluoroethyl ether, trifluoropropyl ether, -C≡CH, -CN;

[0037] R 7 Selected from C1-C8 alkyl;

[0038] The substituted pyrimidine contains 1-3 substituents, and the substituents of the substituted pyrimidine are selected from amino, C1-C5 alkyl, and halogen;

[0039] The substituted pyridine contains 1 to 3 substituents, and the substituents of the substituted pyridine are selected from C1-C8 alkyl, Difluorobenzene ring, methyl ether group;

[0040] R 8 Selected from hydrogen, substituted pyridine, C1-C8 alkyl.

[0041] In certain embodiments, the substituted benzene ring is selected from toluene, ethylbenzene, and p-fluorotoluene.

[0042] In certain embodiments, the pyrazolo[1,5-a]pyridine derivative may also be a compound represented by the general formula (I), and an optical isomer thereof or a pharmaceutically acceptable salt thereof: 13.

[0044]

[0045] In the above general formula (I),

[0046] R 1Selected from: hydrogen atom, carboxyl, nitro, amino, cyano, acyl, alkyl, alkoxy, alkenyl, alkynyl, halogen, isotope, haloalkenyl, heteroalkyl, arylalkyl, cycloalkyl, aryl, heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkenyl, alkoxyalkyl, alkenyloxy, alkynyloxy, alkylamino, aminoalkyl, alkylaminocarbonyl, sulfonyl, alkylsulfonyl, alkylsulfinyl, or aminosulfonyl; any of the above groups can be independently substituted by one or more substituents, including but not limited to halogen, isotope, amino, carboxyl, phenyl, benzyl, phenyloxy, =O, -CF 3 , haloalkyl, alkyl, heteroalkyl, alkenyl, alkynyl, hydroxy, hydroxyalkyl, alkoxy, and alkoxyalkyl;

[0047] L 3 Selected from: covalent bond, alkylene, heteroalkylene, carbonyl, O, NH, CO-NH, NH-CO, alkyl-CO-NH, alkyl-NH-CO, alkyl-NH, alkyl-O; any of the above groups may be independently substituted by one or more substituents, including hydrogen atoms and alkyl groups;

[0048] R 3 Selected from: hydrogen atom, alkyl, heteroalkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, aryl, heteroaryl, heteroarylalkyl, heterocycloalkyl, alkyl ether, heteroalkyl ether, arylalkyl ether, cycloalkyl ether, aryl ether, heteroaryl ether, heteroarylalkyl ether, heterocycloalkyl ether, alkylamino, heteroalkylamino, arylalkylamino, cycloalkylamino, arylamino, heteroarylamino, heteroarylalkylamino, heterocycloalkylamino, carboxyl, carboxylalkylaminocarbonyl, cycloalkylaminocarbonyl, heteroarylaminocarbonyl, heteroarylaminocarbonyl; any of the above groups may be independently unsubstituted or may be substituted by one or more substituents, and these substituents are not limited to halogen, isotope, amino, carboxyl, phenyl, benzyl, phenyloxy, =O, -CF 3 , haloalkyl, alkyl, alkenyl, alkynyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, heteroalkyl, arylalkyl, cycloalkyl, aryl, heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkenyl, alkoxyalkyl, alkenyloxy, alkynyloxy, alkylamino, aminoalkyl, alkylaminocarbonyl, sulfonyl, alkylsulfonyl, alkylsulfinyl, or aminosulfonyl;

[0049] Among them, L 1 -L 2 -R 2 Selected from:

[0050]

[0051] Among them, R 9 ,R10 ,R 11 , and R 12 independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, aryl, heteroaryl, heteroarylalkyl, heterocycloalkyl, alkylether, heteroalkylether, arylalkylether, cycloalkylether, arylether, heteroarylether, heteroarylalkylether, heterocycloalkylether, alkylamino, heteroalkylamino, arylalkylamino, cycloalkylamino, arylamino, heteroarylamino, heteroarylalkylamino, heterocycloalkylamino, carboxyl, carboxylalkylaminocarbonyl, cycloalkylaminocarbonyl, heteroarylaminocarbonyl, heteroarylaminocarbonyl; any of the above groups independently may be unsubstituted or may be substituted by one or more substituents, including but not limited to halogen, isotope, amino, carboxyl, phenyl, benzyl, phenyloxy, =O, -CF 3 , haloalkyl, alkyl, alkenyl, alkynyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, heteroalkyl, arylalkyl, cycloalkyl, aryl, heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkenyl, alkoxyalkyl, alkenyloxy, alkynyloxy, alkylamino, aminoalkyl, alkylaminocarbonyl, sulfonyl, alkylsulfonyl, alkylsulfinyl, or aminosulfonyl.

[0052] In certain embodiments, in the general formula (I), its structure can be selected from one of the following structures, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, solvates, metabolites, pharmaceutically acceptable salts or prodrugs:

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] The present invention also discloses a method for preparing the above-mentioned pyrazolo[1,5-a]pyridine derivative. 2 =CO-NH, the preparation method comprises:

[0062] S1, select I as a raw material, remove the methyl group under the action of a catalyst to obtain compound II;

[0063] S2. Under catalysis, compound II reacts with boron compound III to obtain IV;

[0064] S3. Under the action of N-phenylbis(trifluoromethanesulfonimide), compound IV is converted into compound V;

[0065] S4, compound VIII obtained by condensation of compound VI and compound VII;

[0066] S5. Compound VIII reacts with compound V to obtain compound IX, which is the target compound represented by general formula (I);

[0067]

[0068] In certain embodiments, compound 1-1 is: The synthesis method of compound I-1 includes:

[0069] A and X are used as raw materials, and after acylation and amination, XIV is obtained;

[0070] B. Under the action of acid, XIV undergoes cyclization to obtain compound XVI;

[0071] C. Introducing a formyl group into compound XVI, which is converted into a hydroxyl group by hydroxylation, and then removing the methyl group under the action of a catalyst to obtain compound I-1;

[0072]

[0073] In certain embodiments, L 1 =CH=CH or -C≡C-, L 2 -R 2 To replace the phenyl group, the preparation method of the pyrazolo[1,5-a]pyridine derivative comprises:

[0074] 1) The iodine compound XIX is used as a raw material, and reacts with XX in the presence of a catalyst to obtain a compound XXI;

[0075] 2) Compound XXI reacts in an alkaline methanol solution to obtain XXII;

[0076] 3) XXII reacts with compound V to obtain compound XXIII;

[0077]

[0078] The present invention also includes a pharmaceutical composition comprising the compound represented by the above general formula (I) or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, solvates, metabolites, pharmaceutically acceptable salts or prodrugs:

[0079] The present invention also includes the combined use of the compound represented by the above general formula (I) or a pharmaceutically acceptable salt thereof and one or more other drugs.

[0080] The present invention also includes the use of the compound represented by the above general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating a disease caused by, or associated with or accompanied by, the destruction of cell proliferation and / or angiogenesis.

[0081] The present invention also includes the use of the compound represented by the above general formula (I) or its pharmaceutically acceptable salt or the above pharmaceutical composition for inhibiting the activity of kinase.

[0082] In certain embodiments, the inhibition of kinase activity is the inhibition of the activity of one or more of TRK or RET, RAF, FGHR, PDGFR, and VEGFR kinases.

[0083] In certain embodiments, the inhibition of kinase activity is inhibition of TRK activity.

[0084] In certain embodiments, the inhibition of kinase activity is inhibition of RET activity.

[0085] The present invention also includes a method for treating a patient's condition caused by, or associated with, or accompanied by the destruction of cell proliferation and / or angiogenesis, the method comprising administering to the patient a therapeutically effective amount of a compound represented by the above-mentioned general formula (I) or a pharmaceutically acceptable salt thereof.

[0086] The present invention also includes a method for treating a disease that can be treated by inhibiting kinase in a patient, the method comprising administering to the patient a therapeutically effective amount of the compound represented by the above general formula (I) or a pharmaceutically acceptable salt thereof.

[0087] In certain embodiments, the disorder is selected from the group consisting of a proliferative disease, such as non-small cell lung cancer, renal cell carcinoma, gastric cancer, hepatocellular carcinoma, colorectal cancer, medullary thyroid cancer, follicular thyroid cancer, anaplastic thyroid cancer, papillary thyroid cancer, brain tumor, peritoneal cavity cancer, solid tumors, other lung cancers, renal cell carcinoma, gastric cancer, head and neck cancer, glioma, neuroblastoma, Von Hipple-Lindau syndrome and renal tumors, breast cancer, fallopian tube cancer, ovarian cancer, transitional cell cancer, prostate cancer, esophageal and esophagogastric junction cancer, biliary tract cancer and adenocarcinoma, and any malignancy with increased activity of any one or more of the kinases TRK, RET, FGHR, PDGFR, VEGFR; neurodegenerative diseases, including Huntington's disease, polyglutamine diseases, Parkinson's disease, Alzheimer's disease, seizures, striatonigral degeneration, progressive supranuclear palsy, torsion dystonia, spasmodic torticollis and movement disorders, familial tremor, Tourette syndrome, diffuse Lewy body disease, Pick's disease, intracranial hemorrhage, primary lateral sclerosis, spinal cord Muscular dystrophy, amyotrophic lateral sclerosis, hypertrophic interstitial polyneuropathy, retinitis pigmentosa, hereditary optic atrophy, hereditary spastic paraplegia, progressive ataxia and Shy-Drager syndrome; metabolic diseases, including type 2 diabetes; ocular degenerative diseases, including glaucoma, age-related macular degeneration, iridovir glaucoma; diseases involving angiogenesis, including cancer, psoriasis; psychological disorders, including bipolar disorder, schizophrenia, mania, depression and dementia; cardiovascular diseases, including heart failure, restenosis and arteriosclerosis; fibrotic diseases, including liver fibrosis, cystic fibrosis and angiofibrosis; infectious diseases, including fungal infections, such as Candida albicans, bacterial infections, viral infections. Examples include herpes simplex, protozoan infections such as malaria, leishmaniasis, Trypanosoma brucei, toxoplasmosis and coccidiosis, and hematopoietic disorders including thalassemia, anemia and sickle cell anemia.

[0088] In certain embodiments, in the above methods, the patient is undergoing surgery or radiotherapy, and the compound is administered to the patient concomitantly with, before, or after the surgery or radiotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 The in vivo antitumor activity of the compounds of the present invention in the TT model. DETAILED DESCRIPTION

[0090] Each group in the compound represented by the general formula (I) of the present invention is defined as follows.

[0091] "Halogen" refers to fluorine, chlorine, bromine and iodine.

[0092] "=O" means oxo.

[0093] “CF 3 ” refers to trifluoromethyl.

[0094] "CO-NH" is "-amide-".

[0095] "NH-CO" is an "aminoacyl group".

[0096] "Carbonyl" means

[0097] "Alkyl" when used as a group or part of a group refers to a straight-chain or branched aliphatic hydrocarbon group in which none or one or more (preferably 1, 2 or 3) carbon atoms are replaced by oxygen, nitrogen, phosphorus, boron, selenium, silicon or sulfur atoms (preferably oxygen, sulfur or nitrogen).

[0098] "Alkyl" is preferably C1-C14 alkyl, more preferably C1-C10 alkyl, and most preferably C1-C6 alkyl, unless otherwise specified. Examples of C1-C6 alkyl include, but are not limited to, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, hexyl, and the like.

[0099] "Alkyl" is most preferably C1-C6, unless otherwise specified. Examples of straight chain or branched C1-C6 alkyl include, but are not limited to: methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, hexyl, etc.

[0100] "Cycloalkyl" refers to a saturated or partially saturated monocyclic, condensed or spirocyclic carbon ring. Preferably, it is a ring consisting of 3-9 carbon atoms. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0101] "Heteroalkyl" refers to a group formed by replacing one or more (preferably 1, 2 or 3) carbon atoms in a linear or branched alkyl group with oxygen, nitrogen, phosphorus, boron, selenium, silicon or sulfur atoms (preferably oxygen, sulfur or nitrogen). Preferably, the heteroalkyl group has 2 to 14 atoms, more preferably 2 to 8 atoms, and particularly preferably 2 to 6 atoms. The heteroalkyl group includes, but is not limited to, groups such as ethers, thioethers, alkyl esters, alkyl secondary amines, alkyl tertiary amines, alkyl sulfinic acids, nitriles, isonitriles, cyanates, thiocyanates, isocyanates, isothiocyanates and alkyl nitrile, and specifically includes, for example, methoxy, trifluoromethoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, methoxymethyl, ethoxymethyl, methoxyethyl, methylamino, ethylamino, dimethylamino, diethylamino, isopropylethylamino, methyl-aminomethyl, ethylaminomethyl, di-isopropylaminoethyl, enol ether, dimethylaminomethyl, dimethylaminoethyl, acetyl, propionyl, butyryloxy, acetoxy, methoxycarbonyl, ethoxy-carbonyl, N-ethyl-N-methylcarbamoyl or N-methylcarbamoyl.

[0102] "Heterocycloalkyl" refers to a group formed by replacing one or more (preferably 1, 2 or 3) carbon atoms in the "cycloalkyl" defined above with oxygen, nitrogen, phosphorus, boron, selenium, silicon or sulfur atoms (preferably oxygen, sulfur or nitrogen). Wherein, heterocycloalkyl and alkyl parts are defined in this document. Preferably, it contains 1-3 heteroatoms. The preferred ring is a 3-14-membered ring (i.e., a 3-14-membered heterocycloalkyl), and the more preferred ring is a 4-7-membered ring (i.e., a 4-7-membered heterocycloalkyl). Heterocycloalkyl includes, but is not limited to: pyrrolidinyl, dihydropyrrolyl, tetrahydropyrrolyl, dihydropyrazolyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, oxirane, azirane or 2-pyrazolinyl, as well as lactams, lactones, cyclic imides and cyclic anhydrides. Heterocycloalkyl may be substituted by one or more substituents.

[0103] "Heterocycloalkylalkyl" refers to a group of (heterocycloalkyl-alkyl)-, wherein the heterocycloalkyl and alkyl parts are as defined herein. Heterocycloalkylalkyl groups include, but are not limited to, (2-tetrahydrofuranyl)methyl, (2-tetrahydrothiofuranyl)methyl, and the like.

[0104] "Alkylamino" includes both monoalkylamino and dialkylamino, unless otherwise specified. "Monoalkylamino" refers to a group of (alkyl-NH)-; "dialkylamino" refers to a group of (alkyl) 2 N)-. The alkyl group is as defined herein. The alkylamino group is preferably a C1-C6 alkylamino group. It should be noted that "C1-C6 alkylamino" refers to an amino group substituted by a "C1-C6 alkyl", examples of which include, but are not limited to: methylamino, ethylamino, isopropylamino, N,N-(diethyl)amino, etc.

[0105] "Heteroalkylamino" refers to both mono-heteroalkylamino and di-heteroalkylamino, unless otherwise specified. Mono-heteroalkylamino refers to a group of (heteroalkyl-)NH-; di-heteroalkylamino refers to a group of (heteroalkyl)NH-; 2 The definition of "heteroalkyl" part thereof can be found in the relevant part of this text.

[0106] "Aminoalkyl" refers to a group of (amino-alkyl)-. The "alkyl" part is defined in this document. The aminoalkyl is preferably amino C1-C6 alkyl. It should be noted that "amino-C1-C6 alkyl" refers to a C1-C6 alkyl substituted by "amino", and its examples include but are not limited to: aminoethyl, 1-aminopropyl, 2-aminopropyl, etc.

[0107] "Arylamino" includes both mono-arylamino and di-arylamino, unless otherwise specified. Mono-arylamino refers to a group of (aryl-)NH-; di-arylamino refers to (aryl) 2 The definition of "aryl" part thereof can be found in the relevant part of this text.

[0108] "Acyl" includes (alkyl-CO)-groups and (aryl-CO)-groups, unless otherwise specified. The "alkyl" or "aryl" part thereof is as defined herein. Examples of acyl include, but are not limited to, acetyl, propionyl, isobutyryl, benzoyl, and the like.

[0109] "Acylamide" includes (alkyl-CONH)-groups and (aryl-CONH)-groups, unless otherwise specified. The "alkyl" or "aryl" part thereof is as defined herein. Examples of acylamide include, but are not limited to, acetylamide, propionylamide, butyramide, isobutyramide, benzylamide, and the like.

[0110] "Alkenyl" as a group or part of a group refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond, which can be straight chain or branched. It is preferably C2-C14 alkenyl, more preferably C2-C12 alkenyl, and most preferably C2-C6 alkenyl. The group may contain multiple double bonds in its main chain and their conformations may be E or Z. Examples of alkenyl groups include, but are not limited to: vinyl, propenyl, etc. In addition, the "alkenyl" of the present invention refers to the group when the "alkenyl" defined above is chain-shaped.

[0111] "Alkynyl" as a group or part of a group refers to an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond, which may be straight chain or branched. It is preferably C2-C14 alkynyl, more preferably C2-C12 alkynyl, and most preferably C2-C6 alkynyl. Examples of the alkynyl include, but are not limited to: ethynyl, prop-1-yn-1-yl, prop-2-yn-1-yl, but-1-yn-1-yl, but-3-yn-1-yl, 1-methylprop-2-yn-1-yl, pent-1-yn-1-yl, pent-4-yn-1-yl, hex-1-yn-1-yl, hex-5-yn-1-yl, etc.

[0112] "Alkoxy" refers to a group of (alkyl-O)-. The "alkyl" part thereof is defined in this document. The alkoxy is preferably a C1-C8 alkoxy, and more preferably a C1-C6 alkoxy. Examples of the alkoxy include, but are not limited to: methoxy, ethoxy, n-propoxy, 1-methylethoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, isopentoxy, neopentoxy, 1-methylbutoxy, 1-ethylpropoxy, n-hexyloxy, isohexyloxy, 3-methylpentoxy, 2-methylpentoxy, 1-methylpentoxy, 3,3-dimethylbutoxy, 2,2-dimethylbutoxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy, 1,3-dimethylbutoxy, 2,3-dimethylbutoxy, 1-ethylbutoxy, 2-ethylbutoxy, etc. In addition, "alkoxycarbonyl" means a group in which the "alkoxy" defined above is bonded to a carbonyl group, for example, methoxycarbonyl, ethoxycarbonyl, etc. can be cited.

[0113] "Alkenyloxy" refers to a group of (alkenyl-O)-, wherein the "alkenyl" part is as defined herein, and preferably C2-C6 alkenyloxy.

[0114] "Alkynyloxy" refers to a group of (alkynyl-O)-, wherein the "alkynyl" part is as defined herein, and preferably is a C2-C6 alkynyloxy group.

[0115] "Alkoxycarbonyl" refers to a group of (alkyl-OC(O))-, wherein alkyl is as defined herein. The preferred alkyl group is C1-C6 alkyl. Examples thereof include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, and the like.

[0116] "Alkylsulfinyl" refers to a group of (alkyl-S(O))-. The "alkyl" part is as defined herein. Preferably, it is a C1-C6 alkylsulfinyl. Examples of alkylsulfinyl include, but are not limited to, methylsulfinyl, ethylsulfinyl, and the like.

[0117] "Alkylsulfonyl" refers to (alkyl-S(O) 2-O)-. The "alkyl" part thereof is defined in the relevant text. It is preferably a C1-C6 alkylsulfonyl group. It should be noted that "C1-C6 alkylsulfonyl group" refers to a sulfonyl group substituted by a "C1-C6 alkyl group", and groups such as methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, isopropylsulfonyl, n-butylsulfonyl, isobutylsulfonyl, sec-butylsulfonyl, tert-butylsulfonyl, n-pentylsulfonyl, isopentylsulfonyl, neopentylsulfonyl, and tert-pentylsulfonyl can be cited.

[0118] "Alkylaminocarbonyl" refers to an alkylamino-carbonyl group, wherein the "alkylamino" portion is as defined herein.

[0119] "Cycloalkylalkyl" refers to a cycloalkyl-alkyl group. The cycloalkyl and alkyl moieties are as defined herein. Monocycloalkylalkyl includes, but is not limited to, cyclopropylmethyl, cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl, and the like.

[0120] The term "heterocycloalkenyl" refers to a group of the above-defined "heterocycloalkyl" that contains at least one double bond.

[0121] "Aryl" as a group or part of a group refers to: (1) an aromatic monocyclic or condensed aromatic hydrocarbon ring group; preferably a 6-12-membered aryl group (also expressed as C6-C12 aryl group), more preferably a 6-10-membered aryl group (also expressed as C6-C10 aryl group), examples of which include but are not limited to: phenyl, naphthyl, anthracenyl and phenanthrenyl; or (2) a partially saturated carbon ring, for example: a phenyl and a C5-7 cycloalkyl or C5-7 cycloalkenyl group are fused to each other to form a ring structure. Examples include but are not limited to: tetrahydronaphthyl, indenyl or hydroindenyl, etc. The aryl group may be substituted by one or more substituents.

[0122] "Arylalkenyl" refers to a group of (aryl-alkenyl)-, where the "aryl" and "alkenyl" parts are as defined herein. Exemplary arylalkenyl groups include, but are not limited to, phenylpropenyl, and the like.

[0123] "Aralkyl" refers to a group of (aryl-alkyl)-, wherein the aryl and alkyl parts are as defined herein. Exemplary aralkyl groups include, but are not limited to, benzyl, phenethyl, 1-naphthylmethyl, and the like.

[0124] "Cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and preferably having 5-10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include, but are not limited to, cyclopentene, cyclohexene or cycloheptene. The cycloalkenyl group may be substituted with one or more substituents.

[0125] "Heteroaryl" refers to a monocyclic or condensed polycyclic aromatic heterocyclic group, preferably an aromatic group having one or more (preferably 3 to 14, more preferably 5 to 10, particularly preferably 5 or 6) carbon atoms, and one or more (preferably 1, 2, 3 or 4) oxygen, nitrogen, phosphorus or sulfur ring atoms (preferably O, S or N) as ring atoms, preferably the aromatic group is a 4-15 membered heteroaryl group, more preferably a 5-7 membered heteroaryl group. Examples of the heteroaryl group include, for example, furanyl, thienyl, pyrrolyl, pyrazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl, benzimidazolyl, pyridinyl, imidazolyl, 3-phenylpyrrolyl, thiazolyl-oxazolyl, tetrazolyl, isoxazolyl, indazolyl, pyridazinyl, quinolyl, purinyl, carbazolyl, acridinyl, pyrimidinyl, 2,3'-bifuranyl and isoquinolyl.

[0126] "Heteroarylalkyl" refers to a group of (heteroaryl-alkyl)-, wherein the heteroaryl and alkyl moieties are as defined herein. Exemplary heteroarylalkyl groups include, but are not limited to, 2-furylmethyl, 3-furylmethyl, 2-pyridylmethyl, and the like.

[0127] "Alkyl ether group" refers to a group of: (alkyl)-O-, wherein the "alkyl" part is as defined herein.

[0128] "Cycloalkyl ether group" refers to a group of: (cycloalkyl)-O-, wherein the "cycloalkyl" part is as defined herein.

[0129] "Heteroalkylether" refers to a group of: (heteroalkyl)-O-, wherein the "heteroalkyl" part is as defined herein.

[0130] "Aryl ether group" refers to a group of: (aryl)-O-, wherein the "aryl" part is as defined herein.

[0131] "Arylalkylether" refers to a group of: (aryl-alkyl)-O-, wherein the "aryl" and "alkyl" parts are as defined herein.

[0132] "Heteroaryl ether group" refers to a group of: (heteroaryl)-O-, wherein the "heteroaryl" part is as defined herein.

[0133] "Heteroarylalkylether" refers to a group of: (heteroaryl-alkyl)-O-, wherein the "heteroaryl" and "alkyl" parts are as defined herein.

[0134] "Heterocycloalkyl ether" refers to a group of: (heterocycloalkyl)-O-, wherein the "heterocycloalkyl" part is as defined herein.

[0135] "Heterocycloalkylamino" refers to both mono-heterocycloalkylamino and di-heterocycloalkylamino, unless otherwise specified. Mono-heterocycloalkylamino refers to a group of (heterocycloalkyl-)NH-; di-heterocycloalkylamino refers to (heterocycloalkyl) 2 The definition of "heterocycloalkyl" part thereof can be found in the relevant part of this text.

[0136] "Arylalkylamino" refers to both mono-arylalkylamino and di-arylalkylamino, unless otherwise specified. Mono-arylalkylamino refers to a group of (aryl-alkyl)-NH-; di-arylalkylamino refers to (aryl-alkyl)-NH-. 2 The definitions of "aryl" and "alkyl" parts are as given in the relevant part of this text.

[0137] "Cycloalkylamino" refers to both mono-cycloalkylamino and di-cycloalkylamino, unless otherwise specified. Mono-cycloalkylamino refers to a group of (cycloalkyl)-NH-; di-arylalkylamino refers to a group of (cycloalkyl)-NH-; 2 The definition of "cycloalkyl" part thereof can be found in the relevant part of this text.

[0138] "Arylamino" refers to both mono-arylamino and di-arylamino, unless otherwise specified. Mono-arylamino refers to a group of (aryl)-NH-; di-arylamino refers to (aryl)-NH-; 2 The definition of "aryl" part thereof can be found in the relevant part of this text.

[0139] "Heteroarylamino" refers to both mono-heteroarylamino and di-heteroarylamino, unless otherwise specified. Mono-heteroarylamino refers to a group of (heteroaryl)-NH-; di-heteroarylamino refers to a group of (heteroaryl)-NH-; 2 The definition of "heteroaryl" part can be found in the relevant part of this text.

[0140] "Heteroarylalkylamino" refers to both mono-heteroarylalkylamino and di-heteroarylalkylamino, unless otherwise specified. Mono-heteroarylalkylamino refers to the group (heteroaryl-alkyl)-NH-; di-heteroarylalkylamino refers to the group (heteroaryl-alkyl)-NH-. 2 The definitions of "heteroaryl" and "alkyl" parts are as given in the relevant part of this text.

[0141] Unless otherwise specified, the subunit of the present invention refers to a divalent group, that is, a group in which one hydrogen atom in a monovalent group is replaced by a valency. For example, "heteroalkylene" refers to a heteroalkyl group in which one of the hydrogen atoms is replaced by a valence; "heterocyclylene" refers to a heterocyclyl group in which one of the hydrogen atoms is replaced by a valence; "arylene" refers to an aryl group in which one of the hydrogen atoms is replaced by a valence; "alkylene" refers to an alkyl group in which one of the hydrogen atoms is replaced by a valence; "alkenylene" refers to an alkenyl group in which one of the hydrogen atoms is replaced by a valence; "cycloalkylene" refers to a cycloalkyl group in which one of the hydrogen atoms is replaced by a valence; "heteroarylene" refers to a heteroaryl group in which one of the hydrogen atoms is replaced by a valence; "heterocycloalkylene" refers to a heterocycloalkyl group in which one of the hydrogen atoms is replaced by a valence; "heterocycloalkenylene" refers to a heterocycloalkenyl group in which one of the hydrogen atoms is replaced by a valence; "alkyleneoxy" refers to an alkoxy group in which one of the hydrogen atoms is replaced by a valence; "alkenyleneoxy" refers to an alkenyloxy group in which one of the hydrogen atoms is replaced by a valence; "alkynyleneoxy" refers to an alkynyloxy group in which one of the hydrogen atoms is replaced by a valence, and the like. Wherein, the definitions of the above-mentioned heterocyclic group, aryl, alkyl, alkenyl, cycloalkyl, heteroaryl, heterocycloalkyl, heterocycloalkenyl, alkoxy, alkenyloxy, alkynyloxy, etc. can be found in the relevant definitions herein.

[0142] The present invention includes compounds represented by the general formula (I) and possible various isomeric forms thereof, including non-mirror isomers, mirror isomers, tautomers, and geometric isomers of "E" or "Z" configuration isomers, etc. Any chemist with a certain foundation can separate the above optically pure or stereoisomerically pure compounds.

[0143] Other groups not defined herein have the usual definitions.

[0144] Preferred embodiments of the present invention include the following.

[0145] In the compounds of the present invention, R 1 Preferably, the alkyl radical is a hydrogen atom, a carboxyl group, a nitro group, an amino group, a cyano group, an acyl group, an alkyl group, an alkoxy group, an alkenyl group, an alkynyl group, a halogen group, an isotope group, a haloalkenyl group, a heteroalkyl group, an arylalkyl group, a cycloalkyl group, an aryl group, a heteroaryl group, a heteroarylalkyl group, a heterocycloalkyl group, a heterocycloalkenyl group, an alkoxyalkyl group, an alkenyloxy group, an alkynyloxy group, an alkylamino group, an aminoalkyl group, an alkylaminocarbonyl group, a sulfonyl group, an alkylsulfonyl group, an alkylsulfinyl group, or an aminosulfonyl group; any of the above groups may be independently substituted with one or more substituents, including but not limited to halogen, isotope group, an amino group, a cyano group, a carboxyl group, a phenyl group, a benzyl group, a phenyloxy group, =O, -CF 3 , haloalkyl, alkyl, alkenyl, alkynyl, hydroxy, hydroxyalkyl, alkoxy, and alkoxyalkyl.

[0146] R1 is more preferably amino, cyano, alkenyl, alkynyl, halogen, isotope, C1-C6 alkyl, C6-C12 aryl, 5-12 membered aryl C1-C6 alkyl, C3-C9 cycloalkyl, 4-15 membered heteroaryl, 4-15 membered heteroaryl C1-C6 alkyl, 3-14 membered heterocycloalkyl, or C1-C6 alkoxy; any of the above groups may be independently substituted by one or more substituents selected from the above substituent group A, and these substituents include but are not limited to halogen, isotope, amino, cyano, carboxyl, =O, -CF 3 , alkyl, hydroxy, hydroxyalkyl, alkoxy.

[0147] R 1 Further preferred are cyano, alkynyl, halogen, isotope, or C1-C6 alkyl, pyrazolyl, furanyl, pyrimidinyl, aminopyrazolyl, morpholinyl, or phenyl; any of the above groups may be independently substituted by one or more substituents, and these substituents include but are not limited to halogen, isotope, amino, cyano, carboxyl, phenyl, benzyl, phenyloxy, =O, -CF3, haloalkyl, alkyl, hydroxyl, hydroxyalkyl, and alkoxy.

[0148] In the compounds of the present invention, L 1 Select -CH 2 CH 2 -, -CH=CH-, or -C≡C-.

[0149] L 1 More preferably, it is -CH=CH- or -C≡C-.

[0150] In the compounds of the present invention, L 2 Preferred are alkylene, carbonyl, -CO-NH-, -alkyl-CO-NH-, -alkyl-NH-, or -alkyl-O-. Any of the above groups may be substituted by one or more substituents, which include hydrogen atoms and alkyl groups.

[0151] L 2 More preferably, it is -CO-NH-, -alkyl-CO-NH-, -alkyl-NH-C(O)-, -alkyl-NH-, or -alkyl-O-. Any of the above groups may be substituted by one or more substituents, and these substituents include hydrogen atoms and alkyl groups.

[0152] L 3 Preferred are: covalent bond, alkylene, -O-, -NH-, -C(O)-NH-, -NH-C(O)-, -alkyl-C(O)-NH-, -alkyl-NH-C(O)-, -alkyl-NH-, -alkyl-O-. Any of the above groups may be substituted by one or more substituents, which include hydrogen atoms and alkyl groups.

[0153] L 3 Preferred are: covalent bond, -O-, -NH-, -C(O)-NH-, -NH-C(O)-.

[0154] In the compounds of the present invention, R 2 Preferred are carboxyl, amino, acyl, alkyl, alkoxy, heteroalkyl, arylalkyl, cycloalkyl, aryl, heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkenyl, alkoxyalkyl, alkenyloxy, alkynyloxy, alkylamino, aminoalkyl, alkylaminocarbonyl, sulfonyl, alkylsulfonyl, alkylsulfinyl, or aminosulfonyl; any of the above groups may be independently substituted with one or more substituents, including but not limited to halogen, isotope, amino, cyano, carboxyl, phenyl, benzyl, phenyloxy, =O, -CF3, haloalkyl, alkyl, alkenyl, alkynyl, hydroxyl, hydroxyalkyl, alkoxy, and alkoxyalkyl.

[0155] R 2 More preferably, it is C1-C6 alkyl, C6-C12 aryl, 5-12 membered aryl C1-C6 alkyl, C3-C9 cycloalkyl, 4-15 membered heteroaryl, 4-15 membered heteroaryl C1-C6 alkyl, 3-14 membered heterocycloalkyl, C1-C6 alkoxy C1-C6 alkyl; any of the above groups may be independently substituted by one or more substituents selected from the above substituent group A, and these substituents include but are not limited to halogen, isotope, amino, cyano, carboxyl, phenyl, =O, -CF3, haloalkyl, alkyl, alkenyl, alkynyl, hydroxyl, hydroxyalkyl, alkoxy, and alkoxyalkyl.

[0156] R 2 Further preferred are pyrazolyl, furanyl, pyrimidinyl, bromopyrimidinyl, aminopyrazolyl, morpholinyl and phenyl; any of the above groups may be independently substituted by one or more substituents, which include but are not limited to halogen, isotope, cyano, alkyl, alkenyl, alkynyl, hydroxyl, hydroxyalkyl, alkoxy, and alkoxyalkyl.

[0157] In the compounds of the present invention, R 3Preferred are: hydrogen atom, alkyl, heteroalkyl, arylalkyl, cycloalkyl, aryl, heteroaryl, heteroarylalkyl, heterocycloalkyl, alkyl ether, heteroalkyl ether, arylalkyl ether, cycloalkyl ether, aryl ether, heteroaryl ether, heteroarylalkyl ether, heterocycloalkyl ether, alkylamino, heteroalkylamino, arylalkylamino, cycloalkylamino, arylamino, heteroarylamino, heteroarylalkylamino, heterocycloalkylamino; any of the above groups independently may be unsubstituted or may be substituted by one or more substituents, and these substituents include but are not limited to halogen, isotope, =O, -CF3, haloalkyl, alkyl, alkenyl, alkynyl, hydroxyl, hydroxyalkyl, alkoxy, alkoxyalkyl, heteroalkyl, arylalkyl, cycloalkyl, aryl, heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkenyl, alkoxyalkyl, alkenyloxy, alkynyloxy, alkylamino, aminoalkyl, alkylaminocarbonyl, sulfonyl, alkylsulfonyl, alkylsulfinyl, or aminosulfonyl.

[0158] R 3 More preferably, it is methyl, ethyl, arylalkyl, cycloalkyl, aryl, heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkenyl, alkoxyalkyl, alkenyloxy, alkynyloxy, alkylamino, or aminoalkyl; any of the above groups may be independently unsubstituted or may be substituted with one or more substituents, including but not limited to halogen, isotope, amino, phenyl, benzyl, phenyloxy, =O, -CF 3 , haloalkyl, alkyl, heteroalkyl, alkenyl, alkynyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, heteroalkyl, arylalkyl, cycloalkyl, aryl, heteroaryl, heteroarylalkyl, heterocycloalkenyl, alkoxyalkyl.

[0159] The present invention includes compounds represented by the general formula (I) and possible various isomeric forms thereof, including non-mirror isomers, mirror isomers, tautomers, and geometric isomers of "E" or "Z" configuration isomers, etc. Those skilled in the art can separate the above optically pure or stereoisomerically pure compounds according to conventional means in the art.

[0160] The present invention includes the compounds represented by the general formula (I) and possible racemates and / or mirror isomers and / or mixtures of diastereomers.

[0161] In addition, the compounds represented by the general formula (I) also encompass the solvated and unsolvated forms of the compounds. Therefore, each form includes the compounds having the specified structure, including hydrates and anhydrates thereof.

[0162] In addition to the compounds represented by the general formula (I), kinase inhibitors of various embodiments include pharmaceutically acceptable salts, prodrugs and active metabolites of these compounds, and pharmaceutically acceptable salts of these metabolites.

[0163] The term "pharmaceutically acceptable salt" refers to certain salts of the above compounds that can maintain the original biological activity and are suitable for medical use. The pharmaceutically acceptable salts of the compounds represented by general formula (I) have two forms: one is a salt formed with an acid; the other is a salt formed with a base or an alkali metal. The acids that form pharmaceutically acceptable salts with the compounds represented by general formula (I) include inorganic acids and organic acids. Suitable inorganic acids include: hydrochloric acid, sulfuric acid and phosphoric acid. Suitable organic acids can be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic acids and sulfonic acid organic acids; examples thereof include but are not limited to: formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, glycine, arginine, citric acid, fumaric acid, alkylsulfonic acid, arylsulfonic acid, etc. Alkali metals that form pharmaceutically acceptable salts with the compounds represented by the general formula (I) include: lithium, sodium, potassium, magnesium, calcium, aluminum, zinc, etc.; bases that form pharmaceutically acceptable salts with the compounds represented by the general formula (I) include: choline, diethanolamine, morpholine, etc.

[0164] "Prodrug" is a derivative of a compound represented by general formula (I), which is converted in vivo (e.g., by hydrolysis, reduction or oxidation) into a compound represented by general formula (I) by means of in vivo metabolism. For example, a compound represented by general formula (I) containing a hydroxyl group can be reacted with an acid to prepare a corresponding ester. The corresponding ester is a prodrug, which can hydrolyze the parent drug in vivo. Acids suitable for preparing "prodrugs" include, but are not limited to, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, oxalic acid, salicylic acid, succinic acid, fumaric acid, maleic acid, methylene-bis-β-hydroxynaphthoic acid, gentisic acid, isethionic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like.

[0165] The kinase inhibitors referred to in the present invention include IC 50 The kinases referred to in the present invention include but are not limited to RET and TRK.

[0166] The administration of the compound represented by general formula (I) can be enteral administration or parenteral administration. Enteral administration refers to oral or rectal administration. Parenteral administration includes subcutaneous, intramuscular, intravenous and intradermal routes. Generally, the active compound represented by general formula (I) can be administered using a pharmaceutically acceptable carrier or diluent.

[0167] "Therapeutically effective amount" or "therapeutic amount" refers to an amount sufficient to produce a therapeutic effect. The effective amount can be administered in one or more doses. Generally, the effective amount is sufficient to alleviate, improve, stabilize, slow down or delay the further development of the disease.

[0168] The compounds of the present invention can be used alone or in combination with one or more other drugs; or used for patients undergoing surgery or radiotherapy, wherein the compounds of the present invention are administered to the patients concomitantly with the surgery or radiotherapy, or before the surgery or radiotherapy, or after the surgery or radiotherapy; or are administered in a certain dosage form with a pharmaceutically acceptable carrier, diluent or excipient. The specific dosage form depends on the route of administration.

[0169] The pharmaceutical formulation for parenteral injection of the present invention comprises a pharmaceutically acceptable sterile aqueous solution or non-aqueous solution, a dispersant, a suspending agent or an emulsifier, and a powder injection which is prepared into an injectable sterile aqueous solution before use.

[0170] If desired, and for more effective distribution, the compounds of the invention can be incorporated into slow-release or targeted-delivery systems such as polymer matrices, liposomes, and microspheres.

[0171] Solid dosage forms for oral administration include capsules, tablets, troches, powders and granules. These solid dosage forms contain an active compound represented by the general formula (I) and at least one inert and pharmaceutically acceptable excipient or carrier. These excipients or carriers include sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and salicylic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) dissolution delaying agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glyceryl monostearate; h) adsorbents such as kaolin and bentonite; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols.

[0172] Solid dosage forms of tablets, dragees, capsules, troches, and granules can be prepared with coatings or shells.

[0173] The active compounds can also be administered in microencapsulated form, with one or more of the above-mentioned excipients, if desired.

[0174] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsifiers, solutions, suspensions, syrups, etc. In addition to the active compound, the liquid dosage form may contain inert diluents commonly used in liquid dosage forms, such as water or other solvents, stabilizers and emulsifiers, such as ethanol, ethyl carbonate, ethyl acetate, benzoic acid alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuran alcohol, polyethylene glycol and fatty acid esters of sorbitan, etc.

[0175] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0176] The suspension may contain, in addition to the active compound, a suspending agent such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters.

[0177] Compositions for rectal or vaginal administration are preferably suppositories, which can be prepared by mixing the compounds of the invention with suitable non-irritating excipients or carriers.

[0178] Dosage forms for topical administration of the compounds of this invention include powders, patches, sprays, ointments and inhalants which can be prepared by mixing the active compound under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers or propellants that are required.

[0179] The preferred dosage range of the compound of the present invention is about 0.01-400 mg per kilogram of body weight per day. A more preferred dosage range is 0.2-100 mg per kilogram of body weight per day. Appropriate doses may also be selected for multiple administrations per day.

[0180] The pyrazolo[1,5-a]pyridine derivatives of the present invention can be used as, but not limited to, kinase inhibitors. The pyrazolo[1,5-a]pyridine derivatives disclosed in the present invention can be used alone or together with other drugs or pharmaceutically acceptable carriers, diluents or excipients, and are suitable for preventing or treating diseases caused by, or associated with, or accompanied by the disruption of cell proliferation and / or angiogenesis. One example of these diseases is cancer.

[0181] The compounds of the present invention can also be used to treat diseases that are regulated at least in part by the activity of any one or more kinases such as TRK, RET, FGHR, PDGFR, VEGFR, etc., wherein TRK and RET activity are known to play a role in promoting the onset of the disease, or such symptoms are known or have been shown to be alleviated by TRK and RET inhibitors. Disorders of this type that are expected to be treated by the compounds of the invention include, but are not limited to, the following: antiproliferative disorders (e.g., cancer); neurodegenerative diseases, including Huntington's disease, polyglutamine diseases, Parkinson's disease, Alzheimer's disease, epileptic seizures, striatonigral degeneration, progressive supranuclear palsy, torsion dystonia, spasmodic torticollis and movement disorders, familial tremor, Tourette syndrome, diffuse Lewy body disease, progressive supranuclear palsy, Pick's disease, intracranial hemorrhage, primary lateral sclerosis, spinal muscular atrophy, amyotrophic lateral sclerosis, hypertrophic interstitial polyneuropathy, retinitis pigmentosa, hereditary optic atrophy, hereditary spastic paraplegia, progressive ataxia, and Shy-Drager syndrome; metabolic diseases, including type 2 diabetes; ocular degenerative diseases, including glaucoma, age-related macular degeneration, rubroiridis glaucoma; inflammatory diseases and / or immune system disorders, including rheumatoid arthritis (RA), osteoarthritis, Arthritis, juvenile chronic arthritis, graft-versus-host disease, psoriasis, asthma, spondyloarthropathies, psoriasis, Crohn's disease, inflammatory bowel disease, colon ulcers, alcoholic hepatitis, diabetes, Sjoegrens syndrome, multiple sclerosis, ankylosing spondylitis, membranous glomerulopathy, disc pain, systemic lupus erythematosus; diseases involving angiogenesis, including cancer, psoriasis, rheumatoid arthritis; psychological disorders, including bipolar disorder, schizophrenia , mania, depression and dementia: cardiovascular diseases including; heart failure, restenosis and arteriosclerosis; fibrotic diseases including: liver fibrosis, cystic fibrosis and angiofibroma; infectious diseases including: fungal infections such as: Candida albicans, bacterial infections, viral infections such as: herpes simplex, protozoan infections such as: malaria, Leishmania infection, Trypanosoma brucei infection, toxoplasmosis and coccidiosis and hematopoietic disorders including: thalassemia, anemia and sickle cell anemia.

[0182] The term "cancer" as used herein generally refers to a broad range of diseases characterized by uncontrolled abnormal growth of cells.

[0183] The compounds of the present invention are expected to be useful in treating a variety of cancers, including but not limited to: bone cancers, including: Ewing's sarcoma, osteosarcoma, chondrosarcoma, etc.; brain and CNS tumors, including: acoustic neuroma, neuroblastoma, glioma and other brain tumors, spinal cord tumors, breast cancer, colorectal cancer, advanced colorectal adenocarcinoma; endocrine cancers, including: adrenal cortical carcinoma, pancreatic cancer, pituitary cancer, thyroid cancer, parathyroid cancer, thymic carcinoma, multiple endocrine tumors; gastrointestinal cancers, including: : Gastric cancer, esophageal cancer, small intestine cancer, renal cell carcinoma, liver cancer, extrahepatic bile duct cancer, gastrointestinal carcinoid tumors, gallbladder cancer; genitourinary cancers, including: testicular cancer, penis cancer, prostate cancer; gynecological cancers, including: cervical cancer, ovarian cancer, vaginal cancer, uterine / endometrial cancer, pudendal cancer, gestational trophoblastic tumor, fallopian tube cancer, uterine sarcoma; head and neck tumors, including: oral cancer, lip cancer, salivary gland cancer, laryngeal cancer, hypopharyngeal cancer, orthopharyngeal cancer, nasal cancer, paranasal sinus cancer, nasopharyngeal cancer; blood cancer Cancer, including: childhood leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, acute promyelocytic leukemia, plasma cell leukemia; bone marrow cancer blood diseases, including: myelodysplastic syndrome, myeloproliferative disorders, aplastic anemia, Fanconi anemia, idiopathic macroglobulinemia; lung cancer, including: small cell lung cancer, non-small cell lung cancer; lymphoma, including: Hodgkin's disease, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, AIDS-related lymphoma; eye cancers, including retinoblastoma, uveal melanoma, skin cancers, including melanoma, non-melanoma skin cancer, Merkel cell carcinoma, soft tissue sarcomas, such as childhood soft tissue sarcoma, adult soft tissue sarcoma, Kaposi's sarcoma, urinary system cancers, including renal cancer, Wilms' tumor, bladder cancer, urethral cancer and metastatic cell carcinoma.

[0184] Cancers that the compounds of the present invention can be used to treat include, but are not limited to, breast cancer, lung cancer, renal cell carcinoma, gastric cancer, ovarian cancer, thyroid cancer, colorectal cancer, prostate cancer, head and neck cancer, kidney cancer, stomach cancer, and brain cancer.

[0185] Preferred cancers that can be treated by the compounds of the invention are solid tumors and hematological malignancies.

[0186] In addition, the compounds of the present invention can be used to treat proliferative diseases that are resistant to other chemotherapeutic treatments; and to treat hyperproliferative diseases, such as leukemia, psoriasis, and the like.

[0187] Example_Synthesis and application of new pyrazolo[1,5-a]pyridine derivatives

[0188] The compound represented by the general formula (I) of the present invention can be synthesized by the synthetic route and synthetic method discussed below. The raw materials used are convenient and easily available. However, the synthetic route and synthetic method used in the present invention can be widely applied to the synthesis of analogs, and only the starting raw materials need to be changed. For example, the synthesis of the compound not described in detail in the examples herein can be synthesized by replacing the starting raw materials with the starting raw materials of the corresponding target compound, and slightly changing the reaction conditions when necessary according to common sense in chemistry to synthesize the desired target compound.

[0189] The reagents of each embodiment can be prepared using the reaction pathways or synthetic processes described below, using the technical means in the art, with available starting materials. The preparation of specific compounds of the embodiments is described in detail in the following examples, but those skilled in the art will know that the chemical reactions described can be applied to prepare a variety of other compounds in different embodiments. For example, the synthesis of non-exemplified compounds can be successfully carried out through modifications that are obvious to those skilled in the art, such as: by appropriately protecting interfering groups, by changing to other appropriate reagents known in the art, or by making routine modifications to reaction conditions. Other reactions disclosed herein or known in the art can be identified as having applicability for preparing other compounds of each embodiment.

[0190] Reagents used to synthesize compounds can be obtained or prepared according to techniques known in the art.

[0191] In the following examples, all temperatures are in degrees Celsius unless otherwise indicated.

[0192] All starting materials and reagents were obtained from commercial suppliers, including but not limited to Aldrich Chemical Company, Lancaster Synthesis Ltd, etc. Commercially available starting materials and reagents were used directly without further purification unless otherwise specified.

[0193] The glassware was oven dried and / or heat dried. The reaction was followed on glass silica gel-60 F254 plates (0.25 mm) (TLC). Analytical thin layer chromatography was developed with appropriate solvent ratios (v / v). The end point of the reaction was when the starting material was exhausted on TLC.

[0194] Usually, the subsequent treatment is to double the volume of the reaction solution with the solvent used in the reaction, and then extract three times with 25% of the total volume of the extraction solvent, unless otherwise specified. The product-containing extract is dehydrated with anhydrous sodium sulfate, filtered on a rotary evaporator, and the solvent is evaporated under reduced pressure and attention is paid to the removal of the solvent in vacuum. Finally, the target compound is separated by flash column chromatography.

[0195] 1 H NMR spectra were measured using a Bruker instrument (400 MHz), and chemical shifts are expressed in ppm. Chloroform was used as a reference standard (7.25 ppm) or tetramethylsilane as an internal standard (0.00 ppm). Other solvents commonly used in NMR may also be used as needed. 1 H NMR notation: s = singlet, d = doublet, t = triplet, m = multiplet, br = broadened, dd = doublet of a doublet, dt = doublet of a triplet. Coupling constants, when given, are given in Hz.

[0196] Mass spectra were obtained using LC / MS, with ESI or APCI ionization. All melting points are uncorrected.

[0197] The following examples are only used to illustrate the synthesis methods of the specific compounds invented. However, there is no limitation on the synthesis methods. Compounds not listed below can also be prepared by the same synthesis routes and methods as below, by selecting appropriate starting materials and slightly adjusting the reaction conditions according to common sense where necessary.

[0198] synthesis

[0199] The compound shown in the general formula (I) is 2 =-C(O)-NH-, the synthesis method is as follows: select a suitable I as a raw material, remove the methyl group under a suitable catalyst and suitable conditions (for example, under the action of 1-dodecanethiol) to obtain compound II. Under catalysis, compound II reacts with boron compound III to obtain IV. Under the action of N-phenylbis(trifluoromethanesulfonimide) (1,1,1-trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamide), compound IV is converted into compound V. On the other hand, compound VIII obtained by condensation of compound VI and compound VII reacts with compound V to obtain compound IX, which is the target compound shown in the same general formula (I) (synthetic route 1).

[0200] Synthetic route 1

[0201]

[0202] When R in the general formula (I) 1=-CN, it can be synthesized using synthetic route 2. In this case, the synthesis method is: select a suitable X as a raw material, and obtain XIV through acylation and amination. Under the action of acid, cyclization is performed to obtain compound XVI. A formyl group is introduced into compound XVI, and the latter is converted into a hydroxyl group through hydroxylation. After removing the methyl group under a suitable catalyst and suitable conditions (for example, under the action of 1-dodecanethiol), compound I-1 is obtained.

[0203] Synthetic route 2

[0204]

[0205] The compound shown in the general formula (I) is 1 =-CH=CH- or -C≡C-, L 2 -R 2 When the aryl group is substituted, the corresponding target compound, i.e., compound XXIII, can be synthesized by the method shown in Synthesis Scheme 3. The method is as follows: select a suitable iodine compound XIX as a raw material, react with XX under a suitable catalyst and suitable conditions to obtain compound XXI. Then remove the silicon protecting group in an alkaline methanol solution to obtain XXII. The latter reacts with compound V to obtain compound XXIII.

[0206] Synthetic route 3

[0207]

[0208] The content of the present invention is further explained below with reference to examples. The purpose is to allow technicians with basic knowledge in the field to more clearly understand and practice the specific content of the present invention. However, the protection scope of the present invention is not limited to these examples.

[0209] Example 1 Synthesis of (S)-3-(3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl)-N-(1-(1-methyl-1H-imidazol-4-yl)methyl)pyrrolidin-3-yl)propanamide (IX-1)

[0210] Step-1 Synthesis of 6-bromo-4-hydroxypyrazolo[1,5-a]pyridine-3-carbonitrile (II-2):

[0211]

[0212] The compound 6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbonitrile (I-1) (100.0 g, 396.7 mmol, 1.0 eq) was added to N,N-dimethylformamide (1000.0 mL, 10.0 Vol), and the system was heated to 40°C to 50°C with stirring. A mixed solution of sodium hydroxide (31.7 g, 793.4 mmol, 2.0 eq) and water (64.9 mL, 0.65 Vol) prepared in advance and cooled to room temperature was added, and the temperature was controlled at 40-50°C. n-Dodecanethiol (160.6 g, 793.4 mmol, 2.0 eq) was slowly added dropwise to the reaction system. After the feeding was completed, the reaction was kept at 45°C to 50°C for 10 to 12 hours. After the reaction was completed by HPLC monitoring, the system was cooled to 10-20°C and kept within this temperature range, water (3000.0mL, 30.0Vol) was slowly added to the system to quench, methyl tert-butyl ether (1000.0mL*2, 10.0Vol*2) was added for extraction, the aqueous phase was separated, the temperature was cooled to 0-10°C and kept within this temperature range, the pre-prepared 10% citric acid solution (about 1200g) was slowly added to the aqueous phase, the pH was adjusted to 5-6, a large amount of solid precipitated, the mixture was kept at 0-10°C and stirred for 10-20min, filtered, the filter cake was washed with water (300.0mL*2, 3.0Vol*2), dried, and the filter cake was transferred to an oven at 50-60°C for drying to obtain an off-white solid II-2 (88.0g, 93.2%). 1H MNR (400MHz, DMSO-d 6 )δ:8.87-8.75(m,1H),8.62-8.51(m,1H),8.03-7.89(m,1H),6.95-6.84(m,1H).ESI-MS(m / z):239.8[M+H]+.

[0213] Step-2 Synthesis of 4-hydroxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile IV-1:

[0214]

[0215] The compound 6-bromo-4-hydroxypyrazolo[1,5-a]pyridine-3-carbonitrile (II-1) (55.0 g, 231.1 mmol, 1.0 eq) and potassium carbonate (63.8 g, 462.1 mmol, 2.0 eq) were added to a mixed solution of dioxane (1100.0 mL, 20.0 Vol) and water (275.0 mL, 5.0 Vol). The catalyst tetrakistriphenylphosphine palladium (13.4 g, 11.6 mmol, 0.05 eq) was added to the reaction system under stirring. The nitrogen was replaced 5 times and the system was heated to 95-100 ° C for 4 to 5 hours. After the reaction is completed by HPLC monitoring, the system is cooled to 10-20°C and controlled within this temperature range, water (3300.0mL, 60.0Vol) is slowly added to the system for quenching, and the filtrate is passed through a diatomaceous earth pad. Methyl tert-butyl ether (1100.0mL*3, 20.0Vol*3) is added to the filtrate for extraction, and the aqueous phase is separated. The temperature is controlled at 15-25°C, and the pre-prepared 1M dilute hydrochloric acid solution is slowly added to the aqueous phase to adjust the pH to 5-6. A large amount of solid precipitates. The mixture is kept at 15-25°C and stirred for 10-20min. The filter cake is washed with water (275.0mL*2, 5.0Vol*2), dried, and the filter cake is transferred to an oven and dried at 50-60°C to obtain a gray solid IV-1 (51.6g, 93.4%). 1 H NMR (400 MHz, DMSO-d 6 )δ:11.36(s,1H),8.74(s,1H),8.50(s,1H),8.21(s,1H),7.94(s,1H),6.96(s,1H),3.88(s,3H).ESI-MS(m / z):240.1[M+H] + .

[0216] Step-3 Synthesis of 3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl trifluoromethanesulfonate (V-1):

[0217]

[0218] The compound 4-hydroxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (IV-1) (73.6 g, 307.6 mmol, 1.0 eq) was dissolved in N,N-dimethylacetamide (1472.0 mL, 20.0 Vol), and the temperature was controlled at 10-20°C. N-phenylbistrifluoromethylsulfonimide (120.9 g, 338.4 mmol, 1.1 eq) was added to the system in batches, and the system was cooled to 10-15°C and maintained within this temperature range. N,N-diisopropylethylamine (79.5 g, 615.2 mmol, 2.0 eq) was slowly added dropwise to the system. After the addition was completed, the temperature was controlled at 10-15°C and the reaction was carried out for 4 to 5 hours. After the reaction is completed by HPLC monitoring, the system is cooled to 0-10°C and controlled within this temperature range. Water (3200.0mL, 43.5Vol) is slowly added to the system for quenching. A large amount of solid precipitates. The mixture is kept at 10-20°C and stirred for 10-20min. The mixture is filtered and the filter cake is washed with water (441.6mL*2, 6.0Vol*2). The mixture is dried and the filter cake is transferred to an oven at 50-60°C to obtain an off-white solid V-1 (112.0g, 98.0%). 1 HNMR (40 0MHz, DMSO-d 6 )δ:9.53-9.28(m,1H),8.90-8.64(m,1H),8.58-8.29(m,1H),8.25-7.89(m,2H),4.01-3.68(m,3H).ESI-MS(m / z):372.0[M+H] + .

[0219] Step-4 Synthesis of tert-butyl 3-(S)-propionylaminopyrrolidine-1-carboxylate (VIII-1):

[0220]

[0221] Compound 3-(S)-aminopyrrolidine-1-carboxylic acid tert-butyl ester (VI-1) (1.8 g, 9.6 mmol, 1.0 eq) was added to dichloromethane (100 mL, 55.5 Vol), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 2.8 g, 11.6 mmol, 1.2 0 eq) and propiolic acid (VII.1, 678 mg, 9.6 mmol 1.0 eq) were added under nitrogen protection, and the temperature was controlled at 20-30°C and stirred for 15-20 hours. After the reaction was completed under TLC monitoring, the system was concentrated, and the residue was purified by silica gel column chromatography with petroleum ether / ethyl acetate = 5 / 1 and petroleum ether / ethyl acetate = 2 / 1 as eluents, and concentrated to obtain yellow solid VIII-1 (1.50 g, 65%). 1H NMR (400 MHz, CDCl 3 )δ:6.37-6.47(d,J=42Hz,1H),4.47-4.51(dd,J=5.4Hz,1H),3.59-3.63(dd,J=6.0Hz,1H),3.45(s,2H),3.25 -3.30(d,J=19.6Hz,1H),2.83(s,1H),2.14-2.19(m,1H),1.83(s,1H),1.47(s,9H).ESI-MS(m / z):237.2[MH] -

[0222] Step-5 Synthesis of 3-(S)-(3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl)-N-(pyrrolidin-3-yl)propanamide (IX-1) hydrochloride:

[0223]

[0224] Under nitrogen protection, 3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl trifluoromethanesulfonate (V-1) (0.82 g, 2.2 mmol, 1.0 eq) and tert-butyl 3-(S)-propionylaminopyrrolidine-1-carboxylate (VIII-1) (0.80 g, 3.4 mmol, 1.5 eq) were dissolved in N,N-dimethylacetamide (50.0 mL, 60.0 Vol), and cuprous iodide (0.042 g, 0.22 mmol, 0.1 eq), N,N-diisopropylethylamine (0.571 g, 4.4 mmol, 2.0 eq) and catalyst bistriphenylphosphine palladium dichloride (0.154 g, 0.22 mmol, 0.1 eq) were added. The atmosphere was replaced with nitrogen 6 times, and the system was heated to 95-100 ° C for reaction for 1 to 2 h. After the reaction was completed as monitored by TLC, the system was poured into water (200.0 mL) for quenching and filtered. The filter cake was purified by silica gel column chromatography using petroleum ether / ethyl acetate = 1 / 1 and ethyl acetate as eluents and concentrated to give a white solid Key Intermediate-1 (0.59 g, 58%). 1 H NMR (400 MHz, CDCl 3)δ:8.71(s,1H),8.30(s,1H),7.79-7.83(d,J=17.6Hz,2H),7.69(s,1H),7.28(s,1H),4.55(s,1H),4.03(s,3H),3.66- 3.70(dd,J=6.4Hz,1H),3.30-3.53(m,3H),2.07(s,1H),1.93-1.98(m,1H),1.50(s,9H).ESI-MS(m / z):404.0[M+H-56] + .

[0225] 3-(S)-(3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl)propionamido)pyrrolidine-1-carboxylic acid tert-butyl ester (Key Intermediate-1) (500 mg, 1.08 mmol, 1.0 eq) was dissolved in ethyl acetate (38.5 mL, 77.0 Vol), 4 M hydrogen chloride ethyl acetate solution (3.85 mL, 7.7 Vol) was added, and the temperature was controlled at 20-30°C and stirred for 15-20 hours. After the reaction was completed under TLC monitoring, the system was filtered, the filter cake was washed with ethyl acetate, and recrystallized with water and ethanol to obtain a yellow solid, namely the target compound 1 (IX-1) (305 mg, 71%). 1 H NMR (400 MHz, DMSO-d 6 )δ:9.42(d,J=11.2Hz,1H),9.22(d,J=6.4Hz,2H),8.75(d,J=8.0Hz,1H),8.39(s,1H),8.29(br,1H),8.13(s,1H),4.43-4.48(q,J=6.4Hz,1 H),3.89(s,3H),3.42-3.46(m,1H),3.21-3.30(m,3H),3.05-3.09(m,1H),2.14-2.23(m,1H),1.87-1.95(m,1H).ESI-MS(m / z):360.2[M+H] + .

[0226] Example 2 Synthesis of 6-(1-methyl-1H-pyrazol-4-yl)-4-(5-methyl-2-(4-methylpiperazin-1-yl)pyridin-4-yl)ethynyl)pyrazolo[1,5-a]pyridine-3-carbonitrile (2)

[0227] Step-1 Synthesis of tert-butyl (mesitylenesulfonyl)oxycarbamate (XII):

[0228]

[0229] A solution of 2,4,6-trimethylbenzene-1-sulfonyl chloride (X) (50.0 g, 0.228 mol) and tert-butyl hydroxycarbamate (XI, 30.4 g, 0.228 mol) in tert-butyl methyl ether (500 mL) was cooled to 0°C. Triethylamine (25.4 g, 0.25 mol) was added dropwise. After the addition, the mixture was stirred at 0°C for 0.5 hours. The mixture was then heated to room temperature and stirred for 2 hours. The mixture was filtered. The filtrate was washed with water (100 mL) and brine (50 mL) and washed with Na 2 SO 4 Drying and concentration gave a residue. The residue was triturated with petroleum ether (50 mL) and filtered. The filter cake was collected and dried to give the title compound XII (60.2 g, yield 84%) as an off-white solid. 1 H NMR (400 MHz, CDCl 3 )δ:7.57(br s,1H),6.99(s,2H),2.68(s,6H),2.32(s,3H),1.32(s,9H).

[0230] Step-2 Synthesis of 1-amino-3-bromo-5-methoxypyridin-1-ium 2,4,6-trimethylbenzenesulfonate (XIV):

[0231]

[0232] Trifluoroacetic acid (100 mL) was cooled to 0°C and tert-butyl (mesitylenesulfonyl)oxycarbamate (XII) (60.0 g, 0.19 mol) was added in batches. After the addition was complete, the mixture was stirred at 20°C for 2 hours. Ice water (300 mL) was added dropwise and the mixture was stirred for 0.5 hours and filtered. The filter cake was washed with water (50 mL × 3) and collected to obtain the corresponding intermediate (58 g, wet weight) as a white solid. The product was used immediately in the next step without further purification.

[0233] The intermediate (58 g, wet weight) was dissolved in dichloromethane (300 mL) and the mixture was heated with Na 2 SO 4 Dry for 5 minutes and filter. Cool the filtrate to 0°C. Add 3-bromo-5-methoxypyridine (XIII) (35.8 g, 0.19 mol) in batches. After addition, stir the mixture at room temperature for 2 hours. Filter the mixture. Wash the filter cake with dichloromethane (50 mL x 2), collect and dry to obtain the title compound (XIV) (48.2 g, yield 63%) as a white solid. 1 H NMR (400 MHz, DMSO-d 6)δ:8.71(s,1H),8.62(s,1H),8.59(s,2H),8.27(s,1H),6.74(s,2H),3.97(s,3H),2.50(s,6H),2.17(s,3H).

[0234] Step-3 Synthesis of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine (XVI):

[0235]

[0236] A solution of 1-amino-3-bromo-5-methoxypyridin-1-yl 2,4,6-trimethylbenzenesulfonate (XIV) (48.0 g, 0.12 mol) in N,N-dimethylformamide (150 mL) was cooled to 0°C. Triethylamine (24.1 g, 0.24 mol) was added dropwise. After the addition, the mixture was stirred at 0°C for 5 minutes. Ethyl propiolate (XV) (23.3 g, 0.24 mol) was added dropwise. After the addition, the mixture was stirred at room temperature overnight. The mixture was poured into water (500 mL) and extracted with ethyl acetate (100 mL×4). The combined organic phase was washed with brine (50 mL) and washed with Na 2 SO 4 Dry and concentrate. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5: 1) to give a crude intermediate (28 g) as a yellow solid. The solid was dissolved in hydrobromic acid (48% aqueous solution, 60 mL) and refluxed for 1.5 hours. The mixture was then stirred at room temperature overnight. The mixture was filtered. The filter cake was washed with water (10 mL × 5) and dried to give the title compound (3.0 g) as a yellow solid. The filtrate was combined and extracted with ethyl acetate (50 mL × 2). The combined organic phase was washed with brine (20 mL) and washed with Na 2 SO 4 Dry and concentrate to obtain a residue. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10: 1) to obtain the title compound (1.20 g) as a white solid. The two batches of products were combined to obtain the title compound (XVI, 4.2 g, yield 16%) as a yellow solid. LC-MS [mobile phase: from 95% water and 5% CH 3 CN to 5%water and 95% CH 3 CN in 2.5min], Rt=1.58min; Purity: 100%; MS Calcd: 226.0; MS Found: 226.9[M+H] + .1H NMR (300MHz, CDCl 3)δ:8.28(s,1H),7.85(d,J=1.8Hz,1H),6.63(d,J=1.5Hz,1H),6.46(s,1H),3.96(s,3H).

[0237] Step-4 Synthesis of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde (XVII):

[0238]

[0239] Phosphorus oxychloride (6.10 g, 39.2 mmol) was added dropwise to a solution of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine (XVI) (3.0 g, 13.2 mmol) in N,N-dimethylformamide (75 mL) at 0 ° C. After the addition, the mixture was stirred at room temperature for 4 hours. The mixture was poured into ice water (100 mL) and stirred for 30 minutes. Sodium hydroxide aqueous solution (1 mol / L) was added dropwise until pH = 9. The mixture was filtered. The filter cake was washed with water (5 mL × 2) and tert-butyl methyl ether (5 mL × 2). The solid was collected and dried to give the title compound XVII (3.2 g, yield 95%) as a white solid. 1 H NMR (400 MHz, DMSO-d 6 )δ:10.21(s,1H),8.94(s,1H),8.47(s,1H),7.31(s,1H),4.08(s,3H).

[0240] Step-5 Synthesis of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde oxime (XVIII):

[0241]

[0242] 6-Bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carboxaldehyde (XVII) (3.2 g, 12.5 mmol) was dissolved in ethanol (80 mL) and stirred for 10 minutes. Water (40 mL) and hydroxylamine hydrochloride (1.3 g, 18.8 mmol) were added. The mixture was stirred at 55 ° C for 4 hours. The mixture was concentrated to remove ethanol. The residue was filtered. The filter cake was washed with water (5 mL × 3) and tert-butyl methyl ether (5 mL × 3). The solid was collected and dried to give the title compound XVIII (2.8 g, yield 83%) as an off-white solid. 1 H NMR (400 MHz, DMSO-d 6)δ: 11.42(br s,1H),8.74(s,1H),8.69(s,1H),7.96(s,1H),7.01(s,1H),4.03(s,3H).

[0243] Step-6 Synthesis of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbonitrile (I-1):

[0244]

[0245] A suspension of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde oxime (XVII) (2.8 g, 10.4 mmol) and acetic anhydride (90 mL) was stirred at 120 ° C overnight. The mixture was cooled and concentrated to give a residue. The residue was triturated with (petroleum ether: ethyl acetate = 1:1, 5 mL) and filtered. The solid was collected and dried to give the title compound (I-1, 2.38 g, yield 91%) as a gray solid. 1 H NMR (400 MHz, DMSO-d 6 )δ: 8.95(s,1H),8.60(s,1H),7.25(s,1H),4.04(s,3H).

[0246] Step-7 Synthesis of tert-butyl 4-(5-methyl-4-(trimethylsilyl)ethynyl)pyridin-2-yl)piperazine-1-carboxylate (XXI-1):

[0247]

[0248] Tert-butyl 4-(4-iodo-5-methylpyridin-2-yl)piperazine-1-carboxylate (XIX-1) (3.3 g, 8.19 mmol), ethynyltrimethylsilane (0.96 g, 9.83 mmol) and triethylamine (1.65 g, 16,14 mmol) were dissolved in tetrahydrofuran (30 mL) solution, followed by the addition of copper (I) iodide (78 mg, 0.41 mmol) and bis(triphenylphosphine)palladium (II) chloride (287 mg, 0.41 mmol). After stirring at 40 ° C under nitrogen for 2 days, the mixture was cooled and poured into water (50 mL). The mixture was extracted with ethyl acetate (30 mL×2). The combined organic layer was washed with brine (20 mL) and washed with Na 2 SO 4 The residue was dried and concentrated to give a residue. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=20:1) to give the title compound XXI-1 (2.80 g, yield 92%) as a brown solid. 1 H NMR (300 MHz, CDCl3 )δ:8.03(s,1H),6.69(s,1H),3.53-3.51(m,4H),3.47–3.43(m,4H),2.25(s,3H),1.48(s,9H),0.26(s,9H).

[0249] Step-8 Synthesis of tert-butyl 4-(4-ethynyl-5-methylpyridin-2-yl)piperazine-1-carboxylate (XXII-1):

[0250]

[0251] Potassium carbonate (4.8 g, 6.95 mmol) was added to a mixture of tert-butyl 4-(5-methyl-4-((trimethylsilyl)ethynyl)pyridin-2-yl)piperazine-1-carboxylate (XXI-1, 2.6 g, 6.95 mmol) in methanol (50 mL). After stirring overnight at room temperature, the mixture was filtered. The filtrate was concentrated to obtain a residue. The residue was diluted with water (30 mL) and extracted with ethyl acetate (30 mL×2). The combined organic layers were washed with brine (20 mL) and washed with Na 2 SO 4 Drying and concentration gave a residue. The residue was triturated with petroleum ether (20 mL) and filtered. The filter cake was dried to give the title compound (XXII-1, 1.42 g, yield 68%) as a brown solid. 1 H NMR (400 MHz, CDCl 3 )δ:8.05(s,1H),6.73(s,1H),3.53-3.44(m,8H),3.33(s,1H),2.27(s,3H),1.48(s,9H).

[0252] Step-9 Synthesis of 6-(1-methyl-1H-pyrazol-4-yl)-4-((5-methyl-2-(4-methylpiperazin-1-yl)pyridin-4-yl)ethynyl)pyrazolo[1,5-a]pyridine-3-carbonitrile (2):

[0253]

[0254] 3-Cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl trifluoromethanesulfonate (V-1, 1.11 g, 2.99 mmol), tert-butyl 4-(4-ethynyl-5-methylpyridin-2-yl)piperazine-1-carboxylic acid butyl ester (XVII-1, 0.90 g, 2.99 mmol), copper iodide (57 mg, 0.30 mmol) and N,N-diisopropylethylamine (772 mg, 5.99 mmol) were suspended in N,N-dimethylacetamide (15 mL), and bis(triphenylphosphine)palladium(II) chloride (210 mg, 0.30 mmol) was added. After stirring at 100°C under nitrogen for 5 hours, the mixture was cooled and poured into water (50 mL). Dichloromethane (50 mL×3) was added to extract the desired compound. The combined organic layers were washed with water (20 mL), brine (20 mL), and Na 2 SO 4 Drying and concentration gave a residue. The residue was triturated with petroleum ether (30 mL) and filtered to give a crude product (1.1 g, crude). The crude product was further triturated with methanol (5 mL) and filtered to give Key Intermediate-2 (920 mg, yield 59%) as a yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ:8.63(d,J=1.2Hz,1H),8.29(s,1H),8.10(s,1H),7.80(s,1H),7.71(d,J=1.2 Hz,2H),7.21(s,1H),4.00(s,3H),3.54-3.52(m,8H),2.41(s,3H),1.49(s,9H).

[0255] Trifluoroacetic acid (5 mL) was added to a mixture of Key Intermediate-2 (900 mg, 1.72 mmol) in dichloromethane (20 mL). After stirring at room temperature for 1 hour, the mixture was concentrated to obtain a residue. The residue was diluted with dichloromethane (100 ml) and washed with a saturated aqueous solution of sodium bicarbonate (50 mL). The aqueous layer was extracted with dichloromethane (50 mL × 2). The combined organic phases were concentrated to obtain a residue. This residue was dissolved in (dichloromethane: methanol = 2: 1, 100 mL). Aqueous formaldehyde solution (37%, 0.5 mL) was added. After stirring at room temperature for 30 minutes, sodium cyanoborohydride (325 mg, 5.16 mmol) was added in batches. The mixture was stirred at room temperature overnight. The mixture was poured into water (30 mL) and extracted with dichloromethane (100 mL × 2). The combined organic phases were concentrated to obtain a residue. The residue was triturated with methanol (5 mL) and filtered to give the title compound (620 mg, yield 83%), a yellow solid with a purity of 95%. The product was recrystallized from (chloroform: methanol = 1: 1, 15 mL) to give the title compound 2 (500 mg, yield 67%), a yellow solid with a purity of 96%. The product was recrystallized from (chloroform: methanol = 3: 1, 15 mL) to give a yellow solid with a purity of 97.3% (230 mg, yield 31%). LC-MS [mobile phase: from 80% water (0.02% NH 4 OAc)and 20% CH 3 CN to 30%water(0.02% NH 4 OAc)and 70% CH 3 CN in 6.5min], Rt=3.552min; Purity: 97.35% (214nm), 97.28% (254nm); MS Calcd: 436.2; MS Found: 437.2[M+H]+. 1 H NMR (400 MHz, CDCl 3 )δ:8.63(d,J=1.2Hz,1H),8.28(s,1H),8.10(s,1H),7.80(s,1H),7.71(s,2H),7.20(s,1 H),4.00(s,3H),3.56(t,J=5.2Hz,4H),2.53(t,J=5.2Hz,4H),2.40(s,3H),2.34(s,3H).

[0256] Example 3-183

[0257] According to the method of Example 1 or Example 2, a wide variety of derivatives can be synthesized by simply changing the appropriate starting materials and the corresponding intermediates. Examples 3-183 are some representative exemplary compounds (see Table 1).

[0258] Table 1 Representative compounds of Formula I

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302] In addition, referring to the method of Example 1 or Example 2, as long as the starting raw materials are appropriately selected, a wider variety of derivatives can be synthesized. For example, the compounds listed in Table 2 are some typical example compounds.

[0303] Table 2 Typical compounds of formula I

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314] Example 241 Biological Experiment and Pharmacodynamic Analysis

[0315] 1. Detection of kinase activity

[0316] There are many literature reports on the test of kinase activity, and there are also related kinase detection kits available. Products from Cisbio can be selected but are not limited to: HTRF kinEASE-STK KIT. Taking the detection of NTRK kinase inhibitory activity using the HTRF (homogeneous time-resolved fluorescence) kinase detection kit as an example, the experimental operation steps are as follows:

[0317] 1. Experimental methods and steps:

[0318] 1.1 Prepare the test compound into a 10 mM (mmol / L) DMSO solution.

[0319] 1.2 The 10mM compound was diluted with kinase buffer to 2.5μM (2.5× compound) working solution, and then 2.5μM was used as the highest concentration, and the 2.5× working solution of the test compound was diluted to 9 concentrations in a 3-fold gradient, and the concentrations were: 2.5, 0.833333, 0.277778, 0.092593, 0.030864, ​​0.010288, 0.003429, 0.001143, 0.000381μM; the 10mM control compound was diluted with kinase buffer to 2.5μM (2.5× compound) working solution, and then 2.5μM was used as the highest concentration, and the working solution was diluted to 9 concentrations in a 3-fold gradient ... The buffer was diluted to a 2.5 μM working solution, and then 2.5 μM was used as the highest concentration and diluted in a 3-fold gradient to continuously dilute the 2.5× working solution of the test compound to 9 concentrations, namely 2.5, 0.833333, 0.277778, 0.092593, 0.030864, ​​0.010288, 0.003429, 0.001143, and 0.000381 μM.

[0320] 1.3 Take 4 μL of 2.5× compound working solution and add it to a 384-well plate (Greiner, Cat#781280), set up Blank wells (no compound and kinase) and Control wells (only kinase without compound), and add 4 μL kinase buffer to the Blank and Control wells.

[0321] 1.4 Prepare the kinase storage solution into the corresponding 5× working solution with kinase buffer, take 2μL of 5× kinase working solution and add it to each well containing compound working solution, add 2μL kinase buffer to the Blank well, and add 2μL 5× kinase working solution to the Control well.

[0322] 1.5 Take 2 μL of 5× substrate storage solution (TK Antibody-Cryptate) and add it to each well containing compound and kinase working solution, add 2 μL kinase buffer to the Blank well, and add 2 μL 5× substrate storage solution to the Control well.

[0323] 1.6 Take 2 μL of ATP working solution (5×) and add it to each detection well.

[0324] 1.7 Cover the 384-well plate with a sealing film and incubate at 37°C for 1 hour, then add 5 μL (4X) of reaction stop solution (Streptavidin-XL665) to each well;

[0325] 1.8 Continue to seal the 384-well plate, incubate at 37°C for 1 hour, and then detect the 665 and 620 signal values ​​on the 2104 EnVision plate reader.

[0326] Preparation of detection system working solution:

[0327]

[0328]

[0329] 2. Data Analysis:

[0330] The inhibition rate (IR) of the test compound was calculated using the following formula: IR (%) = (RLUCTR (665 / 620) – RLU compound (665 / 620)) / (RLU CTR (665 / 620) – RLU BLANK (665 / 620)) * 100%. The inhibition rates of different concentrations of the compound were calculated in Excel, and then the IC was calculated using GraphPad Prism 5 software. 50 .

[0331] 3. Activity inhibition test results of TRKa, TRKb, and TRKc kinases

[0332] Some biological activity test results are shown in Table 3

[0333] Table 3 Inhibitory activity of compounds on kinases

[0334] compound <![CDATA[TRKa IC 50 (μM)]]> <![CDATA[TRKb IC 50 (μM)]]> <![CDATA[TRKc IC 50 (μM)]]> 1 0.098 0.019 0.155 2 0.049 0.010 0.341 3 0.020 0.014 0.784

[0335] Similarly, other compounds were also tested to inhibit the activity of TRKa, TRKb, and TRKc kinases, showing good inhibitory effects.

[0336] At the same time, the compounds of the present invention have obvious inhibitory effects on other kinases, such as TRK, RET, RAF, FGHR, PDGFR, VEGFR and other kinases.

[0337] Example 242 Tumor cell inhibitory activity GI 50 Determination of value

[0338] The cell activity test uses CTG (CELL TITER-GLO) luminescence method to test the activity of the target compound. The principle is: ATP adenine nucleoside triphosphate (ATP for short) participates in a variety of enzymatic reactions in the body and is an indicator of living cell metabolism. Its content directly reflects the number and state of cells. During the experiment, an equal volume of CellTiter-Glo was added to the cell culture medium. TMReagents are used to measure the luminescence value. In the light signal and system, the luminescence value is proportional to the amount of ATP, which is positively correlated with the number of living cells and inversely proportional to the activity of anti-tumor drugs. By detecting the fluorescence signal of ATP, the anti-proliferation activity data of the compound on tumor cells can be obtained according to the calculation formula.

[0339] The test of tumor cell inhibitory activity was carried out in two parts: using kinase as the target, the target compound was examined for its effect on human thyroid ductal carcinoma cells (TT) and human colon cancer cells (KM12); in order to determine its activity on NTRK Fusion, the growth inhibitory activity of the target compound on NTRK fusion cells was specifically tested.

[0340] Reagents used in the experiment: F-12K basal medium (ATCC, 30-2004), fetal bovine serum (Corning, 35-076-CV), double antibody (GIBCO, 15240-062), trypsin (GIBCO, 25200072), DMSO (SIGMA, D2650), DMEM basal medium (Corning, 10-013-CV), fetal bovine serum (Gibco, 10091-148).

[0341] 1. Activity determination of compounds on human thyroid ductal carcinoma cells and human colon cancer cells

[0342] The following method is used to determine the effect of compounds on tumor cell proliferation by using the CTG luminescence method.

[0343] Specific experimental operation methods and processes:

[0344] 1.1. Cell recovery:

[0345] Immediately place the frozen cells from the liquid nitrogen storage tank in a 37°C constant temperature water bath and shake for 2 minutes. After the cell freezing solution is completely thawed, transfer the cell suspension into a 15mL centrifuge tube, slowly add 4mL of culture solution, centrifuge (1000r / min, 5min), discard the supernatant, aspirate the original solution, add 5mL of the above culture medium, gently blow to a single cell suspension, transfer it to a culture bottle, and place it in an incubator for culture.

[0346] 1.2 Cell culture:

[0347] The cells were incubated with complete medium at 37°C, 5% CO 2 The cells were cultured in an incubator at 4 °C. The cells were subcultured regularly and the cells in the logarithmic growth phase were used for plating.

[0348] 1.3. Cell plating:

[0349] The cells were stained with trypan blue and the living cells were counted. The cell concentration was adjusted to the appropriate medium plate concentration (TT: 50000 cell / mL, KM12: 35000 cell / mL). 90 μL of cell suspension was added to each well of a 96-well culture plate (Corning, 3599), and blank control wells and solvent control wells were set up. The culture medium containing cells was added to the blank control wells, and the culture medium without cells was added to the solvent control wells. The culture plate was then placed at 37°C and 5% CO 2 , and cultured in an incubator at 100% relative humidity overnight.

[0350] 1.4. Compound preparation:

[0351] Weigh the compound and prepare a 10mM stock solution with DMSO. Dilute the stock solution of the compound to be tested with serum-free medium to a final concentration of 100μM 10× compound working solution (including reference substance) on a dispensing plate (Beaver, Suzhou). Dilute with serum-free medium in a 3-fold concentration gradient to obtain 9 concentration gradients of 10× compound working solutions, with compound concentrations of 100, 33.33, 11.11, 3.70, 1.23, 0.411, 0.137, 0.046, and 0.015μM, respectively.

[0352] 1.5. Addition of Compounds

[0353] Add 10× compound working solution of different concentration gradients to the 96-well cell culture plate, 10 μL / well, add 10 μL DMSO-cell culture solution mixture to the solvent control well and blank control well, the final DMSO concentration is 0.1%, and set 2 replicates for each concentration. Return the 96-well cell plate to the 37°C, 5% carbon dioxide incubator for 5 days.

[0354] 1.6. CTG test:

[0355] The cell culture plate was taken out and placed for 30 minutes to equilibrate to room temperature, 50 μL (equal to half the volume of the cell culture medium in each well) of CellTiter-Glo working solution was added to each well, the cell plate was wrapped with aluminum foil to avoid light, and the culture plate was shaken on an orbital shaker for 2 minutes to induce cell lysis. The culture plate was placed at room temperature for 10 minutes to stabilize the luminescent signal, and the luminescent signal was detected on a 2104 EnVision plate reader.

[0356] 1.7 Data Analysis

[0357] The inhibition rate (IR) of the test compound was calculated using the following formula: IR (%) = (1-(RLU compound-RLU blank control) / (RLU solvent control-RLU blank control))*100%. Finally, nonlinear regression analysis was performed using the logarithm of compound concentration-inhibition rate in Graphpadprism 5 software to obtain the IC value of the compound for inhibiting cell proliferation. 50 value.

[0358] Results The test results are shown in Table 4.

[0359] Table 4 Results of the inhibitory activity test on TT cells and KM12 cells obtained by the compounds

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366] Example 243 Activity determination of compound on fusion tumor cell line

[0367] In addition to using relevant tumor cell lines, NTRK fusion engineered cell lines were also used to specifically detect the activity of the target compound on NTRK Fusion. The following method is used to determine the effect of the compound on the proliferation of NTRK fusion engineered cells, which is determined by using the CTG luminescence method. For NTRK fusion, the BaF3 ETV6-NTRK3 fusion engineered cell line (constructed by Precedo) was used. Culture according to the corresponding conditions.

[0368] Reagents used in the experiment: RPMI-1640 basal medium (GIBCO, 22400-089), fetal bovine serum (SH30084.03, SH30084.03), double antibody (GIBCO, 15240-062), trypsin (GIBCO, 25200072), DMSO (SIGMA, D2650).

[0369] The experimental operation method is as follows:

[0370] 2.1. Cell recovery:

[0371] Immediately place the frozen cells from the liquid nitrogen storage tank in a 37°C constant temperature water bath and shake for 2 minutes. After the cell freezing solution is completely thawed, transfer the cell suspension into a 15mL centrifuge tube, slowly add 4mL of culture solution, centrifuge (1000r / min, 5min), discard the supernatant, aspirate the original solution, add 5mL of the above culture medium, gently blow to a single cell suspension, transfer it to a culture bottle, and place it in an incubator for culture.

[0372] 2.2 Cell culture:

[0373] Ba / F3-ETV6-NTRK3 engineered cells were cultured in complete medium (RPMI-1640 + 10% FBS + 1% P / S) at 37°C, 5% CO 2 The cells were cultured in an incubator at 4 °C. The cells were subcultured regularly and the cells in the logarithmic growth phase were used for plating.

[0374] 2.3. Cell plating:

[0375] The cells were stained with trypan blue and the living cells were counted. The cell concentration was adjusted to 30,000 cells / mL of cell solution. 90 μL of cell suspension was added to each well of a 96-well culture plate (Corning, 3599). A blank control well and a solvent control well were set up. The culture solution containing cells was added to the blank control well, and the culture solution without cells was added to the solvent control well. The culture plate was then placed at 37°C and 5% CO 2 , and cultured in an incubator at 100% relative humidity overnight.

[0376] 2.4. Compound preparation:

[0377] Weigh the compound and prepare a 10mM stock solution with DMSO. Dilute the stock solution of the compound to be tested with serum-free medium to a final concentration of 100μM 10× compound working solution (including reference substance) on a dispensing plate (Beaver, Suzhou). Dilute with serum-free medium in a 3-fold concentration gradient to obtain 9 concentration gradients of 10× compound working solutions, with compound concentrations of 100, 33.33, 11.11, 3.70, 1.23, 0.411, 0.137, 0.046, and 0.015μM, respectively.

[0378] 2.5. Addition of compounds:

[0379] Add 10× compound working solution of different concentration gradients to the 96-well cell culture plate, 10 μL / well, add 10 μL DMSO-cell culture solution mixture to the solvent control well and blank control well, the final DMSO concentration is 0.1%, and set 2 replicates for each concentration. Return the 96-well cell plate to the 37°C, 5% carbon dioxide incubator for 5 days.

[0380] 2.6. CTG test:

[0381] The cell culture plate was removed and placed for 30 minutes to equilibrate to room temperature. 50 μL (equal to half the volume of the cell culture medium in each well) of CellTiter-Glo working solution was added to each well. The cell plate was wrapped with aluminum foil to avoid light. The culture plate was shaken on an orbital shaker for 2 minutes to induce cell lysis. The culture plate was placed at room temperature for 10 minutes to stabilize the luminescent signal, and the luminescent signal was detected on a 2104En Vision plate reader.

[0382] 2.7 Data Analysis

[0383] The inhibition rate (IR) of the test compound was calculated using the following formula: IR (%) = (1-(RLU compound-RLU blank control) / (RLU solvent control-RLU blank control))*100%. Finally, the IC of the compound for inhibiting cell proliferation was obtained by nonlinear regression analysis using the logarithm of compound concentration-inhibition rate in Graphpadprism5 software. 50 value.

[0384] Results The test results are shown in Table 5.

[0385] Table 5 Results of the inhibitory activity test on the growth of fusion tumor cells obtained by the obtained compounds

[0386]

[0387]

[0388] The results showed that the target compound had significant inhibitory activity against NTRK Fusion. Data for the remaining compounds were not provided, but they all had significant inhibitory effects.

[0389] Example 244 In vivo antitumor activity of the compounds of the present invention:

[0390] Some compounds with strong in vitro activity and low toxicity were selected to determine the maximum tolerated dose (MTD) in mice. The anti-tumor activity of the compounds of the present invention in vivo was determined on a human cancer nude mouse xenograft tumor model to explore the dosage, route of administration, frequency and cycle of administration of the test compounds to produce pharmacodynamic effects.

[0391] Female BALB / C nude mice aged 5-6 weeks, weighing approximately 18-20 g, were raised.

[0392] The efficacy of the subcutaneous cell transplanted tumors was studied and evaluated in the BALB / c nude mouse model.

[0393] Construction of human cancer nude mouse xenograft tumor model: TT (human thyroid cancer cell) culture, monolayer cultured tumor cells were digested and detached from the wall, collected and resuspended in serum-free culture medium, and adjusted to a concentration of 5×10 6 / 0.2mL, put it in an ice box and bring it to the animal room. Use a syringe with a No. 6 needle to directly take 0.2mL of cell suspension and transplant it subcutaneously in the scapula behind the left armpit of nude mice. 5×10 6 / 0.2mL / mouse, measure the tumor volume every 2-3 days, select nude mice with vigorous tumor growth and no ulceration, remove the tumor under sterile conditions, cut the tumor tissue into pieces with a diameter of about 2-3mm, and inoculate it subcutaneously at the scapula behind the left armpit of the nude mouse. After three generations, when the tumor volume grows to 100-150mm 3 Nude mice with tumors that were too large or too small were removed and randomly divided into groups for drug administration.

[0394] Randomly divided into 5 groups, including negative control group (solvent), positive control group (Blu-667, 10 mg / kg), high, medium and low dose treatment groups (5 mg / kg, 10 mg / kg, 40 mg / kg, respectively, of which the high dose is lower than MTD), 5 nude mice in each group, intraperitoneal injection, twice a day, for 3 consecutive weeks. During this period, the animal weight, tumor volume and animal death were detected every 3 days. The animals were killed 24 hours after the last administration, and the tumor volume, tumor weight and nude mouse weight were measured. The tumor volume growth curve, nude mouse weight growth curve and tumor inhibition rate, animal mortality rate were drawn, and the relative tumor proliferation rate T / C (%) was calculated according to the formula T / C (%) = TRTV / CRTV*100%. (TRTV: treatment group RTV; CRTV: negative control group RTV, relative tumor volume RTV = Vt / V0, where V0 is the tumor volume at the time of group administration, and Vt is the tumor volume after administration). The in vivo anti-tumor efficacy of the compounds of the present invention is that the relative tumor proliferation rate T / C (%) is ≤40%, and the TGI% is greater than 80% (see: Figure 1 The in vivo antitumor activity of compound 171 in the TT model was statistically significant, with obvious pharmacodynamic effects and good in vivo tumor inhibition. At the same time, the body weight of animals in different dose groups of compound 171 of the present invention did not decrease significantly, showing good tolerance.

[0395] In this experiment, the in vivo efficacy of compound 171 on TT cell xenograft tumor model was evaluated, and Blu-667 was used as a control. The experimental results showed that compound 171 was administered orally at a dose of 30 mg / kg, with a T / C ratio of less than 40%, showing a good in vivo tumor inhibition effect. Similarly, other compounds also had similar in vivo tumor inhibition effects.

[0396] In addition to using relevant tumor cell lines, relevant engineered strains, such as KIF5B-RET fusion tumor cell lines, should also be used for in vivo anti-tumor inhibitory activity.

[0397] The details of the specific examples described in the present invention are not intended to be construed as limiting thereof. Various changes, synonyms and modifications may be made without departing from the essence and scope of the present invention, and it is known that the specific embodiments of these changes, synonyms and modifications are part of the present invention.

Claims

1. A pyrazolo[1,5-a]pyridine derivative, characterized in that: The pyrazolo[1,5-a]pyridine derivative is a compound represented by the general formula (I), and an optical isomer thereof or a pharmaceutically acceptable salt thereof: In the above general formula (I), R 1 is cyano; L 1 Selected from: -CH=CH-, -C≡C-; L 2 Selected from: covalent bond, C1-C6 alkylene, pyridine, Carbonyl, -C(O)-NH-, -C1-C6 alkyl-C(O)-NH-, -C1-C6 alkyl-NH-C(O)-; L 3 for: R 2 Selected from: 6-12 membered aryl C1-C6 alkyl, 3-9 membered cycloalkyl, 6-12 membered aryl, 5-7 membered heteroaryl, 4-15 membered heteroaryl C1-C6 alkyl, 3-14 membered heterocycloalkyl; any of the above groups are independently unsubstituted or substituted by one or more substituents selected from halogen, amino, -CF3, C1-C6 haloalkyl, C1-C6 alkyl, hydroxy, C1-C6 alkoxy, 3-9 membered cycloalkyl, 6-12 membered aryl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C6 alkyl, 3-14 membered heterocycloalkyl, C1-C6 alkylaminocarbonyl, sulfonyl, C1-C6 alkylsulfonyl, C1-C6 alkylsulfinyl or aminosulfonyl; R 3 Selected from C1-C5 alkyl; any of the above groups are independently unsubstituted or substituted by one or more substituents, and these substituents are halogen.

2. A pyrazolo[1,5-a]pyridine derivative, characterized in that: The pyrazolo[1,5-a]pyridine derivative is a compound represented by the general formula (I), and an optical isomer thereof or a pharmaceutically acceptable salt thereof: In the above general formula (I), R 1 is cyano; L 1 Selected from: -CH=CH-, -C≡C-; L 2 Selected from: covalent bond, C1-C6 alkylene, pyridine, Carbonyl, -C(O)-NH-, -C1-C6 alkyl-C(O)-NH-, -C1-C6 alkyl-NH-C(O)-; L 3 for: R 2 Selected from: Amino, Benzene ring, substituted benzene ring, methylpyridine, CO-NH-R 7 , -CH2-CN, -NH-OH、 R 5 Selected from hydrogen, R 6 Selected from hydrogen, Benzene ring, substituted benzene ring, pyrimidine; The substituted benzene ring contains 1-3 substituents, and the substituents of the substituted benzene ring are selected from amino, C1-C5 alkyl, halogen, trifluoromethyl, trichloromethyl, tribromomethyl, trifluoroethyl, trifluoropropyl, Methyl ether, ethyl ether, propyl ether, Trifluoromethyl ether, trifluoroethyl ether, trifluoropropyl ether; R 7 Selected from C1-C8 alkyl; R 3 Selected from C1-C5 alkyl; any of the above groups are independently unsubstituted or substituted by one or more substituents, and these substituents are halogen.

3. A pyrazolo[1,5-a]pyridine derivative, the structure of which is selected from one of the following structures, or an optical isomer or a pharmaceutically acceptable salt thereof:

4. The method for preparing a pyrazolo[1,5-a]pyridine derivative according to any one of claims 1 to 3, characterized in that: L 2 =-CO-NH-, the preparation method comprises: S1, select I as a raw material, remove the methyl group under the action of a catalyst to obtain compound II; S2. Under catalysis, compound II reacts with boron compound III to obtain IV; S3. Under the action of N-phenylbis(trifluoromethanesulfonimide), compound IV is converted into compound V; S4, compound VIII obtained by condensation of compound VI and compound VII; S5. Compound VIII reacts with compound V to obtain compound IX, which is the target compound represented by general formula (I); 5. The method for preparing a pyrazolo[1,5-a]pyridine derivative according to claim 4, characterized in that: When compound I is: hour; The synthesis method of compound I comprises: A and X are reacted as raw materials with XⅠ to obtain XⅡ; XⅡ and XⅢ react to obtain XIV; B. Under the action of acid, XIV and XV are cyclized to obtain compound XVI; C. Introducing a formyl group into compound XVI to obtain XVII, and then hydroxylating XVII to generate compound XVIII. XVIII is then reacted under the action of a catalyst to obtain compound I; 6. A pharmaceutical composition comprising the pyrazolo[1,5-a]pyridine derivative according to any one of claims 1 to 3, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition may also include another drug or multiple drugs for combined therapy.

8. Use of the pyrazolo[1,5-a]pyridine derivative according to any one of claims 1 to 3 or its optical isomer or a pharmaceutically acceptable salt thereof in inhibiting the activity of TRK kinase.

9. Use of the pyrazolo[1,5-a]pyridine derivative according to any one of claims 1 to 3 or its optical isomer or its pharmaceutically acceptable salt in the preparation of a drug for treating cancer.

10. The use according to claim 9, characterized in that The cancer is selected from the group consisting of non-small cell lung cancer, renal cell carcinoma, gastric cancer, hepatocellular carcinoma, colorectal cancer, medullary thyroid cancer, follicular thyroid cancer, anaplastic thyroid cancer, papillary thyroid cancer, brain tumors, peritoneal cavity cancer, head and neck cancer, glioma, neuroblastoma, Von Hipple-Lindau syndrome, breast cancer, fallopian tube cancer, ovarian cancer, prostate cancer, cancer of the esophagus and esophagogastric junction, and biliary tract cancer.

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