CN109983015B

CN109983015B - 6-pyrazole- [1,2,4] triazolo [4,3-a ] pyridine-3-amide derivatives, preparation method and medical application thereof

Info

Publication number: CN109983015B
Application number: CN201880004354.XA
Authority: CN
Inventors: 杨方龙; 张羚; 贺峰; 陶维康
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2017-03-22
Filing date: 2018-03-21
Publication date: 2021-09-03
Anticipated expiration: 2038-03-21
Also published as: WO2018171611A1; TW201835079A; CN109983015A

Abstract

6-pyrazole- [1,2,4] of the general formula (I)]Triazolo [4,3-a]Pyridine-3-amide derivatives, preparation method thereof, pharmaceutical compositions containing the derivatives, and application of the derivatives as therapeutic agents, especially as TGF-beta receptor kinase inhibitors and in preparation of drugs for treating, preventing or reducing tumors mediated by TGF-beta overexpression.

Description

6-pyrazole- [1,2,4] triazolo [4,3-a ] pyridine-3-amide derivatives, preparation method and medical application thereof

Technical Field

The invention belongs to the field of medicines, and relates to a novel 6-pyrazole- [1,2,4] triazolo [4,3-a ] pyridine-3-amide derivative, a preparation method thereof, a pharmaceutical composition containing the derivative, application of the derivative as a therapeutic agent, especially as an inhibitor of TGF-beta receptor kinase, and application of the derivative in preparing a medicament for treating, preventing or reducing tumors mediated by TGF-beta overexpression.

Background

Transforming Growth Factor TGF- β (Transforming Growth Factor β) is a member of the dimeric polypeptide Growth Factor superfamily, which includes, for example, activins, inhibins, Bone Morphogenetic Proteins (BMPs), Growth and Differentiation Factors (GDFs) and Mullerian-inhibiting substructures (MIS).

TGF-beta has three subtypes, TGF-beta 1, TGF-beta 2 and TGF-beta 3, which are involved in regulation of cell proliferation and differentiation, wound healing, extracellular matrix production and immunosuppression. See, e.g., Massague, j.ann.rev, cell.biol.6: 594 (1990); roberts, a.b. peptide Growth Factor and theory receptors, 95: 419-472 Berlin: Springer-Verlag (1990); roberts, a.b. and Sporn m.b. growth Factor 8: 1-9 (1993); and Alexandrow, m.g., Moses, h.l.cancer res.55: 1452-1457(1995). Three subtypes of TGF-. beta.are present in most cells along with their receptors. Each TGF-. beta.subtype is synthesized as a precursor protein that is cleaved intracellularly into the C-terminal region (LAP) and the N-terminal portion, referred to as mature or active TGF-. beta.s. LAP is typically linked to mature TGF-. beta.in a non-covalent manner prior to secretion from the cell. The LAP-TGF-beta complex is unable to bind to the TGF-beta receptor and is biologically inactive. TGF-. beta.s are typically released (and active) from the complex by a variety of mechanisms including, for example, interaction with thrombospondin-1 or plasmin. TGF-. beta.1 transduces signals through two highly conserved single transmembrane serine/threonine kinases, the type I (ALK5) and the type II TGF-. beta.receptor. Once ligand-induced oligomerization, type II receptors hyperphosphorylate serine/threonine residues in the ALK5GS region, leading to activation of ALK5 by creating a binding site for Smad proteins. The activated ALK5 in turn phosphorylates Smad2 and Smad3 proteins at the C-terminal SSXS-motif, causing them to dissociate from the receptor and form heterocomplexes with Smad4 (heterocomplex). Smad complexes are readily localized to the nucleus, assemble with specific DNA-binding cofactors and coregulators, and ultimately activate transcription of extracellular matrix components and matrix-degrading protease inhibitors.

Hyperactive TGF- β signaling pathway is responsible for many human diseases such as excessive deposition of extracellular matrix, abnormally high levels of inflammatory response, fibrotic disorders, and progressive cancer. In the advanced stages of various tumors, tumor cells and stromal cells within the tumor typically overexpress TGF-. beta.s. This causes stimulation of angiogenesis and cellular motility, suppression of the immune system, and increased interaction of tumor cells with the extracellular matrix (e.g., Hojo, M. et al, Nature 397: 530-. Therefore, tumor cells become more aggressive, metastasizing to distal organs such as, for example, Maehara, y, et al, j.clin.oncol.17: 607-614 (1999); picon, a. et al, Cancer epidemol. biomakers prev.7: 497-504(1998)).

A number of experimental animal studies have demonstrated associations between TGF-. beta.glomerular expression and fibrosis, including the Thy-1 rat model of proliferative glomerulonephritis, the rabbit anti-GBM glomerulonephritis and the 5/6 nephrectomized rat model of focal segmental glomerulosclerosis, recently reviewed (e.g., Bitzer, M. et al, Kidney Blood Press. Res.21: 1-12 (1998)). Neutralizing antibodies to TGF-. beta.improve glomerular histology in a model of Thy-1 nephritis (e.g., Border, W.A. et al, Nature 346: 371-374 (1990)).

TGF-. beta.1 and its receptors are overexpressed in damaged blood vessels and fibroproliferative vascular injury, causing overproduction of extracellular matrix (e.g., Saltis, J. et al, Clin. Exp. Pharmacol. physiol.23: 193- "200 (1996); McCaffrey, T.A. et al, J.Clin. invest.96: 2667-" 2675 (1995)).

TGF-. beta.2 levels are increased in most aqueous humor tumor eyes with juvenile glaucoma and in almost half of eyes with Primary Open Angle Glaucoma (POAG) (e.g., Picht, G., et al, Graefes Arch. Clin. exp. Ophthalmol. 239: 199- "207 (2001)).

It would therefore be desirable to develop inhibitors of TGF- β family members to prevent and/or treat diseases that include such signaling pathways. Published patent applications for modulators (e.g., antagonists) of TGF- β family member receptors include WO2004111046, WO2012000595, WO2012002680, WO2013009140, WO 2016106266.

In order to achieve the purpose of better treatment effect and better meet the market demand, the invention provides a TGF-beta receptor kinase inhibitor with a novel structure, which is highly efficient and low in toxicity, and finds that the compound with the structure has the characteristic of good pharmacokinetic property by introducing an amide group.

Disclosure of Invention

The invention aims to provide a compound shown in a general formula (I) or a tautomer, a meso form, a racemate, an enantiomer, a diastereomer or a mixture form thereof or a pharmaceutically acceptable salt form thereof,

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof,

wherein:

ring a is aryl or heteroaryl;

R¹selected from the group consisting of hydrogen atoms, alkyl groups, haloalkyl groups, hydroxyl groups, hydroxyalkyl groups, amino groups, cycloalkyl groups, heterocyclyl groups, aryl groups, heteroaryl groups, -C (O) OR⁶、-C(O)R⁶、-S(O)_mR⁶、-NR⁷R⁸、-S(O)_mNR⁷R⁸and-C (O) NR⁷R⁸Wherein said alkyl, cycloalkyl, or a substituted cycloalkyl,Heterocyclyl, aryl and heteroaryl are each independently optionally selected from halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) OR⁶、-C(O)R⁶、-S(O)_mR⁶、-NR⁷R⁸、-S(O)_mNR⁷R⁸and-C (O) NR⁷R⁸Is substituted with one or more substituents of (1);

R²are the same OR different and are each independently selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) OR⁶、-C(O)R⁶、-S(O)_mR⁶、-NR⁷R⁸、-S(O)_mNR⁷R⁸and-C (O) NR⁷R⁸；

R³The same or different, and each is independently selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl;

R⁴and R⁵Each independently selected from the group consisting of hydrogen atoms, alkyl groups, haloalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups, wherein said alkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups are each independently optionally substituted with one or more substituents selected from the group consisting of alkyl groups, alkoxy groups, halogens, amino groups, cyano groups, nitro groups, hydroxyl groups, hydroxyalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups;

or, said R⁴And R⁵Forming a heterocyclic group together with a linking nitrogen atom, wherein the heterocyclic group contains 1-2 same or different heteroatoms selected from N, O and S, and the heterocyclic group is optionally substituted with one or more substituents selected from alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;

R⁶selected from hydrogen atom, alkyl, amino, halogenated alkyl, cycloalkyl,Heterocyclyl, aryl and heteroaryl;

R⁷and R⁸Each independently selected from the group consisting of hydrogen atoms, alkyl groups, haloalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups, wherein said alkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups are each independently optionally substituted with one or more substituents selected from the group consisting of alkyl groups, alkoxy groups, halogens, amino groups, cyano groups, nitro groups, hydroxyl groups, hydroxyalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups;

or, said R⁷And R⁸Forming a heterocyclic group together with a linking nitrogen atom, wherein the heterocyclic group contains 1-2 same or different heteroatoms selected from N, O and S, and the heterocyclic group is optionally substituted with one or more substituents selected from alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;

n is 0, 1,2, 3 or 4;

s is 0, 1,2 or 3; and is

m is 0, 1 or 2.

In a preferred embodiment of the present invention, the compound of formula (I) wherein ring a is heteroaryl, preferably 5-or 6-membered heteroaryl, more preferably pyridyl.

In a preferred embodiment of the present invention, the compound represented by the general formula (I) is a compound represented by the general formula (II):

wherein: r¹～R⁵N and s are as defined in formula (I).

In a preferred embodiment of the present invention, the compound represented by the general formula (I) wherein R is⁴And R⁵Are all hydrogen atoms。

In a preferred embodiment of the present invention, the compound represented by the general formula (I) is a compound represented by the general formula (III):

wherein: r¹And R²As defined in formula (I).

In a preferred embodiment of the present invention, the compound represented by the general formula (I) wherein R is¹Selected from the group consisting of hydrogen atom, alkyl group, cycloalkyl group and heterocyclic group, preferably hydrogen atom, C_1-6An alkyl group, a 3-to 6-membered cycloalkyl group, a 3-to 6-membered heterocyclic group, more preferably a hydrogen atom, a methyl group, an ethyl group, an isopropyl group, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a tetrahydrofuryl group or a tetrahydropyranyl group.

In a preferred embodiment of the present invention, the compound represented by the general formula (I) wherein R is²Is a hydrogen atom or an alkyl group, preferably a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group or a butyl group.

In a preferred embodiment of the present invention, the compound represented by the general formula (I) wherein R is³Is a hydrogen atom.

The compounds of the present invention include all conformational isomers thereof, such as cis and trans isomers; and all optical isomers and stereoisomers thereof and mixtures thereof. The compounds of the invention have asymmetric centers and thus exist as different enantiomers and diastereomers. The present invention relates to the use of the compounds of the present invention, and all pharmaceutical compositions and methods of treatment which may be employed and which contain them. The present invention relates to the use of all such isomers and mixtures thereof.

Typical compounds of the invention include, but are not limited to:

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention relates to a method of preparing a compound of formula (I), the method comprising:

reacting a compound of formula (I-A) with a compound of formula (I-B) to give a compound of formula (I),

wherein:

w is a boronic acid group or a 4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl group;

x is halogen, preferably bromine;

ring A, R¹～R⁵N and s are as defined in formula (I).

reacting a compound of formula (I-Aa) with a compound of formula (I-Bb) to give a compound of formula (I),

wherein:

x is halogen, preferably bromine;

ring A, R¹～R⁵N and s are as defined in formula (I).

Another aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients. The invention also relates to a method for preparing the composition, which comprises mixing the compound shown in the general formula (I) or the tautomer, the mesomer, the racemate, the enantiomer, the diastereomer or the mixture form thereof, or the pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable carriers, diluents or excipients.

The invention further relates to a compound shown in the general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture form thereof, or a pharmaceutically acceptable salt or a pharmaceutical composition containing the compound, and application of the compound in preparing medicines for inhibiting TGF-beta (particularly human TGF-beta) signaling pathways.

The invention further relates to a compound shown in the general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture form thereof, or a pharmaceutically acceptable salt or a pharmaceutical composition containing the compound, and an application of the compound in preparing medicines for treating, preventing or reducing the metastasis of tumor cells (particularly human tumor cells).

The invention further relates to the use of a compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for the manufacture of a medicament for the treatment, prevention or reduction of tumors mediated by TGF- β overexpression, in particular for the treatment, prevention or reduction of tumors mediated by TGF- β overexpression by inhibiting the human TGF- β signaling pathway.

In the context of the present invention, said diseases to be treated, prevented or alleviated (in particular in humans) are selected from: cardiovascular diseases, various types of inflammation, tumors, fibrosis of various etiologies, vascular injury, renal diseases, liver disorders, lung diseases, adult respiratory distress syndrome, intimal thickening, ocular diseases, excessive or hypertrophic dermal scar or keloid formation occurring during wound healing caused by trauma or surgical wounds, peritoneal and subcutaneous adhesions, scleroderma, fibrosis, progressive systemic sclerosis, osteoporosis, ulcers, reduced nervous system function, male erectile dysfunction, pelmet's disease, dupuytren's contracture, alzheimer's disease, and raynaud's syndrome.

The invention further relates to the use of a compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same, for the preparation of a medicament for the treatment, prevention or alleviation, in particular of humans, of a disease as defined above.

The present invention further relates to a method for treating, preventing or reducing metastasis of human tumor cells, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same.

The present invention further relates to a method of treating, preventing or reducing tumors mediated by TGF- β overexpression, in particular by inhibiting the TGF- β signalling pathway, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same.

The invention further relates to a method for the treatment, prevention or alleviation, in particular of a human being, of a disease selected from the above, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising same.

The invention further relates to a method of inhibiting the TGF- β (in particular human TGF- β) signalling pathway comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same.

The invention further relates to a compound shown in the general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same, which acts as a medicament.

The invention further relates to a compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, which acts as a TGF-beta receptor kinase inhibitor, in particular a TGF-beta receptor I (TGF-beta RI) kinase inhibitor.

The invention further relates to a compound shown in the general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the compound, which is used for treating, preventing or reducing the metastasis of tumor cells (particularly human tumor cells).

The invention further relates to a compound shown in the general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the compound, which is used for treating, preventing or reducing tumors mediated by TGF-beta overexpression, particularly treating, preventing or reducing tumors mediated by TGF-beta overexpression by inhibiting a TGF-beta signaling pathway.

The invention further relates to a compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use in the treatment, prevention, or alleviation of the aforementioned diseases, in particular of humans.

The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Oral compositions may be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may contain one or more ingredients selected from the group consisting of: sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide a pleasant to the eye and palatable pharmaceutical preparation. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be inert excipients, granulating agents, disintegrating agents, binding agents, and lubricating agents. These tablets may be uncoated or they may be coated by known techniques which mask the taste of the drug or delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

Oral formulations may also be provided in soft gelatin capsules wherein the active ingredient is mixed with an inert solid diluent or wherein the active ingredient is mixed with a water soluble carrier or an oil vehicle.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending, dispersing or wetting agents. Aqueous suspensions may also contain one or more preservatives, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents.

Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, or in a mineral oil. The oil suspension may contain a thickener. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable preparation. These compositions can be preserved by the addition of antioxidants.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, or a mineral oil or a mixture thereof. Suitable emulsifiers may be naturally occurring phospholipids, and the emulsions may also contain sweetening, flavoring, preservative and antioxidant agents. Such formulations may also contain a demulcent, a preservative, a colorant and an antioxidant.

The pharmaceutical compositions of the present invention may be in the form of a sterile injectable aqueous solution. Among the acceptable vehicles or solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase, and the injection or microemulsion may be injected into the bloodstream of a patient by local mass injection. Alternatively, it may be desirable to administer the solutions and microemulsions in a manner that maintains a constant circulating concentration of the compounds of the present invention. To maintain such a constant concentration, a continuous intravenous delivery device may be used. An example of such a device is an intravenous pump model Deltec CADD-PLUS. TM.5400.

The pharmaceutical compositions of the present invention may be in the form of sterile injectable aqueous or oleaginous suspensions for intramuscular and subcutaneous administration. The suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally-acceptable, non-toxic diluent or solvent. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. Any blend fixed oil may be used for this purpose. In addition, fatty acids can also be prepared into injections.

The compounds of the present invention may be administered in the form of suppositories for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid in the rectum and therefore will melt in the rectum to release the drug.

As is well known to those skilled in the art, the dosage of a drug administered depends on a variety of factors, including, but not limited to: the activity of the particular compound employed, the age of the patient, the weight of the patient, the health condition of the patient, the behavior of the patient, the diet of the patient, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, and the like; in addition, the optimal treatment regimen, such as the mode of treatment, the daily amount of compound (I) of the formula or the type of pharmaceutically acceptable salt, can be verified according to conventional treatment protocols.

Detailed description of the invention

Unless stated to the contrary, terms used in the specification and claims have the following meanings.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-dimethylpentyl, 2, 4-dimethylpentyl, 2-dimethylpentyl, 3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2, 3-dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 2-dimethylhexyl, 3-dimethylhexyl, 4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-dimethylpentyl, 2-dimethylhexyl, 3-dimethylpentyl, 2-ethylhexyl, 3-dimethylhexyl, 2-ethylhexyl, 2-dimethylhexyl, 2-ethylhexyl, 2-dimethylhexyl, 2-dimethylhexyl, 2-dimethylhexyl, 2-ethylhexyl, 2-ethyl, 2-2, 2-2, 2-2, or, 2, 2-diethylpentyl, n-decyl, 3-diethylhexyl, 2-diethylhexyl, and various branched isomers thereof. More preferred are lower alkyl groups having 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropylPropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl and the like. Alkyl groups may be substituted OR unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, C (O) OR⁶、-C(O)R⁶、-S(O)_mR⁶、-NR⁷R⁸、-S(O)_mNR⁷R⁸and-C (O) NR⁷R⁸Is substituted with one or more substituents.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably from 3 to 10 carbon atoms, and most preferably from 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl and the like, with cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl being preferred; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups.

The term "spirocycloalkyl" refers to a 5 to 20 membered polycyclic group sharing one carbon atom (referred to as a spiro atom) between monocyclic rings, which may contain one or more double bonds, but none of the rings have a completely conjugated pi-electron system. Preferably 6 to 14, more preferably 7 to 10. Spirocycloalkyl groups are classified into a single spirocycloalkyl group, a double spirocycloalkyl group or a multi spirocycloalkyl group, preferably a single spirocycloalkyl group and a double spirocycloalkyl group, according to the number of spiro atoms shared between rings. More preferably 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered. Non-limiting examples of spirocycloalkyl groups include:

the term "fused cyclic alkyl" refers to a 5 to 20 membered all carbon polycyclic group in which each ring in the system shares an adjacent pair of carbon atoms with other rings in the system, wherein one or more of the rings may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system. Preferably 6 to 14, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, tetracyclic or polycyclic fused ring alkyls according to the number of constituent rings, preferably bicyclic or tricyclic, more preferably 5-or 6-membered bicycloalkyl. Non-limiting examples of fused ring alkyl groups include:

the term "bridged cycloalkyl" refers to a 5 to 20 membered all carbon polycyclic group in which any two rings share two carbon atoms not directly attached, which may contain one or more double bonds, but none of the rings have a completely conjugated pi-electron system. Preferably 6 to 14, more preferably 7 to 10. They may be classified as bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl groups, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic, depending on the number of constituent rings. Non-limiting examples of bridged cycloalkyl groups include:

the cycloalkyl ring may be fused to an aryl, heteroaryl or heterocycloalkyl ring, where the ring to which the parent structure is attached is cycloalkyl, non-limiting examples of which include indanyl, tetrahydronaphthyl, benzocycloheptanyl, and the like. Cycloalkyl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocydoalkylCycloalkoxy, cycloalkylthio, heterocycloalkylthio, C (O) OR⁶、-C(O)R⁶、-S(O)_mR⁶、-NR⁷R⁸、-S(O)_mNR⁷R⁸and-C (O) NR⁷R⁸Is substituted with one or more substituents.

The term "heterocyclyl" refers to a saturated and/or partially unsaturated mono-or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms wherein one or more of the ring atoms is selected from nitrogen, oxygen, or S (O)_m(wherein m is an integer from 0 to 2) but excludes the ring moiety of-O-O-, -O-S-, or-S-S-, the remaining ring atoms being carbon. Preferably 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, heterocyclyl contains 3 to 10 ring atoms, and most preferably heterocyclyl contains 3 to 6 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydropyranyl, pyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and

etc., preferably azetidinyl, oxetanyl, pyrrolyl and piperidinyl; polycyclic heterocyclic groups include spiro, fused and bridged heterocyclic groups.

The term "spiroheterocyclyl" refers to a 5-to 20-membered polycyclic heterocyclic group in which one atom (referred to as the spiro atom) is shared between monocyclic rings, and in which one or more ring atoms is selected from nitrogen, oxygen, or S (O)_m(wherein m is an integer of 0 to 2) and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a completely conjugated pi-electron system. Preferably 6 to 14, more preferably 7 to 10. The spiro heterocyclic group is classified into a mono-spiro heterocyclic group, a di-spiro heterocyclic group or a multi-spiro heterocyclic group according to the number of spiro atoms shared between rings, and preferably into a mono-spiro cycloalkyl group and a di-spiro cycloalkyl group. More preferred are 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered mono spiroheterocyclic groups. Non-limiting examples of spiro heterocyclic groups include:

the term "fused heterocyclyl" refers to a 5 to 20 membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system, one or more rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron system in which one or more ring atoms is selected from nitrogen, oxygen or S (O)_m(wherein m is an integer of 0 to 2) and the remaining ring atoms are carbon. Preferably 6 to 14, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups according to the number of constituent rings, preferably bicyclic or tricyclic, more preferably 5-or 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclic groups include:

the term "bridged heterocyclyl" refers to a 5 to 14 membered polycyclic heterocyclic group in which any two rings share two atoms not directly attached which may contain one or more double bonds, but none of the rings have a fully conjugated pi-electron system in which one or more of the ring atoms is selected from nitrogen, oxygen or S (O)_m(wherein m is an integer of 0 to 2) and the remaining ring atoms are carbon. Preferably 6 to 14, more preferably 7 to 10. 7 to 10 yuan. They may be classified into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic groups according to the number of constituent rings, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

the heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring to which the parent structure is attached is heterocyclyl, non-limiting examples of which include:

the heterocyclyl group may be optionally substituted OR unsubstituted, and when substituted, the substituents are preferably one OR more groups independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, C (O) OR⁶、-C(O)R⁶、-S(O)_mR⁶、-NR⁷R⁸、-S(O)_mNR⁷R⁸and-C (O) NR⁷R⁸Is substituted with one or more substituents.

The term "aryl" refers to a 6 to 14 membered all carbon monocyclic or fused polycyclic (i.e., rings which share adjacent pairs of carbon atoms) group having a conjugated pi-electron system, preferably 6 to 10 membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, non-limiting examples of which include:

aryl groups may be substituted OR unsubstituted, and when substituted, the substituents are preferably one OR more groups independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, C (O) OR⁶、-C(O)R⁶、-S(O)_mR⁶、-NR⁷R⁸、-S(O)_mNR⁷R⁸and-C (O) NR⁷R⁸Is substituted with one or more substituents.

The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl is preferably 5 to 10 membered, more preferably 5 or 6 membered, for example furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, pyrazolyl, tetrazolyl and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring joined together with the parent structure is a heteroaryl ring, non-limiting examples of which include:

heteroaryl groups may be optionally substituted OR unsubstituted, and when substituted, the substituents are preferably one OR more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, C (O) OR⁶、-C(O)R⁶、-S(O)_mR⁶、-NR⁷R⁸、-S(O)_mNR⁷R⁸and-C (O) NR⁷R⁸Is substituted with one or more substituents.

The term "alkoxy" refers to-O- (alkyl) and-O- (unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. Alkoxy may be optionally substituted OR unsubstituted, and when substituted, the substituents are preferably one OR more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, amino, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, C (O) OR⁶、-C(O)R⁶、-S(O)_mR⁶、-NR⁷R⁸、-S(O)_mNR⁷R⁸and-C (O) NR⁷R⁸Is substituted with one or more substituents.

The term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group, wherein alkyl is as defined above.

The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

The term "hydroxy" refers to an-OH group.

The term "mercapto" refers to the-SH group.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "amino" refers to the group-NH₂。

The term "cyano" refers to — CN.

The term "nitro" means-NO₂。

The term "oxo" refers to ═ O.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "a heterocyclic group optionally substituted with an alkyl" means that an alkyl may, but need not, be present, and the description includes the case where the heterocyclic group is substituted with an alkyl and the heterocyclic group is not substituted with an alkyl.

"substituted" means that one or more, preferably up to 5, more preferably 1 to 3, hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (experimentally or theoretically) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable in combination with carbon atoms having unsaturated (e.g., olefinic) bonds.

"pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof in admixture with other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

"pharmaceutically acceptable salts" refers to salts of the compounds of the present invention which are safe and effective for use in the body of a mammal and which possess the requisite biological activity.

m and R⁶～R⁸As defined in formula (I).

Synthesis of the Compounds of the invention

In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

scheme one

The invention relates to a method for preparing a compound shown as a general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereoisomer or a mixture form thereof, or a pharmaceutically acceptable salt form thereof, which comprises the following steps:

the first step, reacting the compound of the general formula (I-1) with a halogenating agent to obtain a compound of the general formula (I-A);

a second step of reacting the compound of the general formula (I-2) with the compound of the general formula (I-3) to obtain a compound of the general formula (I-4);

in the third step, a compound of the formula (I-4) and NHR⁴R⁵To obtain a compound of the general formula (I-Bb);

fourthly, reacting the compound of the general formula (I-Bb) with a borane compound under alkaline conditions in the presence of a catalyst to obtain a compound of the general formula (I-B);

fifthly, carrying out Suzuki reaction on the compound of the general formula (I-A) and the compound of the general formula (I-B) under alkaline conditions in the presence of a catalyst to obtain the compound of the general formula (I); r¹In the case of tetrahydropyranyl, the tetrahydropyranyl group can be removed under acidic conditions;

the reagents that provide basic conditions include organic bases including, but not limited to, triethylamine, N-diisopropylethylamine, N-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamide, potassium acetate, sodium t-butoxide, or potassium t-butoxide, and inorganic bases including, but not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium acetate, cesium carbonate, sodium hydroxide, and lithium hydroxide;

reagents that provide acidic conditions include, but are not limited to, hydrogen chloride, 1, 4-dioxane solution of hydrogen chloride, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, p-toluenesulfonic acid, Me₃SiCl and TMSOT_f；

Halogenated agents include, but are not limited to, liquid bromine, hydrogen bromide, N-bromosuccinimide (NBS), PBr₃、POBr₃Pyridine Perbromide Hydrobromide (PHP), Tetrabromocycloketone (TBCO), diethyl bromomalonate, tetrabutylammonium bromide, N-chlorosuccinimide, PCl₃And POCl₃；

Catalysts include, but are not limited to, palladium on carbon, raney nickel, tetrakis-triphenylphosphine palladium, palladium dichloride, palladium acetate, [1, 1 ' -bis (diphenylphosphino) ferrocene ] palladium dichloride, 1 ' -bis (dibenzylideophosphonium) dichlorodipentaerythritol palladium, tris (dibenzylideneacetone) dipalladium, or 2-dicyclohexylphosphine-2 ', 6 ' -dimethoxybiphenyl, preferably [1, 1 ' -bis (diphenylphosphino) ferrocene ] palladium dichloride or 2-dicyclohexylphosphine-2 ', 6 ' -dimethoxybiphenyl;

boranes include, but are not limited to, 4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan, 4,4, 4 ', 4 ', 5,5, 5 ', 5 ' -octamethyl-2, 2 ' -bis (1, 3, 2-dioxaborolan), neopentyl glycol diborate, B (OB)_u-n)₃Or B (OP)_r-i)₃；

The above reaction is preferably carried out in a solvent including, but not limited to: acetic acid, methanol, ethanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, N-hexane, dimethyl sulfoxide, 1, 4-dioxane, water, N-dimethylformamide and a mixture thereof;

wherein:

w is

X is halogen, preferably bromine;

R⁹is an alkyl group; preferably C_1-6An alkyl group;

ring A, R¹～R⁵N and s are as defined in formula (I).

Scheme two

secondly, reacting the compound of the general formula (I-A) with a borane compound under alkaline conditions in the presence of a catalyst to obtain a compound of the general formula (I-Aa);

thirdly, reacting the compound of the general formula (I-2) with the compound of the general formula (I-3) to obtain a compound of the general formula (I-4);

the fourth step, a compound of the formula (I-4) and NHR⁴R⁵To obtain a compound of the general formula (I-Bb);

fifthly, carrying out Suzuki reaction on the compound of the general formula (I-Bb) and the compound of the general formula (I-Aa) under alkaline conditions in the presence of a catalyst to obtain the compound of the general formula (I); r¹In the case of tetrahydropyranyl, the tetrahydropyranyl group can be removed under acidic conditions;

Catalysts include, but are not limited to, palladium on carbon, raney nickel, tetrakis-triphenylphosphine palladium, palladium dichloride, palladium acetate, [1, 1 ' -bis (diphenylphosphino) ferrocene ] palladium dichloride, 1 ' -bis (dibenzylphosphine) dichlorodiamantapalladium, or tris (dibenzylideneacetone) dipalladium, preferably [1, 1 ' -bis (diphenylphosphino) ferrocene ] palladium dichloride;

wherein:

w is

X is halogen, preferably bromine;

R⁹is an alkyl group; preferably, it isC_1-6An alkyl group;

ring A, R¹～R⁵N and s are as defined in formula (I).

Detailed Description

Examples

The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or/and Mass Spectrometry (MS). NMR shift (. delta.) of 10^-6The units in (ppm) are given. NMR was measured using a Bruker AVANCE-400 NMR spectrometer using deuterated dimethyl sulfoxide (DMSO-d)₆) Deuterated chloroform (CDCl)₃) Deuterated methanol (CD)₃OD), internal standard Tetramethylsilane (TMS).

MS was determined using a FINNIGAN LCQAD (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

HPLC was carried out using an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18150X 4.6mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18150X 4.6mm column).

Chiral HPLC analytical determination using LC-10Avp (Shimadzu) or SFC-analytical (Berger Instruments Inc.);

the thin layer chromatography silica gel plate adopts HSGF254 of tobacco yellow sea or GF254 of Qingdao, the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.15 mm-0.2 mm, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm.

The column chromatography generally uses 200-300 mesh silica gel of the Tibet Huanghai silica gel as a carrier.

Chiral preparative column chromatography used Prep Star SD-1(Varian Instruments Inc.) or SFC-multigram (Berger Instruments Inc.).

The CombiFlash rapid preparation instrument uses CombiFlash Rf200(TELEDYNE ISCO).

Average inhibition rate of kinase and IC₅₀The values were determined with a NovoStar microplate reader (BMG, Germany).

Known starting materials of the present invention may be synthesized by or according to methods known in the art, or may be purchased from companies such as ABCR GmbH & Co.KG, Acros Organics, Aldrich Chemical Company, Shao Yuan Chemical technology (Accela ChemBio Inc), Darri Chemicals, and the like.

In the examples, the reaction can be carried out in an argon atmosphere or a nitrogen atmosphere, unless otherwise specified.

An argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a balloon of argon or nitrogen with a volume of about 1L.

The hydrogen atmosphere refers to a reaction flask connected with a hydrogen balloon with a volume of about 1L.

The pressure hydrogenation reaction used a hydrogenation apparatus of Parr 3916EKX type and a hydrogen generator of Qinglan QL-500 type or a hydrogenation apparatus of HC2-SS type.

The hydrogenation reaction was usually evacuated and charged with hydrogen and repeated 3 times.

The microwave reaction was carried out using a CEM Discover-S908860 type microwave reactor.

In the examples, the solution means an aqueous solution unless otherwise specified.

In the examples, the reaction temperature is, unless otherwise specified, from 20 ℃ to 30 ℃ at room temperature.

The monitoring of the progress of the reaction in the examples employed Thin Layer Chromatography (TLC), a developing solvent used for the reaction, a system of eluents for column chromatography used for purifying compounds and a developing solvent system for thin layer chromatography including: a: dichloromethane/methanol system, B: n-hexane/ethyl acetate system, C: petroleum ether/ethyl acetate system, D: the volume ratio of petroleum ether/ethyl acetate/methanol and solvent is regulated according to the different polarity of the compound, and a small amount of basic or acidic reagents such as triethylamine, acetic acid and the like can be added for regulation.

Example 1

6- (1-cyclopropyl-3- (6-methylpyridin-2-yl) -1H-pyrazol-4-yl) - [1,2,4] triazolo [4,3-a ] pyridine-3-carboxamide 1

First step of

2- (1-cyclopropyl-1H-pyrazol-3-yl) -6-methylpyridine 1c

2-methyl-6- (1H-pyrazol-3-yl) pyridine 1a (700mg, 4.40mmol, prepared by a known method "Bioorganic and Medicinal Chemistry, 2015, 23(6), 1260-. After the completion of the reaction, filtration was carried out, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography with eluent system B to obtain the title compound 1c (440mg, yield: 50.2%).

MS m/z(ESI)：200.2[M+1]

Second step of

2- (4-bromo-1-cyclopropyl-1H-pyrazol-3-yl) -6-methylpyridine 1d

Compound 1c (330mg, 1.66mmol) was dissolved in 8mL of dichloromethane, N-bromosuccinimide (295mg, 1.66mmol) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, water was added to the reaction solution, followed by extraction with dichloromethane (10 mL. times.3), the organic phases were combined, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system A to obtain the title compound 1d (335mg, yield: 72.6%).

The third step

2- (1-cyclopropyl-4- (4, 4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-pyrazol-3-yl) -6-methylpyridine 1f

Compound 1d (400mg, 1.44mmol), 4,5, 5-tetramethyl-1, 3, 2-dioxaborolan 1e (1.84g, 14.4mmol), 2-dicyclohexylphosphine-2 ', 6' -dimethoxybiphenyl (89mg, 0.216mmol), triethylamine (364mg, 3.6mmol) and bis (cyanobenzene) palladium dichloride (18.6mg, 0.072mmol) were dissolved in toluene and reacted with microwaves at 90 ℃ for 1 hour. The reaction mixture was added with water, extracted with ethyl acetate (30 mL. times.2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was collected, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with eluent system A to give the title compound 1f (300mg, yield: 64.1%).

MS m/z(ESI)：244.4[M-82+1]

The fourth step

6-bromo- [1,2,4] triazolo [4,3-a ] pyridine-3-carboxylic acid ethyl ester 1h

1g (4g, 21.27mmol, prepared by the method disclosed in the patent application "US 20140134133") of 5-bromo-2-hydrazinopyridine and 2-oxoethyl acetate (2.17g, 21.27mmol) were dissolved in 60mL of methanol under argon atmosphere, reacted at 60 ℃ for 1 hour, the temperature of the reaction solution was cooled to room temperature, concentrated under reduced pressure, 60mL of 1, 4-dioxane was added to the concentrated solution, and iodobenzene diethyl ester (8.22g, 25.53mmol) was added to the solution, and reacted for 18 hours. Concentrated under reduced pressure, applied to a column, and the residue was purified by silica gel chromatography using eluent system D to give the title compound 1h (4.5g, yield: 70.49%).

MS m/z(ESI)：270.3[M+1]

The fifth step

6-bromo- [1,2,4] triazolo [4,3-a ] pyridine-3-carboxamide 1i

Compound 1h (500mg, 1.85mmol) was dissolved in 7N methanolic ammonia (8.37mL, 58.58mmol) and the reaction stirred for 1 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography with eluent system A to give the title compound 1i (450mg, yield: 99%).

MS m/z(ESI)：241.3[M+1]

The sixth step

Compound 1f (75mg, 0.23mmol), compound 1i (55.59mg, 0.23mmol), bis diphenylphosphinoferrocene (12.79mg, 0.02mmol), [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (16.87mg, 0.02mmol) and potassium carbonate (63.75mg, 0.46mmol) were dissolved in this order in 12mL of a mixed solution of 1, 4-dioxane and water under argon (V/V ═ 5: 1). The reaction was stirred at 80 ℃ for 18 hours. The reaction solution was cooled to room temperature, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with eluent system I to give title compound 1(35mg, yield: 40%).

MS m/z(ESI)：360.4[M+1]

¹H NMR(400MHz，CD₃OD)δ9.32(s，1H)，8.13(s，1H)，7.79-7.72(m，2H)，7.61-7.56(m，2H)，7.24-7.22(d，1H)，3.83-3.79(m，1H)，2.40(s，3H)，1.28-1.23(m，2H)，1.15-1.10(m，2H).

Example 2

6- (1-cyclobutyl-3- (6-methylpyridin-2-yl) -1H-pyrazol-4-yl) - [1,2,4] triazolo [4,3-a ] pyridine-3-carboxamide 2

First step of

(3-carbamoyl- [1,2,4] triazolo [4,3-a ] pyridin-6-yl) boronic acid 2a

Compound 1i (4.2g, 17.42mmol), 4,4, 4 ', 4 ', 5,5, 5 ', 5 ' -octamethyl-2, 2 ' -bis (1, 3, 2-dioxaborolan) (6.64g, 26.14 mmol), prepared by the well-known method "Journal of the American Chemical Society, 2009, 131(5), 1656-. The reaction was stopped, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with eluent system A to give the title compound 2a (2.6g, yield: 36.2%).

MS m/z(ESI)：207.4[M+1]

Second step of

2- (1-cyclobutyl-1H-pyrazol-3-yl) -6-methylpyridine 2c

Compound 1a (2.01g, 12.6mmol), cyclobutyl 4-methylbenzenesulfonate 2b (4.29g, 18.9mmol, prepared by the known method "Journal of the American Chemical Society, 1980, 102(11), 3863-3870"), and cesium carbonate (8.23g, 25.3mmol) were successively added to 100mL of N, N-dimethylformamide and, after completion of the addition, stirred at 60 ℃ for 12 hours. The reaction solution was cooled to room temperature, water was added, extraction was performed with ethyl acetate (20mL × 3), the organic phases were combined, washed with a saturated sodium chloride solution (10mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was collected, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with eluent system B to give the title compound 2c (1.81g, yield: 67.3%).

MS m/z(ESI)：214.4[M+1]

The third step

2- (4-bromo-1-cyclobutyl-1H-pyrazol-3-yl) -6-methylpyridine 2d

Compound 2c (1.81g, 8.49mmol) was dissolved in 18mL of dichloromethane, N-bromosuccinimide (1.51g, 8.49mmol) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography with eluent system B to obtain the title compound 2d (2.13g, yield: 85.9%).

MS m/z(ESI)：292.3[M+1]

The fourth step

Compound 2a (78.25mg, 0.38mmol), compound 2d (74mg, 0.25mmol), potassium carbonate (70.01mg, 0.51mmol), bis-diphenylphosphinoferrocene (14.04mg, 0.03mmol) and [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (18.53mg, 0.03mmol) were dissolved in this order in a mixed solution of 11mL of 1, 4-dioxane and water under argon (V/V ═ 10: 1). The reaction was stirred at 80 ℃ for 12 hours. The reaction solution was cooled to room temperature, 20mL of water was added to the reaction solution, extraction was performed with ethyl acetate (10mL × 3), the organic phases were combined, washed with saturated sodium chloride solution (10mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was collected, the filtrate was concentrated under reduced pressure, the residue was purified by silica gel column chromatography with eluent system a, and the obtained crude product was purified by high performance liquid chromatography to obtain the title compound 2(4mg, yield: 4.2%).

MS m/z(ESI)：374.5[M+1]

¹H NMR(400MHz，CDCl₃)δ9.34(s，1H)，7.76(d，1H)，7.72(s，1H)，7.64-7.62(m，2H)，7.54(d，1H)，7.42(brs，1H)，7.11(d，1H)，5.76(brs，1H)，4.93-4.84(m，1H)，2.66-2.56(m，4H)，2.46(s，3H)，1.96-1.88(m，2H).

Example 3

6- (1-methyl-3- (6-methylpyridin-2-yl) -1H-pyrazol-4-yl) - [1,2,4] triazolo [4,3-a ] pyridine-3-carboxamide 3

First step of

2-methyl-6- (1-methyl-1H-pyrazol-3-yl) pyridine 3a

Compound 1a (1g, 6.28mmol), methyl iodide (1.07g, 7.54mmol) and potassium carbonate (1.74g, 12.56mmol) were added to 10mL of N, N-dimethylformamide and reacted for 12 hours. After the completion of the reaction, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with eluent system A to give the title compound 3a (320mg, yield: 29.4%).

MS m/z(ESI)：174.4[M+1]

Second step of

2- (4-bromo-1-methyl-1H-pyrazol-3-yl) -6-methylpyridine 3b

Compound 3a (240mg, 1.39mmol) and N-bromosuccinimide (294.29mg, 1.66mmol) were added to 10mL of dichloromethane, and the reaction was stirred for 12 hours. After the reaction, 10mL of saturated sodium bicarbonate solution was added to the reaction solution, followed by extraction with ethyl acetate, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude title compound 3b (349mg), which was used in the next reaction without purification.

MS m/z(ESI)：253.1[M+1]

The third step

Crude 3b (300mg, 1.19mmol), compound 2a (294.11mg, 1.43mmol), potassium carbonate (328.92mg, 2.38mmol) and [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (88.27mg, 0.119mmol) were dissolved in 10.2mL of a mixed solution of 1, 4-dioxane and water in that order under argon (V/V ═ 50: 1). The reaction was stirred at 90 ℃ for 12 hours. Celite was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography to give title compound 3(120mg, yield: 30.3%).

MS m/z(ESI)：334.4[M+1]

¹H NMR(400MHz，CDCl₃)δ9.39(s，1H)，7.80(d，1H)，7.67(s，1H)，7.63(t，1H)，7.58-7.56(dd，2H)，7.40(s，1H)，7.12(d，1H)，5.71(s，1H)，4.08(s，3H)，2.47(s，3H).

Example 4

6- (1-Ethyl-3- (6-methylpyridin-2-yl) -1H-pyrazol-4-yl) - [1,2,4] triazolo [4,3-a ] pyridine-3-carboxamide 4

First step of

2- (1-Ethyl-1H-pyrazol-3-yl) -6-methylpyridine 4a

Compound 1a (1.2g, 7.54mmol), iodoethane (1.18g, 7.54mmol) and potassium carbonate (2.08g, 15.08mmol) were added to 5mL of N, N-dimethylformamide and reacted for 12 hours. After the completion of the reaction, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with eluent system A to give the title compound 4a (520mg, yield: 36.8%).

MS m/z(ESI)：188.4[M+1]

Second step of

2- (4-bromo-1-ethyl-1H-pyrazol-3-yl) -6-methylpyridine 4b

Compound 4a (460mg, 2.46mmol) and N-bromosuccinimide (521.80mg, 2.95mmol) were added to 10mL of dichloromethane, and the reaction was stirred for 12 hours. After the reaction was completed, 10mL of a saturated sodium bicarbonate solution was added to the reaction solution, followed by extraction with ethyl acetate, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude title compound 4b (650mg), which was used in the next reaction without purification.

MS m/z(ESI)：267.2[M+1]

The third step

Crude 4b (300mg, 1.13mmol), compound 2a (278.61mg, 1.35mmol), potassium carbonate (311.58mg, 2.25mmol) and [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (83.62mg, 0.112mmol)) were dissolved in succession in 10.2mL of a mixed solution of 1, 4-dioxane and water (V/V ═ 50: 1) under argon. The reaction was stirred at 90 ℃ for 12 hours. Celite was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography to give title compound 4(125mg, yield: 31.9%).

MS m/z(ESI)：348.4[M+1]

¹H NMR(400MHz，CDCl₃)δ9.39(s，1H)，7.79(d，1H)，7.70(s，1H)，7.63(t，1H)，7.59-7.56(dd，2H)，7.39(s，1H)，7.12(d，1H)，5.68(s，1H)，4.34(q，2H)，2.46(s，3H)，1.65(t，3).

Example 5

6- (1-isopropyl-3- (6-methylpyridin-2-yl) -1H-pyrazol-4-yl) - [1,2,4] triazolo [4,3-a ] pyridine-3-carboxamide 5

First step of

2- (1-isopropyl-1H-pyrazol-3-yl) -6-methylpyridine 5a

Compound 1a (1.2g, 7.54mmol), 2-bromopropane (927.14mg, 7.54mmol) and potassium carbonate (2.08g, 15.08mmol) were added to 5mL of N, N-dimethylformamide and reacted for 12 hours. After the completion of the reaction, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with eluent system A to give the title compound 5a (520mg, yield: 36.8%).

MS m/z(ESI)：202.4[M+1]

Second step of

2- (4-bromo-1-isopropyl-1H-pyrazol-3-yl) -6-methylpyridine 5b

Compound 5a (250mg, 1.24mmol) and N-bromosuccinimide (263.82mg, 1.49mmol) were added to 10mL of dichloromethane, and the reaction was stirred for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography with eluent system A to give the title compound 5b (348mg, yield: 100%).

MS m/z(ESI)：281.2[M+1]

The third step

Crude 5b (300mg, 1.07mmol), compound 2a (330.82mg, 1.61mmol), potassium carbonate (295.99mg, 2.14mmol) and [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (79.43mg, 107.08. mu. mol) were dissolved in succession in 10.2mL of a mixed solution of 1, 4-dioxane and water (V/V ═ 50: 1) under argon. The reaction was stirred at 90 ℃ for 12 hours. Celite was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography to give title compound 5(132mg, yield: 34.1%).

MS m/z(ESI)：362.5[M+1]

¹H NMR(400MHz，CDCl₃)δ9.39(t，1H)，7.80-7.78(dd，1H)，7.71(s，1H)，7.64(s，1H)，7.63(d，1H)，7.59-7.56(d，1H)，7.41(s，1H)，7.12-7.10(m，1H)，5.69(s，1H)，4.71-4.64(m，1H)，2.45(s，3H)，1.64(d，6H).

Example 6

6- (3- (6-methylpyridin-2-yl) -1H-pyrazol-4-yl) - [1,2,4] triazolo [4,3-a ] pyridine-3-carboxamide 6

First step of

2- (4-bromo-1H-pyrazol-3-yl) -6-methylpyridine 6a

Compound 1a (1g, 6.28mmol) and N-bromosuccinimide (1.12g, 6.28mmol) were added to 30mL of dichloromethane, and the reaction was stirred for 1.5 hours. After the reaction was completed, 10mL of a saturated potassium carbonate solution was added to the reaction solution, followed by extraction with methylene chloride (10 mL. times.3), the organic phases were combined, the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with eluent system A to give the title compound 6a (1.45g, yield: 87.3%).

MS m/z(ESI)：238.5[M+1]

Second step of

2- (4-bromo-1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-3-yl) -6-methylpyridine 6b

Compound 6a (1.4g, 5.88mmol) and 3, 4-dihydro-2H-pyran (1.12g, 6.28mmol) were added to 20mL of toluene under argon atmosphere, trifluoroacetic acid (0.03g, 0.29mmol) was added to the above reaction solution, and the reaction was stirred at 80 ℃ for 48 hours. After the reaction, 10mL of saturated sodium carbonate solution was added to the reaction solution, followed by extraction with ethyl acetate (20 mL. times.3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain title compound 6b (1.45g, yield: 87.26%).

MS m/z(ESI)：322.5[M+1]

The third step

6- (3- (6-methylpyridin-2-yl) -1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) - [1,2,4] triazolo [4,3-a ] pyridine-3-carboxamide 6c

Compound 6b (300mg, 0.93mmol), compound 2a (9.59mg, 0.05mmol), potassium carbonate (386.06mg, 2.79mmol), and [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (690.67mg, 0.93mmol) were dissolved in the order of 16.5mL of a mixed solution of 1, 4-dioxane and water (V/V ═ 10: 1) under argon atmosphere. The reaction was stirred at 80 ℃ for 18 hours. Ethyl acetate was added, washed with water, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography to give the title compound 6c (150mg, yield: 35.94%).

MS m/z(ESI)：404.5[M+1]

The fourth step

Compound 6c (20mg, 0.05mmol) was added to 2mL of 1, 4-dioxane under argon atmosphere, and 4mL of 4N hydrogen chloride 1, 4-dioxane solution was added dropwise to the reaction mixture at 0 ℃ and reacted for 2 hours after the addition. The reaction solution was concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography to give the title compound 6(5mg, yield: 26.9%).

MS m/z(ESI)：427.2[M+1]

¹H NMR(400MHz，CD₃OD)δ9.38(s，1H)，8.03-8.07(m，1H)，7.83-7.85(m，1H)，7.74-7.78(m，1H)，7.64-7.67(m，1H)，7.49-7.54(m，1H)，7.27-7.29(m，1H)，3.50(m，1H)，3.15(m，1H)，2.48(s，3H)

Example 7

(R) -6- (3- (6-methylpyridin-2-yl) -1- (tetrahydrofuran-3-yl) -1H-pyrazol-4-yl) - [1,2,4] triazolo [4,3-a ] pyridine-3-carboxamide 7

First step of

(R) -2-methyl-6- (1- (tetrahydrofuran-3-yl) -1H-pyrazol-3-yl) pyridine 7b

Compound 1a (637mg, 4.0mmol) was dissolved in 20mL of N, N-dimethylformamide, and (S) -tetrahydrofuran-3-yl 4-methylbenzenesulfonate 7a (1.45g, 6.0mmol, prepared by the method disclosed in patent application "WO 2014049133") and cesium carbonate (2.61g, 8.0mmol) were added and the reaction was stirred at 60 ℃ for 16 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by using CombiFlash flash prep with eluent system B to give the title compound 7B (610mg, yield: 66.5%).

MS m/z(ESI)：230.4[M+1]

Second step of

(R) -2- (4-bromo-1- (tetrahydrofuran-3-yl) -1H-pyrazol-3-yl) -6-methylpyridine 7c

Compound 7b (600mg, 2.62mmol) was dissolved in 30mL of dichloromethane, N-bromosuccinimide (466mg, 2.62mmol) was added, and the mixture was stirred at room temperature for 16 hours. After completion of the reaction, concentration was performed under reduced pressure, and the resulting residue was purified by using CombiFlash flash Rapid prep with eluent system B to obtain the title compound 7c (795mg, yield: 98.6%).

MS m/z(ESI)：310.3[M+1]

The third step

Compound 7c (115mg, 0.37mmol), compound 2a (307.45mg, 1.49mmol), bis diphenylphosphinoferrocene (20.69mg, 0.04mmol), [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (27.31mg, 0.040mmol) and potassium carbonate (103.15mg, 0.75mmol) were dissolved in this order in 16.5mL of a mixed solution of 1, 4-dioxane and water (V/V ═ 10: 1) under argon. The reaction was stirred at 80 ℃ for 18 hours. The reaction solution was cooled to room temperature, 30mL of water was added to the reaction solution, followed by extraction with ethyl acetate (20mL × 3), the organic phases were combined, washed with saturated sodium chloride solution (10mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was collected, the filtrate was concentrated under reduced pressure, the residue was purified by silica gel chromatography with eluent system a, and the resulting crude product was purified by high performance liquid chromatography to give title compound 7(8mg, yield: 5.5%).

MS m/z(ESI)：390.4[M+1]

¹H NMR(400MHz，CDCl₃)δ9.31(s，1H)，7.82-7.77(m，3H)，7.58(d，1H)，7.51(d，1H)，7.35(brs，1H)，7.26(s，1H)，5.64(brs，1H)，5.17-5.12(m，1H)，4.28-4.22(m，2H)，4.14-4.10(m，1H)，4.02-3.97(m，1H)，2.63(s，3H)，2.61-2.54(m，2H).

Example 8

(S) -6- (3- (6-methylpyridin-2-yl) -1- (tetrahydrofuran-3-yl) -1H-pyrazol-4-yl) - [1,2,4] triazolo [4,3-a ] pyridine-3-carboxamide 8

First step of

(S) -2-methyl-6- (1- (tetrahydrofuran-3-yl) -1H-pyrazol-3-yl) pyridine 8b

Compound 1a (328mg, 2.06mmol) was dissolved in 10mL of N, N-dimethylformamide, and (R) -tetrahydrofuran-3-yl 4-methylbenzenesulfonate 8a (500mg, 2.06mmol, prepared by the method disclosed in patent application "WO 2016021192") and cesium carbonate (1.3g, 4.12mmol) were added and the reaction was stirred at 60 ℃ for 2 hours. The reaction solution was concentrated under reduced pressure, water was added to the residue, extraction was performed with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with eluent system a to give the title compound 8b (175mg, yield: 37.0%).

MS m/z(ESI)：230.4[M+1]

Second step of

(S) -2- (4-bromo-1- (tetrahydrofuran-3-yl) -1H-pyrazol-3-yl) -6-methylpyridine 8c

Compound 8b (370mg, 1.6mmol) was dissolved in 18mL of dichloromethane, N-bromosuccinimide (341.76mg, 1.92mmol) was added, and the mixture was stirred at room temperature for 12 hours. After the reaction was completed, water was added to the reaction solution, followed by extraction with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with eluent system A to obtain the title compound 8c (325.5mg, yield: 65.1%).

The third step

Compound 8c (100mg, 0.32mmol), compound 2a (300mg, 1.46mmol), bis diphenylphosphinoferrocene ((17.99mg, 0.0300mmol), [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (23.74mg, 0.03mmol) and potassium carbonate (89.7mg, 0.65mmol) were dissolved in this order in a mixed solution of 11mL of 1, 4-dioxane and water (V/V ═ 10: 1) under argon atmosphere, stirred for reaction for 12 hours, the reaction solution was cooled to room temperature, 30mL of water was added to the reaction solution, extraction was performed with ethyl acetate (10mL × 3), the organic phases were combined, anhydrous sodium sulfate was dried, filtered, the filtrate was collected, the filtrate was concentrated under reduced pressure, the residue was purified with silica gel chromatography system a, and the resulting crude product was purified by high performance liquid chromatography to give title compound 8(14mg, yield: 11.1%).

MS m/z(ESI)：390.4[M+1]

¹H NMR(400MHz，CDCl₃)δ9.39(s，1H)，7.81(d，1H)，7.78(s，1H)，7.62(d，1H)，7.57(d，1H)，7.42(s，1H)，7.14(d，1H)，5.75-5.69(m，1H)，5.19-5.13(m，1H)，4.29-4.23(m，2H)，4.18-4.12(m，1H)，4.06-4.00(m，1H)，2.66-2.55(m，1H)，2.48(s，3H).

Test example:

biological evaluation

Test example 1 measurement of inhibitory Effect of the Compound of the present invention on TGF-. beta.RI (ALK5) kinase Activity

Inhibition of TGF β RI (ALK5) kinase activity in vitro was tested by the following method.

The inhibitory effect of the compounds of the present invention on the activity of TGF β RI kinase ALK5 was determined by the following experimental method:

enzymatic Activity detection Using TGF-. beta.RI kinase detection kit (V4093, Promega), 2. mu.l of a reaction buffer (40mM Tris pH7.5, 20mM MgCl. sub.L) was sequentially added to a 384-well plate (4514, Corning)₂0.1mg/ml BSA), 1. mu.l of a 3-fold gradient diluted compound dissolved in 5% DMSO, 2. mu.l of a mixed solution of ATP and TGF. beta. RI substrate polypeptide (ATP final concentration of 50. mu.M, substrate final concentration of 0.2. mu.g/. mu.L), and after reacting at 27 ℃ for 2.5 hours, 5. mu.l of ADP-Glo solution in the kit was added to each well, and allowed to stand at 27 ℃ for 40 minutes, 10. mu.l of a kinase assay reagent was added to each well, and allowed to stand at 27 ℃ for 30 minutes. Chemiluminescence signal values were detected using a Victor 3(PerkinElmer) multifunctional microplate reader. Calculating IC of the inhibitory effect of the compound on enzyme by Graphpad prism software according to each concentration of the compound and corresponding signal value₅₀The value is obtained.

The biological activity of the compounds of the invention is determined by the above assay, the IC determined₅₀The values are given in table 1 below.

TABLE 1 Activity of the Compounds of the invention on TGF-. beta.RI kinase ALK5Sex-inhibited IC₅₀

And (4) conclusion: the compounds of the embodiment of the invention have obvious inhibition effect on the activity of TGF beta RI kinase ALK 5.

Test example 2 determination of inhibitory Effect of the Compounds of the present invention on VEGFR2 kinase Activity

The inhibition of VEGFR2 kinase activity in vitro was tested by the following method.

The following assay was used to determine the inhibitory effect of the compounds of the invention on VEGFR2 kinase activity:

the enzyme activity detection method

Mu.l of reaction buffer (50mM HEPES pH7.5, 10mM MgCl. sub.1) was sequentially added to a 384-well plate (4513, Corning) using the Kit of Kinase Assay Kit-Tyrosine 1 Peptide (PV3190, Invitrogen)₂1mM EGTA, 0.05% BRIJ-35) and VEGFR2 substrate polypeptide (final enzyme concentration of 0.14 ng/. mu.L and final substrate concentration of 2. mu.M in the reaction system), 2.5. mu.L of 2-fold gradient diluted compound dissolved in 5% DMSO, 2.5. mu.L of ATP solution (final ATP concentration of 50. mu.M), 5. mu.L of detection reagent was added to each well after 2 hours of reaction at 25 ℃, and after 1 hour of reaction at 25 ℃, fluorescence signals with emission wavelengths of 445nm and 520nm were measured using a NOVOstar (BMG) multifunctional plate reader. Calculating IC of the inhibitory effect of the compound on enzyme by Graphpad prism software according to each concentration of the compound and corresponding signal value₅₀The value is obtained.

The biological activity of the compounds of the invention is determined by the above assay, the IC determined₅₀The values are given in Table 2 below.

TABLE 2 inhibition of VEGFR2 kinase Activity by Compounds of the inventionIC of₅₀

Example numbering	IC₅₀(nM)
		1	＞10000
6	1001

And (4) conclusion: the compounds of the embodiments of the invention have weak inhibition effect on VEGFR2 kinase activity, which shows that the compounds of the embodiments of the invention have selective inhibition effect on TGF beta RI kinase.

Test example 3 determination of the inhibitory Effect of the Compound of the present invention on the p 38. alpha. kinase Activity

Inhibition of p38 α kinase activity in vitro was tested by the following method.

The following assay was used to determine the inhibition of p38 α kinase activity by the compounds of the present invention:

enzyme Activity detection Using p 38. alpha. kinase detection kit (V9591, Promega), 2. mu.L of reaction buffer (40mM Tris pH7.5, 20mM MgCl. sub.L) was sequentially added to a 384-well plate (4514, Corning)₂0.1mg/mL BSA), 1. mu.L of a 3-fold gradient diluted compound dissolved in 5% DMSO, 2. mu.L of a mixed solution of ATP and p38 substrate polypeptide (ATP final concentration of 50. mu.M, substrate final concentration of 0.2. mu.g/. mu.L), and after 2.5 hours of reaction at 27 ℃, 5. mu.L of ADP-Glo solution in the kit was added to each well, and the mixture was left at 27 ℃ for 40 minutes, 10. mu.L of a kinase assay reagent was added to each well, and left at 27 ℃ for 30 minutes. Chemiluminescence signal values were detected using a Victor 3(PerkinElmer) multifunctional microplate reader. With Graphpad prism software calculates IC of the compound on enzyme inhibition according to each concentration of the compound and corresponding signal value₅₀The value is obtained.

The biological activity of the compounds of the invention is determined by the above assay, the IC determined₅₀The values are given in Table 3 below.

TABLE 3 IC of inhibition of p38 alpha kinase activity by the compounds of the invention₅₀

Example numbering	IC₅₀(nM)
		1	1368
6	2613

And (4) conclusion: the compounds of the examples of the invention have weak inhibition effect on the activity of p38 alpha kinase, which shows that the compounds of the examples of the invention have selective inhibition effect on TGF beta RI kinase.

Test example 4 inhibition assay of NIH3T3 cell proliferation by Compounds of the invention

The following in vitro assays were used to determine the inhibitory activity of the compounds of the invention on the proliferation of NIH3T3 cells.

The following assay was used to determine the inhibitory effect of the compounds of the invention on the proliferation of NIH3T3 cells:

100. mu.L of NIH3T3 cells (GNM6, cell bank of the national academy of sciences type culture Collection) were seeded in 96-well transparent bottom plates (3903, Corning) in a medium of DMEM (SH30243.01, GE) containing 10% FBS at a density of 2000 cells/well at 37 ℃ in 5% CO₂Culturing overnight under the condition. After overnight incubation, each well was replaced with 90. mu.L of DMEM medium containing 0.5% FBS, then 10. mu.L of compound diluted in 3-fold gradient in DMEM medium containing 0.5% FBS was added and left at 37 ℃ with 5% CO₂The cells were cultured in a cell incubator for 72 hours. Finally 50. mu.L of CellTiter-Glo (G7573, Promega) was added to each well and the chemiluminescent signal was read using a Victor3 microplate reader (Perkinelmer) after 10 minutes incubation at room temperature. IC of the compound was calculated from each concentration of the compound and the corresponding signal value using Graphpad Prism software₅₀The value is obtained.

The biological activity of the compounds of the invention is determined by the above analysis and the calculated IC₅₀The values are given in Table 4 below:

TABLE 4 IC of inhibition of NIH3T3 cell proliferation by compounds of the invention₅₀

Example numbering	IC₅₀(nM)
		1	246
2	357
		5	203
6	40

And (4) conclusion: the compound of the invention has obvious inhibitory activity on NIH3T3 cell proliferation.

Test example 5 measurement of inhibitory Activity of the Compound of the present invention on Smad Signaling pathway of TGF-. beta.RI

The following in vitro assays were used to determine the inhibitory activity of the compounds of the invention on the Smad signaling pathway of TGF β RI.

The following experimental methods were used to determine the inhibitory activity of the compounds of the invention on the Smad signaling pathway of TGF β RI:

mu.L of HepG2 (TCtu 72, cell bank of the culture Collection for type culture Collection of China academy of sciences) cells were seeded in 96-well plates at a density of 2.5X 10 with 10% FBS-containing EMEM medium (42360-⁴Cells/well, cells at 37 ℃ 5% CO₂Incubated under conditions overnight. The fresh EMEM medium containing 10% FBS was replaced and transfected with 0.1. mu.g of 3TP-lux plasmid (11767, proeg Biotech (Beijing) Ltd.) per well and the cells were continued at 37 ℃ with 5% CO₂Incubated under conditions for 24 hours. 90 μ L of EMEM medium containing 0.5% FBS was replaced per well and starved for 6 hours. Compounds were prepared as 20mM stock, diluted to 400X concentration with a 100% DMSO gradient, and diluted 40-fold with EMEM containing 0.5% FBS. The cell culture plate was removed, 10. mu.L of diluted compound or control (0.25% DMSO) was added to each well, mixed by gentle shaking, and left at 37 ℃ with 5% CO₂Culturing in incubator for 18 hr, and adding 100 μ L detection reagent ONE-Glo into each well^TMLuciferase Assay (E6110, Promega) was left at room temperature for 10 minutes in the absence of light and the chemiluminescent signal was read using Victor3.0 (Perkinelmer). IC of the compound was calculated from each concentration of the compound and the corresponding signal value using Graphpad Prism software₅₀The value is obtained.

The biological activity of the compounds of the invention is determined by the above analysis and the calculated IC₅₀The values are given in Table 5 below:

TABLE 5 Smad signaling pathway inhibition of TGF-beta RI by compounds of the invention IC₅₀

Example numbering	IC₅₀(nM)
		1	167
2	40
		6	34

And (4) conclusion: the compounds of the invention all have obvious inhibitory activity on Smad signaling pathway of TGF beta RI.

Pharmacokinetic evaluation

Test example 6 pharmacokinetic testing of the Compound of the invention

1. Abstract

Using rats as test animals, the drug concentrations in plasma at different times after gavage administration of the compound of example 1 were determined by LC/MS/MS method. The pharmacokinetic behavior of the compounds of the invention in rats was studied and their pharmacokinetic profile was evaluated.

2. Test protocol

2.1 test drugs

The compound of example 1.

2.2 test animals

Healthy adult SD rats, 4 male and female halves, purchased from shanghai jequirity laboratory animals limited, animal production license number: SCXK (Shanghai) 2013 and 0006.

2.3 pharmaceutical formulation

A certain amount of the drug is weighed, and then 5% volume of DMSO, 5% volume of Tween 80 and 90% volume of physiological saline are added to prepare colorless clear transparent liquid of 0.2 mg/mL.

2.4 administration

SD rats are subjected to gastric lavage after being fasted overnight, the administration dose is 2.0mg/kg, and the administration volume is 10.0 mL/kg.

3. Operation of

Rats were gavaged with the compound of example 1, 0.2mL of blood was collected from the orbit before and after administration at 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 11.0, 24.0 hours, placed in heparinized tubes, centrifuged at 4 ℃ and 3500 rpm for 10 minutes to separate plasma, stored at-20 ℃ and fed 2 hours after administration.

Determining the content of the compound to be tested in rat plasma after the drug with different concentrations is administered by gastric lavage: rat plasma at each time post-dose was taken at 25. mu.L, added with camptothecin as an internal standard solution at 80. mu.L (100ng/mL), acetonitrile at 200. mu.L, vortexed for 5 minutes, centrifuged for 10 minutes (4000 rpm), and plasma samples were taken at 1.0. mu.L of supernatant for LC/MS/MS analysis.

4. Pharmacokinetic parameter results

The pharmacokinetic parameters of the compounds of the invention are as follows:

and (4) conclusion: the compound of the invention has better drug absorption and pharmacokinetic advantage.

Claims

1. A compound of the general formula (I):

wherein:

ring a is aryl or heteroaryl;

R¹selected from the group consisting of hydrogen atoms, alkyl groups, haloalkyl groups, hydroxyalkyl groups, cycloalkyl groups, and heterocyclyl groups, wherein the alkyl groups, cycloalkyl groups, and heterocyclyl groups are each independently optionally substituted with one or more substituents selected from the group consisting of halogen, alkyl groups, alkoxy groups, haloalkyl groups, hydroxy groups, hydroxyalkyl groups, cyano groups, amino groups, and nitro groups;

R²the same or different, and each is independently selected from the group consisting of hydrogen atom, halogen, alkyl group, alkoxy group, haloalkyl group, hydroxyl group, hydroxyalkyl group, cyano group, amino group, nitro group, and cycloalkyl group;

R³the same or different, and each is independently selected from the group consisting of a hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, and nitro;

R⁴and R⁵Each independently selected from the group consisting of a hydrogen atom, an alkyl group and a haloalkyl group, wherein the alkyl group is optionally substituted with one or more substituents selected from the group consisting of an alkoxy group, an amino group, a cyano group, a nitro group and a hydroxyl group;

n is 0, 1,2, 3 or 4;

s is 0, 1,2 or 3;

the alkyl is C_1-6An alkyl group;

the haloalkyl is C_1-6A haloalkyl group;

the hydroxyalkyl is C_1-6A hydroxyalkyl group;

the alkoxy is C_1-6An alkoxy group;

the aryl is 6-membered aryl;

the heteroaryl is a 5 or 6 membered heteroaryl;

the cycloalkyl is 3-to 6-membered cycloalkyl; and is

The heterocyclic group is a 3-to 6-membered heterocyclic group.

2. The compound of formula (I) according to claim 1, wherein ring a is pyridyl.

3. The compound of formula (I) according to any one of claims 1 or 2, which is a compound of formula (II):

wherein: r¹～R⁵N and s are as defined in claim 1.

4. A compound of formula (I) according to claim 1, wherein R⁴And R⁵Are all hydrogen atoms.

5. The compound of formula (I) according to claim 1, which is a compound of formula (III):

wherein: r¹And R²As defined in claim 1.

6. A compound of formula (I) according to claim 1, wherein R¹Selected from hydrogen atoms, C_1-6Alkyl, 3-to 6-membered cycloalkyl and 3-to 6-membered heterocyclyl.

7. A compound of formula (I) according to claim 1, wherein R²Is a hydrogen atom or C_1-6An alkyl group.

8. A compound of formula (I) according to claim 1, wherein R³Is a hydrogen atom.

9. A compound of formula (I) according to claim 1, selected from:

10. a process for the preparation of a compound of formula (I) according to claim 1, which process comprises:

wherein:

x is halogen;

ring A, R¹～R⁵N and s are as defined in claim 1.

11. A process for the preparation of a compound of formula (I) according to claim 1, which process comprises:

wherein:

x is halogen;

ring A, R¹～R⁵N and s are as defined in claim 1.

12. The production method according to claim 10 or 11, wherein X is bromine.

13. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) according to any one of claims 1 to 9, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

14. Use of a compound of general formula (I) according to any one of claims 1 to 9, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 13, for the preparation of a medicament for the treatment, prevention, or reduction of tumor cell metastasis.

15. Use of a compound of general formula (I) according to any one of claims 1 to 9, or a tautomer, racemate, enantiomer, or diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 13, for the preparation of a medicament for the treatment, prevention, or reduction of tumors mediated by TGF- β overexpression.

16. Use of a compound of general formula (I) according to any one of claims 1 to 9 or a tautomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 13, for the preparation of a medicament for the treatment, prevention, or alleviation of a disease selected from: cardiovascular diseases, various types of inflammation, tumors, fibrosis of various etiologies, vascular injury, renal diseases, liver disorders, lung diseases, adult respiratory distress syndrome, intimal thickening, ocular diseases, excessive or hypertrophic dermal scar or keloid formation occurring during wound healing caused by trauma or surgical wounds, peritoneal and subcutaneous adhesions, scleroderma, fibrosis, progressive systemic sclerosis, osteoporosis, ulcers, reduced nervous system function, male erectile dysfunction, pelmet's disease, dupuytren's contracture, alzheimer's disease, and raynaud's syndrome.

17. Use of a compound of general formula (I) according to any one of claims 1 to 9, or a tautomer, racemate, enantiomer, or diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 13, for the preparation of a medicament for inhibiting the TGF- β signaling pathway.