CN114315899B

CN114315899B - 3- (Aromatic ring imidazole) pyrazolopyrimidine derivative and application thereof

Info

Publication number: CN114315899B
Application number: CN202011056455.7A
Authority: CN
Inventors: 任峰; 王显连; 陈春麟; 周南梅; 蔡金娜; 潘桂梅; 周举
Original assignee: Shanghai Medicilon Inc; Medicilon Preclinical Research Shanghai LLC
Current assignee: Shanghai Medicilon Inc; Medicilon Preclinical Research Shanghai LLC
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2024-09-03
Anticipated expiration: 2040-09-30
Also published as: CN114315899A

Abstract

The invention relates to a novel 3- (aromatic ring imidazole) pyrazolopyrimidine derivative, a preparation method thereof and application thereof in medicines. In particular, the invention relates to novel 3- (aromatic ring imidazole) pyrazolopyrimidine derivative shown in a general formula (I), a preparation method thereof and application of the derivative or a pharmaceutical composition containing the derivative as a therapeutic agent, particularly as a Tropomyosin Related Kinase (TRK) inhibitor in treating or preventing related diseases mediated by TRK, such as tumors. Wherein each substituent (R ¹、R²、R³) and group (X) in the general formula (I) is as defined in the specification.

Description

3- (Aromatic ring imidazole) pyrazolopyrimidine derivative and application thereof

Technical Field

The invention relates to a novel 3- (aromatic ring imidazole) pyrazolopyrimidine derivative, a preparation method thereof and application of the derivative or a pharmaceutical composition containing the derivative as a therapeutic agent, particularly as a Tropomyosin Related Kinase (TRK) inhibitor.

Background

Tropomyosin-related kinase (TRK, also known as "tropomyosin receptor kinase") is a class of nerve growth factor receptor that exists in a variety of tissues and plays an important role in the proliferation, differentiation, and survival of cells. TRK belongs to the family of receptor tyrosine kinases, and mainly comprises TRKA, TRKB and TRKC, and is encoded by NTRK1, NTRK2 and NTRK3 genes respectively.

Each TRK has a neurotrophic factor ligand corresponding thereto. The ligands for TRKA are NGF (nerve growth factor), the ligands for TRKB include BDGF (brain-derived growth factor) and NT-4/5 (neurotophin-4/5), and the ligand for TRKC is NT-3. These neurotrophins bind specifically to TRKs, triggering receptor dimerization and phosphorylation of kinase-specific tyrosine residues, thereby activating downstream signaling pathways including Ras/MAPK, plcγ/PKC and PI3K/AKT, which in turn regulate a range of physiological processes such as proliferation, differentiation, metabolism, apoptosis, etc. of cells.

TRK signaling is usually precisely regulated, and when it is abnormally activated by gene fusion, overexpression of proteins, or single nucleotide mutation, it leads to the occurrence of various tumors and is independent of the tissue source and type of tumor. The rapid development of genomics has led to the discovery of more and more NTRK fusion genes, such as ETV6-NTRK3, MPRIP-NTRK1, CD74-NTRK1, etc. The TRK inhibitor can be developed to treat various tumors of NTRK fusion protein, such as lung cancer, malignant hematopathy, prostate cancer, breast cancer, ovarian cancer, glioma, pancreatic cancer, hepatobiliary tract cancer, papillary thyroid cancer, colon cancer, head and neck squamous cell carcinoma, melanoma and the like, and has great potential and broad market prospect. However, it has been found during the clinical trial phase that some patients develop drug resistance upon sustained administration and have been shown to be caused by NTRK mutations, such as the G595R or G667C mutations of NTRK 1. The development of new generation TRK kinase inhibitors is expected to solve these problems.

Disclosure of Invention

In view of the above problems, the present invention aims to develop a new generation of TRK kinase inhibitor compounds with novel structural types and better patentability. The compound with the structure shown in the general formula (I) or diastereoisomers and pharmaceutically acceptable salts thereof are found to have excellent effect and action as a new generation TRK kinase inhibitor.

In a first aspect, the present invention provides a compound of formula (I) or a tautomer, enantiomer, diastereomer, and pharmaceutically acceptable salt thereof:

Wherein:

X is selected from N or CH;

R ¹、R² is independently selected from a hydrogen atom, halogen, C _1～3 alkyl or C _1～3 alkoxy;

R ³ is selected from a hydrogen atom, halogen, cyano, C _1～3 alkyl, C _3～6 cycloalkyl or C _1～3 alkoxy.

Preferably, X is selected from N or CH;

R ¹、R² is independently selected from a hydrogen atom or halogen;

R ³ is selected from a hydrogen atom, halogen or cyano.

Further preferably, X is selected from N or CH;

R ¹、R² is independently selected from a hydrogen atom, a fluorine atom, or a chlorine atom;

R ³ is selected from a hydrogen atom, a fluorine atom or a cyano group.

Still more preferably, the compound is selected from:

(R) - (2- (5- (2- (2-, 2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1H-benzo [ d ] imidazol-6-yl) dimethylphosphine oxide;

(R) -2- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -6- (dimethylphosphoryl) -1H-benzo [ d ] imidazole-5-carbonitrile;

(R) - (2- (5- (2- (2-, 2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -5-fluoro-1H-benzo [ d ] imidazol-6-yl) dimethylphosphine oxide;

(R) - (2- (5- (2- (3-chlorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1H-benzo [ d ] imidazol-6-yl) dimethylphosphine oxide;

(R) - (2- (5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1H-benzo [ d ] imidazol-6-yl) dimethylphosphine oxide;

(R) -6- (dimethylphosphoryl) -2- (5- (2- (3-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1H-benzo [ d ] imidazole-5-carbonitrile;

(R) - (2- (5- (2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -3H-imidazo [4,5-b ] pyridin-6-yl) dimethylphosphine oxide.

In a second aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) or a tautomer, enantiomer, diastereomer, and pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

In a third aspect, the present invention provides the use of a compound of formula (I) or a diastereomer thereof, and a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above, in the manufacture of a medicament for the prevention and/or treatment of tropomyosin-related kinase mediated diseases.

The medicament may be a tropomyosin-related kinase inhibitor. Also, the medicament can be used for preventing and/or treating the related diseases of tropomyosin related kinase dysfunction caused by tropomyosin related kinase gene amplification, over-expression, mutation or fusion. Also, the disease includes, but is not limited to, cancers such as lung cancer, hematological malignancy, prostate cancer, breast cancer, ovarian cancer, glioma, pancreatic cancer, hepatobiliary tract cancer, papillary thyroid cancer, colon cancer, head and neck squamous cell carcinoma, melanoma.

Detailed Description

The invention is further illustrated by the following embodiments, which are to be understood as merely illustrative of the invention and not limiting thereof.

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

"Alkyl" refers to a saturated aliphatic hydrocarbon group, including straight or branched chain groups of 1 to 10 carbon atoms. Alkyl groups having 1 to 5 carbon atoms are preferred. More preferably an alkyl group having 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl.

The carbon atom content of the various carbon-hydrogen containing moieties is represented by the prefix of the moiety, which is labeled with the minimum and maximum number of carbon atoms, i.e., prefix C _i～j represents that the number of carbon atoms of the moiety is an integer from "i" to "j" (including i and j). Thus, for example, a C _1～3 alkyl group refers to an alkyl group of 1 to 3 carbon atoms (including 1 and 3).

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

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

Unless otherwise indicated, all compounds appearing in the present invention are intended to include all possible isomers, such as tautomers, enantiomers, diastereomers, and mixtures thereof.

The term "compound of the present invention" refers to a compound represented by the general formula (I). The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compound of formula (I).

The term "pharmaceutically acceptable salt" refers to salts of the compounds of the invention with acids or bases that are suitable for use as medicaments. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is the salts of the compounds of the present invention with acids. Suitable salts forming acids include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, and the like; acidic amino acids such as aspartic acid and glutamic acid.

The term "pharmaceutically acceptable carrier" means a carrier that can be used to prepare pharmaceutical compositions, which are generally safe, nontoxic, not biologically or otherwise undesirable, and includes carriers that are pharmaceutically acceptable to animals and humans. As used in the specification and claims, a "pharmaceutically acceptable carrier" includes one or more such carriers.

The terms "comprising," "including," or "comprising" mean that the various ingredients can be used together in a mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "containing.

The term "preventing" refers, for example, to the prevention of progression of clinical symptoms of a disease in a mammal that may be exposed to or pre-treated for the disease but has not yet experienced or displayed symptoms of the disease.

The term "treating" may refer to inhibiting a disease, e.g., preventing or reducing the progression of a disease or a clinical symptom thereof, or alleviating a disease, e.g., degrading a disease or a clinical symptom thereof.

Compounds of the general formula (I)

Wherein:

X is selected from N or CH;

In some embodiments of the invention, R ¹、R² is independently selected from a hydrogen atom or halogen. In a more preferred embodiment, R ¹、R² is independently selected from a hydrogen atom, a fluorine atom, or a chlorine atom.

In some embodiments of the invention, R ³ is selected from a hydrogen atom, halogen, or cyano. In a more preferred embodiment, R ³ is selected from a hydrogen atom, a fluorine atom, or a cyano group.

In some embodiments of the invention, the compound of formula (I) is selected from the compounds shown in table 1.

TABLE 1

The preparation method of the compound of the general formula (I) is concretely described in the examples.

General synthetic route

In the above synthetic route, the compounds are labeled as the compound of formula I-1, the compound of formula I-2, the compound of formula I-3 and the compound of formula I-4 in order from left to right, and each step is step (a), step (b) and step (c) in order from left to right.

Wherein X, R ¹、R²、R³ is defined as described above.

In step (a), the compound of formula I-1 is reacted with the corresponding fragment (labeled A) of the synthetic route to give the compound of formula I-2.

The molar ratio of the compound of formula I-1 to A may be 1: (0.8-2.0). The reaction solvent can be ethanol, acetonitrile, acetone, tetrahydrofuran, dioxane, N-dimethylformamide, etc. The reaction temperature in step (a) may be suitably set by a person skilled in the art, for example (50 to 120 ℃).

In the step (b), the compound of the formula I-2 is hydrolyzed to remove ethyl to obtain the compound of the formula I-3. The reaction conditions may be those commonly used in the art for the hydrolysis of esters. For example, ethyl groups are removed in an ethanol solution using an aqueous lithium hydroxide solution. The molar ratio of the compound of formula I-2 to lithium hydroxide is 1: (1.0-10.0).

In step (c), the compound of formula I-3 is reacted with the corresponding fragment of the synthetic route (labeled B) to provide the compound of formula I-4.

The molar ratio of the compound of formula I-3 to B may be 1: (1.0-2.0). The reaction solvent may be acetonitrile, tetrahydrofuran, dioxane, N-dimethylformamide, etc. Step (c) may be performed in the presence of phosphorus oxychloride. The volume ratio of phosphorus oxychloride to acetonitrile can be (0.1-1.0): 1. the reaction temperature in step (c) may be set appropriately by those skilled in the art, and may be, for example, 80 to 100 ℃.

Use of compounds of formula (I)

The compounds of formula (I) are useful as inhibitors of tropomyosin-related kinase (TRK).

The compounds of the general formula (I) are useful for the prevention and/or treatment of diseases associated with abnormal TRK function caused by amplification, overexpression, mutation or fusion of the TRK gene.

The compounds of formula (I) are useful for the prevention and/or treatment of TRK mediated diseases including, but not limited to, cancers such as lung cancer, hematological malignancies, prostate cancer, breast cancer, ovarian cancer, glioma, pancreatic cancer, hepatobiliary tract cancer, papillary thyroid cancer, colon cancer, squamous cell carcinoma of the head and neck, melanoma.

Pharmaceutical composition

The pharmaceutical composition of the present invention comprises an effective amount of a compound of formula (I) or a tautomer, enantiomer, diastereomer, and mixture thereof, and pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier or excipient or diluent thereof.

By "effective amount" is meant a compound of the present invention: (i) treating a particular disease, condition, or disorder, (ii) reducing, ameliorating, or eliminating one or more symptoms of a particular disease, condition, or disorder, or (iii) preventing or delaying the onset of one or more symptoms of a particular disease, condition, or disorder described herein.

Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, and the like), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, and the like), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, and the like), emulsifiers (e.g.80，20 Wetting agents (such as sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include, but are not limited to: oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

The compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

Another aspect of the present invention relates to a method of inhibiting tropomyosin-related kinase (TRK) comprising administering to a patient in need thereof an effective amount of a compound of formula (I) or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

The structure of the compound is determined by Nuclear Magnetic Resonance (NMR) or Mass Spectrometry (MS), and the purity of the compound is determined by liquid phase high pressure chromatography (HPLC). NMR measurements were performed using a Bruker AVANCE-400 NMR apparatus with deuterated dimethyl sulfoxide (DMSO-d 6) or deuterated methanol (MeOH-d 4), with internal standard Tetramethylsilane (TMS), and chemical shifts in ppm. MS was determined using an agilent 6120 mass spectrometer. HPLC was determined using an agilent 1200DAD high pressure liquid chromatograph.

In the case of the invention, which is not specified, the eluent gradient refers to the volume ratio of the eluent.

In the case where the present invention is not specifically described, concentrated sulfuric acid refers to a 98% by mass concentrated sulfuric acid aqueous solution.

In the case where the present invention is not specifically described, 10% palladium on carbon means Pd/c (10%), that is, the mass fraction of palladium is 10%.

Example 1

(R) - (2- (5- (2- (2-, 2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1H-benzo [ d ] imidazol-6-yl) dimethylphosphine oxide

First step (4-aminophenyl) dimethylphosphine oxide

4-Iodoaniline (32.0 g,148.1 mmol), phosphorus dimethyl oxide (12.1 g,155.6 mmol), potassium phosphate (47.1 g,222.2 mmol), palladium acetate (1.66 g,7.405 mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (8.6 g,14.81 mmol) were dissolved in a mixture of dioxane (400 mL) and water (40 mL) and replaced three times with nitrogen at room temperature. The reaction solution was then stirred at 120℃for 14 hours under nitrogen protection. After completion of the reaction, the reaction mixture was filtered, dried and concentrated, and the residue was purified by column chromatography (eluent gradient, dichloromethane: methanol=20:1) to give (4-aminophenyl) dimethylphosphine oxide (4.15 g, yield 16.4%) as a brown solid .¹H NMR(400MHz,DMSO-d6)δ:7.38-7.32(2H,m),6.62-6.58(2H,m),5.61(2H,br s),1.51(6H,d).MS m/z(ESI):170.2[M+1]⁺.

Second step (4-amino-3-nitrophenyl) dimethylphosphine oxide

(4-Aminophenyl) dimethylphosphine oxide (4.10 g,24.23 mmol) was added to a solution of concentrated sulfuric acid (20 mL) cooled to 0deg.C, followed by the addition of potassium nitrate (2.96 g,29.27 mmol) in portions. After the addition was completed, the reaction was continued at 0℃for 30 minutes until completion. The reaction solution was poured into ice water, extracted with ethyl acetate, the organic phases combined, dried over anhydrous sodium sulfate, filtered, the filtrate concentrated under reduced pressure, and the residue purified by silica gel column chromatography (eluent gradient, petroleum ether: ethyl acetate=10:1) to give 4-amino-3-nitrophenyl) dimethylphosphine oxide (1.83 g, yield 35.1%) as a yellow solid. MS m/z (ESI) 214.3[ M+1] ⁺.

Third step (3, 4-diaminophenyl) dimethylphosphine oxide

4-Amino-3-nitrophenyl) dimethylphosphine oxide (1.80 g,7.46 mmol) was dissolved in methanol (15 mL), 10% palladium on charcoal (200 mg) was added, hydrogen was replaced three times, and the reaction mixture was stirred overnight under a hydrogen atmosphere. The solid residue was filtered off and the residue was washed with methanol, the resulting filtrates were combined and dried, and the residue was purified by preparative thin layer chromatography (eluent gradient, dichloromethane: methanol=5:1) to give (3, 4-diaminophenyl) dimethylphosphine oxide (850 mg, yield 61.8%) as a brown solid. MS m/z (ESI): 185.1[ M+1] ⁺.

Fourth step (R) -5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester

(R) -2- (2, 5-difluorophenyl) pyrrolidine (1.31 g,7.13 mmol) and ethyl 5-chloropyrazolo [1,5-a ] pyrimidine-3-carboxylate (1.61 g,7.13 mmol) were dissolved in ethanol (20 mL), triethylamine (2.16 g,21.4 mmol) was added at room temperature, and the reaction was heated at 80℃overnight. After cooling to room temperature, the reaction solution was poured into ice water, extracted with dichloromethane, the combined organic phases were dried over anhydrous sodium sulfate, filtered, the organic phases were concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent gradient, ethyl acetate: petroleum ether=1:5) to give (R) -5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester (2.0 g, yield 75.4%) as a white solid. MS m/z (ESI): 372.9[ M+1] ⁺.

Fifth step (R) -5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid

(R) - (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylate (2.0 g,5.37 mmol) was suspended in ethanol (30 mL) and heated at 90℃for 20 min, aqueous LiOH (11.4 mL,1M,11.4 mmol) was added and the reaction mixture was heated at 90℃for 15 h after cooling, the reaction mixture was concentrated, diluted with water and washed with ethyl acetate to remove any unreacted starting material, the ethyl acetate phase was discarded, the aqueous layer was then acidified to pH=1 using 2M hydrochloric acid, the acidified aqueous phase was extracted with ethyl acetate, the organic fraction was combined, dried over anhydrous sodium sulfate, filtered and concentrated to give (R) -5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylate (1.65 g, 89.3%) as pale yellow solid ¹H-NMR(DMSO-d₆, 400 MHz: 11.45 (1H, 8.5 s) and 0.59.5 s 8.77(0.5H,s),8.15-8.19(1H,m),6.99-7.33(3H,m),6.67(0.5H,s),6.07(0.5H,s),5.53(0.5H,s),5.32(0.5H,s),4.00(1H,br.s),3.77(0.5H,br.s),3.64(0.5H,br.s),2.33-2.60(1H,br.s),1.92-2.04(3H,m).MS m/z(ESI):345.0[M+1]⁺.

Sixth step (R) - (2- (5- (2- (2-, 2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1H-benzo [ d ] imidazol-6-yl) dimethylphosphine oxide

(R) -5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid (173 mg,0.50 mmol) was dissolved in acetonitrile (15 mL), and (3, 4-diaminophenyl) dimethylphosphine oxide (138 mg,0.75 mmol) and phosphorus oxychloride (1.5 mL) were added and stirred at 100deg.C for 16 hours. After the completion of the reaction, the organic phase was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate, followed by washing with a saturated sodium hydrogencarbonate solution and then with a saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography (eluent gradient, ethyl acetate: petroleum ether=1:5) to give (R) - (2- (5- (2- (2-, 2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1H-benzo [ d ] imidazol-6-yl) dimethylphosphine oxide (97 mg, yield 39.5%) as a pale yellow solid .¹H NMR(400MHz,MeOD-d4)δ8.73(s,1H),8.24(d,J＝8.0Hz,1H),7.73-7.66(m,2H),7.52-7.17(m,3H),7.08(d,J＝8.0Hz,1H),6.81-6.65(m,2H),3.66-3.57(m,1H),2.45-2.35(m,2H),2.20-2.10(m,2H),1.93-1.84(m,2H),1.70(s,6H).MS m/z(ESI):495.6[M+1]⁺.

Example 2

(R) -2- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -6- (dimethylphosphoryl) -1H-benzo [ d ] imidazole-5-carbonitrile

First step 5-amino-2- (dimethylphosphoryl) benzonitrile

5-Amino-2-iodobenzonitrile (3.60 g,15 mmol), phosphorus dimethyl oxide (1.56 g,20 mmol), potassium phosphate (4.14 g,20 mmol), palladium acetate (166 mg,0.74 mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (860 mg,1.48 mmol) were dissolved in a mixture of dioxane (50 mL) and water (5 mL) and replaced three times with nitrogen at room temperature. The reaction solution was then stirred at 120℃for 14 hours under nitrogen protection. After completion of the reaction, the reaction mixture was filtered, dried and concentrated, and the residue was purified by column chromatography (eluent gradient, dichloromethane: methanol=20:1) to give 5-amino-2- (dimethylphosphoryl) benzonitrile (1.32 g, yield 45.3%) as a brown solid. MS m/z (ESI): 195.1[ M+1] ⁺.

Second step 5-amino-2- (dimethylphosphoryl) -4-nitrobenzonitrile

5-Amino-2- (dimethylphosphoryl) benzonitrile (1.30 g,6.7 mmol) was added to a solution of concentrated sulfuric acid (20 mL) cooled to 0deg.C, followed by the addition of potassium nitrate (800 mg,8.0 mmol) in portions. After the addition was completed, the reaction was continued at 0℃for 30 minutes until completion. The reaction solution was poured into ice water, extracted with ethyl acetate, the organic phases combined, dried over anhydrous sodium sulfate, filtered, the filtrate concentrated under reduced pressure, and the residue purified by silica gel column chromatography (eluent gradient, petroleum ether: ethyl acetate=10:1) to give 5-amino-2- (dimethylphosphoryl) -4-nitrobenzonitrile (510 mg, yield 31.8%) as a yellow solid. MS m/z (ESI) 240.3[ M+1] ⁺.

Third step 4, 5-diamino-2- (dimethylphosphoryl) benzonitrile

5-Amino-2- (dimethylphosphoryl) -4-nitrobenzonitrile (510 mg,2.13 mmol) was dissolved in methanol (15 mL), 10% palladium on charcoal (200 mg) was added thereto, the hydrogen gas was replaced three times, and the reaction solution was stirred overnight under a hydrogen gas atmosphere. The solid residue was filtered off and the residue was washed with methanol, the resulting filtrates were combined and dried, and the residue was purified by preparative thin layer chromatography (eluent gradient, dichloromethane: methanol=5:1) to give 4, 5-diamino-2- (dimethylphosphoryl) benzonitrile (350 mg, yield 78.6%) as a yellow solid. MS m/z (ESI) 210.1[ M+1] ⁺.

Fourth step (R) -2- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -6- (dimethylphosphoryl) -1H-benzo [ d ] imidazole-5-carbonitrile

(R) -5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid (173 mg,0.50 mmol) was dissolved in acetonitrile (15 mL) and 4, 5-diamino-2- (dimethylphosphoryl) benzonitrile (158 mg,0.75 mmol) and phosphorus oxychloride (1.5 mL) were added and stirred at 100deg.C for 16 hours. After the completion of the reaction, the organic phase was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate, followed by washing with a saturated sodium hydrogencarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography (eluent gradient, ethyl acetate: petroleum ether=1:5) to give (R) -2- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -6- (dimethylphosphoryl) -1H-benzo [ d ] imidazole-5-carbonitrile (51 mg, yield 19.7%) as a pale yellow solid .¹H NMR(400MHz,MeOD-d4)δ8.71(s,1H),8.54(d,J＝8.0Hz,1H),7.75-7.70(m,1H),7.42-7.10(m,3H),7.01(d,J＝8.0Hz,1H),6.84-6.69(m,2H),3.96-3.87(m,1H),2.54-2.50(m,2H),2.22-2.16(m,2H),1.94-1.89(m,2H),1.71(s,6H).MS m/z(ESI):518.1[M+1]⁺.

Example 3

(R) - (2- (5- (2- (2-, 2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -5-fluoro-1H-benzo [ d ] imidazol-6-yl) dimethylphosphine oxide

First step (4-amino-2-fluorophenyl) dimethylphosphine oxide

3-Fluoro-4-iodoaniline (3.56 g,15 mmol), phosphorus dimethyl oxide (1.56 g,20 mmol), potassium phosphate (4.14 g,20 mmol), palladium acetate (166 mg,0.74 mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (860 mg,1.48 mmol) were dissolved in a mixture of dioxane (50 mL) and water (5 mL) and replaced three times with nitrogen at room temperature. Then stirring the reaction solution for 14 hours at 120 ℃ under the protection of nitrogen; after completion of the reaction, the reaction mixture was filtered, dried and concentrated. The residue was purified by column chromatography (eluent gradient, dichloromethane: methanol=20:1) to give (4-amino-2-fluorophenyl) dimethylphosphine oxide (1.55 g, yield 55.1%) as a brown solid. MS m/z (ESI): 188.1[ M+1] ⁺.

Second step (4-amino-2-fluoro-5-nitrophenyl) dimethylphosphine oxide

(4-Amino-2-fluorophenyl) dimethylphosphine oxide (1.50 g,8.3 mmol) was added to a solution of concentrated sulfuric acid (20 mL) cooled to 0deg.C, followed by the addition of potassium nitrate (1.24 g,12.4 mmol) in portions. After the addition was completed, the reaction was continued at 0℃for 30 minutes until completion. The reaction solution was poured into ice water, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (gradient of eluent, petroleum ether: ethyl acetate=10:1) to give (4-amino-2-fluoro-5-nitrophenyl) dimethylphosphine oxide (630 mg, yield 32.7%) as a yellow solid. MS m/z (ESI): 233.3[ M+1] ⁺.

Third step (4, 5-diamino-2-fluorophenyl) dimethylphosphine oxide

(4-Amino-2-fluoro-5-nitrophenyl) dimethylphosphine oxide (630 mg,2.71 mmol) was dissolved in methanol (15 mL), 10% palladium on charcoal (200 mg) was added, hydrogen was replaced three times, and the reaction solution was stirred overnight under a hydrogen atmosphere. The solid residue was filtered off and the residue was washed with methanol, the resulting filtrates were combined and dried, and the residue was purified by preparative thin layer chromatography (eluent gradient, dichloromethane: methanol=5:1) to give (4, 5-diamino-2-fluorophenyl) dimethylphosphine oxide (380 mg, yield 69.4%) as a yellow solid. MS m/z (ESI) 203.1[ M+1] ⁺.

Fourth step (R) - (2- (5- (2- (2-, 2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -5-fluoro-1H-benzo [ d ] imidazol-6-yl) dimethylphosphine oxide

(R) -5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid (173 mg,0.50 mmol) was dissolved in acetonitrile (15 mL), and (4, 5-diamino-2-fluorophenyl) dimethylphosphine oxide (150 mg,0.75 mmol) and phosphorus oxychloride (1.5 mL) were added and stirred at 100℃for 16 hours. After the completion of the reaction, the organic phase was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate, followed by washing with a saturated sodium hydrogencarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography (eluent gradient, ethyl acetate: petroleum ether=1:5) to give (R) - (2- (5- (2- (2-, 2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -5-fluoro-1H-benzo [ d ] imidazol-6-yl) dimethylphosphine oxide (65 mg, yield 24.9%) as a pale yellow solid .¹H NMR(400MHz,MeOD-d4)δ8.76(s,1H),8.14(d,J＝8.0Hz,1H),7.74-7.70(m,1H),7.24-7.10(m,3H),7.07(d,J＝8.0Hz,1H),6.85-6.71(m,2H),4.02-3.87(m,1H),2.55-2.51(m,2H),2.32-2.17(m,2H),1.94-1.88(m,2H),1.73(s,6H).MS m/z(ESI):511.2[M+1]⁺.

Example 4

(R) - (2- (5- (2- (3-chlorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1H-benzo [ d ] imidazol-6-yl) dimethylphosphine oxide

First step (R) -5- (2- (3-chlorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester

(R) -2- (3-chlorophenyl) pyrrolidine (1.30 g,7.13 mmol) and 5-chloropyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester (1.61 g,7.13 mmol) were dissolved in ethanol (20 mL), triethylamine (2.16 g,21.4 mmol) was added at room temperature, and the reaction was heated at 80℃overnight with stirring. After cooling to room temperature, the reaction solution was poured into ice water, extracted with dichloromethane, the combined organic phases were dried over anhydrous sodium sulfate, filtered, the organic phases were concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent gradient, ethyl acetate: petroleum ether=1:5) to give ethyl (R) -5- (2- (3-chlorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylate (2.0 g, yield 75.9%) as a white solid. MS m/z (ESI) 371.8[ M+1] ⁺.

Second step (R) -5- (2- (3-chlorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid

(R) -5- (2- (3-chlorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester (2.0 g,5.37 mmol) was suspended in ethanol (30 mL) and heated at 90℃for 20 minutes. Aqueous LiOH (11.4 ml,1m,11.4 mmol) was added and the reaction mixture was heated at 90 ℃ for 15 hours. After cooling, the reaction mixture was concentrated, diluted with water and washed with ethyl acetate to remove any unreacted starting material, and the ethyl acetate phase was discarded. The aqueous layer was then acidified to ph=1 or so using 2M hydrochloric acid. After extraction with ethyl acetate, the combined organic portions were dried over anhydrous sodium sulfate, filtered and concentrated to give (R) -5- (2- (3-chlorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid (1.65 g, 89.9% yield) as a pale yellow solid. MS m/z (ESI): 343.0[ M+1] ⁺.

Third step (R) - (2- (5- (2- (3-chlorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1H-benzo [ d ] imidazol-6-yl) dimethylphosphine oxide

(R) -5- (2- (3-chlorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid (173 mg,0.50 mmol) was dissolved in acetonitrile (15 mL), and (3, 4-diaminophenyl) dimethylphosphine oxide (138 mg,0.75 mmol) and phosphorus oxychloride (1.5 mL) were added and stirred at 100℃for 16 hours. After the completion of the reaction, the organic phase was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate, followed by washing with a saturated sodium hydrogencarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography (eluent gradient, ethyl acetate: petroleum ether=1:5) to give (R) - (2- (5- (2- (3-chlorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1H-benzo [ d ] imidazol-6-yl) dimethylphosphine oxide (47 mg, yield 19.1%) as a yellow solid .¹H NMR(400MHz,MeOD-d4)δ8.75(s,1H),8.24(d,J＝8.0Hz,1H),7.75-7.70(m,3H),7.24-7.13(m,3H),7.09(d,J＝8.0Hz,1H),6.95-6.75(m,2H),3.93-3.85(m,1H),2.55-2.51(m,2H),2.36-2.27(m,2H),1.95-1.88(m,2H),1.74(s,6H).MS m/z(ESI):491.0[M+1]⁺.

Example 5

(R) - (2- (5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1H-benzo [ d ] imidazol-6-yl) dimethylphosphine oxide

First step (R) -5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester

(R) -2- (5-chloro-2-fluorophenyl) pyrrolidine (1.42 g,7.13 mmol) and ethyl 5-chloropyrazolo [1,5-a ] pyrimidine-3-carboxylate (1.61 g,7.13 mmol) were dissolved in ethanol (20 mL), triethylamine (2.16 g,21.4 mmol) was added at room temperature, and the reaction was heated at 80℃under stirring overnight. After cooling to room temperature, the reaction solution was poured into ice water, extracted with dichloromethane, the combined organic phases were dried over anhydrous sodium sulfate, filtered, the organic phases were concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent gradient, ethyl acetate: petroleum ether=1:5) to give (R) -5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester (2.0 g, yield 72.3%) as a white solid. MS m/z (ESI): 389.4[ M+1] ⁺.

Second step (R) -5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid

(R) -5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester (2.0 g,5.16 mmol) was suspended in ethanol (30 mL) and heated at 90℃for 20 minutes. Aqueous LiOH (11.4 ml,1m,11.4 mmol) was added and the reaction mixture was heated at 90 ℃ for 15 hours. After cooling, the reaction mixture was concentrated, diluted with water and washed with ethyl acetate to remove any unreacted starting material, and the ethyl acetate phase was discarded. The aqueous layer was then acidified to ph=1 or so using 2M hydrochloric acid. After extraction with ethyl acetate, the combined organic portions were dried over anhydrous sodium sulfate, filtered and concentrated to give (R) -5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid (1.70 g, yield 91.3%) as a pale yellow solid. MS m/z (ESI): 361.2[ M+1] ⁺.

Third step (R) - (2- (5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1H-benzo [ d ] imidazol-6-yl) dimethylphosphine oxide

(R) -5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid (180 mg,0.50 mmol) was dissolved in acetonitrile (15 mL), and (3, 4-diaminophenyl) dimethylphosphine oxide (138 mg,0.75 mmol) and phosphorus oxychloride (1.5 mL) were added and stirred at 100℃for 16 hours. After the completion of the reaction, the organic phase was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate, followed by washing with a saturated sodium hydrogencarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography (eluent gradient, ethyl acetate: petroleum ether=1:5) to give (R) - (2- (5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1H-benzo [ d ] imidazol-6-yl) dimethylphosphine oxide (50 mg, yield 19.6%) as a pale yellow solid .¹H NMR(400MHz,MeOD-d4)δ8.56(s,1H),8.24(d,J＝8.0Hz,1H),7.75-7.70(m,3H),7.34-7.23(m,2H),7.12(d,J＝8.0Hz,1H),6.85-6.73(m,2H),3.83-3.75(m,1H),2.65-2.57(m,2H),2.31-2.25(m,2H),1.97-1.87(m,2H),1.72(s,6H).MS m/z(ESI):509.2[M+1]⁺.

Example 6

(R) -6- (dimethylphosphoryl) -2- (5- (2- (3-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1H-benzo [ d ] imidazole-5-carbonitrile

First step (R) -5- (2- (3-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester

(R) -2- (3-fluorophenyl) pyrrolidine (1.18 g,7.13 mmol) and 5-chloropyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester (1.61 g,7.13 mmol) were dissolved in ethanol (20 mL), triethylamine (2.16 g,21.4 mmol) was added at room temperature, and the reaction was heated at 80℃under stirring overnight. After cooling to room temperature, the reaction solution was poured into ice water, extracted with dichloromethane, the combined organic phases were dried over anhydrous sodium sulfate, filtered, the organic phases were concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent gradient, ethyl acetate: petroleum ether=1:5) to give ethyl (R) -5- (2- (3-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylate (2.0 g, yield 79.2%) as a white solid. MS m/z (ESI): 355.1[ M+1] ⁺.

Second step (R) -5- (2- (3-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid

(R) -5- (2- (3-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester (2.0 g,5.65 mmol) was suspended in ethanol (30 mL) and heated at 90℃for 20 minutes. Aqueous LiOH (11.4 ml,1m,11.4 mmol) was added and the reaction mixture was heated at 90 ℃ for 15 hours. After cooling, the reaction mixture was concentrated, diluted with water and washed with ethyl acetate to remove any unreacted starting material, and the ethyl acetate phase was discarded. The aqueous layer was then acidified to ph=1 using 2M hydrochloric acid. After extraction with ethyl acetate, the combined organic portions were dried over anhydrous sodium sulfate, filtered and concentrated to give (R) -5- (2- (3-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid (1.60 g, 86.9%) as a pale yellow solid. MS m/z (ESI): 327.0[ M+1] ⁺.

Third step (R) -6- (dimethylphosphoryl) -2- (5- (2- (3-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1H-benzo [ d ] imidazole-5-carbonitrile

(R) -5- (2- (3-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid (163 mg,0.50 mmol) was dissolved in acetonitrile (15 mL), 4, 5-diamino-2- (dimethylphosphoryl) benzonitrile (158 mg,0.75 mmol) and phosphorus oxychloride (1.5 mL) were added and stirred at 100℃for 16 hours. After the completion of the reaction, the organic phase was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate, followed by washing with a saturated sodium hydrogencarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography (eluent gradient, ethyl acetate: petroleum ether=1:5) to give (R) -6- (dimethylphosphoryl) -2- (5- (2- (3-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1H-benzo [ d ] imidazole-5-carbonitrile (35 mg, yield 14.0%) as a pale yellow solid .¹H NMR(400MHz,MeOD-d4)δ8.76(s,1H),8.34(d,J＝8.0Hz,1H),7.77-7.72(m,3H),7.44-7.33(m,2H),7.22(d,J＝8.0Hz,1H),6.84-6.73(m,2H),3.87-3.75(m,1H),2.65-2.57(m,2H),2.31-2.25(m,2H),1.97-1.87(m,2H),1.71(s,6H).MS m/z(ESI):500.0[M+1]⁺.

Example 7

(R) - (2- (5- (2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -3H-imidazo [4,5-b ] pyridin-6-yl) dimethylphosphine oxide

First step (5, 6-diaminopyridin-3-yl) dimethylphosphine oxide

2, 3-Diamino-5-iodopyridine (3.52 g,15 mmol), phosphorus dimethyl oxide (1.56 g,20 mmol), potassium phosphate (4.14 g,20 mmol), palladium acetate (166 mg,0.74 mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (860 mg,1.48 mmol), and a mixture of dioxane (50 mL) and water (5 mL) were dissolved in a nitrogen atmosphere and replaced three times at room temperature. Then stirring the reaction solution for 14 hours at 120 ℃ under the protection of nitrogen; after completion of the reaction, the reaction mixture was filtered, dried and concentrated. The residue was purified by column chromatography (eluent gradient, dichloromethane: methanol=10:1) to give (5, 6-diaminopyridin-3-yl) dimethylphosphine oxide (1.05 g, yield 37.8%) as a brown solid. MS m/z (ESI): 186.1[ M+1] ⁺.

Second step (R) - (2- (5- (2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -3H-imidazo [4,5-b ] pyridin-6-yl) dimethylphosphine oxide

(R) -5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid (173 mg,0.50 mmol) was dissolved in acetonitrile (15 mL), and (5, 6-diaminopyridin-3-yl) dimethylphosphine oxide (138 mg,0.75 mmol) and phosphorus oxychloride (1.5 mL) were added and stirred at 100℃for 16 hours. After the completion of the reaction, the organic phase was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate, followed by washing with a saturated sodium hydrogencarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography (eluent gradient, ethyl acetate: petroleum ether=1:5) to give (R) - (2- (5- (2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -3H-imidazo [4,5-b ] pyridin-6-yl) dimethylphosphine oxide (51 mg, yield 19.7%) as a yellow solid .¹H NMR(400MHz,MeOD-d4)δ8.77(s,1H),8.54(d,J＝8.0Hz,1H),7.67-7.62(m,2H),7.54-7.43(m,2H),7.22(d,J＝8.0Hz,1H),6.84-6.73(m,2H),3.87-3.75(m,1H),2.67-2.57(m,2H),2.33-2.29(m,2H),1.87-1.83(m,2H),1.70(s,6H).MS m/z(ESI):494.1[M+1]⁺.

Test example: determination of TRKA kinase Activity inhibitory Effect of Compounds

The following experiments were performed to determine the inhibition of TRKA kinase activity by the compounds of the present invention.

1. Experimental materials

TABLE 2

2. Experimental instrument

Full-automatic multi-functional multichannel enzyme-labeled appearance: MD, spectraMax i3x

3. Reagent preparation

(1) 1-Fold preparation of Kinase Buffer working solution (1 XKinase Buffer): 200. Mu.l of a 5-fold Kinase Buffer (5 XKinase Buffer), 5. Mu.l of 1M MgCl ₂ stock solution and 1. Mu.l of 1M DTT stock solution were taken, dissolved in 794. Mu.l of double distilled water and mixed well to obtain a 1-fold Kinase Buffer.

(2) 5. Mu.M tyrosine kinase Substrate solution (Substrate-TK solution) was prepared: the Substrate-TK was diluted to 5. Mu.M with 1 XKinase Buffer. The reaction plate was charged with 4. Mu.l/well and the final concentration of the Substrate-TK was 1. Mu.M.

(3) Compound solution preparation: a2.5 Xsolution of the compound was prepared with a 1 XKinase Buffer. Add 4. Mu.l/well to the reaction plate.

(4) 75. Mu.M ATP: ATP was diluted 75. Mu.M with 1 XKinase Buffer. The reaction plate was charged with 4. Mu.l/well and the final ATP concentration was 15. Mu.M.

(5) Preparing TRKA: TRKA enzyme was formulated at 1.5 ng/. Mu.l with 1 XKinase Buffer. The reaction plate was charged with 2. Mu.l/well and the TRKA final concentration was 3 ng/well.

(6) Preparing streptavidin: the fluorescein XL166 labeled Streptavidin (strepavidin-XL 66) stock was diluted to 500nM with Detection Buffer. Add to the reaction plate at 5 μl/well.

(7) Preparing tyrosine phosphorylation antibody: the Cryptate-labeled tyrosine phosphorylate antibody (TK-Ab-Cryptate) stock was diluted 100-fold with Detection Buffer. Add to the reaction plate at 5 μl/well.

3. Experimental procedure

(1) And (3) preparation of a reagent: the reagent was prepared as described above, and TRKA kinase enzyme (Carna, cat. No. 08-186) was placed at 4℃for use, and the remaining reagents were equilibrated to room temperature.

(2) Substrate phosphorylation: the substrate TK-biotin, ATP, enzyme and a certain concentration of the compound are uniformly mixed in a1 XKinase Buffer (the volume percentage concentration of DMSO in the reaction system is controlled to be 1 percent), and the mixture is reacted for 40 minutes at room temperature.

(3) Specific binding reaction: to each reaction well, 10. Mu.l of a mixture of Strepitavidin-XL 665 and TK antibody europi. Mu. M CRYPTATE diluted with a Detection Buffer was added, and the mixture was reacted at room temperature for 1 hour.

(4) And (3) detecting: fluorescence signals (excitation light 320nm, emission light 315 nm, 6615 nm) were detected using M [ mu ] ltilabel Reader (MD, spectraMax i3 x) multifunction.

(5) Data analysis and mapping: subtracting the background signal value from the background signal hole, and comparing the background signal value with a full-active enzyme active hole (solvent control hole), and calculating the inhibition effect of each compound on TRKA enzyme activity; and plotted using the plotting analysis software PRISM 6.0.

4. Data analysis

Excitation Ratio (ER) =665 nm emission optical signal value/615 nm emission optical signal value

Inhibition = (ER _{Solvent set}―ER_{Sample of})/(ER_{Solvent set}―ER_{Blank space}) ×100%

5. Experimental results

The inhibition ratios (IC ₅₀) of the compounds of the examples are shown in table 3.

TABLE 3 Table 3

As can be seen from Table 3, the compounds of the present invention have excellent TRKA kinase inhibitory activity.

Claims

1. A compound of formula (I) or a tautomer, enantiomer, diastereomer or a pharmaceutically acceptable salt thereof,

；

Wherein:

x is selected from N or CH;

R ³ is selected from a hydrogen atom, a fluorine atom or a cyano group.

2. The compound of claim 1, or a tautomer, enantiomer, diastereomer, and pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

3. A pharmaceutical composition comprising a compound of claim 1 or 2, or a tautomer, enantiomer, diastereomer, and pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

4. Use of a compound according to claim 1 or 2, or a tautomer, enantiomer, diastereomer or pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention and/or treatment of tropomyosin-related-kinase mediated disease.

5. The use according to claim 4, wherein the medicament is a tropomyosin-related kinase inhibitor.

6. The use according to claim 4, wherein the disease is a related disease of tropomyosin-related kinase dysfunction caused by tropomyosin-related kinase gene amplification, overexpression, mutation, or fusion.

7. The use according to claim 4, wherein the disease is a cancer selected from lung cancer, hematological malignancy, prostate cancer, breast cancer, ovarian cancer, brain glioma, pancreatic cancer, hepatobiliary tract liver cancer, papillary thyroid cancer, colon cancer, head and neck squamous cell carcinoma and melanoma.