CN114315900B - 3-aryl pyrazolopyrimidine derivative and application thereof - Google Patents
3-aryl pyrazolopyrimidine derivative and application thereof Download PDFInfo
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Abstract
The invention relates to a novel 3-aryl pyrazolopyrimidine derivative, a preparation method thereof and application thereof in medicines. In particular, the invention relates to a novel 3-aryl 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) in the general formula (I) 1 、R 2 、R 3 ) And the group (X) is as defined in the specification.
Description
Technical Field
The present invention relates to a new class of 3-arylpyrazolopyrimidine derivatives, to processes for their preparation and to their use as therapeutic agents, in particular as tropomyosin-related kinase (TRK) inhibitors, as such or as pharmaceutical compositions containing such derivatives.
Background
Tropomyosin-receptor kinase (TRK), which is a type of nerve growth factor receptor, exists in various 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 (neurotrophin-4/5), and the ligands for TRKC are 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 、R 2 independently selected from hydrogen atoms, halogen, C 1～3 Alkyl or C 1～3 An alkoxy group;
R 3 selected from hydrogen atom, halogen, cyano, C 1～3 Alkyl, C 3～6 Cycloalkyl or C 1～3 An alkoxy group.
Preferably, X is selected from N or CH;
R 1 、R 2 independently selected from a hydrogen atom or a halogen;
R 3 selected from a hydrogen atom, a halogen or a cyano group.
Further preferably, X is selected from N or CH;
R 1 、R 2 independently selected from a hydrogen atom, a fluorine atom, or a chlorine atom;
R 3 selected from a hydrogen atom, a fluorine atom or a cyano group.
Still more preferably, the compound is selected from:
(R) - (3- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) phenyl) dimethylphosphine oxide;
(R) -4- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile;
(R) -5- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile;
(R) - (4- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2-fluorophenyl) dimethylphosphine oxide;
(R) -2- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -5- (dimethylphosphoryl) isonicotinnitrile;
(R) -5- (5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile;
(R) -5- (5- (2- (3-chlorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile.
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 hydrocarbon-containing moieties is determined by the minimum and maximum number of carbon atoms in the moietyPrefix representation, i.e. prefix C i～j The number of carbon atoms representing the moiety is an integer from "i" to "j" (including i and j). Thus, for example, C 1～3 Alkyl refers to alkyl groups 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;
R 1 、R 2 independently selected from hydrogen atoms, halogen, C 1～3 Alkyl or C 1～3 An alkoxy group;
R 3 selected from hydrogen atom, halogen, cyano, C 1～3 Alkyl, C 3～6 Cycloalkyl or C 1～3 An alkoxy group.
In some embodiments of the invention, R 1 、R 2 Independently selected from hydrogen atoms or halogen. In a more preferred embodiment, R 1 、R 2 Independently selected from a hydrogen atom, a fluorine atom or a chlorine atom.
In some embodiments of the invention, R 3 Selected from a hydrogen atom, a halogen or a cyano group. In a more preferred embodiment, R 3 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 synthetic route, the compounds are marked as a compound of formula I-1, a compound of formula I-2 and a compound of formula I-3 from left to right, and each step is a step (a) and a step (b) from left to right.
Therein, X, R 1 、R 2 、R 3 Is defined as 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: (0.8-3.0). The reaction solvent may be dimethyl sulfoxide, 1, 4-dioxane, N-dimethylformamide, etc. The reaction temperature of step (a) may be suitably set by a person skilled in the art, for example 80 to 130 ℃. The reaction may be carried out in the presence of potassium fluoride. The molar ratio of the compound of formula I-1 to potassium fluoride is 1: (2.0-6.2).
In step (B), the compound of formula I-2 is reacted with the corresponding fragment of the synthetic route (labeled B) to provide the compound of formula I-3.
The molar ratio of the compound of formula I-2 to B may be 1: (0.8-3.0). The reaction solvent may be 1, 4-dioxane, N-dimethylformamide, etc. Step (b) may be performed in the presence of sodium carbonate and [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride. The molar ratio of the compound of formula I-2, sodium carbonate and [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride may be 1: (1.0-4.0): (0.05-0.1). The reaction temperature in step (b) may be set appropriately by those skilled in the art and may be, for example, 80 to 120 ℃.
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.
Example 1 (R) - (3- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) phenyl) dimethylphosphine oxide
First step (3-bromophenyl) dimethylphosphine oxide
1-bromo-3-iodobenzene (2.81 g,10.0 mmol), dimethylphosphine oxide (1.17 g,15.0 mmol), potassium phosphate (4.24 g,20.0 mmol), tris (dibenzylideneacetone) dipalladium(458 mg,0.5 mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (579 mg,1.0 mmol), dissolved in 1, 4-dioxane (40 mL), and replaced three times with nitrogen at room temperature. The reaction solution was then stirred at 110℃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, petroleum ether: ethyl acetate=10:1) to give (3-bromophenyl) dimethylphosphine oxide (1.50 g, yield 64.3%) as a white solid. MS m/z (ESI): 233.0[ M+1 ]] + 。
Second step dimethyl (3- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) phenyl) phosphine oxide in a 100mL eggplant type bottle, (3-bromophenyl) dimethylphosphine oxide (1.50 g,6.44 mmol), pinacol biborate (2.6 g,10.2 mmol), potassium acetate (2.5 g,25.4 mmol) and [1,1' -bis (diphenylphosphine) ferrocene were sequentially added]Palladium dichloride dichloromethane complex (208 mg,0.254 mmol) and 1, 4-dioxane (40 mL), displaced argon 3 times, were reacted overnight in an oil bath at 80 ℃. After the reaction was completed, the reaction solution was cooled, poured into water (20 mL), and then extracted with ethyl acetate (100 ml×3). The organic phases were combined and washed with saturated brine (50 ml x 3), then dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography (eluent gradient, petroleum ether: ethyl acetate=5:1) to give dimethyl (3- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) phenyl) phosphine oxide (850 mg, yield 47.0%) as a colorless oil. MS m/z (ESI): 281.0[ M+1 ]] + 。
Third step (R) -3-bromo-5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine
(R) -2- (2, 5-difluorophenyl) pyrrolidine (17 g,92.8 mmol) was added to 3-bromo-6-chloroimidazo [1,2-b]A solution of pyridazine (11 g,47.3 mmol) and potassium fluoride (27 g, 460 mmol) in DMSO (100 mL) was heated to 130℃and stirred overnight. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into ice water (500 mL), and extracted with ethyl acetate (200 ml×3). The combined organic phases were washed with saturated aqueous sodium chloride (100 ml×2), dried over anhydrous sodium sulfate, filtered and concentrated to give an orange solid. The solid was separated by column chromatography (eluent gradient, petroleum ether: ethyl acetate=5:1)Isolating to obtain (R) -3-bromo-5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a]Pyrimidine (16 g, 66.8% yield) as a pale yellow solid. MS m/z (ESI): 379.2[ M+1 ]] + 。
Fourth step (R) - (3- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) phenyl) dimethylphosphine oxide
(R) -3-bromo-5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a]Pyrimidine (380 mg,1 mmol), sodium carbonate (318 mg,3 mmol) and dimethyl (3- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) phenyl) phosphine oxide (280 mg,1 mmol) were added to a solution of 1, 4-dioxane/water (10 mL/2 mL), followed by Pd (dppf) Cl 2 (73 mg,0.1 mmol). After the reaction liquid nitrogen is replaced by air for deoxidization, the reaction liquid nitrogen is heated to 100 ℃ under the protection of nitrogen and reacts overnight. After the reaction, the reaction solution was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (100 mL), then washed with saturated aqueous sodium chloride (100 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid. The solid was separated by column chromatography (eluent gradient, dichloromethane: methanol=10:1) to give (R) - (3- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1, 5-a)]Pyrimidin-3-yl) phenyl) dimethylphosphine oxide (45 mg, 9.95% yield) as a white solid. 1 H NMR(400MHz,MeOD-d4)δ8.77(s,1H),8.14(d,J＝8.0Hz,1H),7.67-7.62(m,4H),7.24-7.13(m,3H),6.62(d,J＝8.0Hz,1H),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):453.2[M+1] + 。
Example 2 (R) -4- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile
First step 4-bromo-2- (dimethylphosphoryl) benzonitrile
4-bromo-2-iodobenzonitrile (3.08 g,10.0 mmol), phosphorus oxide dimethyl (1.17 g,15.0 mmol), potassium phosphate (4.24 g,20.0 mmol), tris (dibenzylideneacetone) dipalladium (458 mg,0.5 mmol), 4, 5-bis-diphenylphosphine-9, 9-di-Methyl xanthene (579 mg,1.0 mmol) was dissolved in a solution of 1, 4-dioxane (40 mL) and replaced three times with nitrogen at room temperature. Then stirring the reaction solution for 14 hours at 110 ℃ under the protection of nitrogen; after completion of the reaction, the reaction mixture was filtered, dried and concentrated, and the residue was purified by column chromatography (eluent gradient, petroleum ether: ethyl acetate=10:1) to give 4-bromo-2- (dimethylphosphoryl) benzonitrile (1.30 g, yield 50.4%) as a white solid. MS m/z (ESI): 258.0[ M+1 ]] + 。
Second step 2- (dimethylphosphoryl) -4- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzonitrile
In a 100mL eggplant type bottle, 4-bromo-2- (dimethylphosphoryl) benzonitrile (1.30 g,5.03 mmol), pinacol biborate (2.6 g,10.2 mmol), potassium acetate (2.5 g,25.4 mmol) and [1,1' -bis (diphenylphosphine) ferrocene were sequentially added]Palladium dichloride dichloromethane complex (208 mg,0.254 mmol) and 1, 4-dioxane (40 mL), displaced argon 3 times, were reacted overnight in an oil bath at 80 ℃. After the reaction was completed, the reaction solution was cooled, poured into water (20 mL), and then extracted with ethyl acetate (100 ml×3). The organic phases were combined and washed with saturated brine (50 ml x 3), then dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography (eluent gradient, petroleum ether: ethyl acetate=10:1) to give 2- (dimethylphosphoryl) -4- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzonitrile (550 mg, yield 35.8%) as a white solid. MS m/z (ESI) 306.1[ M+1 ]] + 。
Third step (R) -4- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile
(R) -3-bromo-5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a]Pyrimidine (380 mg,1 mmol), sodium carbonate (318 mg,3 mmol) and 2- (dimethylphosphoryl) -4- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzonitrile (305 mg,1 mmol) were added to a solution of dioxane/water (10 mL/2 mL), followed by Pd (dppf) Cl 2 (73 mg,0.1 mmol). After the reaction liquid nitrogen is replaced by air for deoxidization, the reaction liquid nitrogen is heated to 100 ℃ under the protection of nitrogen and reacts overnight. After the reaction, the reaction solution was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (100)mL), then washed with a saturated aqueous sodium chloride solution (100×2 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid. The solid was separated by column chromatography (eluent gradient: dichloromethane: methanol=10:1) to give (R) -4- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1, 5-a)]Pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile (55 mg, 11.5% yield) as a white solid. 1 H NMR(400MHz,MeOD-d4)δ8.85(s,1H),8.24(d,J＝8.0Hz,1H),7.77-7.72(m,3H),7.28-7.23(m,3H),6.67(d,J＝8.0Hz,1H),4.05-3.98(m,1H),2.77-2.67(m,2H),2.35-2.29(m,2H),1.85-1.83(m,2H),1.71(s,6H)。MS m/z(ESI):478.2[M+1] + 。
Example 3 (R) -5- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile
/>
First step 5-bromo-2- (dimethylphosphoryl) benzonitrile
5-bromo-2-iodobenzonitrile (3.08 g,10.0 mmol), phosphorus oxide (1.17 g,15.0 mmol), potassium phosphate (4.24 g,20.0 mmol), tris (dibenzylideneacetone) dipalladium (458 mg,0.5 mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (579 mg,1.0 mmol) were dissolved in a solution of 1, 4-dioxane (40 mL) and replaced three times with nitrogen at room temperature. Then stirring the reaction solution for 14 hours at 110 ℃ under the protection of nitrogen; after completion of the reaction, the reaction mixture was filtered, dried and concentrated, and the residue was purified by column chromatography (eluent gradient, petroleum ether: ethyl acetate=10:1) to give 5-bromo-2- (dimethylphosphoryl) benzonitrile (1.55 g, yield 60.1%) as a white solid. MS m/z (ESI): 258.0[ M+1 ]] + 。
Second step 2- (dimethylphosphoryl) -5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzonitrile
Sequentially in 100mL eggplant type bottleTo which 5-bromo-2- (dimethylphosphoryl) benzonitrile (1.55 g,6.01 mmol), pinacol ester of diboronic acid (2.6 g,10.2 mmol), potassium acetate (2.5 g,25.4 mmol) and [1,1' -bis (diphenylphosphine) ferrocene were added]Palladium dichloride dichloromethane complex (208 mg,0.254 mmol) and 1, 4-dioxane (40 mL), displaced argon 3 times, were reacted overnight in an oil bath at 80 ℃. After the reaction was completed, the reaction solution was cooled, poured into water (20 mL), and then extracted with ethyl acetate (100 ml×3). The organic phases were combined and washed with saturated brine (50 ml x 3), then dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography (eluent gradient, petroleum ether: ethyl acetate=5:1) to give 2- (dimethylphosphoryl) -5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzonitrile (600 mg, yield 32.7%) as a white solid. MS m/z (ESI) 306.1[ M+1 ]] + 。
Third step (R) -5- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile
(R) -3-bromo-5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a]Pyrimidine (380 mg, 1-mmol), sodium carbonate (318 mg,3 mmol) and 2- (dimethylphosphoryl) -5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzonitrile (305 mg,1 mmol) were added to a solution of dioxane/water (10 mL/2 mL), followed by Pd (dppf) Cl 2 (73 mg,0.1 mmol). After the reaction liquid nitrogen is replaced by air for deoxidization, the reaction liquid nitrogen is heated to 100 ℃ under the protection of nitrogen and reacts overnight. After the reaction, the reaction solution was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (100 mL), then washed with saturated aqueous sodium chloride (100 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid. The solid was separated by column chromatography (eluent gradient, dichloromethane: methanol=10:1) to give (R) -5- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1, 5-a)]Pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile (65 mg, 13.6% yield) as a white solid. 1 H NMR(400MHz,MeOD-d4)δ8.86(s,1H),8.24(d,J＝8.0Hz,1H),7.75-7.72(m,3H),7.25-7.23(m,3H),6.67(d,J＝8.0Hz,1H),4.05-3.98(m,1H),2.77-2.67(m,2H),2.35-2.29(m,2H),1.85-1.83(m,2H),1.73(s,6H)。MS m/z(ESI):478.2[M+1] + 。
Example 4 (R) - (4- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2-fluorophenyl) dimethylphosphine oxide
First step (4-bromo-2-fluorophenyl) dimethylphosphine oxide
4-bromo-2-fluoro-1-iodobenzene (3.00 g,10.0 mmol), phosphorus oxide (1.17 g,15.0 mmol), potassium phosphate (4.24 g,20.0 mmol), tris (dibenzylideneacetone) dipalladium (458 mg,0.5 mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (579 mg,1.0 mmol) were dissolved in a solution of 1, 4-dioxane (40 mL) and replaced three times with nitrogen at room temperature. Then stirring the reaction solution for 14 hours at 110 ℃ under the protection of nitrogen; after completion of the reaction, the reaction mixture was filtered, dried and concentrated, and the residue was purified by column chromatography (eluent gradient, petroleum ether: ethyl acetate=10:1) to give (4-bromo-2-fluorophenyl) dimethylphosphine oxide (1.60 g, yield 63.4%) as a white solid. MS m/z (ESI): 252.0[ M+1 ]] + 。
Second step (2-fluoro-5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) phenyl) dimethylphosphine oxide
(4-bromo-2-fluorophenyl) dimethylphosphine oxide (1.50 g,5.95 mmol), pinacol biboronate (2.6 g,10.2 mmol), potassium acetate (2.5 g,25.4 mmol) and [1,1' -bis (diphenylphosphine) ferrocene were sequentially added to a 100mL eggplant type bottle]Palladium dichloride dichloromethane complex (208 mg,0.254 mmol) and 1, 4-dioxane (40 mL), displaced argon 3 times, were reacted overnight in an oil bath at 80 ℃. After the reaction was completed, the reaction solution was cooled, poured into water (20 mL), and then extracted with ethyl acetate (100 ml×3). The organic phases were combined and washed with saturated brine (50 ml x 3), then dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography (eluent gradient, petroleum ether: ethyl acetate=5:1) to give (2-fluoro-5- (4,4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) phenyl) dimethylphosphine oxide (600 mg, 33.8% yield) as a white solid. MS m/z (ESI) 299.1[ M+1 ]] + 。
Third step (R) - (4- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2-fluorophenyl) dimethylphosphine oxide
(R) -3-bromo-5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a]Pyrimidine (380 mg, 1-mmol), sodium carbonate (318 mg, 3-mmol) and (2-fluoro-5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) phenyl) dimethylphosphine oxide (300 mg,1 mmol) were added to a solution of dioxane/water (10 mL/2 mL), followed by Pd (dppf) Cl 2 (73 mg,0.1 mmol). After the reaction liquid nitrogen is replaced by air for deoxidization, the reaction liquid nitrogen is heated to 100 ℃ under the protection of nitrogen and reacts overnight. After the reaction, the reaction solution was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (100 mL), then washed with saturated aqueous sodium chloride (100 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid. The solid was separated by column chromatography (eluent gradient, dichloromethane: methanol=10:1) to give (R) - (4- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1, 5-a)]Pyrimidin-3-yl) -2-fluorophenyl) dimethylphosphine oxide (55 mg, 11.7% yield) as a white solid. 1H NMR (400 MHz, meOD-d 4) delta 8.46 (s, 1H), 8.24 (d, J=8.0 Hz, 1H), 7.77-7.71 (m, 3H), 7.25-7.23 (m, 3H), 6.67 (d, J=8.0 Hz, 1H), 4.05-3.98 (m, 1H), 2.77-2.67 (m, 2H), 2.35-2.29 (m, 2H), 1.85-1.83 (m, 2H), 1.75 (s, 6H). MS m/z (ESI): 471.2[ M+1 ]] + 。
Example 5 (R) -2- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -5- (dimethylphosphoryl) isonicotinic nitrile
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First step 2-amino-5-iodoisonicotinic nitrile
2-AmmoniaThe isonicotinic nitrile (11.9 g,10.0 mmol) was dissolved in DMF (30 mL), and after the addition of N-iodosuccinimide (2.48 g,11.0 mmol), it was stirred at room temperature for 3 hours. A saturated aqueous sodium bicarbonate solution (30 mL) was then added to the reaction mixture, and the mixture was filtered through celite, and the solid was rinsed with ethyl acetate (50 mL). The filtrate was added with saturated aqueous sodium sulfite (5 mL), the organic phase was separated, the aqueous phase was back-extracted once with ethyl acetate (50 mL), the organic phases were combined and washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid. The solid was separated by column chromatography (eluent gradient, petroleum ether: ethyl acetate=5:1) to give 2-amino-5-iodoisonicotinic nitrile (9.5 g, yield 37.3%) as a yellow solid. MS m/z (ESI) 256.2[ M+1 ]] + 。
Second step 2-amino-5- (dimethylphosphoryl) isonicotinyl nitrile
2-amino-5-iodoisonicotinnitrile (2.55 g,10.0 mmol), phosphorus oxide (1.17 g,15.0 mmol), potassium phosphate (4.24 g,20.0 mmol), tris (dibenzylideneacetone) dipalladium (458 mg,0.5 mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (579 mg,1.0 mmol) were dissolved in a solution of 1, 4-dioxane (40 mL) and replaced three times with nitrogen at room temperature. Then stirring the reaction solution for 14 hours at 110 ℃ under the protection of nitrogen; after completion of the reaction, the reaction mixture was filtered, dried and concentrated, and the residue was purified by column chromatography (eluent gradient, petroleum ether: ethyl acetate=10:1) to give 2-amino-5- (dimethylphosphoryl) isonicotinic nitrile (1.10 g, yield 56.1%) as a yellow solid. MS m/z (ESI) 196.2[ M+1 ]] + 。
Third step 2-bromo-5- (dimethylphosphoryl) isonicotinic nitrile
2-amino-5- (dimethylphosphoryl) isonicotinyl nitrile (604 mg,3.1 mmol) was slowly added to a mass fraction of 48% hydrobromic acid (1.9 mL). The mixture was cooled to-2deg.C and treated with bromine (0.5 mL,9.5 mmol) followed by an aqueous solution of sodium nitrite (0.55 g,8.0 mmol) (10 mL). After stirring for 20 minutes, 30% by mass sodium hydroxide solution (6 mL) was added. The mixture was extracted with diethyl ether (10 mL. Times.3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid. The solid medicine is preparedColumn chromatography (eluent gradient, dichloromethane: methanol=10:1) afforded 2-bromo-5- (dimethylphosphoryl) isonicotinyl nitrile (310 mg, 38.6% yield) as a yellow solid. MS m/z (ESI): 259.1[ M+1 ]] + 。
Fourth step 5- (dimethylphosphoryl) -2- (trimethylstannyl) isonicotinyl nitrile
2-bromo-5- (dimethylphosphoryl) isonicotinnitrile (260 mg,1.0 mmol) was dissolved in toluene (6.0 mL), and hexamethyl-ditin (264 mg,2.0 mmol) and tetraphenylpalladium phosphate (116 mg,0.1 mmol) were added. After oxygen removal by argon replacement, the reaction solution was stirred at 110℃for 5 hours under argon protection. After completion of the reaction, the reaction mixture was filtered, dried and concentrated, and the residue was purified by column chromatography (eluent gradient, petroleum ether: ethyl acetate=3:1) to give 5- (dimethylphosphoryl) -2- (trimethylstannyl) isonicotinic nitrile (150 mg, yield 43.7%) as a yellow solid. MS m/z (ESI): 345.2[ M+1 ]] + 。
Fifth step (R) -2- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -5- (dimethylphosphoryl) isonicotinic nitrile
5- (dimethylphosphoryl) -2- (trimethylstannyl) isonicotinyl nitrile (150 mg,0.44 mmol) and (R) -3-bromo-5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a]Pyrimidine (190 mg,0.50 mmol) was dissolved in toluene (6.0 mL) and tetraphenylpalladium phosphate (58 mg,0.05 mmol) was added. After oxygen removal by argon replacement, the reaction solution was stirred at 110℃for 5 hours under argon 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=10:1) to give (R) -2- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a]Pyrimidin-3-yl) -5- (dimethylphosphoryl) isonicotinyl nitrile (15 mg, yield 7.1%) as a yellow solid. 1 H NMR(400MHz,MeOD-d4)δ8.86(s,1H),8.54(d,J＝8.0Hz,1H),7.97-7.79(m,2H),7.65-7.43(m,3H),6.77(d,J＝8.0Hz,1H),4.05-3.98(m,1H),2.77-2.67(m,2H),2.35-2.29(m,2H),1.85-1.83(m,2H),1.74(s,6H)。MS m/z(ESI):479.2[M+1] + 。
Example 6 (R) -5- (5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile
First step (R) -3-bromo-5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a]Pyrimidine (R) -2- (5-chloro-2-fluorophenyl) pyrrolidine (1.86 g,9.30 mmol) was added to 3-bromo-6-chloroimidazo [1,2-b]A solution of pyridazine (2.16 g,9.30 mmol) and potassium fluoride (1.62 g,27.9 mmol) in DMSO (50 mL) was heated to 130℃and stirred overnight. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into ice water (50 mL), and extracted with ethyl acetate (20 mL. Times.3). The combined organic phases were washed with saturated aqueous sodium chloride (10 ml×2), dried over anhydrous sodium sulfate, filtered and concentrated to give an orange solid. The solid was separated by column chromatography (eluent gradient, petroleum ether: ethyl acetate=5:1) to give (R) -3-bromo-5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a]Pyrimidine (2.5 g, 68.0% yield) was a pale yellow solid. MS m/z (ESI): 395.2[ M+1 ]] + 。
Second step (R) -5- (5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile
(R) -3-bromo-5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a]Pyrimidine (390 mg, 1-mmol), sodium carbonate (318 mg,3 mmol) and 2- (dimethylphosphoryl) -5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzonitrile (305 mg, 1-mmol) were added to a solution of dioxane/water (10 mL/2 mL), followed by Pd (dppf) Cl 2 (73 mg,0.1 mmol). After the reaction liquid nitrogen is replaced by air for deoxidization, the reaction liquid nitrogen is heated to 100 ℃ under the protection of nitrogen and reacts overnight. After the reaction, the reaction solution was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (100 mL), then washed with saturated aqueous sodium chloride (100 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid. The solid was separated by column chromatography (eluent gradient, dichloromethane: methanol=10:1) to give (R) -5- (5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1, 5-a)]Pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile (65 mg, 12.9% yield) as white colorA solid. 1H NMR (400 MHz, meOD-d 4) delta 8.56 (s, 1H), 8.34 (d, J=8.0 Hz, 1H), 7.87-7.71 (m, 3H), 7.35-7.28 (m, 3H), 6.97 (d, J=8.0 Hz, 1H), 4.05-3.98 (m, 1H), 2.77-2.67 (m, 2H), 2.35-2.29 (m, 2H), 1.85-1.83 (m, 2H), 1.71 (s, 6H). MS m/z (ESI): 494.1[ M+1 ]] + 。
Example 7 (R) -5- (5- (2- (3-chlorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile
First step (R) -3-bromo-5- (2- (3-chlorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidine
(R) -2- (3-chlorophenyl) pyrrolidine (1.69 g,9.30 mmol) was added to 3-bromo-6-chloroimidazo [1,2-b]A solution of pyridazine (2.16 g,9.30 mmol) and potassium fluoride (1.62 g,27.9 mmol) in DMSO (50 mL) was heated to 130℃and stirred overnight. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into ice water (50 mL), and extracted with ethyl acetate (20 mL. Times.3). The combined organic phases were washed with saturated aqueous sodium chloride (10 ml×2), dried over anhydrous sodium sulfate, filtered and concentrated to give an orange solid. The solid was separated by column chromatography (eluent gradient, petroleum ether: ethyl acetate=5:1) to give (R) -3-bromo-5- (2- (3-chlorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a]Pyrimidine (2.0 g, 56.9% yield) as a pale yellow solid. MS m/z (ESI): 377.2[ M+1 ]] + 。
Second step (R) -5- (5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile
(R) -3-bromo-5- (2- (3-chlorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a]Pyrimidine (378 mg,1 mmol), sodium carbonate (318 mg,3 mmol) and 2- (dimethylphosphoryl) -5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzonitrile (305 mg,1 mmol) were added to a solution of dioxane/water (10 mL/2 mL), followed by Pd (dppf) Cl 2 (73 mg,0.1 mmol). After the reaction liquid nitrogen is replaced by air for deoxidization, the reaction liquid nitrogen is heated to 100 ℃ under the protection of nitrogen and reacts overnight. After the reaction, the reaction solution was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (100 mL)After that, the mixture was washed with a saturated aqueous sodium chloride solution (100 mL. Times.2), dried over anhydrous sodium sulfate, filtered and concentrated to give a pale yellow solid. The solid was separated by column chromatography (eluent gradient, dichloromethane: methanol=10:1) to give (R) -5- (5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1, 5-a)]Pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile (52 mg, yield 10.9%) as a white solid. 1 H NMR(400MHz,MeOD-d4)δ8.76(s,1H),8.44(d,J＝8.0Hz,1H),7.67-7.51(m,3H),7.35-7.28(m,4H),6.90(d,J＝8.0Hz,1H),4.05-3.98(m,1H),2.77-2.67(m,2H),2.35-2.29(m,2H),1.85-1.83(m,2H),1.73(s,6H)。MS m/z(ESI):476.1[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 μl of 5-fold Kinase Buffer (5 XKinase Buffer) and 1M MgCl were taken 2 Stock 5. Mu.l, 1. Mu.l 1M DTT stock, was dissolved in 794. Mu.l double distilled water and mixed well to give 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 a 1 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 an M.mu.Multilabel 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 ratio = (ER Solvent set ―ER Sample of )/(ER Solvent set ―ER Blank space )×100％
5. Experimental results
Compounds of the examplesInhibition ratio (IC) 50 ) Shown in table 3.
TABLE 3 Table 3
| Numbering of compounds | IC 50 (nM) |
| Example 1 | 102 |
| Example 2 | 2.5 |
| Example 3 | 4.7 |
| Example 4 | 16 |
| Example 5 | 92 |
| Example 6 | 2.8 |
| Example 7 | 9.5 |
As can be seen from Table 3, the compounds of the present invention have excellent TRKA kinase inhibitory activity.
Claims (7)
1. A compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof,
wherein:
x is selected from N or CH;
R 1 、R 2 independently selected from a hydrogen atom, a fluorine atom or a chlorine atom;
R 3 selected from a hydrogen atom, a fluorine atom or a cyano group.
2. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:
(R) - (3- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) phenyl) dimethylphosphine oxide;
(R) -4- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile;
(R) -5- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile;
(R) - (4- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2-fluorophenyl) dimethylphosphine oxide;
(R) -2- (5- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -5- (dimethylphosphoryl) isonicotinnitrile;
(R) -5- (5- (2- (5-chloro-2-fluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile;
(R) -5- (5- (2- (3-chlorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2- (dimethylphosphoryl) benzonitrile.
3. A pharmaceutical composition comprising a compound of claim 1 or 2, or a 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 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.
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| CN110734437A (en) * | 2018-07-19 | 2020-01-31 | 金秋 | Pyrazolopyrimidine compounds, pharmaceutical compositions and uses thereof |
| CN111718349A (en) * | 2019-03-19 | 2020-09-29 | 华中师范大学 | Fluorine-containing pyrazolopyrimidine compound, pharmaceutical composition and application thereof |
| CN111718350A (en) * | 2019-03-19 | 2020-09-29 | 华中师范大学 | Pyrazole-substituted pyrazolopyrimidine compounds, pharmaceutical compositions and uses thereof |
| CN110194773A (en) * | 2019-07-26 | 2019-09-03 | 上海美迪西生物医药股份有限公司 | Pyrrolo-pyrazole analog derivative, preparation method and its application in medicine |
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