Compound with TRK kinase inhibitory activity, preparation method, composition and application
The technical field is as follows:
the present invention relates to novel compounds, pharmaceutical compositions comprising such compounds, processes for preparing such compounds and the use of such compounds in therapy. More particularly, the present invention relates to certain substituted [1,2,4] triazolo [4,3-a ] pyrazine derivatives which exhibit TRK kinase inhibitory activity and are useful in the treatment of pain, inflammation, cancer and certain infectious diseases.
Background art:
cancer is a disease resulting from abnormal growth of tissues. Some cancers have the potential to invade local tissues and also metastasize to distant organs. This disease may develop into a wide variety of different organs, tissues and cell types. Thus, the term "cancer" refers to a collection of more than one thousand different diseases.
The neurotrophin tyrosine kinase receptor, also known as tropomyosin-related kinase (Trk), is a soluble growth factor-activated high affinity receptor for Neurotrophins (NTs). The NTRK receptor family has 3 members: NTRK1 (also known as TrkA), NTRK2 (also known as TrkB), NTRK3 (also known as TrkC). NT includes the following proteins: nerve Growth Factor (NGF) activating NTRK1, brain-derived neurotrophic factor (BDNF) and NT-4/5 activating NTRK2 and NT3 activating NTRK 3. Each NTRK receptor contains an extracellular domain (ligand binding site), a transmembrane domain, and an intracellular domain (containing a kinase domain). Each kinase catalyzes autophosphorylation upon binding to a ligand and then activates downstream signaling pathways.
Normally, NTRK is widely expressed in nerve continence, plays an important role in the maintenance and survival of these cells, and plays an important role in the development and function of the nervous system (Current Opinion in Neurobiology,11, 272-. A large body of recent literature has described that overexpression, activation, amplification, fusion gene formation or mutation of NTRK is associated with neuroblastoma (PediatrBlood Cancer,59, 226-.
Trk kinases are activated in malignant tumors by a variety of mechanisms, mainly structural rearrangements and alterations in expression. The encoding gene NTRK of Trk kinase is rearranged to generate chimeric oncogene, which leads to the constitutive activation of Trk kinase, is no longer regulated and controlled by nerve growth factor ligand and has the risk of carcinogenesis. In colon cancer KM12 cells, the tropomyosin-3 gene TPM3 located on the long arm of chromosome 1 and NTRK1 undergo gene fragment rearrangement, TPM3 is broken between exons 7 and 8, NTRK1 is broken inside exon 8, and then TPM3 is directly linked with NTRK1 expressing an intracellular structural region, thereby abnormally expressing a TPM3-TRKA chimeric protein. This gene alteration disrupts the interaction of the ligand NGF with TrkA in cells, intracellular TRKA is in an over-expressed and sustained activation state, and the downstream three signaling pathways of PI3K/AKT, Ras/MAPK and PLC gamma are also in an abnormal active state.
Selective NTRK tyrosine kinase inhibitors have been reported, including CEP-751, CEP-701(cancer research,59,2395-2341(1999)), indolocarbazole compounds (WO01/14380), oxindole compounds (WO2/20479, WO02/20513), pyrazolyl fused ring compounds (Japanese patent laid-open No. 15-231687), isothiazole compounds (bioorg. Med. chem. Lett.,16,3444-3448(2006)), and other types of compounds (WO2005/049033, WO2005/103010, WO2006/082392, WO2006/087530, WO 2006087538). Imidazopyridazine backbone compounds (WO2007/013673, WO2007/025540, WO2007/147646, WO2008/052734, WO 2012/125667). The Trk kinase inhibitor is designed by simulating the structure of ATP and competing with ATP for binding sites to achieve the purpose of inhibiting the catalytic activity of the kinase.
The invention aims to improve the compound with the inhibiting effect on TRK, thereby solving the problem of drug resistance of the existing anticancer drugs and improving the targeting effect.
Disclosure of Invention
The investigators of the present invention have discovered TRK kinase inhibitory activity of compounds represented by structural formula I. Accordingly, it is an object of the present invention to provide compounds represented by formula I, stereoisomers, and pharmaceutically acceptable salts, hydrates or solvates thereof.
Wherein the content of the first and second substances,
R1is optionally substituted by CN, OH, OMe, NH2、NHMe、N(Me)2、Cl、Br、F、CF3、CONH2、SO2NH2、NHSO2(C1-C4 alkyl) mono-or polysubstituted phenyl or arylheterocyclyl;
r2 is selected from absent, H or C1-C4 alkyl;
r3 is optionally substituted by CN, OH, OMe, NH2、NHMe、N(Me)2、Cl、Br、F、CF3、CONH2、 SO2NH2SO2(C1-C4 alkyl), CO (C1-C4 alkyl), NHSO2(C1-C4 alkyl) mono-or poly-substituted phenyl, aryl heterocyclyl;
or R2 and R3 form a substituted four-membered ring, five-membered ring or six-membered ring together.
Preferably, R1 is optionally substituted by OH, OMe, Cl, Br, F, CF3、CONH2、SO2NH2、NHSO2(C1-C4 alkyl) mono-or polysubstituted phenyl.
Preferably, R1 is optionally substituted by OH, OMe, Cl, Br, F, CF3、CONH2、SO2NH2、NHSO2(C1-C4 alkyl) monosubstituted or polysubstituted aromatic heterocyclic radical.
Preferably, R is H or absent.
Preferably, R3 is optionally substituted CN, OH, OMe, NH2、NHMe、N(Me)2、Cl、Br、F、CF3、 CONH2、SO2NH2SO2(C1-C4 alkyl), CO (C1-C4 alkyl), NHSO2(C1-C4 alkyl) mono-or poly-substituted phenyl and aromatic heterocyclic radical.
Preferably, R2 and R3 together form a substituted four-membered, five-membered, six-membered ring.
Preferably, R2 and R3 together form a substituted four-, five-, six-membered ring containing 1-2 heteroatoms.
Preferably, R2 and R3 form a substituted four-membered ring, five-membered ring and six-membered ring together, and the substituents of the four-membered ring, five-membered ring and six-membered ring are selected from C-C4 alkyl, CN, OH, OMe and NH2、NHMe、N(Me)2、 Cl、Br、F、CF3、CONH2、SO2NH2SO2(C1-C4 alkyl), CO (C1-C4 alkyl), NHSO2(C1-C4 alkyl) the substitution being mono-or polysubstituted.
These compounds may be, but are not limited to, compounds of the following formulae (1) to (36):
the second purpose of the invention is to provide a pharmaceutical composition containing the compound. The present invention provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and at least one compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, or solvate thereof.
A third object of the present invention is to achieve the goal of treating tumors by inhibiting TRK kinase activity, which comprises administering to a patient in need of such treatment or prevention a therapeutically effective amount of one or more compounds of the present invention, or pharmaceutically acceptable salts, stereoisomers, or tautomers thereof.
The tumor is selected from breast cancer, skin cancer, bladder cancer, ovarian cancer, stomach cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, rectal cancer, esophageal cancer, tongue cancer, stomach cancer, kidney cancer, renal parenchymal cancer, cervical cancer, endometrial cancer, testicular cancer, urinary cancer, melanoma, astrocytic cancer, meningioma, hodgkin's lymphoma, non-hodgkin's lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, adult T-cell leukemia lymphoma, hepatocellular carcinoma, bronchial cancer, small cell lung cancer, non-small cell lung cancer, multiple myeloma, basal cell tumor, seminoma, rhabdomyosarcoma, chondrosarcoma, myosarcoma, fibrosarcoma.
The fourth object of the present invention is to provide a method for preparing the compound, stereoisomer, isotopic compound of formula I and pharmaceutically acceptable salts thereof, or hydrates or solvates thereof, comprising the steps of:
(1) synthesis of intermediate IV
Performing a ring closing reaction on the compound II and the compound III under the action of alkali, wherein the reaction is performed at the temperature of 20-100 ℃, and the alkali is selected from triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine, potassium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide and lithium hydroxide; the reaction solvent is ethanol, isopropanol, dioxane, tetrahydrofuran and toluene.
(2) Synthesis of intermediate VI
Reacting a compound IV with a compound V under the action of a catalyst and a ligand, wherein the reaction is carried out at the temperature of between 20 and 100 ℃ by base catalysis, and the catalyst is selected from PdCl2(PPh3)2、Pd(PPh3)4、Pd(dba)2、Pd(OAc)2、 Pd(dppf)2Cl2(ii) a The ligand is selected from 2-dicyclohexylphosphine-2 ',4',6' -triisopropylbiphenyl (XPhos), 2-dicyclohexylphosphine-2 ',6' -dimethoxybiphenyl (SPhos), 4, 5-bisdiphenylphosphine-9, 9-dimethylxanthene (XantPhos); the base is selected from cesium carbonate, sodium tert-butoxide, potassium phosphate, sodium acetate; the reaction solvent is dioxane, DMF or toluene.
(3) Synthesis of intermediate VII
Reacting compound VI under the action of a base, wherein the reaction is carried out at the temperature of 20-100 ℃, and the base is selected from lithium hydroxide, sodium hydroxide and potassium hydroxide; the reaction solvent is methanol, ethanol, tetrahydrofuran, methanol/water, ethanol/water, tetrahydrofuran/water.
(4) Synthesis of intermediate VIII
Reacting a compound VII under the action of DPPA, wherein the reaction is carried out at a temperature of 80-100 ℃, and the base is selected from triethylamine and diisopropylethylamine; the reaction solvent was toluene.
(4) Reacting a synthetic compound VIII of a final product I with a compound IX under the action of a condensing agent, wherein the reaction is carried out at the temperature of 20-100 ℃ in a base catalysis mode, and the condensing agent is selected from CDI and phenyl chloroformate; the base is selected from triethylamine, diisopropylethylamine, N-methylmorpholine and DMAP; the reaction solvent is dichloromethane, DMF, tetrahydrofuran and toluene.
Definition of
"pharmaceutically acceptable salts" refers to those salts that retain the biological effectiveness and properties of the parent compound. The salt comprises: acid addition salts obtained by reaction of the free base of the parent compound with an inorganic acid or with an organic acid; such as hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid, sulfuric acid, perchloric acid, and the like; such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, benzenesulfonic acid (benzenesulfonate), benzoic acid, camphorsulfonic acid, citric acid, fumaric acid, gluconic acid, glutamic acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, mucic acid, pamoic acid, pantothenic acid, succinic acid, tartaric acid, malonic acid, or the like; preferably hydrochloric acid or (L) -malic acid; or when the acid proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, alkaline earth ion or aluminum ion, or coordinated with an organic base, a salt is formed; such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like.
By "excipient" is meant an inert substance added to a pharmaceutical composition to further facilitate administration of the compound. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like.
"pharmaceutical composition" refers to a mixture of one or more of the compounds described herein or a physiologically acceptable salt thereof with other chemical ingredients such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to the organism.
"carrier" when used herein refers to a carrier or diluent that does not produce a significant stimulus to an organism and does not abrogate the biological activity and properties of the administered compound.
"method" refers to manners, means, techniques and procedures for accomplishing a specified task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of chemistry, pharmacy, biology, biochemistry and medicine.
"alkyl" refers to saturated aliphatic hydrocarbons, including straight or branched chain saturated aliphatic hydrocarbons.
"aryl" refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. Preferably, the aryl group has 6 to 12 carbon atoms in the ring.
The compounds of the invention may have one or more asymmetric centers; the compounds can thus be prepared as individual (R) -stereoisomers or (S) -stereoisomers or as mixtures thereof. Unless otherwise indicated, the description or designation of a particular compound in the specification and claims is intended to include the individual enantiomers as well as racemic or other mixtures thereof. Methods for determining stereochemical configuration and separating stereoisomers are well known in the art (see the discussion in chapter 4 of "Advanced Organic Chemistry", 4 th edition, j. march, John Wiley and Sons, new york, 1992). Thus, the present invention also covers any stereoisomeric form, its corresponding enantiomers (d-and l-or (+) and (-) isomers) and its diastereoisomers and mixtures thereof having the ability to modulate the activity of c-Met and is not limited to any one stereoisomeric form.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples.
Example 1
(S) -N- (6- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3-hydroxypyrrolidine-1-carbamoyl
The first step is as follows:
dissolving the compound 1a (14.5g,100mmol) and the compound 1b (12.3g,100mmol) in a mixed solvent of toluene (100ml) and pyridine (20ml), heating to 100 ℃, stirring for 6 hours, detecting the reaction by TLC, adding diluted hydrochloric acid to quench the reaction after the reaction is finished, drying an organic layer, filtering, concentrating, and carrying out column chromatography to obtain 18.7g of yellow oily matter with the yield of 88.0%.
The second step is that:
compound 1c (18.5g, 87.0mmol), Compound 1d (15.9g, 87.0mmol), Cesium carbonate (42.0g, 130.5mmol), Pd (dba)2(2.6g,4.4mmol) and 4, 5-bis (diphenylphosphine) -9, 9-dimethylxanthene (2.6g,4.4mmol) are dissolved in DMF (100ml), the temperature is raised to 100 ℃, the mixture is stirred and reacted for 12 hours, the reaction is detected by TLC, after the reaction is finished, the reaction solution is poured into water (100ml), ethyl acetate (200ml X2) is extracted, the organic phases are combined and dried, filtered, concentrated and separated by column chromatography to obtain 20.2g of off-white solid, and the yield is 64.6%.
The third step:
dissolving compound 1e (20.0g,55.7mmol) in ethanol (100ml) and purified water (50ml), adding sodium hydroxide (6.7g,167.0mmol) at room temperature, stirring for 12 hours, detecting by TLC, removing ethanol under reduced pressure after reaction, adding ethyl acetate (200ml), adjusting pH to 5-6 with dilute hydrochloric acid, drying the organic layer, filtering, concentrating, and separating by column chromatography to obtain off-white solid 15.3g with yield 79.7%.
The fourth step:
dissolving compound 1f (2.0g, 5.8mmol), DPPA (2.1g, 7.5mmol), DIPEA (1.1g, 8.7mmol) in toluene (100ml), refluxing with stirring for 12 hours, detecting by TLC, adding water (20ml) after the reaction is finished, stirring for 1 hour, stopping the reaction, separating layers, drying the organic layer, filtering, concentrating, and separating by column chromatography to obtain an off-white solid 1.1g with a yield of 60.1%.
The fifth step
After 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) were placed in toluene (50ml) and stirred at room temperature for 1 hour, compound 1h (278mg, 3.2mmol) was added and stirred for 6 hours, and TLC detection was performed, after completion of the reaction, concentration was performed under reduced pressure, and column chromatography was performed to obtain 0.6g of off-white solid with a yield of 43.8%. MS-ESI (M/z is 430.2[ M +1]
Example 2
(R) -1- (4-acetylphenyl) -3- (6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) urea
Referring to example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, compound 4-acetanilide (432mg, 3.2mmol) was added, stirred for 8 hours, TLC detected, concentrated under reduced pressure after completion of the reaction, and column chromatography was performed to obtain off-white solid 0.7g with a yield of 45.8%. MS-ESI (M/z 478.2[ M +1]
Example 3
(R) -N- (6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -4-methylpiperazine-1-aminocarboxamide
Referring to the procedure of example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound 1-methylpiperazine (320mg, 3.2mmol) was added, stirred for 5 hours, detected by TLC, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give off-white solid 0.9g, yield 63.5%, MS-ESI: M/z 443.2[ M +1]
Example 4
(R) -N- (6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -4-ethylpiperazine-1-aminocarboxamide
Referring to the procedure of example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound 1-ethylpiperazine (365mg, 3.2mmol) was added, stirred for 7 hours, detected by TLC, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give off-white solid 0.8g, yield 54.7%, MS-ESI: M/z 457.2[ M +1]
Example 5
(R) -N- (6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -4-isopropylpiperazine-1-aminocarboxamide
Referring to example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound 1-isopropylpiperazine (410mg, 3.2mmol) was added, stirred for 6 hours, TLC detected, after completion of the reaction, concentrated under reduced pressure, and separated by column chromatography to give off-white solid 1.0g, yield 66.2%, MS-ESI: M/z 471.2[ M +1]
Example 6
N- (6- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3-methylpiperazine-1-aminocarboxamide
Referring to the procedure of example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound 2-methylpiperazine (320mg, 3.2mmol) was added, stirred for 5 hours, detected by TLC, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give off-white solid 1,1g, yield 77.6%, MS-ESI: M/z 443.2[ M +1]
Example 7
(R) -4-amino-N- (6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) piperazine-1-carbamoyl
Referring to example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml) and stirred at room temperature for 1 hour, then compound 4-tert-butoxycarbonylaminopiperidine (640mg, 3.2mmol) was added and stirred for 5 hours, TLC detection was performed, after completion of the reaction, concentration was performed under reduced pressure, and column chromatography was performed to obtain 1.6g of off-white solid.
Adding trifluoroacetic acid (2ml) into the solid solution dichloromethane (20ml) obtained above at room temperature, stirring for reaction for 2 hours, adding saturated sodium bicarbonate solution (10ml) to quench the reaction, standing, layering, drying the organic phase, and separating by column chromatography to obtain off-white solid 1.0g, wherein the total yield of the two steps is 70.5%. MS-ESI (M/z 443.2[ M +1]
Example 8
(S) -N- (6- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3-hydroxypiperidine-1-carbamoyl
Referring to example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound 3-hydroxypiperidine (323mg, 3.2mmol) was added, stirred for 6 hours, TLC detected, concentrated under reduced pressure after completion of the reaction, and column chromatography was performed to obtain off-white solid 0.7g with yield 49.3%.
MS-ESI:m/z=444.2[M+1]
Example 9
(S) -3-amino-N- (6- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) pyrrolidine-1-carbamoyl
Referring to example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml) and stirred at room temperature for 1 hour, then compound 3-tert-butoxycarbonylaminopyrrolidine (595mg, 3.2mmol) was added and stirred for 4 hours, the reaction was detected by TLC, after completion of the reaction, it was concentrated under reduced pressure and separated by column chromatography to give 1.5g of off-white solid.
Adding trifluoroacetic acid (2ml) into the solid solution dichloromethane (20ml) obtained above at room temperature, stirring for reaction for 2 hours, adding saturated sodium bicarbonate solution (10ml) to quench the reaction, standing, layering, drying the organic phase, and separating by column chromatography to obtain off-white solid 0.9g, wherein the two-step total yield is 65.5%. MS-ESI (M/z 429.2[ M +1]
Example 10
(R) -N- (6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3, 3-difluoropyrrolidine-1-carbamoyl
Referring to example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound 3, 3-difluoropyrrolidine (342mg, 3.2mmol) was added, stirred for 7 hours, TLC detected, after completion of the reaction, concentrated under reduced pressure, and column chromatography was performed to obtain off-white solid 0.8g, yield 55.6%.
MS-ESI:m/z=450.2[M+1]
Example 11
1- (6- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3- ((1R,3S) -3-hydroxycyclopentyl) urea
Referring to the procedure of example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound (1S,3R) -3-aminocyclopentan-1-ol (323mg, 3.2mmol) was added, stirred for 5 hours, TLC detected, after completion of the reaction, concentrated under reduced pressure, and separated by column chromatography to give off-white solid 0.6g, yield 42.3%. MS-ESI (M/z 444.2[ M +1]
Example 12
1- (6- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3- ((R) -3-oxocyclopentyl) urea
Referring to example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound (R) -3-aminocyclopentan-1-one (317mg, 3.2mmol) was added, stirred for 5 hours, detected by TLC, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give off-white solid 0.8g, yield 56.6%, MS-ESI: M/z 442.2[ M +1]
Example 13
N- (6- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3, 5-dimethylpiperidin-1-carbamoyl
Referring to the procedure of example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) were placed in toluene (50ml), stirred at room temperature for 1 hour, then compound 2, 6-dimethylpiperazine (365mg, 3.2mmol) was added, stirred for 5 hours, detected by TLC, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give off-white solid 1.0g, yield 68.4%, MS-ESI 457: M/z: 457.2[ M +1]
Example 14
(R) -N- (6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -4-oxopiperidine-1-carbamoyl
Referring to example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then piperidine-4-one (317mg, 3.2mmol) was added, stirred for 6 hours, TLC detected, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give off-white solid 1.1g, yield 77.8%, MS-ESI: M/z 442.2[ M +1]
Example 15
(R) -N- (6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3-oxopyrrolidine-1-carbamoyl
Referring to example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then pyrrolidine-3-one (272mg, 3.2mmol) was added, stirred for 4 hours, TLC detected, after completion of the reaction, concentrated under reduced pressure, and separated by column chromatography to give off-white solid 0.7g, yield 51.1%, MS-ESI: M/z 428.2[ M +1]
Example 16
1- ((1R,3S) -3-aminocyclopentyl) -3- (6- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) urea
Referring to example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound (1R,3S) -cyclopentane-1, 3-diamine (320mg, 3.2mmol) was added, stirred for 8 hours, TLC detected, concentrated under reduced pressure after completion of the reaction, and column chromatography was performed to give off-white solid 0.8g, yield 56.4%, MS-ESI: M/z 443.2[ M +1]
Example 17
1- ((R) -3, 3-Difluorocyclopentyl) -3- (6- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) urea
Referring to example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml) and stirred at room temperature for 1 hour, then compound 3-tert-butoxycarbonylaminopyrrolidine (595mg, 3.2mmol) was added and stirred for 5 hours, the reaction was detected by TLC, after completion of the reaction, it was concentrated under reduced pressure and separated by column chromatography to give 1.5g of off-white solid.
Adding trifluoroacetic acid (2ml) into the solid solution dichloromethane (20ml) obtained above at room temperature, stirring for reaction for 2 hours, adding saturated sodium bicarbonate solution (10ml) to quench the reaction, standing, layering, drying the organic phase, and separating by column chromatography to obtain off-white solid 0.8g, wherein the total yield of two steps is 53.9%. MS-ESI (M/z 464.2M + 1)
Example 18
(R) -1- (6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3- (4- (methylsulfonyl) phenyl) urea
Referring to example 1, compound 1g (1.0g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound p-methylsulfonylaniline (547mg, 3.2mmol) was added, stirred for 8 hours, TLC detected, concentrated under reduced pressure after completion of the reaction, and column chromatography was performed to obtain off-white solid 1.2g, yield 72.9%, MS-ESI: M/z 514.2[ M +1]
Example 19
1- (6- ((R) -2- (2, 5-difluorophenyl) -4, 4-difluoropyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3- ((1R,3S) -3-hydroxycyclopentanyl) urea
Referring to the procedure of example 1, the compound (R) -6- (2- (2, 5-difluorophenyl) -4, 4-difluoropyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-amine (prepared by the procedure of reference compound 1 g) (1.1g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), and reacted with stirring at room temperature for 1 hour, then, compound (1S,3R) -3-aminocyclopentane-1-ol (323mg, 3.2mmol) was added, and the reaction was stirred for 6 hours, TLC detection reaction, decompression concentration after the reaction, column chromatography separation to obtain white solid 0.9g, yield 58.6%, MS-ESI: and M/z is 480.2[ M +1 ].
Example 20
1- (6- ((R) -2- (2, 5-difluorophenyl) -4, 4-difluoropyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3- ((R) -3-oxocyclopentyl) urea
Referring to the procedure of example 1, compound (R) -6- (2- (2, 5-difluorophenyl) -4, 4-difluoropyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-amine (1.1g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound (R) -3-aminocyclopentan-1-one (317mg, 3.2mmol) was added, stirred for 5 hours, detected by TLC, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give an off-white solid 0.8g, yield 52.3%, MS-ESI: M/z ═ 478.2[ M +1]
Example 21
(S) -N- (6- ((R) -2- (3-fluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3-hydroxypyrrolidine-1-carbamoyl
Referring to the procedure of example 1, compound (R) -6- (2- (3-fluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-amine (0.95g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound (S) -pyrrolidin-3-ol (278mg, 3.2mmol) was added, stirred for 7 hours, TLC detected, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give off-white solid 0.9g, yield 68.2%, MS-ESI: M/z 412.2[ M +1]
Example 22
(R) -1- (4-acetylphenyl) -3- (6- (2- (3-fluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) urea
Referring to the procedure of example 1, compound (R) -6- (2- (3-fluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-amine (0.95g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound 4-acetanilide (432mg, 3.2mmol) was added, stirred for 6 hours, checked by TLC, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give off-white solid 0.7g, yield 47.6%, MS-ESI: M/z 460.2[ M +1]
Example 23
(R) -N- (6- (2- (3-fluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -4-methylpiperazine-1-carbamoyl
Referring to the procedure of example 1, the compound (R) -6- (2- (3-fluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-amine (0.95g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then the compound 1-methylpiperazine (320mg, 3.2mmol) was added, stirred for 8 hours, detected by TLC, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give 1.0g of off-white solid in 73.5% yield, MS-ESI: M/z: 425.2[ M +1]
Example 24
(R) -N- (6- (2- (2-chloro-5-fluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -4-ethylpiperazine-1-carbamoyl
Referring to the procedure of example 1, compound (R) -6- (2- (2-chloro-5-fluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-amine (1.06g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound 1-methylpiperazine (320mg, 3.2mmol) was added, stirred for 5 hours, detected by TLC, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give off-white solid 0.9g, yield 61.2%, MS-ESI: M/z ═ 459.2[ M +1]
Example 25
(R) -N- (6- (2- (2-chloro-5-fluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -4-ethylpiperazine-1-carbamoyl
Referring to the procedure of example 1, compound (R) -6- (2- (2-chloro-5-fluorophenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-amine (1.06g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound 1-ethylpiperazine (365mg, 3.2mmol) was added, stirred for 5 hours, TLC detected, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give off-white solid 1.0g, yield 66.1%, MS-ESI: M/z ═ 473.2[ M +1]
Example 26
N- (6- ((R) -2- (2, 5-difluorophenyl) -4-oxopyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3-methylpiperazine-1-carbamoyl
Referring to the procedure of example 1, the compound (R) -1- (3-amino- [1,2,4] triazolo [4,3-a ] pyrazin-6-yl) -5- (2, 5-difluorophenyl) pyrrolidin-3-one (1.06g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then the compound 2-methylpiperazine (320mg, 3.2mmol) was added, stirred for 5 hours, checked by TLC, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give an off-white solid 0.7g, yield 47.8%, MS-ESI: M/z ═ 457.2[ M +1]
Example 27
(R) -4-amino-N- (6- (2- (2, 5-difluorophenyl) -4-oxopyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) piperidine-1-carbamoyl
Referring to the procedure of example 1, the compound (R) -1- (3-amino- [1,2,4] triazolo [4,3-a ] pyrazin-6-yl) -5- (2, 5-difluorophenyl) pyrrolidin-3-one (1.06g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then the compound tert-butyl piperidin-4-ylcarboxylate (640mg, 3.2mmol) was added, stirred for 5 hours, checked by TLC, and after completion of the reaction, concentrated under reduced pressure, and isolated by column chromatography to give 1.5g of an off-white solid.
Adding trifluoroacetic acid (2ml) into the solid solution dichloromethane (20ml) at room temperature, stirring for 2 hours, adding saturated sodium bicarbonate solution (10ml) to quench the reaction, standing, layering, drying the organic phase, and separating by column chromatography to obtain 1.1g of off-white solid with a two-step total yield of 75.2%, and MS-ESI (M/z: 457.2[ M +1]
Example 28
(S) -N- (6- ((R) -2- (2, 5-difluorophenyl) -4, 4-dimethylpyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3-hydroxypiperidine-1-carbamoyl
Referring to the procedure of example 1, compound (R) -6- (2- (2, 5-difluorophenyl) -4, 4-dimethylpyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-amine (1.1g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound (S) -piperidin-3-ol (323mg, 3.2mmol) was added, stirred for 8 hours, checked by TLC, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give off-white solid 0.9g, yield 59.7%, MS-ESI: M/z 471.2[ M +1]
Example 29
(S) -3-amino-N- (6- ((R) -2- (2, 5-difluorophenyl) -4, 4-dimethylpyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) pyrrolidine-1-carbamoyl
Referring to the procedure of example 1, compound (R) -6- (2- (2, 5-difluorophenyl) -4, 4-dimethylpyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-amine (1.1g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound (S) -pyrrolidine-3-carboxylic acid tert-butyl ester (595mg, 3.2mmol) was added, stirred for 5 hours, detected by TLC, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give 1.3g of an off-white solid.
Adding trifluoroacetic acid (2ml) into the solid solution dichloromethane (20ml) at room temperature, stirring for 2 hr, adding saturated sodium bicarbonate solution (10ml), quenching, standing, layering, drying, and separating by column chromatography to obtain off-white solid 0.6g with two-step total yield of 41.0%, MS-ESI (M/z: 457.2[ M +1]
Example 30
(R) -N- (6- (2- (2, 5-difluorophenyl) -4, 4-dimethylpyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3, 3-difluoropyrrolidine-1-carbamoyl
Referring to the procedure of example 1, compound (R) -6- (2- (2, 5-difluorophenyl) -4, 4-dimethylpyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-amine (1.1g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound 3, 3-difluoropyrrolidine (342mg, 3.2mmol) was added, stirred for 7 hours, checked by TLC, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give an off-white solid 1.0g, yield 63.4%, MS-ESI: M/z 478.2[ M +1]
Example 31
(R) -N- (6- (2- (5-fluoro-2-methoxyphenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3, 5-dimethylpiperazine-1-carbamoyl
Referring to the procedure of example 1, compound (R) -6- (2- (5-fluoro-2-methoxyphenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-amine 1.05g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound 2, 6-dimethylpiperazine (365mg, 3.2mmol) was added, stirred for 7 hours, checked by TLC, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give off an off-white solid 1.2g, yield 80.0%, MS-ESI: M/z 469.2[ M +1]
Example 32
(R) -N- (6- (2- (5-fluoro-2-methoxyphenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -4-oxopiperidine-1-carbamoyl
Referring to the procedure of example 1, the compound (R) -6- (2- (5-fluoro-2-methoxyphenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-amine 1.05g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then the compound piperidin-4-one (317mg, 3.2mmol) was added, stirred for 6 hours, detected by TLC, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give an off-white solid 0.9g, yield 62.0%, MS-ESI: M/z ═ 454.2[ M +1]
Example 33
(R) -N- (6- (2- (5-fluoro-2-methoxyphenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) -3-oxopyrrolidine-1-carbamoyl
Referring to the procedure of example 1, compound (R) -6- (2- (5-fluoro-2-methoxyphenyl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-amine 1.05g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound pyrrolidin-3-one (272mg, 3.2mmol) was added, stirred for 6 hours, detected by TLC, concentrated under reduced pressure after completion of the reaction, and separated by column chromatography to give off-white solid 0.8g, yield 56.8%, MS-ESI: M/z ═ 440.2[ M +1]
Example 34
1- ((R) -3, 3-Difluorocyclopentyl) -3- (6- ((R) -2- (6-methoxypyridin-2-yl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) urea
Referring to the procedure of example 1, compound (R) -6- (2- (6-methoxypyridin-2-yl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-amine (0.96g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then compound (R) -3, 3-difluorocyclopentane-1-amine (387mg, 3.2mmol) was added, stirred for 6 hours, checked by TLC, concentrated under reduced pressure after completion of the reaction, and isolated to give an off-white solid 1.1g, yield 74.9%, MS-ESI: M/z 459.2[ M +1]
Example 35
1- ((1R,3S) -3-Aminocyclopentyl) -3- (4- (methylsulfonyl) phenyl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) urea referring to the procedure of example 1, the compound (R) -6- (2- (6-methoxypyridin-2-yl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-amine (0.96g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then the compound 4-methylsulfonylaniline (547mg, 3.2mmol) was added, stirred for 6 hours, checked by TLC, after the reaction, the reaction mixture was concentrated under reduced pressure and subjected to column chromatography to obtain 1.0g of an off-white solid in a yield of 61.4% and MS-ESI (MS-ESI) wherein M/z is 509.2[ M +1]
Example 36
1- ((1R,3S) -3-aminocyclopentyl) -3- (6- ((R) -2- (6-methoxypyridin-2-yl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-yl) urea
Referring to the procedure of example 1, the compound (R) -6- (2- (6-methoxypyridin-2-yl) pyrrolidin-1-yl) - [1,2,4] triazolo [4,3-a ] pyrazin-3-amine (0.96g, 3.2mmol), CDI (518mg, 3.2mmol), DIPEA (1.2g, 9.5mmol) was placed in toluene (50ml), stirred at room temperature for 1 hour, then the compound 3-t-butoxycarbonylaminopyrrolidine (595mg, 3.2mmol) was added, stirred for 4 hours, TLC detected, concentrated under reduced pressure after completion of the reaction, and isolated by column chromatography to give 1.5g of an off-white solid.
Adding trifluoroacetic acid (2ml) into the solid solution dichloromethane (20ml) obtained above at room temperature, stirring for reaction for 2 hours, adding saturated sodium bicarbonate solution (10ml) to quench the reaction, standing, layering, drying the organic phase, and separating by column chromatography to obtain off-white solid 0.8g, wherein the two-step total yield is 57.1%. MS-ESI (M/z 438.2[ M +1]
EXAMPLE 37 determination of biological Activity
TrkA kinase inhibitory Activity evaluation
An enzyme-linked immunosorbent (ELISA) assay was used to estimate the kinase activity of TrkA in the presence of the inhibitor. Immulon 4HBX 384 microtiter plates were coated with 0.025mg/ml solutions of poly (Glu, Ala, Tyr,6:3: 1). Various concentrations of test compound, 2.5nm TrkA and 500. mu.M ATP were incubated in microtiter plates at ambient temperature for 25 minutes with shaking, the test buffer having a 25nm MOPS pH of 7.5, consisting of 0.005% (V/V) TritonX-100 and 5nmMgCl2, and the reaction mixture was removed from the plates by washing with PBS containing 0.1% (V/V) Tween 20. Phosphorylated reaction products were detected using 0.2. mu.g/mL phosphotyrosine-specific monoclonal antibody conjugated to horseradish peroxidase and TMB peroxidase substrate System (KPL), the color intensity of chromogenic substrates was quantified by adsorption at 450nm after addition of 1M phosphoric acid, and IC was calculated using S-curve50Values, specific results are shown in table 1:
the above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention.