CN113527304A

CN113527304A - Preparation method of quinoline TGF-beta 1 inhibitor

Info

Publication number: CN113527304A
Application number: CN202110414826.2A
Authority: CN
Inventors: 陈程; 杜爽; 赵立文; 陈宏雁
Original assignee: Nanjing Shenghe Pharmaceutical Research And Development Co ltd
Current assignee: Nanjing Shenghe Pharmaceutical Research And Development Co ltd
Priority date: 2020-04-17
Filing date: 2021-04-17
Publication date: 2021-10-22

Abstract

The invention belongs to the field of medicinal chemistry, relates to a preparation method of quinoline TGF-beta 1 inhibitor, and specifically relates to 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1,2, 4-dihydro- [1]Triazolo [4,3-a]A process for the preparation of pyrazin-7 (8H) -yl) quinoline or a salt, hydrate, solvate or crystal thereof,

Description

Preparation method of quinoline TGF-beta 1 inhibitor

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to a preparation method of 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazole-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazine-7 (8H) -yl) quinoline or a salt, hydrate, solvate or crystal thereof.

Background

TGF-beta (transforming growth factor beta) is an important class of cytokines, and up to now 6 different subtypes (TGF-beta 1-6) have been found to be different in homology to each other, and only 3 subtypes, i.e., TGF-beta 1, TGF-beta 2, and TGF-beta 3, are expressed in mammals. It is a multifunctional growth factor superfamily, has extensive biological activity, and is involved in early embryonic development, cartilage and bone formation, synthesis of extracellular matrix, inflammation, interstitial fibrosis, regulation of immune and endocrine functions, and formation and development of tumors. Meanwhile, the 3 isomers have similar structures, and the amino acid sequences of the isomers have high homology, but show completely different phenotypes in respective knockout mouse models, which indicates that each isomer has specific and non-crossed functions in vivo. The TGF- β family of ligands can bind to receptors on the membrane surface, initiating the transmission of downstream signals within the cell.

TGF-beta 1 is the most common and important subtype of TGF, is the most abundantly expressed subtype in liver, is also known as the strongest hepatic fibrosis induction factor, and plays a pivotal role in the progression from chronic liver Disease to end-stage liver Disease (Yamazaki, et al, diagnostic Disease,2011,29: 284-288). Several studies have shown that TGF- β 1 and TGF β receptors are often highly expressed in diseased liver organs, blood vessels and extracellular mediators. In the classic TGF beta-TGF beta R-Smads pathway, TGF beta 1 activates TGF beta R1 (transforming growth factor beta receptor 1, ALK5) in the signal pathway, thereby regulating the whole signal pathway and realizing the regulation of the expression of a series of target genes related to fibrosis and tumorigenesis development. Currently, it is widely believed that TGF- β has a promoting effect on liver cancer, which is mainly manifested in promoting tumor cell metastasis, enhancing tumor cell immune escape, and inducing angiogenesis (Ling, et al. current Pharmaceutical Biotechnology,2011,12: 2190-.

The research on the medicines targeting the TGF-beta pathway has been carried out for many years, but TGF beta R1 inhibitors such as Galunesertib and the like show certain cardiotoxicity (such as bleeding, function degradation, inflammatory injury and the like) on animal models, and the reason is that the medicines have low target selectivity and specificity, and have stronger inhibition effect (such as p38 alpha) on other proteins with the same kinase region while inhibiting the TGF beta R1 kinase activation site, thereby generating a plurality of unexpected off-target toxic and side effects. Thus, there remains a need to develop more selective inhibitors of TGF β R1 in order to specifically modulate the TGF- β signaling pathway for use in the treatment of TGF- β related diseases.

Disclosure of Invention

The inventor of the invention finds that a quinoline TGF-beta 1 inhibitor, the structure of which is shown in the following formula (I), and the chemical name of which is 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazin-7 (8H) -yl) quinoline (hereinafter referred to as the compound of the formula (I)):

the inventor of the invention researches and discovers that the compound shown as the formula (I) or the hydrate, solvate or crystal thereof shows remarkable inhibitory activity on TGF-beta R1 kinase, and is very hopeful to be used as a therapeutic agent for TGF-beta R1 related diseases.

It is well known that for human administration, national and international regulatory agencies have very low limits for unidentified or poorly toxic impurities in drug Substances (APIs) for safety reasons. Impurities in a drug substance may be generated by degradation itself or may originate from a production process, for example, including unreacted starting materials, chemical derivatives of impurities contained in the starting materials, synthesis by-products, and the like. Therefore, research on a preparation method of the compound of formula (I) or a derivative thereof is needed to obtain a method for preparing the compound of formula (I) or a pharmaceutically acceptable salt, isomer, solvate or crystal thereof, which has mild reaction conditions, stable process, easy purification, easy operation and is advantageous for industrial mass production.

The inventors of the present invention have conducted research and research on a method for preparing a compound represented by formula (I), and an attempted reaction route includes the following reaction steps:

1) protecting the hydroxyl group of the 7-chloro-4-hydroxyquinoline of formula (1) to form a compound of formula (2);

2) carrying out buchward coupling reaction on the compound of the formula (2) and 3- (trifluoromethyl) -5,6,7, 8-tetrahydro- [1,2,4] triazolo [4,3-a ] pyrazine hydrochloride to obtain a compound of a formula (3);

3) removing a hydroxyl protecting group from the compound of the formula (3) under an acidic condition to obtain a compound of a formula (4);

4) the hydroxyl group of the compound of the formula (4) is chlorinated by phosphorus oxychloride to obtain a compound of a formula (5);

5) the compound of the formula (5) reacts with 1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazole-4-ol under the action of an acid-binding agent to obtain the compound of the formula (I).

Route one step 2) in the preparation of the compound of formula (3) by buchward coupling, the compound of formula (2) is partially deprived of BOC (t-butyloxycarbonyl), and the compound of formula (1) after BOC removal has a deactivation effect on the buchward coupling reaction due to the electron donating effect of the hydroxyl group, so that once BOC is removed, the obtained raw material cannot participate in the reaction, and the conversion rate in this reaction step is reduced. Later attempts to solve the problem of low conversion rate of other hydroxyl protecting groups such as methoxymethyl ether (MOM), trimethylsilyl ethoxymethyl (SEM) and the like have the defects that raw materials are remained, and a large amount of chlorine-removed by-products are generated if the dosage of a palladium reagent is increased.

An object of the present invention is to provide a method for producing a compound represented by the formula (I) or a salt, hydrate, solvate or crystal thereof, which comprises the step of reacting a compound of the formula (II) wherein X is a leaving group with a compound of the formula (III),

in some embodiments, the leaving group X is selected from halogen, hydroxy, amino, alkoxy, acyloxy, aryloxy, heteroaryloxy, sulfonyloxy, optionally substituted alkylsulfonyloxy, optionally substituted alkenylsulfonyloxy, optionally substituted arylsulfonyloxy, acyl, diazo moieties, and active esters of hydroxy groups, such as carboxylate, sulfonate, phosphate, or borate. In some specific embodiments, the leaving group X is chloro, iodo, bromo, fluoro, acetoxy, mesyloxy, tosyloxy, trifluormesyloxy, nitrobenzenesulfonyloxy, or bromo-benzenesulfonyloxy.

In some preferred embodiments, the present invention provides a process for the preparation of a compound of formula (I) of the present invention, or a salt, hydrate, solvate or crystal thereof, wherein X is selected from the group consisting of halogen and an active ester of hydroxy; further preferably, X is selected from the group consisting of fluorine, chlorine, bromine, iodine, carboxylate, sulfonate, phosphate and borate; still further preferably, X is selected from the group consisting of fluoro, chloro, bromo, iodo, mesylate, triflate, benzenesulfonate, p-toluenesulfonate, p-bromophenylsulfonate and p-nitrobenzenesulfonate. In some embodiments, the method of the present invention for preparing a compound of formula (I) comprises the step of reacting a compound of formula (II) and a compound of formula (III) in the presence of a catalyst, preferably wherein the catalyst is a transition metal catalyst. In some embodiments, preferably, the transition metal catalyst is a palladium catalyst. In other embodiments, the transition metal catalyst is a copper catalyst. In other embodiments, the transition metal catalyst is a nickel or rhodium catalyst. In other embodiments, the transition metal catalyst is a manganese catalyst, such as MnCl₂. The palladium catalyst comprises a source of Pd (0) and P (II). In some embodiments, palladium on carbon may be used as the catalyst. In some embodiments, a palladium catalyst species may be used that generally includes one or more ligands bound to palladium metal. In some specific embodiments, the palladium catalyst is selected from (Ph)₃P)₄Pd、(Ph₃P)₂PdCl₂、(CH₃CN)₂PdCl₂、Pd₂(dba)₃、(dppf)PdCl₂And Pd (OAc)₂. A wide variety of ligands are known in the art, including the generally preferred phosphine ligands (see, e.g., C.Amatore and A.Juttand, Coord.chem.Rev.1998,178-180 and 511-528). Phosphine ligands useful in the process of the present invention include, but are not limited to, triphenylphosphine, tri (o-tolyl) phosphineTris (2-furyl) phosphine, 1, 2-bis (diphenylphosphino) ethane (dppe), 1, 4-bis (diphenylphosphino) butane (dppb), 2, 3-bis (diphenylphosphino) butane (chiralphos), 4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene (Xantphos), 1, 2-bis (2, 5-dimethylphospholylethyl) benzene (Me-DuPhos), diphenylphosphino) ferrocenyl]Ethyldicyclohexylphosphine (Josiphos), bis (diphenylphosphino) methane (dppm), 1, 3-bis (diphenylphosphino) propane (dppp), 1, 2-bis (dicyclohexylphosphino) ethane (dcpe), tricyclohexylphosphine, tributylphosphine, tri-tert-butylphosphine, tris (pentafluorophenyl-phosphine), tris (2,4, 6-trimethylphenyl) phosphine, 2 '-bis (diphenylphosphino) -1,1' -binaphthyl (binap), (2-biphenyl) di-tert-butylphosphine, (2-biphenyl) dicyclohexylphosphine, 2-di-tert-butylphosphino-2 ',4',6 '-triisopropylbiphenyl ("tert-butylXPhos"), 2-dicyclohexylphosphino-2', 6 '-dimethoxybiphenyl ("Sphos"), 2-dicyclohexylphosphino-2' - (N), n-dimethylamino) biphenyl ("DavePhos"), 2-dicyclohexylphosphino-2 ',4',6' -triisopropylbiphenyl ("Xphos"), and the like. In some embodiments, the process for the preparation of compounds of formula (I) according to the present invention, wherein the ligand is selected from the group consisting of 2-dicyclohexylphosphine-2 ',6' -diisopropoxy-1, 1 '-biphenyl (Ruphos), 2-dicyclohexylphosphino-2' - (N, N-dimethylamine) -biphenyl (Davephos), 2-dicyclohexylphosphine-2 ',4',6 '-triisopropylbiphenyl (X-Phos), 2- (di-tert-butylphosphine) biphenyl (Johnphos), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (Xantphos), and 2-dicyclohexylphosphine-2', 6 '-diisopropoxy-1, 1' -biphenyl (Ruphos); preferably, the ligand is selected from the group consisting of 2-dicyclohexylphosphine-2 ',6' -diisopropyloxy-1, 1 '-biphenyl (Ruphos), 2-dicyclohexylphosphino-2' - (N, N-dimethylamine) -biphenyl (Davephos) and 4, 5-bisdiphenylphosphine-9, 9-dimethylxanthene (xanthphos).

In addition, palladium coupling reactions can be carried out with N-heterocyclic carbene ligands (see, e.g., Hillier, a.c. et al, j. organomet. chem.2002,69-82), including but not limited to 1, 3-bis (2,4, 6-trimethylphenyl) imidazolium chloride, 1, 3-bis (2, 6-diisopropylphenyl) imidazolidinium tetrafluoroborate, 1, 3-bis (2,4, 6-trimethylphenyl) imidazolidinium tetrafluoroborate, and the like. The catalyst may be derived from a preformed complex, for example (Ph)₃P)₄Pd、(Ph₃P)₂PdCl₂、(CH₃CN)₂PdCl₂、Pd₂(dba)₃、(dppf)PdCl₂([1,1' -bis (diphenylphosphino) ferrocene)]Palladium (II) dichloride), or the like, or the catalyst may be selected from the group including, but not limited to, PdCl₂、Pd(OAc)₂、Pd(dba)₂And combinations of palladium sources and suitable ligands. In some embodiments, the catalyst is Pd₂(dba)₃. In other embodiments, an amine base such as diisopropylethylamine, triethylamine, and the like may be added to the reaction mixture to stabilize the catalyst. In other embodiments, the palladium catalyst may be palladium on carbon. Various types of palladium on carbon catalysts are commercially available from Johnson-Matthey and other sources. In other embodiments, the catalyst is a supported palladium catalyst. These catalysts comprise a metal, such as palladium, supported on a polymeric support comprising a metal binding moiety. In some embodiments, the supported polymer is palladium on polymer substrate fibers including, but not limited to, polyolefin substrate fibers such as from Johnson-Matthey

A polyolefin base fiber. In other embodiments, the supported catalyst is a polymer-immobilized homogeneous catalyst in which palladium metal is covalently bound to a polymer chain, which may be further attached to inert polyolefin fibers that are insoluble in common organic solvents. Suitable supported catalysts include Johnson-Matthey, trade name

Those sold, in particular, by Johnson-Matthey

Supported polymer 1000 series. Of course, other types of palladium catalysts supported on a polymer support may be used, including but not limited to polystyrene-based supported catalysts and the like. In certain embodiments, it is desirable to remove the solvent from and/or containOxygen is removed from the solution of the catalyst to avoid oxidation of the ligand and destabilization of the catalyst. This may be accomplished in any manner known in the art, such as by alternately applying a vacuum to the mixture followed by introduction of nitrogen or another suitable inert gas to degas the mixture. Alternatively, nitrogen or another inert gas may be bubbled through the solvent or catalyst-containing solution. In some specific embodiments, the method for preparing a compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to the present invention comprises the step of reacting a compound of formula (II) and a compound of formula (III) in the presence of a palladium catalyst, a phosphine ligand. In some specific embodiments, the palladium coupling reaction is generally carried out in a solvent that does not interfere with the reaction. Useful solvents include, but are not limited to, hydrocarbon solvents, aromatic solvents, ethers, halogenated solvents, ester solvents, ketone solvents, amide solvents, nitrile solvents, and the like. The hydrocarbon solvent includes 1, 4-dioxane, heptane, cyclohexane, methylcyclohexane, isooctane, etc.; aromatic solvents include, but are not limited to, toluene, xylene, ethylbenzene, anisole, and the like. Ethers include, but are not limited to, tetrahydrofuran, 2-methyltetrahydrofuran, ethyl ether, methyl tert-butyl ether, dioxane, and the like. Ester solvents include alkyl esters such as ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, and the like. Nitrile solvents include acetonitrile and the like. The ketone solvent includes acetone, methyl ethyl ketone, methyl isobutyl ketone, methyl isopropyl ketone, and the like. The amide solvent includes dimethylformamide, dimethylacetamide, N-methylpyrrolidone, and the like. In some embodiments, according to the present invention, a suitable base may be added to the reaction mixture in addition to the catalyst, in the preparation method of the compound of formula (I) or a salt, hydrate, solvate or crystal thereof. Suitable bases include, but are not limited to, alkali metal hydroxides or alkoxides such as NaOH, LiOH, and KOH; alkaline earth metal hydroxides or alkoxides, alkali metal carbonates including sodium, potassium and cesium carbonates, alkaline earth metal carbonates, alkali metal and alkaline earth metal phosphates such as K₃PO₄Alkali metal acetates, alkaline earth metal acetates, and amine bases such as trialkylamines, including but not limited to triethylamine, diisopropylethylamine, 1, 8-diazabicyclo [ 5.4.0%]Undecane-7-ene (DBU); 1, 5-diazabicyclo [4.3.0]]Non-3-ene (DBN); 1, 4-diazabicyclo [2.2.2]Octane (DABCO), and the like. In some specific embodiments, the inventors of the present invention examined that under otherwise identical conditions, tris (dibenzylideneacetone) dipalladium Pd₂(dba)₃The specific data are shown in table 1, and the experimental data show that when tris (dibenzylideneacetone) dipalladium is selected as the metal catalyst and different ligands are selected, the two ligands of X-phos and Johnphos can not completely react. The present invention therefore provides a process for the preparation of a compound of formula (I) according to the invention, wherein said process comprises contacting a ligand, preferably, wherein the ligand is selected from the group consisting of 2-dicyclohexylphosphine-2 ',6' -diisopropoxy-1, 1 '-biphenyl (Ruphos), 2-dicyclohexylphosphino-2' - (N, N-dimethylamine) -biphenyl (Davephos), 2-dicyclohexylphosphine-2 ',4',6 '-triisopropylbiphenyl (X-Phos), 2- (di-t-butylphosphino) biphenyl (Johnphos), 4, 5-bisdiphenylphosphino-9, 9-dimethylxanthene (Xantphos), and 2-dicyclohexylphosphine-2', 6 '-diisopropoxy-1, 1' -biphenyl (Ruphos); preferably, the ligand is selected from the group consisting of 2-dicyclohexylphosphine-2 ',6' -diisopropyloxy-1, 1 '-biphenyl (Ruphos), 2-dicyclohexylphosphino-2' - (N, N-dimethylamine) -biphenyl (Davephos) and 4, 5-bisdiphenylphosphine-9, 9-dimethylxanthene (xanthphos).

TABLE 1

Ligands	Compound of formula (II) (%)	Compound of formula (I) (%)
			Ruphos	0.15	87.98
Davephos	0.24	80.80
			X-phos	17.08	68.27
Johnphos	66.24	24.55
			Xantphos	2.18	80.86

In other specific embodiments, the inventors of the present invention examined the amount of the metal catalyst and the experimental data showed that the reaction became more and more complete with the increase of the amount of tris (dibenzylideneacetone) dipalladium, and when the amount of tris (dibenzylideneacetone) dipalladium is too large, the debromination of the compound of formula (II) to the hybrid compound-1 of formula (II); if the amount of tris (dibenzylideneacetone) dipalladium is too small, the problems of incomplete reaction, too long reaction time and the like are caused, and part of experimental data are shown in table 2. Accordingly, the present invention provides a process for the preparation of a compound of formula (I) according to the present invention, wherein the molar ratio of the compound of formula (II) and tris (dibenzylideneacetone) dipalladium is about 1:0.005 to 1:0.04, preferably about 1:0.009 to 1:0.02, further preferably about 1: 0.01.

TABLE 2

In other specific embodiments, the inventors of the present invention examined the effect of reaction temperature and time in the case of using tris (dibenzylideneacetone) dipalladium catalyst. The reaction is carried out at 60 ℃, 80 ℃ and 100 ℃, reaction liquid is taken for 2 hours, 3 hours, 4 hours, 8 hours and 20 hours for HPLC detection, the change relationship of the compound of the formula (I) and the compound of the formula (II) along with time is examined at different temperatures, and the tolerance of the compound of the formula (I) in the reaction liquid is also examined, and the specific data are shown in Table 3. The experimental data show that the reaction rate increases with increasing temperature over the same time period, and the relationship between the remaining amount of the compound of formula (II) and the reaction temperature shows a clear correlation. The compound of formula (II) does not react completely at a temperature of 60 ℃ and 80 ℃ for the same reaction time, and even if the reaction time is prolonged, the reaction rate is not accelerated significantly. HPLC detection shows that the compound of formula (II) is completely reacted after 3 hours at 100 ℃, and the impurities and purity in the reaction solution are not obviously changed along with the prolonging of the reaction time. Under the same conditions, the reaction end time is prolonged by lowering the temperature, and the impurity compound-1 of the formula (II) is increased by lowering the temperature and increasing the dosage of the catalyst tris (dibenzylideneacetone) dipalladium. The amount and temperature of tris (dibenzylideneacetone) dipalladium therefore have an influence on the key quality properties of the product, and the invention therefore provides a process for the preparation of the compounds of the formula (I) according to the invention, wherein the reaction temperature is from about 60 to 120 c, preferably from about 80 to 110 c, and more preferably from about 90 to 100 c.

TABLE 3

In a specific embodiment, the method for preparing the compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to the present invention comprises the step of reacting the compound of formula (II) and the compound of formula (III) in the presence of a palladium catalyst tris (dibenzylideneacetone) dipalladium, a phosphine ligand.

In some specific embodiments, the process for the preparation of a compound of formula (I), or a salt, hydrate, solvate or crystal thereof, of the invention further comprises the step of reacting a compound of formula (IV) with a compound of formula (V) to produce a compound of formula (II), wherein X is a leaving group (as defined above),

in some specific embodiments, the method of preparing the compound of formula (II) above comprises the step of reacting the compound of formula (IV) with the compound of formula (V) in a suitable solvent such as a water-soluble solvent, e.g., acetonitrile, ethanol, acetone, alkanol, alcohol, ether, propylene glycol, glycerol, triacetin, poly (propylene glycol), PVP (poly (vinyl pyrrolidone)), dimethylsulfoxide, N-dimethylformamide, formamide, N-dimethylacetamide, pyridine, propanol, N-methylacetamide, butanol, soluphor (2-pyrrolidone), pharmasolve (N-methyl-2-pyrrolidone), or the like, preferably acetonitrile, methanol, or ethanol, to produce the compound of formula (II).

In some specific embodiments, the process for the preparation of the compound of formula (II) according to the present invention, wherein the reaction solvent is acetonitrile. The inventors of the present invention conducted an exemplary experiment using acetonitrile as a solvent, and found that this step is a heterogeneous reaction, and if the amount of the solvent is too large, the reaction contact surface is reduced to slow down the reaction rate; if the amount of solvent is too small, it may cause explosion during the post-treatment. Experiments show that when the amount of acetonitrile used is about 10V to 20V (the ratio between the volume of the solvent (mL) and the mass of the compound of formula (IV) (g)), the compound of formula (II) is not lost by cooling precipitation, and the risk of explosion of the compound of formula (II) during hot filtration and reduction of the reaction rate due to excessive reaction solvent is avoided. Thus, in some preferred embodiments, the present invention provides a process for the preparation of a compound of formula (II) according to the present invention using acetonitrile as reaction solvent, wherein the ratio of acetonitrile to the compound of formula (IV) is about 10-20:1 (ratio between acetonitrile volume (mL) and mass (g) of the compound of formula (IV)), preferably about 10-17:1 (ratio between acetonitrile volume (mL) and mass (g) of the compound of formula (IV)). In a specific embodiment, the present invention provides a process for the preparation of a compound of formula (II) of the present invention using acetonitrile as reaction solvent, wherein the ratio of acetonitrile to compound of formula (IV) is about 14:1 (ratio between volume of acetonitrile (mL) and mass of compound of formula (IV) (g)).

In some embodiments, the process for the preparation of a compound of formula (I) or a salt, hydrate, solvate or crystal thereof, further comprises the step of reacting a compound of formula (VI) to produce a compound of formula (V) wherein X is a leaving group (as defined above),

in some specific embodiments, the present invention provides a process for the preparation of the compounds of formula (V) of the present invention, wherein the reaction solvent is selected from water-soluble solvents such as acetonitrile, ethanol, acetone, alkanols, alcohols, ethers, propylene glycol, glycerol, triacetin, poly (propylene glycol), PVP (poly (vinyl pyrrolidone)), dimethylsulfoxide, N-dimethylformamide, formamide, N-dimethylacetamide, pyridine, propanol, N-methylacetamide, butanol, soluphor (2-pyrrolidone), pharmasolve (N-methyl-2-pyrrolidone), and the like, preferably from acetonitrile, methanol and ethanol.

In some specific embodiments, the method for preparing a compound of formula (I) or a salt, hydrate, solvate or crystal thereof of the present invention further comprises a step of dissociating the compound of formula (VII) into a compound of formula (III) under the action of a basic agent,

in some embodiments, the present invention provides a process for the preparation of the compounds of formula (III) of the present invention, wherein the alkaline agent includes, but is not limited to, alkali metal hydroxides or alkoxides such as NaOH, LiOH, and KOH; alkaline earth metal hydroxides or alkoxides, alkali metal carbonates including sodium, potassium and cesium carbonates, alkaline earth metal carbonates, alkali and alkaline earth metal phosphates, alkali metal acetates, alkaline earth metal acetates, and amine bases such as trialkylamines, including but not limited to triethylamine, diisopropylethylamine, 1, 8-diazabicyclo [5.4.0] undec-7-ene (DBU); 1, 5-diazabicyclo [4.3.0] non-3-ene (DBN); 1, 4-diazabicyclo [2.2.2] octane (DABCO), and the like. In some specific embodiments, the alkaline agent is selected from sodium carbonate, potassium carbonate, and sodium hydroxide.

In some specific embodiments, the process for the preparation of the compound of formula (III) according to the invention, wherein acetonitrile is used as reaction solvent. In some preferred embodiments, the process for the preparation of the compound of formula (III) according to the invention, wherein a mixture of acetonitrile and water is used as reaction solvent. Preferably, a catalytic amount of water is used. In some specific embodiments, the process for the preparation of the compound of formula (III) according to the present invention, wherein the reaction solvent is acetonitrile containing about 0.1-1.0% water. In some specific embodiments, the process for the preparation of the compound of formula (III) according to the present invention, wherein the reaction solvent is acetonitrile containing about 0.2-0.6% water. In some specific embodiments, the process for the preparation of the compound of formula (III) according to the present invention, wherein the reaction solvent is acetonitrile containing about 0.2-0.4% water. In some specific embodiments, the process for the preparation of the compound of formula (III) according to the present invention, wherein the reaction solvent is acetonitrile containing about 0.3% water.

In some specific embodiments, the present invention provides a process for the preparation of a compound of formula (IV) of the present invention, comprising the steps of:

1) reacting the compound of the formula (IV-1) with bromine under acidic conditions to obtain a compound of a formula (IV-2);

2) reacting the compound of the formula (IV-2) with benzoic acid under alkaline conditions to obtain a compound of a formula (IV-3);

3) reacting the compound of the formula (IV-3) with N, N-dimethylformamide dimethyl acetal (DMF-DMA) to obtain a compound of a formula (IV-4);

4) preparing a compound of a formula (IV-5) by using the compound of the formula (IV-4) and hydrazine hydrate under the action of glacial acetic acid;

5) reacting the compound of the formula (IV-5) with cyclopropylboronic acid in 1, 2-dichloromethane to produce a compound of the formula (IV-6);

6) the compound of formula (IV-6) is reacted under alkaline conditions to produce the compound of formula (IV).

In some specific embodiments, the present invention provides a process for the preparation of a compound of formula (I) or a salt, hydrate, solvate or crystal thereof, wherein the process comprises the steps of:

1) reacting the compound of the formula (VI-1) to obtain a compound of a formula (V-1);

2) reacting the compound of the formula (V-1) with 1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazole-4-ol of the formula (IV) to prepare a compound of a formula (II-1);

3) the 3- (trifluoromethyl) -5,6,7, 8-tetrahydro- [1,2,4] triazolo [4,3-a ] pyrazine hydrochloride of the formula (VII) is dissociated into a compound of a formula (III) under the action of an alkaline reagent;

4) reacting the compound of the formula (II-1) with the compound of the formula (III) to obtain the compound of the formula (I).

This route greatly simplifies the experimental procedure compared to route one. In which chloroquinoline is replaced by bromoquinoline, so that the buchward coupling can prepare the compound of the formula (I) with higher yield. The reaction sequence is changed into nucleophilic substitution and then coupling, so that the problems of hydroxyl passivation, hydroxyl protecting group screening, protecting group removal and the like can be solved. The route has the advantages of few steps, simple and convenient operation, suitability for industrial production, more environmental friendliness and higher yield.

In some preferred embodiments, the present invention provides a purification method of a compound of formula (I) or a salt, hydrate, solvate or crystal thereof, wherein the method comprises dissolving the compound of formula (I) or a salt, hydrate, solvate or crystal thereof in a solvent, and crystallizing at reduced temperature; preferably, the present invention provides a method for purifying a compound of formula (I) or a salt, hydrate, solvate or crystal thereof, wherein the method comprises dissolving the compound of formula (I) or a salt, hydrate, solvate or crystal thereof in a solvent, adding a metal scavenger, activated carbon, filtering, cooling and crystallizing; further preferably, the present invention provides a purification process of the compound of formula (I) or a salt, hydrate, solvate or crystal thereof, wherein the process comprises dissolving the compound of formula (I) or a salt, hydrate, solvate or crystal thereof in a solvent, adding a metal scavenger, filtering, adding an anti-solvent, and crystallizing; wherein the solvent is preferably selected from one or more of dichloromethane, acetonitrile, water, esters with the carbon number less than 6, alcohols with the carbon number less than 6 and ketones with the carbon number less than 6, and is more preferably selected from one or more of ethyl acetate, acetonitrile, methanol, ethanol, propanol, butanol, sec-butanol, isopropanol, dichloromethane, acetone and water; wherein the antisolvent is selected from the group consisting of methane, ethane, propane, butane, pentane, and heptane.

The inventor of the invention finds that the preparation method of the compound shown in the formula (I) or the salt, hydrate, solvate or crystal thereof has the advantages of few reaction route steps, simple and convenient operation, environmental friendliness, higher yield and purity, mild reaction conditions, easy purification, stable process and easy operation, and can meet the requirements of industrial scale production and application.

Description of the terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The "leaving group" of the present invention has the ordinary meaning in the art and refers to a group that can be easily displaced, an active functional group on a molecule undergoing a displacement reaction from the molecule when a new bond is formed. Groups having this function are well known to those skilled in the art, and specific examples thereof can be further referred to organic synthesis manuals common in the art, such as "advanced organic Chemistry," jerry macch (JerryMarch), 5 th edition, p 351-357, john wileyandsons, n.y. For example, the leaving group may be a halogen atom, an amino group, an alkoxy group, an acyloxy group, an aryloxy group, a heteroaryloxy group, an alkylsulfonyloxy group, an arylsulfonyloxy group, a hydroxyl group, an active ester of a hydroxyl group, such as a carboxylate, a sulfonate, a phosphate, or a borate.

The "acid scavenger" of the present invention has the meaning generally used in the art, preferably wherein said acid scavenger is selected from the group consisting of alkoxide bases, alkali metal alkoxides, and carbonate bases; further preferably, the acid scavenger is selected from potassium tert-butoxide, sodium tert-butoxide, potassium tert-amylate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, cesium bicarbonate, potassium phosphate, and dipotassium hydrogen phosphate.

The "hydroxyl-protecting Groups" according to the invention are suitable Groups known in the art for hydroxyl protection, see the literature ("Protective Groups in Organic Synthesis", 5)^Th Ed.T.W.Greene&P.g.m.wuts). By way of example, the hydroxyl protecting group may preferably be (C)_1-10Alkyl or aryl)₃Silane groups, for example: triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl and the like; may be C_1-10Alkyl or substituted alkyl, for example: methyl, t-butyl, allyl, benzyl, methoxymethyl, ethoxyethyl, 2-Tetrahydropyranyl (THP), etc.; may be (C)_1-10Alkyl or aryl) acyl groups, such as: formyl, acetyl, benzoyl and the like; may be (C)_1-6Alkyl or C_6-10Aryl) sulfonyl; or (C)_1-6Alkoxy or C_6-10Aryloxy) carbonyl.

The "salt" of the present invention may be any salt, especially pharmaceutically acceptable salts. As used herein, "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of a compound of the invention with an "acid" or "acidic agent" of the invention, which may be selected from hydrochloric acid, hydrobromic acid, phosphoric acid, sulfamic acid, nitric acid, p-toluenesulfonic acid, benzenesulfonic acid, sulfanilic acid, sulfuric acid, acetic acid, oxalic acid, phenylacetic acid, propionic acid, malonic acid, trifluoroacetic acid, succinic acid, glycolic acid, stearic acid, ascorbic acid, pamoic acid, hydroxymaleic acid, glutamic acid, benzoic acid, salicylic acid, 2-acetoxybenzoic acid, fumaric acid, ethanedisulfonic acid, oxalic acid, isethionic acid, citric acid, D-gluconic acid, lactic acid, L-malic acid, succinic acid, L-tartaric acid, fumaric acid, alpha-ketoglutaric acid, hippuric acid, succinic acid, alpha-ketoglutaric acid, succinic acid, acetic acid, succinic acid, alpha-beta-hydroxy acids, and mixtures thereof, Maleic acid, D-tartaric acid, methane sulfonic acid, or the like. "pharmaceutically acceptable salts" of the compounds of the present invention can be synthesized from the compounds of the present invention which contain acidic or basic moieties by conventional chemical methods, and typically, salts of basic compounds can be prepared by, for example, exchange chromatography or by reacting the free base with a stoichiometric amount or an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents. Similarly, salts of acidic compounds may be formed by reaction with a suitable inorganic or organic base.

The "basic agent" according to the invention is a compound capable of deprotonating a hydroxyl or amino group. Examples of bases include, but are not limited to, (C) in combination with an alcohol solvent_1-6Alkyl oxide ((C)_1-6Alkyl) OM), wherein (C)_1-6Alkyl) oxides include, but are not limited to, MeO-, EtO-, n-PrO-, i-PrO-, t-BuO-, i-AmO- (isopentyloxy), and the like, and wherein M is an alkali metal cation, e.g., Li⁺、Na⁺、K⁺And the like. The alcohol solvent comprises (C)_1-6Alkyl) OH, such as, for example, methanol, ethanol, n-propanol, isopropanol, tert-butanol, isoamyl alcohol, and the like. Non-alkoxy bases such as sodium hydroxide, potassium hydroxide, sodium hydride, sodium hexamethyldisilylamine, lithium diisopropylamide, calcium hydride, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, DBU (1, 8-diazabicyclo [5.4.0] can also be used]Undec-7-ene), DBN (1, 5-diazabicyclo [ 4.3.0)]Non-5-ene), Grignard reagents such as (C)_1-6Alkyl) Mg (halogen) including, but not limited to, methyl magnesium chloride, methyl magnesium bromide, tert-butyl magnesium chloride, tert-butyl magnesium bromide, and the like.

The term "solvate" refers to a form of a compound of the present invention that forms a solid or liquid complex by coordination with a solvent molecule. Hydrates are a special form of solvates in which coordination occurs with water. Within the scope of the present invention, the solvate is preferably a hydrate.

The term "crystalline" refers to the various solid forms formed by the compounds of the present invention, including crystalline forms, amorphous forms.

The "hydrogen", "carbon" and "oxygen" in the compounds of the present invention include all isotopes thereof. Isotopes are understood to include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include protium, tritium, and deuterium, and isotopes of carbon include¹³C and¹⁴c, isotopes of oxygen including¹⁶O and¹⁸o, and the like.

Detailed Description

The following representative examples are intended to better illustrate the present invention and are not intended to limit the scope of the present invention. The materials used in the following examples are all commercially available unless otherwise specified.

Example 14 preparation of- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazin-7 (8H) -yl) quinoline

Step 1: preparation of 2-bromo-1- (tetrahydro-2H-pyran-4-yl) ethan-1-one

Under the protection of nitrogen, methanol (100mL) and 1- (tetrahydro-2H-pyran-4-yl) ethanone (20.0g,156mmol) are sequentially added into a 1000mL three-neck flask, the temperature is reduced to below-15 ℃, liquid bromine is slowly dropped, and the temperature is kept below-15 ℃. Heating to 0 deg.C after dripping, reacting for 45min, heating to 10 deg.C, reacting for 45min, and maintaining11mol/L sulfuric acid (55mL) was slowly added dropwise thereto at a temperature below room temperature, and the reaction was allowed to proceed overnight at room temperature. After the reaction was monitored, ethyl acetate and aqueous sodium chloride solution were added for extraction, the organic layers were combined, the organic layer was adjusted to pH 7-8 with saturated sodium bicarbonate, the organic layers were combined and concentrated under reduced pressure to give the title compound as a pale yellow solid, 28.5g in total, yield: 87.5 percent; MS (ESI) M/z 207.0[ M + H ]]⁺。

Step 2: preparation of 2-oxo-2- (tetrahydro-2H-pyran-4-yl) ethyl benzoate

Benzoic acid (18.5g, 151.4mmol) was dissolved in N, N-dimethylformamide (DMF,495mL), potassium carbonate (38g, 275.2mmol) was added, and 2-bromo-1- (tetrahydro-2H-pyran-4-yl) ethan-1-one (28.5g, 137.6mmol) was added to the system and reacted at room temperature overnight. The mixture was diluted with ethyl acetate and washed with an aqueous sodium chloride solution. The organic phases were combined. Concentration under reduced pressure gave the title compound as a pale yellow solid, 30.0g in total, yield: 88.2 percent; LC-MS M/z [ M + H ]]⁺＝249。

And step 3: preparation of (Z) -1- (dimethylamino) -3-oxo-3- (tetrahydro-2H-pyran-4-yl) prop-1-ene-2-benzoic acid ester

To 1, 1-dimethoxy-N, N-dimethylmethylamine (795.15mL,5975.8mmol), 2-oxo-2- (tetrahydro-2H-pyran-4-yl) ethyl benzoate (95.0g,383mmol) was added, the mixture was heated to 100 ℃ to react for 2 hours, the temperature was raised to 106 ℃ to react for 2 hours, the temperature was returned to room temperature after monitoring the completion of the reaction, the mixture was concentrated to dryness under reduced pressure, ethyl acetate was added to the system, the mixture was washed with brine, and the organic phase was dried over anhydrous sodium sulfate. The organic phase was filtered and concentrated under reduced pressure to give the title compound as a red solid, 107.9g in total, yield: 93.1%, used directly in the next step. LC-MS M/z [ M + H ]]⁺＝304。

And 4, step 4: preparation of 3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazole-4-benzoic acid ester

To a 1000mL reaction flask was added acetic acid (376.1mL), (Z) -1- (dimethylamino) -3-oxo-3- (tetrahydro-2H-pyran-4-yl) prop-1-en-2-ylbenzoate (32.56g, 107.46mmol) in that order, 80% hydrazine hydrate (37.6mL) was slowly added dropwise over ice, and after completion of dropwise addition, the mixture was stirred at room temperature overnight. When the reaction is monitored to be over, ethyl acetate is added into the reaction liquid, the mixture is washed by water, organic phases are combined, a saturated sodium bicarbonate solution is washed until the pH value is 7-8, the organic phase is dried by anhydrous sodium sulfate and filtered to obtain a yellow oily substance after the organic phase is concentrated under reduced pressure, and the yellow oily substance is placed at room temperature overnight to obtain the title compound of a yellow solid, wherein the total amount is 28.0g, and the yield is as follows: 95.8 percent; LC-MS M/z [ M + H ]]⁺＝273。

And 5: preparation of 1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazole-4-benzoic acid ester

To 1, 2-dichloroethane (250mL) was added 2,2' -bipyridine (17.7g,113.2mmol), copper acetate (20.6g,113.2mmol) in this order, reacted at 75 ℃ for 30min, cooled to room temperature, and then a solution of cyclopropylboronic acid (17.5g,205.9mmol), sodium carbonate (21.8g,205.9mmol) and 3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazole-4-carboxylic acid ester (28.0g,102.9mmol) in 1, 2-dichloroethane (250mL) was added in this order, and reacted at 75 ℃ for 4H under an oxygen atmosphere. The reaction was monitored to the end, cooled to room temperature, filtered through celite, the filter cake was washed with ethyl acetate, and the filtrate was concentrated under reduced pressure to give the title compound as a tan oil, 32.0g total, yield: 98.5%, used directly in the next step; LC-MS M/z [ M + H ]]⁺＝313。

Step 6: preparation of 1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-ol

To methanol (308mL) was added 1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazole-4-benzoic acid ester (32.0g, 102.6mmol) and reacted at room temperature for 2H. Monitoring to the end of the reaction, decompressing and concentrating to remove part of methanol, adjusting the pH value to 6-7 by 1mol/L dilute hydrochloric acid, extracting by dichloromethane, and combining organic layers. Concentrated to dryness under reduced pressure and purified by column chromatography to give the title compound as a yellow solid, 9.0g in total, yield: 45 percent; LC-MS M/z [ M + H ]]⁺＝209。

And 7: preparation of 7-bromo-4-chloroquinoline

Adding 4.50kg of acetonitrile into a 50L vertical jacket reaction kettle, starting stirring, sequentially adding 7-bromo-4-hydroxyquinoline (1.80kg,8.03mol) and 4.00kg of acetonitrile, dropwise adding phosphorus oxychloride (1.85kg,12.05mol) when the internal temperature is reduced to be lower than 10 ℃, heating up, refluxing and stirring for 1-3 hours after dropwise adding is finished, monitoring to the end point of the reaction, cooling the system to be below 5 ℃, dropwise adding 4mol/L of NaOH solution to adjust the pH to be 7-8, adding 42.00kg of water, stirring for 1-2 hours at room temperature, centrifuging the feed liquid, washing a filter cake by 5.00kg of water, drying in vacuum, collecting and weighing solids to obtain a crude product of the title compound of brown solid, wherein the total amount of the crude product is 1.73kg, and the yield is as follows: 88.8 percent.

Adding 14.4kg of methyl tert-butyl ether and 1.73kg of crude 7-bromo-4-chloroquinoline into a 50L vertical jacket reaction kettle in sequence, stirring at 50 +/-5 ℃ for 1-3 hours, filtering while hot, washing a filter cake by using the methyl tert-butyl ether, concentrating the filtrate under reduced pressure until the filtrate is dry, drying the obtained solid in vacuum, collecting and weighing the solid after the drying is finished, thus obtaining the refined product of the title compound of yellow solid, wherein the total amount is 1.54kg, and the yield is 89.0%.

And 8: preparation of 7-bromo-4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) quinoline

Adding 8.40kg of acetonitrile into a 30L glass reaction kettle, starting stirring, sequentially adding 7-bromo-4-chloroquinoline (1.26kg,5.18mol), cesium carbonate (1.69kg,5.18mol), 1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-ol (0.90kg,4.32 mol), 4.00kg of acetonitrile for washing, heating to 70 +/-5 ℃, stirring for 2-3 hours, raising the temperature to reflux for 2-3 hours, cooling to 70 +/-5 ℃, filtering while hot, washing filter residues with dichloromethane, concentrating the filtrate under reduced pressure to a viscous state, adding 14.00kg of water, stirring, centrifuging, washing a filter cake with 3.00kg of water, drying at 50 +/-5 ℃ for 10-20 hours in vacuum, collecting and weighing the solid after drying to obtain 1.88kg of crude title compound of brown solid, wherein the total weight is 1.88kg, the crude product yield was 100.0%.

6.40kg of heptane and 1.88kg of crude 7-bromo-4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) quinoline 1 are added into a 10L four-neck flask, and stirred at 25 +/-5 ℃ for 3-5 hours. Filtering, washing a filter cake by 1.40kg of heptane, and vacuum drying for 3-20 hours at 50 +/-5 ℃. After drying, the solid was collected and weighed to give crude title compound 2 as a dark yellow solid, totaling 1.68kg, 89.4% yield.

Adding 4.70kg of absolute ethyl alcohol and 1.68kg of 7-bromo-4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) quinoline crude product 2 into a 10L four-neck flask, heating to reflux, stirring for 0.5-1 hour after clearing, closing and heating, naturally cooling for crystallization, starting an external circulation when the internal temperature is lower than 30 ℃, and stirring for 1-3 hours at-10 to-5 ℃. And (3) centrifuging the feed liquid, washing a filter cake by 0.4kg of cold ethanol (-10 to-5 ℃), and performing vacuum drying for 10-20 hours at the temperature of 50 +/-5 ℃. After the drying, the solid was collected and weighed to obtain a purified product of the title compound as a pale yellow solid, which amounted to 1.35kg and had a yield of 80.4%.

And step 9: preparation of 3- (trifluoromethyl) -5,6,7, 8-tetrahydro- [1,2,4] triazolo [4,3-a ] pyrazine

Adding 3- (trifluoromethyl) -5,6,7, 8-tetrahydro- [1,2,4] triazolo [4,3-a ] pyrazine hydrochloride (1.20kg,5.25mol) and 11.8kg of acetonitrile into a 20L four-neck flask, starting stirring, adding sodium hydroxide (0.42kg,10.50mol) and 36.00g of water, and vigorously stirring at 25 +/-5 ℃ for 3-15 hours; filtering, washing with acetonitrile, concentrating the filtrate under reduced pressure to dryness, adding 0.5g of seed crystal, solidifying and vacuum drying. After drying, the solid was collected and weighed to give the title compound as a white solid in a total of 0.92kg with a yield of 91.2%.

Step 10: preparation of 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazin-7 (8H) -yl) quinoline

Into a 20L four-necked flask was charged 7.10kg of 1, 4-dioxane, and with stirring turned on, 7-bromo-4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) quinoline (1.34kg,3.23mol), 3- (trifluoromethyl) -5,6,7, 8-tetrahydro- [1,2,4] oxy) was added in the order named]Triazolo [4,3-a]Pyrazine (0.75kg,3.88mol), potassium phosphate (K)₃PO₄1.37kg,6.47mol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (Xantphos,37.40g,65.00mmol), tris (dibenzylideneacetone) dipalladium (Pd)₂(dba)₃29.6g,32.00 mmol). And (5) protecting with nitrogen and maintaining the nitrogen atmosphere. Heating to 95 +/-5 ℃, stirring for 3-15 hours, turning off heating, filtering when the internal temperature is reduced to 70-80 ℃, washing by 1.6kg of hot 1, 4-dioxane (70-80 ℃), slowly pouring the mother liquor into ice water, adding N-acetyl-L-cysteine, stirring for 1-1.5 hours, centrifuging, washing by 5.00kg of water, and drying in vacuum. After drying, the solid was collected and weighed to give crude title compound 1 as a yellow solid in a total of 1.41kg with a yield of 83.1%.

Step 11: purification of 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazin-7 (8H) -yl) quinoline

Adding 3.8kg of ethyl acetate and 1.40kg of crude 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazine-7 (8H) -yl) quinoline 1 into a 10L four-neck flask, heating to 55 +/-5 ℃ under the protection of nitrogen, stirring for 2-4 hours, naturally cooling to the internal temperature of below 30 ℃, stirring and crystallizing for 1-3 hours at the temperature of minus 5-minus 10 ℃, centrifuging, washing by 0.5kg of cold ethyl acetate (-10-minus 5 ℃), and drying in vacuum to obtain the crude title compound 2 of a light yellow solid. The total amount was 1.21kg, and the yield was 85.8%.

A30L glass reactor was charged with 14.30kg of anhydrous methanol, 1.20kg of 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1,2, 4-d]Triazolo [4,3-a]Heating up, refluxing and dissolving the pyrazine-7 (8H) -yl) quinoline crude product 2 under the protection of nitrogen, adding 0.12kg of active carbon and 0.12kg of active carbon

Thiol. Refluxing for 1-1.5 hours, filtering while the solution is hot, washing with hot methanol (50-60 ℃), concentrating the filtrate under reduced pressure, cooling and crystallizing at-10 to-5 ℃ for 1-3 hours, centrifuging, and washing with cold methanol (minus 10 to-5 ℃). Vacuum drying to obtain white solid of the title compound crude product 3 total 1.03 kg. The yield thereof was found to be 85.1%.

Into a 10L four-necked flask were charged 9.50kg of methylene chloride, 1.02kg of 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1,2,4]Triazolo [4,3-a]Pyrazine-7 (8H) -yl) quinoline crude 3, start stirring. Adding 0.20kg of the mixture after dissolving

Thiol, stirring, filtering, washing with dichloromethane, slowly dropwise adding 26.20kg of heptane into the filtrate, stirring for 10-15 hours, filtering, washing a filter cake with 1.60kg of heptane, and drying in vacuum to obtain the refined product of the title compound as a white solid, wherein the total amount is 0.99 kg. The yield thereof was found to be 97.1%. ESI-MS [ M + H ]]⁺m/z:526.3,¹H NMR(400MHz,DMSO)δ8.59(d,J＝5.2Hz,1H),8.17(d,J＝11.2Hz,1H),7.94(s,1H),7.64(dd,J＝11.2,2.4Hz,1H),7.45(d,J＝2.4Hz,1H),6.56(d,J＝5.2Hz,1H),4.91(s,2H),4.34(t,J＝5.1Hz,2H),4.02(t,J＝5.2Hz,2H),3.88–3.71(m,2H),3.73–3.63(m,1H),3.30–3.19(m,2H),2.82–2.65(m,1H),1.78–1.59(m,4H),1.12–1.01(m,2H),1.01–0.88(m,2H).

Experimental example 1 evaluation of ALK5 kinase Activity in vitro with Compound

1. Experimental Material

1.1 Compounds

The compound of formula (I) of the present invention of example 1 was prepared in DMSO at 10mM, and then diluted to 3.333. mu.M, 1.111. mu.M, 370nM, 123nM, 41nM, 14nM, 4.6nM, 1.5nM, 0.5nM in that order.

1.2 reagents and instruments

Reagent: ALK5, available from Carna corporation, Cat. No. 09-141; p38 α was obtained from Carna corporation, Cat.No. 04-152; TGF β R1 peptide was purchased from SignalChem, cat.no. t 36-58; dimethyl sulfoxide (DMSO), available from Sigma, usa; EDTA, available from Sigma, USA; ADP-Glo Kinase Assay available from Promega, Cat. No. v9102/3, 1 Xkinase buffer (40mM Tris, pH 7.5, 0.10% BSA, 20mM MgCl)₂1mM DTT), prepared immediately prior to use.

The instrument comprises the following steps: 2104Multilabel Reader, available from Perkin Elmer, USA.

2. Experimental methods

2.1 preparation of 1 Xkinase buffer

1x assay buffer

40mM Tris,pH 7.5

20mM MgCl₂

0.10％BSA

1mM DTT

2.2 preparation of the Compound

2.2.1 dilution of the Compound

2.2.1.1 formulation of 50-fold compound: the final concentration of the compound tested was 10 μ M, configured at a 50-fold concentration, i.e. 500 μ M: a1000. mu.M solution of the compound was prepared by adding 95. mu.l of 100% DMSO to the second well of a 96-well plate and then adding 5. mu.l of a 10mM compound solution. Additional wells were added with 60. mu.l of 100% DMSO. Mu.l of compound from the second well was added to the third well and diluted sequentially 3-fold further down for a total of 10 concentrations.

Dilution apparatus: an automatic micropore pipette (Precision PRC 384U).

2.2.1.2 transfer 100nl of compound to the reaction plate with echo.

2.3 kinase reaction

2.3.1 preparation of 2-fold kinase solution

The kinase was added to 1 fold kinase buffer to form a 2 fold enzyme solution. 100nl of 100% DMSO-solubilized compound was present in 384 well plates. To a 384-well reaction plate, 2.5. mu.l of a 2-fold enzyme solution was added. Incubate for 10 minutes at room temperature.

2.3.2 preparation of 2-fold substrate solution

FAM-labeled polypeptide and ATP were added to 1-fold kinase buffer to form a 2-fold substrate solution. To a 384 well reaction plate 2.5. mu.l of a 2-fold substrate solution was added.

2.4 kinase reaction

The 384 well plates were incubated at 28 degrees for 120 minutes,

2.5 detection of reaction results

2.5.1 equilibrate ADP-Glo reagent to room temperature.

2.5.2 transfer 5. mu.l of reaction to a new 384-well plate reaction well.

2.5.3 transfer 5. mu.l of ADP-Glo reagent to 384 well plate reaction wells to stop the reaction.

2.5.4 incubate at 28 ℃ for 120 minutes.

2.5.5 mu.l of the kinase detection reagent was transferred to each reaction well, shaken for 1 minute, and allowed to stand at room temperature for 30 minutes.

2.6 data reading

The luminescence values of the samples were read on Envision.

2.7 Curve fitting

2.7.1 copying data of luminescence readings from Envision program

2.7.2 the value of the luminescence reading is converted to a percentage inhibition by a formula.

"min" is the fluorescence reading for the control where the reaction was run without enzyme addition; "max" is the sample fluorescence reading with DMSO added as a control.

2.7.3 data were imported into MS Excel and curve-fitted using XLFit Excel add-in version 5.4.0.8, the fit being Y ═ Bottom + (Top-Bottom)/(1+ (IC)₅₀/X) ^ HillSlope) and the results are shown in Table 4.

TABLE 4

The experimental results show that the compound has good inhibitory activity on ALK5 kinase, low inhibitory action on p38 alpha and high selectivity. The compound of the invention has lower side effect while generating higher curative effect.

Experimental example 2 in vitro evaluation of cellular luciferase with Compounds

1. Experimental Material

Test compounds: the compound of formula (I) of the invention of example 1, formulated in DMSO to 4mM, is then diluted sequentially 4-fold in 20000.00nM, 5000.00nM, 1250.00nM, 312.5nM, 78.125nM, 19.53nM, 4.88nM, 1.22 nM.

Luc-Smad2/3-NIH3T3 mouse fibroblasts (engineered to overexpress SMAD2, 3-responsive promoter) were gifted by the university of Chinese medicine laboratory.

Reagent: DMEM, available from Invitrogen, usa; FBS, available from Invitrogen, usa; DMSO, available from Sigma, usa; glo Lysis Buffer, available from Progema, usa; Bright-Glo Luciferase assay system available from Promega, USA; TGF β, available from PeproTech, USA.

The instrument comprises the following steps: MD SpectraMax M3 multifunctional microplate reader, available from Molecular Devices, USA.

2. Experimental methods

2.1 cell culture:

cell recovery: the cells were lysed in a 37 ℃ water bath, transferred to 15mL of pre-warmed medium, centrifuged at 1000rpm for 5 minutes, the medium was discarded, the cells were resuspended in 15mL of fresh medium, transferred to a 10cm petri dish, placed at 37 ℃ in 5% CO₂The culture was performed in the incubator (1), and after 24 hours, the cells were replaced with fresh medium.

Cell passage: transferring the recovered cells into a 50mL sterile centrifuge tube, centrifuging at 1000rpm for 5 minutes, discarding the culture medium,counting the uniformly dispersed cells, adjusting the appropriate cell concentration to 15mL of fresh medium, adding into a 10cm culture dish, placing at 37 deg.C and 5% CO₂Cultured in an incubator.

2.2 Experimental procedures:

day 1: cell spreading (bottom 96 pore plate)

Culturing Luc-Smad2/3-NIH3T3 cells in a 10cm culture dish normally until the confluency reaches 80% -90%, collecting the cells into a 15mL centrifuge tube after digestion, centrifuging for 5 minutes at 1000Xg, removing supernatant, suspending the cells in 1mL culture medium, diluting by 10 times for counting, diluting the cells according to the counting result, and adding 4X10³The number of cells per well was transferred to a 96-well plate (100. mu.l of resuspended cells per well).

Day 2: cell administration

The drug was weighed 1-2mg (weighed in advance) and prepared in 4mM stock solution using DMSO. After 24 hours, the medium was removed. The drug was diluted in 2% FBS medium and 100. mu.l of 1 Xdrug solution was added to give final concentrations of 20000.00nM, 5000.00nM, 1250.00nM, 312.5nM, 78.125nM, 19.53nM, 4.88nM, 1.22nM, respectively, and a final concentration of 4ng/mL of TGF β 1 per well, along with the compound diluted in 2% FBS medium.

Day 3: fluorescence detection experiment

The Glo Lysis Buffer and Bright-glociferase assay system and cells were equilibrated to room temperature, the cell supernatant was removed, 100. mu.l Glo Lysis Buffer was added to each well, the cells were uniformly lysed by gentle shaking, and 5mins were lysed at room temperature. Then, 100. mu.l of Bright-fluorescence assay system was added to each well, incubated at room temperature for 5 minutes, shaken for 2 minutes, and 180. mu.l of the supernatant was transferred to a 96-well white-bottomed plate, and a chemiluminescent signal was detected under 1s conditions.

2.3 data processing: nonlinear curve fitting and data analysis are carried out by using Graphpad Prism 5 software, and IC is obtained by fitting₅₀The results are shown in Table 5.

TABLE 5

From the above experiments, the compound of the invention shows good inhibitory activity on TGF beta-ALK 5-SMAD2/3 signal channels in NIH3T3 cells, and is very promising to be a therapeutic agent for various cancer-related diseases.

Although the present invention has been described in detail above, those skilled in the art will appreciate that various modifications and changes can be made to the present invention without departing from the spirit and scope of the invention. The scope of the invention is not to be limited by the above detailed description but is only limited by the claims.

Claims

1. A process for the preparation of a compound of formula (I) or a salt, hydrate, solvate or crystal thereof, comprising the step of reacting a compound of formula (II) wherein X is a leaving group with a compound of formula (III),

2. a process for the preparation of a compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to claim 1 wherein X is selected from the group consisting of halogen, hydroxy, active ester of hydroxy, amino, alkoxy, acyloxy, aryloxy, heteroaryloxy, sulfonyloxy, optionally substituted alkylsulfonyloxy, optionally substituted alkenylsulfonyloxy, optionally substituted arylsulfonyloxy, acyl, diazo moieties; further preferably, X is selected from the group consisting of fluorine, chlorine, bromine, iodine, carboxylate, sulfonate, phosphate and borate; still further preferably, X is selected from the group consisting of fluoro, chloro, bromo, iodo, mesylate, triflate, benzenesulfonate, p-toluenesulfonate, p-bromophenylsulfonate and p-nitrobenzenesulfonate.

3. The process for producing a compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to claim 1 or 2, wherein the process for producing comprises the step of reacting a compound of formula (II) with a compound of formula (III) in the presence of a catalyst.

4. A process for the preparation of a compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to claim 3 wherein the catalyst is a transition metal catalyst, preferably the catalyst is a palladium catalyst.

5. A process for the preparation of a compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to claim 4 wherein the catalyst is selected from (Ph)₃P)₄Pd、(Ph₃P)₂PdCl₂、(CH₃CN)₂PdCl₂、Pd₂(dba)₃、(dppf)PdCl₂And Pd (OAc)₂。

6. A method for producing a compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to any one of claims 1 to 5, wherein the production method comprises a step of reacting a compound of formula (II) and a compound of formula (III) in the presence of a palladium catalyst and a phosphine ligand.

7. The method for preparing a compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to claim 6, wherein the phosphine ligand is selected from the group consisting of 2-dicyclohexylphosphine-2 ',6' -diisopropyloxy-1, 1 '-biphenyl, 2-dicyclohexylphosphino-2' - (N, N-dimethylamine) -biphenyl, 2-dicyclohexylphosphine-2 ',4',6 '-triisopropylbiphenyl, 2- (di-t-butylphosphine) biphenyl, 4, 5-bisdiphenylphosphine-9, 9-dimethylxanthene and 2-dicyclohexylphosphine-2', 6 '-diisopropyloxy-1, 1' -biphenyl.

8. A process for the preparation of a compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to any one of claims 2 to 7, wherein the molar ratio of compound of formula (II) to catalyst is from about 1:0.005 to 1:0.04, preferably from about 1:0.009 to 1:0.02, further preferably from about 1: 0.01; for example, the molar ratio of the compound of formula (II) to tris (dibenzylideneacetone) dipalladium is about 1:0.005 to 1:0.04, preferably about 1:0.009 to 1:0.02, and more preferably about 1: 0.01; the molar ratio of the compound of formula (II) and tris (dibenzylideneacetone) dipalladium is about 1:0.005 to 1:0.04, preferably about 1:0.009 to 1:0.02, and more preferably about 1: 0.01.

9. A process for the preparation of a compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to any one of claims 1 to 8 further comprising the step of reacting a compound of formula (IV) with a compound of formula (V) to produce a compound of formula (II) wherein X is a leaving group,

10. the method for producing a compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to claim 9, further comprising the step of reacting a compound of formula (VI) in a reaction solvent to produce a compound of formula (V) wherein X is a leaving group,

11. the process for the preparation of a compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to any one of claims 1 to 10, which further comprises a step of liberating the compound of formula (VII) into the compound of formula (III) under the action of a basic agent,