Preparation method of quinoline TGF-β1 inhibitor

Abstract

The present invention belongs to the field of medicinal chemistry and relates to a method for preparing a quinoline TGF-β1 inhibitor. Specifically, it relates to a method for preparing 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 of formula (I) or a salt, hydrate, solvate or crystal thereof.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and specifically relates to a method for preparing 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 or its salt, hydrate, solvate or crystal. Background Art

[0002] Transforming growth factor β (TGF-β) is an important cytokine. To date, six distinct isoforms (TGF-β1–6) have been identified, each with varying degrees of homology. In mammals, only three isoforms are expressed: TGF-β1, TGF-β2, and TGF-β3. It is a multifunctional growth factor superfamily with a wide range of biological activities, including involvement in early embryonic development, cartilage and bone formation, extracellular matrix synthesis, inflammation, interstitial fibrosis, regulation of immune and endocrine function, and tumor formation and progression. While these three isoforms are structurally similar and share high amino acid sequence homology, they exhibit distinct phenotypes in knockout mouse models, suggesting that each isoform has specific and independent functions in vivo. TGF-β ligands can bind to membrane receptors, initiating downstream signaling within the cell.

[0003] TGF-β1 is the most common and important isoform of TGFβ, the most abundant isoform expressed in the liver and the strongest known inducer of liver fibrosis. It plays a crucial role in the progression of chronic liver disease to end-stage liver disease (Yamazaki et al. Digestive Disease, 2011, 29:284-288). Multiple studies have shown that TGF-β1 and its receptor are often highly expressed in liver diseased organs, blood vessels, and the extracellular matrix. In the classic TGFβ-TGFβR-Smads pathway, TGF-β1 activates TGFβR1 (transforming growth factor β receptor 1, ALK5), which in turn regulates the entire signaling pathway, ultimately modulating the expression of a series of target genes associated with fibrosis and tumor development. It is generally believed that TGF-β promotes liver cancer mainly by promoting tumor cell metastasis, enhancing tumor cell immune escape and inducing angiogenesis (Ling, et al. Current Pharmaceutical Biotechnology, 2011, 12: 2190-2202).

[0004] Research on drugs targeting the TGF-β pathway has been ongoing for many years, but TGFβR1 inhibitors such as Galunisertib have demonstrated certain cardiotoxic effects (such as bleeding, functional degradation, and inflammatory damage) in animal models. This is due to the low target selectivity and specificity of these drugs. While inhibiting the TGFβR1 kinase activation site, the drugs also have a strong inhibitory effect on other proteins with the same kinase region (such as p38α), resulting in numerous unexpected off-target side effects. Therefore, there is still a need to develop more selective TGFβR1 inhibitors that can specifically regulate the TGF-β signaling pathway for the treatment of TGF-β-related diseases. Summary of the Invention

[0005] The inventors of the present invention have discovered a quinoline TGF-β1 inhibitor, the compound structure of which is shown in the following formula (I), and the chemical name 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 formula (I)"):

[0006]

[0007] The inventors of the present invention have found that the compound of formula (I) or its hydrate, solvate or crystal exhibits significant inhibitory activity against TGF-βR1 kinase and is very promising as a therapeutic agent for TGF-βR1-related diseases.

[0008] As is well known, for human drug use, domestic and international regulatory agencies have set very low limits for unidentified or undetermined toxicity impurities in active pharmaceutical ingredients (APIs) for safety reasons. Impurities in APIs may arise from the API's own degradation or from the preparation process, for example, including unreacted starting materials, chemical derivatives of impurities contained in the starting materials, and synthetic by-products. Therefore, it is necessary to develop methods for preparing compounds of formula (I) or their derivatives in order to obtain methods for preparing compounds of formula (I) or their pharmaceutically acceptable salts, isomers, solvates, or crystals that have mild reaction conditions, stable processes, easy purification, and ease of operation, and are conducive to large-scale industrial production.

[0009] The inventors of the present invention have conducted research and exploration on the preparation method of the compound represented by formula (I), and the attempted reaction route 1 includes the following reaction steps:

[0010]

[0011] 1) Protecting the hydroxyl group of 7-chloro-4-hydroxyquinoline of formula (1) to generate a compound of formula (2);

[0012] 2) Buchward coupling reaction of the compound of formula (2) with 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine hydrochloride to obtain a compound of formula (3);

[0013] 3) removing the hydroxy protecting group from the compound of formula (3) under acidic conditions to obtain a compound of formula (4);

[0014] 4) chlorinating the hydroxyl group of the compound of formula (4) with phosphorus oxychloride to obtain a compound of formula (5);

[0015] 5) The compound of formula (5) reacts with 1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-ol in the presence of an acid-binding agent to obtain a compound of formula (I).

[0016] In step 2 of Route 1, during the Buchward coupling to prepare the compound of formula (3), the compound of formula (2) partially removes the BOC (tert-butyloxycarbonyl) group. However, the electron-donating effect of the hydroxyl group in the compound of formula (1) after BOC removal has a passivating effect on the Buchward coupling reaction. As a result, once the BOC is removed, the resulting raw material cannot participate in the reaction, resulting in a low conversion rate in this reaction step. Subsequent attempts at other hydroxyl protecting groups, such as methoxymethyl ether (MOM) and (trimethylsilyl)ethoxymethyl (SEM), all suffered from low conversion rates and residual raw materials. Increasing the amount of palladium reagent produced a large amount of chlorine-depleted byproducts.

[0017] One object of the present invention is to provide a method for preparing a compound represented by formula (I) or a salt, hydrate, solvate or crystal thereof, comprising the step of reacting a compound represented by formula (II) with a compound represented by formula (III), wherein X is a leaving group,

[0018]

[0019] 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 moiety and active ester of hydroxy, such as carboxylate, sulfonate, phosphate or borate. In some specific embodiments, the leaving group X is chloro, iodo, bromo, fluoro, acetoxy, methanesulfonyloxy, toluenesulfonyloxy, trifluoromethanesulfonyloxy, nitrobenzenesulfonyloxy or bromo-benzenesulfonyloxy.

[0020] In some preferred embodiments, the present invention provides a method for preparing a compound of formula (I) of the present invention or a salt, hydrate, solvate or crystal thereof, wherein X is selected from an active ester of a halogen and a hydroxyl group; further preferably, X is selected from fluorine, chlorine, bromine, iodine, carboxylate, sulfonate, phosphate and borate; further preferably, X is selected from fluorine, chlorine, bromine, iodine, methanesulfonate, trifluoromethanesulfonate, benzenesulfonate, p-toluenesulfonate, p-bromobenzenesulfonate and p-nitrobenzenesulfonate. In some embodiments, the method for preparing a compound of formula (I) of the present invention includes 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. Palladium catalysts include Pd(0) and P(II) sources. In some embodiments, palladium / carbon can be used as a catalyst. In some embodiments, palladium catalyst species can be used, which generally include one or more ligands that are bound to palladium metal. In some specific embodiments, the palladium catalyst is selected from (Ph3P)4Pd, (Ph3P)2PdCl2, (CH3CN)2PdCl2, Pd2(dba)3, (dppf)PdCl2 and Pd(OAc)2. A wide variety of ligands are known in the art, including generally preferred phosphine ligands (see, for example, C.Amatore and A.Jutand, Coord.Chem.Rev.1998,178-180 and 511-528).The phosphine ligands used in the method of the present invention include, but are not limited to, triphenylphosphine, tri(o-tolyl)phosphine, tri(2-furyl)phosphine, 1,2-bis(diphenylphosphino)ethane (dppe), 1,4-bis(diphenylphosphino)butane (dppb), 2,3-bis(diphenylphosphino)butane (Chiraphos), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos), 1,2-bis(2,5-dimethylphosphacyclopentyl)benzene (Me-DuPhos), diphenylphosphino)ferrocenyl]ethyldicyclohexylphosphine (Josiphos), bis(diphenylphosphino)methane (dppm), 1,3-bis(diphenylphosphino)propane (dppp), 1,2-bis(dicyclopentyl)benzene (dppb), 1,3-bis(diphenylphosphino)propane (dppp), 1,2-bis(dicyclopentyl)benzene (dppb), 1,3-bis(diphenylphosphino)propane (dppp), 1,3-bis(diphenylphosphino)propane ... phosphino), 2-di-tert-butylphosphine, ... In some embodiments, according to the preparation method of the compound of formula (I) of the present invention, the ligand is selected from 2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl (Ruphos), 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)-biphenyl (Davephos), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (X-Phos), 2-(di-tert-butylphosphino)biphenyl (Johnphos), 4,5-bis(diphenylphosphine) -9,9-dimethylxanthene (Xantphos), and 2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl (RuPhos); preferably, the ligand is selected from 2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl (Ruphos), 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)-biphenyl (Davephos) and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos).

[0021] In addition, palladium coupling reactions can be carried out with N-heterocyclic carbene ligands (see, for example, Hillier, AC 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)imidazolium chloride, 1,3-bis(2,6-diisopropylphenyl)imidazolidinium tetrafluoroborate, 1,3-bis(2,4,6-trimethylphenyl)imidazolidinium tetrafluoroborate, and the like. The catalyst can be derived from a preformed complex, such as (Ph3P)4Pd, (Ph3P)2PdCl2, (CH3CN)2PdCl2, Pd2(dba)3, (dppf)PdCl2 ([1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride), etc., or the catalyst can be formed in situ from a combination of a palladium source including, but not limited to, PdCl2, Pd(OAc)2, Pd(dba)2, etc., and a suitable ligand. In some embodiments, the catalyst is Pd2(dba)3. In other embodiments, an amine base such as diisopropylethylamine, triethylamine, etc. can be added to the reaction mixture to stabilize the catalyst. In other embodiments, the palladium catalyst can 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 include a metal, such as palladium, supported on a polymer support that includes a metal binding moiety. In some embodiments, the supported polymer is palladium on a polymeric substrate fiber, including but not limited to polyolefin substrate fibers such as those from Johnson-Mathey. Polyolefin substrate fibers. In other embodiments, the supported catalyst is a polymer-immobilized homogeneous catalyst in which palladium metal is covalently bound to polymer chains, which can be further linked to inert polyolefin fibers that are insoluble in common organic solvents. Suitable supported catalysts include those sold by Johnson-Mathey under the trade name Those sold, especially those sold by Johnson-Matthey Supported polymer 1000 series. Of course, other types of palladium catalysts supported on polymer supports can be used, including but not limited to supported catalysts based on polystyrene, etc. In certain embodiments, it is desirable to remove oxygen from a solvent and / or a solution containing a catalyst to avoid ligand oxidation and the destabilization of the catalyst. This can be accomplished in any manner known in the art, such as by alternately applying a vacuum to the mixture, subsequently introducing nitrogen or other suitable inert gases to degas the mixture. Alternatively, nitrogen or other inert gases can be bubbled through a solvent or a solution containing a catalyst. In some specific embodiments, the compound of formula (I) of the present invention, or its salt, hydrate, solvate or crystallization method is included in the presence of a palladium catalyst, a phosphine ligand, and the step of reacting the compound of formula (II) and the compound of formula (III). 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, etc. Hydrocarbon solvents include 1,4-dioxane, heptane, cyclohexane, methylcyclohexane, isooctane, and the like; 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, 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. Ketone solvents include acetone, methyl ethyl ketone, methyl isobutyl ketone, and methyl isopropyl ketone, and the like. Amide solvents include dimethylformamide, dimethylacetamide, N-methylpyrrolidone, and the like. In some embodiments, according to the method for preparing the compound of formula (I) or its salt, hydrate, solvate, or crystal according to the present invention, in addition to the catalyst, a suitable base may be added to the reaction mixture. 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 carbonate, potassium carbonate, and cesium carbonate, alkaline earth metal carbonates, alkali metal and alkaline earth metal phosphates such as K3PO4, 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 inventors of the present invention have investigated the residual amount of compound (I) and the amount of compound (I) produced when tris (dibenzylideneacetone) dipalladium Pd2 (dba) 3 is used as a metal catalyst and only the ligand is different when other conditions are the same. The specific data are shown in Table 1. The experimental data show that when tris (dibenzylideneacetone) dipalladium is selected as a metal catalyst and different ligands are selected, it is found that the two ligands X-phos and Johnphos cannot react completely. Therefore, the present invention provides a method for preparing a compound of formula (I) of the present invention, wherein the preparation method includes a ligand, preferably, wherein the ligand is selected from 2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl (Ruphos), 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)-biphenyl (Davephos), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (X-Phos), 2-(di-tert-butylphosphino)biphenyl (Johnphos), 4 ,5-bis(diphenylphosphine)-9,9-dimethyloxanthene (Xantphos), and 2-dicyclohexylphosphine-2',6'-diisopropoxy-1,1'-biphenyl (RuPhos); preferably, the ligand is selected from 2-dicyclohexylphosphine-2',6'-diisopropoxy-1,1'-biphenyl (Ruphos), 2-dicyclohexylphosphine-2'-(N,N-dimethylamino)-biphenyl (Davephos) and 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthene (Xantphos).

[0022] Table 1

[0023] ligand Compound of formula (II) (%) Compounds 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

[0024] In other specific embodiments, the inventors of the present invention have investigated the amount of metal catalyst used. Experimental data show that as the amount of tris(dibenzylideneacetone)dipalladium increases, the reaction becomes more and more complete. When the amount of tris(dibenzylideneacetone)dipalladium is too large, the compound of formula (II) is debrominated to form the impurity compound of formula (II)-1; if the amount of tris(dibenzylideneacetone)dipalladium is too small, the reaction is incomplete and the reaction time is too long. Some experimental data are shown in Table 2. Therefore, the present invention provides a method for preparing a compound of formula (I) of the present invention, wherein 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.

[0025] Table 2

[0026]

[0027] In other specific embodiments, the inventors of the present invention have investigated the effects of reaction temperature and time when using tris(dibenzylideneacetone)dipalladium catalyst. Reactions were carried out at 60°C, 80°C and 100°C respectively, and the reaction liquid was taken for HPLC detection at 2 hours, 3 hours, 4 hours, 8 hours and 20 hours to investigate the relationship between the change of the compound of formula (I) and the compound of formula (II) over time at different temperatures. The tolerance of the compound of formula (I) in the reaction liquid was also investigated. Specific data are shown in Table 3. Experimental data show that within the same time, the reaction rate accelerates as the temperature increases, and the relationship between the remaining amount of the compound of formula (II) and the reaction temperature shows a significant correlation. When the temperature is 60°C and 80°C, the compound of formula (II) cannot react completely within the same reaction time. Even if the reaction time is extended, the reaction rate is not significantly accelerated. At 100°C, the compound of formula (II) is completely reacted after 3 hours by HPLC detection. As the reaction time is extended, the impurities and purity in the reaction liquid do not change significantly. Under the same conditions, lowering the temperature prolongs the reaction completion time, while lowering the temperature and increasing the amount of the catalyst tris(dibenzylideneacetone)dipalladium instead increases the amount of the impurity compound-1 of formula (II). Therefore, both the amount of tris(dibenzylideneacetone)dipalladium and the temperature affect the key quality attributes of the product. Therefore, the present invention provides a method for preparing the compound of formula (I) of the present invention, wherein the reaction temperature is about 60-120°C, preferably about 80-110°C, and more preferably about 90-100°C.

[0028] Table 3

[0029]

[0030]

[0031] In a specific embodiment, the preparation method of the compound of formula (I) of the present invention or its salt, hydrate, solvate or crystal 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 and a phosphine ligand.

[0032] In some specific embodiments, the method for preparing the compound of formula (I) or its salt, hydrate, solvate or crystal of the present invention further comprises the step of reacting the compound of formula (IV) with the compound of formula (V) to form the compound of formula (II), wherein X is a leaving group (as defined above),

[0033]

[0034] In some specific embodiments, the preparation method of the compound of formula (II) above includes the step of reacting a compound of formula (IV) with a compound of formula (V) in a suitable solvent such as a water-soluble solvent, for example, acetonitrile, ethanol, acetone, alkanol, alcohol, ether, propylene glycol, glycerol, triacetin, poly(propylene glycol), PVP (poly(vinyl pyrrolidone)), dimethyl sulfoxide, N,N-dimethylformamide, formamide, N,N-dimethylacetamide, pyridine, propanol, N-methylacetamide, butanol, soluphor (2-pyrrolidone) or pharmasolve (N-methyl-2-pyrrolidone), etc., preferably acetonitrile, methanol or ethanol, to produce a compound of formula (II).

[0035] In some specific embodiments, according to the preparation method of the compound of formula (II) of the present invention, wherein the reaction solvent is acetonitrile. The inventors of the present invention used acetonitrile as a solvent to carry out exemplary experiments and found that this step is a heterogeneous reaction. If the amount of solvent is too large, the reaction contact surface is reduced, resulting in a slow reaction rate; if the amount of solvent is too little, it will cause explosive analysis during post-processing. Experiments show that when the amount of acetonitrile is about 10V-20V (the ratio between the volume of solvent (mL) and the mass (g) of the compound of formula (IV)), the cooling and precipitation of the compound of formula (II) will not cause losses, while also avoiding the risk of explosive analysis of the compound of formula (II) and excessive reaction solvent reducing the reaction rate during hot filtration. Therefore, in some preferred embodiments, the present invention provides a method for preparing a compound of formula (II) of the present invention, using acetonitrile as a reaction solvent, wherein the ratio of acetonitrile to the compound of formula (IV) is about 10-20:1 (the ratio between the volume of acetonitrile (mL) and the mass of the compound of formula (IV) (g)), preferably about 10-17:1 (the ratio between the volume of acetonitrile (mL) and the mass of the compound of formula (IV) (g)). In a specific embodiment, the present invention provides a method for preparing a compound of formula (II) of the present invention, using acetonitrile as a reaction solvent, wherein the ratio of acetonitrile to the compound of formula (IV) is about 14:1 (the ratio between the volume of acetonitrile (mL) and the mass of the compound of formula (IV) (g)).

[0036] In some specific embodiments, the method for preparing the compound of formula (I) or its salt, hydrate, solvate or crystal of the present invention further comprises the step of reacting the compound of formula (VI) to produce a compound of formula (V), wherein X is a leaving group (as defined above),

[0037]

[0038] In some specific embodiments, the present invention provides a method for preparing a compound of formula (V) of the present invention, wherein the reaction solvent is selected from a water-soluble solvent, such as acetonitrile, ethanol, acetone, alkanol, alcohol, ether, propylene glycol, glycerol, triacetin, poly(propylene glycol), PVP (poly(vinyl pyrrolidone)), dimethyl sulfoxide, N,N-dimethylformamide, formamide, N,N-dimethylacetamide, pyridine, propanol, N-methylacetamide, butanol, soluphor (2-pyrrolidone), pharmasolve (N-methyl-2-pyrrolidone), etc., preferably selected from acetonitrile, methanol and ethanol.

[0039] In some specific embodiments, the method for preparing the compound of formula (I) or its salt, hydrate, solvate or crystal of the present invention further comprises the step of freeing the compound of formula (VII) to the compound of formula (III) under the action of an alkaline reagent.

[0040]

[0041] In some embodiments, the present invention provides a method for preparing a compound 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 carbonate, potassium carbonate and cesium carbonate, alkaline earth metal carbonates, alkali metal 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), etc. In some specific embodiments, the alkaline agent is selected from sodium carbonate, potassium carbonate and sodium hydroxide.

[0042] In some specific embodiments, according to the preparation method of the compound of formula (III) of the present invention, acetonitrile is used as reaction solvent. In some preferred embodiments, according to the preparation method of the compound of formula (III) of the present invention, a mixture of acetonitrile and water is used as reaction solvent. Preferably, a catalytic amount of water is used. In some specific embodiments, according to the preparation method of the compound of formula (III) of the present invention, wherein the reaction solvent is the acetonitrile containing about 0.1-1.0% water. In some specific embodiments, according to the preparation method of the compound of formula (III) of the present invention, wherein the reaction solvent is the acetonitrile containing about 0.2-0.6% water. In some specific embodiments, according to the preparation method of the compound of formula (III) of the present invention, wherein the reaction solvent is the acetonitrile containing about 0.2-0.4% water. In some specific embodiments, according to the preparation method of the compound of formula (III) of the present invention, wherein the reaction solvent is the acetonitrile containing about 0.3% water.

[0043] In some specific embodiments, the present invention provides a method for preparing the compound of formula (IV) of the present invention, comprising the following steps:

[0044]

[0045] 1) The compound of formula (IV-1) reacts with bromine under acidic conditions to produce a compound of formula (IV-2);

[0046] 2) the compound of formula (IV-2) reacts with benzoic acid under alkaline conditions to produce a compound of formula (IV-3);

[0047] 3) reacting the compound of formula (IV-3) with N,N-dimethylformamide dimethyl acetal (DMF-DMA) to produce a compound of formula (IV-4);

[0048] 4) The compound of formula (IV-4) and hydrazine hydrate are reacted with glacial acetic acid to obtain a compound of formula (IV-5);

[0049] 5) reacting the compound of formula (IV-5) with cyclopropylboronic acid in 1,2-dichloromethane to produce a compound of formula (IV-6);

[0050] 6) The compound of formula (IV-6) reacts under alkaline conditions to prepare the compound of formula (IV).

[0051] In some specific embodiments, the present invention provides a method for preparing a compound of formula (I) or a salt, hydrate, solvate or crystal thereof, wherein the method comprises the following steps:

[0052]

[0053] 1) reacting a compound of formula (VI-1) to obtain a compound of formula (V-1);

[0054] 2) reacting a compound of formula (V-1) with 1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-ol of formula (IV) to produce a compound of formula (II-1);

[0055] 3) 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine hydrochloride of formula (VII) is liberated into a compound of formula (III) under the action of an alkaline reagent;

[0056] 4) The compound of formula (II-1) reacts with the compound of formula (III) to prepare the compound of formula (I).

[0057] Compared to Route 1, this route significantly simplifies the experimental steps. Substituting chloroquinoline for bromoquinoline allows for Buchward coupling to produce compounds of Formula (I) in higher yields. Changing the reaction sequence to nucleophilic substitution followed by coupling can address numerous issues, including hydroxyl passivation, screening for hydroxyl protecting groups, and removal of protecting groups. This route offers fewer steps, simple operation, suitability for industrial production, greater environmental friendliness, and higher yields.

[0058] In some preferred embodiments, the present invention provides a method for purifying a compound of formula (I) or its salt, hydrate, solvate or crystal, wherein the method comprises dissolving the compound of formula (I) or its salt, hydrate, solvate or crystal in a solvent, cooling and crystallizing; preferably, the present invention provides a method for purifying a compound of formula (I) or its salt, hydrate, solvate or crystal, wherein the method comprises dissolving the compound of formula (I) or its salt, hydrate, solvate or crystal in a solvent, adding a metal scavenger and activated carbon, filtering, cooling and crystallizing; further preferably, the present invention provides a compound of formula (I) or its salt A method for refining 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, filtering, and adding an anti-solvent for crystallization; wherein the solvent is preferably selected from one or more of dichloromethane, acetonitrile, water, esters with less than 6 carbon atoms, alcohols with less than 6 carbon atoms and ketones with less than 6 carbon atoms, and more preferably selected from one or more of ethyl acetate, acetonitrile, methanol, ethanol, propanol, butanol, sec-butanol, isopropanol, dichloromethane, acetone and water; and wherein the anti-solvent is selected from methane, ethane, propane, butane, pentane and heptane.

[0059] The inventors of the present invention have found that the preparation method of the compound of formula (I) or its salt, hydrate, solvate or crystal provided by the present invention has a reaction route with fewer steps, simple operation, is more environmentally friendly, has a higher yield and purity, mild reaction conditions, easy purification, stable process, and is easy to operate, and can meet the requirements of industrial-scale production and application.

[0060] Terminology

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the invention pertains. "Leaving group" of the present invention, having the ordinary meaning of this area, refers to a group that can be easily displaced, and when a new bond is formed, a reactive functional group on a molecule that undergoes a displacement reaction by a molecule. Groups with this function are well known to those skilled in the art, and their specific examples can be further referenced to the common organic synthesis handbooks in this area, such as "Advanced Organic Chemistry," Jerry March, 5th edition, pages 351-357, John Wiley and Sons, NY. For example, the leaving group can 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.

[0062] The "acid binding agent" of the present invention has the usual meaning in this field. Preferably, the acid binding agent is selected from alkoxide base, alkali metal alkoxide and carbonate base; further preferably, the acid binding agent 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.

[0063] The "hydroxy protecting group" of the present invention is a suitable group for protecting a hydroxy group known in the art, as shown in the literature ("Protective Groups in Organic Synthesis", 5 Th Ed.TWGreene & P.GM Wuts) in the hydroxyl protecting group. As an example, preferably, the hydroxyl protecting group can be (C 1-10 Alkyl or aryl) 3 silyl, for example: triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, etc.; can be C 1-10 Alkyl or substituted alkyl, for example: methyl, tert-butyl, allyl, benzyl, methoxymethyl, ethoxyethyl, 2-tetrahydropyranyl (THP), etc.; can be (C 1-10 Alkyl or aromatic) acyl, for example: formyl, acetyl, benzoyl, etc.; can be (C 1-6 Alkyl or C6-10 aryl)sulfonyl; can also be (C 1-6 Alkoxy or C 6-10 aryloxy)carbonyl.

[0064] The "salt" of the present invention can be any salt, in particular a pharmaceutically acceptable salt. In this article, "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt formed by the compound of the present invention and the "acid" or "acidic agent" of the present invention, wherein the "acid" or "acidic agent" of the present invention can be selected from hydrochloric acid, hydrobromic acid, phosphoric acid, sulfamic acid, nitric acid, p-toluenesulfonic acid, benzenesulfonic acid, p-aminobenzenesulfonic 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, α-ketoglutaric acid, hippuric acid, maleic acid, D-tartaric acid, methanesulfonic acid or its analogues. "Pharmaceutically acceptable salts" of the compounds of the present invention can be synthesized from compounds of the present invention containing an acidic or basic moiety by conventional chemical methods. Generally, salts of basic compounds can be prepared by ion 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 can be formed by reaction with a suitable inorganic or organic base.

[0065] The "alkaline reagent" of the present invention refers to a compound that can deprotonate a hydroxyl group or an amino group. Examples of bases include, but are not limited to, (C 1-6 Alkyl) oxide ((C 1-6 alkyl)OM), wherein (C 1-6 Alkyl) oxides include but are not limited to MeO-, EtO-, n-PrO-, i-PrO-, t-BuO-, i-AmO- (isopentyloxy), etc., and wherein M is an alkali metal cation, such as Li + 、Na + , K + Etc. Alcohol solvents include (C 1-6 Alkyl) OH, for example, such as methanol, ethanol, n-propanol, isopropanol, tert-butanol, isopentanol, etc. Non-alkoxy bases such as sodium hydroxide, potassium hydroxide, sodium hydride, sodium hexamethyldisilazide, lithium hexamethyldisilazide, lithium diisopropylamide, calcium hydride, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, DBU (1,8-diazabicyclo [5.4.0] undec-7-ene), DBN (1,5-diazabicyclo [4.3.0] non-5-ene), Grignard reagents such as (C 1-6Alkyl)Mg(halogen), which includes but is not limited to methylmagnesium chloride, methylmagnesium bromide, tert-butylmagnesium chloride, tert-butylmagnesium bromide, etc.

[0066] The term "solvate" refers to a form of the compound of the present invention which forms a complex in the solid or liquid state by coordination with solvent molecules. 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.

[0067] The term "crystalline" refers to various solid forms of the compounds of the present invention, including crystalline forms and amorphous forms.

[0068] The "hydrogen", "carbon" and "oxygen" in the compounds of the present invention include all isotopes thereof. Isotopes should be 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 include 13 C and 14 C, oxygen isotopes include 16 O and 18 O etc. DETAILED DESCRIPTION

[0069] The following representative examples are provided to better illustrate the present invention, but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the materials used in the following examples were all commercially available.

[0070] Example 1 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

[0071]

[0072] Step 1: Preparation of 2-bromo-1-(tetrahydro-2H-pyran-4-yl)ethan-1-one

[0073]

[0074] Under nitrogen, methanol (100 mL) and 1-(tetrahydro-2H-pyran-4-yl)ethanone (20.0 g, 156 mmol) were added sequentially to a 1000 mL three-necked flask. The temperature was lowered to below -15°C, and liquid bromine was slowly added dropwise, maintaining the temperature below -15°C. After the addition was complete, the temperature was raised to 0°C and the reaction was continued for 45 minutes. Then, the temperature was raised to 10°C and the reaction was continued for 45 minutes. Maintaining the internal temperature below room temperature, 11 mol / L sulfuric acid (55 mL) was slowly added dropwise. After the addition was complete, the reaction was allowed to proceed overnight at room temperature. The reaction was monitored for completion, extracted with ethyl acetate and aqueous sodium chloride solution, and the organic layers were combined. The pH of the organic layers was adjusted to 7-8 with saturated sodium bicarbonate. The combined organic layers were concentrated under reduced pressure to afford the title compound as a pale yellow solid, 28.5 g in 87.5% yield; MS (ESI) m / z 207.0 [M+H]. + .

[0075] Step 2: Preparation of 2-oxo-2-(tetrahydro-2H-pyran-4-yl)ethyl benzoate

[0076]

[0077] Benzoic acid (18.5 g, 151.4 mmol) was dissolved in N,N-dimethylformamide (DMF, 495 mL), potassium carbonate (38 g, 275.2 mmol) was added, and 2-bromo-1-(tetrahydro-2H-pyran-4-yl)ethan-1-one (28.5 g, 137.6 mmol) was added to the system. The reaction was allowed to react at room temperature overnight. Ethyl acetate was added to dilute the mixture, and the mixture was washed with sodium chloride solution. The organic phases were combined and concentrated under reduced pressure to obtain the title compound as a light yellow solid, 30.0 g in total, with a yield of 88.2%; LC-MS m / z [M+H] + =249.

[0078] Step 3: Preparation of (Z)-1-(dimethylamino)-3-oxo-3-(tetrahydro-2H-pyran-4-yl)prop-1-en-2-yl benzoate

[0079]

[0080] To 1,1-dimethoxy-N,N-dimethylmethanamine (795.15 mL, 5975.8 mmol) was added 2-oxo-2-(tetrahydro-2H-pyran-4-yl)ethyl benzoate (95.0 g, 383 mmol). The mixture was heated to 100°C for 2 h, then raised to 106°C for 2 h. After monitoring the reaction, the mixture was returned to room temperature and concentrated under reduced pressure to dryness. Ethyl acetate was added to the mixture, and the mixture was washed with brine. The organic phase was dried over anhydrous sodium sulfate. The organic phase was filtered and concentrated under reduced pressure to obtain the title compound as a red solid, 107.9 g in total (93.1% yield), which was used directly in the next step. LC-MS m / z [M+H]+ =304.

[0081] Step 4: Preparation of 3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl benzoate

[0082]

[0083] To a 1000 mL reaction flask, acetic acid (376.1 mL) and (Z)-1-(dimethylamino)-3-oxo-3-(tetrahydro-2H-pyran-4-yl)prop-1-en-2-ylbenzoate (32.56 g, 107.46 mmol) were added sequentially. 80% hydrazine hydrate (37.6 mL) was slowly added dropwise under an ice bath. After completion, the mixture was stirred at room temperature overnight. The reaction was monitored until endpoint, and ethyl acetate was added to the reaction solution. The mixture was washed with water, and the organic phases were combined and washed with saturated sodium bicarbonate solution until the pH reached 7-8. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a yellow oil. The mixture was allowed to stand at room temperature overnight to obtain the title compound as a yellow solid, 28.0 g in total, yield: 95.8%; LC-MS m / z [M+H] + =273.

[0084] Step 5: Preparation of 1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-ylbenzoate

[0085]

[0086] To 1,2-dichloroethane (250 mL) were added 2,2'-bipyridine (17.7 g, 113.2 mmol) and copper acetate (20.6 g, 113.2 mmol) in sequence, and the mixture was reacted at 75 ° C for 30 min. After cooling to room temperature, cyclopropylboronic acid (17.5 g, 205.9 mmol), sodium carbonate (21.8 g, 205.9 mmol) and a solution of 3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-ylbenzoate (28.0 g, 102.9 mmol) in 1,2-dichloroethane (250 mL) were added in sequence, and the mixture was reacted at 75 ° C for 4 h under an oxygen atmosphere. Monitor the reaction until the end point, cool to room temperature, filter with celite, wash the filter cake with ethyl acetate, and concentrate the filtrate under reduced pressure to obtain the title compound as a reddish-brown oil, 32.0 g in total, yield: 98.5%, which was used directly in the next step; LC-MS m / z [M+H] + =313.

[0087] Step 6: Preparation of 1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-ol

[0088]

[0089] 1-Cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-ylbenzoate (32.0 g, 102.6 mmol) was added to methanol (308 mL) and allowed to react at room temperature for 2 h. The reaction was monitored until endpoint and concentrated under reduced pressure to remove some of the methanol. The pH was adjusted to 6-7 with 1 mol / L dilute hydrochloric acid, and the mixture was extracted with dichloromethane. The organic layers were combined. The mixture was concentrated under reduced pressure to dryness and purified by column chromatography to afford the title compound as a yellow solid, 9.0 g in a 45% yield; LC-MS m / z [M+H] + =209.

[0090] Step 7: Preparation of 7-bromo-4-chloroquinoline

[0091]

[0092] To a 50L vertical jacketed reactor was added 4.50kg of acetonitrile and stirring was started. 7-bromo-4-hydroxyquinoline (1.80kg, 8.03mol) and 4.00kg of acetonitrile were added in sequence. When the internal temperature dropped below 10°C, phosphorus oxychloride (1.85kg, 12.05mol) was added dropwise. After the addition was complete, the temperature was raised to reflux and stirred for 1 to 3 hours. The reaction was monitored until the end point. The system was cooled to below 5°C and 4mol / LNaOH solution was added dropwise to adjust the pH to 7 to 8. 42.00kg of water was added and stirred at room temperature for 1 to 2 hours. The feed liquid was centrifuged, the filter cake was washed with 5.00kg of water, and vacuum dried. The solid was collected and weighed to give a crude brown solid of the title compound, totaling 1.73kg, with a yield of 88.8%.

[0093] To a 50 L vertical jacketed reactor, 14.4 kg of methyl tert-butyl ether and 1.73 kg of crude 7-bromo-4-chloroquinoline were added in sequence and stirred at 50 ± 5 ° C for 1 to 3 hours. The mixture was filtered while hot and the filter cake was washed with methyl tert-butyl ether. The filtrate was concentrated to dryness under reduced pressure and the resulting solid was vacuum dried. After drying, the solid was collected and weighed to give 1.54 kg of the refined title compound as a yellow solid with a yield of 89.0%.

[0094] Step 8: Preparation of 7-bromo-4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)oxy)quinoline

[0095]

[0096] 8.40 kg of acetonitrile was added to a 30 L glass reactor, stirring was started, and 7-bromo-4-chloroquinoline (1.26 kg, 5.18 mol), cesium carbonate (1.69 kg, 5.18 mol), 1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-ol (0.90 kg, 4.32 mol, 4.00 kg of acetonitrile was added in sequence. Nitrogen protection was applied, and the temperature was raised to 70 ± 5 The mixture was stirred at 400°C for 2-3 hours, then the temperature was raised to reflux for 2-3 hours. The mixture was cooled to 70±5°C and filtered while hot. The filter residue was washed with dichloromethane. The filtrate was concentrated under reduced pressure until it became viscous. 14.00 kg of water was added, stirred, and centrifuged. The filter cake was washed with 3.00 kg of water and dried under vacuum at 50±5°C for 10-20 hours. After drying, the solid was collected and weighed to obtain 1.88 kg of crude brown solid 1, with a crude product yield of 100.0%.

[0097] To a 10L four-necked flask, add 6.40 kg of heptane and 1.88 kg of crude 7-bromo-4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)oxy)quinoline 1. Stir at 25±5°C for 3-5 hours. Filter, wash the filter cake with 1.40 kg of heptane, and dry under vacuum at 50±5°C for 3-20 hours. After drying, collect and weigh the solid to yield 1.68 kg of crude title compound 2 as a dark yellow solid, for a yield of 89.4%.

[0098] To a 10L four-necked flask, add 4.70kg of anhydrous ethanol and 1.68kg of 7-bromo-4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)oxy)quinoline crude product 2, raise the temperature to reflux, stir for 0.5 to 1 hour after dissolving, turn off the heating, cool naturally and crystallize, and when the internal temperature is lower than 30°C, open the external circulation and stir at -10 to -5°C for 1 to 3 hours. The liquid was centrifuged, the filter cake was washed with 0.4kg of cold ethanol (-10 to -5°C), and vacuum dried at 50±5°C for 10 to 20 hours. After drying, the solid was collected and weighed to obtain a refined product of the title compound as a light yellow solid, totaling 1.35kg, with a yield of 80.4%.

[0099] Step 9: Preparation of 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine

[0100]

[0101] To a 20L four-necked flask, add 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. Stirring was initiated, followed by the addition of sodium hydroxide (0.42kg, 10.50mol) and 36.00g of water. Stir vigorously at 25±5°C for 3-15 hours. Filter, wash with acetonitrile, and concentrate the filtrate to dryness under reduced pressure. Add 0.5g of seed crystals, and vacuum dry after solidification. After drying, collect and weigh the solid to yield the title compound as a white solid, 0.92kg in total, for a yield of 91.2%.

[0102] 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

[0103]

[0104] To a 20 L four-necked flask, add 7.10 kg of 1,4-dioxane and start stirring. Then, add 7-bromo-4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)oxy)quinoline (1.34 kg, 3.23 mol), 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine (0.75 kg, 3.88 mol), potassium phosphate (K3PO4, 1.37 kg, 6.47 mol), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (Xantphos, 37.40 g, 65.00 mmol), and tris(dibenzylideneacetone)dipalladium (Pd2(dba)3, 29.6 g, 32.00 mmol) in that order. Maintain a nitrogen atmosphere with a nitrogen blanket. Heat to 95±5°C and stir for 3-15 hours. Turn off the heat. When the internal temperature drops to 70-80°C, filter while hot and wash with 1.6 kg of hot 1,4-dioxane (70-80°C). Slowly pour the mother liquor into ice water, add N-acetyl-L-cysteine, stir for 1-1.5 hours, centrifuge, wash with 5.00 kg of water, and dry under vacuum. After drying, collect and weigh the solid to obtain 1.41 kg of crude yellow solid title compound 1, with a yield of 83.1%.

[0105] 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

[0106] To a 10L four-necked flask, add 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]pyrazin-7(8H)-yl)quinoline 1. Under nitrogen, heat to 55±5°C and stir for 2-4 hours. Cool naturally to an internal temperature below 30°C, stir at -5-10°C to crystallize for 1-3 hours, centrifuge, wash with 0.5kg of cold ethyl acetate (-10-5°C), and dry in vacuo to obtain the crude title compound 2 as a pale yellow solid. The total weight is 1.21kg, with a yield of 85.8%.

[0107] To a 30L glass reactor, 14.30kg of anhydrous methanol and 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]triazolo[4,3-a]pyrazine-7(8H)-yl)quinoline crude product 2 were added, and the mixture was heated to reflux under nitrogen protection to dissolve the mixture. 0.12kg of activated carbon and 0.12kg of Thiol. Reflux for 1-1.5 hours, filter while hot, wash with hot methanol (50-60°C), concentrate the filtrate under reduced pressure, cool at -10--5°C to crystallize for 1-3 hours, centrifuge, and wash with cold methanol (-10--5°C). Vacuum dry to obtain 1.03 kg of crude title compound 3 as an off-white solid. Yield: 85.1%.

[0108] Add 9.50 kg of dichloromethane and 1.02 kg 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]pyrazin-7(8H)-yl)quinoline 3 to a 10L four-necked flask and start stirring. After dissolving, add 0.20 kg of Thiol, stirred, filtered, washed with dichloromethane, and 26.20 kg of heptane was slowly added dropwise to the filtrate. Stirred for 10-15 hours, filtered, and the filter cake washed with 1.60 kg of heptane. Vacuum drying gave a purified white solid of the title compound, 0.99 kg in total. Yield: 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).

[0109] Experimental Example 1 Evaluation of ALK5 kinase activity of compounds in vitro

[0110] 1. Experimental Materials

[0111] 1.1 Compounds

[0112] The compound of formula (I) of the present invention in Example 1 was prepared with DMSO to 10 mM and then diluted to 3.333 μM, 1.111 μM, 370 nM, 123 nM, 41 nM, 14 nM, 4.6 nM, 1.5 nM and 0.5 nM in sequence.

[0113] 1.2 Reagents and Instruments

[0114] Reagents: ALK5, purchased from Carna, Cat. No. 09-141; p38α, purchased from Carna, Cat. No. 04-152; TGFβR1 peptide, purchased from SignalChem, Cat. No. T36-58; dimethyl sulfoxide (DMSO), purchased from Sigma, USA; EDTA, purchased from Sigma, USA; ADP-Glo Kinase Assay, purchased from Promega, Cat. No. v9102 / 3, 1× kinase buffer (40 mM Tris, pH 7.5, 0.10% BSA, 20 mM MgCl2, 1 mM DTT), prepared before use.

[0115] Instrument: 2104 Multilabel Reader, purchased from Perkin Elmer, USA.

[0116] 2. Experimental Methods

[0117] 2.1 Prepare 1x kinase buffer

[0118] 1x assay buffer

[0119] 40 mM Tris, pH 7.5

[0120] 20mM MgCl2

[0121] 0.10% BSA

[0122] 1mM DTT

[0123] 2.2 Compound preparation

[0124] 2.2.1 Compound dilution

[0125] 2.2.1.1 Prepare 50-fold Compound: The final assay concentration of the compound is 10 μM. Prepare the compound to a 50-fold concentration, i.e., 500 μM: Add 95 μl of 100% DMSO to the second well of a 96-well plate, followed by 5 μl of a 10 mM compound solution, to a 1000 μM solution. Add 60 μl of 100% DMSO to the remaining wells. Add 30 μl of compound from the second well to the third well, and continue with the following 3-fold dilutions, for a total of 10 dilutions.

[0126] Dilution instrument: Automatic micropipette (Precision PRC384U).

[0127] 2.2.1.2 Use echo to transfer 100 nl of compound to the reaction plate.

[0128] 2.3 Kinase reaction

[0129] 2.3.1 Preparation of 2x kinase solution

[0130] Add kinase to 1x kinase buffer to create a 2x enzyme solution. Add 2.5 μl of the 2x enzyme solution to a 384-well plate containing 100 nl of compound dissolved in 100% DMSO. Incubate at room temperature for 10 minutes.

[0131] 2.3.2 Preparation of 2x substrate solution

[0132] Add FAM-labeled peptide and ATP to 1x kinase buffer to form a 2x substrate solution. Add 2.5 μl of the 2x substrate solution to a 384-well reaction plate.

[0133] 2.4 Kinase reaction

[0134] The 384-well plate was incubated at 28°C for 120 minutes.

[0135] 2.5 Detection of reaction results

[0136] 2.5.1 Equilibrate ADP-Glo reagent to room temperature.

[0137] 2.5.2 Transfer 5 μl of the reaction solution to a new reaction well of a 384-well plate.

[0138] 2.5.3 Transfer 5 μl of ADP-Glo reagent to the reaction wells of a 384-well plate to terminate the reaction.

[0139] 2.5.4 Incubate at 28°C for 120 minutes.

[0140] 2.5.5 Transfer 10 μl of kinase assay reagent to each reaction well, shake for 1 minute, and let stand at room temperature for 30 minutes.

[0141] 2.6 Data Reading

[0142] The sample luminescence values were read on Envision.

[0143] 2.7 Curve Fitting

[0144] 2.7.1 Copying Luminescence Reading Data from Envision

[0145] 2.7.2 Convert the luminescence readings to inhibition percentage using the formula.

[0146] Percent inhibition = (max-sample RLU) / (max-min)*100. “Min” is the fluorescence reading of the control sample without enzyme; “max” is the fluorescence reading of the sample with DMSO added as a control.

[0147] 2.7.3 The data were imported into MS Excel and curve fitting was performed using XLFit Excel add-in version 5.4.0.8. The fitting formula was: Y = Bottom + (Top - Bottom) / (1 + (IC 50 / X)^HillSlope), the results are shown in Table 4.

[0148] Table 4

[0149]

[0150] From the above experimental results, it can be seen that the compounds of the present invention have good inhibitory activity against ALK5 kinase and low inhibitory effect against p38α with high selectivity, indicating that the compounds of the present invention have higher efficacy and lower side effects.

[0151] Experimental Example 2 Evaluation of Compounds by In Vitro Cell Luciferase Assay

[0152] 1. Experimental Materials

[0153] Test compound: the compound of formula (I) of the present invention in Example 1, prepared with DMSO to 4 mM, and then diluted 4-fold to 20000.00 nM, 5000.00 nM, 1250.00 nM, 312.5 nM, 78.125 nM, 19.53 nM, 4.88 nM, and 1.22 nM.

[0154] Luc-Smad2 / 3-NIH3T3 mouse fibroblasts (engineered to overexpress SMAD2,3-responsive promoter) were kindly provided by the laboratory of China Pharmaceutical University.

[0155] Reagents: DMEM, purchased from Invitrogen, USA; FBS, purchased from Invitrogen, USA; DMSO, purchased from Sigma, USA; Glo Lysis Buffer, purchased from Promega, USA; Bright-Glo Luciferase assay system, purchased from Promega, USA; TGFβ, purchased from PeproTech, USA.

[0156] Instrument: MD SpectraMax M3 multi-function microplate reader, purchased from Molecular Devices, USA.

[0157] 2. Experimental Methods

[0158] 2.1 Cell culture:

[0159] Cell recovery: Dissolve cells in a 37°C water bath, then transfer to 15 mL of prewarmed culture medium. Centrifuge at 1000 rpm for 5 minutes. Discard the culture medium and resuspend the cells in 15 mL of fresh culture medium. Transfer the cells to a 10 cm dish and culture in a 37°C, 5% CO2 incubator. Replace the cells with fresh culture medium after 24 hours.

[0160] Cell passaging: Transfer the revived cells to a 50 mL sterile centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the culture medium, count the evenly dispersed cells, adjust the cell concentration to an appropriate level in 15 mL of fresh culture medium, add the culture medium to a 10 cm culture dish, and culture in a 37°C, 5% CO2 incubator.

[0161] 2.2 Experimental steps:

[0162] Day 1: Cell plating (transparent bottom 96-well plate)

[0163] Luc-Smad2 / 3-NIH3T3 cells were cultured normally in a 10 cm culture dish until the confluence reached 80%-90%. After digestion, the cells were collected into a 15 mL centrifuge tube and centrifuged at 1000 × g for 5 minutes. The supernatant was removed and the cells were resuspended in 1 mL of culture medium. The cells were diluted 10-fold and counted. The cells were diluted according to the counting results. 4 × 10 3 The number of cells per well was transferred into a 96-well plate (100 μl of resuspended cells was added to each well).

[0164] Day 2: Cell administration

[0165] Weigh 1-2 mg of drug (pre-weighed) and prepare a 4 mM stock solution in DMSO. After 24 hours, remove the culture medium. Dilute the drug with 2% FBS medium and add 100 μl of the 1x drug solution to achieve final drug concentrations of 20,000.00 nM, 5,000.00 nM, 1,250.00 nM, 312.5 nM, 78.125 nM, 19.53 nM, 4.88 nM, and 1.22 nM, respectively. The final TGFβ1 concentration in each well is 4 ng / mL. Dilute the drug with the compound in 2% FBS medium.

[0166] Day 3: Fluorescence detection experiment

[0167] Equilibrate Glo Lysis Buffer, Bright-glo luciferase assay system, and cells to room temperature. Remove the cell supernatant and add 100 μl of Glo Lysis Buffer to each well. Gently shake to evenly lyse the cells. Lyse at room temperature for 5 minutes. Then, add 100 μl of Bright-glo luciferase assay system to each well. Incubate at room temperature for 5 minutes, shake for 2 minutes, and transfer 180 μl of the supernatant to a white-bottomed 96-well plate. Detect the chemiluminescent signal using a 1-second detection condition.

[0168] 2.3 Data processing: Graphpad Prism 5 software was used for nonlinear curve fitting and data analysis, and the IC 50 , the experimental results are shown in Table 5.

[0169] Table 5

[0170]

[0171] From the above experiments, it can be seen that the compounds of the present invention exhibit good inhibitory activity on the TGFβ-ALK5-SMAD2 / 3 signaling pathway in NIH3T3 cells and are very promising as therapeutic agents for various cancer-related diseases.

[0172] Although the present invention has been described in detail above, it will be appreciated by those skilled in the art that various modifications and variations may be made to the present invention without departing from the spirit and scope of the present invention. The scope of the present invention is not limited to the detailed description above, but rather to the claims.

Claims

1. A method for preparing a compound of formula (I) or a salt thereof, comprising reacting a compound of formula (II) with a compound of formula (III) in the presence of a palladium catalyst and a phosphine ligand, wherein X is selected from halogen, The palladium catalyst is Pd2(dba)3, and the phosphine ligand is 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene.

2. The method for preparing a compound of formula (I) or a salt thereof according to claim 1, wherein the molar ratio of the compound of formula (II) to the palladium catalyst Pd2(dba)3 is 1:0.005 to 1:0.

04.

3. The method for preparing a compound of formula (I) or a salt thereof according to claim 1 or 2, 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 selected from halogen, 4. The method for preparing a compound of formula (I) or a salt thereof according to claim 3, 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 selected from halogen, 5. The method for preparing a compound of formula (I) or a salt thereof according to claim 1 or 2, further comprising the step of freeing the compound of formula (VII) to a compound of formula (III) under the action of an alkaline reagent,

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