CN113527306A

CN113527306A - Crystal forms of quinoline TGF-beta 1 inhibitor

Info

Publication number: CN113527306A
Application number: CN202110414834.7A
Authority: CN
Inventors: 陈程; 杜爽; 赵立文; 王成
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 medical chemistry, relates to a crystal form of quinoline TGF-beta 1 inhibitor, a preparation method and application thereof, and particularly relates to 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazole-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1,2, 4-dihydro- [1 ] or (4-hydroxy-1-methyl) -5]Triazolo [4,3-a]A crystal form of pyrazine-7 (8H) -yl) quinoline and a preparation method thereof, the crystal form can be used for preparing medicaments for treating and/or preventing cancers, tissue proliferation diseases, fibrosis or inflammatory diseases,

Description

Crystal forms of quinoline TGF-beta 1 inhibitor

Technical Field

The invention belongs to the field of medical chemistry, and particularly relates to a crystal form 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, and a preparation method and application 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 a quinoline TGF-beta 1 inhibitor, the structure of the compound of which is shown in the following formula (I), and the chemical name of the inhibitor 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.

Those skilled in the art know that the crystal structure of the pharmaceutically active compound often affects the chemical stability, solubility and other properties, and therefore, extensive research is needed to find a crystal form suitable for pharmaceutical use.

The invention aims to provide a crystal form of a quinoline TGF-beta 1 inhibitor with good water solubility, high bioavailability and high stability. Specifically, the invention provides a 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 crystal form shown in a formula (I),

the inventor of the invention detects the crystal form of the compound shown in the formula (I) by X-ray powder diffraction, differential scanning thermal analysis (DSC), thermogravimetric analysis (TGA) and the like.

In some embodiments, the compound of formula (I) form a of the present invention has an X-ray powder diffraction pattern, see fig. 1, with characteristic peaks at about 3.7 ± 0.2, 9.5 ± 0.2, 14.2 ± 0.2, 14.7 ± 0.2, expressed in terms of 2 θ using Cu-Ka radiation.

Furthermore, the X-ray powder diffraction pattern of the crystal form A of the compound shown as the formula (I) has characteristic peaks at about 3.7 +/-0.2, 9.5 +/-0.2, 14.2 +/-0.2, 14.7 +/-0.2, 18.9 +/-0.2, 24.1 +/-0.2 and 25.9 +/-0.2.

Furthermore, the X-ray powder diffraction pattern of the crystal form A of the compound shown as the formula (I) has characteristic peaks at about 3.7 +/-0.2, 6.2 +/-0.2, 6.6 +/-0.2, 7.0 +/-0.2, 7.4 +/-0.2, 9.5 +/-0.2, 14.2 +/-0.2, 14.7 +/-0.2, 16.3 +/-0.2, 18.9 +/-0.2, 21.6 +/-0.2, 22.2 +/-0.2, 23.0 +/-0.2, 24.1 +/-0.2 and 25.9 +/-0.2.

Furthermore, the X-ray powder diffraction pattern of the crystal form A of the compound shown as the formula (I) has characteristic peaks at about 3.7 +/-0.2, 6.2 +/-0.2, 6.6 +/-0.2, 7.0 +/-0.2, 7.4 +/-0.2, 9.5 +/-0.2, 14.2 +/-0.2, 14.7 +/-0.2, 16.3 +/-0.2, 18.2 +/-0.2, 18.5 +/-0.2, 18.9 +/-0.2, 21.6 +/-0.2, 22.2 +/-0.2, 23.0 +/-0.2, 24.1 +/-0.2, 25.9 +/-0.2, 27.2 +/-0.2, 27.8 +/-0.2, 29.7 +/-0.2, 30.4 +/-0.2, 32.9 +/-0.2 and 34.0 +/-0.2.

Without limitation, in a particular embodiment, the compound of formula (I) form a of the present invention has an X-ray powder diffraction pattern as shown in figure 1.

Without limitation, in a particular embodiment, the DSC profile of form a of the compound of formula (I) of the present invention (see figure 2) shows a sharp endothermic peak at 215.6 ℃ for the sample.

Without limitation, in a specific embodiment, the compound of formula (I) of the present invention, form a, has a thermogravimetric analysis (TGA) profile as shown in figure 3, which shows no significant weight loss before 300 ℃. The compound of the formula (I) of the invention has very good stability in the crystal form A.

The invention provides a preparation method of a 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 crystal form A shown in a formula (I), which comprises the following steps of preparing a crystal form A of 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1, a step of subjecting 2,4] triazolo [4,3-a ] pyrazin-7 (8H) -yl) quinoline to an organic solvent and filtration, wherein the starting material, 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, is present in a form not particularly limited, and any crystalline or amorphous solid can be used. In some embodiments, the organic solvent is selected from the group consisting of dichloromethane, methyl isobutyl ketone (MIBK), isopropyl acetate, ethanol, isopropanol, toluene, tetrahydrofuran, chloroform, 1, 4-dioxane, acetonitrile, N-heptane, dimethyl sulfoxide, N-dimethylacetamide, methyl tert-butyl ether (MTBE), ethyl formate. In some specific embodiments, the process for the preparation of compound of formula (I) form a according to the present invention, wherein the organic solvent is dichloromethane and heptane. In some specific embodiments, the process for the preparation of compound of formula (I) form a according to the present invention, wherein the organic solvent is dichloromethane and butyl acetate. In some specific embodiments, the process for the preparation of compound of formula (I) form a according to the invention, wherein the organic solvent is tetrahydrofuran and MTBE. In some specific embodiments, the process for the preparation of compound of formula (I) form a according to the present invention, wherein the organic solvent is ethyl acetate and heptane. In some specific embodiments, the process for the preparation of the compound of formula (I) form a according to the present invention, wherein the organic solvent is dichloromethane and heptane, the volume ratio (V/V) of dichloromethane and heptane is from about 1:1 to about 1:10, such as about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, etc. In some specific embodiments, form a is prepared by anti-solvent dropwise addition according to the preparation method of form a of the compound of formula (I) of the present invention. In some specific embodiments, the process for the preparation of the compound of formula (I) form a of the present invention comprises the steps of adding dichloromethane to the compound of formula (I) followed by the addition of n-heptane. In some specific embodiments, the process for the preparation of the compound of formula (I) form a of the present invention comprises the steps of adding dichloromethane to the compound of formula (I) followed by the addition of n-heptane at a temperature of 40 ± 15 ℃, preferably 45 ± 10 ℃, more preferably 45 ± 5 ℃. In some specific embodiments, the preparation method of the compound of formula (I) in the form of crystal form a of the present invention comprises the steps of adding dichloromethane to the compound of formula (I), and then adding n-heptane dropwise at a speed of 50-150 mL/min, preferably 60-120 mL/min, more preferably 80-100 mL/min at a temperature of 40 ± 15 ℃, preferably 45 ± 10 ℃, more preferably 45 ± 5 ℃.

In some embodiments, the compound of formula (I) form B of the present invention has an X-ray powder diffraction pattern, see fig. 4, with characteristic peaks at about 3.9 ± 0.2, 15.8 ± 0.2, expressed in terms of 2 θ using Cu-Ka radiation.

Furthermore, the X-ray powder diffraction pattern of the crystal form B of the compound shown in the formula (I) has characteristic peaks at about 3.9 +/-0.2, 7.8 +/-0.2, 10.0 +/-0.2, 11.8 +/-0.2 and 15.8 +/-0.2.

Furthermore, the X-ray powder diffraction pattern of the crystal form B of the compound shown in the formula (I) has characteristic peaks at about 3.9 +/-0.2, 7.8 +/-0.2, 10.0 +/-0.2, 11.8 +/-0.2, 15.1 +/-0.2, 15.8 +/-0.2 and 23.9 +/-0.2.

Furthermore, the X-ray powder diffraction pattern of the crystal form B of the compound shown as the formula (I) has characteristic peaks at about 3.9 +/-0.2, 7.8 +/-0.2, 10.0 +/-0.2, 11.8 +/-0.2, 15.1 +/-0.2, 15.8 +/-0.2, 18.8 +/-0.2, 19.2 +/-0.2, 19.8 +/-0.2, 20.9 +/-0.2, 21.4 +/-0.2, 22.6 +/-0.2, 23.2 +/-0.2, 23.9 +/-0.2, 25.0 +/-0.2, 28.0 +/-0.2, 28.7 +/-0.2, 32.0 +/-0.2 and 37.7 +/-0.2.

Without limitation, in a specific embodiment, the compound of formula (I) of the present invention in crystalline form B has an X-ray powder diffraction pattern as shown in figure 4.

The invention provides a preparation method of a 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 crystal form B shown in a formula (I), which comprises the following steps of preparing a crystal form B of 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1, a step of placing 2,4] triazolo [4,3-a ] pyrazin-7 (8H) -yl) quinoline in an organic solvent, and filtering. Among them, the existing form of the starting material 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 is not particularly limited, and any crystalline or amorphous solid can be used. In some embodiments, the organic solvent is selected from the group consisting of methyl isobutyl ketone (MIBK) and isopropyl acetate.

In some embodiments, the compound of formula (I) form C of the present invention has an X-ray powder diffraction pattern, see fig. 5, with characteristic peaks at about 8.2 ± 0.2, 12.0 ± 0.2, expressed in terms of 2 θ using Cu-Ka radiation.

Furthermore, the X-ray powder diffraction pattern of the crystal form C of the compound shown as the formula (I) has characteristic peaks at about 8.2 +/-0.2, 12.0 +/-0.2, 16.6 +/-0.2, 19.2 +/-0.2 and 24.1 +/-0.2.

Without limitation, in a particular embodiment, the compound of formula (I) form C of the present invention has an X-ray powder diffraction pattern as shown in figure 5.

The invention provides a preparation method of a 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 crystal form C shown in a formula (I), which comprises the following steps of preparing 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1, a step of placing 2,4] triazolo [4,3-a ] pyrazin-7 (8H) -yl) quinoline in a solvent and filtering; wherein the existing form of the starting material 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 is not particularly limited, and any crystalline or amorphous solid can be used; wherein the solvent is selected from water and water saturated ethyl acetate.

In some embodiments, an X-ray powder diffraction pattern of form D of the compound of formula (I) of the present invention, see fig. 6, using Cu-Ka radiation, expressed as an X-ray powder diffraction pattern at 2 Θ angles, has characteristic peaks at about 7.1 ± 0.2, 10.1 ± 0.2, 14.2 ± 0.2, 20.2 ± 0.2, 21.4 ± 0.2.

Furthermore, the X-ray powder diffraction pattern of the crystal form D of the compound shown in the formula (I) has characteristic peaks at about 7.1 +/-0.2, 8.3 +/-0.2, 10.1 +/-0.2, 14.2 +/-0.2, 15.1 +/-0.2, 20.2 +/-0.2, 21.4 +/-0.2 and 22.6 +/-0.2.

Furthermore, the X-ray powder diffraction pattern of the crystal form D of the compound shown as the formula (I) has characteristic peaks at about 7.1 +/-0.2, 8.3 +/-0.2, 10.1 +/-0.2, 11.0 +/-0.2, 14.2 +/-0.2, 15.1 +/-0.2, 15.9 +/-0.2, 16.1 +/-0.2, 17.4 +/-0.2, 18.9 +/-0.2, 20.2 +/-0.2, 21.4 +/-0.2, 22.6 +/-0.2, 23.4 +/-0.2, 23.8 +/-0.2, 24.6 +/-0.2, 26.8 +/-0.2, 27.4 +/-0.2, 28.2 +/-0.2 and 30.6 +/-0.2.

Without limitation, in a particular embodiment, the compound of formula (I) form D of the present invention has an X-ray powder diffraction pattern as shown in figure 6.

The invention provides a preparation method of a 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 crystal form D shown in a formula (I), which comprises the following steps of preparing 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1, a step of dissolving 2,4] triazolo [4,3-a ] pyrazin-7 (8H) -yl) quinoline in an organic solvent 1, and then adding an organic solvent 2; wherein the existing form of the starting material 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 is not particularly limited, and any crystalline or amorphous solid can be used; the organic solvent 1 is selected from chloroform, and the organic solvent 2 is selected from methyl tert-butyl ether (MTBE), toluene and n-heptane. Preferably, the volume ratio of the organic solvent 1 to the organic solvent 2 is about 1:0.8-1: 2; further preferably, the volume ratio of the organic solvent 1 to the organic solvent 2 is about 1:1.1-1: 1.2; even more preferably, the volume ratio of the organic solvent 1 to the organic solvent 2 is about 3: 3.5.

In some embodiments, the compound of formula (I) form E of the present invention has an X-ray powder diffraction pattern, see fig. 7, with characteristic peaks at about 4.9 ± 0.2, 9.9 ± 0.2, 15.0 ± 0.2, 18.7 ± 0.2, expressed in terms of 2 θ using Cu-Ka radiation.

Furthermore, the X-ray powder diffraction pattern of the crystal form E of the compound shown as the formula (I) has characteristic peaks at about 4.9 +/-0.2, 6.9 +/-0.2, 9.9 +/-0.2, 15.0 +/-0.2 and 18.7 +/-0.2.

Furthermore, the X-ray powder diffraction pattern of the crystal form E of the compound shown as the formula (I) has characteristic peaks at about 4.9 +/-0.2, 6.9 +/-0.2, 9.9 +/-0.2, 15.0 +/-0.2, 18.7 +/-0.2, 20.1 +/-0.2, 21.2 +/-0.2 and 30.4 +/-0.2.

Furthermore, the X-ray powder diffraction pattern of the crystal form E of the compound shown as the formula (I) has characteristic peaks at about 4.9 +/-0.2, 6.9 +/-0.2, 9.9 +/-0.2, 15.0 +/-0.2, 18.7 +/-0.2, 20.1 +/-0.2, 21.2 +/-0.2, 23.6 +/-0.2, 30.4 +/-0.2, 31.9 +/-0.2 and 38.9 +/-0.2.

Without limitation, in a particular embodiment, the compound of formula (I) of the present invention in crystalline form E has an X-ray powder diffraction pattern as shown in figure 7.

The invention provides a preparation method of a 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 crystal form E shown in a formula (I), which comprises the following steps of preparing a crystal form E of 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1, a step of dissolving 2,4] triazolo [4,3-a ] pyrazin-7 (8H) -yl) quinoline in an organic solvent 3, and then adding an organic solvent 4; wherein the existing form of the starting material 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 is not particularly limited, and any crystalline or amorphous solid can be used; wherein the organic solvent 3 is selected from dichloromethane and the organic solvent 4 is selected from methyl tert-butyl ether (MTBE). Preferably, the volume ratio of the organic solvent 3 to the organic solvent 4 is about 1:3 to 1: 7; further preferably, the volume ratio of the organic solvent 3 to the organic solvent 4 is about 1:4 to 1: 6; even more preferably, the volume ratio of the organic solvent 3 to the organic solvent 4 is about 1: 5.

The crystal forms A-E prepared by the method of the invention do not contain or contain low content of residual solvent, meet the limit requirement of residual solvent of related medical products specified by national formulary, and can be better used as medical active ingredients.

In another aspect of the invention there is provided an amorphous form of the compound of formula (I).

Without limitation, a typical example of an amorphous form of a compound of formula (I) according to the invention has an X-ray powder diffraction pattern as shown in figure 8.

The invention provides a preparation method of amorphous 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 shown in a formula (I), wherein the preparation method comprises the steps of preparing amorphous 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1, a step of dissolving 2,4] triazolo [4,3-a ] pyrazin-7 (8H) -yl) quinoline in an organic solvent 5, and then adding it to a solvent 6; preferably, the organic solvent 5 is selected from 1, 4-dioxane; the solvent 6 is selected from water.

Another aspect of the invention provides a crystalline composition wherein 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 crystalline form A, B, C, D or E comprises more than 50%, preferably more than 80%, more preferably more than 90%, most preferably more than 95% by weight of the crystalline composition.

In another aspect of the invention, there is provided a pharmaceutical composition comprising the crystalline form 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 and a pharmaceutically acceptable carrier, preferably comprising 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 in crystalline form A, B, C, D or E and a pharmaceutically acceptable carrier.

Another aspect of the invention provides a crystalline form 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 or a pharmaceutical composition comprising a crystalline form of the above compound, in particular 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) -7- (3- (trifluoromethyl) -5, 6-dihydro- [1, use of 2,4] triazolo [4,3-a ] pyrazin-7 (8H) -yl) quinoline form A, B, C, D or E or a pharmaceutical composition comprising the compound form A, B, C, D or E for the preparation of a medicament for the treatment and/or prevention of cancer, a tissue proliferative disorder, fibrosis or inflammatory disease, wherein the cancer, tissue proliferative disorder, fibrosis or inflammatory disease condition includes, but is not limited to: melanoma, papillary thyroid tumors, cholangiocarcinoma, colon cancer, ovarian cancer, lung cancer, malignant lymphoma, carcinomas and sarcomas of the liver, kidney, bladder, prostate, breast and pancreas, as well as primary and recurrent solid tumors of the skin, colon, thyroid, lung and ovary or leukemia, glioblastoma (glioma), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), Acute Myelogenous Leukemia (AML), sarcoma, non-small cell lung cancer, chondrosarcoma, cholangiocarcinomas or immunoblastic lymphomas, liver fibrosis and chronic kidney disease.

It is specifically stated herein that the X-ray powder diffraction pattern is characteristic for a particular crystalline form. To determine if it is the same as the known crystal type, care should be taken with respect to the relative positions of the peaks (i.e., 2 θ) rather than their relative intensities. This is because the relative intensities of the spectra (especially at low angles) vary due to the dominant orientation effects resulting from differences in crystal conditions, particle size or other measurement conditions, and the relative intensities of the diffraction peaks are not characteristic for the determination of the crystalline form. In addition, the 2 theta value of the same crystal form may have slight error, which is about +/-0.2 degrees. Therefore, this error should be taken into account when determining each crystalline structure. Peak positions are typically expressed in XRPD patterns in terms of 2 θ angles or crystal plane distances d, with a simple conversion between the two: d ═ λ/2sin θ, where the value of d represents interplanar spacing, λ represents the wavelength of the X-rays, and θ is the diffraction angle. It should also be noted that in the identification of mixtures, where partial loss of diffraction lines is caused by, for example, a reduction in the amount of the compound, one band may be characteristic of a given crystal without relying on all bands observed in a high purity sample.

DSC measures the transition temperature when a crystal absorbs or releases heat due to a change in its crystal structure or melting of the crystal. For the same crystal form of the same compound, the thermal transition temperature and melting point errors are typically within about 5 ℃ in a continuous analysis. When we say that a compound has a given DSC peak or melting point, this means that the DSC peak or melting point ± 5 ℃. It is noted that the DSC peak or melting point for the mixture may vary over a larger range. Furthermore, the melting temperature is related to the rate of temperature rise due to decomposition that accompanies the process of melting the substance.

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 "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.

Drawings

FIG. 1 is an X-ray diffraction pattern of form A of the compound of formula (I);

FIG. 2 is a DSC profile of form A of the compound of formula (I);

FIG. 3 is a TGA profile of form A of the compound of formula (I);

FIG. 4 is an X-ray diffraction pattern of form B of the compound of formula (I);

FIG. 5 is an X-ray diffraction pattern of compound of formula (I) in crystalline form C;

FIG. 6 is an X-ray diffraction pattern of form D of the compound of formula (I);

FIG. 7 is an X-ray diffraction pattern of crystalline form E of the compound of formula (I);

FIG. 8 is an X-ray diffraction pattern of an amorphous compound of formula (I).

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.

Test instrument for experiment

X-ray powder diffraction Spectroscopy (XRPD)

The instrument model is as follows: switzerland D8 Advance X-ray diffractometer

And (3) testing conditions are as follows: copper target, 40kV in pipe pressure and 40mA in pipe flow

2. Thermogravimetric analysis (TGA)

The instrument model is as follows: model PERKINELMER TGA4000 thermogravimetric analyzer 4000

Temperature range: 30-300 deg.C

The heating rate is as follows: 10 ℃/min

3. Differential Scanning Calorimetry (DSC)

The instrument model is as follows: NETZSCH DSC 204 type differential thermal analyzer

Temperature range: 40-250 deg.C

The heating rate is as follows: 10 ℃/min

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, sequentially adding into a 1000mL three-neck bottleAdding methanol (100mL) and 1- (tetrahydro-2H-pyran-4-yl) ethanone (20.0g,156mmol), cooling to below-15 deg.C, slowly adding dropwise liquid bromine, and keeping the temperature below-15 deg.C. After the completion of the dropwise addition, the temperature was raised to 0 ℃ for 45min, then to 10 ℃ for 45min, and 11mol/L sulfuric acid (55mL) was slowly added dropwise while maintaining the internal temperature at room temperature, after the completion of the dropwise addition, the reaction was carried out 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. Filtering to obtainThe organic phase was 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).

The compound of formula (I) of example 1 above was subjected to crystal form screening experiments under more than 50 conditions, including volatilization experiments, slurry experiments, cooling crystallization experiments, diffusion experiments, and the like. A plurality of better crystal forms with good stability, high solubility and small particle size are found, and the specific details are shown in the following examples.

Example 2: preparation of compound of formula (I) in crystal form A

1: 60mg of the compound of the formula (I) is weighed respectively, 0.5mL of each of ethanol, isopropanol, toluene, tetrahydrofuran, chloroform, 1, 4-dioxane, acetonitrile or n-heptane is added, the mixture is stirred for 12 hours at 50 ℃, the mixture is filtered, and the solid is taken for XRPD characterization, and the characterization result shows that the experimental crystal forms are the crystal form A.

2: respectively weighing a proper amount of a compound shown as a formula (I), dissolving the compound in a solvent A according to the formula 1, then dripping a solvent B in proportion, separating out a solid, stirring for about 5 hours, filtering, drying the solid for 12 hours at room temperature, taking the solid for XRPD characterization, and displaying that the experimental crystal forms are crystal forms A according to characterization results.

TABLE 1

3: respectively weighing 60mg of the compound of the formula (I), adding 0.5mL of toluene, tetrahydrofuran, chloroform or 1, 4-dioxane respectively, stirring at 50 ℃ for 12 hours, filtering, placing the filtered mother liquor in an open manner, volatilizing the solvent, drying the solid in vacuum at room temperature, sampling, and performing XRPD characterization, wherein the characterization result shows that each experimental crystal form is the crystal form A.

4: adding ethanol, a methanol/chloroform mixed solvent (the volume ratio of methanol to chloroform is 3:2), an isopropanol/acetonitrile mixed solvent (the volume ratio of isopropanol to acetonitrile is 1:1) or an acetonitrile/water mixed solvent (the volume ratio of acetonitrile to water is 9:1) into the compound shown in the formula (I), stirring at 50 ℃, standing, taking a supernatant, stirring at 5 ℃ for crystallization for 4 hours, filtering, drying, sampling, and carrying out XRPD characterization, wherein the characterization result shows that all experimental crystal forms are crystal form A.

5: weighing about 600mg of the compound shown in the formula (I), adding dichloromethane, dropwise adding heptane (the ratio (V/V) of the added dichloromethane to the heptane is 1:2 (20V in total volume), 1:3 (27V in total volume), 1:4 (34V in total volume) or 1:5 (42V in total volume) at the temperature of 45 +/-5 ℃ at the speed of 80-100 mL/min, stirring for 10-20 hours, filtering, taking the solid, and performing XRPD characterization, wherein the characterization result shows that the experimental crystal forms are all crystal form A.

In addition, the crystalline form obtained was prepared as form a using dichloromethane/butyl acetate, tetrahydrofuran/MTBE or ethyl acetate/heptane, respectively, as solvents.

An X-ray diffraction pattern (see figure 1) of a compound of a formula (I) in a crystal form A shows an X-ray powder diffraction pattern at an angle of 2 theta by using Cu-Ka radiation, and has characteristic peaks at about 3.7, 6.2, 6.6, 7.0, 7.4, 9.5, 14.2, 14.7, 16.3, 18.2, 18.5, 18.9, 21.6, 22.2, 23.0, 24.1, 25.9, 27.2, 27.8, 29.7, 30.4, 32.9 and 34.0. The 2 θ in FIG. 1 and the relative intensities of the peaks are shown in Table 2.

The DSC characterization result of the compound of the formula (I) in the form of the crystal form A is shown in figure 2, and the test result shows that the sample has a sharp endothermic peak at 215.6 ℃.

The TGA characterization of form a of the compound of formula (I) is shown in figure 3, which shows that no significant weight loss is seen before 300 ℃.

Table 2: details of the XRPD pattern of form A of the compound of formula (I)

Form a can be obtained by reverse dropwise addition. Experiments have shown that these solvents have good solubility for the compound of formula (I), especially dichloromethane in combination with heptane. The stability and solubility of form a are very good.

Example 3: preparation of compound of formula (I) in crystalline form B

1: weighing about 500mg of the compound shown as the formula (I), pulping by ethyl acetate, filtering, adding 5mL of isopropanol into the obtained solid, heating to 90 ℃, supplementing 5mL of isopropanol until the mixture is clear, closing heating, and naturally cooling for crystallization. And filtering, drying a filter cake at room temperature in vacuum, and performing XRPD characterization on the obtained solid, wherein the characterization result shows that the experimental crystal form is the crystal form B.

2: 60mg of the compound shown as the formula (I) is weighed respectively, 0.5mL of methyl isobutyl ketone or isopropyl acetate is added, the mixture is stirred for 12 hours at the temperature of 50 ℃, the mixture is filtered, the solid is taken for XRPD characterization, and the characterization result shows that the experimental crystal form is the crystal form B.

An X-ray diffraction pattern (see figure 4) of a compound of a formula (I) in a crystal form B, using Cu-Ka radiation and expressing an X-ray powder diffraction pattern at an angle of 2 theta, wherein the X-ray powder diffraction pattern has characteristic peaks at about 3.9, 7.8, 10.0, 11.8, 15.1, 15.8, 18.8, 19.2, 19.8, 20.9, 21.4, 22.6, 23.2, 23.9, 25.0, 28.0, 28.7, 32.0 and 37.7. The 2 θ in FIG. 4 and the relative intensities of the peaks are shown in Table 3.

Table 3: details of the XRPD pattern of form B of the compound of formula (I)

Example 4: preparation of compound of formula (I) in crystalline form C

1: weighing 60mg of the compound of formula (I), and adding 0.5mL of water; or weighing 60mg of the compound shown in the formula (I), and adding a water saturated ethyl acetate solution; and then stirring for 12 hours at 50 ℃, filtering, taking the solid for XRPD characterization, wherein the characterization result shows that the experimental crystal form is the crystal form C.

2: weighing a proper amount of a compound shown as a formula (I), dissolving the compound in chloroform, then dropwise adding isopropyl acetate, wherein the volume ratio of chloroform to isopropyl acetate is 3:3, stirring for about 5 hours after solid is separated out, filtering, drying the solid for 12 hours at room temperature, taking the solid for XRPD characterization, and displaying the experimental crystal form as a crystal form C according to characterization results.

An X-ray diffraction pattern (see figure 5) of a compound of formula (I) in a crystal form C, using Cu-Ka radiation and representing an X-ray powder diffraction pattern with characteristic peaks at about 8.2, 12.0, 16.6, 19.2 and 24.1 degrees. The 2 θ in FIG. 5 and the relative intensities of the peaks are shown in Table 4.

Table 4: details of the XRPD pattern of form C of the compound of formula (I)

Example 5: preparation of compound of formula (I) in crystalline form D

1: weighing a proper amount of a compound shown as a formula (I), dissolving the compound in chloroform, then dropwise adding methyl tert-butyl ether (MTBE) in a volume ratio of 3:3.5, precipitating a solid, stirring for about 5 hours, filtering, drying the solid at room temperature for 12 hours, taking the solid, and carrying out XRPD characterization, wherein the characterization result shows that the crystal form in the experiment is crystal form D.

2: weighing a proper amount of a compound shown as a formula (I), dissolving the compound in chloroform, then dropwise adding toluene, wherein the volume ratio of the chloroform to the toluene is 3:4, stirring for about 5 hours after solid is separated out, filtering, drying the solid at room temperature for 12 hours, taking the solid for XRPD characterization, and displaying the characterization result that the crystal form in the experiment is crystal form D.

3: weighing a proper amount of a compound shown as a formula (I), dissolving the compound in chloroform, then dropwise adding n-heptane, wherein the volume ratio of chloroform to n-heptane is 3:6, stirring for about 5 hours after solid is separated out, filtering, drying the solid for 12 hours at room temperature, taking the solid for XRPD characterization, and displaying the characterization result that the crystal form in the experiment is crystal form D.

An X-ray diffraction pattern (see figure 6) of a compound of formula (I) in a crystal form D, wherein Cu-Ka radiation is used, and an X-ray powder diffraction pattern is expressed by 2 theta angles, and the X-ray powder diffraction pattern has characteristic peaks at about 7.1, 8.3, 10.1, 11.0, 14.2, 15.1, 15.9, 16.1, 17.4, 18.9, 20.2, 21.4, 22.6, 23.4, 23.8, 24.6, 26.8, 27.4, 28.2 and 30.6. The 2 θ in FIG. 6 and the relative intensities of the peaks are shown in Table 5.

Table 5: details of the XRPD pattern of form D of the compound of formula (I)

Example 6: preparation of Compound of formula (I) in crystalline form E

Weighing a proper amount of a compound shown as a formula (I), dissolving the compound in dichloromethane, then dropwise adding MTBE (methyl tert-butyl ether), wherein the volume ratio of dichloromethane to MTBE is 1:5, stirring for about 5 hours after solid is separated out, filtering, drying the solid for 12 hours at room temperature, taking the solid for XRPD characterization, and displaying the characterization result that the crystal form in the experiment is crystal form E.

An X-ray diffraction pattern (see figure 7) of a crystal form E of the compound shown as the formula (I) shows an X-ray powder diffraction pattern by using Cu-Ka radiation and using an angle of 2 theta, and characteristic peaks exist at about 4.9, 6.9, 9.9, 15.0, 18.7, 20.1, 21.2, 23.6, 30.4, 31.9 and 38.9. The 2 θ in FIG. 7 and the relative intensities of the peaks are shown in Table 6.

Table 6: details of the XRPD pattern of form E of Compound of formula (I)

Example 7: preparation of amorphous form

Weighing a proper amount of the compound shown in the formula (I), dissolving the compound in 1, 4-dioxane with 5 times of volume, heating to dissolve the compound, sucking the 1, 4-dioxane solution of the compound shown in the formula (I) by a suction pipe in batches, dripping the solution into water, taking the solid, and performing XRPD characterization on the solid, wherein the characterization result shows that the crystal form in the experiment is an amorphous form, and an X-ray diffraction spectrogram of the amorphous form is shown in figure 8.

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

1. Experimental Material

1.1 Compounds

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

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 fromPromega, 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 7.

TABLE 7

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 example 1 was formulated in DMSO at 4mM, then sequentially diluted 4-fold at 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 5min, discarding the culture medium, counting the uniformly dispersed cells, adjusting the appropriate cell concentration to 15mL fresh culture medium, adding into a 10cm culture dish, placing at 37 deg.C with 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 8.

TABLE 8

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. A4- ((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 crystal form A is characterized in that an X-ray powder diffraction spectrum has characteristic peaks at 3.7 +/-0.2, 9.5 +/-0.2, 14.2 +/-0.2 and 14.7 +/-0.2 by using a 2 theta angle.

2. Form A according to claim 1, characterized by an X-ray powder diffraction spectrum having characteristic peaks expressed in terms of 2 θ at 3.7 ± 0.2, 9.5 ± 0.2, 14.2 ± 0.2, 14.7 ± 0.2, 18.9 ± 0.2, 24.1 ± 0.2, 25.9 ± 0.2.

3. A4- ((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 crystal form B is characterized in that an X-ray powder diffraction spectrum has characteristic peaks at 3.9 +/-0.2 and 15.8 +/-0.2 by using a 2 theta angle.

4. A4- ((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 crystal form C is characterized in that an X-ray powder diffraction spectrum has characteristic peaks at 8.2 +/-0.2 and 12.0 +/-0.2 by using a 2 theta angle.

5. A4- ((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 crystal form D is characterized in that an X-ray powder diffraction spectrum has characteristic peaks at 7.1 +/-0.2, 10.1 +/-0.2, 14.2 +/-0.2, 20.2 +/-0.2 and 21.4 +/-0.2 in terms of 2 theta angles.

6. A4- ((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 crystal form E is characterized in that an X-ray powder diffraction spectrum has characteristic peaks at 4.9 +/-0.2, 9.9 +/-0.2, 15.0 +/-0.2 and 18.7 +/-0.2 by using a 2 theta angle.

7. An amorphous form 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 having an X-ray powder diffraction spectrum substantially as shown in figure 8.

8. A crystalline composition wherein the crystalline form a 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 of claim 1 or 2, the crystalline form 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 form B, 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 form C according to claim 4, 4- ((1-cyclopropyl-3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) oxy) -7- (3- (trifluoromethyl) -5, crystalline form D of 6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazin-7 (8H) -yl) quinoline or crystalline form E 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 according to claim 6 comprises more than 50%, preferably more than 80%, more preferably more than 90%, most preferably more than 95% by weight of the crystalline composition.

9. A pharmaceutical composition comprising the crystalline form a 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 of claim 1 or 2, or the crystalline form B of claim 3, or the crystalline form C of claim 4, or the crystalline form D of claim 5, or the crystalline form E of claim 6, or the amorphous form of claim 7, and a pharmaceutically acceptable carrier.

10. Use of the crystalline form a 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 according to claim 1 or 2, or the crystalline form B according to claim 3, or the crystalline form C according to claim 4, or the crystalline form D according to claim 5, or the crystalline form E according to claim 6, or the amorphous according to claim 7, or the crystalline composition according to claim 8, or the pharmaceutical composition according to claim 9, for the preparation of a medicament for the treatment and/or prevention of cancer, for the treatment and/or prophylaxis of cancer, for the treatment of cancer, or for the treatment of cancer, or for the treatment of cancer, or for the treatment of cancer, for the treatment of the, The application of the medicine for treating tissue hyperplasia diseases, fibrosis or inflammatory diseases.