Pharmaceutically acceptable salts of quinoline TGF-β1 inhibitors and their preparation methods

Abstract

The present invention belongs to the field of medicinal chemistry and relates to a pharmaceutically acceptable salt of a quinoline-based TGF-β1 inhibitor or its hydrate, solvate or crystal, and a preparation method and use thereof. Specifically, the present invention relates to a pharmaceutically acceptable salt 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 formula (I) or its hydrate, solvate or crystal and a preparation method thereof. The pharmaceutically acceptable salt or its hydrate, solvate or crystal can be used for preparing drugs for treating and / or preventing cancer, tissue hyperplasia diseases, fibrosis or inflammatory diseases.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and particularly relates to a pharmaceutically acceptable salt 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 hydrate, solvate or crystal thereof, and a preparation method and use thereof. Background Art

[0002] TGF-β (transforming growth factor-β) is an important class of cytokines. So far, 6 different subtypes (TGF-β1-6) have been discovered, with different homologies to each other, and only 3 subtypes are expressed in mammals, namely TGF-β1, TGF-β2 and TGF-β3. It is a multifunctional growth factor superfamily with a wide range of biological activities, participating in early embryonic development, cartilage and bone formation, extracellular matrix synthesis, inflammation, interstitial fibrosis, regulation of immune and endocrine functions, and tumor formation and development. At the same time, these 3 isomers have similar structures and highly homologous amino acid sequences, but in their respective gene knockout mouse models, they show completely different phenotypes, suggesting that each isomer has specific and non-overlapping functions in vivo. Ligands of the TGF-β family can bind to receptors on the cell surface and initiate the transmission of downstream signals within the cell.

[0003] TGF-β1 is the most common and important subtype of TGFβ, the most abundant subtype expressed in the liver, and also the strongest known liver fibrosis inducer, playing a crucial role in the development of chronic liver disease to end-stage liver disease (Yamazaki, et al. Digestive Disease, 2011, 29:284-288). A number of studies have shown that TGF-β1 and TGFβ receptors are usually highly expressed in liver-lesioned organs, blood vessels and extracellular matrix. In the classical TGFβ-TGFβR-Smads pathway, TGF-β1 activates TGFβR1 (transforming growth factor-β receptor 1, ALK5) in the signal pathway, and then regulates the entire signal pathway to achieve the regulation of the expression of a series of target genes related to fibrosis and tumorigenesis and development. It is generally believed that the promoting effect of TGF-β on liver cancer is mainly manifested in promoting tumor cell metastasis, enhancing tumor cell immune escape and inducing angiogenesis, etc. (Ling, et al. Current Pharmaceutical Biotechnology, 2011, 12:2190-2202).

[0004] Drug research targeting the TGF-β pathway has been carried out for many years. However, TGFβR1 inhibitors such as Galunisertib have shown certain cardiotoxicity in animal models (such as bleeding, functional degradation, inflammatory damage, etc.). The reason is that the target selectivity and specificity of such drugs are relatively low. While the drugs inhibit the kinase activation site of TGFβR1, they also have a strong inhibitory effect on other proteins with the same kinase region (such as p38α), thereby producing many unexpected off-target toxic side effects. Therefore, there is still a need to develop TGFβR1 inhibitors with higher selectivity to 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-based TGF-β1 inhibitor compound, the structure of which is shown in the following formula (I), and its 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 "compound of formula (I)"):

[0006]

[0007] The inventors of the present invention have studied and 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 to be a therapeutic agent for TGF-βR1-related diseases.

[0008] An object of the present invention is to provide a pharmaceutically acceptable salt of a TGF-βR1 inhibitor or its hydrate, solvate or crystal with high crystallinity, good water solubility, high thermal stability and high bioavailability. Specifically, the present invention provides a pharmaceutically acceptable salt 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 as shown in formula (I) or its hydrate, solvate or crystal,

[0009]

[0010] In some embodiments, the pharmaceutically acceptable salts of the compounds of formula (I) provided by the present invention, or their hydrates, solvates or crystals are inorganic salts or organic salts of the compounds of formula (I). Preferably, the pharmaceutically acceptable salts are hydrochloride, hydrobromide, phosphate, aminosulfonate, nitrate, p-toluenesulfonate, benzenesulfonate, p-aminobenzenesulfonate, mesylate, sulfate, acetate, oxalate, phenylacetate, propionate, malonate, trifluoroacetate, succinate, glycolate, stearate, ascorbate, pamoate, hydroxymaleate, glutamate, benzoate, salicylate, 2-acetoxybenzoate, fumarate, ethanedisulfonate, oxalate, isethionate, citrate, D-gluconate, lactate, malate, adipate, gentisate, 1-hydroxy-2-naphthoate, ethanedisulfonate, succinate, L-tartrate, fumarate, α-ketoglutarate, hippurate, nicotinate, maleate or D-tartrate. Further preferably, the pharmaceutically acceptable salts are hydrochloride, sulfate, phosphate, mesylate, maleate, adipate, oxalate, fumarate, acetate, hippurate, glycolate, malonate, oxalate, benzoate, lactate, nicotinate, p-toluenesulfonate, succinate, malate, citrate or tartrate. Even more preferably, the pharmaceutically acceptable salts are hydrochloride, sulfate, phosphate, mesylate, maleate, malonate, adipate, oxalate or fumarate. In some embodiments, the present invention provides the hydrochloride, sulfate, phosphate, mesylate, maleate, malonate, adipate, oxalate or fumarate of the compound of formula (I), wherein the chemical ratio of the compound of formula (I) to hydrochloric acid, sulfuric acid, phosphoric acid, mesylic acid, maleic acid, malonic acid, adipic acid, oxalic acid or fumaric acid is about 1:0.1 to about 1:2, preferably about 1:0.5 to about 1:1.2, and more preferably about 1:0.8 to about 1:1.

[0011] Another object of the present invention is to provide a method for preparing the pharmaceutically acceptable salt 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 its hydrate, solvate or crystal, which comprises the step of reacting the free base compound of formula (I) with an acid to form a salt.

[0012] The above reaction can be carried out in an organic solvent or a mixed solvent of an organic solvent and water. Preferably, the organic solvent is selected from one or more of alcohols, ketones, esters, ethers, nitrile solvents, and furan solvents having less than 6 carbon atoms, more preferably selected from one or more of methanol, ethanol, n-propanol, isopropanol, n-butanol, n-pentanol, n-hexanol, acetone, methyl ethyl ketone, methyl isobutyl ketone, ethyl formate, ethyl acetate, acetonitrile, and tetrahydrofuran, and even more preferably selected from one or more of methanol, ethanol, isopropanol, acetone, ethyl formate, ethyl acetate, acetonitrile, and tetrahydrofuran.

[0013] A method for preparing a pharmaceutically acceptable salt or a hydrate, solvate, or crystal thereof 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 the present invention, comprising the following steps:

[0014] 1. A step of preparing 7-bromo-4-chloroquinoline;

[0015] 2. A step of using 7-bromo-4-chloroquinoline in step 1 to prepare 7-bromo-4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)oxy)quinoline;

[0016] 3. A step of preparing 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine;

[0017] 4. Using 7-bromo-4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)oxy)quinoline in step 2 and 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine in step 3 to prepare 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.

[0018] According to the method for preparing a pharmaceutically acceptable salt or a hydrate, solvate, or crystal thereof 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 the present invention, 7-bromo-4-chloroquinoline prepared in step 1 is added to methyl tert-butyl ether, and then washed with methyl tert-butyl ether for further purification.

[0019] In some preferred embodiments, a method for preparing a pharmaceutically acceptable salt of a compound of formula (I) according to the present invention, or a hydrate, solvate or crystal thereof, wherein the molar ratio of the compound of formula (I) to the acid is about 1:0.5 to about 1:5, more preferably about 1:0.8 to about 1:3, and even more preferably about 1:0.9 to about 1:2.

[0020] In some preferred embodiments, the present invention provides a method for preparing a sulfate salt of a compound of formula (I) of the present invention, wherein the molar ratio 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 to sulfuric acid is about 1:0.5 to about 1:5, more preferably 1:0.8 to about 1:3, and even more preferably about 1:0.9 to about 1:2. In a specific embodiment, the molar ratio 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 to sulfuric acid is about 1:1.

[0021] Another aspect of the present invention provides a solvate of a pharmaceutically acceptable salt of a compound of formula (I), preferably a hydrate of a pharmaceutically acceptable salt of a compound of formula (I), or a cyclohexane, petroleum ether, ethyl acetate, isopropyl acetate, acetone, methanol, ethanol, chloroform, benzene, tetrahydrofuran, dichloromethane, ether, isopropyl ether, isopropanol, n-hexane, n-heptane, n-butane, n-pentane, isopentane or acetonitrile complex of a pharmaceutically acceptable salt of a compound of formula (I).

[0022] Another aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable salt 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 hydrate, solvate or crystal thereof and a pharmaceutically acceptable carrier.

[0023] Another aspect of the present invention provides the use of a pharmaceutically acceptable salt 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 hydrate, solvate or crystal thereof, or a pharmaceutical composition comprising the above-mentioned pharmaceutically acceptable salt or a hydrate, solvate or crystal thereof, in the preparation of a drug for treating and / or preventing cancer, tissue hyperplasia diseases, fibrosis or inflammatory diseases. The present invention provides the use of a pharmaceutically acceptable salt of the present invention, or a hydrate, solvate, crystal thereof, or a pharmaceutical composition comprising them, in the preparation of a drug for treating and / or preventing cancer, tissue hyperplasia diseases, fibrosis or inflammatory diseases, wherein the symptoms of the cancer, tissue hyperplasia diseases, fibrosis or inflammatory diseases include but are not limited to: melanoma, papillary thyroid tumor, cholangiocarcinoma, colon cancer, ovarian cancer, lung cancer, malignant lymphoma, carcinoma and sarcoma of the liver, kidney, bladder, prostate, breast and pancreas, and primary and recurrent solid tumors of the skin, colon, thyroid, lung and ovary, or leukemia, glioblastoma (glioma), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), acute myeloid leukemia (AML), sarcoma, non-small cell lung cancer, chondrosarcoma, cholangiocarcinoma or angioimmunoblastic lymphoma, liver fibrosis and chronic kidney disease.

[0024] Unless otherwise defined, 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.

[0025] The term "solvate" refers to a form of the compound of the present invention that forms a solid or liquid complex by coordinating with solvent molecules. A hydrate is a special form of a solvate, in which coordination occurs with water. Within the scope of the present invention, the solvate is preferably a hydrate.

[0026] The term "crystal" refers to various solid forms formed by the compounds described in the present invention, including crystal forms and amorphous forms.

[0027] "Hydrogen", "carbon" and "oxygen" in the compounds of the present invention include all their isotopes. 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, isotopes of carbon include 13 C and 14 C, and isotopes of oxygen include 16 O and 18 O, etc.

[0028] The "free base" of the present invention refers to 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.

[0029] The hydrochloride, sulfate, phosphate, mesylate, maleate, adipate, oxalate, fumarate, acetate, hippurate, glycolate, malonate, ethanedioate, benzoate, lactate, nicotinate, p-toluenesulfonate, succinate, malate, citrate or tartrate of the present invention refers to the hydrochloride, sulfate, phosphate, mesylate, maleate, adipate, oxalate, fumarate, acetate, hippurate, glycolate, malonate, ethanedioate, benzoate, lactate, nicotinate, p-toluenesulfonate, succinate, malate, citrate or tartrate 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.

[0030] "Equivalent to 1.0" in the present invention means that the ratio of the molar amount of the acid of the present invention to the molar amount of the free base is 1.0. Description of the Drawings

[0031] Figure 1 XRPD pattern of crystalline form A of the hydrochloride salt of the compound of formula (I);

[0032] Figure 2 XRPD pattern of crystalline form A of the sulfate salt of the compound of formula (I);

[0033] Figure 3 XRPD pattern of crystalline form A of the phosphate salt of the compound of formula (I);

[0034] Figure 4 XRPD pattern of crystalline form B of the phosphate salt of the compound of formula (I);

[0035] Figure 5 XRPD pattern of crystalline form A of the mesylate salt of the compound of formula (I);

[0036] Figure 6 XRPD pattern of crystalline form A of the maleate salt of the compound of formula (I);

[0037] Figure 7 XRPD pattern of crystalline form A of the adipate salt of the compound of formula (I);

[0038] Figure 8 XRPD pattern of crystalline form A of the oxalate salt of the compound of formula (I);

[0039] Figure 9 XRPD pattern of oxalate crystal form B of the compound of formula (I);

[0040] Figure 10 XRPD pattern of fumarate crystal form A of the compound of formula (I);

[0041] Figure 11 XRPD pattern of fumarate crystal form B of the compound of formula (I). Detailed implementation manners

[0042] The following representative examples are for better illustrating the present invention, rather than for limiting the protection scope of the present invention. Materials used in the following examples are commercially available unless otherwise specified.

[0043] I. Preparation of the compound of formula (I)

[0044] 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

[0045]

[0046] Step 1: Preparation of 2-bromo-1-(tetrahydro-2H-pyran-4-yl)ethan-1-one

[0047]

[0048] Under nitrogen protection, in a 1000 mL three-necked flask, methanol (100 mL) and 1-(tetrahydro-2H-pyran-4-yl)ethanone (20.0 g, 156 mmol) were successively added, cooled to below -15 °C, and liquid bromine was slowly added dropwise while maintaining the temperature below -15 °C. After the addition, the temperature was raised to 0 °C and the reaction was carried out for 45 min, then the temperature was raised to 10 °C and the reaction was carried out for 45 min. While maintaining the internal temperature below room temperature, 11 mol / L sulfuric acid (55 mL) was slowly added dropwise. After the addition, the reaction was carried out overnight at room temperature. After monitoring the reaction to completion, ethyl acetate and aqueous sodium chloride solution were added for extraction, and the organic layers were combined. The organic layer was adjusted to pH 7 - 8 with saturated sodium bicarbonate, and the organic layers were combined and concentrated under reduced pressure to obtain 28.5 g of the title compound as a pale yellow solid, with a yield of 87.5%; MS(ESI) m / z 207.0 [M+H] + .

[0049] Step 2: Preparation of 2-oxo-2-(tetrahydro-2H-pyran-4-yl)ethyl benzoate

[0050]

[0051] Dissolve benzoic acid (18.5 g, 151.4 mmol) in N,N-dimethylformamide (DMF, 495 mL), add potassium carbonate (38 g, 275.2 mmol), and then add 2-bromo-1-(tetrahydro-2H-pyran-4-yl)ethan-1-one (28.5 g, 137.6 mmol) to the system. React at room temperature overnight. Dilute with ethyl acetate and wash with aqueous sodium chloride solution. Combine the organic phases. Concentrate under reduced pressure to obtain the title compound as a pale yellow solid, totaling 30.0 g, yield: 88.2%; LC-MS m / z [M+H] + = 249.

[0052] Step 3: Preparation of (Z)-1-(dimethylamino)-3-oxo-3-(tetrahydro-2H-pyran-4-yl)prop-1-en-2-yl benzoate

[0053]

[0054] Add 2-oxo-2-(tetrahydro-2H-pyran-4-yl)ethyl benzoate (95.0 g, 383 mmol) to 1,1-dimethoxy-N,N-dimethylmethanamine (795.15 mL, 5975.8 mmol), heat to 100 °C, react for 2 h, then raise the temperature to 106 °C and react for 2 h. After monitoring the reaction to completion, cool to room temperature and concentrate to dryness under reduced pressure. Add ethyl acetate to the system and wash with brine. Dry the organic phase with anhydrous sodium sulfate. Filter to obtain the organic phase and concentrate under reduced pressure to obtain the title compound as a red solid, totaling 107.9 g, yield: 93.1%, and directly use it for the next step. LC-MS m / z [M+H] + = 304.

[0055] Step 4: Preparation of 3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl benzoate

[0056]

[0057] 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-yl benzoate (32.56 g, 107.46 mmol) were added successively. 80% hydrazine hydrate (37.6 mL) was slowly added dropwise under an ice bath. After the addition, the mixture was stirred at room temperature overnight. Monitoring until the reaction reached the end point, ethyl acetate was added to the reaction solution, followed by washing with water. The organic phases were combined, washed with saturated sodium bicarbonate solution until the pH was 7 - 8, dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated under reduced pressure to obtain a yellow oil. The yellow oil was left standing at room temperature overnight to obtain the yellow solid title compound, with a total of 28.0 g and a yield of 95.8%; LC-MS m / z [M + H] + = 273.

[0058] Step 5: Preparation of 1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl benzoate

[0059]

[0060] To 1,2-dichloroethane (250 mL), 2,2'-bipyridine (17.7 g, 113.2 mmol) and copper(II) acetate (20.6 g, 113.2 mmol) were added successively. The reaction was carried out 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-yl benzoate (28.0 g, 102.9 mmol) in 1,2-dichloroethane (250 mL) were added successively. The reaction was carried out at 75 °C for 4 h under an oxygen atmosphere. Monitoring until the reaction reached the end point, the reaction mixture was cooled to room temperature, filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure to obtain the reddish-brown oily title compound, with a total of 32.0 g and a yield of 98.5%, which was directly used in the next step; LC-MS m / z [M + H] + = 313.

[0061] Step 6: Preparation of 1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-ol

[0062]

[0063] To methanol (308 mL), add 1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl benzoate (32.0 g, 102.6 mmol), and react at room temperature for 2 h. Monitor until the reaction reaches the end point, concentrate under reduced pressure to remove part of the methanol, adjust the pH to 6 - 7 with 1 mol / L dilute hydrochloric acid, extract with dichloromethane, and combine the organic layers. Concentrate to dryness under reduced pressure and purify by column chromatography to obtain the yellow solid title compound, a total of 9.0 g, yield: 45%; LC-MS m / z + = 209.

[0064] Step 7: Preparation of 7-bromo-4-chloroquinoline

[0065]

[0066] Add 4.50 kg of acetonitrile to a 50 L vertical jacketed reactor, start stirring, and successively add 7-bromo-4-hydroxyquinoline (1.80 kg, 8.03 mol) and 4.00 kg of acetonitrile. When the internal temperature drops below 10 °C, add phosphorus oxychloride (1.85 kg, 12.05 mol) dropwise. After the addition is complete, raise the temperature to reflux and stir for 1 - 3 hours. Monitor until the reaction reaches the end point, cool the system to below 5 °C, adjust the pH to 7 - 8 by dropwise adding 4 mol / L NaOH solution, add 42.00 kg of water, stir at room temperature for 1 - 2 hours, centrifuge the feed liquid, wash the filter cake with 5.00 kg of water, and dry in vacuo. Collect and weigh the solid to obtain the crude product of the title compound as a brown solid, a total of 1.73 kg, yield: 88.8%.

[0067] Add 14.4 kg of methyl tert-butyl ether and 1.73 kg of the crude product of 7-bromo-4-chloroquinoline to a 50 L vertical jacketed reactor in sequence, stir at a temperature range of 50 ± 5 °C for 1 - 3 hours, filter while hot, wash the filter cake with methyl tert-butyl ether, concentrate the filtrate to dryness under reduced pressure, dry the obtained solid in vacuo, collect and weigh the solid after drying to obtain the refined product of the title compound as a yellow solid, a total of 1.54 kg, yield 89.0%.

[0068] Step 8: Preparation of 7-bromo-4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)oxy)quinoline

[0069]

[0070] Add 8.40 kg of acetonitrile to a 30 L glass reactor, start stirring, and sequentially add 7-bromo-4-chloroquinoline (1.26 kg, 5.18 mol), cesium carbonate (1.69 kg, 5.18 mol), and 1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-ol (0.90 kg, 4.32 mol). Wash with 4.00 kg of acetonitrile. Under nitrogen protection, heat to 70 ± 5 °C and stir for 2 - 3 hours, then raise the temperature to reflux for 2 - 3 hours. Cool to 70 ± 5 °C and filter while it is hot. Wash the filter cake with dichloromethane. Concentrate the filtrate under reduced pressure to a viscous state, add 14.00 kg of water, stir, centrifuge. Wash the filter cake with 3.00 kg of water and dry it under vacuum at 50 ± 5 °C for 10 - 20 hours. After drying, collect and weigh the solid to obtain 1.88 kg of the crude product of the title brown solid compound 1, and the crude product yield is 100.0%.

[0071] Add 6.40 kg of heptane and 1.88 kg of the crude product 1 of 7-bromo-4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)oxy)quinoline to a 10 L four-necked flask, and stir at 25 ± 5 °C for 3 - 5 hours. Filter, wash the filter cake with 1.40 kg of heptane, and dry it under vacuum at 50 ± 5 °C for 3 - 20 hours. After drying, collect and weigh the solid to obtain 1.68 kg of the crude product 2 of the title deep yellow solid compound, and the yield is 89.4%.

[0072] Add 4.70 kg of absolute ethanol and 1.68 kg of the crude product 2 of 7-bromo-4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)oxy)quinoline to a 10 L four-necked flask, heat to reflux, stir for 0.5 - 1 hour after it becomes clear, turn off the heating, and let it cool and crystallize naturally. When the internal temperature is lower than 30 °C, start the external circulation and stir at -10 - -5 °C for 1 - 3 hours. Centrifuge the material liquid, wash the filter cake with 0.4 kg of cold ethanol (-10 - -5 °C), and dry it under vacuum at 50 ± 5 °C for 10 - 20 hours. After drying, collect and weigh the solid to obtain 1.35 kg of the refined product of the title pale yellow solid compound, and the yield is 80.4%.

[0073] Step 9: Preparation of 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine

[0074]

[0075] Add 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine hydrochloride (1.20 kg, 5.25 mol) and 11.8 kg of acetonitrile to a 20 L four-necked flask. Start stirring, add sodium hydroxide (0.42 kg, 10.50 mol), 36.00 g of water, and stir vigorously at 25 ± 5 °C for 3 - 15 hours; filter, wash with acetonitrile, concentrate the filtrate under reduced pressure to dryness, add 0.5 g of seed crystals, and vacuum dry after solidification. After drying, collect and weigh the solid to obtain 0.92 kg of the white solid title compound, with a yield of 91.2%.

[0076] 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

[0077]

[0078] Add 7.10 kg of 1,4-dioxane to a 20 L four-necked flask. Start stirring, and successively 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(diphenylphosphino)-9,9-dimethylxanthene (Xantphos, 37.40 g, 65.00 mmol), and tris(dibenzylideneacetone)dipalladium (Pd2(dba)3, 29.6 g, 32.00 mmol). Protect with nitrogen and maintain a nitrogen atmosphere. Heat to 95 ± 5 °C and stir for 3 - 15 hours, turn off the heating, filter while hot when the internal temperature drops to 70 - 80 °C, 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 vacuum dry. After drying, collect and weigh the solid to obtain 1.41 kg of the crude product 1 of the yellow solid title compound, with a yield of 83.1%.

[0079] 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

[0080] Add 3.8 kg of ethyl acetate and 1.40 kg of crude product 1 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 to a 10 L four-necked flask. Under nitrogen protection, heat to 55 ± 5 °C and stir for 2 - 4 hours. Naturally cool to an internal temperature below 30 °C, stir and crystallize at -5 to -10 °C for 1 - 3 hours, centrifuge, wash with 0.5 kg of cold ethyl acetate (-10 to -5 °C), and dry in vacuo to obtain 1.21 kg of crude product 2 of the title compound as a pale yellow solid. The yield is 85.8%.

[0081] Add 14.30 kg of anhydrous methanol and 1.20 kg of crude product 2 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 to a 30 L glass reactor. Under nitrogen protection, heat to reflux until clear. Add 0.12 kg of activated carbon and 0.12 kg Reflux for 1 - 1.5 hours, filter while hot, wash with hot methanol (50 - 60 °C), concentrate the filtrate under reduced pressure, cool and crystallize at -10 to -5 °C for 1 - 3 hours, centrifuge, and wash with cold methanol (-10 to -5 °C). Dry in vacuo to obtain 1.03 kg of crude product 3 of the title compound as an off-white solid. The yield is 85.1%.

[0082] Add 9.50 kg of dichloromethane and 1.02 kg of crude product 3 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 to a 10 L four-necked flask and start stirring. After it becomes clear, add 0.20 kg Stir, filter, wash with dichloromethane. Slowly add 26.20 kg of heptane dropwise to the filtrate, stir for 10 - 15 hours, filter, wash the filter cake with 1.60 kg of heptane, and dry in vacuo to obtain 0.99 kg of the refined product of the title compound as a white solid. The yield is 97.1%. ESI-MS [M + H] + m / z: 526.3, 11H NMR (400 MHz, DMSO) δ 8.59 (d, J = 5.2 Hz, 1H), 8.17 (d, J = 11.2 Hz, 1H), 7.94 (s, 1H), 7.64 (dd, J = 11.2, 2.4 Hz, 1H), 7.45 (d, J = 2.4 Hz, 1H), 6.56 (d, J = 5.2 Hz, 1H), 4.91 (s, 2H), 4.34 (t, J = 5.1 Hz, 2H), 4.02 (t, J = 5.2 Hz, 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).

[0083] II. Preparation of salts 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

[0084] Example 2: 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 hydrochloride

[0085] Dissolve 50 mg of the free base sample in 0.75 mL of acetone. Slowly add hydrochloric acid dilution (molar ratio 1:1) dropwise at room temperature, stir at room temperature for 20 hours, filter, and dry overnight under vacuum at room temperature to obtain a yellow powdery solid, a total of 43 mg of hydrochloride crystal form A, with a yield of 81%. XRPD is shown in Figure 1 。

[0086] Example 3: 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 sulfate

[0087] Dissolve 50 mg of the free base sample in 0.3 mL of ethyl formate. Slowly add sulfuric acid dilution (diluted with 0.2 mL of ethyl formate) dropwise at room temperature, and the sulfuric acid equivalent is 1.0 (i.e., the molar amount of sulfuric acid is the same as that of the free base). Stir at room temperature for 20 hours, filter, and dry overnight under vacuum at room temperature to obtain a yellow powdery solid, a total of 52 mg of sulfate crystal form A, with a yield of 88%. 1The \(^1H\) NMR results showed that the chemical ratio of the compound of formula (I) to sulfuric acid was 1:1. XRPD is shown in Figure 2 。

[0088] Example 4: 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 phosphate

[0089] Dissolve 50 mg of the free base sample in 0.75 mL of acetone, slowly add phosphoric acid dropwise at room temperature, and the phosphoric acid equivalent is 1.0. Stir at room temperature for 20 hours, filter, and dry overnight under vacuum at room temperature to obtain 45 mg of yellow powdery solid, namely phosphate crystal form A, with a yield of 76%. XRPD is shown in Figure 3 。

[0090] Dissolve 50 mg of the free base sample in 0.5 mL of ethyl formate, slowly add phosphoric acid dropwise at room temperature, and the phosphoric acid equivalent is 1.0. Stir at room temperature for 20 hours, filter, and dry overnight under vacuum at room temperature to obtain 37 mg of yellow powdery solid, namely phosphate crystal form B, with a yield of 63%. XRPD is shown in Figure 4 。

[0091] Example 5: 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 methanesulfonate

[0092] Dissolve 50 mg of the free base sample in 0.75 mL of acetone, slowly add methanesulfonic acid dropwise at room temperature, and the methanesulfonic acid equivalent is 1.0. Stir at room temperature for 20 hours, filter, and dry overnight under vacuum at room temperature to obtain 45 mg of yellow powdery solid, namely methanesulfonate crystal form A, with a yield of 76%. XRPD is shown in Figure 5 。

[0093] Example 6: 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 maleate

[0094] Dissolve 60 mg of the free base sample in 6 mL of ethyl formate, add maleic acid at room temperature, and the maleic acid equivalent is 1.0. Stir at room temperature for 20 hours, filter, and dry overnight under vacuum at room temperature to obtain 56 mg of yellow powdery solid, namely maleate crystal form A, with a yield of 77%. XRPD is shown in Figure 6 。

[0095] Example 7: 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 adipate

[0096] Dissolve 60 mg of the free base sample in 6 mL of ethyl formate. Add adipic acid at room temperature with an adipic acid equivalent of 1.0. Stir at room temperature for 20 hours, filter, and dry overnight under vacuum at room temperature to obtain a yellow powdery solid, a total of 56 mg of adipate crystal form A, with a yield of 73%. XRPD is shown in Figure 7 。

[0097] Example 8: 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 oxalate

[0098] Dissolve 60 mg of the free base sample in 0.9 mL of acetone. Add oxalic acid at room temperature with an oxalic acid equivalent of 1.0. Stir at room temperature for 20 hours, filter, and dry overnight under vacuum at room temperature to obtain a yellow powdery solid, a total of 44 mg of oxalate crystal form A, with a yield of 63%. 1 The HNMR results show the presence of a solvent peak of acetone in the NMR. XRPD is shown in Figure 8 。

[0099] Dissolve 60 mg of the free base sample in 0.6 mL of ethyl formate. Add oxalic acid at room temperature with an oxalic acid equivalent of 1.0. Stir at room temperature for 20 hours, filter, and dry overnight under vacuum at room temperature to obtain a yellow powdery solid, a total of 69 mg of oxalate crystal form B, with a yield of 98%. XRPD is shown in Figure 9 。

[0100] Example 9: 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 fumarate

[0101] Dissolve 60 mg of the free base sample in 0.9 mL of acetone. Add fumaric acid at room temperature with a fumaric acid equivalent of 1.0. Stir at room temperature for 20 hours, filter, and dry overnight under vacuum at room temperature to obtain a yellow powdery solid, a total of 53 mg of fumarate crystal form A, with a yield of 72%. XRPD is shown in Figure 10 。

[0102] 60 mg of the free base sample was dissolved in 0.6 mL of ethyl formate, and fumaric acid was added at room temperature with a fumaric acid equivalent of 1.0. Stir at room temperature for 20 hours, filter, and dry overnight under vacuum at room temperature to obtain a yellow powdery solid, a total of 50 mg of fumarate crystal form B with a yield of 68%. XRPD is shown in Figure 11 。

[0103] Example 10: Preparation of other salt forms 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

[0104] 60 mg of the free base sample was taken and dissolved in the corresponding solvent as shown in Table 1, and the corresponding acid was added at room temperature with an acid equivalent of 1.0. After the acid was dissolved, stir at room temperature for 20 hours. The experimental results are shown in Table 1.

[0105] Table 1

[0106]

[0107]

[0108] III. Activity 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 compound

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

[0110] 1. Experimental materials

[0111] 1.1 Compound

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

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

[0116] 2. Experimental methods

[0117] 2.1 Preparation of 1x kinase buffer

[0118] 1x assay buffer

[0119] 40 mM Tris, pH 7.5

[0120] 20 mM MgCl2

[0121] 0.10% BSA

[0122] 1 mM DTT

[0123] 2.2 Compound preparation

[0124] 2.2.1 Compound dilution

[0125] 2.2.1.1 Preparation of 50-fold compound: The final detection concentration of the compound is 10 μM, and it is configured into a 50-fold concentration, that is, 500 μM: Add 95 μl of 100% DMSO to the second well of the 96-well plate, and then add 5 μl of 10 mM compound solution to prepare a 1000 μM compound solution. Add 60 μl of 100% DMSO to other wells. Take 30 μl of the compound from the second well and add it to the third well, and perform 3-fold dilution downwards in turn, with a total of 10 concentrations diluted.

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

[0127] 2.2.1.2 Transfer 100 nl of the compound to the reaction plate using echo.

[0128] 2.3 Kinase reaction

[0129] 2.3.1 Preparation of 2× kinase solution

[0130] Add kinase to 1× kinase buffer to form 2× enzyme solution. There is already 100 nl of compound dissolved in 100% DMSO in the 384-well reaction plate. Add 2.5 μl of 2× enzyme solution to the 384-well reaction plate. Incubate at room temperature for 10 minutes.

[0131] 2.3.2 Preparation of 2× substrate solution

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

[0133] 2.4 Kinase reaction

[0134] Incubate the 384-well plate at 28 °C for 120 minutes

[0135] 2.5 Detection of reaction results

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

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

[0138] 2.5.3 Transfer 5 μl of ADP-Glo reagent to the well of the 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 detection reagent to each well, shake for 1 minute, and let stand at room temperature for 30 minutes.

[0141] 2.6 Data reading

[0142] Read the luminescence value of the sample on Envision.

[0143] 2.7 Curve fitting

[0144] 2.7.1 Copy the data of the luminescence readings from the Envision program

[0145] 2.7.2 Convert the luminescence reading values to the percentage of inhibition through a formula.

[0146] Percent inhibition=(max-sample RLU) / (max-min)*100. "min" is the fluorescence reading of the control sample without adding enzyme for the reaction; "max" is the fluorescence reading of the sample with DMSO as the control.

[0147] 2.7.3 Import the data into MS Excel and perform curve fitting using the XLFit excel add-in version 5.4.0.8. The fitting formula is: Y = Bottom + (Top - Bottom) / (1 + (IC 50 / X)^HillSlope), and the results are shown in Table 2.

[0148] Table 2

[0149]

[0150] It can be seen from the above experimental results that the compound of the present invention has good inhibitory activity against ALK5 kinase, while having low inhibitory effect on p38α and high selectivity. It shows that the compound of the present invention has lower side effects while producing higher efficacy.

[0151] Experimental Example 2 Evaluation of the compound in vitro cell luciferase assay

[0152] 1. Experimental materials

[0153] Test compound: The compound of formula (I) of the present invention in Example 1 was formulated into 4 mM with DMSO, and then serially 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, 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 was purchased from Invitrogen, USA; FBS was purchased from Invitrogen, USA; DMSO was purchased from Sigma, USA; Glo Lysis Buffer was purchased from Progema, USA; Bright-Glo Luciferase assay system was purchased from Promega, USA; TGFβ was purchased from PeproTech, USA.

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

[0157] 2. Experimental method

[0158] 2.1 Cell culture:

[0159] Cell Resuscitation: Place the cells in a 37°C water bath to dissolve, then transfer them to 15 mL of pre-warmed medium, centrifuge at 1000 rpm for 5 minutes, discard the medium, resuspend the cells with 15 mL of fresh medium, transfer to a 10 cm culture dish, and culture in an incubator at 37°C and 5% CO2. Replace the fresh medium after 24 hours.

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

[0161] 2.2 Experimental Procedures:

[0162] Day1: Seed cells (clear-bottom 96-well plate)

[0163] Luc-Smad2 / 3-NIH3T3 cells are normally cultured in a 10 cm culture dish until the confluence reaches 80%-90%. After digestion, collect them into a 15 mL centrifuge tube, centrifuge at 1000 xg for 5 minutes, remove the supernatant, resuspend with 1 mL of medium, dilute 10 times for counting, dilute the cells according to the counting result, and transfer 4x10 3 cells per well into the 96-well plate (add 100 μl of resuspended cells to each well).

[0164] Day2: Administer drugs to cells

[0165] Weigh 1-2 mg of the drug (pre-weighed), prepare a 4 mM stock solution using DMSO. After 24 hours, remove the medium. Dilute the drug with 2% FBS medium, add 100 μl of 1x drug solution, so that the final concentrations of the drug are 20000.00 nM, 5000.00 nM, 1250.00 nM, 312.5 nM, 78.125 nM, 19.53 nM, 4.88 nM, 1.22 nM respectively, and the final concentration of TGFβ1 in each well is 4 ng / mL. Dilute with 2% FBS medium together with the compound.

[0166] Day3: Fluorescence detection experiment

[0167] Equilibrate the Glo Lysis Buffer, Bright-glo luciferase assay system and the cells to room temperature. Remove the cell supernatant, add 100 μl of Glo Lysis Buffer to each well, gently shake to dissolve the cells evenly, and lyse the cells at room temperature for 5 mins. Then add 100 μl of Bright-glo luciferase assay system to each well, incubate at room temperature for 5 minutes, shake for 2 minutes, transfer 180 μl of the supernatant to a white-bottom 96-well plate, and detect the chemiluminescence signal with a detection condition of 1 s.

[0168] 2.3 Data processing: Use Graphpad Prism 5 software for non-linear curve fitting and data analysis, and fit the IC 50 , and the experimental results are shown in Table 3.

[0169] Table 3

[0170]

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

[0172] IV. Related properties of the pharmaceutically acceptable salts 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 compounds

[0173] Experimental Example 3: Study on the water solubility and stability of the new salt forms prepared in Examples 2-10

[0174] Water solubility experiment: When the solubility is greater than 10 mg / mL, it is considered to have good water solubility; if it is less than 1 mg / mL, it is considered poor; if it is between the two, it is considered relatively poor. The water solubility of each salt 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 prepared in Examples 2-10 was detected using a conventional method. The experimental results showed that 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 hydrochloride, crystalline form A of sulfate, crystalline form A of phosphate, crystalline form B of phosphate, crystalline form A of mesylate, crystalline form A of maleate, crystalline form A of malonate, crystalline form A of adipate, crystalline form A of oxalate, crystalline form B of oxalate, crystalline form A of fumarate, and crystalline form B of fumarate have good water solubility.

[0175] Influence factors of 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 hydrochloride, crystalline form A of sulfate, crystalline form A of phosphate, crystalline form B of phosphate, crystalline form A of mesylate, crystalline form A of maleate, crystalline form A of malonate, crystalline form A of adipate, crystalline form A of oxalate, crystalline form B of oxalate, crystalline form A of fumarate, and crystalline form B of fumarate were investigated (under the conditions of light, 40 °C or humidity of 75%). The change rate of total impurities at day 0 was detected. The experimental results found that the stability order of each salt form was: sulfate > malonate > maleate > adipate > fumarate ≈ oxalate > hydrochloride > phosphate > mesylate.

[0176] Although the present invention has been described in detail above, those skilled in the art understand that various modifications and changes can be made to the present invention without departing from the spirit and scope of the present invention. The scope of the rights of the present invention is not limited to the detailed description above, but should be attributed to the claims.

Claims

1. A pharmaceutically acceptable salt 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 formula (I). Wherein the pharmaceutically acceptable salt is hydrochloride, phosphate, mesylate, sulfate, oxalate, adipate, fumarate or maleate.

2. The pharmaceutically acceptable salt according to claim 1, wherein the chemical ratio 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 to hydrochloric acid, sulfuric acid, phosphoric acid, mesylic acid, maleic acid, adipic acid, oxalic acid or fumaric acid is 1:0.1 to 1:

2.

3. The pharmaceutically acceptable salt according to claim 2, wherein the chemical ratio 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 to hydrochloric acid, sulfuric acid, phosphoric acid, mesylic acid, maleic acid, adipic acid, oxalic acid or fumaric acid is 1:0.5 to 1:1.

2.

4. The pharmaceutically acceptable salt according to claim 3, wherein the chemical ratio 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 to hydrochloric acid, sulfuric acid, phosphoric acid, mesylic acid, maleic acid, adipic acid, oxalic acid or fumaric acid is 1:0.8 to 1:

1.

5. A method for preparing the pharmaceutically acceptable salt according to any one of claims 1-4, the method comprising the step of reacting 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 with an acid to form a salt.

6. The method according to claim 5, wherein the salt-forming reaction is carried out in an organic solvent or a mixed solvent of an organic solvent and water, and the organic solvent is selected from one or more of alcohols, ketones, esters, ethers, nitrile solvents and furan solvents having less than 6 carbon atoms.

7. The method according to claim 5 or 6, wherein the molar ratio 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 to the acid is 1:0.5 to 1:

5.

8. The method according to claim 7, wherein the molar ratio 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 to the acid is from 1:0.8 to 1:

3.

9. The method according to claim 8, wherein the molar ratio 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 to the acid is from 1:0.9 to 1:

2.

10. A pharmaceutical composition comprising a pharmaceutically acceptable salt according to any one of claims 1-4 and a pharmaceutically acceptable carrier.

11. Use of a pharmaceutically acceptable salt according to any one of claims 1-4 or a composition according to claim 10 in the preparation of a medicament for the treatment and / or prevention of cancer, tissue hyperplasia diseases, fibrosis or inflammatory diseases.

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