CN115232124A

CN115232124A - Crystal form of ATX inhibitor and preparation method thereof

Info

Publication number: CN115232124A
Application number: CN202210429914.4A
Authority: CN
Inventors: 吴琪; 杨俊然; 杜振兴
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2021-04-23
Filing date: 2022-04-22
Publication date: 2022-10-25

Abstract

The present disclosure relates to a crystalline form of an ATX inhibitor and a process for its preparation. In particular, the disclosure relates to a crystal form A of a compound shown in formula I and a preparation method thereof. The crystal form has good stability.

Description

Crystal form of ATX inhibitor and preparation method thereof

Technical Field

The disclosure relates to a crystal form of an ATX inhibitor and a preparation method thereof, and particularly provides a crystal form of a compound shown in a formula (I) and a preparation method thereof.

Background

Autotaxin (ATX), also known as ENPP2, is a secreted enzyme that is highly expressed in cancer cells, bronchial epithelial cells in the lung, and alveolar macrophages. ATX was first isolated from melanoma cells in 1992 (Stracke, m.l. et al, j.biol.chem.1992,267, 2524-2529), belonging to one of seven members of the ENPP family, among which ENPP1 and ENPP3 are closest to ATX (Albers, h.m.h.g. et al, chem.rev.2012,112, 2593-2603). ATX is the only lysophospholipase D (lysoPLD) activity in ENPP enzymes and primarily converts LPC to the bioactive lipid lysophosphatidic acid (LPA). LPA is a lipid, mainly LPA 16, LPA 18. LPA functions via six receptor proteins on the cell surface (LPA 1-6), namely protein coupled receptors (GPCRs) (Lin, M.E. et al Prostagladins Other Lipid Mediators 2010,91,130-138). The LPA receptor family can be further divided into two broad categories: (1) the EDG receptor family, including LPA1-3; (2) the non-EDG receptor family LPA4-6. Both have a similarity of less than 40% (Zhao, y. Et al Cell Signalling 2009,21,367-377). Each LPA receptor mediates a series of cellular signaling cascades through specific G body proteins. The major signaling pathways include protein kinase (MAPK) activation, inhibition of the adenylate cyclase pathway, arachidonic acid release, activation of the PI3K-AKT pathway, regulation of apoptosis and survival, activation of the Rho, rock, rac and Ras signaling pathways (Mills, nat. Rev. Cancer 2003,3,582-591, et al). The ATX-LPA signaling pathway is involved in many physiological and pathological processes, leading to its important association with a number of serious diseases, mainly including cancer, fibrotic diseases, pain, immune diseases, inflammatory nervous system and cardiovascular diseases (Nicolas, d. Et al, US8993590B 2). Experiments have shown that ATX is involved in the invasion and metastasis process of tumor cells, and over-expression of ATX can be observed in tumor tissues of ovarian Cancer (Vidot, s., et al Cell Signal,2010,22,926-935), breast Cancer (panupuntu, n. Et al British Journal of Cancer 2010,102, 941-946), prostate Cancer (Nouh, m.a. Et al Cancer sci.2009,100, 1631-1638), hepatocellular carcinoma (Wu, j. Et al Mol Cancer,2010,9,71), and lung Cancer (Xu, x. Et al Cancer,2010,116, 1739-1750). And LPA produced by it promotes tumor formation by increasing cell motility and invasiveness. Therefore, ATX inhibitors may prevent LPA production and have potential in the treatment of a variety of diseases.

Compared with the traditional kinase inhibitor, the ATX inhibitor regulates and controls signal paths related to cell proliferation, survival, apoptosis and migration by inhibiting the formation of LPA, can be potentially used for treating various cancers, and is an important target for researching novel fibrotic diseases because the signal paths of LPA are closely related to fibrosis of various organs.

WO2021078227 provides a compound of the formula which has been found to have strong inhibitory activity against ATX.

Disclosure of Invention

The present disclosure provides crystalline forms of a compound of formula (I).

The present disclosure provides amorphous forms of the compound of formula (I) having an X-ray powder diffraction pattern with diffraction angles 2 theta in the range of 3-45 ° without distinct characteristic peaks.

The present disclosure further provides a method of preparing an amorphous form of a compound of formula (I), method 1, comprising the steps of: mixing the compound shown in the formula (I) with a solvent I, and stirring for crystallization. In certain embodiments, the solvent I is selected from water.

The present disclosure further provides an amorphous method of preparing a compound of formula (I), method 2, comprising the steps of: mixing the compound shown in the formula (I) with a solvent II, and volatilizing for crystallization. In certain embodiments, the solvent II is selected from one or more of acetonitrile, methanol.

In certain embodiments, the volume (. Mu.l) used for solvent I or II according to the present disclosure may be 1 to 200 times the mass (mg) of the compound represented by formula (I), and in non-limiting embodiments may be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200. In certain embodiments, the methods of making described in the present disclosure further comprise a centrifugation, washing, or drying step.

The present disclosure further provides crystalline form a of the compound of formula (I) having characteristic peaks at 12.777, 18.719, 22.502, 25.747, and 26.517 in an X-ray powder diffraction pattern expressed in diffraction angle 2 theta angles. In certain embodiments, form a of the compound of formula (I) has characteristic peaks at 6.355, 12.777, 18.719, 19.232, 22.502, 25.003, 25.747, 26.517, and 27.735. In certain embodiments, form a of the compound of formula (I) has characteristic peaks at 6.355, 12.777, 14.274, 16.915, 18.719, 19.232, 22.502, 23.401, 23.777, 25.003, 25.747, 26.517, and 27.735. In certain embodiments, form a of the compound of formula (I) has characteristic peaks at 6.355, 12.777, 14.274, 15.987, 16.915, 18.719, 19.232, 22.502, 23.401, 23.777, 25.003, 25.747, 26.517, 27.735, and 29.175. In certain embodiments, the compound of formula (I) has an X-ray powder diffraction pattern for form a as shown in figure 2, expressed as diffraction angle 2 Θ angles.

The present disclosure further provides a process for preparing form a of the compound of formula (I), process 1, comprising the steps of: mixing the compound shown in the formula (I) with a solvent III, and stirring at room temperature for crystallization. In certain embodiments, the solvent III is preferably selected from one or more of an alcohol solvent, a ketone solvent, an ester solvent, a haloalkane solvent, an alkane solvent, and an aromatic hydrocarbon solvent; the alcohol solvent is preferably selected from methanol, ethanol and isopropanol; the ketone solvent is preferably selected from acetone and 2-butanone; the ester solvent is preferably selected from ethyl acetate and isopropyl acetate; the haloalkane solvent is preferably selected from dichloromethane; the alkane solvent is preferably selected from n-heptane; the aromatic hydrocarbon solvent is preferably selected from toluene.

The present disclosure further provides a process, process 2, for preparing form a of the compound of formula (I) comprising the steps of: mixing the compound shown in the formula (I) with a solvent IV, and cooling for crystallization. In certain embodiments, the solvent IV is selected from acetonitrile.

The present disclosure further provides a process for preparing form a of the compound of formula (I), process 3, comprising the steps of: mixing the compound shown in the formula (I) with a solvent V, and volatilizing for crystallization. In certain embodiments, the solvent V is selected from dioxane.

The present disclosure further provides a process for preparing form a of the compound of formula (I), process 4, comprising the steps of: mixing the compound shown in the formula (I) with a solvent VI, dissolving, adding a solvent VII, and crystallizing and precipitating. In certain embodiments, the solvent VI solvent is preferably selected from one or more of acetone, tetrahydrofuran, dichloromethane; the solvent VII is preferably one or more selected from cyclohexane, water and ethyl acetate.

In certain embodiments, the solvents III-VII described in the present disclosure may be used in a volume (μ l) of 1 to 200 times the mass (mg) of the compound of formula (I), and in non-limiting embodiments may be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200. In certain embodiments, the methods of making described in the present disclosure further comprise a centrifugation, washing, or drying step.

The present disclosure also provides pharmaceutical compositions prepared from crystalline forms of the compounds described by the foregoing formula (I).

The disclosure also provides a pharmaceutical composition comprising a crystalline form of the compound and optionally a pharmaceutically acceptable carrier, diluent or excipient.

The present disclosure also provides a process for preparing a pharmaceutical composition comprising the step of mixing a crystalline form of the aforementioned compound with a pharmaceutically acceptable carrier, diluent or excipient.

The disclosure also provides a use of a crystalline form of the aforementioned compound, or the aforementioned composition, or a composition prepared by the aforementioned method, in the preparation of a medicament for inhibiting ATX.

The present disclosure also provides a crystalline form of the aforementioned compound, or the aforementioned composition, or a composition made by the aforementioned method, for use in preventing and/or treating a fibrotic disease, cancer, a proliferative disease, an inflammatory disease, an autoimmune disease, a respiratory disease, a cardiovascular disease, a neurodegenerative disease, a dermatological disease, a metabolic disease, a myelodysplastic syndrome, an abnormal angiogenesis-related disease, or pain. In certain embodiments, for the prevention and/or treatment of fibrotic diseases, cancer, in certain embodiments, for the prevention and/or treatment of pulmonary fibrosis, idiopathic pulmonary fibrosis, liver fibrosis, scleroderma, kidney cancer or pancreatic cancer.

The "2 theta or 2 theta angle" referred to in this disclosure means the diffraction angle, theta being the bragg angle in degrees or degrees; the error range of each characteristic peak 2 θ is ± 0.2 (including the case where a number exceeding 1 decimal is rounded), and may be-0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, -0.09, -0.08, -0.07, -0.06, -0.05, -0.04, -0.03, -0.02, -0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20.

The precipitation mode of the present disclosure includes, but is not limited to, stirring, cooling, volatilizing, pulping, and precipitating.

According to the guiding principle of moisture-attracting property of 9103 medicament in 2015 th edition of four parts of Chinese pharmacopoeia and the definition of moisture-attracting weight increment,

deliquescence: absorbing sufficient water to form a liquid;

has very moisture-wicking property: the moisture-inducing weight is not less than 15%;

moisture absorption: the moisture-inducing weight is less than 15% but not less than 2%;

slightly hygroscopic: the moisture-inducing weight is less than 2% but not less than 0.2%;

no or little hygroscopicity: the moisture-drawing weight gain is less than 0.2 percent.

The differential scanning calorimetry or DSC in the present disclosure refers to measuring the temperature difference and the heat flow difference between the sample and the reference during the temperature rise or constant temperature process of the sample to characterize all the physical changes and chemical changes related to the thermal effect and obtain the phase change information of the sample.

In the present disclosure, the drying temperature is generally 25 ℃ to 100 ℃, preferably 40 ℃ to 70 ℃, and the drying may be performed under normal pressure or under reduced pressure.

"pharmaceutical composition" means a mixture comprising one or more compounds of formula (I) as described herein or a pharmaceutically acceptable salt thereof, in admixture with other chemical components, as well as other components such as pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

Drawings

FIG. 1: an XRPD pattern of amorphous crystals of the compound of formula (I).

FIG. 2 is a schematic diagram: an XRPD pattern of a crystal form A of the compound shown as the formula (I).

FIG. 3: a DVS data profile for form a of the compound of formula (I).

FIG. 4: XRPD patterns before and after crystal form A DVS of the compound shown in the formula (I).

FIG. 5: a DSC data pattern of a crystal form A of the compound shown as the formula (I).

FIG. 6: TGA data pattern of the A crystal form of the compound shown as the formula (I).

Detailed Description

The present invention will be explained in more detail with reference to examples or experimental examples, which are only used to illustrate the technical solution of the present invention and are not intended to limit the spirit and scope of the present invention.

The reagents used in the present invention are commercially available.

The test conditions of the apparatus used in the experiment of the present invention were as follows:

1. differential Scanning Calorimeter (DSC)

The instrument model is as follows: mettler Toledo DSC 3+ STARe System

And (3) purging gas: nitrogen gas; nitrogen purging speed: 50mL/min

The heating rate is as follows: 10.0 ℃/min

Temperature range: 25-350 deg.C

2. X-ray Powder Diffraction Spectroscopy (XRPD)

The instrument model is as follows: BRUKER D8 Discover X-ray powder diffractometer

Ray: monochromatic Cu-Ka rays (λ = 1.5406)

The scanning mode is as follows: θ/2 θ, scan range (2 θ range): 3 to 50 DEG

Voltage: 40kV, current: 40mA

3. Thermogravimetric Analyzer (TGA)

The instrument model is as follows: mettler Toledo TGA2

And (3) purging gas: nitrogen gas; nitrogen purging speed: 50mL/min

The heating rate is as follows: 10.0 ℃/min

Temperature range: 30-400 deg.C

4. DVS for dynamic moisture adsorption

The detection adopts Surface Measurement Systems adaptation 2, the humidity is increased from 50-95-0-95-50% RH at 25 ℃, the stepping is 10%, the judgment standard is that the dM/dT of each gradient mass change is less than 0.002%, TMAX is 360min, and the cycle is two circles.

5. The average kinase inhibition and IC50 values were determined using a NovoStar microplate reader (BMG, germany).

6. The monitoring of the progress of the reaction in the examples employed Thin Layer Chromatography (TLC), a developing solvent used for the reaction, a system of eluents for column chromatography used for purifying compounds and a developing solvent system for thin layer chromatography including: a: dichloromethane/methanol system, B: n-hexane/ethyl acetate system. The thin layer chromatography silica gel plate adopts HSGF254 of tobacco yellow sea or GF254 of Qingdao, the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.15 mm-0.2 mm, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm. Silica gel column chromatography generally uses 200-300 mesh silica gel of the Litsea crassirhizomes as a carrier.

7. The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or/and Mass Spectrometry (MS). NMR shifts (. Delta.) are given in units of 10-6 (ppm).

NMR was measured using Bruker AVANCE NEO 500M, deuterated dimethyl sulfoxide (DMSO-d 6), deuterated chloroform (CDCl 3), deuterated methanol (CD 3 OD) and internal standard Tetramethylsilane (TMS).

MS was measured using an Agilent 1200/1290 DAD-6110/6120 Quadrupole MS LC MS (manufacturer: agilent, MS model: 6110/6120 Quadrupole MS). waters ACQuity UPLC-QD/SQD (manufacturer: waters, MS model: waters ACQuity Qda Detector/waters SQ Detector). THERMO Ultimate 3000-Q active (manufacturer: THERMO, MS model: THERMO Q active)

8. Known starting materials of the present invention may be synthesized by or according to methods known in the art, or may be purchased from companies such as ABCR GmbH & Co.KG, acros Organics, aldrich Chemical Company, shao Yuan Chemical technology (Accela ChemBio Inc), darri Chemicals, and the like.

9. HPLC measurements were performed using Agilent 1260DAD high performance liquid chromatography (ACE Excel C18X 4.6mm column) and Thermo Dionex Ultimate 3000 high pressure liquid chromatography (Waters Xbridge C18X 4.6mm column).

Example 1: preparation and Activity test of Compounds of formula (I)

First step of

2- ((2,3-dihydro-1H-inden-2-yl) amino) -6,7-dihydro-5H-cyclopenta [ d ] pyrimidine-6-carboxylic acid Ib

Methyl 3- ((dimethylamino) methylidene) -4-oxocyclopentane-1-carboxylate Ia (3.7 g,18.76mmol, prepared by the method disclosed in patent application "intermediate 63 on page 96 of the specification in WO 2013028474 Al) and 1- (2,3-dihydro-1H-inden-2-yl) guanidine hydrochloride (4.77g, 22.51mmol, prepared by the method disclosed in patent application" intermediate 1 on page 6 of the specification in US20140200231 A1) were dissolved in anhydrous methanol (50 mL), sodium methoxide (2.46g, 45.5mmol, adamas) was added and stirred at 80 ℃ for 16 hours. Quenching was performed by adding water (10 mL), followed by adjusting pH to 3-4 with 1N diluted hydrochloric acid, followed by extraction with N-butanol, drying, concentration under reduced pressure, and purification of the residue by silica gel column chromatography with eluent system B to obtain the title product Ib (3.3 g, yield: 59.6%). MS m/z (ESI): 296.0[ M ] +1].

Second step of

3- (2- (2- ((2,3-dihydro-1H-inden-2-yl) amino) -6,7-dihydro-5H-cyclopenta [ d ] pyrimidine-6-carbonyl) hydrazino) -3-oxopropanoic acid ethyl ester Ic

Compound Ib (170mg, 0.58mmol) and the compound ethyl 3-hydrazino-3-oxopropionate (371mg, 0.87mmol) were dissolved in N, N-dimethylformamide (10 mL), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (362mg, 0.70mmol, shaoshao) and N, N-diisopropylethylamine (225mg, 1.74mmol, adamas) were added and the reaction stirred at room temperature for 3 hours. Water was added thereto for quenching, followed by extraction with ethyl acetate, drying, concentration under reduced pressure and purification of the resulting residue by silica gel column chromatography with eluent system B to give the title product Ic (120 mg, yield: 48.9%). MS m/z (ESI): 424.0[ M ] +1].

The third step

Ethyl 2- (5- (2- ((2,3-dihydro-1H-inden-2-yl) amino) -6,7-dihydro-5H-cyclopenta [ d ] pyrimidin-6-yl) -1,3,4-oxadiazol-2-yl) acetate Id

Compound Ic (120mg, 0.28mmol) was dissolved in tetrahydrofuran (5 mL), bougies' reagent (267mg, 1.12mmol, adamas) was added, and the mixture was stirred at 80 ℃ for 4 hours while closing the tube. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography with eluent system B to give the title product Id (56 mg, yield: 49.3%). MS m/z (ESI): 406.0[ M ] +1].

The fourth step

2- (5- (2- ((2,3-dihydro-1H-inden-2-yl) amino) -6,7-dihydro-5H-cyclopenta [ d ] pyrimidin-6-yl) -1,3,4-oxadiazol-2-yl) acetic acid Ie

Compound Id (56mg, 0.14mmol) was dissolved in THF (4 mL) and water (1.0 mL), and lithium hydroxide monohydrate (23.5mg, 0.56mmol, guyao) was added at 0 ℃ to stir for 1 hour. The organic solvent was removed by concentration under reduced pressure, the pH was adjusted to 3 to 4 with 1N dilute hydrochloric acid, and a solid was precipitated, which was filtered and dried to obtain the title product Ie (50 mg, yield: 94.6%). MS m/z (ESI): 378.0[ M ] +1].

The fifth step

2- (5- (2- ((2,3-dihydro-1H-inden-2-yl) amino) -6,7-dihydro-5H-cyclopenta [ d ] pyrimidin-6-yl) -1,3,4-oxadiazol-2-yl) -1- (3,4,6,7-tetrahydro-5H- [1,2,3] triazolo [4,5-c ] pyridin-5-yl) ethan-1-one If

Compound Ie (120mg, 0.32mmol) and 4,5,6,7-tetrahydro-3H- [1,2,3]Triazolo [4,5-c]Pyridine hydrochloride (67mg, 0.42mmol, prepared by the method disclosed in the patent application "WO 2018212534A1, intermediate im-7 on page 53"), was dissolved in N, N-dimethylformamide (5.0 mL), N-diisopropylethylamine (124mg, 0.96mmol, adamas) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (249mg, 0.48mmol, adamas) were added and the reaction stirred for 1 hour. Water (10 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (10 mL), concentration under reduced pressure, and purification of the residue by high performance liquid chromatography (Sharpsil-T C18 Column 21.2 x 150mm 5um, elution system: water (10 mmoL/L ammonium acetate), acetonitrile) gave the title product If (54 mg, yield: 35.1%). MS m/z (ESI): 484.0[ M ] +1]。 ¹ H NMR(400MHz,CD ₃ OD)δ8.15(s,1H),7.23-7.13(m,4H),4.85-4.69(m,3H),4.05-3.92(m,3H),3.39-3.36(m,4H),3.33-3.14(m,4H),2.98-2.85(m,4H)。

The sixth step

(R) -2- (5- (2- ((2,3-dihydro-1H-inden-2-yl) amino) -6,7-dihydro-5H-cyclopenta [ d ] pyrimidin-6-yl) -1,3,4-oxadiazol-2-yl) -1- (3,4,6,7-tetrahydro-5H- [1,2,3] triazolo [4,5-c ] pyridin-5-yl) ethan-1-one I

Compound If (54mg, 0.11mmol) was subjected to chiral preparation (separation conditions: chiral preparative column CHIRALPAK OJ,5.0cm I.D.. Times.25cm, 10 μm; mobile phase: methanol (100%), flow rate: 50 mL/min), and the corresponding fractions were collected and concentrated under reduced pressure to give (S), (R) configuration compound (12mg, 13mg), respectively.

Single configuration compound (shorter retention time):

MS m/z(ESI)：484.0[M+1]。

chiral HPLC analysis: retention time 7.04 min, chiral purity: 100% (column: CHIRALPAK OJ-H,0.46cm I.D. times 15cm; mobile phase: methanol (100%)). ¹ H NMR(400MHz,CD ₃ OD)δ8.15(s,1H),7.23-7.13(m,4H),4.85-4.69(m,3H),4.05-3.92(m,3H),3.39-3.36(m,4H),3.33-3.14(m,4H),2.98-2.85(m,4H)。

Single configuration compound (longer retention time):

MS m/z(ESI)：484.0[M+1]。

chiral HPLC analysis: retention time 9.84 min, chiral purity: 99.4% (column: CHIRALPAK OJ-H,0.46cm I.D. times 15cm; mobile phase: methanol (100%)). ¹ H NMR(400MHz,CD ₃ OD)δ8.15(s,1H),7.23-7.13(m,4H),4.85-4.69(m,3H),4.05-3.92(m,3H),3.39-3.36(m,4H),3.33-3.14(m,4H),2.98-2.85(m,4H)。

Wherein the single configuration compound corresponding to the longer retention time (9.84 minutes) is the compound shown in the formula (I) of the disclosure.

Test example 1 enzymatic test of the Compound of formula (I) of this disclosure

ATX (autotaxin) catalyzes a substrate Lysophosphatidylcholine (LPC) to generate choline, the choline is oxidized by choline oxidase to generate betaine and hydrogen peroxide, and the peroxidase catalyzes a substrate 2-hydroxy-3-m-toluidine sodium propanesulfonate (TOOS) and 4-aminoantipyrine to react and develop color in the presence of the hydrogen peroxide and has absorption at 555 nm. The measured light absorption value is positively correlated with the amount of choline released by the first step of enzyme-catalyzed reaction, thereby reflecting the inhibition effect of the compound on the activity of ATX enzyme.

1) Purpose of experiment

The compound shown in the formula (I) is screened in vitro by utilizing the characteristic that the compound can inhibit the activity of ATX enzyme.

2) Experimental methods

And (3) buffer solution A:50mM Tris-HCl pH8.5 (Beijing Tiannzze organism, # 101207-250), 500mM NaCl (gumbo, # 10019318), 5mM KCl (gumbo, # 10016318), 10mM Ca chloride (gumbo, # 10005861) and 0.1% bovine serum albumin (Sigma, # B2064).

And (3) buffer solution B:50mM Tris-HCl pH8.5, 500mM NaCl, 5mM KCl, 10mM calcium chloride, 0.1% BSA and 20mM EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid, sigma, # E3889).

The compound of formula (I) was prepared in dimethylsulfoxide (Sigma, # D2650) at an initial concentration of 500. Mu.M, diluted 7-fold, and dosed at 8 doses. ATX (R & D, # 5255-EN) was formulated with buffer A to a final concentration of 0.5 ng/. Mu.l, and LPC 16 (Sigma, # 855675P) was formulated to a final concentration of 150uM. Mu.l ATX, 1ul compound and 30. Mu.l LPC were added to a 96-well plate (Corning, # 3799) in this order and incubated at 37 ℃ for 3 hours.

A test solution containing 0.6U/ml choline oxidase (Sigma, # C5896), 0.6U/ml peroxidase (Sigma, # P8375), 1.8mM TOOS (Sigma, # 04340) and 1.2mM 4-aminoantipyrine (Sigma, # A4382) was prepared in buffer B. The detection solution was added at 50. Mu.l/well to a 96-well plate after 3-hour incubation, and after shaking at room temperature for 15 minutes, the OD value at 555nm was read with a microplate reader (Molecular Devices, flexstation 3).

3) Test results

IC for inhibiting ATX enzymatic Activity by Compounds of formula (I) ₅₀ The value was 1.4nM and the maximum inhibition was 97%.

Test example 2 Effect of the Compounds of formula (I) of the present disclosure on IL-6 secretion induced by TGF-. Beta.transforming growth factor beta

1) Purpose of experiment

The compounds of formula (I) of the present disclosure were tested for their inhibitory effect on the secretion of IL-6 (interleukin 6) by TGF-beta (transforming growth factor beta) induced human skin fibroblasts.

2) Experimental methods

Primary human skin fibroblasts (NHDF, promocell, # C-12303) were plated into 96-well plates (Corning, # 3799) at 37 ℃ with 5% CO after resuspension to 8000 cells/well in FGM Medium2 (Fibroplast Growth Medium, promocell, # C-23020) ₂ Cultured in an incubator (thermo scientific, # STERI-CYCLEI 160) for 48 hours. Recombinant human TGF-. Beta.was prepared at 10ng/ml using FGM Medium (fiber Growth Medium2, # Promocell, # C-23020). The compound of formula (I) to be tested was formulated with FGM medium at an initial concentration of 100. Mu.M, 10-fold diluted, and 8 doses total. Removing the culture medium from the cell plate, adding 80. Mu.l of fresh FGM culture medium and 10. Mu.l of test compound solution at different concentrations, respectively, and subjecting to 37 deg.C, 5% CO ₂ Incubate for 1.5 hours in the incubator. Adding 10ul of TGF-beta solution, standing at 37 deg.C, 5% ₂ The incubation was continued in the incubator. Cell supernatants were harvested after 24 hours, assayed for IL-6 content and IC calculated by ELISA (Xinbo Cheng Shengwu, # EHC 007.96) ₅₀ The value is obtained.

3) Data analysis

The IC of the compound shown in the formula (I) in the disclosure for inducing IL-6 secretion of human skin fibroblasts by TGF-beta ₅₀ The value was 1.2nM and the maximum inhibition was 103%.

Test example 3 in vitro human plasma LPA inhibition of Compounds of formula (I) of the present disclosure

1) Purpose of experiment

Compounds of formula (I) of the present disclosure were tested for their inhibitory effect on LPA 18 levels in healthy human plasma by inhibiting ATX enzyme activity.

2) Experimental methods

Blood from healthy volunteers was collected into heparin blood collection tubes (BD, # 367886), centrifuged at 3000rpm for 15 minutes at 4 ℃ and the supernatant was collected. Plasma was dispensed into 96-well plates (Corning, # 3788) at 99. Mu.l/well. The compound of formula (I) was formulated with dimethyl sulfoxide (Sigma, # D2650) at an initial concentration of 100. Mu.M, 10-fold dilution, and 7 doses. Mu.l of each was added to a plasma plate and incubated at 37 ℃ for 2 hours. Plasma LPA 18 content was detected with Xevo TQ-S triple quadrupole tandem mass spectrometer and ACQUITY UPLC ultra performance liquid chromatography system (Waters). Relative amounts were evaluated based on peak areas of LPA 18.

3) Test results

The inhibitory effect IC of the compounds of formula (I) of the present disclosure on LPA 18 levels in healthy human plasma ₅₀ The value was 1.4nM and the maximum inhibition was 92%.

Test example 4 blocking Effect of the Compound of formula (I) of this disclosure on hERG Potassium Current

1) Purpose of experiment

The blocking effect of the compound shown in formula (I) on the hERG potassium current was tested on a stable cell line transfected with the hERG potassium channel by using a fully automatic patch clamp.

2) Experimental methods

2.1 Experimental materials and instruments

Experimental materials:

an experimental instrument:

2.2 Full-automatic patch clamp experimental procedure

HEK293-hERG stable cell lines (constructed internally according to known techniques) were subcultured according to 1:4 density in DMEM/HIGH glucose medium (10% FBS, 1.5. Mu.g/ml puromycin dihydrochloride) and fully automated patch clamp experiments were performed within 48-72 hours of culture. On the day of the experiment, cells were digested with 0.25% trypsin, collected by centrifugation, and resuspended in extracellular fluid to prepare a cell suspension. The cell suspension was placed on the cell bank of the Patchliner instrument, which applied the cells to the chip (NPC-16) using a negative pressure controller, which draws individual cells to the wells of the chip. After the whole cell mode is formed, the apparatus will obtain hERG current according to the set hERG current voltage program, and then the apparatus automatically carries out compound perfusion from low concentration to high concentration. The current at each concentration of compound and the blank control current were analyzed by data analysis software provided by the peak Patchmaster, peak EPC10 patch clamp amplifier (Nanion) and pathlarsoft ware and Pathcontrol HT software.

2.3 Results of the test)

The blocking effect of the compounds of formula (I) on hERG potassium current was determined by the above assay, and the measured IC ₅₀ Has a value of>30μM。

And (4) conclusion: the compounds of formula (I) of the present disclosure have weak hERG inhibitory effects and can reduce side effects caused by the hERG pathway.

Example 2: amorphous preparation of a Compound of formula (I)

About 10mg of the compound represented by the formula (I) was weighed, 1.0mL of water was added, and after stirring at room temperature for two days, centrifugation and vacuum drying were carried out to obtain a solid. The product was amorphous as detected by X-ray powder diffraction, and the XRPD pattern is shown in figure 1.

Example 3: amorphous preparation of a Compound of formula (I)

About 10mg of the compound represented by the formula (I) was weighed, and added to 0.05mL of an acetonitrile/methanol (1, v/v) mixed solvent, followed by evaporation and crystallization. The product was amorphous as detected by X-ray powder diffraction.

Example 4: preparation of crystal form A of compound shown as formula (I)

About 10mg of the compound represented by the formula (I) was weighed, 1.0mL of methanol was added, and the mixture was stirred at room temperature for crystallization and dried in vacuo to obtain a solid. The product is of A crystal form through X-ray powder diffraction detection, an XRPD spectrogram is shown in figure 2, and the characteristic peak positions are shown in table 1. The DSC spectrum shows an endothermic peak of 201.61 ℃. The TGA spectrum shows that the weight loss is 0.93 percent at the temperature of 30-240 ℃.

TABLE 1

Example 5: preparation of crystal form A of compound shown as formula (I)

About 10mg of the compound represented by the formula (I) was weighed, added to 1.0mL of ethanol, stirred at room temperature for crystallization, centrifuged, and vacuum-dried to obtain a solid. The product is of A crystal form through X-ray powder diffraction detection.

Example 6: preparation of crystal form A of compound shown as formula (I)

About 10mg of the compound represented by the formula (I) was weighed, added to 1.0mL of isopropyl alcohol, stirred at room temperature for crystallization, centrifuged, and dried in vacuum to obtain a solid. The product is of A crystal form through X-ray powder diffraction detection.

Example 7: preparation of crystal form A of compound shown as formula (I)

About 10mg of the compound represented by the formula (I) was weighed, 0.2mL of acetone was added, and the mixture was stirred at room temperature for crystallization, centrifuged, and dried in vacuo to obtain a solid. The product is of A crystal form through X-ray powder diffraction detection.

Example 8: preparation of crystal form A of compound shown as formula (I)

About 10mg of the compound represented by the formula (I) was weighed, 0.15mL2-butanone was added, and the mixture was stirred at room temperature for crystallization, centrifuged, and dried in vacuo to obtain a solid. The product is of A crystal form through X-ray powder diffraction detection.

Example 9: preparation of crystal form A of compound shown as formula (I)

About 10mg of the compound represented by the formula (I) was weighed, 1.0mL of acetonitrile was added, and the mixture was stirred at room temperature for a while, stirred at 5 ℃ overnight for crystallization, centrifuged, and dried in vacuo to obtain a solid. The product is of A crystal form through X-ray powder diffraction detection.

Example 10: preparation of crystal form A of compound shown as formula (I)

About 10mg of the compound represented by the formula (I) was weighed, and 1.0mL of ethyl acetate was added thereto, followed by stirring at room temperature for crystallization, centrifugation and vacuum drying to obtain a solid. The product is of A crystal form through X-ray powder diffraction detection.

Example 11: preparation of crystal form A of compound shown as formula (I)

About 10mg of the compound represented by the formula (I) was weighed, added with 1.0mL of isopropyl acetate, stirred at room temperature for two days, centrifuged, and vacuum-dried to obtain a solid. The product is of A crystal form through X-ray powder diffraction detection.

Example 12: preparation of crystal form A of compound shown as formula (I)

About 10mg of the compound represented by the formula (I) was weighed, 0.05mL of dichloromethane was added to dissolve it, and a white solid was precipitated by stirring, centrifuged, and dried in vacuo to obtain a solid. The product is of A crystal form through X-ray powder diffraction detection.

Example 13: preparation of crystal form A of compound shown as formula (I)

About 10mg of the compound represented by the formula (I) was weighed, 0.05mL of dioxane was added, and crystallization was performed by volatilization to obtain a solid. The product is of A crystal form through X-ray powder diffraction detection.

Example 14: preparation of crystal form A of compound shown as formula (I)

About 10mg of the compound represented by the formula (I) was weighed, and 1.0mL of n-heptane was added thereto, followed by stirring at room temperature for two days, centrifugation and vacuum drying to obtain a solid. The product is of A crystal form through X-ray powder diffraction detection.

Example 15: preparation of crystal form A of compound shown as formula (I)

About 10mg of the compound represented by the formula (I) was weighed, 1.0mL of toluene was added, and after stirring at room temperature for two days, the mixture was centrifuged and vacuum-dried to obtain a solid. The product is of A crystal form through X-ray powder diffraction detection.

Example 16: preparation of crystal form A of compound shown as formula (I)

About 30mg of the compound represented by the formula (I) was weighed, 0.6mL of acetone was added to the solution, 6.0mL of cyclohexane was added to the solution to precipitate a solid, and the solid was centrifuged and dried in vacuo. The product is of A crystal form through X-ray powder diffraction detection.

Example 17: preparation of crystal form A of compound shown as formula (I)

About 20mg of the compound represented by the formula (I) was weighed, dissolved in 0.5mL of methylene chloride, and added with 5.0mL of water to precipitate a solid, which was centrifuged and dried in vacuo to obtain a solid. The product is of A crystal form through X-ray powder diffraction detection.

Example 18: preparation of crystal form A of compound shown as formula (I)

About 15mg of the compound represented by the formula (I) was weighed, and after 0.4mL of tetrahydrofuran was added to dissolve it, 4.0mL of ethyl acetate was added to precipitate a solid, which was centrifuged and dried in vacuo to obtain a solid. The product is of A crystal form through X-ray powder diffraction detection.

Example 19: hygroscopicity study of form a

Adopting Surface Measurement Systems intrinsic DVS, observing the humidity range of 0-95% at 25 ℃, stepping 10%, judging the standard that the dM/dT of each gradient mass change is less than 0.002%, TMAX 360min, and circulating for two circles.

TABLE 2

The free form of form A sample has a slightly hygroscopic property at 25 ℃ in a range of 0% RH to 80.0% RH, a water absorption amount increasing with an increase in humidity, a weight change of 0.27%, and a hygroscopic weight gain of less than 2% but not less than 0.2%. Water absorption of about 0.19% under normal storage conditions (i.e., 60% humidity at 25 ℃); under accelerated test conditions (i.e., 70% humidity), the water absorption was about 0.22%; under extreme conditions (i.e., 90% humidity), the water uptake is about 0.35%.

In the process of 0-95% humidity change, the adsorption and desorption curves are basically coincided; x-ray powder diffraction contrast graphs before and after DVS show that crystal forms before and after DVS are consistent.

Example 20: stability study of influencing factors of crystal form A

The free crystal form A is placed in an open and flat way, the stability of the sample is respectively inspected under the conditions of illumination (4500 Lux), high temperature (40 ℃, 60 ℃) and high humidity (RH 75 percent and RH 92.5 percent), and the sampling inspection period is 30 days.

TABLE 3

Influence factor stability experimental results show that: 40 ℃, 60 ℃, 75% RH, 93% RH and light conditions of 30 days, good physical and chemical stability.

Experimental example 21: crystal form a long term/accelerated stability study

Stability of form A was investigated by placing it in the conditions of 25 ℃/60% RH and 40 ℃/75% RH

TABLE 4

Long term/accelerated stability experiments show that: form A has good physical and chemical stability after being left for 6 months at 25 ℃/60% RH and 40 ℃/75% RH.

Claims

1. A crystal form A of a compound shown as a formula (I),

characterized in that the X-ray powder diffraction pattern expressed by the angle of diffraction 2 theta has characteristic peaks at 12.777, 18.719, 22.502, 25.747 and 26.517; preferably, there are characteristic peaks at 6.355, 12.777, 18.719, 19.232, 22.502, 25.003, 25.747, 26.517 and 27.735; preferably, there are characteristic peaks at 6.355, 12.777, 14.274, 16.915, 18.719, 19.232, 22.502, 23.401, 23.777, 25.003, 25.747, 26.517 and 27.735; preferably characteristic peaks at 6.355, 12.777, 14.274, 15.987, 16.915, 18.719, 19.232, 22.502, 23.401, 23.777, 25.003, 25.747, 26.517, 27.735, and 29.175; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2 θ is shown in FIG. 2.

2. The crystalline form a of the compound of formula (I) as claimed in claim 1, wherein the 2 Θ angle error range is ± 0.2.

3. A process for the preparation of the compound of formula (I) in the form of form a according to claim 1, characterized in that it comprises the steps of: mixing a compound shown in a formula (I) with a solvent III, and stirring at room temperature to crystallize, wherein the solvent III is preferably one or more of an alcohol solvent, a ketone solvent, an ester solvent, a halogenated alkane solvent, an alkane solvent and an aromatic hydrocarbon solvent; the alcohol solvent is preferably selected from methanol, ethanol and isopropanol; the ketone solvent is preferably selected from acetone and 2-butanone; the ester solvent is preferably selected from ethyl acetate and isopropyl acetate; the haloalkane solvent is preferably selected from dichloromethane; the alkane solvent is preferably selected from n-heptane; the aromatic hydrocarbon solvent is preferably selected from toluene.

4. A process for the preparation of the compound of formula (I) in the form of form a according to claim 1, characterized in that it comprises the steps of: mixing the compound shown in the formula (I) with a solvent IV, and cooling for crystallization, wherein the solvent IV is preferably acetonitrile.

5. A process for the preparation of the compound of formula (I) in the form of form a according to claim 1, characterized in that it comprises the steps of: mixing the compound shown in the formula (I) with a solvent V, and volatilizing for crystallization, wherein the solvent V is preferably dioxane.

6. A process for the preparation of the compound of formula (I) in the form of form a according to claim 1, characterized in that it comprises the steps of: mixing a compound shown in a formula (I) with a solvent VI, dissolving, adding a solvent VII, and crystallizing to separate out, wherein the solvent VI is preferably one or more of acetone, tetrahydrofuran and dichloromethane; the solvent VII is preferably one or more selected from cyclohexane, water and ethyl acetate.

7. A pharmaceutical composition prepared from the compound of formula (I) as described in claim 1 in crystal form a.

8. A pharmaceutical composition comprising the form a of the compound of formula (I) as claimed in claim 1 and optionally a pharmaceutically acceptable carrier, diluent or excipient.

9. A process for the preparation of a pharmaceutical composition comprising the step of mixing form a of the compound of formula (I) as claimed in claim 1 with a pharmaceutically acceptable carrier, diluent or excipient.

10. Use of the crystalline form a of a compound of formula (I) as described in claim 1, or the composition as described in claim 7 or 8, or prepared by the process of claim 9, for the preparation of a medicament for inhibiting ATX (autotaxin).

11. Use according to claim 10, said ATX-related disease being a fibrotic disease, a cancer, a proliferative disease, an inflammatory disease, an autoimmune disease, a respiratory disease, a cardiovascular disease, a neurodegenerative disease, a dermatological disease, a metabolic disease, a myelodysplastic syndrome, an aberrant angiogenesis-related disease or pain, preferably a fibrotic disease, a cancer, preferably pulmonary fibrosis, idiopathic pulmonary fibrosis, hepatic fibrosis, scleroderma, renal cancer or pancreatic cancer.