Crystal form of enpp1 inhibitor

Abstract

The invention provides a compound in a crystal form as shown in a formula (I), a specific crystal form of the compound, a pharmaceutical composition containing the compound and application of the compound. The compound in the crystal form as shown in the formula (I) and the specific crystal form of the compound have good crystallinity and chemical purity, are easy and convenient to prepare and high in yield and have good patent medicine potential. # imgabs0 #

Description

A crystal form of an ENPP1 inhibitor

[0001] This application claims priority to a prior application filed with the State Intellectual Property Office of China on June 15, 2023, with patent application number 202310706966.6, entitled “A Crystalline Form of an ENPP1 Inhibitor.” The entire text of the aforementioned application is incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of medicinal chemistry, discloses a crystal form of an ENPP1 inhibitor, and also includes application of the crystal form in preparing drugs for treating ENPP1-related diseases. Background Art

[0003] ENPP1 is a type II transmembrane glycoprotein with nucleotide pyrophosphatase and phosphodiesterase activities. It belongs to the extracellular nucleotide pyrophosphatase / phosphodiesterase (Enpp) family, which consists of seven functionally distinct proteins (1-7). The ENPP1 structure comprises two N-terminal SMB domains (SMB1 and SMB2), two linker regions (L1 and L2), a catalytic domain, and a nuclease-like domain. ENPP1 is differentially expressed in immune cells, with low levels in NK cells, DCs, and macrophages, and high levels in neutrophils. ENPP1 is also expressed in a small subset of B cells, which may be involved in regulating T cell activity. ENPP1 expression is elevated in M2 subtype macrophages, which play a role in tumor promotion. ENPP1 expression is increased in astrocytic tumors, breast cancer, and head and neck tumors. ENPP1 expression varies significantly among different tumor tissues. ENPP1 is specifically overexpressed in some tumors and may be a driver of tumor immune escape and metastasis.

[0004] ENPP1 plays a crucial role in immune responses to various stimuli through the STING pathway, selectively activating the STING signaling pathway in tumor cells and other cells within the tumor microenvironment, achieving high selectivity. ENPP1 also catalyzes the hydrolysis of ATP into PPi and AMP, promoting the production of adenosine, a potent immunosuppressive agent. Inhibiting ENPP1 can alleviate tumor immunosuppression by inhibiting ATP hydrolysis and reducing adenosine production.

[0005] While STING agonists indiscriminately activate STING in both cancer cells and host cells, ENPP1 restricts STING activation to tumor tissue and the tumor microenvironment, enhancing anti-tumor immunity. Furthermore, ENPP1 is selectively upregulated in metastatic and chromosomally unstable tumor cells. Systemic administration of ENPP1 inhibitors can interfere with the ability of disseminated tumor cells to evade immune surveillance, circumventing the technical difficulties of intratumoral administration of STING agonists.

[0006] ENPP1 inhibitors combined with radiotherapy have achieved good results in animal models and have also shown synergistic effects when combined with various anti-tumor drugs, including immune checkpoint inhibitors such as PD-1 and PARP inhibitors. Currently, the development of ENPP1 inhibitors is in the preclinical stage, but several pharmaceutical companies have published patents related to ENPP1 inhibitors, such as WO2021061803A1, WO2021158829A1, WO2020190912A1, WO2019177971A1, and WO2019046778A1.

[0007] Although several small molecule ENPP1 inhibitors have been disclosed in the prior art, no small molecule drugs are currently available for clinical use. Therefore, the development of new compounds with potential for market adoption and improved efficacy and pharmacokinetic properties remains urgent. This paper designs a series of compounds with novel structures represented by the general formula and finds that these compounds exhibit excellent efficacy and effects, which has positive implications for the development of ENPP1 inhibitors.

[0008] Summary of the Invention

[0009] In a first aspect, the present invention provides a compound of formula (I) in crystalline form,

[0010] In a preferred aspect of the present invention, the compound represented by formula (I) in the crystalline form is Form A, and its X-ray powder diffraction pattern using Cu-Kα radiation has diffraction peaks (±0.2°) at the following 2θ angles: 24.9, 25.1, 25.7.

[0011] In some embodiments of the present invention, the crystalline form A of the compound of formula (I) above, using Cu-Kα radiation, has an X-ray powder diffraction pattern having diffraction peaks (±0.2°) at the following 2θ angles: 12.8, 14.0, 16.0, 18.5, 24.9, 25.1, 25.7.

[0012] In some embodiments of the present invention, the crystalline form A of the compound of formula (I) above, using Cu-Kα radiation, has an X-ray powder diffraction pattern having diffraction peaks (±0.2°) at the following 2θ angles: 12.8, 14.0, 16.0, 18.5, 24.9, 25.1, 25.7, 26.9, 28.5.

[0013] In some embodiments of the present invention, the crystalline form A of the compound of formula (I) above, using Cu-Kα radiation, has an X-ray powder diffraction pattern having diffraction peaks (±0.2°) at the following 2θ angles: 12.8, 14.0, 16.0, 18.5, 24.9, 25.1, 25.7, 26.9, 28.5, 29.8.

[0014] In some embodiments of the present invention, the crystalline form A of the compound of formula (I) above, using Cu-Kα radiation, has an X-ray powder diffraction pattern having diffraction peaks (±0.2°) at the following 2θ angles: 12.8, 14.0, 16.0, 17.0, 18.5, 20.5, 24.9, 25.1, 25.7, 26.9, 28.5, 29.8.

[0015] In some embodiments of the present invention, the XRPD pattern of Form A of the compound of formula (I) above using Cu-Kα radiation is shown in FIG1 or FIG7 .

[0016] In some embodiments of the present invention, the XRPD pattern analysis of Form A of the compound of formula (I) using Cu-Kα radiation is shown in Table 1 or Table 2.

[0017] In some embodiments of the present invention, the crystal form A of the compound of formula (I) has an endothermic peak at 233.23±4° C. in its differential scanning calorimetry curve.

[0018] In some embodiments of the present invention, the crystal form A of the compound of formula (I) has an exothermic peak at 234.3±4°C in its differential scanning calorimetry curve.

[0019] In some embodiments of the present invention, the crystal form A of the compound of formula (I) has a differential scanning calorimetry (DSC) spectrum substantially as shown in FIG2 .

[0020] In some embodiments of the present invention, the thermogravimetric analysis curve of the crystal form A of the compound of formula (I) above shows a weight loss of 13.50±1% between about 105°C and 254°C, for example, a weight loss of 13.5111%.

[0021] In some embodiments of the present invention, the thermogravimetric analysis curve of the crystal form A of the compound of formula (I) above shows a weight loss of 13.50±1% at 254.00±3°C, for example, a weight loss of 13.5111%.

[0022] In some embodiments of the present invention, the crystal form A of the compound of formula (I) has a thermogravimetric analysis (TGA) spectrum substantially as shown in FIG3 .

[0023] The present invention also provides a crystalline form B of the compound of formula (I), which has an X-ray powder diffraction pattern using Cu-Kα radiation and has diffraction peaks (±0.2°) at the following 2θ angles: 19.19, 27.18, 13.44, 22.55.

[0024] In some embodiments of the present invention, the crystalline form B of the compound of formula (I) above, using Cu-Kα radiation, has an X-ray powder diffraction pattern having diffraction peaks (±0.2°) at the following 2θ angles: 26.19, 20.76, 4.42, 22.84, 19.19, 27.18, 13.44, 22.55.

[0025] In some embodiments of the present invention, the XRPD pattern of Form B of the compound of formula (I) above using Cu-Kα radiation is shown in FIG4 .

[0026] In some embodiments of the present invention, the XRPD pattern analysis of Form B of the compound of formula (I) using Cu-Kα radiation is shown in Table 3.

[0027] In some embodiments of the present invention, the crystalline form B of the compound of formula (I) is a DMF solvate of the compound of formula (I).

[0028] In some embodiments of the present invention, in the crystalline form B of the compound of formula (I), the ratio of the compound of formula (I) to the DMF solvent is 1:1.

[0029] The present invention also provides a crystalline form C of the compound of formula (I), which has an X-ray powder diffraction pattern using Cu-Kα radiation and has diffraction peaks (±0.2°) at the following 2θ angles: 22.11, 22.55, 20.96, 26.63.

[0030] In some embodiments of the present invention, the crystalline form C of the compound of formula (I) above, using Cu-Kα radiation, has an X-ray powder diffraction pattern having diffraction peaks (±0.2°) at the following 2θ angles: 22.11, 22.55, 20.96, 26.63, 15.01, 25.68, 26.38, 28.54, 13.22, 18.68.

[0031] In some embodiments of the present invention, the XRPD pattern of Form C of the compound of formula (I) above using Cu-Kα radiation is shown in FIG5 .

[0032] In some embodiments of the present invention, the XRPD pattern analysis of Form C of the compound of formula (I) using Cu-Kα radiation is shown in Table 4.

[0033] In some embodiments of the present invention, the crystalline form C of the compound of formula (I) is a DMSO solvate of the compound of formula (I).

[0034] The present invention also provides a crystalline form D of the compound of formula (I), which has an X-ray powder diffraction pattern using Cu-Kα radiation having diffraction peaks (±0.2°) at the following 2θ angles: 22.71, 18.92, 22.96, 26.4, 24.44, 16.62, 13.15, 17.79, 27.12.

[0035] In some embodiments of the present invention, the crystalline form D of the compound of formula (I) above, using Cu-Kα radiation, has an X-ray powder diffraction pattern having diffraction peaks (±0.2°) at the following 2θ angles: 22.71, 18.92, 22.96, 26.4, 24.44, 16.62, 13.15, 17.79, 27.12, 24.17, 28.81, 19.95, 28.42, 24.85, 14.78, 17.36, 29.16.

[0036] In some embodiments of the present invention, the XRPD pattern of Form D of the compound of formula (I) above using Cu-Kα radiation is shown in FIG6 .

[0037] In some embodiments of the present invention, the XRPD pattern analysis of Form D of the compound of formula (I) above using Cu-Kα radiation is shown in Table 5.

[0038] In some embodiments of the present invention, the crystalline form D of the compound of formula (I) is an NMP solvate of the compound of formula (I).

[0039] In some embodiments of the present invention, in the crystalline form D of the compound of formula (I), the ratio of the compound of formula (I) to the NMP solvent is 1:1.

[0040] In a second aspect, the present application provides a method for preparing the crystalline form of the compound of formula (I) as described in the first aspect, comprising method 1:

[0041] The compound of formula I is added to organic solvent A and dissolved, and the solution is then added to organic solvent B to precipitate crystals.

[0042] The organic solvent A is selected from one or more of DMF (N,N-dimethylformamide), DMSO (dimethyl sulfoxide), and NMP (N-methylpyrrolidone); the organic solvent B is selected from one or more of water, methanol, ethanol, MTBE, toluene, and dichloromethane.

[0043] In some embodiments of the present invention, when the compound of formula (I) is crystalline form A, in the method 1, the organic solvent A is selected from DMF, and the organic solvent B is selected from one or two of water and ethanol; or the organic solvent A is selected from DMSO, and the organic solvent B is selected from one or two of toluene and methanol.

[0044] In some embodiments of the present invention, when the compound of formula (I) is in crystalline form B, in the method 1, the organic solvent A is selected from DMF, and the organic solvent B is selected from one or both of MTBE and toluene.

[0045] In some embodiments of the present invention, when the compound of formula (I) is in crystalline form D, in the method 1, the organic solvent A is selected from NMP, and the organic solvent B is selected from one or more of water, dichloromethane, and MTBE.

[0046] The method for preparing the crystalline compound of formula (I) comprises method 2:

[0047] The compound of formula I is added to an organic solvent C and dissolved, and then an organic solvent D is added to precipitate crystals.

[0048] The organic solvent C is selected from one or more of DMF, DMSO, and NMP, and the organic solvent D is selected from one or more of acetone, ethylene glycol methyl ether, water, dichloromethane, ethyl acetate, MTBE, toluene, tetrahydrofuran, dioxane, ethanol, trifluoroethanol, acetonitrile, ethylene glycol dimethyl ether, isopropanol, and butanone.

[0049] In some embodiments of the present invention, when the compound of formula (I) is crystalline form A, in the method 1, the organic solvent C is selected from DMF, and the organic solvent D is selected from one or more of acetone, ethylene glycol methyl ether, water, and dichloromethane; or the organic solvent C is selected from DMSO, and the organic solvent D is selected from one or more of ethanol, trifluoroethanol, ethylene glycol methyl ether, acetonitrile, dioxane, ethylene glycol dimethyl ether, and butanone; or the organic solvent C is selected from NMP, and the organic solvent D is selected from one or more of water, acetonitrile, dichloromethane, and ethylene glycol dimethyl ether.

[0050] In some embodiments of the present invention, when the compound of formula (I) is in crystalline form B, in the second method, the organic solvent C is selected from DMF, and the organic solvent D is selected from one or more of ethyl acetate, MTBE, toluene, tetrahydrofuran, dioxane, and butanone.

[0051] In some embodiments of the present invention, when the compound of formula (I) is in crystalline form C, in the second method, the organic solvent C is selected from DMSO, and the organic solvent D is selected from one or more of ethyl acetate, water, and toluene.

[0052] In some embodiments of the present invention, when the compound of formula (I) is in crystalline form D, in the second method, the organic solvent C is selected from NMP, and the organic solvent D is selected from one or more of isopropanol, MTBE, toluene, tetrahydrofuran, and butanone.

[0053] The method for preparing the crystalline compound of formula (I) comprises method three:

[0054] The compound of formula I is added to an organic solvent E for suspension, slurrying, and filtered to obtain crystals.

[0055] The organic solvent E is selected from one or more of methanol, ethanol, isopropanol, ethyl acetate, n-heptane, MTBE, ethylene glycol methyl ether, water, acetonitrile, toluene, dichloromethane, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, butanone, dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, and acetone.

[0056] In some embodiments of the present invention, when the compound of formula (I) is crystalline form A, in the method three, the organic solvent E is selected from one or more of methanol, ethanol, isopropanol, ethyl acetate, n-heptane, MTBE, ethylene glycol methyl ether, water, acetonitrile, toluene, dichloromethane, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, and butanone.

[0057] In some embodiments of the present invention, when the compound of formula (I) is in crystalline form B, in the method three, the organic solvent E is selected from DMF.

[0058] In some embodiments of the present invention, when the compound of formula (I) is in crystalline form C, in the method three, the organic solvent E is selected from DMSO.

[0059] In some embodiments of the present invention, when the compound of formula (I) is in crystalline form D, in the method three, the organic solvent E is selected from NMP.

[0060] In a third aspect, the present application provides a crystalline composition comprising a crystalline form of the compound of formula (I) as described in the first aspect. Preferably, in the crystalline composition, the crystalline form of the compound of formula (I) is selected from one or more of Form A, Form B, Form C, and Form D.

[0061] In some embodiments of the present application, the crystalline form of the compound of formula (I) accounts for more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or more than 99.5% by weight of the crystalline composition.

[0062] Preferably, the compound of formula (I) in crystalline form comprises more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or more than 99.5% by weight of the crystalline composition.

[0063] In some embodiments of the present application, the crystalline composition comprises Form A of the compound of formula (I).

[0064] In some embodiments of the present application, the crystalline form A accounts for more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or more than 99.5% of the weight of the crystalline composition.

[0065] Preferably, the crystalline form A accounts for more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or more than 99.5% by weight of the crystalline composition.

[0066] In a third aspect, the present application provides a pharmaceutical composition comprising a crystalline compound of formula (I) and an optional pharmaceutically acceptable carrier. Preferably, in the pharmaceutical composition, the crystalline compound of formula (I) is selected from one or more of Form A, Form B, Form C, and Form D.

[0067] In some embodiments of the present application, the pharmaceutical composition comprises the crystalline form A of the compound represented by formula (I) and an optional pharmaceutically acceptable carrier.

[0068] In a fourth aspect, the present application provides a pharmaceutical composition comprising the crystalline composition as described in the second aspect and an optional pharmaceutically acceptable carrier.

[0069] In a fifth aspect, the present application provides the use of the compound of formula (I) in crystalline form as described in the first aspect, the crystalline composition as described in the second aspect, or the pharmaceutical composition as described in the third aspect as a drug or in the preparation of a drug.

[0070] In some embodiments of the present application, the drug is used to prevent and / or treat ENPP1-mediated diseases.

[0071] In some embodiments of the present application, the drug is used to prevent and / or treat cancer or tumor-related diseases, or cardiovascular diseases.

[0072] In some embodiments of the present application, the pharmaceutical composition contains another, two or more drugs for treating cancer or tumors.

[0073] In some embodiments of the present application, the pharmaceutical composition is used in combination with another, two or more drugs or treatment methods for treating cancer or tumors.

[0074] Preferably, the anti-tumor drug is a chemotherapy drug, a targeted therapy drug or an immunotherapy drug; preferably, the drug for preventing and / or treating cancer or tumors includes but is not limited to: cell signal transduction inhibitors, chlorambucil, melphalan, cyclophosphamide, ifosfamide, busulfan, carmustine, lomustine, streptozotocin, cisplatin, carboplatin, oxaliplatin, dacarbazine, temozolomide, procarbazine, methotrexate, fluorouracil, cytarabine, gemcitabine, mercaptopurine, fludarabine, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, topotecan, irinotecan, etoposide, trabectedin, dactinomycin, doxorubicin, Biclin, epirubicin, daunorubicin, mitoxantrone, bleomycin, mitomycin C, ixabepilone, tamoxifen, flutamide, gonadorelin analogs, megestrol acetate, prednisone, dexamethasone, methylprednisolone, thalidomide, interferon alfa, leucovorin, sirolimus, temsirolimus, everolimus, afatinib, alisertib, amuvatinib, apatinib, axitinib, bortezomib, bosutinib, brivanib, cabozantinib, cediranib, crenolanib, crizotinib, dabrafenib, dacomitinib, danusetib, dasatinib, dovitinib, erlotinib, foretinib , ganetespib, gefitinib, ibrutinib, icotinib, imatinib, iniparib, lapatinib, lenvatinib, linifanib, linsitinib, masitinib, momelotinib, motesanib, neratinib, nilotinib, niraparib, oprozomib, olaparib, pazopanib, pictilisib, ponatinib, quizartinib, regorafenib, rigosertib, rucaparib, ruxolitinib, sacatinib, saridegib, sorafenib, sunitinib, tiratinib, tivantinib, Wozanib, tofacitinib, trametinib, vandetanib, veliparib, vemurafenib, vismodegib, volasertib, alemtuzumab, bevacizumab, berentuzumab vedotin, catumaxomab, cetuximab, denosumab, gemtuzumab, ipilimumab, nimotuzumab, ofatumumab, panitumumab, rituximab, tositumomab, trastuzumab, PI3K inhibitors, CSF1R inhibitors, A2A and / or A2B receptor antagonists, IDO inhibitors, anti-PD-1 antibodies, anti-PD-L1 antibodies, LAG3 antibodies, TIM-3 antibodies, TIGIT antibodies, CD47 antibodies, CLAUDIN 18.2 antibodies, anti-CTLA-4 antibodies or any combination thereof; preferably, the treatment method is radiotherapy or surgery.

[0075] In some embodiments of the present application, the drug is used to prevent and / or treat skin cancer, bladder cancer, ovarian cancer, breast cancer, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, neuroblastoma, rectal cancer, colon cancer, familial adenomatous polyposis carcinoma, hereditary non-polyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cancer, cervical cancer, uterine body cancer, endometrial cancer, choriocarcinoma, testicular cancer, urinary cancer, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumor, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, leukemia, acute lymphoid leukemia (ALL), chronic lymphoid leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), adult T-cell leukemia lymphoma, diffuse large B-cell lymphoma (DLBCL), hepatocellular carcinoma, gallbladder cancer, bronchogenic carcinoma, small cell lung cancer, non-small cell lung cancer, multiple myeloma, basal cell tumor, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myeloma, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmacytoma.

[0076] In some embodiments of the present application, the cardiovascular disease treated and / or prevented is the treatment and / or prevention of myocardial infarction (MI), heart failure (HF), cardiac trauma, abnormal scar formation in cardiac tissue, cardiomyopathy, myocardial cell death, enhanced cardiac repair, and the release of pro-inflammatory molecules in myocardial cells.

[0077] Definition and Description

[0078] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings: A particular phrase or term should not be considered ambiguous or unclear without a specific definition, but should be understood according to its ordinary meaning.

[0079] The "crystalline form of the compound of formula (I)" referred to in this application refers to the compound of formula (I) in crystalline form, including an anhydrous and solvent-free form, a hydrate form, and a solvate form of the compound of formula (I). The crystalline form is preferably an anhydrous and solvent-free form or a hydrate form; more preferably an anhydrous and solvent-free form.

[0080] The term "solvate" or "solvate" refers to an association formed by solvent molecules in stoichiometric or non-stoichiometric ratios and the compound represented by formula (I) of the present application, including associations containing both water molecules and one or more other solvent molecules, and associations containing only one or more other solvent molecules.

[0081] The term "hydrate" refers to an association formed by water molecules in a stoichiometric ratio or a non-stoichiometric ratio and the compound represented by formula (I) of the present application.

[0082] The "anhydrous and solvent-free form" means that there are no water molecules or solvent molecules in the unit cell or crystal lattice, or that water molecules or solvent molecules coexist with the compound represented by formula (I) in a manner that is not bound by intermolecular forces, such as by adsorption.

[0083] The term "crystalline composition" refers to a solid form comprising the Form A mentioned herein. Furthermore, in addition to the crystalline form described herein, the crystalline composition may optionally comprise other crystalline forms or other amorphous forms of the compound represented by Formula (I) or its salt, or impurities other than these. Those skilled in the art will appreciate that the sum of the contents of the various components in the crystalline composition should be 100%.

[0084] The "room temperature" is the room temperature conventionally used in the art, generally 10-30°C, preferably 25°C±5°C.

[0085] In the context of an X-ray powder diffraction pattern, the term "substantially" or "substantially as shown" refers to a substantially pure crystalline form, wherein at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% of the peaks in the powder X-ray diffraction pattern appear in the given pattern. Furthermore, as the content of a crystalline form in a product decreases, some of the diffraction peaks attributable to that form in its X-ray powder diffraction pattern may become less frequent due to instrument sensitivity. Furthermore, for any given crystalline form, the positions of peaks may vary slightly, as is well known in the art of crystallography. For example, peak positions may shift due to temperature changes, sample movement, or instrument calibration during sample analysis, resulting in a 2θ value with an error of approximately ±0.3° and typically approximately ±0.2°. Therefore, when determining each crystalline structure, this error should be taken into account, and the term "substantially" or "substantially as shown in the accompanying drawings" is also intended to cover such differences in the diffraction peak positions, which refers to ±0.3°, preferably ±0.2°, and further preferably ±0.1°.

[0086] In a DSC spectrum, the term "substantially" or "substantially as shown" means that for the same crystalline form of the same compound, in consecutive analyses, the error of the thermal transition onset temperature, the peak temperature of the endothermic peak, the peak temperature of the exothermic peak, the melting point, etc. is typically within about 8°C, usually about 5°C, and usually about 3°C. When a compound is described as having a given thermal transition onset temperature, peak temperature of the endothermic peak, peak temperature of the exothermic peak, melting point, etc., it refers to that temperature ±5°C.

[0087] As used herein, the term "prevent" or "prevent" refers to reducing the frequency of symptoms of a medical condition or delaying the onset of a medical condition in a subject when used for a disease or condition (e.g., a viral disease) compared to a subject to which the compound or drug (e.g., the combination product claimed herein) is not administered.

[0088] As used herein, the term "treat," ...

[0089] The pharmaceutical composition of the present application can be prepared by conventional methods in the art.

[0090] In the context of this application, the term "pharmaceutically acceptable carrier" or "excipient" or "pharmaceutically acceptable excipient" or "pharmaceutically acceptable excipient" refers to those excipients that have no significant irritating effect on organisms and do not impair the biological activity and performance of the active compound. The term "pharmaceutically acceptable excipient" includes: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc. Those skilled in the art can select specific pharmaceutically acceptable excipients according to actual needs. The knowledge about excipients is well known to those skilled in the art, for example, reference may be made to "Pharmaceutics" (edited by Cui Fude, 5th edition, People's Medical Publishing House, 2003).

[0091] The word "comprise" or "comprises" and its English variations such as comprises or comprising should be understood as having an open and non-exclusive meaning, ie, "including but not limited to".

[0092] In the scope of this application, various options of any feature can be combined with various options of other features to form many different embodiments. This application is intended to include all possible embodiments composed of various options of all technical features.

[0093] The intermediate compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present application.

[0094] The chemical reactions described in the specific embodiments of the present application are carried out in a suitable solvent that is compatible with the chemical transformations described herein and the reagents and materials required. To obtain the compounds described herein, it may sometimes be necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0095] The present application will be described in detail below through examples, which are not intended to limit the present application in any way.

[0096] All reagents and solvents used in this application are commercially available and can be used without further purification.

[0097] Technical Effects

[0098] The compound of formula (I) of the present invention has good PK properties and oral bioavailability, its crystal form is stable, and its drug development prospects are good. The compound of formula (I) exhibits good inhibitory activity against ENPP1 and has good pharmacokinetic indicators.

[0099] The crystalline form of the compound of formula (I), and the specific crystal forms provided by the present invention, have one or more of the following beneficial effects: (1) the crystalline form of the compound of formula (I) has good properties and is easy to weigh, transfer, separate, purify and store; (2) the crystalline form of the compound of formula (I) and the specific crystal forms have good crystallinity; (3) the crystalline form of the compound of formula (I) and the specific crystal forms have excellent operability, for example, they are easy to purify, filter and separate, especially the crystal form A, which is simple to prepare and has a high yield; (4) the preferred crystal forms have good physical and chemical stability, especially the crystal form A, which has high purity and can be directly used as a raw material drug, and has good pharmaceutical prospects.

[0100] 1. Instruments and analytical methods

[0101] 1.1. X-ray powder diffractometer (XRPD)

[0102] Instrument model: D8 Advance

[0103] Test conditions: Chinese Pharmacopoeia Part IV General Chapter 0451

[0104] X-ray generator: copper target

[0105] Tube voltage: 40KV

[0106] Scattering slit: 0.6mm

[0107] Detector slit: 10.5mm

[0108] Anti-scatter slit: 2.5°

[0109] Scanning range: 3-50°

[0110] Step size: 0.1s / step

[0111] Step size: 0.02°

[0112] 1.2. Differential Scanning Calorimeter (DSC)

[0113] Instrument model: DSC 3

[0114] Test conditions: starting temperature 30°C, heating to 300°C at a heating rate of 10°C / min; purge gas flow rate: 50 ml / min;

[0115] Drying gas flow rate: 150 ml / min; Purge gas: N2; Crucible: aluminum crucible.

[0116] 1.3. Thermogravimetric Analysis (TGA)

[0117] Instrument model: TGA 2

[0118] Test conditions: starting temperature 30℃, heating to 350℃ at a heating rate of 10℃ / min; balance shielding gas flow rate:

[0119] 20ml / min; reaction gas flow rate: 50ml / min; purge gas: N2; crucible: alumina crucible.

[0120] 1.4. Dynamic Gas Sorption Analyzer (DVS)

[0121] Instrument model: DVS Intrinsic

[0122] Test method: 25℃ 0%RH-90%RH-0%RH; refer to Chinese Pharmacopoeia Part IV General Chapter 0103

[0123] Balanced dm / dt: 0.002

[0124] RH (%) measurement gradient: 10% per step

[0125] RH (%) measurement gradient range: 0% to 90%

[0126] Description of hygroscopic characteristics and definition of hygroscopic weight gain:

[0127] 1.5 Crystal stability test in water and biological media

[0128] The preparation process of the biological medium is shown in the table below. The crystalline sample was added to the biological medium and water and shaken at 37°C for 24 hours. Samples were taken at 1 hour, 4 hours, and 24 hours. The sampled solutions were filtered through a 0.22μm water filter. Some samples with higher concentrations were appropriately diluted with diluent. The signal peak area of ​​the solution was measured by HPLC. Finally, the concentration of the compound in the solution was calculated based on the peak area, the HPLC standard curve of the raw material, and the dilution factor. In addition, samples were taken at different time points to test their pH value, and the remaining solid was analyzed by XRPD.

[0129] Note: FaSSIF: simulates the intestinal fluid in the small intestine of humans in a hungry state before a meal; FeSSIF: simulates the intestinal fluid in the small intestine of humans in a full state after a meal; FaSSGF: simulates the gastric fluid in an empty stomach when humans are hungry. Description of the drawings:

[0130] FIG1 is an XRPD pattern of Form A of the compound of formula (I) prepared in Example 2.

[0131] FIG2 is a DSC spectrum of Form A of the compound of formula (I).

[0132] FIG3 is a TGA spectrum of crystal form A of the compound of formula (I).

[0133] FIG4 is an XRPD pattern of Form B of the compound of formula (I) prepared in Example 4.

[0134] Figure 5 is the XRPD pattern of Form C of the compound of formula (I) prepared in Example 5.

[0135] FIG6 is an XRPD pattern of Form D of the compound of formula (I) prepared in Example 6.

[0136] FIG7 is an XRPD pattern of Form A of the compound of formula (I) prepared in Example 3.

[0137] Figure 8 shows cardiac function indicators and body weight changes in the acute myocardial ischemia model in mice treated with the compound of formula (I), including: A) left ventricular ejection fraction in mice; B) left ventricular fractional shortening in mice; and C) body weight changes in mice.

[0138] Figure 9 shows the changes in myocardial fibrosis in the acute myocardial ischemia model in mice treated with the compound of formula (I); D) Masson-stained pathological sections of mouse heart tissue; E) the degree of myocardial fibrosis in mice;

[0139] Figure 10 shows cardiac function indicators and body weight changes in rats with acute myocardial ischemia model treated with the compound of formula (I), including: a) left ventricular fractional shortening of rats; b) left ventricular ejection fraction of rats; and c) body weight changes of rats.

[0140] Figure 11 shows the changes in myocardial fibrosis in the acute myocardial ischemia model in rats treated with the compound of formula (I); d) Masson-stained pathological sections of rat heart tissue; e) the degree of myocardial fibrosis in rats;

[0141] FIG12 is a comparison of XRPD patterns of Form A before and after the stability test.

[0142] Note: "Compound 1" in the diagram is the compound of formula (I). DETAILED DESCRIPTION

[0143] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments, but the specific implementation methods are not intended to limit the content of the present invention.

[0144] Example 1 Synthesis of the compound of formula (I)

[0145] Step 1: Preparation of ethyl 4-chloro-7-methoxy-1,8-naphthylidene-3-carboxylate

[0146] 1,3-Diethyl 2-(((6-methoxypyridin-2-yl)amino)methylene)malonate (12 g, 22.7 mmol, 1 eq.) was dissolved in phosphorus oxychloride (60 mL) and stirred at 110°C under nitrogen for 4 hours. After completion of the reaction, the solvent was removed by distillation under reduced pressure. The resulting residue was dissolved in ethyl acetate (60 mL) and quickly washed with water (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was dried by rotary evaporation. The crude product was purified by flash chromatography (Silica gel, DCM:EA = 1:1) to obtain the title compound (1.5 g, 12% yield). LCMS (ESI) [M+H] + =266.0.

[0147] Step 2: Preparation of ethyl 4-((4-bromo-2,6-difluorobenzyl)amino)-7-methoxy-1,8-naphthyridine-3-carboxylate

[0148] Ethyl 4-chloro-7-methoxy-1,8-naphthyridine-3-carboxylate (3.0 g, 18.75 mmol, 1 eq.) was dissolved in acetonitrile (80 mL), followed by the addition of potassium carbonate (5.2 g, 37.5 mmol, 2.0 eq.) and (4-bromo-2,6-difluorophenyl)methanamine (4.37 g, 19.69 mmol, 1.05 eq.). The reaction was carried out at 40°C under nitrogen for 20 hours. LCMS monitored the reaction completion. The reaction solution was concentrated, diluted with ethyl acetate, washed with water, and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was spin-dried. The crude product was purified by flash chromatography (Silica gel, DCM:EA = 4:1) to yield the title compound (3.5 g, 69.2% yield). LCMS (ESI) [M+H] + =452.0.

[0149] Step 3: Preparation of (4-((4-bromo-2,6-difluorobenzyl)amino)-7-methoxy-1,8-naphthyridin-3-yl)methanol

[0150] Ethyl 4-((4-bromo-2,6-difluorobenzyl)amino)-7-methoxy-1,8-naphthyridine-3-carboxylate (120 mg, 22.7 mmol, 1 eq.) was dissolved in ethanol (10 mL) and sodium borohydride (134 mg, 5 eq.) was slowly added. The reaction was stirred at 50°C under nitrogen for 16 hours. LCMS monitored the reaction completion, and the solvent was removed by rotary evaporation. The resulting residue was quenched with saturated sodium chloride solution (5 mL) and extracted with ethyl acetate (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was rotary evaporation. The crude product was purified by flash chromatography (Silica gel, DCM:MeOH = 2:1) to obtain the title compound (120 mg, 38% yield). LCMS (ESI) [M+H] + =410.0; 1 H NMR (400MHz, CDCl3) δ8.43(s,1H),8.35(d,J=9.2Hz,1H),7.09(d,J=6.8Hz,2H),6.90(d,J=9.2Hz,1H),4.75(s,2H),4.69(s,2H),4.12(s,3H).

[0151] Step 4: Preparation of 1-(4-bromo-2,6-difluorobenzyl)-8-methoxy-1,4-dihydro-2H-[1,3]oxazino[5,4-c][1,8]phthalazin-2-one

[0152] (4-((4-Bromo-2,6-difluorobenzyl)amino)-7-methoxy-1,8-naphthyridin-3-yl)methanol (300 mg, 0.73 mmol, 1 eq.) was dissolved in dichloromethane (15 mL), and N,N-diisopropylethylamine (473 mg, 3.66 mmol, 5 eq.) and triphosgene (651 mg, 2.19 mmol, 3 eq.) were added, and the mixture was reacted at 0°C for 2 hours.

[0153] The reaction was complete as monitored by LCMS. The reaction solution was washed with water, dried over anhydrous sodium sulfate, filtered, and the filtrate was spun down to dryness. The crude product was separated and purified by flash silica gel column chromatography (Silica gel, MeOH:DCM = 5:95) to obtain the title compound (250 mg, yield 78.51%). LCMS (ESI) [M+H] + =436.0.

[0154] Step 5: Preparation of 1-(2,6-difluoro-4-((4-methoxybenzyl)thio)benzyl)-8-methoxy-1,4-dihydro-2H-[1,3]oxazino[5,4-c][1,8]phthalazin-2-one

[0155] 1-(4-Bromo-2,6-difluorobenzyl)-8-methoxy-1,4-dihydro-2H-[1,3]oxazino[5,4-c][1,8]phthalazin-2-one (200 mg, 0.46 mmol, 1 eq.) was dissolved in dioxane (10 mL), and (4-methoxyphenyl)methanethiol (141 mg, 0.92 mmol, 2 eq.), [5-(diphenylphosphino)-9,9-dimethyl-9H-xanthogen-4-yl]diphenylphosphine (53 mg, 0.09 mmol, 0.2 eq.), tris(1,5-diphenylpenta-1,4-dien-3-one)dipalladium (84 mg, 0.09 mmol, 0.2 eq.) and N,N-diisopropylethylamine (178 mg, 1.38 mmol, 3 eq.) were added. The reaction was allowed to proceed at 90°C for 2 hours. LCMS monitored the reaction to be complete. The reaction solution was concentrated to dryness and then purified by flash silica gel column chromatography (MeOH:DCM = 5:95) to obtain the title compound (110 mg, yield 47.09%). LCMS (ESI) [M+H] + =510.1.

[0156] Step 6: Preparation of 3,5-difluoro-4-((8-methoxy-2-oxo-2H-[1,3]oxazino[5,4-c][1,8]naphthyridin-1(4H)-yl)methyl)benzenesulfonamide

[0157] 1-(2,6-difluoro-4-((4-methoxybenzyl)thio)benzyl)-8-methoxy-1,4-dihydro-2H-[1,3]oxazino[5,4-c][1,8]phthalazin-2-one (100 mg, 0.2 mmol, 1.0 eq.), acetic acid (83 mg, 1.37 mmol, 7.0 eq.), and water (50 mg, 2.75 mmol, 14.0 eq.) were dissolved in tetrahydrofuran (15 mL). Dichlorohydantoin (116 mg, 0.59 mmol, 3 eq.) was added at 0°C, and the mixture was reacted at 25°C for 1 hour. Aqueous ammonia (0.5 mL) was added and the mixture was reacted at 25°C for 10 minutes. LCMS confirmed the complete reaction of the starting material. The reaction solution was concentrated and purified by preparative HPLC to obtain the title compound (14.6 mg, 17.05% yield). LCMS (ESI) [M+H] + =437.1; 1 HNMR (400MHz, MeOD-d4) δ8.71(s,1H),8.59(d,J=9.2Hz,1H),7.46(d,J=7.6Hz,2H),7.13(d,J=9.6Hz,1H),5.57(s,2H),5.30(s,2H),4.12(s,3H).

[0158] Example 2 Preparation of Crystalline Form A of Compound (I)

[0159] To a reaction flask, the compound of formula (I) prepared in Example 1 (100 mg) and dimethyl sulfoxide (0.3 mL) were added sequentially. The mixture was slowly heated to dissolve, and then slowly added to water (1 mL) at 80°C. The mixture was slowly cooled to room temperature, stirred for one hour, filtered, and the filter cake was washed with an appropriate amount of water. The filter cake was dried under reduced pressure at 65°C to obtain crystalline form A of compound of formula (I).

[0160] Table 1 XRPD pattern analysis data of Form A of the compound of formula (I)

[0161] Example 3 Preparation of Crystalline Form A of Compound of Formula (I)

[0162] The compound of formula (I) prepared in Example 1 (100 mg) was added to the reaction flask, followed by acetone (1 mL). The mixture was stirred at room temperature for 24 hours to slurry the suspension. The filter cake was dried under reduced pressure at 45°C to obtain crystalline form A of the compound of formula (I).

[0163] Table 2 XRPD pattern analysis data of Form A of the compound of formula (I)

[0164] Example 4 Preparation of Crystalline Form B of Compound (I)

[0165] Weigh 260 mg of sample and dissolve it in 10 mL of DMF at room temperature. Add approximately 8 mL of methyl tert-butyl ether (MTBE) dropwise to 0.8 mL of the solution. After stirring at room temperature for a period of time, centrifuge the system to separate the precipitated solid, and vacuum dry the solid at room temperature to obtain Form B.

[0166] Table 3 XRPD pattern analysis data of Form B of the compound of formula (I)

[0167] Example 5 Preparation of Crystalline Form C of Compound of Formula (I)

[0168] Weigh 260 mg of sample and dissolve it in 4 mL of DMSO at room temperature. Take 0.3 mL of the solution and add approximately 3 mL of toluene dropwise. After stirring at room temperature for a period of time, centrifuge the system to separate the precipitated solid, and then vacuum dry the solid at room temperature to obtain Form C.

[0169] Table 4 XRPD pattern analysis data of Form C of the compound of formula (I)

[0170] Example 6 Preparation of Crystalline Form D of Compound of Formula (I)

[0171] Weigh 260 mg of sample and dissolve it in 10 mL of NMP at room temperature. Add approximately 8 mL of methyl tert-butyl ether (MTBE) dropwise to 0.8 mL of the solution. After stirring at room temperature for a period of time, centrifuge the system to separate the precipitated solid, and vacuum dry the solid at room temperature to obtain Form D.

[0172] Table 5 XRPD pattern analysis data of Form D of the compound of formula (I)

[0173] Test Example 1 Study on the Hygroscopicity of Crystalline Forms

[0174] Experimental instrument: DVS Intrinsic

[0175] Experimental method: 25-35 mg of the crystalline form A of the compound of formula (I) prepared in Example 2 was placed in a DVS sample tray for testing.

[0176] Experimental conclusion: The crystal form A of compound (I) showed a weight gain of ΔW = 0.4461% at 25°C and 80% humidity, indicating slight hygroscopicity. The crystal form did not change after DVS testing.

[0177] Test Example 2: Solid Stability Study

[0178] A 20 mg sample of Form A of Compound (I) prepared in Example 2 was weighed into a vial and placed open under high temperature (60°C), long-term (25°C / 60% RH), and accelerated (40°C / 75% RH) conditions for 7 days. The samples were then subjected to purity testing and X-ray powder diffraction analysis to investigate the stability of Example 2 (Form A) under different conditions. The results are shown in Table 6 and Figure 12.

[0179] Table 6 Solid stability test results

[0180] The data show that Example 2 (Form A) can maintain chemical stability and crystal stability in the solid stability test.

[0181] Test Example 3: Stability test of the crystalline form A of the compound of formula (I) in biological solvents

[0182] The stability of the sample of Example 2 (Form A) in three biological media (FaSSIF, FeSSIF, and FaSSGF) and water was tested. The remaining sample was subjected to X-ray powder diffraction after 24 hours. The results are shown in Table 7:

[0183] Table 7 Stability test in biosolvent medium

[0184] The data show that Example 2 (Form A) can maintain a stable crystalline form in all biological solvents.

[0185] Test Example 4 In vitro ENPP1 enzymatic inhibition test

[0186] ENPP1 is a transmembrane glycoprotein that hydrolyzes nucleotides and derivatives with nucleotide-5'-monophosphate structures. ENPP1 hydrolyzes synthetic para-nitrophenyl 5'-monophosphate (TMP-pNP) into nucleotide 5'-monophosphate and para-nitrophenol, the latter being a chromogenic product. The amount of para-nitrophenol produced can be directly measured by its absorbance at 405 nm, which is proportional to the enzyme activity.

[0187] Experimental procedures

[0188] Compounds were prepared in DMSO by 100x serial dilutions, starting at 1 mM and proceeding through four-fold serial dilutions, with 10 concentration points. Using an Echo, 300 nL of the compound mixture was transferred to a 384-well assay plate (starting at a final concentration of 10 μM and followed by four-fold serial dilutions, with 10 concentration points). To each well, 15 μL of 0.2 ng / μL hENPP1 enzyme (2x final concentration) prepared in assay buffer (250 mM NaCl, 50 mM Tris, pH 9.5) was added. Then, 15 μL of 400 μM TMP-pNP (2x final concentration) prepared in assay buffer was added. After incubation at 37°C for 0.5 hours, the OD at 405 nm was read on a microplate reader. Percent inhibition was calculated using the formula: % inhibition = (OD high signal control - OD sample well) / (OD high signal control - OD low signal control) * 100. IC50 values ​​were calculated using a four-parameter fit, where the high signal control was the DMSO-free group and the low signal control was the blank group. The data of the compound of formula (I) prepared in Example 1 are shown in Table 8.

[0189] Table 8 ENPP1 enzyme inhibitory activity

[0190] Test Example 5: Pharmacokinetic Study of Compound Concentrations in Mice Using LC-MS / MS

[0191] Test principle: Use LC-MS / MS to determine the drug concentration of the target drug in plasma at different times and draw the pharmacokinetic curve of the target compound in the body.

[0192] Test Method: The test compound (the compound of formula (I) prepared in Example 1) was dissolved in DMSO to a final concentration of 20 mg / mL. The compound was then dissolved in a solvent containing 5% DMSO (Sigma-Aldrich, SHBJ2847), 45% PEG400 (Sigma-Aldrich, BCCC0015), and 50% ddH2O to a final concentration of 1 mg / mL. CD-1 male mice (JH Laboratory Animal Co., Ltd.) were used. Nine mice per group were dosed with 10 mg / kg PO. Blood was collected from three mice at 0.25, 0.5, 1, 2, 4, 8, and 24 hours post-incubation. 110 μL of whole blood (anticoagulated with K2EDTA) was collected and immediately centrifuged at 2000 g for 5 minutes at 4°C. Serum was collected and stored at -70°C. Blood drug concentrations were determined using a Triple-quadrupole MS system (SCIEX), including the preparation of standard curves, quality control curves, and samples. Standard curves and quality control curves were prepared by diluting a working solution of MeOH:H₂O (1:1) and adding 3 μL of these working solutions to 57 μL of blank plasma. Sample preparation involved adding 200 μL of internal standard solution (propranolol, 40 ng / mL) to 30 μL of plasma sample. The mixture was mixed for 1 minute and centrifuged at 5800 rpm for 10 minutes. 100 μL of the supernatant was transferred to a new plate for injection and analysis. Chromatographic conditions, including mobile phase composition, elution gradient, flow rate, and retention time, were optimized for each sample. A Waters BEH C₁₀ column (2.1 × 50 mm, 1.7 μm) was used, and the injection volume was 1 μL. Mass spectrometry was performed using a TuREo spray ionization source in positive ion detection mode and multi-channel reaction monitoring (MRM) mode. Pharmacokinetic parameters, including peak concentration (Cmax), time to peak concentration (Tmax), area under the concentration-time curve (AUC), and elimination half-life (t½), were calculated using WinNonlin 8.2 software using a non-compartmental model based on drug concentration-time data. AUC was calculated using the linear up-log down method.

[0193] Table 9 pharmacokinetic parameters are as follows:

[0194] The test results show that the compound of formula (I) of the present invention has good in vivo pharmacokinetics and has drug-forming potential.

[0195] Test Example 6: Pharmacodynamic evaluation of the compound of formula (I) in the mouse pancreatic cancer Pan02 model

[0196] 1. Experimental purpose: To evaluate the in vivo efficacy of the test drug in a mouse pancreatic cancer Pan02 cell subcutaneous transplant tumor model.

[0197] 2. Test method:

[0198] 2.1 Cell Culture: Mouse pancreatic cancer Pan02 cells were cultured as monolayers in DMEM supplemented with 10% fetal bovine serum, 0.01 mg / ml sinsulin, and 1% penicillin / streptomycin / amphotericin B in a 37°C, 5% CO2 incubator. When cell saturation reached 80%-90% and the desired number of cells was reached, the cells were harvested, counted, and plated.

[0199] 2.2 Animals: C57BL / 6 female mice, 6-8 weeks old, weighing 18-22 g. They were provided by Shanghai Xipu-Bikai Laboratory Animal Co., Ltd. or other suppliers.

[0200] 2.3 Tumor inoculation and animal grouping and administration: 0.1 mL (5×10 6 Pan02 cells were subcutaneously inoculated on the right back of each mouse, and the average tumor volume reached approximately 100-200 mm 3 The animals were randomly divided into groups according to the tumor volume, with 8 mice in each group. The control group was given the vehicle: 5% DMSO + 45% PEG400 + 50% H2O (1% CMC-Na). The experimental group was given 20 mg / kg by oral gavage, with a dosing volume of 10 μl / g based on the mouse body weight, twice a day. The health status of the animals was observed daily. If the tumor volume exceeded 3,000 mm 3 , or have serious illness, or pain, weight loss greater than 20%, and continued deterioration, must be euthanized.

[0201] 2.4 Tumor growth inhibition rate: Tumor diameter was measured twice a week using a vernier caliper. The formula for calculating tumor volume was: V = 0.5a × b 2 a and b represent the long and short diameters of the tumor, respectively. The tumor inhibitory efficacy (TGI) of the compound (%) = [(1 - (average tumor volume of the treated group after dosing - average tumor volume of the treated group at the start of dosing)) / (average tumor volume of the solvent control group before treatment - average tumor volume of the solvent control group after treatment)] × 100%. Data were analyzed using the T-test for comparisons between two groups; p < 0.05 was considered significant.

[0202] 3. Experimental results:

[0203] The compound of formula (I) prepared in Example 1 of the present invention demonstrated activity in the Pan02 mouse pancreatic cancer model. Administration of the compound of formula (I) for 16 days significantly inhibited tumor growth, with a TGI of 43.4%, significantly different from the control group (p < 0.05). It also showed no effect on mouse body weight, demonstrating good safety. The crystalline form of the compound of formula (I) (e.g., Form A of the compound of formula (I)) exhibited substantially the same efficacy.

[0204] Test Example 7: Pharmacodynamic evaluation of the compound in a mouse myocardial ischemia model

[0205] 1. Purpose of the experiment

[0206] The purpose of this study was to evaluate the efficacy of the compounds in a C57 BL / 6 mouse myocardial ischemia model.

[0207] 2. Test methods

[0208] After the animals arrived at the facility and adapted for one week, they were divided into four groups according to their weight one day before surgery, with 6 animals in the sham group and 8 animals in the model group (MI). The next day, they underwent myocardial ischemia surgery (mice in the sham group only underwent thoracotomy and suture threading without ligation). The model was established by ligating the left anterior descending coronary artery (LAD). After model establishment, they were treated with penicillin (20,000 units / mouse im) for 3 days and disinfected to prevent infection.

[0209] One hour before surgery, each group of animals was given the corresponding compound. The control group (Vehicle) was given the solvent: 5% DMSO + 45% PEG400 + 50% H2O (1% CMC-Na), and the experimental groups were given 4 mg / kg and 20 mg / kg, respectively, and were given orally by gavage twice a day for 28 consecutive days. The body weight of the animals was monitored daily after administration, and cardiac ultrasound imaging indicators were measured on the 1st, 7th and 28th days. The main indicators of echocardiography evaluation were left ventricular fractional shortening (FS) and left ventricular ejection fraction (LVEF). After the echocardiography measurement on the 28th day, the heart tissue was collected in tissue fixative for Masson trichrome staining pathological analysis.

[0210] 3. Experimental Results

[0211] The compound (I) of Example 1 of the present invention showed good cardiac function-improving activity in a mouse myocardial infarction model. The compound of formula (I) of the present invention had a protective effect on cardiac function after pre-administration one day before modeling. As shown in Figure 5, on day 1, 4 mpk and 20 mpk increased the left ventricular ejection fraction (LVEF) of mice by 15% and 10% (Figure A) and the left ventricular short-axis shortening rate (FS) by 7% and 5% (Figure B) respectively compared with the model vehicle control group. On day 7, the left ventricular ejection fraction (LVEF) of mice was increased by 23.4% and 23.3% respectively, and the left ventricular short-axis shortening rate (FS) was increased by 12.2% and 12.3% respectively. On day 28, the LVEF and FS of the model vehicle control group further deteriorated, and compound (I) was able to further maintain the left ventricular LVEF and FS of mice, and had no significant effect on body weight (Figure C). Statistical significance was analyzed using two-way analysis of variance / one-way analysis of variance / t-test, and p<0.05 was considered to be significantly different.

[0212] The compound of formula (I) of Example 1 of the present invention showed an improving effect on myocardial fibrosis in mice with myocardial infarction. As shown in Figure 6, Figure D is an electron micrograph of a Masson-stained pathological section of mouse heart tissue. Figure E is a statistical result of the degree of fibrosis in mouse heart tissue. As shown in the figure, in the sham-operated group, the red myocardial fibers of the mice were neatly arranged, and blue collagen fibers were rare. In the MI model control group, myocardial cell necrosis, myocardial tissue structure was disordered, and large areas of blue collagen fibers were visible in the infarcted area. The myocardial tissue structure of mice in the group administered with the compound of formula (I) was improved to a certain extent, with red myocardial cells and blue collagen in the infarcted area alternating and collagen fibers significantly reduced, indicating that the compound of formula (I) can reduce the degree of myocardial fibrosis after MI when administered for 28 days, and can reduce the degree of fibrosis by 8.8% and 9.6% respectively compared with the model solvent control group (Figure E). The crystalline form A of the compound of formula (I) has substantially the same pharmacological efficacy.

[0213] Test Example 8: Pharmacodynamic evaluation of the compound in a rat myocardial ischemia model

[0214] 1. Experimental purpose: The purpose of this study is to evaluate the efficacy of the compound in the SD rat myocardial ischemia model.

[0215] 2. Test methods

[0216] After the animals arrived at the facility and adapted for one week, the model was established by ligating the left anterior descending coronary artery (LAD). After the model was established, penicillin (80,000 units / rat im) was given for treatment for 3 days and disinfection was performed to prevent infection.

[0217] One day after model establishment, the animals were randomly divided into four groups based on baseline body weight and cardiac ultrasound imaging parameters: a sham group (4 animals per group) and a myocardial infarction (MI) group (6 animals per group). Drug administration began. The control group received a blank vehicle (5% DMSO + 45% PEG400 + 50% H2O (1% CMC-Na). The experimental groups received oral gavage at doses of 30 mg / kg and 75 mg / kg, respectively, twice daily for 28 consecutive days. Animal body weight was monitored daily after drug administration, and cardiac ultrasound imaging parameters were measured on days 7 and 28. The main echocardiographic parameters evaluated were left ventricular fractional shortening (FS) and left ventricular ejection fraction (LVEF). After echocardiographic measurements on day 28, cardiac tissue was collected and fixed in tissue fixative for pathological analysis using Masson's trichrome staining.

[0218] 3. Experimental Results

[0219] The compound of formula (I) prepared in Example 1 of the present invention showed good cardiac function-improving activity in a rat myocardial infarction model (Figures 7a and b). The compound of formula (I) of the present invention significantly improved the left ventricular ejection fraction (LVEF) and left ventricular short axis shortening index (FS) of rats on the 7th day of administration. The 30mpk and 75mpk groups increased the LVEF by 11.43% and 18.85%, respectively, and the left ventricular short axis shortening index (FS) by 9.73% and 12.62%, respectively. On the 28th day, the 30mpk and 75mpk dosage groups increased the LVEF by 18.75% and 23.8%, respectively, and the left ventricular short axis shortening index (FS) by 13.01% and 15.88%, respectively. Moreover, it showed a dose-dependent trend and had no significant effect on body weight (Figure c). Statistical significance was analyzed using a t-test, and p<0.05 was considered to be significantly different.

[0220] The compound of formula (I) prepared in Example 1 of the present invention showed an improving effect on myocardial fibrosis in rats with myocardial infarction. Figure d is an electron micrograph of a Masson-stained pathological section of rat heart tissue. Figure e is a statistical result of the degree of fibrosis in rat heart tissue. As shown in Figure 8, in the sham-operated group, the red myocardial fibers of the rats were neatly arranged, and blue collagen fibers were rare. In the MI model control group, myocardial cell necrosis, myocardial tissue structure was disordered, and large areas of blue collagen fibers were visible in the infarcted area. The myocardial tissue structure of rats in the group administered with the compound of formula (I) was improved to a certain extent, with red myocardial cells and blue collagen in the infarcted area distributed alternately, and collagen fibers were significantly reduced, indicating that the compound of formula (I) can reduce the degree of myocardial fibrosis after MI when administered for 28 days, and can reduce the degree of fibrosis by 5.2% and 6.5% respectively compared with the model solvent control group (Figure e). The crystalline form A of the compound of formula (I) has substantially the same pharmacodynamics.

Claims

1. A compound of formula (I) in crystalline form, 2. The compound of formula (I) in crystalline form according to claim 1, characterized in that The compound of formula (I) in the crystalline form is Form A, and its X-ray powder diffraction pattern using Cu-Kα radiation includes diffraction peaks at the following 2θ angles (±0.2°): 24.9, 25.1, 25.7; Alternatively, using Cu-Kα radiation, its X-ray powder diffraction pattern comprises diffraction peaks at the following 2θ angles (±0.2°): 12.8, 14.0, 16.0, 18.5, 24.9, 25.1, 25.7; Alternatively, using Cu-Kα radiation, its X-ray powder diffraction pattern comprises diffraction peaks at the following 2θ angles (±0.2°): 12.8, 14.0, 16.0, 18.5, 24.9, 25.1, 25.7, 26.9, 28.5; Alternatively, using Cu-Kα radiation, its X-ray powder diffraction pattern comprises diffraction peaks at the following 2θ angles (±0.2°): 12.8, 14.0, 16.0, 18.5, 24.9, 25.1, 25.7, 26.9, 28.5, 29.8; Alternatively, using Cu-Kα radiation, its X-ray powder diffraction pattern comprises diffraction peaks at the following 2θ angles (±0.2°): 12.8, 14.0, 16.0, 17.0, 18.5, 20.5, 24.9, 25.1, 25.7, 26.9, 28.5, 29.8; Alternatively, it has an X-ray powder diffraction pattern substantially as shown in FIG. 1 or FIG. 7 Alternatively, using Cu-Kα radiation, the XRPD pattern analysis is shown in Table 1 or Table 2; Alternatively, its differential scanning calorimetry curve has an endothermic peak at 233.23±4°C; Alternatively, its differential scanning calorimetry curve has an exothermic peak at 234.3±4°C; Alternatively, it has a differential scanning calorimetry (DSC) spectrum substantially as shown in FIG2 . Alternatively, the thermogravimetric analysis curve thereof shows a weight loss of 13.50±1% between about 105° C. and 254° C., for example, a weight loss of 13.5111%. Alternatively, the thermogravimetric analysis curve thereof shows a weight loss of 13.50±1% at 254.00±3° C., for example, a weight loss of 13.5111%. Alternatively, the Form A of the compound of formula (I) above has a thermogravimetric analysis (TGA) spectrum substantially as shown in FIG3 . Alternatively, the compound of formula (I) in the crystalline form is Form B, and using Cu-Kα radiation, its X-ray powder diffraction pattern has diffraction peaks (±0.2°) at the following 2θ angles: 19.19, 27.18, 13.44, 22.55; Alternatively, the crystalline form B, using Cu-Kα radiation, has an X-ray powder diffraction pattern with diffraction peaks (±0.2°) at the following 2θ angles: 26.19, 20.76, 4.42, 22.84, 19.19, 27.18, 13.44, 22.55; Alternatively, the crystal form B, using Cu-Kα radiation, has an XRPD pattern as shown in FIG4 ; Alternatively, the crystal form B, using Cu-Kα radiation, has an XRPD pattern analysis as shown in Table 3; Alternatively, the crystalline form B is a DMF solvate of the compound of formula (I); Alternatively, the compound of formula (I) in the crystalline form is Form C, and its X-ray powder diffraction pattern using Cu-Kα radiation has diffraction peaks (±0.2°) at the following 2θ angles: 22.11, 22.55, 20.96, 26.63; Alternatively, the crystalline form C, using Cu-Kα radiation, has an X-ray powder diffraction pattern having diffraction peaks (±0.2°) at the following 2θ angles: 22.11, 22.55, 20.96, 26.63, 15.01, 25.68, 26.38, 28.54, 13.22, 18.68; Alternatively, the crystal form C, using Cu-Kα radiation, has an XRPD pattern as shown in FIG5 ; Alternatively, the XRPD pattern analysis of the crystal form C using Cu-Kα radiation is shown in Table 4; Alternatively, the crystalline form C is a DMSO solvate of the compound of formula (I); Alternatively, the compound of formula (I) in the crystalline form is Form C, and using Cu-Kα radiation, its X-ray powder diffraction pattern has diffraction peaks (±0.2°) at the following 2θ angles: 22.71, 18.92, 22.96, 26.4, 24.44, 16.62, 13.15, 17.79，27.12； Alternatively, the crystalline form D, using Cu-Kα radiation, has an X-ray powder diffraction pattern having diffraction peaks (±0.2°) at the following 2θ angles: 22.71, 18.92, 22.96, 26.4, 24.44, 16.62, 13.15, 17.79, 27.12, 24.17, 28.81, 19.95, 28.42, 24.85, 14.78, 17.36, 29.16; Alternatively, the crystal form D, using Cu-Kα radiation, has an XRPD pattern as shown in FIG6 ; Alternatively, the XRPD pattern analysis of the crystal form D using Cu-Kα radiation is shown in Table 5; Alternatively, the crystalline form D is an NMP solvate of the compound of formula (I).

3. A method for preparing a crystalline compound of formula (I) according to claim 1 or 2, comprising method 1: The compound of formula I is added to organic solvent A to dissolve, and then the solution is added to organic solvent B to precipitate crystals. The organic solvent A is selected from one or more of DMF (N,N-dimethylformamide), DMSO (dimethyl sulfoxide), and NMP (N-methylpyrrolidone); the organic solvent B is selected from one or more of water, methanol, ethanol, MTBE, toluene, and dichloromethane; In some embodiments of the present invention, when the compound of formula (I) is crystalline form A, in the method 1, the organic solvent A is selected from DMF, and the organic solvent B is selected from one or two of water and ethanol; or the organic solvent A is selected from DMSO, and the organic solvent B is selected from one or two of toluene and methanol; In some embodiments of the present invention, when the compound of formula (I) is in crystalline form B, in the method 1, the organic solvent A is selected from DMF, and the organic solvent B is selected from one or two of MTBE and toluene; In some embodiments of the present invention, when the compound of formula (I) is in crystalline form D, in the method 1, the organic solvent A is selected from NMP, and the organic solvent B is selected from one or more of water, dichloromethane, and MTBE; Or including method 2: The compound of formula I is added to an organic solvent C to dissolve, and then an organic solvent D is added to precipitate crystals; The organic solvent C is selected from one or more of DMF, DMSO, and NMP, and the organic solvent D is selected from one or more of acetone, ethylene glycol methyl ether, water, dichloromethane, ethyl acetate, MTBE, toluene, tetrahydrofuran, dioxane, ethanol, trifluoroethanol, acetonitrile, ethylene glycol dimethyl ether, isopropanol, and butanone; Preferably, when the compound of formula (I) is crystalline form A, in the method 1, the organic solvent C is selected from DMF, and the organic solvent D is selected from one or more of acetone, ethylene glycol methyl ether, water, and dichloromethane; or the organic solvent C is selected from DMSO, and the organic solvent D is selected from one or more of ethanol, trifluoroethanol, ethylene glycol methyl ether, acetonitrile, dioxane, ethylene glycol dimethyl ether, and butanone; or the organic solvent C is selected from NMP, and the organic solvent D is selected from one or more of water, acetonitrile, dichloromethane, and ethylene glycol dimethyl ether; Preferably, when the compound of formula (I) is crystalline form B, in the method 2, the organic solvent C is selected from DMF, and the organic solvent D is selected from one or more of ethyl acetate, MTBE, toluene, tetrahydrofuran, dioxane, and butanone; Preferably, when the compound of formula (I) is crystalline form C, in the method 2, the organic solvent C is selected from DMSO, and the organic solvent D is selected from one or more of ethyl acetate, water, and toluene; Preferably, when the compound of formula (I) is crystalline form D, in the method 2, the organic solvent C is selected from NMP, and the organic solvent D is selected from one or more of isopropanol, MTBE, toluene, tetrahydrofuran, and butanone; Or including method three: The compound of formula I is added to an organic solvent E for suspension, slurrying, and filtering to obtain crystals; The organic solvent E is selected from one or more of methanol, ethanol, isopropanol, ethyl acetate, n-heptane, MTBE, ethylene glycol methyl ether, water, acetonitrile, toluene, dichloromethane, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, butanone, dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, and acetone; Preferably, when the compound of formula (I) is crystalline form A, in the method three, the organic solvent E is selected from one or more of methanol, ethanol, isopropanol, ethyl acetate, n-heptane, MTBE, ethylene glycol methyl ether, water, acetonitrile, toluene, dichloromethane, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, and butanone; Preferably, when the compound of formula (I) is in crystalline form B, in the method three, the organic solvent E is selected from DMF; Preferably, when the compound of formula (I) is crystalline form C, in the method three, the organic solvent E is selected from DMSO; Preferably, when the compound of formula (I) is in crystalline form D, in the method three, the organic solvent E is selected from NMP.

4. A crystalline composition comprising a compound of formula (I) in a crystalline form according to any one of claims 1 to 3; preferably, in the crystalline composition, the compound of formula (I) in a crystalline form is selected from one or more of crystalline form A, crystalline form B, crystalline form C, and crystalline form D; Preferably, the compound of formula (I) in crystalline form accounts for more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or more than 99.5% by weight of the crystalline composition; Preferably, the compound of formula (I) in crystalline form accounts for more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or more than 99.5% by weight of the crystalline composition.

5. A pharmaceutical composition comprising the compound of formula (I) in crystalline form as claimed in claim 1 or 2 or the crystalline composition as claimed in claim 4, and optionally a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, comprising another, two or more drugs for treating cancer or tumor.

7. Use of the compound of formula (I) in crystalline form according to claim 1 or 2, the crystalline composition according to claim 4, or the pharmaceutical composition according to claim 5 or 6 as a drug or in the preparation of a drug for preventing and / or treating ENPP1-mediated diseases.

8. Use of the compound of formula (I) in crystalline form according to claim 1 or 2, the crystalline composition according to claim 4, or the pharmaceutical composition according to claim 5 or 6 as a drug or in the preparation of a drug for preventing and / or treating cancer or tumor-related diseases, or cardiovascular diseases; Preferably, the compound of formula (I) in crystalline form as described in claim 1 or 2, the crystalline composition as described in claim 4, or the pharmaceutical composition as described in claim 5 or 6 is used in combination with another, two or more drugs or treatment methods for treating cancer or tumors.

9. The pharmaceutical composition according to claim 6 or the use according to claim 8, wherein: The anti-tumor drug is a chemotherapy drug, a targeted therapy drug or an immunotherapy drug; Preferably, the drug for preventing and / or treating cancer or tumor is selected from: cell signal transduction inhibitors, chlorambucil, melphalan, cyclophosphamide, ifosfamide, busulfan, carmustine, lomustine, streptozotocin, cisplatin, carboplatin, oxaliplatin, dacarbazine, temozolomide, procarbazine, methotrexate, fluorouracil, cytarabine, gemcitabine, mercaptopurine, fludarabine, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, topotecan, irinotecan, etoposide, trabectedin, dactinomycin, doxorubicin, epirubicin, daunorubicin, mitoxantrone, bleomycin, mitomycin C, ixabepilone, tamoxifen, flutamide, gonadorelin analogs, megestrol acetate, prednisone, dexamethasone, methylprednisolone, thalidomide, interferon α, subacute Calcium folic acid, sirolimus, temsirolimus, everolimus, afatinib, alisertib, amuvatinib, apatinib, axitinib, bortezomib, bosutinib, brivanib, cabozantinib, cediranib, crenolanib, crizotinib, dabrafenib, dacomitinib, danusetib, dasatinib, dovitinib, erlotinib, foretinib, ganetespib, gefitinib, ibrutinib, icotinib, imatinib, iniparib, lapatinib, lenvatinib, linifanib, linsitinib, masitinib, momelotinib, motesanib, neratinib, nilotinib, niraparib, oprozomib, olaparib, pazopa albuterol, pictilisib, ponatinib, quizartinib, regorafenib, rigosertib, rucaparib, ruxolitinib, saracutinib, saridegib, sorafenib, sunitinib, tiratinib, tivantinib, tivozanib, tofacitinib, trametinib, vandetanib, veliparib, vemurafenib, vismodegib, volasertib, alemtuzumab, bevacizumab, belentuzumab, vedotinib, Catumaxomab, cetuximab, denosumab, gemtuzumab, ipilimumab, nimotuzumab, ofatumumab, panitumumab, rituximab, tositumomab, trastuzumab, PI3K inhibitor, CSF1R inhibitor, A2A and / or A2B receptor antagonist, IDO inhibitor, anti-PD-1 antibody, anti-PD-L1 antibody, LAG3 antibody, TIM-3 antibody, TIGIT antibody, CD47 antibody, CLAUDIN 18.2 antibody, anti-CTLA-4 antibody or any combination thereof; preferably, the treatment method is radiotherapy or surgery.

10. The use according to claim 8, characterized in that: The cancer or tumor is selected from the group consisting of skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, neuroblastoma, rectal cancer, colon cancer, familial adenomatous polyposis cancer, hereditary non-polyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cancer, cervical cancer, uterine corpus cancer, endometrial cancer, choriocarcinoma, testicular cancer, urinary cancer, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumor, Hodgkin lymphoma, Non-Hodgkin lymphoma, Burkitt lymphoma, leukemia, acute lymphoid leukemia (ALL), chronic lymphoid leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), adult T-cell leukemia lymphoma, diffuse large B-cell lymphoma (DLBCL), hepatocellular carcinoma, gallbladder cancer, bronchial carcinoma, small cell lung cancer, non-small cell lung cancer, multiple myeloma, basal cell tumor, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmacytoma; The cardiovascular disease is selected from myocardial infarction (MI), heart failure (HF), cardiac trauma, abnormal scarring of cardiac tissue, cardiomyopathy, myocardial cell death, enhanced cardiac repair, and release of pro-inflammatory molecules in myocardial cells.

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