Hemisuccinate crystal form of protein tyrosine kinase inhibitor

Abstract

Disclosed in the present invention is a hemisuccinate crystal form of a protein tyrosine kinase inhibitor. The inhibitor has the ability to inhibit anti-angiogenic tyrosine kinases. In addition to effectively antagonizing VEGFR1, VEGFR2, VEGFR3 tyrosine kinase activity, the inhibitor has high selectivity in inhibiting EGFR tyrosine kinase activity, thereby effectively reducing and avoiding side effects. The hemisuccinate crystal form of a protein tyrosine kinase inhibitor exhibits excellent solid-state chemical stability and physical stability, shows almost no hygroscopicity, and thus can serve as an advantageous crystal form in drug research and development, and offers considerable application value in downstream development and industrialization.

Description

A semisuccinic salt crystalline form of a protein tyrosine kinase inhibitor TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a semisuccinic salt crystalline form of a protein tyrosine kinase inhibitor, its preparation method and use. BACKGROUND

[0002] Receptor tyrosine kinases play an important role in developmental biology, tissue homeostasis and cancer biology. Receptor tyrosine kinases consist of an extracellular ligand-binding domain, a transmembrane domain and an intracellular catalytic domain. Dimerization of two receptor tyrosine kinases upon ligand binding leads to autophosphorylation of tyrosine residues in the intracellular catalytic domain, which results in an active conformation and subsequent activation of the intracellular signaling cascade. Due to their important influence on cells, tyrosine kinases are highly regulated. When these kinases are constitutively activated by mutation or overexpression and are independent of ligand, many diseases (e.g. cancer or ocular diseases such as diabetic retinopathy) develop through unregulated cell proliferation and other mechanisms. For this reason, tyrosine kinase inhibitors can be used to treat some diseases by interfering with this unregulated process. The development of inhibitors targeting proangiogenic receptor tyrosine kinases, mainly the vascular endothelial growth factor receptor (VEGFR) family, has significantly improved the prognosis of some types of cancer (such as renal cell carcinoma, hepatocellular carcinoma and colorectal cancer), becoming an effective treatment for tumor-related angiogenesis.

[0003] Many proliferative disorders (e.g. ocular diseases, tumors and cancers) involve overexpression or upregulation of receptor tyrosine kinase (RTK) activity. Receptor tyrosine kinases are a class of kinase enzymes that modify proteins by chemically adding a phosphate group (phosphorylation). Phosphorylation often leads to a functional change in the target protein by altering enzyme activity, cellular localization, or binding to other proteins. Kinases are known to regulate most cellular pathways, especially those involved in signal transduction. To date, one of the methods to inhibit the VEGF pathway is to inhibit receptor tyrosine kinase (RTK) activity. At the same time, the importance of VEGFR as a proangiogenic inducer in tumor growth, invasion and extravasation makes it an excellent therapeutic target for a variety of cancers. However, existing VEGFR receptor tyrosine kinase inhibitors (RTKi) usually also inhibit EGFR activity. This cross-reactivity can lead to adverse side effects by inhibiting biological functions associated with one or more of these off-target receptors. This problem greatly affects the use of VEGFR receptor tyrosine kinase inhibitors for the treatment of ocular diseases. SUMMARY

[0004] To overcome the deficiencies of the prior art, the inventors prepared a protein tyrosine kinase inhibitor compound (as described in patent application PCT / CN2023 / 142499 (which is incorporated herein by reference), for example, (S)-1-(3-(5-(cyclopropylcarbamoyl)-2-fluorobenzyloxy(4- carbamoylisothiazol-5-yl)-3-(3-(3-fluoropyrrolidin-1-yl)propyl)urea, Compound I), on this basis, further research obtained the semisuccinate salt crystal form C of the compound, which has good solid chemical stability and physical stability, and almost no hygroscopicity, can be used as an advantage crystal form for drug research and development.

[0005] Specifically, the technical solutions of the present application are as follows:

[0006] In the first aspect of the present application, a crystal form C of a semisuccinate salt of Compound I is provided, which has characteristic peaks (main characteristic diffraction peaks) at at least three (at least four, at least five, at least six, at least seven, or all) of the positions of diffraction angle 2θ values of 14.2°±0.2°, 18.3°±0.2°, 18.6°±0.2°, 20.9°±0.2°, 22.2°±0.2°, 22.9°±0.2°, 23.2°±0.2°, 25.0°±0.2° using Cu-Kα radiation, and the Compound I has the following structure:

[0007] In the semisuccinate salt, the molar ratio of Compound I to succinic acid is 1:0.4-0.6, for example, 1:0.45-0.55, 1:0.5.

[0008] In some embodiments of the present application, the semisuccinate salt of Compound I has the following structure:

[0009] Chemical formula: C 23 H 29 F2N6O4S·1 / 2C4H4O4

[0010] Molecular weight: 581.62.

[0011] Further, the XRPD pattern of the crystal form also has characteristic peaks at at least one (at least two, at least three, at least four, or all) of the positions of diffraction angle 2θ values of 12.2°±0.2°, 18.5°±0.2°, 22.0°±0.2°, 25.4°±0.2°, 28.6°±0.2°.

[0012] Further, the XRPD pattern of the crystal form further comprises at least one (at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all) of the peaks at diffraction angles 2Q values of 12.4°±0.2°, 13.0°±0.2°, 13.2°±0.2°, 21.4°±0.2°, 23.5°±0.2°, 24.6°±0.2°, 26.2°±0.2°, 27.2°±0.2°, 29.8°±0.2°, 32.0°±0.2°.

[0013] In some embodiments of the application, the XRPD pattern of the crystal form comprises the peaks set forth in Table 8.

[0014] Specifically, the crystal form is triclinic with a space group of Pl and cell parameters of a = 8. 1 1 (1) A, b = 9. 1 1 (1) A, c = 1 1. 1 1 (1) A, a = 1 1. 1 1 (1)°, b = 1 1. 1 1 (1)°, g = 1 1. 1 1 (1)°, V = 1 1 1 1 (1) A3, C = 1. 1 1 (1) g / cm3, F(000) = 48, Z = 1. a = 73.087(2)°, b = 77.109(2)°, g = 88.448(2)°, Z = 1,

[0015] In some embodiments of the application, the crystal form has an XRPD pattern substantially as set forth in FIG. 1.

[0016] Further, the DSC pattern of the crystal form has an endothermic peak at about 175.8 °C.

[0017] In some embodiments of the application, the crystal form has a DSC pattern substantially as set forth in FIG. 2.

[0018] Further, the crystal form loses about 0.5% ± 0.05% weight when heated from room temperature to 163 °C.

[0019] In some embodiments of the application, the crystal form has a TGA pattern substantially as set forth in FIG. 3.

[0020] Further, the crystal form is an anhydrate.

[0021] In a second aspect of the application, there is provided a process for preparing the crystal form C of the first aspect.

[0022] Specifically, the process can be selected from the group consisting of one or more of room temperature suspension stirring, 50 °C suspension stirring, temperature cycling, slow cooling, anti-solvent addition, and rapid evaporation.

[0023] Specifically, for room temperature suspension stirring and 50 °C suspension stirring, the process comprises mixing Compound I with 0.5-1 equivalent (e.g. 0.5, 0.55, 0.6, 0.65, 0.7, 0.8 equivalent) of succinic acid, a solvent, and stirring the suspension at room temperature or 50 °C.

[0024] More specifically, the solvent can be selected from the group consisting of water, methanol, ethanol, acetone, methyl isobutyl ketone, methyl tert-butyl ether, acetonitrile, ethyl acetate, 2-methyltetrahydrofuran, dimethylsulfoxide / isopropyl acetate (e.g. in a ratio of 1 / 2, v / v), tetrahydrofuran / water (e.g. 4 / 1, v / v), methanol / water (e.g. 1 / 1, v / v).

[0025] Specifically, for slow cooling, the preparation method comprises: mixing Compound I with 0.5-1 equivalent (e.g. 0.5, 0.55, 0.6, 0.65, 0.7, 0.8 equivalent) of succinic acid, solvent, warming the resulting mixture to 45-55°C, stirring, and then slowly cooling to 10-25°C, stirring.

[0026] More specifically, the solvent can be selected from the group consisting of water, methanol, ethanol, acetone, methyl isobutyl ketone, methyl tert-butyl ether, acetonitrile, ethyl acetate, 2-methyltetrahydrofuran, dimethylsulfoxide / isopropyl acetate (e.g. in a ratio of 1 / 2, v / v), tetrahydrofuran / water (e.g. 4 / 1, v / v), methanol / water (e.g. 1 / 1, v / v).

[0027] Specifically, for slow cooling, the preparation method comprises: mixing Compound I with 0.5-1 equivalent (e.g. 0.5, 0.55, 0.6, 0.65, 0.7, 0.8 equivalent) of succinic acid, solvent, warming the resulting mixture to 45-55°C, stirring, and then slowly cooling to 10-25°C, stirring.

[0028] In one embodiment of the present application, the solvent is DCM or acetone.

[0029] Specifically, the preparation method further comprises a step of isolating the crystal form, for example by filtration, centrifugation or a combination of both.

[0030] Specifically, the method further comprises a step of drying the crystal form.

[0031] In a third aspect of the present application, a pharmaceutical composition comprising the crystal form C of the first aspect, and one or more pharmaceutically acceptable excipients is provided.

[0032] Specifically, the pharmaceutically acceptable excipients can be selected from one or more of a disintegrant, a binder, a lubricant, a suspending agent, a stabilizer, a filler, an absorption enhancer, a surfactant, a flavoring agent, an antioxidant, a preservative, a pH adjuster, an osmotic pressure adjuster, a buffer, a solubilizer, etc.

[0033] In particular, the crystalline form C of the first aspect can be used alone or in combination with other kinds of active ingredients in the pharmaceutical composition.

[0034] In particular, the pharmaceutical composition can employ any suitable administration route, such as gastrointestinal administration (e.g., oral, rectal administration) or non-gastrointestinal administration (e.g., intravenous, intramuscular, intranasal, intraocular, intracerebral, intravaginal, intraperitoneal, transdermal, subcutaneous, intradermal, respiratory tract administration, etc.). In some embodiments of the present application, the pharmaceutical composition employs an intraocular administration route (e.g., intraocular administration, ocular surface administration, periocular administration, specifically, intravitreal injection administration, anterior chamber injection, vitreous implantation administration; eye drop administration, eye ointment administration, eye gel administration; subconjunctival injection administration, retrobulbar injection, periocular administration, subtenon administration, etc.).

[0035] In particular, the pharmaceutical composition can be any suitable dosage form, such as a gastrointestinal administration dosage form, for example, including, but not limited to, tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, etc.; a non-gastrointestinal administration dosage form, for example, an injection administration dosage form: such as an injection (e.g., for subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), a respiratory tract administration dosage form: such as a spray, an aerosol, a powder spray, etc., a skin administration dosage form: such as a topical solution, a lotion, an ointment, a plaster, a paste, a patch, etc., a mucosal administration dosage form: such as an eye drop, an eye ointment, a nose drop, a gargle, a sublingual tablet, etc., a cavity administration dosage form: such as a suppository, an aerosol, an effervescent tablet, a drop, a drop pill, etc., for rectum, vagina, urethra, nasal cavity, ear canal, etc. In some embodiments of the present application, the pharmaceutical composition is an eye preparation, such as an eye drop (solution, emulsion, suspension), an eye ointment, an eye gel. In some embodiments of the present application, the pharmaceutical composition is a solid dosage form, such as, but not limited to, tablets, pills, powders, granules, capsules, powders, lozenges, films, etc.

[0036] In particular, the various dosage forms of the pharmaceutical composition can be prepared according to conventional production methods in the pharmaceutical field. For example, the active ingredient is mixed with one or more pharmaceutically acceptable excipients, and then it is formed into the desired dosage form.

[0037] In particular, the weight percentage of the crystalline form C of the first aspect in the pharmaceutical composition can be 0.1-99.5%, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, in particular, 0.1-30%.

[0038] In the fourth aspect of the present application, the use of the crystalline Form C of the first aspect in the preparation of a medicament for preventing and / or treating a disease related to protein tyrosine kinases is provided.

[0039] In particular, the tyrosine kinase is VEGFR, for example one or more of VEGFR1, VEGFR2, VEGFR3.

[0040] In particular, the disease is a disease that can be beneficially prevented and / or treated by inhibiting protein tyrosine kinases, for example, a tumor, an autoimmune disease, an inflammatory disease, a disease associated with pathogen infection, a neurodegenerative disease, a cardiovascular disease, a metabolic disease, a fibrotic disease, an ocular disease, in particular, a proliferative disease mediated by protein tyrosine kinases.

[0041] In some embodiments of the present application, the disease is a tumor, in particular a malignant tumor (cancer), including, but not limited to, breast cancer, lung cancer (in particular non-small cell lung cancer), colorectal cancer, renal cancer, liver cancer, pancreatic cancer, ovarian cancer, prostate cancer, glioblastoma, glioma, myeloid dysplasia, mesothelioma, sarcoma, myelodysplastic syndrome, hematological malignancies.

[0042] In particular, the hematological malignancies include leukemia, lymphoma, multiple myeloma (MM).

[0043] In particular, the leukemia can be chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML), acute lymphocytic leukemia (ALL), acute myelocytic leukemia (AML), acute monocytic leukemia.

[0044] In particular, the lymphoma can be Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL) (e.g., diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma, primary mediastinal B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and primary central nervous system (CNS) lymphoma and T-cell NHL, such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma, NK / T-cell lymphoma, in particular diffuse large B-cell lymphoma (DLBCL). In particular, in the application, the treatment of the tumor comprises killing the tumor, preventing metastatic spread of the tumor and growth of micrometastases.

[0045] In some embodiments of the application, the tumor is selected from the group consisting of: renal cell carcinoma, hepatocellular carcinoma, colorectal cancer, lung cancer, gastric cancer, leukemia.

[0046] In some embodiments of the application, the disease is an ocular disease, including, but not limited to, diabetic retinopathy (including nonproliferative (background) diabetic retinopathy, proliferative diabetic retinopathy, and diabetic macular edema); age-related macular degeneration (AMD) (including neovascular (wet / exudative) AMD, dry AMD, and geographic atrophy); pathologic choroidal neovascularization (CNV) from any pathologic mechanism (i.e., high myopia, trauma, sickle cell (anemia) disease; ocular histoplasmosis, angioid streaks, traumatic choroidal rupture, optic nerve drusen, and certain retinal dystrophies); pathologic retinal neovascularization from any pathologic mechanism (i.e., sickle cell retinopathy, Eales disease, ocular ischemic syndrome, carotid-cavernous fistula, familial exudative vitreoretinopathy, hyperviscosity syndrome, idiopathic retinal vasculitis, birdshot retinochoroidopathy, retinal vasculitis, sarcoid, or toxoplasmosis); uveitis; retinal vein occlusion (central or branch); ocular trauma; surgical-induced edema; surgical-induced neovascularization; cystoid macular edema; ocular ischemia; retinopathy of prematurity; Coat's disease (Coats' disease, also known as exudative retinopathies, retinal telangiectasia); sickle cell retinopathy and / or neovascular glaucoma, retinoblastoma.

[0047] In one embodiment of the application, the disease is diabetic retinopathy, including nonproliferative (background) diabetic retinopathy, proliferative diabetic retinopathy, and diabetic macular edema.

[0048] In another embodiment of the application, the disease is age-related macular degeneration (AMD), including neovascular (wet / exudative) AMD, dry AMD, geographic atrophy.

[0049] In some embodiments of the application, the disease is an autoimmune disease, including, but not limited to: organ-specific autoimmune diseases, systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, autoimmune thyroid disease, ulcerative colitis, and the like.

[0050] In some embodiments of the present application, the disease is an inflammatory disease, including but not limited to: osteoarthritis, acute gout, multiple sclerosis, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), neuroinflammation, asthma, chronic obstructive airway disease, lung inflammation, myositis, eczema, dermatitis, acne, cellulitis, occlusive disease, thrombosis, alopecia, nephritis, vasculitis, retinitis, uveitis, scleritis, sclerosing cholangitis, hypophysitis, thyroiditis, septic shock, systemic inflammatory response syndrome (SIRS), toxic shock syndrome, acute lung injury, ARDS (adult respiratory distress syndrome), acute kidney failure, burn, pancreatitis (e.g., acute pancreatitis), postoperative syndrome, sarcoidosis, Herxheimer reaction, encephalitis, myelitis, meningitis, malaria, and the like.

[0051] In some embodiments of the present application, the disease is a neurodegenerative disease, including but not limited to: Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease, and the like.

[0052] In some embodiments of the present application, the disease is a pathogen infection related disease, including but not limited to: influenza, SARS, COVID-19, viral hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, hepatitis D, and the like), AIDS, rabies, dengue fever, Ebola virus disease, and the like.

[0053] In some embodiments of the present application, the disease is a cardiovascular disease, including but not limited to: coronary heart disease, peripheral arterial disease, atherosclerosis, ischemic heart disease, ischemic cardiomyopathy, myocardial infarction, heart failure, angina pectoris, myocarditis, hypercholesterolemia, hypertension, ischemia-reperfusion injury, cerebrovascular ischemia (stroke), embolism (e.g., pulmonary embolism, renal embolism, hepatic embolism, gastrointestinal embolism, or peripheral limb embolism), or myocardial ischemia, and the like.

[0054] In some embodiments of the present application, the disease is a metabolic disease, including but not limited to: diabetes (e.g., type I diabetes, type II diabetes, or gestational diabetes), obesity, fatty liver (NASH or others), cachexia, hypercholesterolemia, gout, and the like.

[0055] In some embodiments of the present application, the disease is a fibrotic disease, including but not limited to: myocardial fibrosis, pulmonary fibrosis, renal fibrosis, post-surgical stenosis, keloid formation, cirrhosis, biliary cirrhosis, scleroderma, and the like.

[0056] In the fifth aspect of the present application, there is provided a method for preventing and / or treating a protein tyrosine kinase-mediated proliferative disease, comprising the step of administering to a subject in need thereof an effective amount of the crystalline Form C of the first aspect, or the pharmaceutical composition of the third aspect.

[0057] In particular, the disease is as described in the fourth aspect of the present application.

[0058] In particular, the subject is a mammal, for example, a human.

[0059] In particular, the administration can employ any suitable route of administration, for example, a gastrointestinal (e.g., oral) or non-gastrointestinal (e.g., intravenous, intramuscular, intranasal, intraocular, intracerebral, intravaginal, intraperitoneal, transdermal, subcutaneous, intradermal, respiratory tract, etc.) route.

[0060] The present application provides a hemi-succinate salt crystalline form of a protein tyrosine kinase inhibitor. During the research and development process, the inventors previously prepared a protein tyrosine kinase inhibitor compound ((S)-1-(3-(5-(cyclopropylcarbamoyl)-2-fluorobenzyloxy(4- carbamoylisothiazol-5-yl)-3-(3-(3-fluoropyrrolidin-1-yl)propyl)urea, Compound I) which has the ability to inhibit anti-angiogenic tyrosine kinases, in addition to effectively antagonizing VEGFR1, VEGFR2, VEGFR3 tyrosine kinase activity, has high selectivity for inhibiting EGFR tyrosine kinase activity, can effectively reduce and avoid side effects. On this basis, the inventors further studied and obtained a hemi-succinate salt crystalline Form C of the compound, which has good solid chemical stability and physical stability, and almost no hygroscopicity, can be used as an advantage crystalline form for drug research and development, and has very good application value in downstream development and industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 shows the XRPD pattern of crystalline Form C.

[0062] Figure 2 shows the DSC pattern of crystalline Form C.

[0063] Figure 3 shows the TGA pattern of crystalline Form C.

[0064] Figure 4 shows the H NMR pattern of crystalline Form C. 1 H NMR pattern.

[0065] Figure 5 shows the single crystal structure Ortep diagram of crystalline Form C.

[0066] Figure 6 shows the asymmetric unit of crystalline Form C.

[0067] Figure 7 shows the intermolecular hydrogen bond interaction of crystalline Form C.

[0068] Figure 8 shows XRPD overlays of Form C samples over 1 week under different conditions.

[0069] Figure 9 shows XRPD overlays of Form C samples over 4 weeks under different conditions. DETAILED DESCRIPTION

[0070] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0071] The disclosures of various publications, patents and published patent specifications, referred to herein are hereby incorporated by reference in their entirety.

[0072] Explanations of some abbreviations used in the present application are as follows:

[0073] XRPD: X-ray powder diffraction

[0074] SCXRD: single crystal X-ray powder diffraction

[0075] DSC: differential scanning calorimetry

[0076] TGA: thermogravimetric analysis

[0077] 1 H HMR: hydrogen nuclear magnetic resonance

[0078] PLM: polarized light microscopy

[0079] DVS: dynamic vapor sorption analysis

[0080] Ortep: Oak Ridge Thermal Ellipsoid Plot

[0081] In the present application, the term "crystal form" is confirmed by X-ray powder diffraction patterns. Those skilled in the art will appreciate that the physicochemical properties discussed herein can be characterized with experimental error that depends on the conditions of the instrument, the preparation of the sample, and the purity of the sample, among other things. In particular, it is well known in the art that X-ray diffraction patterns will vary somewhat depending on the conditions of the instrument. In particular, it is noted that the relative intensities of the X-ray powder diffraction pattern can vary with experimental conditions, and therefore the order of peak intensities should not be used as an absolute or exclusive indicator. In fact, the relative intensities of the diffraction peaks in an XRPD pattern are related to the preferred orientation of the crystals, and the peak intensities shown herein are illustrative and not intended for absolute comparison. In addition, the experimental error in peak angles is typically 5% or less, and this error in angles should be taken into account, typically allowing for a ± 0.2° error. In addition, there can be a general shift in peak angles due to experimental factors such as sample thickness, and some shift is typically allowed. Thus, those skilled in the art will appreciate that the X-ray powder diffraction pattern of a crystal form of the present application need not be identical to the X-ray powder diffraction pattern of the examples set forth herein, and that "XRPD pattern identical" does not mean absolutely identical, but that the peak positions can differ by ± 0.2° and that the peak intensities allow for some variability. Any crystal form having a pattern that is the same or similar to the characteristic peaks of these patterns is within the scope of the present application. Those skilled in the art will be able to compare the patterns set forth herein with the patterns of an unknown crystal form to confirm whether the two sets of patterns reflect the same or different crystal forms.

[0082] In some embodiments, the crystal forms of the present application are pure, single, and substantially free of any other crystal form. In the present application, "substantially free of" when used in reference to a new crystal form indicates that the crystal form contains less than 20% by weight of another crystal form, more particularly less than 10% by weight of another crystal form, more particularly less than 5% by weight of another crystal form, more particularly less than 1% by weight of another crystal form.

[0083] It is noted that the numerical values and ranges recited in this application are not intended to be narrowly limited to the precise values or range recited, but rather are intended to encompass variations which are apparent to those skilled in the art. In the present application, such variations are contemplated to be within the scope of the present application, and are to be understood to be "about" the numerical values recited. When the term "about" is used in connection with a numerical value recited in the present application, and refers to that value, it means within ± 10% of that value, preferably within ± 5% of that value, more preferably within ± 2% of that value, and more preferably within ± 1% of that value. For example, "about 10" is to be interpreted as meaning 9-11, preferably 9.5-10.5, more preferably 9.8-10.2, and more preferably 9.9-10.1.

[0084] In the present invention, the term "room temperature" refers to the temperature of an article being close to or the same as the temperature of the space in which the article is located, such as the location of a fume hood. Typically, room temperature is about 20 °C to about 30 °C, or about 22 °C to 27 °C, or about 25 °C.

[0085] Anti-solvent addition (also known as anti-solvent crystallization, precipitation crystallization, salting-out or antecrystallization) is a method of crystallization by adding one or more anti-solvents to a solution in which the target product is dissolved in a good solvent, the product is in a slightly soluble state in the solution, so that the solution is in a supersaturated state after the addition of anti-solvents, and then the product is crystallized.

[0086] The anti-solvent has a poorer solubility for the target product than the good solvent, such as more than 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% poorer, so the anti-solvent in the system is relative. The good solvent and the anti-solvent can be polar solvents or non-polar solvents, such as one or more selected from dimethylformamide (DMF), dimethyl sulfoxide (DMSO), water, alcohol solvents, ether solvents, ketone solvents, ester solvents, alkane solvents, aromatic hydrocarbon solvents, nitrile solvents. Among them, the alcohol solvent includes but is not limited to methanol, ethanol, propanol, isopropanol or 1,3-propanediol, 1,2-propanediol or trichloro-tert-butyl alcohol or a combination thereof; the ether solvent includes but is not limited to, such as tetrahydrofuran, methyl tert-butyl ether or 1,4-dioxane or a combination thereof; the ketone solvent includes but is not limited to acetone, methyl ethyl ketone or 4-methyl-2-pentanone or a combination thereof; the ester solvent includes but is not limited to ethyl acetate, isopropyl acetate, n-butyl acetate or tert-butyl acetate or a combination thereof; the alkane solvent includes but is not limited to dichloromethane, chloroform, n-hexane, cyclohexane or pentane or n-heptane or a combination thereof; the aromatic hydrocarbon solvent includes but is not limited to benzene, toluene or a combination thereof; the nitrile solvent includes but is not limited to acetonitrile, malononitrile.

[0087] The anti-solvent addition and anti-anti-solvent addition can be carried out by batch, semi-batch or continuous crystallization operation. The anti-solvent is added to the solution (anti-solvent crystallization) or the product solution is added to the anti-solvent (anti-anti-solvent crystallization), which can be added at a constant rate, or slowly at the beginning and then gradually increase the rate.

[0088] The technical solutions of the present invention will be described clearly and completely below in combination with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present invention.

[0089] The instruments and parameters used by the detection means involved in the following examples are as follows

[0090] 1. XRPD:

[0091] Instrument: Bruker D8 Advance

[0092] X-ray optics: Reflection mode

[0093] Detector: LYNXEYE_XE_T (1D mode)

[0094] Opening angle: 2.9° (max)

[0095] Radiation source: Cu / K-Alpha1

[0096] X-ray source power: 40 kV, 40 mA

[0097] Primary optics slit: Double primary motorized slit: 10.0 mm; Primary soller slit: 2.5°

[0098] Secondary optics slit: Secondary soller slit: 2.5°

[0099] Scan mode: Continuous scan

[0100] Scan type: Two-theta mode

[0101] Step size: 0.02°

[0102] Step time: 0.4 s / step

[0103] Scan range: 2° to 40°

[0104] Sample rotation speed: 15 rpm

[0105] Sample plate: Single crystal silicon wafer, flat plate

[0106] 2. DSC:

[0107] Instrument: TA Instruments Discovery 2500

[0108] Sample pan: Tzero pan and Tzero hermetic lid with ~0.7 mm diameter hole

[0109] Temperature range: 0 to 250 °C or until decomposition

[0110] Heating rate: 10 °C / min

[0111] Nitrogen flow rate: 50 mL / min

[0112] Sample size: ~0.5-5 mg

[0113] 3. TGA:

[0114] Instrument: TA Instruments Discovery 5500

[0115] Sample pan: aluminum pan, open

[0116] Starting temperature: ambient temperature (less than 35°C)

[0117] Ending temperature: 300°C or less than 80% of initial sample weight remains

[0118] Heating rate: 10°C / min

[0119] Nitrogen flow rate: 10 mL / min for equilibration; 25 mL / min for sample chamber

[0120] Sample size: about 2-10 mg

[0121] 4. PLM:

[0122] Instrument: Leica DM4 P

[0123] Method: crossed polarized light, drop of silicone oil

[0124] 5. NMR:

[0125] Instrument: Bruker Avance-AV 400M

[0126] Frequency: 400 MHz

[0127] Probe: 5 mm PABBO BB / 19F-1H / D Z-GRD Z108618 / 0406

[0128] Number of scans: 8

[0129] Temperature: 297.6 K

[0130] Relaxation delay: 1 s

[0131] 6. HPLC:

[0132] Instrument: Agilent 1260 infinity II Binary Pump

[0133] Method:

[0134] Wavelength: 254 nm

[0135] Column: Agilent poroshell 120 EC-C18 (150 mm*4.6 mm, 2.7 μm) Detector: DAD

[0136] Column temperature: 25 °C

[0137] Flow rate: 0.8 mL / min

[0138] Mobile phase A: 0.1% formic acid in water, v / v

[0139] Mobile phase B: 0.1% formic acid in ACN, v / v

[0140] Diluent: Acetonitrile: water = (1 : 1, v:v)

[0141] Injection volume: 2 pL

[0142] Table 1 Gradient

[0143] 7. DVS:

[0144] Instrument: Intrinsic

[0145] Total gas flow: 200 seem

[0146] Temperature: 25 °C

[0147] Solvent: Water

[0148] Method: Cycle: 40-0-95-0-40% RH

[0149] Step: 10% RH

[0150] Equilibration: 0.002 dm / dt (% / min)

[0151] Minimum dm / dt hold time: 60 min

[0152] Maximum dm / dt hold time: 360 min

[0153] Sample size: ~5-50 mg

[0154] 8. SCXRD:

[0155] Instrument: Bruker D8 Venture

[0156] Detector: CMOS area detector

[0157] Test temperature: 170 K

[0158] Radiation source: Cu / K-Alpha 1

[0159] X-ray source power: 50 kV, 1.2 mA

[0160] Sample to detector distance: 40 mm

[0161] Exposure time: 10 seconds

[0162] Resolution:

[0163] Structure solution and refinement were performed with reference to Dolomanov, O. V., Bourhis, L. J., Gildea, R. J., Howard, J. A. K. & Puschmann, H. (2009). J. Appl. Cryst. 42, 339-341; Sheldrick, G. M. (2015). Acta Cryst. A71, 3-8. ORTEP program was used to obtain the single crystal analysis structure ellipsoid plot.

[0164] The cell parameters obtained from the crystal structure determination have the following meanings:

[0165] a, b, c are the edge lengths of the unit cell; a, b, g are the angles between the unit cell axes; V is the volume of the unit cell; Z is the number of formula units in the unit cell.

[0166] Example 1: Synthesis of compound I

[0167] The synthesis route is as follows:

[0168] First step

[0169] 3-[(3S)-3-fluoropyrrolidin-1-yl]propanenitrile

[0170] 3-[(3S)-3-fluoropyrrolidin-1-yl]propanenitrile

[0171] (3S)-3-fluoropyrrolidine hydrochloride (5 g, 39.8 mmol) was dissolved in acetonitrile (48.7 mL), 3-bromopropionitrile (5.3 g, 39.82 mmol) and potassium carbonate (16.5 g, 119.5 mmol) were added, and the reaction system was stirred at 60°C for 16 hours. The reaction liquid was poured into water (50 mL), extracted with ethyl acetate (50 mL x 3), the organic phases were combined and washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product, which was purified by neutral alumina column chromatography (DCM) to obtain the colorless oily product 3-[(3S)-3-fluoropyrrolidin-1-yl]propanenitrile (IM-1) (3.4 g, 23.9 mmol, 60.06% yield).

[0172] The product was confirmed by LCMS and H-NMR

[0173] 1H NMR (400 MHz, CDC13): δ (ppm) 5.28 - 5.06 (m, 1H), 3.00 - 2.73 (m, 5H), 2.64 - 2.44 (m, 3H), 2.26 - 1.97 (m, 2H).

[0174] Second step

[0175] 3-[(3S)-3-fluoropyrrolidin-1-yl]propan-1-amine

[0176] 3-[(3S)-3-fluoropyrrolidin-1-yl]propan-1-amine

[0177] 3-[(3S)-3-fluoropyrrolidin-1-yl]propan-1-amine

[0178] The product was confirmed by LCMS and H-NMR.

[0179] 1 H NMR (400 MHz, CDC13): δ (ppm) 5.83 - 4.93 (m, 1H), 2.93 - 2.34 (m, 7H), 2.17 - 1.48 (m, 6H).

[0180] Third step

[0181] methyl

[0182] 3-[[5-(cyclopropylcarbamoyl)-2-fluoro-phenyl]methoxy]-5-[3-[(3S)-3-fluoropyrrolidin-1-yl]propylcarbamoylamino]isothiazole-4-carboxylate

[0183] 3-[[5-(cyclopropylcarbamoyl)-2-fluoro-phenyl]methoxy]-5-[3-[(3S)-3-fluoropyrrolidin-1-yl]propylcarbamoylamino]isothiazole-4-carboxylate

[0184] To a solution of 3-[(3S)-3-fluoropyrrolidin-1-yl]propylamine (IM-2) (196.1 mg, 1.3 mmol) in anhydrous THF (5 mL) was added CDI (217.5 mg, 1.3 mmol) under N2protection. The reaction was stirred at 25 °C for 1.0 h. DMSO (5 mL) was added, THF was removed under reduced pressure, 5-amino-3-[[5-(cyclopropylcarbamoyl)-2-fluoro-phenyl]methoxy]isothiazole-4-carboxylic acid methyl ester (IM-3) (350 mg, 957.9 μmol) and potassium carbonate (397.2 mg, 2.9 mmol) were added. The reaction was stirred at 25 °C for 1.0 h. The reaction was poured into water (60 mL), the mixture was extracted with ethyl acetate (20 mL x 3), the organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product. The crude product was purified by preparative thin layer chromatography (DCM / MeOH = 10 / 1) to give 3-[[5-(cyclopropylcarbamoyl)-2-fluorophenyl]methoxy]-5-[3-[(3S)-3-fluoropyrrolidin-1-yl]propylcarbamoylamino]isothiazole-4-carboxylic acid methyl ester (IM-4) (175 mg, 325.5 μmol, 33.98% yield) as a yellow solid.

[0185] The product was confirmed by LCMS.

[0186] Fourth step

[0187] 3-[[5-(cyclopropylcarbamoyl)-2-fluoro-phenyl]methoxy]-5-[3-[(3S)-3-fluoropyrrolidin-1-yl]propylcarbamoylamino]isothiazole-4-carboxamide

[0188] 3-[[5-(cyclopropylcarbamoyl)-2-fluoro-phenyl]methoxy]-5-[3-[(3S)-3-fluoropyrrolidin-1-yl]propylcarbamoylamino]isothiazole-4-carboxamide

[0189] Methyl 3-[[5-(cyclopropylcarbamoyl)-2-fluorophenyl]methoxy]-5-[3-[(3S)-3- fluoropyrrolidin-1-yl]propylcarbamoylamino]isothiazole-4-carboxylate (IM-4) (175 mg, 325.5 μmol) was dissolved in NH3 / MeOH (10 mL, 10 mol / L) and DMSO (0.3 mL). The mixture was stirred at 40 °C for 16 h. The reaction solution was concentrated under reduced pressure to give the crude product, which was purified by preparative thin layer chromatography (DCM / MeOH = 10 / 1) to give the product 3-[[5-(cyclopropylcarbamoyl)-2-fluorophenyl]methoxy]-5-[3-[(3S)-3- fluoropyrrolidin-1-yl]propylcarbamoylamino]isothiazole-4-carboxamide (Compound I) as a white solid (40 mg, 76.55 μmol, 23.51% yield).

[0190] The product was confirmed by LCMS, H-NMR, C-NMR and F-NMR.

[0191] 1 H NMR (400 MHz, CD3OD): δ (ppm) 8.00 (dd, J = 7.2, 2.4 Hz, 1H), 7.88-7.82 (m, 1H), 7.24 (t, J = 7.2 Hz, 1H), 5.56 (s, 2H), 5.26-5.06 (m, 1H), 3.27 (t, J = 6.8 Hz, 2H), 3.02-2.88 (m, 2H), 2.86-2.79 (m, 1H), 2.72-2.64 (m, 2H), 2.64-2.53 (m, 2H), 2.46-2.36 (m, 1H), 2.27-2.11 (m, 1H), 2.09-1.91 (m, 1H), 1.83-1.70 (m, 1H), 0.84-0.75 (m, 2H), 0.67-0.57 (m, 2H).

[0192] 13 C NMR (101 MHz, CD3OD) δ 170.35, 167.16, 165.71, 163.19, 162.97, 156.10, 132.09, 131.57, 131.52, 131.18, 131.08, 125.08, 124.93, 116.86, 116.63, 98.73, 95.18, 93.44, 65.16, 61.76, 61.53, 54.71, 53.40, 40.43, 39.41, 33.57, 33.34, 29.60, 24.07, 6.55.

[0193] 19F NMR (400 MHz, CD3OD) δ (ppm) -115.43, -168.98.

[0194] Example 2: Detection of the ability of compounds to inhibit tyrosine kinases

[0195] The assay uses Mobility Shift Assays / IMAP TM The ability of compounds to inhibit kinase phosphorylation was monitored directly by measuring unphosphorylated and phosphorylated substrates in the kinase reaction. The reagents and instruments used are shown in the table below.

[0196] Table 2 Reagents and instruments

[0197] Experimental procedure:

[0198] The kinase and DTT were added to 1x kinase buffer to form a 2.5x kinase solution; 10 microliters of the 2.5x kinase solution was transferred to the 384 well plate reaction plate, and 1x kinase buffer was added to the negative control well. Incubate at room temperature for 10 minutes; the FAM labeled peptide, MgCl2, ATP, etc. were added to 1x kinase buffer to form a 2.5x substrate solution; 10 microliters of the 2.5x substrate solution was transferred to the 384 well plate reaction plate; incubate at 28°C for different time, and 25 microliters of stop solution was added to stop the reaction. Read the conversion rate data on the Caliper EZ Reader II. Convert the conversion rate to inhibition rate data.

[0199] "Min" is the reading of the control well without enzyme; "Max" is the reading of the control well with DMSO. IC50values were fitted using XLFit excel add-in version 5.4.0.8. 50

[0200] X is the log value of the concentration of the compound, and Y is the inhibition rate (% inhibition).

[0201] IC50of Compound I against tyrosine kinases 50 The results are shown in the table below, with the compound PAN90806 (commercial product) as the positive control.

[0202] Table 3 Experimental results

[0203] ​Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) are known as the most potent vascular permeability agents and endothelial cell specific, playing a key role in the proliferation, migration and angiogenesis of endothelial cells. Angiogenesis is an important mechanism in many physiological and pathological processes, involving the proliferation, migration and survival of endothelial cells, which in turn leads to further capillary formation, ultimately promoting the formation of blood vessels. Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) play an important role in angiogenesis associated with pathologies such as tumor development and ocular neovascular diseases. For example, the expression level of VEGF is positively correlated with the degree of vascularization of tumor tissue. VEGF acts on VEGFR receptors, activates the phosphorylation of VEGFR receptor tyrosine kinase and leads to abnormal cell signal transduction, thereby promoting the proliferation of endothelial cells and the formation of new blood vessels, and is the main participant in many different cancers and eye diseases accompanied by pathological neovascularization. However, although people have tried to design VEGFR receptor-specific molecules, it is inevitable to have some cross-reactions with other "off-target" receptors, such as inhibition of TIE2 receptor (i.e. TEK tyrosine kinase), or inhibition of EGFR receptor activity. It has been clinically observed that receptor tyrosine kinase inhibitors, because of the inhibition of VEGFR, also inhibit EGFR, affecting EGFR-mediated corneal epithelial wound healing, thus causing adverse side effects on the eye. In addition, TIE2 plays a crucial role in maintaining vascular integrity, and its inhibition can lead to weakened endothelial cell connections, promoting fluid leakage.

[0204] As can be seen from the results shown in Table 3, compound I has a significant improvement in selectivity in inhibiting TIE2 and EGFR. Not only does it improve the antagonistic activity of all VEGFR receptors (VEGFR1, VEGFR2, VEGFR3) tyrosine kinase, but it also significantly improves the selectivity of inhibiting TIE2 and EGFR receptor activity, which is beneficial to reduce and avoid side effects.

[0205] Example 3: Test of the ability of the compound to inhibit VEGFR2 phosphorylation in HUVEC cells

[0206] VEGF is a potent growth factor that promotes angiogenesis and vascular permeability. Vascular endothelial growth factor receptor 2 (VEGFR2) is the primary receptor for VEGF-induced endothelial cell signaling. Upon ligand binding, VEGFR2 undergoes autophosphorylation and is activated. The primary function from VEGFR2 signaling is to induce vascular formation during development and after tissue injury, and to bypass blocked blood vessels. Pathologically, VEGF and VEGFR2 are involved in tumor angiogenesis and vascular leakage. They are considered to be the primary players in many different cancers and ocular diseases that are accompanied by pathological neovascularization. This assay measures the ability of compounds to inhibit VEGF-induced VEGFR2 autophosphorylation (pVEGFR2) in HUVEC cells using an ELISA. The reagents and instruments used are shown in the table below.

[0207] Table 4 Reagents and Instruments

[0208] Experimental Procedure:

[0209] Prior to seeding cells, cells were brought to 60-80% confluency in cell culture flasks. Cell suspensions were diluted to 4.5 x 10 6cells / mL. 80 microliters of cell suspension was added to all wells of a 96-well cell plate (Corning-3599); incubated overnight at 37°C with 5% CO2. Compound powder was dissolved in DMSO to 10 mM and stored in a nitrogen cabinet. Compound dilution master plate: in a 96V-well plate (Axygen-WIPP02280), add reference compound Ponatinib or test compound at concentrations, starting from 5 mM (5 microliters of 10 mM compound solution plus 5 microliters of DMSO) with 3-fold gradient dilution, total of 8 concentration points. For 100% inhibition control, reference compound Ponatinib was at a concentration of 2.5 mM (3 microliters of 10 mM compound solution plus 9 microliters of DMSO) in the V-well plate. 20 microliters of diluted compound was transferred from the middle plate to the cell plate. The cell plate was then incubated at 37°C with 5% CO2for 1 hour. After 1 hour of compound treatment, 25 microliters of 250 ng / mL human VEGF was added to the 96-well plate, with a final concentration of 50 ng / mL human VEGF; after 5 minutes of incubation at 37°C with 5% CO2, centrifuged at 3000 rpm for 10 minutes, the medium was removed and the cells were washed once with 300 microliters / well of pre-cooled DPBS. Then centrifuged at 4000 rpm for 10 minutes, remove DPBS and add 110 microliters / well of pre-cooled 1X cell lysis buffer in the 96-well plate. The plate was shaken at 4°C for 40 minutes. Then the plate was frozen in the -80°C refrigerator. The next day, the cell plate was taken out from -80°C, after thawing, centrifuged at 3500 rpm for 10 minutes, 100 microliters / well of supernatant was added to the ELISA detection plate. Seal the plate and incubate at 37°C for 2 hours. Wash the wells 4 times with 1X Wash Buffer. Add 100 microliters of recombinant detection antibody to each well. Seal the plate, after incubating the plate at 37°C for 1 hour, repeat the plate washing steps. Add 100 microliters of secondary antibody to each well. Seal the plate, after incubating the plate at 37°C for 30 minutes, repeat the plate washing steps. Add 100 microliters of TMB to each well (protect from light). Seal the plate, after incubating the plate at 37°C for 30 minutes, add 100 microliters of stop solution to each well. Gently shake for 30 seconds, and read the optical density at 450 nm within 30 minutes using a 340PC 384Absorbance Microplate Reader to measure absorbance, reading the optical density at 450 nm.

[0210] “Human VEGF stimulated cells with DMSO” is the negative control (0% inhibition), “Human VEGF stimulated cells with 5 mM Ponatinib” is the positive control (100% inhibition), all other values are expressed as a percentage of this control (inhibition).

[0211] IC50values were determined using the 4 Parameter Logistic Model or Sigmoidal Dose-Response Model for curve fitting 50

[0212] X is the compound concentration log value, Y is the inhibition rate (% inhibition), Top is the negative control well reading; Bottom is the positive control well reading.

[0213] IC50of compound I on pVEGFR2 50 The results are shown in Table 6, with compound PAN90806 (commercial product) as positive control.

[0214] Example 4: Detection of the inhibitory effect of compounds on VEGF-induced HUVEC cell proliferation

[0215] This experiment used human umbilical vein endothelial cells (HUVEC, Lonza). HUVEC cells are primary cells isolated from umbilical vein. This cell line is a model system for studying endothelial cell function. Endothelial cell proliferation in response to VEGF plays an important role in angiogenesis associated with pathologies such as tumor development and ocular neovascular diseases.

[0216] This assay used the luciferase bioluminescence assay (CellTiter GLO TM , Promega) to determine the inhibitory effect of compounds on VEGF-induced HUVEC cell proliferation and viability. CellTiter GLO TM is a homogeneous method for quantifying the number of viable cells in culture based on ATP, an indicator of metabolically active cells. The assay incubated the compounds with HUVEC cells for 72 hours, and then measured the resulting inhibitory effect of the compounds on VEGF-induced HUVEC cell proliferation on an EnVision plate reader.

[0217] The reagents and instruments used are shown in the following table.

[0218] Table 5 Reagents and instruments

[0219] Experimental procedure:

[0220] ​HUVEC cell proliferation assay was performed in RPMI-1640 medium (with 5% FBS, 1% Penicillin-Streptomycin). Cell suspension was added to 96-well plates, 100 microliter per well, i.e. 4000 HUVEC cells per well, and incubated at 37°C, 5% CO2 incubator overnight (Serum starvation). After 24 hours, 96 microliter of assay medium was added to the cell wells, and then 2 microliter of compound was added to the cell wells; 1 hour after the addition of compound, 2 microliter of 2 microgram / milliliter VEGF was added to the cell wells. Wells with cells plus 0.1% DMSO and 20 nanogram / milliliter VEGF were used as high reading control wells. Wells without cells but with medium were used as low reading control wells. The final concentration of the tested compounds was: 1000, 333, 111, 37, 12.3, 4.12, 1.37, 0.46, 0.15, 0 nM. After incubation of the cell plates in the incubator for 72 hours, the cell plates were taken out of the incubator and placed at room temperature, and 100 microliter of medium was discarded from each well. 100 microliter of CTG reagent (CellTiter Glo kit) was added to each well, and the plate was placed on a rapid shaker for 3 minutes. The plate was incubated at room temperature for 30 minutes in the dark. The chemiluminescence signal was read using an Envision instrument. The IC50 (half maximal inhibitory concentration) of the tested compounds was calculated using GraphPad Prism 8 software with the following non-linear fitting equation 4 Parameter Logistic Model or Sigmoidal Dose-Response Model: 50 50

[0221] X is the log value of the concentration of the compound, Y is the inhibition rate (% inhibition), Top is the reading of the high reading control wells, and Bottom is the reading of the low reading control wells.

[0222] IC50 of Compound I on HUVEC cell proliferation 50 The results are shown in the following table, with compound PAN90806 (commercial product) as the positive control.

[0223] Table 6 Experimental results

[0224] VEGFR2 is the main receptor for VEGF-induced endothelial cell signaling. After the binding of ligand VEGF and receptor during development and / or after tissue damage, VEGFR2 is autophosphorylated and activated, inducing angiogenesis and bypassing blocked blood vessels. Clinical treatment targeting the vascular endothelial growth factor VEGF-A / VEGFR2 signaling pathway has been proven to be an effective approach to treat ocular neovascular diseases such as wet AMD.​​

[0225] As shown in Table 6, compound I has a significant inhibitory ability on VEGF-induced VEGFR2 autophosphorylation (pVEGFR2) in human endothelial cells, so as to block the signal transduction of abnormal cells and thereby inhibit neovascularization. The main function from VEGFR receptor signaling is to promote the proliferation of endothelial cells and neovascularization, and compound I shows an inhibitory ability on VEGF-induced human endothelial cell proliferation at a nanomolar concentration level. Therefore, compound I is a novel tyrosine kinase inhibitor, which can be applied not only to the treatment of neovascular age-related macular degeneration and diabetic retinopathy, but also to tumor indication therapy, by blocking the tumor neovascularization, blocking the blood and nutrient supply required for tumor growth, and leading to tumor cell death.

[0226] Example 5: Solubility test of compound

[0227] The reagents used are shown in the following table.

[0228] Table 7 Reagents

[0229] Experimental procedure:

[0230] Accurately weigh 3 portions of about 2 mg of compound, and add a suitable volume of buffer with pH of 2.0, 6.5 and 7.4 respectively to obtain a solution with a concentration of 2 mg / ml. After ultrasonic treatment for 10 minutes, fix on a shaker and shake at room temperature for 8 hours. After shaking, ultrasonic treatment for 10 minutes, centrifugation at 13000 rpm for 15 minutes. Take 0.1 ml of supernatant into a new tube, shake and rinse with 5 ml, and then discard the liquid. Again transfer 0.5 ml of supernatant into a new tube, centrifuge at 13000 rpm for 15 minutes, and then take the supernatant (if necessary, dilute with water) for LC-MS injection analysis. The sample concentration is quantified by 3-5 point fitting calibration curve.

[0231] Experimental results:

[0232] The solubility of compound I is 1085.50 ug / ml, while the solubility of the control compound PAN90806 (commercial product) is only 124.50 ug / ml.

[0233] Example 6: Preparation of crystal form

[0234] 1. Experimental method

[0235] (1) Add DCM (5V) to the reaction bottle;

[0236] (2) Add compound I to the reaction bottle;

[0237] (3) Add succinic acid (1.0 eq.) to the reaction bottle;

[0238] (4) Heat the reaction flask to 45-55℃;

[0239] (5) Stir at 45-55℃ for 2 hours;

[0240] (6) Slowly cool to 10-25℃ and stir for 16 hours;

[0241] (7) Filtration: The filter cake is washed with 1-2V DCM and then dried to obtain the product.

[0242] Samples were taken and analyzed using XRPD, SCXRD, DSC, TGA, and... 1 Further characterization was performed using H-NMR and other methods.

[0243] 2. Experimental Results

[0244] The crystalline product was prepared, and its test results are shown below.

[0245] The purity of the obtained sample (HPLC detection) was 99.6%.

[0246] The XRPD pattern of the obtained sample is shown in Figure 1, and its XRPD data is shown in the table below, indicating that it is a crystal and named crystal form C.

[0247] Table 8 XRPD Data for Crystal Form C

[0248] The DSC spectrum of crystal form C is shown in Figure 2, where the sample has a sharp endothermic peak at approximately 175.8 °C (peak temperature).

[0249] The TGA spectrum of crystal form C is shown in Figure 3, in which the sample lost approximately 0.5% weight when heated from approximately 33°C to 163°C.

[0250] Crystal form C 1 The H NMR spectrum is shown in Figure 4. The results show that the molar ratio of free state (free base) to succinic acid is 1:0.5 (the resulting crystal form is the crystal form of the hemisuccinate of compound I, which is sometimes simply referred to as crystal form C in this invention).

[0251] The single-crystal structure diagram of crystal form C is shown in Figure 5-7, and the relevant measurement data are shown in Table 9. The results show that crystal form C belongs to the triclinic crystal system, space group P1. At 170K, R... intThe value is 5.7%, the final R1[I>2s(I)] is 6.2%, and the Flack parameter is 0.08(3). The asymmetric unit (Figure 6) contains 2 molecules of Compound I and 1 molecule of succinic acid, and the transfer of a proton from the carboxyl group of succinic acid to the nitrogen atom on the pyrrolidine of Compound I can be observed (Figure 7). The Ortep diagram in the single crystal data is shown in Figure 5, which confirms the absolute configuration of C2 (adjacent to F) in the structure of Compound I. The chirality of the carbon atoms is determined according to the Cahn-Ingold-Prelog rule and verified by ChemDraw Professional 16.0 software. The XRPD spectrum obtained based on the single crystal data simulation is consistent with the measured XRPD spectrum of Form C (Figure 1). Therefore, the obtained single crystal structure represents the crystal structure of the hemisuccinate salt of Compound I Form C.

[0252] Table 9 Crystal structure data

[0253] The inventors further investigated the solid stability, solubility and hygroscopicity of Form C.

[0254] Example 7: Investigation of the solid stability of Form C

[0255] 1. Experimental method

[0256] Open containers containing Form C (prepared in Example 6) were placed at 25°C / 92.5% RH and 40°C / 75% RH for 1 week and 4 weeks, respectively. Closed containers containing Form C were placed at 60°C for 1 week and 4 weeks. The stability samples under these conditions were detected by XRPD, HPLC and the color change of the samples was observed.

[0257] 2. Experimental results

[0258] The experimental results are shown in the following table.

[0259] Table 10 Investigation results of solid stability Note: A: no color change; B: slight discoloration; C: moderate discoloration; D: severe discoloration

[0260] From the results in the above table, Form C showed good physical and chemical stability under the above accelerated stability conditions for 1 week and 4 weeks.

[0261] Example 8: Investigation of the solubility of Form C

[0262] 1. Experimental method

[0263] Take 22.6 mg of Form C (prepared in Example 6) (equivalent to 20 mg of anhydrous free state) into a 2 ml glass bottle. Add 1 ml of solvent. Adjust the pH of ES 1-ES 3 samples to 6.0, 6.5 and 7.0 respectively using 2M KOH solution or phosphoric acid solution. The time for adjusting the pH is about 30 minutes. The resulting suspension is stirred at 25 °C for 2 hours and 24 hours at 400 rpm. Then the resulting suspension is centrifuged at 25 °C for 5 minutes at 14,000 rpm. The solubility of the supernatant is determined by HPLC, the pH of the supernatant is determined by pH meter, and the crystal form of the residual solid (wet product) is confirmed by XRPD.

[0264] 2. Experimental results

[0265] The experimental results are shown in the following table.

[0266] Table 11 Solubility investigation results Note: " / / ": not carried out.

[0267] From the results in the above table, the solubility of Form C in aqueous system is pH dependent. For the solubility samples in different pH buffers, the pH of the system is adjusted back to the target pH after the addition of Form C. The solubility of Form C in pH 6.0 buffer is 16.8 mg / mL, and the end point pH is 5.9; the solubility in pH 6.5 buffer is 16.7 mg / mL, and the end point pH is 6.3; the solubility in pH 7.0 buffer is 1.2 mg / mL, and the end point pH is 6.7; the solubility in water is 9.0 mg / mL, and the end point pH is 6.3.

[0268] Example 9: Investigation of hygroscopicity of Form C

[0269] 1. Experimental method

[0270] The water absorption and dehydration behavior of Form C (prepared in Example 6) was investigated by DVS test at 25 °C. The sample after DVS test was subjected to XRPD test to determine whether a crystal form transition occurred.

[0271] 2. Experimental results

[0272] The experimental results are shown in the following table.

[0273] Table 12 Water adsorption and desorption test results Note: " / / ": not tested

[0274] No or almost no hygroscopicity: hygroscopic weight gain less than 0.2%

[0275] Slightly hygroscopic: hygroscopic weight gain less than 2% but not less than 0.2%

[0276] Hygroscopic: hygroscopic weight gain less than 15% but not less than 2%

[0277] Extremely hygroscopic: hygroscopic weight gain not less than 15%

[0278] Deliquescent: absorbs sufficient moisture to form a liquid

[0279] Water uptake = water uptake at specific RH (80% to 95%) - water uptake at 40% RH

[0280] From the results of the above table, it can be seen that Form C is almost non-hygroscopic, as it adsorbs about 0.1% moisture between 40% RH and 80% RH at 25°C. After DVS testing, the resulting sample is still Form C. In summary, Form C obtained by the present application exhibits good solid chemical stability and physical stability, and is almost non-hygroscopic, which is an advantageous crystal form for downstream development.

[0281] The above description is merely preferred embodiments of the present application, but not to limit the present application. Any modification, equivalent replacement, and the like made in the spirit and principle of the present application shall fall within the scope of the present application.

[0282] The foregoing examples and methods described in the present application can vary based on the ability, experience and preference of a person skilled in the art.

[0283] The steps of the method in the present application are only listed in a certain order, which does not constitute any limitation on the order of the steps of the method.

Claims

1. A crystal form C of a hemisuccinate of compound I, the XRPD pattern of which has characteristic peaks at at least three (at least four, at least five, at least six, at least seven, or all) positions at diffraction angles 2θ of 14.2°±0.2°, 18.3°±0.2°, 18.6°±0.2°, 20.9°±0.2°, 22.2°±0.2°, 22.9°±0.2°, 23.2°±0.2°, and 25.0°±0.2°, wherein compound I has the following structure:

2. The crystal form C as described in claim 1, characterized in that, The XRPD pattern of crystal form C also has characteristic peaks at at least one of the following positions: diffraction angle 2θ values ​​of 12.2°±0.2°, 18.5°±0.2°, 22.0°±0.2°, 25.4°±0.2°, and 28.6°±0.2°. Preferably, the XRPD pattern of crystal form C also has characteristic peaks at at least one of the following positions with diffraction angles 2θ: 12.4°±0.2°, 13.0°±0.2°, 13.2°±0.2°, 21.4°±0.2°, 23.5°±0.2°, 24.6°±0.2°, 26.2°±0.2°, 27.2°±0.2°, 29.8°±0.2°, and 32.0°±0.2°. More preferably, the crystal form C has an XRPD pattern substantially as shown in FIG1.

3. The crystal form C as described in claim 1, characterized in that, The crystal form C is triclinic, with space group P1 and cell parameters as follows: α=73.087(2)°, β=77.109(2)°, γ=88.448(2)°, Z=1, 4. The crystal form C as described in claim 1, characterized in that, The DSC spectrum of the crystal form C has an endothermic peak at approximately 175.8 °C; Preferably, the crystal form C has a DSC pattern substantially as shown in FIG2.

5. The crystal form C as described in claim 1, characterized in that, The crystal form C loses approximately 0.5% ± 0.05% weight when heated from room temperature to 163°C; Preferably, the crystal form C has a TGA pattern substantially as shown in FIG3.

6. The crystal form C as described in claim 1, characterized in that, The crystal form C is an anhydrous compound.

7. A pharmaceutical composition comprising crystal form C as described in any one of claims 1-5, and one or more pharmaceutically acceptable excipients; Preferably, the pharmaceutical composition is an ophthalmic preparation, preferably selected from: eye drops, ophthalmic ointments, and ophthalmic gels; or, The pharmaceutical composition is in the form of a solid dosage form, preferably tablets, pills, powders, granules, capsules, powders, lozenges, or films.

8. The use of crystal form C according to any one of claims 1-6 in the preparation of a medicament for the prevention and / or treatment of diseases related to protein tyrosine kinase; Preferably, the tyrosine kinase is VEGFR, and more preferably one or more of VEGFR1, VEGFR2, and VEGFR3; More preferably, the disease is selected from: tumors, autoimmune diseases, inflammatory diseases, diseases related to pathogen infection, neurodegenerative diseases, cardiovascular diseases, metabolic diseases, fibrotic diseases, and eye diseases.

9. The application as described in claim 8, characterized in that, The tumors are selected from: breast cancer, lung cancer, colorectal cancer, kidney cancer, liver cancer, pancreatic cancer, ovarian cancer, prostate cancer, glioblastoma, glioma, unrelated myeloid metaplasia, mesothelioma, sarcoma, myelodysplastic syndrome, and hematologic malignancies, preferably from: renal cell carcinoma, hepatocellular carcinoma, colorectal cancer, lung cancer, gastric cancer, and leukemia.

10. The application as described in claim 8, characterized in that, The eye diseases mentioned are selected from: diabetic retinopathy, age-related macular degeneration (AMD), pathological choroidal neovascularization (CNV) of any pathological mechanism, pathological retinal neovascularization of any pathological mechanism, uveitis, retinal vein occlusion, ocular trauma, surgical edema, surgical neovascularization, cystoid macular edema, ocular ischemia, retinopathy of prematurity, Coats' disease, sickle cell retinopathy and / or neovascular glaucoma, retinoblastoma, preferably from: diabetic retinopathy, age-related macular degeneration (AMD), and Coats' disease.

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