Crystal form of free base of protein tyrosine kinase inhibitor

Abstract

Disclosed in the present invention is a crystal form of the free base of a protein tyrosine kinase inhibitor. The inhibitor not only antagonizes the activity of VEGFR1, VEGFR2 and VEGFR3 tyrosine kinases, but also has a high selectivity in inhibiting the activity of an EGFR tyrosine kinase, and can effectively reduce and avoid side effects. The crystal form of the free base of the protein tyrosine kinase inhibitor has a good solid-state chemical stability and physical stability, is almost non-hygroscopic, can be used as an advantageous crystal form for drug research and development, exhibits broad application prospects in downstream development and industrial production, and is expected to provide safer and more effective drug selections for the treatment of related diseases.

Description

A crystalline form of a protein tyrosine kinase inhibitor free base TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a crystalline form of a protein tyrosine kinase inhibitor free base, a preparation method and application thereof. BACKGROUND

[0002] Receptor tyrosine kinases are the largest class of enzyme-linked receptors, with functions of growth factor receptors and enzymatic activities of catalyzing phosphorylation of downstream target proteins. The activation process depends on the binding of ligands and the dimerization of receptors. In normal physiological conditions, receptor tyrosine kinases are essential for the maintenance of cell functions, but in tumor environments, they can promote tumor development. Therefore, regulating the activity of receptor tyrosine kinases plays a key role in preventing abnormal cell functions.

[0003] As a family of receptor tyrosine kinases, vascular endothelial growth factor (VEGF) has a variety of important physiological functions, including but not limited to increasing vascular permeability, promoting extracellular matrix degeneration, etc. Since the growth and metastasis of tumors are highly dependent on angiogenesis, blocking the binding of VEGF and VEGFR to inhibit angiogenesis has become one of the effective strategies for tumor treatment.

[0004] Nowadays, VEGF inhibitors are developed to regulate multiple targets to inhibit the growth and reproduction of pathological sites, thereby blocking their growth and reproduction. They have achieved good results in lung cancer, renal cell carcinoma, and colon cancer, etc. However, the multi-target effect also brings some adverse reactions to the body, such as increasing the risk of nephritis and / or cardiovascular disease. SUMMARY

[0005] 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), and further studied to obtain a free base crystal form B of the compound, which has good solid chemical stability and physical stability, and almost no hygroscopicity, and can be used as an advantage crystal form for drug research and development.

[0006] Specifically, the technical solutions of the present application are as follows:

[0007] In a first aspect of the application, there is provided a crystalline form B of Compound I free base having an XRPD pattern with at least three (or all) of the following characteristic peaks (major characteristic diffraction peaks) at diffraction angles 2Θ values of 5.7°±0.2°, 8.8°±0.2°, 17.1°±0.2°, 19.6°±0.2° using Cu-Ka radiation, said Compound I having the following structure:

[0008] Further, the XRPD pattern of said crystalline form also has characteristic peaks at at least one (at least two, at least three, at least four, or all) of the following positions at diffraction angles 2Θ values of 3.6°±0.2°, 15.4°±0.2°, 16.1°±0.2°, 17.0°±0.2°, 19.1°±0.2°.

[0009] Further, the XRPD pattern of said crystalline form also has characteristic peaks at at least one (at least two, at least three, at least four, at least five, or all) of the following positions at diffraction angles 2Θ values of 13.8°±0.2°, 15.1°±0.2°, 19.9°±0.2°, 21.8°±0.2°, 22.9°±0.2°, 26.7°±0.2°.

[0010] Further, the XRPD pattern of said crystalline form also has characteristic peaks at 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 following positions at diffraction angles 2Θ values of 8.5°±0.2°, 11.7°±0.2°, 18.3°±0.2°, 18.5°±0.2°, 20.1°±0.2°, 23.4°±0.2°, 24.0°±0.2°, 25.2°±0.2°, 25.6°±0.2°, 28.3°±0.2°, 32.1°±0.2°.

[0011] In some embodiments of the application, the XRPD pattern of said crystalline form has the characteristic peaks shown in Table 7.

[0012] In some embodiments of the application, said crystalline form has an XRPD pattern substantially as shown in Figure 1.

[0013] Further, said crystalline form has a DSC pattern with an endothermic peak at about 181.9 °C.

[0014] In some embodiments of the application, said crystalline form has a DSC pattern substantially as shown in Figure 2.

[0015] Further, said crystalline form has a weight loss of about 0.4% ± 0.05% when heated from room temperature to 160 °C.

[0016] In some embodiments of the present application, the crystalline form has a TGA pattern substantially as shown in Figure 3.

[0017] Further, the crystalline form is an anhydrate.

[0018] In a second aspect of the present application, there is provided a method for preparing the crystalline form B of the first aspect.

[0019] Specifically, the method for preparing can be selected from a combination of one or more of anti-solvent addition, anti-anti-solvent addition, gas-solid diffusion, room temperature suspension stirring, 5°C suspension stirring, 50°C suspension stirring, slow evaporation, slow cooling, gas-liquid diffusion, polymer induction.

[0020] In some embodiments of the present application, the method for preparing employs 50°C suspension stirring; specifically, the method comprises mixing the compound I with an organic solvent, stirring the suspension at 50°C (e.g. for 1 week), and then isolating the solid.

[0021] Specifically, the organic solvent can be selected from methyl isobutyl ketone, ethyl acetate, 2-methyltetrahydrofuran.

[0022] Specifically, the solid isolation can employ filtration, centrifugation, or a combination of both. In some embodiments of the present application, the solid isolation step comprises centrifuging the resulting suspension at 14000 rpm using a 0.45 μιη nylon filter tube.

[0023] In a third aspect of the present application, there is provided a pharmaceutical composition comprising the crystalline form B of the first aspect, and one or more pharmaceutically acceptable excipients.

[0024] Specifically, the pharmaceutically acceptable excipients can be selected from one or more of solvents, co-solvents, emulsifiers, disintegrants, binders, stabilizers, flavoring agents, preservatives, surfactants, pH adjusting agents, antioxidants, penetration enhancers, buffers, plasticizers, solubilizers, and the like.

[0025] Specifically, in the pharmaceutical composition, the crystalline form B of the first aspect can be used alone, or in combination with other kinds of active ingredients.

[0026] Specifically, 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, and the like).

[0027] In particular, the pharmaceutical composition can be in any suitable dosage form, for example, a gastrointestinal administration dosage form, for example, including, but not limited to, a tablet, a pill, a powder, a granule, a capsule, a lozenge, a syrup, a liquid, an emulsion, a suspension, etc.; a non-gastrointestinal administration dosage form, for example, an injection administration dosage form: such as an injection (for example, for subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), a respiratory 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 mucous membrane 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, for example, 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, for example, but not limited to, a tablet, a pill, a powder, a granule, a capsule, a powder, a lozenge, a film, etc.

[0028] In particular, the various dosage forms of the pharmaceutical composition can be prepared according to the conventional production method in the pharmaceutical field. For example, the active ingredient is mixed with one or more pharmaceutically acceptable excipients, and then it is made into the desired dosage form.

[0029] In particular, in the pharmaceutical composition, the weight percentage of the crystalline form B of the first aspect 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%.

[0030] In the fourth aspect of the present application, the use of the crystalline form B of the first aspect in the preparation of a medicament for preventing and / or treating a disease related to protein tyrosine kinase is provided.

[0031] In particular, the tyrosine kinase is VEGFR, for example, one or more of VEGFR1, VEGFR2, VEGFR3.

[0032] In particular, the disease is a disease that can be beneficial to its prevention and / or treatment by inhibiting protein tyrosine kinase, 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 kinase.

[0033] In some embodiments of the application, the disease is a tumor, in particular a malignant tumor (cancer), including, but not limited to, advanced renal cell carcinoma, lung cancer, breast cancer, colon cancer, ovarian cancer, pancreatic cancer, prostate cancer, pseudovasculoma, soft tissue sarcoma, melanoma, idiopathic pulmonary fibrosis, myelodysplastic syndrome, hematological malignancies.

[0034] In particular, the hematological malignancies include leukemia, lymphoma, multiple myeloma (MM).

[0035] In particular, the leukemia can be chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML), acute lymphocytic leukemia (ALL), acute myelocytic leukemia (AML), hairy cell leukemia, prolymphocytic leukemia.

[0036] 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 includes killing the tumor, preventing metastatic spread of the tumor and growth of micrometastases.

[0037] In some embodiments of the application, the tumor is selected from the group consisting of renal cell carcinoma, hepatocellular carcinoma, colorectal cancer, lung cancer, breast cancer, leukemia.

[0038] 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 pathogenic 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 pathogenic 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.

[0039] 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 diseases of the thyroid, ulcerative colitis, and the like.

[0040] In some embodiments of the 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, pneumonitis, 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 renal failure, burns, pancreatitis (e.g., acute pancreatitis), post-surgical syndrome, sarcoidosis, Herxheimer reaction, encephalitis, myelitis, meningitis, and malaria, and the like.

[0041] 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, etc.

[0042] 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 (such as hepatitis A, hepatitis B, hepatitis C, hepatitis D, etc.), AIDS, rabies, dengue fever, Ebola virus disease, etc.

[0043] 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, cerebral ischemia (stroke), embolism (such as pulmonary embolism, renal embolism, hepatic embolism, gastrointestinal embolism or peripheral limb embolism) or myocardial ischemia, etc.

[0044] In some embodiments of the present application, the disease is a metabolic disease, including but not limited to diabetes (such as type I diabetes, type II diabetes or gestational diabetes), obesity, hyperglycemic hyperosmolar syndrome, cachexia, hypercholesterolemia, scurvy, etc.

[0045] 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, etc.

[0046] In the fifth aspect of the present application, a method for preventing and / or treating a protein tyrosine kinase-mediated proliferative disease is provided, which comprises the step of administering to a subject in need thereof an effective amount of the crystalline form B of the first aspect, or the pharmaceutical composition of the third aspect.

[0047] Specifically, the disease is as described in the fourth aspect of the present application.

[0048] Specifically, the subject is a mammal, such as a human.

[0049] Specifically, the administration can employ any suitable administration route, such as a gastrointestinal administration (e.g., oral administration) or a non-gastrointestinal administration (e.g., intravenous, intramuscular, intranasal, intraocular, intracerebral, intravaginal, intraperitoneal, transdermal, subcutaneous, intradermal, respiratory tract administration, etc.).

[0050] The present application provides a free base crystal form of a protein tyrosine kinase inhibitor. In the process of research and development, 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 tyrosine kinases against angiogenesis, 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 to obtain a free base crystal form B 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, and has very good application value in downstream development and industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 shows the XRPD pattern of crystal form B.

[0052] Figure 2 shows the DSC pattern of crystal form B.

[0053] Figure 3 shows the TGA pattern of crystal form B.

[0054] Figure 4 shows the 1 H NMR pattern of crystal form B.

[0055] Figure 5 shows the XRPD superimposition of crystal form B samples under different conditions for 1 week.

[0056] Figure 6 shows the XRPD superimposition of crystal form B samples under different conditions for 4 weeks. DETAILED DESCRIPTION

[0057] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0058] Unless otherwise defined, all scientific and technical terms used in the present application have the same meanings as commonly understood by those skilled in the art to which the present application relates.

[0059] The disclosures of various publications, patents and published patent specifications, cited herein are hereby incorporated by reference in their entireties.

[0060] The explanations of some abbreviations involved in the present application are as follows:

[0061] XRPD: X-ray powder diffraction

[0062] DSC: differential scanning calorimetry

[0063] TGA: thermogravimetric analysis

[0064] 1 H NMR: proton nuclear magnetic resonance

[0065] PLM: polarized light microscopy

[0066] DVS: dynamic vapor sorption analysis

[0067] 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 relied upon as the sole or controlling factor. 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 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 as 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 pattern of an unknown crystal form to confirm whether the two sets of patterns reflect the same or different crystal forms.

[0068] 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" when used in reference to a new crystal form indicates that the crystal form contains less than about 20% by weight of another crystal form, particularly less than about 10% by weight of another crystal form, more particularly less than about 5% by weight of another crystal form, and even more particularly less than about 1% by weight of another crystal form.

[0069] It should be noted that the numerical values and numerical ranges mentioned in the present application should not be understood as being strictly limited to the numerical values or numerical ranges themselves, but a person skilled in the art should understand that they can fluctuate around the specific numerical values depending on the specific technical environment, without departing from the spirit and principles of the present application. In the present application, such a fluctuation range that can be foreseen by a person skilled in the art is often indicated by the term "about". When the term "about" is used in front of a numerical value of the present application and refers to the numerical value, it means any value within the range of ±10% of the value, preferably within the range of ±5%, more preferably within the range of ±2%, preferably within the range of ±1%. For example, "about 10" should be interpreted to mean 9-11, preferably 9.5-10.5, more preferably 9.8-10.2, more preferably 9.9-10.1.

[0070] In the present application, 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 place of a fume hood. Generally, the room temperature is about 20°C to about 30°C, or about 22°C to 27°C, or about 25°C.

[0071] The anti-solvent addition (also known as anti-solvent crystallization, precipitation crystallization, salting-out or crystallization under pressure) method is generally a method of adding one or more anti-solvents to a solution in which the target product is dissolved with a good solvent, the product being in a slightly soluble state in the solution, so that the solution is oversaturated and the product is precipitated. Anti-anti-solvent addition is generally a method of adding one or more anti-solvents to a solution in which the target product is dissolved with a good solvent, the product being in a slightly soluble state in the solution, so that the solution is oversaturated and the product is precipitated.

[0072] The anti-solvent has a poorer ability to dissolve 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.

[0073] Anti-solvent addition, anti-anti-solvent addition can be through batch, semi-batch or continuous crystallization operation. 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 dropwise at a constant rate, or slowly added dropwise at the beginning, and then gradually increase the rate.

[0074] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0075] The instruments and parameters used in the detection means involved in the following embodiments are as follows

[0076] 1. XRPD:

[0077] Instrument: Bruker D8 Advance

[0078] X-ray path: reflection mode

[0079] Detector: LYNXEYE_XE_T (1D mode)

[0080] Opening angle: 2.9° (max)

[0081] Radiation source: Cu / K-Alpha1

[0082] X-ray source power: 40 kV, 40 mA

[0083] Primary light path slit: double primary motorized slit: 10.0 mm; primary soller slit: 2.5°

[0084] Secondary light path slit: secondary soller slit: 2.5°

[0085] Scan mode: continuous scan

[0086] Scan type: dual path mode

[0087] Step size: 0.02°

[0088] Time per step: 0.4 s / step

[0089] Scan range: 2° to 40°

[0090] Sample rotation speed: 15 rpm

[0091] Sample plate: single crystal silicon wafer, flat plate

[0092] 2. DSC:

[0093] Instrument: TA Instruments Discovery 2500

[0094] Sample pan: Tzero pan and Tzero hermetic lid with ~0.7 mm diameter hole

[0095] Temperature range: 0 to 250 °C or until decomposition

[0096] Heating rate: 10 °C / min

[0097] Nitrogen flow rate: 50 mL / min

[0098] Sample amount: ~0.5-5 mg

[0099] 3. TGA:

[0100] Instrument: TA Instruments Discovery 5500

[0101] Sample pan: aluminum pan, open

[0102] Starting temperature: ambient temperature (less than 35 °C)

[0103] Ending temperature: 300 °C or until the remaining sample weight is less than 80% of the initial

[0104] Heating rate: 10 °C / min

[0105] Nitrogen flow rate: 10 mL / min equilibration; 25 mL / min sample chamber

[0106] Sample amount: ~2-10 mg

[0107] 4. PLM:

[0108] Instrument: Leica DM4P

[0109] Method: Crossed polarized light, drop of silicone oil

[0110] 5. NMR:

[0111] Instrument: Bruker Avance-AV 400M

[0112] Frequency: 400 MHz

[0113] Probe: 5 mm PABBO BB / 19F-1H / D Z-GRD Z108618 / 0406

[0114] Number of scans: 8

[0115] Temperature: 297.6 K

[0116] Relaxation delay: 1 s

[0117] 6. HPLC:

[0118] Instrument: Agilent 1260 infinity II Binary Pump

[0119] Method:

[0120] Wavelength: 254 nm

[0121] Column: Agilent poroshell 120 EC-C18 (150 mm*4.6 mm, 2.7 pm)

[0122] Detector: DAD

[0123] Column temperature: 25 °C

[0124] Flow rate: 0.8 mL / min

[0125] Mobile phase A: 0.1% formic acid in water, v / v

[0126] Mobile phase B: 0.1% formic acid in ACN, v / v

[0127] Diluent: Acetonitrile: water = (1 : 1, v:v)

[0128] Injection volume: 2 pL

[0129] Table 1 Gradient

[0130] 7. DVS:

[0131] Instrument: Intrinsic

[0132] Total gas flow: 200 seem

[0133] Temperature: 25 °C

[0134] Solvent: Water

[0135] Method: Cycle: 40-0-95-0-40% RH

[0136] Step: 10% RH

[0137] Equilibration: 0.002 dm / dt (% / min)

[0138] Minimum dm / dt hold time: 60 min

[0139] Maximum dm / dt hold time: 360 min

[0140] Sample amount: ~5-50 mg

[0141] Example 1 Synthesis of compound I

[0142] The synthesis route is as follows:

[0143] First step

[0144] 3-[(3S)-3-fluoropyrrolidin-1-yl]propanenitrile

[0145] 3-[(3S)-3-fluoropyrrolidin-1-yl]propanenitrile

[0146] (3S)-3-fluoropyrrolidine hydrochloride (5 g, 39.8 mmol) was dissolved in acetonitrile (48.7 mL), 3-bromopropanenitrile (5.3 g, 39.82 mmol) and potassium carbonate (16.5 g, 119.5 mmol) were added, 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 phase was combined and washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure to obtain the crude product, which was purified by neutral alumina column chromatography (DCM) to obtain the product 3-[(3S)-3-fluoropyrrolidin-1-yl]propanenitrile (IM-1) (3.4 g, 23.9 mmol, 60.06% yield) as colorless oil.

[0147] The product was confirmed by LCMS and H-NMR

[0148] 1 H 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).

[0149] Second step

[0150] 3-[(3S)-3-fluoropyrrolidin-1-yl]propan-1-amine

[0151] 3-[(3S)-3-fluoropyrrolidin-1-yl]propan-1-amine

[0152] To a solution of 3-[(3S)-3-fluoropyrrolidin-1-yl]propionitrile (IM-1) (3 g, 21.1 mmol) in methanol (15 mL) was added Raney nickel (1.4 g, 23.2 mmol) and the reaction mixture was stirred under hydrogen atmosphere at 25 °C for 3 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to obtain the product 3-[(3S)-3-fluoropyrrolidin-1-yl]propylamine (IM-2) (3 g, 20.52 mmol, 97.24 % yield) as colorless oil.

[0153] The product was confirmed by LCMS and H-NMR.

[0154] 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).

[0155] Third step

[0156] methyl

[0157] 3-[[5-(cyclopropylcarbamoyl)-2-fluoro-phenyl]methoxy]-5-[3-[(3S)-3-fluoropyrrolidin-1-yl]propylcarbamoylamino]isothiazole-4-carboxylate

[0158] 3-[[5-(cyclopropylcarbamoyl)-2-fluoro-phenyl]methoxy]-5-[3-[(3S)-3-fluoropyrrolidin-1-yl]propylcarbamoylamino]isothiazole-4-carboxylate

[0159] 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.

[0160] The product was confirmed by LCMS.

[0161] Fourth step

[0162] 3-[[5-(cyclopropylcarbamoyl)-2-fluoro-phenyl]methoxy]-5-[3-[(3S)-3-fluoropyrrolidin-1-yl]propylcarbamoylamino]isothiazole-4-carboxamide

[0163] 3-[[5-(cyclopropylcarbamoyl)-2-fluoro-phenyl]methoxy]-5-[3-[(3S)-3-fluoropyrrolidin-1-yl]propylcarbamoylamino]isothiazole-4-carboxamide

[0164] 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).

[0165] The product was confirmed by LCMS, H-NMR, C-NMR and F-NMR.

[0166] 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).

[0167] 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.

[0168] 19F NMR (400 MHz, CD3OD) δ (ppm) -115.43, -168.98.

[0169] Example 2: Detection of the ability of compound I to inhibit tyrosine kinase

[0170] 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.

[0171] Table 2 Reagents and instruments

[0172] Experimental procedure:

[0173] 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, and other reagents 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 add 25 microliters of stop solution to stop the reaction. Read the conversion rate data on the Caliper EZ Reader II. Convert the conversion rate to inhibition rate data.

[0174] "Min" is the reading of the control well without enzyme; "Max" is the reading of the control well with DMSO. IC 50 values were fitted using XLFit excel add-in version 5.4.0.8.

[0175] X is the log value of the concentration of the compound, and Y is the inhibition rate (% inhibition).

[0176] IC50 of compound I against tyrosine kinases 50 The results are shown in the table below, with compound PAN90806 (commercial product) as the positive control.

[0177] Table 3 Experimental results

[0178] 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.

[0179] 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.

[0180] Example 3: Detection of the ability of compound I to inhibit VEGFR2 phosphorylation in HUVEC cells

[0181] 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.

[0182] Table 4 Reagents and Instruments

[0183] Experimental Procedure:

[0184] 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. XL Fit software was used for data analysis:

[0185] “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).

[0186] Curve fitting IC using 4 Parameter Logistic Model or Sigmoidal Dose-Response Model 50 Values

[0187] 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.

[0188] IC of compound I on pVEGFR2 50 The results are shown in Table 6, with compound PAN90806 (commercial product) as the positive control.

[0189] Example 4: Detection of the inhibitory effect of compound I on VEGF-induced HUVEC cell proliferation

[0190] 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.

[0191] This assay used a luciferase bioluminescence assay (CellTiter GLO TM , Promega) to measure 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.

[0192] The reagents and instruments used are shown in the following table.

[0193] Table 5 Reagents and instruments

[0194] Experimental procedures:

[0195] 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

[0196] 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.

[0197] 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.

[0198] Table 6 Experimental results

[0199] 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 treating ocular neovascular diseases such as wet AMD.​​

[0200] 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 thus inhibit neovascularization. The main function from VEGFR receptor signal transduction is to promote the proliferation of endothelial cells and neovascularization, and compound I shows a nanomolar concentration level of inhibitory ability on VEGF-induced human endothelial cell proliferation. 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.

[0201] Example 5: Preparation of crystal form

[0202] 1. Experimental method

[0203] 40 mg of compound I (prepared in Example 1) was weighed and added into 0.2 mL of ethyl acetate, and then suspended at 50°C under magnetic stirring at a speed of 300-400 rpm for 1 week.

[0204] The obtained suspension was centrifuged and filtered with a 0.45 μm nylon filter tube at 14000 rpm, and the obtained solid part (wet product) was characterized by XRPD. If the obtained sample shows different X-ray diffraction peaks, further characterization such as DSC, TGA and 1H-NMR was carried out.

[0205] 2. Experimental results

[0206] The crystal form product was prepared, and the test results are shown as follows.

[0207] The purity (detected by HPLC) of the obtained product was 99.4%.

[0208] The XRPD spectrum of the obtained product is shown in Figure 1, and the XRPD data is shown in the following table, indicating that it is a crystal, which is named as crystal form B.

[0209] Table 7 XRPD data table of crystal form B

[0210] (Referring to the foregoing experimental method, methyl isobutyl ketone and 2-methyl tetrahydrofuran were used to replace ethyl acetate as solvents, and crystal form B was also obtained)

[0211] The DSC result of crystal form B is shown in Figure 2, and DSC shows that it has a melting peak with an enthalpy of 94 J / g at T onset 181.9°C.

[0212] TGA results of Form B are shown in Figure 3. TGA showed a weight loss of about 0.4% at about 160°C.

[0213] TGA results of Form B are shown in Figure 3. TGA showed a weight loss of about 0.4% at about 160°C. 1 H-NMR results are shown in Figure 4. 1H-NMR showed no solvent residue was detected.

[0214] The physicochemical properties of Form B are summarized in the following table.

[0215] Table 8 Physicochemical property characterization of Form B

[0216] Example 6: Solid stability investigation of Form B

[0217] 1. Experimental method

[0218] Open containers containing Form B (prepared in Example 5) were placed at 25°C / 92.5% RH and 40°C / 75% RH for 1 week and 4 weeks, respectively. Closed containers containing Form B (prepared in Example 5) were placed at 60°C for 1 week and 4 weeks. The stability samples under these conditions were subjected to XRPD, HPLC detection and observation of color change of the samples.

[0219] 2. Experimental results

[0220] The experimental results are shown in the following table.

[0221] Table 9 Solid stability of Form B

[0222] Note A: no color change; B: slight color change

[0223] C: moderate color change; D: severe color change

[0224] From the results in the above table, Form B showed good physical and chemical stability under the above accelerated stability conditions for 1 week and 4 weeks.

[0225] Example 7: Solubility investigation of Form B

[0226] 1. Experimental method

[0227] 4 mg of Form B (prepared in Example 5) was weighed into an 8 mL glass bottle. 2 mL of water was added. The resulting suspension was stirred at 25°C for 2 h and 24 h at a rotation speed of 400 rpm, and then the resulting suspension was centrifuged at 25°C at a rotation speed of 14,000 rpm for 5 min. The solubility of the supernatant was detected by HPLC, the pH of the supernatant was determined by a pH meter, and the residual solid (wet product) was detected by XRPD to confirm the crystal form.

[0228] Take 2 mg of Form B (prepared in Example 5) into a 2 mL glass bottle. Add 0.1 mL of 0.02 M citric acid aqueous solution. The resulting sample is a clear solution within 24 hours.

[0229] 2. Experimental results

[0230] The experimental results are shown in the following table.

[0231] Table 10 Solubility of Form B in water and citric acid aqueous solution

[0232] Note " / / ": not tested.

[0233] From the results in the above table, it can be seen that the solubility of Form B is stable in both neutral and acidic aqueous systems.

[0234] Example 8: Investigation of hygroscopicity of Form B

[0235] 1. Experimental method

[0236] The water absorption and dehydration behavior of Form B (prepared in Example 5) was studied by DVS testing at 25°C. The sample after DVS testing was subjected to XRPD detection to determine whether a crystal form transition had occurred.

[0237] 2. Experimental results

[0238] The experimental results are shown in the following table.

[0239] Table 11 Results of water adsorption and desorption experiments

[0240] Note " / / ": not tested.

[0241] No or almost no hygroscopicity: hygroscopic weight gain less than 0.2%

[0242] Slightly hygroscopic: hygroscopic weight gain less than 2% but not less than 0

[0243] Hygroscopic: hygroscopic weight gain less than 15% but not less than 2%

[0244] Extremely hygroscopic: hygroscopic weight gain not less than 15%

[0245] Deliquescent: absorbs sufficient moisture to form a liquid

[0246] Water absorption amount = water absorption amount at specific RH (80% to 95%) - water absorption amount at 40% RH

[0247] This standard is partially adjusted in reference to the description of hygroscopicity in the Chinese Pharmacopoeia

[0248] From the results of the above table, it can be seen that Form B has almost no hygroscopicity, and it adsorbs about 0.1% moisture at 25°C between 40% RH and 80% RH. After DVS testing, the obtained sample is still Form B.

[0249] In summary, the Form B obtained in the present application exhibits good solid chemical stability and physical stability, and almost no hygroscopicity, and is an advantageous crystal form for downstream development.

[0250] The above only describes the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

[0251] The foregoing embodiments and methods described in the present application can be different based on the ability, experience and preference of the person skilled in the art. The fact that the steps of the method are listed in a certain order in the present application does not constitute any limitation on the order of the steps of the method.

Claims

1. A free base of compound I, crystal form B, whose XRPD pattern shows characteristic peaks at at least three or all of the positions at diffraction angles 2θ of 5.7°±0.2°, 8.8°±0.2°, 17.1°±0.2°, and 19.6°±0.2°, wherein compound I has the following structure:

2. The crystalline Form B of claim 1, characterized by, the XRPD pattern of said crystalline Form B further comprises at least one characteristic peak at a diffraction angle 2Q value of 3.6°±0.2°, 15.4°±0.2°, 16.1°±0.2°, 17.0°±0.2°, 19.1°±0.2°; Preferably, the XRPD pattern of said crystalline Form B further comprises at least one characteristic peak at a diffraction angle 2Q value of 13.8°±0.2°, 15.1°±0.2°, 19.9°±0.2°, 21.8°±0.2°, 22.9°±0.2°, 26.7°±0.2°; More preferably, said crystalline Form B has an XRPD pattern substantially as shown in Figure 1.

3. The crystalline Form B of claim 1, characterized by, The DSC pattern of said crystalline Form B has an endothermic peak at about 181.9°C; Preferably, said crystalline Form B has a DSC pattern substantially as shown in Figure 2.

4. The crystalline Form B of claim 1, characterized by, Said crystalline Form B loses about 0.4%±0.05% weight from room temperature to 160°C; Preferably, said crystalline Form B has a TGA pattern substantially as shown in Figure 3.

5. The crystalline Form B of claim 1, characterized by, Said crystalline Form B is an anhydrate.

6. A method for preparing the crystalline Form B of any one of claims 1-5, which employs 50°C suspension stirring; Preferably, the method comprises: mixing said compound I with an organic solvent, stirring suspension at 50°C, and then separating the solid. More preferably, said organic solvent is selected from the group consisting of methyl isobutyl ketone, ethyl acetate, 2-methyltetrahydrofuran.

7. A pharmaceutical composition comprising the crystalline Form B of any one of claims 1-5, and one or more pharmaceutically acceptable excipients; Preferably, said pharmaceutical composition is an ophthalmic preparation, preferably selected from the group consisting of eye drops, eye ointment, eye gel; or, Said pharmaceutical composition is a solid dosage form, preferably selected from the group consisting of tablets, pills, powders, granules, capsules, dispersions, lozenges, films.

8. Use of the crystalline Form B of any one of claims 1-5 in the preparation of a medicament for preventing and / or treating a disease associated with protein tyrosine kinase. Preferably, said tyrosine kinase is VEGFR, preferably selected from one or more of VEGFR1, VEGFR2, VEGFR3; More preferably, said disease is selected from the group consisting of tumor, autoimmune disease, inflammatory disease, disease associated with pathogen infection, neurodegenerative disease, cardiovascular disease, metabolic disease, fibrotic disease, ocular disease.

9. Use according to claim 8, wherein the compound is ###0002### Said tumor is selected from the group consisting of advanced renal cell carcinoma, lung cancer, breast cancer, colon cancer, ovarian cancer, pancreatic cancer, prostate cancer, pseudovascular tumor, soft tissue sarcoma, melanoma, idiopathic pulmonary fibrosis, myelodysplastic syndrome, hematological malignancy, preferably selected from the group consisting of renal cell carcinoma, hepatocellular carcinoma, colorectal cancer, lung cancer, breast cancer, leukemia.

10. The use according to claim 8, wherein the compound is ###0002### The ocular disease is selected from the group consisting of diabetic retinopathy, age-related macular degeneration (AMD), pathologic choroidal neovascularization (CNV) from any pathomechanism, pathologic retinal neovascularization from any pathomechanism, uveitis, retinal vein occlusion, ocular trauma, surgery-induced edema, surgery-induced neovascularization, cystoid macular edema, ocular ischemia, retinopathy of prematurity, Coats' disease, sickle cell retinopathy and / or neovascular glaucoma, retinoblastoma, preferably from the group consisting of diabetic retinopathy, age-related macular degeneration (AMD), Coats' disease.

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