Use of salt forms of compound and crystal forms thereof in treatment of heart failure with preserved ejection fraction

Abstract

The present invention relates to the use of salt forms of a compound and crystal forms thereof in the treatment of heart failure with preserved ejection fraction. Specifically provided are salts of the compound as shown in formula I, wherein the salts are sodium salts, potassium salts, calcium salts, tert-butylamine salts, ammonium salts, choline salts, L-lysine salts, diethylamine salts, dimethylethanolamine salts, betaine salts or ethylenediamine salts of the compound as shown in formula I.

Description

Use of salt crystal form of compound for treating heart failure with preserved ejection fraction Technical Field

[0001] The present invention relates to the field of medicine, and in particular to use of a salt-type crystal form of a compound for treating heart failure with preserved ejection fraction. Background Art

[0002] Cardiovascular disease represents one of the most challenging medical issues. Heart failure (HF) is the leading cause of death in patients with CVD and a major clinical challenge. HF is divided into heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF).

[0003] Currently, HFpEF accounts for approximately 50% of all heart failure patients, and its prevalence is increasing at an alarming rate. It is also the main cause of rising cardiovascular mortality. HFpEF is a complex disease involving multiple organ disorders, with symptoms and consequences caused by the heart, lungs, kidneys, bones, immunity, inflammation, metabolism and other components, often accompanied by symptoms such as obesity, hypertension, myocardial hypertrophy, diabetes or atrial fibrillation. HFpEF is a syndrome with high morbidity and mortality. According to clinical statistics, the mortality rate due to HF is 35%, and the proportion of deaths caused by HFpEF accounts for 57%. However, to date, there are few drug therapies or medical devices that have been proven to change the disease progression and prognosis of HFpEF patients. At present, the field urgently needs to develop a drug and / or treatment method that can effectively treat HFpEF.

[0004] Jun is a transcription factor and a member of the AP1 family. It has chromatin binding activity and binding activity to transcriptional cis-regulatory regions, participating in the regulation of processes including organ development, protein phosphorylation, and cell proliferation. Jun inhibitors are those that can inhibit Jun gene expression, reduce Jun binding activity to DNA, decrease the level of Jun gene expression products, or prevent or block Jun signal transduction. Studies have shown that Jun inhibitors have therapeutic effects in animal models of endometriosis, breast cancer, and sepsis. Summary of the Invention

[0005] The inventors of the present invention have discovered for the first time that inhibiting elevated Jun expression using a Jun inhibitor (e.g., T5524) can prevent and treat HFpEF. Based on this discovery, the inventors further screened salt forms and crystal forms of T5524, obtaining salt forms and crystal forms that exhibit high stability, good solubility, and high bioavailability, as well as excellent therapeutic efficacy for HFpEF, demonstrating high application value.

[0006] To this end, in the first aspect of the present invention, the present invention provides a salt of the compound represented by formula I, wherein the salt is a sodium salt, potassium salt, calcium salt, tert-butylamine salt, ammonium salt, choline salt, L-lysine salt, diethylamine salt, dimethylethanolamine salt, betaine salt or ethylenediamine salt of the compound represented by formula I,

[0007] In some embodiments, the salt is a sodium salt, potassium salt, tert-butylamine salt, choline salt, L-lysine salt, diethylamine salt, dimethylethanolamine salt, betaine salt or ethylenediamine salt of the compound represented by Formula I.

[0008] In some embodiments, the salt is a choline salt, a diethylamine salt, or a betaine salt of the compound represented by Formula I.

[0009] In some embodiments, the salt is a choline salt or a betaine salt of the compound represented by Formula I.

[0010] In some embodiments, the salt is a choline salt of the compound of Formula I.

[0011] In some embodiments, the sodium salt of the compound of Formula I is Form A, wherein the sodium salt of the compound of Formula I, Form A, has an X-ray powder diffraction pattern substantially the same as that in FIG4 .

[0012] In some embodiments, the sodium salt of the compound of Formula I is in Form B, wherein the sodium salt of the compound of Formula I in Form B has an X-ray powder diffraction pattern substantially the same as that in FIG6 .

[0013] In some embodiments, the potassium salt of the compound of Formula I is Form A, wherein the potassium salt of the compound of Formula I Form A has an X-ray powder diffraction pattern substantially the same as that in FIG10 .

[0014] In some embodiments, the potassium salt of the compound of Formula I is in Form B, wherein the potassium salt of the compound of Formula I in Form B has an X-ray powder diffraction pattern substantially the same as that in FIG12 .

[0015] In some embodiments, the potassium salt of the compound of Formula I is Form C, wherein the potassium salt of the compound of Formula I, Form C, has an X-ray powder diffraction pattern substantially the same as that in FIG16 .

[0016] In some embodiments, the potassium salt of the compound of Formula I is Form D, wherein the potassium salt of the compound of Formula I, Form D, has an X-ray powder diffraction pattern substantially the same as that in FIG18 .

[0017] In some embodiments, the calcium salt of the compound represented by Formula I is in Form A, wherein the calcium salt of the compound represented by Formula I in Form A has an X-ray powder diffraction pattern substantially the same as that in FIG19 .

[0018] In some embodiments, the tert-butylamine salt of the compound represented by Formula I is in Form A, wherein the Form A of the tert-butylamine salt of the compound represented by Formula I has an X-ray powder diffraction pattern substantially the same as that in FIG20 .

[0019] In some embodiments, the tert-butylamine salt of the compound represented by Formula I is in Form B, wherein the tert-butylamine salt of the compound represented by Formula I in Form B has an X-ray powder diffraction pattern substantially the same as that in FIG. 22 .

[0020] In some embodiments, the ammonium salt of the compound represented by Formula I is in Form A, wherein the ammonium salt of the compound represented by Formula I in Form A has an X-ray powder diffraction pattern substantially the same as that in FIG. 26 .

[0021] In some embodiments, the ammonium salt of the compound represented by Formula I is in Form B, wherein the ammonium salt of the compound represented by Formula I in Form B has an X-ray powder diffraction pattern substantially the same as that in FIG. 27 .

[0022] In some embodiments, the L-lysine salt of the compound represented by Formula I is in Form A, wherein the Form A of the L-lysine salt of the compound represented by Formula I has an X-ray powder diffraction pattern substantially the same as that in FIG32 .

[0023] In some embodiments, the diethylamine salt of the compound represented by Formula I is in Form A, wherein the diethylamine salt of the compound represented by Formula I in Form A has an X-ray powder diffraction pattern substantially the same as that in FIG. 36 .

[0024] In some embodiments, the diethylamine salt of the compound represented by Formula I is in Form B, wherein the diethylamine salt of the compound represented by Formula I in Form B has an X-ray powder diffraction pattern substantially the same as that in FIG40 .

[0025] In some embodiments, the diethylamine salt of the compound represented by Formula I is a crystalline form C, wherein the X-ray powder diffraction 2θ of the diethylamine salt crystalline form C of the compound represented by Formula I has characteristic peaks at 4.7±0.2°, 6.4±0.2°, 9.4±0.2°, 13.8±0.2°, 15.0±0.2°, 16.8±0.2°, 19.7±0.2° and 20.4±0.2°.

[0026] In some embodiments, the X-ray powder diffraction 2θ of the diethylamine salt form C of the compound represented by Formula I has characteristic peaks at 4.7±0.2°, 6.4±0.2°, 9.4±0.2°, 13.1±0.2°, 13.8±0.2°, 14.1±0.2°, 15.0±0.2°, 16.8±0.2°, 18.0±0.2°, 18.9±0.2°, 19.4±0.2°, 19.7±0.2°, 20.4±0.2°, 23.0±0.2°, 23.3±0.2° and 24.9±0.2°.

[0027] In some embodiments, the 2θ, d, and peak intensity of the X-ray powder diffraction of Form C of the diethylamine salt of the compound represented by Formula I are as shown in Table C.

[0028] In some embodiments, the diethylamine salt Form C of the compound represented by Formula I has an X-ray powder diffraction pattern substantially the same as that in FIG. 62 .

[0029] In some embodiments, the dimethylethanolamine salt of the compound represented by Formula I is in Form A, wherein the dimethylethanolamine salt of the compound represented by Formula I in Form A has an X-ray powder diffraction pattern substantially the same as that in FIG44 .

[0030] In some embodiments, the betaine salt of the compound represented by Formula I is in Form A, wherein the X-ray powder diffraction 2θ of the betaine salt Form A of the compound represented by Formula I has characteristic peaks at 8.7±0.2°, 11.8±0.2°, 15.4±0.2°, 16.3±0.2°, 19.6±0.2°, 21.4±0.2° and 23.4±0.2°.

[0031] In some embodiments, the X-ray powder diffraction 2θ of the betaine salt form A of the compound represented by Formula I has characteristic peaks at 8.1±0.2°, 8.7±0.2°, 11.8±0.2°, 15.4±0.2°, 16.3±0.2°, 18.4±0.2°, 19.6±0.2°, 20.4±0.2°, 21.4±0.2°, 22.3±0.2°, 23.4±0.2°, 24.2±0.2° and 25.9±0.2°.

[0032] In some embodiments, the 2θ, d, and peak intensity of the X-ray powder diffraction of Form A of the betaine salt of the compound represented by Formula I are as shown in Table A.

[0033] In some embodiments, the betaine salt Form A of the compound represented by Formula I has an X-ray powder diffraction pattern substantially the same as that in FIG. 48 .

[0034] In some embodiments, the ethylenediamine salt of the compound represented by Formula I is in Form A, wherein the ethylenediamine salt of the compound represented by Formula I in Form A has an X-ray powder diffraction pattern substantially the same as that in FIG52 .

[0035] In some embodiments, the ethylenediamine salt of the compound represented by Formula I is in Form B, wherein the ethylenediamine salt of the compound represented by Formula I in Form B has an X-ray powder diffraction pattern substantially the same as that in FIG54 .

[0036] In some embodiments, the choline salt of the compound represented by Formula I is in Form C, wherein the X-ray powder diffraction 2θ of the choline salt of the compound represented by Formula I in Form C has characteristic peaks at 4.7±0.2°, 6.4±0.2°, 9.4±0.2°, 15.0±0.2°, 16.8±0.2°, 19.7±0.2° and 20.4±0.2°.

[0037] In some embodiments, the choline salt of the compound represented by Formula I has characteristic peaks in X-ray powder diffraction 2θ of Form C at 4.7±0.2°, 6.4±0.2°, 9.4±0.2°, 13.1±0.2°, 13.8±0.2°, 15.0±0.2°, 16.8±0.2°, 18.9±0.2°, 19.7±0.2°, 20.4±0.2°, 22.4±0.2°, 22.9±0.2°, 23.2±0.2° and 24.9±0.2°.

[0038] In some embodiments, the 2θ, d, and peak intensity of the X-ray powder diffraction of Form C of the choline salt of the compound represented by Formula I are as shown in Table E.

[0039] In some embodiments, the choline salt Form C of the compound represented by Formula I has an X-ray powder diffraction pattern substantially the same as that in FIG. 70 .

[0040] In some embodiments, the choline salt of the compound represented by Formula I is in Form A, wherein the X-ray powder diffraction 2θ of the choline salt of the compound represented by Formula I in Form A has characteristic peaks at 6.9±0.2°, 14.0±0.2°, 19.7±0.2°, 21.2±0.2°, 22.8±0.2°, 26.5±0.2° and 27.0±0.2°.

[0041] In some embodiments, the choline salt form A of the compound represented by formula I has characteristic peaks in X-ray powder diffraction 2θ at 6.9±0.2°, 11.7±0.2°, 14.0±0.2°, 15.0±0.2°, 17.7±0.2°, 18.6±0.2°, 19.1±0.2°, 19.7±0.2°, 20.9±0.2°, 21.2±0.2°, 22.8±0.2°, 26.3±0.2°, 26.5±0.2°, 27.0±0.2° and 27.6±0.2°.

[0042] In some embodiments, the 2θ, d, and peak intensity of the X-ray powder diffraction of the choline salt crystal form A of the compound represented by Formula I are as shown in Table B.

[0043] In some embodiments, the choline salt Form A of the compound represented by Formula I has an X-ray powder diffraction pattern substantially the same as that in FIG. 28 .

[0044] In some embodiments, the choline salt of the compound represented by Formula I is in Form B, wherein the X-ray powder diffraction 2θ of the choline salt of the compound represented by Formula I in Form B has characteristic peaks at 8.9±0.2°, 17.0±0.2°, 17.4±0.2°, 19.5±0.2°, 20.4±0.2°, 22.2±0.2°, 22.7±0.2° and 27.2±0.2°.

[0045] In some embodiments, the choline salt form B of the compound represented by formula I has characteristic peaks in X-ray powder diffraction 2θ at 8.9±0.2°, 16.2±0.2°, 17.0±0.2°, 17.4±0.2°, 19.5±0.2°, 19.9±0.2°, 20.4±0.2°, 20.9±0.2°, 21.8±0.2°, 22.2±0.2°, 22.5±0.2°, 22.7±0.2°, 26.5±0.2°, 27.2±0.2° and 27.5±0.2°.

[0046] In some embodiments, the 2θ, d, and peak intensity of the X-ray powder diffraction of Form B of the choline salt of the compound represented by Formula I are as shown in Table D.

[0047] In some embodiments, the choline salt Form B of the compound represented by Formula I has an X-ray powder diffraction pattern substantially the same as that in FIG. 66 .

[0048] In the second aspect of the present invention, the present invention provides a pharmaceutical composition comprising the aforementioned salt; optionally, further comprising a pharmaceutically acceptable excipient.

[0049] In the third aspect of the present invention, the present invention provides use of the aforementioned salt in the preparation of a medicament for treating and / or preventing heart failure with preserved ejection fraction;

[0050] Alternatively, the aforementioned salt is provided for use in treating and / or preventing heart failure with preserved ejection fraction;

[0051] Alternatively, provided is a method for treating and / or preventing heart failure with preserved ejection fraction, comprising administering an effective amount of the aforementioned salt to a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1: Construction of the HFpEF model. A: Schematic diagram of the experimental process; B: Systolic function test results of mice after 5 weeks of feeding; C: Diastolic function test results of mice after 5 weeks of feeding.

[0053] Figure 2: Relative expression of Jun in cardiomyocytes after 15 weeks of HFD+L-NAME feeding, indicating that Jun is highly expressed in HFpEF model mice.

[0054] Figure 3: T-5224 effectively alleviates the development and progression of HFpEF. A: Systolic function test results of mice at different time points; B: Diastolic function test results of mice at different time points; C: Changes in body weight of mice treated with different treatments; D: Changes in Jun expression in cardiomyocytes of mice treated with different treatments.

[0055] Figure 4: XRPD spectrum of T-5224 sodium salt Form A.

[0056] Figure 5: T-5224 sodium salt form A 1 H-NMR spectrum.

[0057] Figure 6: XRPD spectrum of T-5224 sodium salt Form B.

[0058] Figure 7: DSC spectrum of T-5224 sodium salt Form B.

[0059] Figure 8: TGA spectrum of T-5224 sodium salt form B.

[0060] Figure 9: T-5224 sodium salt form B 1 H-NMR spectrum.

[0061] Figure 10: XRPD spectrum of T-5224 potassium salt Form A.

[0062] Figure 11: T-5224 potassium salt form A 1 H-NMR spectrum.

[0063] Figure 12: XRPD spectrum of T-5224 potassium salt Form B.

[0064] Figure 13: DSC spectrum of T-5224 potassium salt Form B.

[0065] Figure 14: TGA spectrum of T-5224 potassium salt form B.

[0066] Figure 15: T-5224 potassium salt form B 1 H-NMR spectrum.

[0067] Figure 16: XRPD spectrum of T-5224 potassium salt Form C.

[0068] Figure 17: T-5224 potassium salt form C 1 H-NMR spectrum.

[0069] Figure 18: XRPD spectrum of T-5224 potassium salt Form D.

[0070] Figure 19: XRPD spectrum of T-5224 calcium salt Form A.

[0071] Figure 20: XRPD spectrum of T-5224 tert-butylamine salt Form A.

[0072] Figure 21: T-5224 tert-butylamine salt Form A 1 H-NMR spectrum.

[0073] Figure 22: XRPD spectrum of T-5224 tert-butylamine salt Form B.

[0074] Figure 23: DSC spectrum of T-5224 tert-butylamine salt Form B.

[0075] Figure 24: TGA spectrum of T-5224 tert-butylamine salt Form B.

[0076] Figure 25: T-5224 tert-butylamine salt Form B 1 H-NMR spectrum.

[0077] Figure 26: XRPD spectrum of T-5224 ammonium salt Form A.

[0078] Figure 27: XRPD spectrum of T-5224 ammonium salt Form B.

[0079] Figure 28: XRPD spectrum of T-5224 choline salt Form A.

[0080] Figure 29: DSC spectrum of T-5224 choline salt Form A.

[0081] Figure 30: TGA spectrum of T-5224 choline salt form A.

[0082] Figure 31: T-5224 choline salt crystal form A 1 H-NMR spectrum.

[0083] Figure 32: XRPD spectrum of T-5224 L-lysine salt Form A.

[0084] Figure 33: DSC spectrum of T-5224 L-lysine salt Form A.

[0085] Figure 34: TGA spectrum of T-5224 L-lysine salt Form A.

[0086] Figure 35: T-5224 L-lysine salt Form A 1 H-NMR spectrum.

[0087] Figure 36: XRPD spectrum of T-5224 diethylamine salt Form A.

[0088] Figure 37: DSC spectrum of T-5224 diethylamine salt Form A.

[0089] Figure 38: TGA spectrum of T-5224 diethylamine salt Form A.

[0090] Figure 39: T-5224 diethylamine salt crystal form A 1 H-NMR spectrum.

[0091] Figure 40: XRPD spectrum of T-5224 diethylamine salt Form B.

[0092] Figure 41: DSC spectrum of T-5224 diethylamine salt Form B.

[0093] Figure 42: TGA spectrum of T-5224 diethylamine salt Form B.

[0094] Figure 43: T-5224 diethylamine salt crystal form B 1 H-NMR spectrum.

[0095] Figure 44: XRPD spectrum of T-5224 dimethylethanolamine salt Form A.

[0096] Figure 45: DSC spectrum of T-5224 dimethylethanolamine salt Form A.

[0097] Figure 46: TGA spectrum of T-5224 dimethylethanolamine salt Form A.

[0098] Figure 47: T-5224 dimethylethanolamine salt crystal form A 1 H-NMR spectrum.

[0099] Figure 48: XRPD spectrum of T-5224 betaine salt Form A.

[0100] Figure 49: DSC spectrum of T-5224 betaine salt Form A.

[0101] Figure 50: TGA spectrum of T-5224 betaine salt Form A.

[0102] Figure 51: T-5224 betaine salt crystal form A 1 H-NMR spectrum.

[0103] Figure 52: XRPD spectrum of T-5224 ethylenediamine salt Form A.

[0104] Figure 53: T-5224 ethylenediamine salt crystal form A 1 H-NMR spectrum.

[0105] Figure 54: XRPD spectrum of T-5224 ethylenediamine salt Form B.

[0106] Figure 55: DSC spectrum of T-5224 ethylenediamine salt Form B.

[0107] Figure 56: TGA spectrum of T-5224 ethylenediamine salt Form B.

[0108] Figure 57: T-5224 ethylenediamine salt crystal form B 1 H-NMR spectrum.

[0109] Figure 58: XRPD spectrum of T-5224 Form A.

[0110] Figure 59: DSC spectrum of T-5224 Form A.

[0111] Figure 60: TGA spectrum of T-5224 Form A.

[0112] Figure 61: T-5224 Form A 1 H-NMR spectrum.

[0113] Figure 62: XRPD spectrum of T-5224 diethylamine salt Form C.

[0114] Figure 63: DSC spectrum of T-5224 diethylamine salt Form C.

[0115] Figure 64: TGA spectrum of T-5224 diethylamine salt Form C.

[0116] Figure 65: T-5224 diethylamine salt crystal form C 1 H-NMR spectrum.

[0117] Figure 66: XRPD spectrum of T-5224 choline salt Form B.

[0118] Figure 67: DSC spectrum of T-5224 choline salt Form B.

[0119] Figure 68: TGA spectrum of T-5224 choline salt Form B.

[0120] Figure 69: T-5224 choline salt crystal form B 1 H-NMR spectrum.

[0121] Figure 70: XRPD spectrum of T-5224 choline salt Form C.

[0122] Figure 71: DSC spectrum of T-5224 choline salt Form C.

[0123] Figure 72: T-5224 choline salt crystal form C 1 H-NMR spectrum.

[0124] Figure 73: T-5224 choline salt form B can effectively inhibit the occurrence and development of HFpEF. DETAILED DESCRIPTION

[0125] It should be understood that the terminology used herein is intended to describe specific embodiments and is not intended to be limiting. In addition, although any method, device, and material similar or equivalent to those described herein may be used for implementing or testing the present invention, preferred methods, devices, and materials are now described.

[0126] As used herein, the term "treat" generally refers to obtaining a desired pharmacological and / or physiological effect. This effect can be prophylactic, in terms of completely or partially preventing a disease or its symptoms; and / or therapeutic, in terms of partially or completely stabilizing or curing a disease and / or causing side effects due to the disease. As used herein, "treat" encompasses any treatment of a disease in a patient, including: (a) preventing the onset of a disease or symptom in a patient who is susceptible to the disease or symptom but has not yet been diagnosed with the disease; (b) suppressing the symptoms of a disease, i.e., arresting its development; or (c) alleviating the symptoms of a disease, i.e., causing the disease or symptom to regress.

[0127] In the present invention, "subject" refers to a vertebrate. In certain embodiments, the vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, the mammal refers to a human.

[0128] As used herein, an "effective amount" refers to an amount effective to achieve the desired therapeutic effect at the dosage and duration necessary. A "therapeutically effective amount" of a substance / molecule of the invention may vary depending on factors such as the individual's disease state, age, sex, and weight, and the ability of the substance / molecule to elicit a desired response in the individual. A therapeutically effective amount also encompasses an amount in which any toxic or deleterious effects of the substance / molecule are outweighed by the therapeutically beneficial effects.

[0129] The pharmaceutical composition of the present invention may contain pharmaceutically acceptable excipients, including but not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, beeswax, lanolin, etc.

[0130] The pharmaceutical composition of the present invention can be prepared into various forms according to different routes of administration. For example, the pharmaceutical composition can be administered by any of the following methods: oral administration, spray inhalation, rectal administration, nasal administration, buccal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion, or administration via an explanted reservoir. Oral and intravenous administration are preferred.

[0131] The compounds of the present invention may optionally be used in combination with one or more other active ingredients, and the dosage and ratio of each can be adjusted by those skilled in the art according to the specific disease and patient conditions and clinical needs.

[0132] The test methods involved in the present invention are all well-known and commonly used test methods in the art, for example, as shown below:

[0133] The present invention will be further explained below with reference to specific embodiments.

[0134] Example A: Preliminary Studies

[0135] 1. Materials and Reagents

[0136] In this example, C57BL / 6N wild-type mice were purchased from Beijing Weitonglihua. The sources of the reagents are shown in the following table.

[0137] In addition to the above, other materials and reagents used in this example are also commercially available products.

[0138] 2. Animal Experiment Guidelines

[0139] In this example, all animal studies were conducted under the guidance of the Laboratory Animal Center, Fuwai Hospital Animal Care and Use Committee, National Center for Cardiovascular Diseases, China. All mice were propagated and housed under the same conditions and randomly assigned to groups during the experiment. Echocardiographic analysis was performed by an independent investigator who was unaware of the study objectives.

[0140] 3. Induction of Heart Failure Model with Preserved Ejection Fraction

[0141] Eight- to ten-week-old male C57BL / 6N wild-type mice were divided into three groups: a normal control group (normal diet and water), a model control group (high-fat diet combined with N-nitro-L-arginine methyl ester), and a model treatment group (high-fat diet combined with N-nitro-L-arginine methyl ester and treatment with T-5224). The model control and model treatment groups were established using the method described in the following literature: Gabriele G. Schiattarella et al., Nitrosative stress drives heart failure with preserved ejection fraction, https: / / doi.org / 10.1038 / s41586-019-1100-z. Specifically, a high-fat diet (HFD) (60% kcal from fat (lard)) and N-nitro-L-arginine methyl ester (L-NAME, 0.5 g / L in drinking water) were used to induce heart failure with preserved ejection fraction, creating an HFpEF animal model.

[0142] The systolic function parameter LVEF of the mice tested in the fifth week of model induction did not change, while the diastolic function parameter (E / E') increased significantly in the fifth week of model induction, indicating that the heart failure model with preserved ejection fraction described in the aforementioned literature has been successfully obtained. At the same time, there was no significant difference in the diastolic function parameter (E / E') between the model control group and the model treatment group at five weeks, and subsequent drug administration was carried out under the same baseline conditions, as shown in Figure 1.

[0143] 4. Jun expression is correlated with HFpEF

[0144] At 15 weeks of model induction, myocardial cells from normal mice and the HFpEF model were extracted and separated using a perfusion method, and quantitative RCR detection was performed. The specific procedures are as follows:

[0145] 4.1. Isolation of adult mouse cardiomyocytes:

[0146] In order to isolate cardiomyocytes from the heart of adult mice, we used the classic perfusion method to isolate cardiomyocytes. Specifically, 100 μl of sodium heparin (1000 units in 50 ml) was injected into the mouse 20 minutes before being killed to prevent heart coagulation during the operation, which increased the difficulty of digestion. After that, the mouse was anesthetized and killed, the heart was removed and transferred to a calcium-free solution for washing. Then, the Langendorff method was used for digestion. The heart was perfused with calcium-free solution for 5 minutes using a Langendorff apparatus, and then digested with a digestive enzyme solution (0.7 mg / ml type II collagenase and 0.7 mg / ml bovine serum albumin calcium-free solution) for about 30 minutes. After about 20 minutes, the heart was constantly touched. When the heart became soft and slippery, it indicated that the digestion was basically completed. Then, the tissue from the ventricle was collected, chopped, and gently blown to dissociate into single cells. The cells were allowed to settle, the supernatant was taken, and the undigested and adherent tissues were removed. 100 g Centrifuge at 4°C for 2 minutes to obtain a myocardial cell pellet. The supernatant is mostly non-myocardial cells. Resuspend the myocardial cells in calcium-free solution containing 10% FBS for subsequent experiments. Non-myocardial cells can be re-selected with culture medium or PBS for subsequent experiments. To obtain purer myocardial cells and non-myocardial cells, centrifuge the cell suspension (100g, 2 minutes at room temperature) three times to separate myocardial cells from non-myocardial cells. Collect myocardial cells for further experiments.

[0147] 4.2. Quantitative PCR detection:

[0148] Total RNA was extracted from cells using a GeneJet RNA purification kit (Thermo Scientific, K0732), and 0.1 μg of total RNA was reverse transcribed using an iScript™ cDNA synthesis kit (Bio-Rad, 1708890) to generate cDNA. qPCR was performed using iTaq Universal SYBR Green supermix (1725121, Bio-Rad) on an ABI Vii7 Real-Time System (Life Technologies, Q6). β-Actin was used for standardized quantitative analysis. As shown in Figure 2, significantly higher expression of Jun was observed in the HFpEF mouse model compared to normal mice. This suggests that Jun expression in mice is correlated with HFpEF and that Jun is highly expressed in HFpEF.

[0149] 5. T-5224 administration method

[0150] After establishing the animal model, starting from the fifth week of HFpEF induction, mice in the treatment group were treated with T-5224, while the control group was treated with a non-drug-containing solvent. Mice that received a normal diet and water throughout the induction process served as negative controls. Mice were treated at five weeks of age. T-5224 was administered to the treatment group at a dose of 250 mg / kg based on body weight, every other day, with 0.8 mg of T-5224 dissolved in 200 μL of 1% PVP solution. Dosing began in the fifth week and continued through the thirteenth week (a total of 15 doses), for a total of 250 mg / kg. The control group received an equal volume of 1% PVP solution, and all other treatments were identical.

[0151] 6. Conventional ultrasonic testing

[0152] All mice were fed under different conditions for five weeks and then underwent routine ultrasound examinations every two weeks until the end of the fifteen-week monitoring period. Specifically, transthoracic echocardiography was performed using a VisualSonics Vevo 2100 system equipped with an MS400 transducer (Visual Sonics). Left ventricular ejection fraction (LVEF) and other systolic function indicators were obtained from short-axis M-mode scans at the level of the mid-ventricle, as indicated by the presence of papillary muscles, in conscious, lightly restrained mice. Apical four-chamber views were obtained in anesthetized mice for diastolic function measurements using pulsed wave and tissue Doppler imaging at the level of the mitral valve. Anesthesia was induced by 2.5% isoflurane and confirmed by the lack of response to firm pressure on one of the hind paws. Isoflurane was reduced to 1.0-1.5% during echocardiographic acquisition (under temperature-controlled conditions) and adjusted to keep the heart rate within 500 beats per minute. Parameters collected included heart rate, left ventricular end-diastolic diameter, left ventricular end-systolic diameter, end-diastolic interventricular septal wall thickness, left ventricular end-diastolic posterior wall thickness, left ventricular fractional shortening, left ventricular ejection fraction (LVEF), peak Doppler velocity across the mitral valve in early diastole, peak Doppler velocity across the mitral valve in late diastole, isovolumetric relaxation time, and tissue Doppler peak relaxation velocity at the mitral annulus during early diastole and early filling deceleration. At the end of the procedure, all mice recovered from anesthesia without any abnormalities. All parameters were measured at least three times, and the mean values ​​are presented. Ultrasound testing included both systolic and diastolic function.

[0153] 7. Experimental results and conclusions

[0154] First, systolic and diastolic function were assessed at 5 weeks. While systolic function remained unchanged, the diastolic function parameter, E / E', increased significantly, demonstrating diastolic dysfunction and the successful establishment of the model described in the aforementioned literature. Furthermore, with the fifth week as the starting point for drug administration, no significant differences in cardiac diastolic function were observed between the control and treatment groups before drug administration (Figures 1A-C). Based on this, drug administration was performed.

[0155] Second, Jun expression was upregulated in the control and normal groups, indicating a correlation between Jun expression and HFpEF. Jun is highly expressed in the HFpEF mouse model (Figure 2). Based on this correlation, it is possible that Jun inhibitors could be used for the prevention and treatment of HFpEF.

[0156] Furthermore, T-5224 was used to verify the efficacy of Jun inhibitors in preventing and treating HFpEF. After confirming the successful model construction and the baseline of the model control and model treatment groups was consistent, the model treatment group was treated with T-5224. Cardiac function tests showed that the development and progression of HFpEF in the model treatment group (after T-5224 administration) were significantly inhibited. Specifically, after T-5224 treatment, diastolic function in mice treated with a high-fat diet combined with L-NAME (HFD + 0.5g / L L-NAME) was significantly improved, and this improvement lasted until the 15th week. However, in the model control group mice not treated with T-5224, diastolic function continued to deteriorate (Figures 3A-B). At the same time, Jun expression in the model treatment group was downregulated compared with the model control group (Figure 3D), and mouse obesity was improved (Figure 3C). This indicates that T-5224, as a Jun inhibitor, can have a preventive and therapeutic effect on HFpEF in the mouse HFpEF model.

[0157] Example 1: Screening and preparation of T-5524 salt

[0158] Based on the experimental results of Example A, the inventors further explored new salt forms and crystal forms of T-5524.

[0159] 1. Suspension method

[0160] Approximately 30-50 mg of compound T-5224 Form A was weighed and placed in a 2 mL glass vial with 0.5, 1, or 2 equivalents of base. Screening was performed using a suspension method with the addition of the screening solvent. The resulting sample was suspended at 50°C for 2 hours, then naturally cooled to 25°C and suspended at 25°C for at least 48 hours.

[0161] The resulting suspension was centrifuged at 14,000 rpm through a 0.45 μm nylon filter, and the resulting solid was dried under vacuum at 50° C. for 2 hours and characterized by XRPD.

[0162] Table 1-1: Preparation of salt by suspension method

[0163] Note: “ / / ” indicates that it has not been carried out

[0164] 2. Cooling method

[0165] The foggy suspension and clear solution obtained by the above suspension were cooled to 5°C to prepare solid samples for characterization. Only jelly or clear solution was obtained.

[0166] Table 1-2: Cooling methods

[0167] Based on the above screening experiments, the sodium, potassium, calcium, tert-butylamine, ammonium, choline, L-lysine, diethylamine, dimethylethanolamine, betaine, and ethylenediamine salts of compound T-5224, as well as their corresponding polymorphic forms, were prepared. The characterization of the crystalline forms of each salt is shown in Figures 4-57. The X-ray powder diffraction (XRPD) data for Form A of the betaine salt and Form A of the choline salt of T-5224 are shown in Tables A and B.

[0168] Table A: X-ray powder diffraction data of T-5224 betaine salt form A

[0169] Table B: X-ray powder diffraction data of T-5224 choline salt form A

[0170] Example 2: Preliminary Study on the Stability and Solubility of T-5524 Salt and Its Crystalline Forms

[0171] The sodium salt crystal form B, potassium salt crystal form B, tert-butylamine salt crystal form B, choline salt crystal form A, L-lysine salt crystal form A, diethylamine salt crystal form A, diethylamine salt crystal form B, dimethylethanolamine salt crystal form A, betaine salt crystal form A and ethylenediamine salt crystal form B prepared in Example 1 have good crystallinity and a reasonable salt formation ratio. Therefore, the stability of the polymorphs of these salts was studied.

[0172] Ten candidate salt forms were exposed to 25°C / 60% RH for three days and 25°C / 80% RH for two days to initially assess their physical stability. Furthermore, their approximate solubility in pH 4.5 acetate buffer was also evaluated. Based on their physical stability and solubility, candidate salt forms were selected for further evaluation. The results are shown in Tables 2-1 and 2-2 below.

[0173] Table 2-1

[0174] Table 2-2

[0175] Among the above alternative salt forms, choline salt form A, diethylamine salt form A, and betaine salt form A exhibit good physical and chemical properties, including good crystallinity and good physical stability. In addition, in the approximate solubility experiment in pH 4.5 acetate buffer solution at 25°C, they also showed potential solubility advantages. Therefore, the above three alternative salt forms were selected as candidate salt forms for scaled-up preparation, and the three candidate salt forms were systematically evaluated and compared with compound T-5224 form A in terms of stability, solubility, and hygroscopicity.

[0176] Example 3: Comparison of the physical and chemical properties of the dominant salt form and its crystal form with T-5524 crystal form A

[0177] 1. Preparation of reference compounds and candidate salt forms

[0178] In this section, scaled-up batches of choline salt Form A and betaine salt Form A were successfully obtained. The candidate salt forms of the scaled-up batches were consistent with the crystal forms of the candidate salt forms obtained in the previous screening. For the diethylamine salt, a new crystal form, diethylamine salt Form C, was obtained even when diethylamine salt Form A seed crystals were added. This indicates that diethylamine salt Form C is more stable than diethylamine salt Form A, so diethylamine salt Form C was systematically characterized subsequently.

[0179] 1.1 Preparation of Control T5224 Form A

[0180] Weigh 500 mg of compound T5224 into an 8 mL glass bottle. Add 4 mL of acetone and stir at 50°C for 5 minutes to obtain a suspension. (Suspension)

[0181] About 3 mg of T-5524 Form A seed crystals were added to the above suspension. (Suspension)

[0182] After stirring at 50°C for 2 h, the suspension was cooled to 25°C and stirred at 25°C for about 3 days. (Suspension)

[0183] The solid portion was collected by centrifugation and dried under vacuum at 50°C for about 2 hours.

[0184] A total of about 253 mg of Form A white powder was obtained, with a yield of 51%.

[0185] The characterization of T-5224 Form A as a control is shown in Figures 58-61.

[0186] 1.2 Preparation of T-5224 Choline Salt Form A

[0187] Weigh 500 mg of compound T5224 and 256 mg (1.05 equivalents) of choline into an 8 mL glass bottle. Add 1 mL of acetone and stir at 50°C for 5 minutes to obtain a clear solution. (Clear solution)

[0188] After stirring at 50°C for 2 h, the solution became turbid and gradually turned into a suspension. (Clear solution → suspension)

[0189] About 3 mg of choline salt Form A seed crystals were added to the above suspension. (Suspension)

[0190] The suspension was cooled to 25°C and stirred at 25°C for about 2 days. (Suspension)

[0191] The suspension was cooled to 5°C and stirring was continued at 5°C for about 1 day. (Suspension)

[0192] The solid portion was collected by centrifugation and dried under vacuum at 50°C for about 2 hours.

[0193] A total of 150 mg of choline salt crystal form A white powder was obtained, with a yield of 20%.

[0194] 1.3 Preparation of T-5224 diethylamine salt Form C

[0195] Weigh 500 mg of compound T5224 and 71 mg (1.05 equivalents) of diethylamine into an 8 mL glass bottle. Add 5.1 mL of acetone and stir at 50°C for 5 minutes to obtain a suspension. (Suspension)

[0196] About 3 mg of diethylamine salt Form A seed crystals were added to the above suspension. (Suspension)

[0197] After stirring at 50°C for 2 h, the suspension was cooled to 25°C and stirred at 25°C for about 2 days. (Suspension)

[0198] The solid portion was collected by centrifugation and dried under vacuum at 50°C for about 2 hours.

[0199] A total of 523 mg of a new crystalline diethylamine salt, form C, as an off-white powder was obtained, with a yield of 92%.

[0200] The characterization of T-5224 diethylamine salt Form C is shown in Figures 62-65 , wherein the X-ray powder diffraction (XRPD) data is shown in Table C.

[0201] Table C: X-ray powder diffraction data of T-5224 diethylamine salt form C

[0202] 1.4 Preparation of T-5224 Betaine Salt Form A

[0203] Weigh 500 mg of compound T5224 and 122 mg (1.05 equivalents) of betaine into an 8 mL glass bottle. Add 4.4 mL of acetone and stir at 50°C for 5 minutes to obtain a suspension. (Suspension)

[0204] About 3 mg of betaine salt Form A seed crystals were added to the above suspension. (Suspension)

[0205] After stirring at 50°C for 2 h, the suspension was cooled to 25°C and stirred at 25°C for about 2 days. (Suspension)

[0206] The solid portion was collected by centrifugation and dried under vacuum at 50°C for about 2 hours.

[0207] A total of 473 mg of betaine salt crystal form A white powder was obtained, with a yield of 76%.

[0208] 2. Stability comparison

[0209] 2.1 Starting chemical purity

[0210] The chemical purities of T5224 crystal form A, choline salt crystal form A, diethylamine salt crystal form C and betaine salt crystal form A were 98.7%, 99.4%, 98.4% and 98.8%, respectively.

[0211] 2.2 Solid stability

[0212] Open containers containing T5224 Form A and three candidate salt forms were placed at 25°C / 92.5% RH for one week. Closed containers containing T5224 Form A and three candidate salt forms were placed at 60°C for one week. After solid stability assessment, samples were characterized by XRPD and HPLC, and color changes were observed.

[0213] Table 3-1: Solid Stability: Purity and Appearance (Color, CL) Note: A: No color change; B: Slight color change; C: Moderate color change; D: Severe color change

[0214] The above physical forms all show good physical and chemical stability.

[0215] 2.3 Solubility

[0216] This study investigated the solubility of Form A of T5224 and three candidate salt forms in water, five aqueous buffers, and simulated biological fluids at 37°C for 2 and 24 hours. The solvents used included 50 mM acetate buffer (pH 4.5) (solubility tested only for 2 hours at 37°C), water, FaSSGF (pH 1.6), FaSSIF-v1 (pH 6.5), and FeSSIF-v1 (pH 5.0). The residual solids from the solubility experiments were characterized by XRPD.

[0217] Accurately weigh 10.0 mg of T5224 Form A, 12.6 mg of choline salt Form A, 11.3 mg of diethylamine salt Form C, or 12.3 mg of betaine salt Form A into 8 mL glass bottles. Add 5 mL of solvent to each. The mass of the salt weighed is equivalent to 10 mg of T5224 Form A. The resulting suspension is stirred at 400 rpm at 37°C, and samples are taken after 2 hours and 24 hours. The samples are centrifuged at 14,000 rpm at 37°C for 5 minutes. The supernatant concentration is determined by HPLC, and the pH of the supernatant is measured using a pH meter. The results are shown in Table 3-2.

[0218] Table 3-2: Solubility Note: “ / / ”: not yet launched

[0219] The solubility of T-5524 Form A and three candidate salt forms is pH-dependent. The three candidate salt forms exhibit comparable solubility to T-5524 in pH 1.6 FaSSGF, pH 4.5 acetate buffer (50 mM), and pH 5.0 FeSSIF-v1. Solubility in pH 1.6 FaSSGF and pH 4.5 acetate buffer (50 mM) is close to or below the limit of detection, while solubility in pH 5.0 FeSSIF-v1 is 15-20 μg / mL.

[0220] The solubility of the three candidate salt forms in FaSSIF-v1 at pH 6.5 (15-35 μg / mL) was slightly higher than that of T-5524 form A (about 7 μg / mL).

[0221] The solubility of choline salt form A and diethylamine salt form C in water (0.9-1 mg / mL) is much higher than that of T-5524 form A and betaine salt form A (1-5 μg / mL).

[0222] In summary, the three candidate salt forms showed good crystallinity, as well as good physical and chemical stability. In addition, the three candidate salt forms had obvious advantages in solubility. However, due to its poor thermal stability (gradual decomposition began at about 120°C), the diethylamine salt had no advantage over the other two salt forms. Choline salt and betaine salt are relatively close in terms of physical and chemical properties, but from the perspective of base safety, choline salt was selected as the preferred salt form for subsequent crystal screening.

[0223] Example 4: Screening of choline salt crystal forms and comparison of physical and chemical properties with T-5524 crystal form A

[0224] 1. Preparation of choline salt crystals

[0225] 1.1 Preparation of Choline Salt Form B

[0226] Weigh 5.0 g of T-5524 Form A and approximately 2.6 g (1.05 equivalents) of choline into an 8 mL glass bottle. Add 5 mL of ethanol and stir at 50°C for 5 minutes to obtain a suspension. (Suspension)

[0227] About 10 mg of choline salt form B seed crystals were added to the above suspension. (Suspension)

[0228] The suspension was cooled to 5°C and stirred at 5°C for about 3 days. (Suspension)

[0229] A portion of the solid was taken for XRPD analysis to obtain a physical mixture of choline salt Form A and free base Form A. About 0.3 g (0.1 equivalent) of choline was further added and stirred for about 1 day.

[0230] A portion of the solid was taken for XRPD analysis, and a physical mixture of choline salt form B and free base form A was obtained.

[0231] The solvent was blown dry, 13 mL of ethyl acetate was added, and the mixture was stirred at 25°C for 5 min to obtain a suspension. (Suspension)

[0232] About 3 mg of choline salt Form B seed crystals were added to the above suspension. (Suspension)

[0233] The suspension was stirred at 25°C for about 1 day. (Suspension)

[0234] The solid portion was collected by centrifugation and dried under vacuum at 50°C for about 2 hours.

[0235] A total of 5.2 g of off-white powder of choline salt crystal form B was obtained with a yield of 66%. The characterization of choline salt crystal form B is shown in Figures 66-69. The X-ray powder diffraction (XRPD) data are shown in Table D.

[0236] Table D: X-ray powder diffraction data of T-5224 choline salt form B

[0237] 1.2 Preparation of Choline Salt Form A

[0238] See Table 1-1 of Example 1.

[0239] 1.3 Preparation of Choline Salt Form C

[0240] Weigh 10-20 mg of choline salt Form A and add 100-300 μL of ethyl acetate. The resulting suspension is suspended at 25°C for 2 days. The solid fraction is collected by centrifugation to obtain choline salt Form C. Characterization of choline salt Form C is shown in Figures 70-72. X-ray powder diffraction (XRPD) data is shown in Table E.

[0241] Table E: X-ray powder diffraction data of T-5224 choline salt form C

[0242] 2. Polymorph Characterization

[0243] Choline salt crystal form A is a hydrate. Choline salt crystal form A has high crystallinity. DSC shows that it begins to dehydrate at about 21 ° C, with a melting point T onset The melting point is 137.8°C and the melting enthalpy is about 50 J / g. TGA shows that it loses 2.6% of its weight at about 80°C and loses about 0.1% of its weight between 80°C and 130°C. 1 H-NMR analysis revealed a stoichiometric ratio of free acid to choline of 1:1.0, with no residual solvent detected. Water activity competition experiments revealed that choline salt Form A transformed into choline salt Form B at 5°C (aw ≤ 0.6) and 25°C (aw ≤ 0.3).

[0244] Choline salt crystal form B is an anhydrous form. Choline salt crystal form B has high crystallinity. DSC shows a melting point of 153.3°C and a melting enthalpy of approximately 62 J / g. TGA shows a weight loss of approximately 0.7% at approximately 140°C. 1H-NMR analysis revealed a stoichiometric ratio of free acid to choline of 1:1.0, with no residual solvent detected. Choline salt Form B is the most stable crystalline form discovered to date. Water activity competition experiments revealed that Form B is thermodynamically more stable than Form A at 5°C (aw ≤ 0.6) and 25°C (aw ≤ 0.3).

[0245] Choline salt crystal form C is an anhydrous crystal form. Choline salt crystal form C has high crystallinity. DSC shows that its melting point is 155.6°C and the melting enthalpy is about 59 J / g. 1 H-NMR showed a stoichiometric ratio of free acid to choline of 1:1.0, and no residual solvent was detected in the sample. Choline salt Form C transformed into choline salt Form B after 2 days under ambient conditions (approximately 20-25°C, 60%-75% RH).

[0246] Table 4-1: Summary of polymorph characterization Note: “ / / ”: Characterization not performed

[0247] To investigate the critical water activity between T-5224 choline salt Form A and Form B, water activity experiments were conducted in an ethanol / water system at 5°C and 25°C. The method is as follows.

[0248] Approximately 2 mg of choline salt Form A, approximately 2 mg of choline salt Form B, and approximately 1 mg of choline salt Form B with additional diffraction peaks were weighed and placed in 0.1 mL of the corresponding saturated ethanol / water solution. The resulting suspension was incubated at 5°C and 25°C for 5 days. The resulting suspension was then centrifuged, and the resulting solid was subjected to XRPD analysis. The results are shown in Table 4-2 below.

[0249] Among them, the preparation method of choline salt crystal form B with additional diffraction peaks is as follows:

[0250] Approximately 10-20 mg of T-5224 choline salt Form B was weighed and thoroughly dissolved in a minimum volume of solvent (ethanol, 2.0 mL) at ambient temperature (approximately 20-25°C). The resulting solution or thin suspension was passed through a 0.45 μm nylon membrane syringe filter to obtain a clear solution. Four to eight volumes of antisolvent (n-heptane, 16 mL) were slowly added to the resulting clear solution. The resulting suspension was centrifuged at 14,000 rpm using a 0.45 μm nylon membrane centrifuge tube, and the resulting solid was characterized by XRPD.

[0251] Table 4-2: Water activity experiment Note: “ / / ”: not performed; “*”: water activity (aw) calculated by UNIFAC method

[0252] 3. Evaluation of the properties of T5224 choline salt form B

[0253] T5224 choline salt crystal form B is the most stable crystal form of choline salts discovered so far. Therefore, a systematic study was conducted on the stability, solubility, and mechanical properties of T5224 choline salt crystal form B.

[0254] 3.1 Solid stability

[0255] In this study, the solid stability of choline salt form B was evaluated under open conditions at 25°C / 92.5% RH and closed conditions at 60°C for 1 week.

[0256] An open container containing T-5224 choline salt Form B was placed at 25°C / 92.5% RH for one week. A sealed container containing T-5224 choline salt Form B was placed at 60°C for one week. Samples under these conditions were analyzed by XRPD and HPLC to observe whether the samples showed any color change.

[0257] Table 4-3: Solid Stability Note: A: No color change; B: Slight color change; C: Moderate color change; D: Severe color change

[0258] Choline salt form B exhibits good physical and chemical stability under the above accelerated stability conditions.

[0259] 3.2 Solubility

[0260] In this study, the solubility of the starting materials, T-5224 Form A and the choline salt Form B, was evaluated in water, FaSSGF solution at pH 1.6, FaSSIF solution at pH 6.5, and FeSSIF solution at pH 5.0 at 37°C for 2 hours and 24 hours. The residual solids from the solubility experiments were characterized by XRPD.

[0261] Accurately weigh 10.0 mg of T-5224 Form A and 12.5 mg of choline salt Form B into separate 8 mL glass vials. Add 5 mL of solvent to each. The mass of salt weighed is equivalent to 10 mg of T-5224 Form A. Stir the resulting suspension at 400 rpm at 37°C, and sample it after 2 hours and 24 hours. Centrifuge the samples at 14,000 rpm at 37°C for 5 minutes. Determine the supernatant concentration by HPLC, and the pH of the supernatant using a pH meter.

[0262] Table 4-4: Solubility

[0263] The solubility of T-5224 Form A and Choline Salt Form B is pH-dependent. The 24-hour solubility of Choline Salt Form B in pH 6.5 FaSSIF-v1 and pH 5.0 FeSSIF-v1 (30-45 μg / mL) is slightly higher than that of T-5224 Form A (20-35 μg / mL). In pH 1.6 FaSSGF, the solubility of both T-5224 Form A and Choline Salt Form B is below the minimum detection limit.

[0264] The solubility of choline salt form B in water (>2 mg / mL) is much higher than that of T-5224 form A (8-9 μg / mL).

[0265] 3.3 Mechanical properties

[0266] In this study, the mechanical properties of choline salt form B were evaluated through simulated tableting, grinding, and simulated granulation experiments.

[0267] 3.3.1 Simulated tableting experiment

[0268] Approximately 10 mg of T-5224 choline salt Form B sample was weighed and pressed at 2 MPa, 5 MPa, and 10 MPa for 5 minutes. XRPD characterization was performed to investigate the crystal transformation and crystallinity changes.

[0269] Table 4-5: Simulated tableting experiment

[0270] 3.3.2 Simulated dry grinding experiment

[0271] About 10 mg of T-5224 choline salt Form B sample was weighed and manually ground for 5 minutes. XRPD characterization was performed to investigate the crystal transformation and crystallinity changes.

[0272] Table 4-6: Simulated dry grinding experiment

[0273] 3.3.3 Simulated wet granulation experiment

[0274] Weigh approximately 10 mg of T-5224 choline salt Form B sample and add ethanol dropwise until thoroughly wet. Gently grind the wetted sample with a pestle and then allow the sample to dry at ambient conditions for 10 minutes. XRPD characterization was performed to investigate the crystal transformation and changes in crystallinity.

[0275] Table 4-7: Simulated wet granulation experiment

[0276] Choline salt Form B exhibited good tolerance to tableting (<2 MPa), dry grinding, and wet grinding, with no crystal transformation and no significant decrease in crystallinity. When the tableting pressure was increased to 5 to 10 MPa, choline salt Form B showed no crystal transformation and only a slight decrease in crystallinity.

[0277] Combined with the above research results, T-5224 choline salt Form B is the most stable crystalline form currently available. It exhibits good chemical stability, physical stability, and mechanical resistance. In terms of solubility, the solubility of choline salt Form B in water, pH 6.5 FaSSIF solution, and pH 5.0 FeSSIF after 24 hours is higher than that of the starting material T-5224 Form A. Therefore, T-5224 choline salt Form B was selected as the preferred crystalline form for subsequent pharmacokinetic studies.

[0278] Example 5: Pharmacokinetic Study and Comparison of T-5224 Salt-Free Form A (i.e., the aforementioned T5224 Form A) and T-5224 Choline Salt Form B

[0279] 1. Dosage Method

[0280] 2. Preparation method

[0281] 3. Detection Methods

[0282] LC-MS / MS

[0283] 1. Instrument

[0284] API-4000Qtrap-Shimadzu Controller-CBM20A

[0285] 2.MS conditions

[0286] Positive ion mode

[0287] 3.LC conditions

[0288] MP-A: 0.1% formic acid

[0289] MP-B:MeOH

[0290] HPLC column: Waters Xbridge C18 3.5um 2.1*50mm

[0291] Gradient

[0292] 4. Sample preparation: a) Transfer 20ul of plasma sample to a 96-well plate.

[0293] b) Add 250 μl of acetonitrile (containing internal standard) to precipitate the protein.

[0294] c) Centrifuge at 4000 rpm at 4°C for 20 min.

[0295] d) Transfer 180 μl of supernatant to a new 96-well plate and mix with 180 μl of water.

[0296] e) Pipette 5 μl of the sample into LC-MS / MS for detection.

[0297] 4. Experimental Results

[0298] 5. Experimental Conclusion

[0299] From the above results, it can be seen that the bioavailability of the choline salt form B of the present application is significantly improved compared with the control T-5524 form A (55.4% vs 17.5%), which is increased by at least 2 times, and has a significant advantage.

[0300] Example 6: Comparison of the therapeutic efficacy of T-5224 salt-free crystal form A (i.e., the aforementioned T5224 crystal form A) and T-5224 choline salt crystal form B in an animal model

[0301] Materials and reagents, animal experiment guidelines, and induction of the heart failure model with preserved ejection fraction were the same as those in Example A.

[0302] The dosage is as follows:

[0303] After the animal model was successfully induced, the mice in the model treatment group were treated with T-5224 salt-free crystal form A and T-5224 choline salt crystal form B, and the model control group was treated with a solvent without drugs. Mice that were given normal diet and drinking water throughout the induction process served as negative controls. When the mice were 5-8 weeks old, the model was successfully established by ultrasound detection and drug treatment was started. For the treatment group, T-5224 salt-free crystal form A was administered at a single dose of 12 mg / kg based on the mouse body weight, specifically every other day, with 0.54 mg of the compound dissolved in 100 uL of 0.5% PVP solution each time (the specific actual dosage process was based on the number of mice, for example, for 16 mice, 9 mg of the drug was dissolved in 1.7 ml of solution, and 100 ul was administered to each mouse). T-5224 choline salt crystal form B was administered at a single dose of 3 mg / kg based on the mouse body weight, specifically every other day, with 0.14 mg of the compound dissolved in 100 uL of 0.5% PVP solution each time (the specific actual dosage process was based on the number of mice, for example, for 16 mice, 2.2 mg of the drug was dissolved in 1.7 ml of solution, and 100 ul was administered to each mouse). Drug treatment began from the successful induction of the model and lasted for 7 weeks. The control group was given an equal volume of 0.5% PVP solution, other treatment methods are the same.

[0304] The experimental results are shown in Figure 73 (wherein the 0 week on the horizontal axis represents the day when the drug administration begins after the modeling is successful, i.e., the starting point of the administration). It can be clearly seen from Figure 73 that at 3 weeks after administration, the diastolic function of mice administered with T-5224 choline salt crystal form B (3 mg / kg) has been significantly improved (E / E' is significantly reduced), but the diastolic function of mice administered with the positive control T-5224 salt-free crystal form A (12 mg / kg) has not changed significantly. It is obvious that compared to the positive control T-5224 salt-free crystal form A, T-5224 choline salt crystal form B can inhibit the occurrence and development of the disease earlier at a significantly lower dose, has obvious advantages, and has broad application prospects. Therefore, the salt-type crystal form of the present invention (especially T-5224 choline salt crystal form B) has a significant therapeutic effect on HFpEF at a lower dose, and the effect is very fast.

Claims

1. A salt of the compound of formula I, wherein, The salt is a sodium salt, potassium salt, calcium salt, tert-butylamine salt, ammonium salt, choline salt, L-lysine salt, diethylamine salt, dimethylethanolamine salt, betaine salt or ethylenediamine salt of the compound represented by formula I, 2. The salt according to claim 1, wherein The salt is the sodium salt, potassium salt, tert-butylamine salt, choline salt, L-lysine salt, diethylamine salt, dimethylethanolamine salt, betaine salt or ethylenediamine salt of the compound represented by formula I.

3. The salt according to any one of claims 1-2, wherein, The salt is the choline salt, diethylamine salt or betaine salt of the compound represented by formula I.

4. The salt according to any one of claims 1-3, wherein, The salt is the choline salt or betaine salt of the compound represented by formula I.

5. The salt according to any one of claims 1 - 4, wherein, The salt is the choline salt of the compound represented by formula I.

6. The salt according to any one of claims 1-5, wherein, The salt further has one or more technical features selected from the following (1)-(20): (1) The sodium salt of the compound represented by formula I is crystalline form A, wherein the sodium salt crystalline form A of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that in Figure 4; (2) The sodium salt of the compound represented by formula I is crystalline form B, wherein the sodium salt crystalline form B of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that in Figure 6; (3) The potassium salt of the compound represented by formula I is crystalline form A, wherein the potassium salt crystalline form A of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that in Figure 10; (4) The potassium salt of the compound represented by formula I is crystalline form B, wherein the potassium salt crystalline form B of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that in Figure 12; (5) The potassium salt of the compound represented by formula I is crystalline form C, wherein the potassium salt crystalline form C of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that in Figure 16; (6) The potassium salt of the compound represented by formula I is crystalline form D, wherein the potassium salt crystalline form D of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that in Figure 18; (7) The calcium salt of the compound represented by formula I is crystalline form A, wherein the calcium salt crystalline form A of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that in Figure 19; (8) The tert-butylamine salt of the compound represented by formula I is crystalline form A, wherein the tert-butylamine salt crystalline form A of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that in Figure 20; (9) The tert-butylamine salt of the compound represented by formula I is crystalline form B, wherein the tert-butylamine salt crystalline form B of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that in Figure 22; (10) The ammonium salt of the compound represented by formula I is crystalline form A, wherein the ammonium salt crystalline form A of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that in Figure 26; (11) The ammonium salt of the compound represented by formula I is crystalline form B, wherein the ammonium salt crystalline form B of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that in Figure 27; (12) The L-lysine salt of the compound represented by formula I is crystalline form A, wherein the L-lysine salt crystalline form A of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that in Figure 32; (13) The diethylamine salt of the compound represented by formula I is crystalline form A, wherein the diethylamine salt crystalline form A of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that in Figure 36; (14) The diethylamine salt of the compound represented by formula I is crystalline form B, wherein the diethylamine salt crystalline form B of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that in Figure 40; The diethylamine salt of the compound represented by formula I is crystalline form C, wherein the X-ray powder diffraction 2θ of the crystalline form C of the diethylamine salt of the compound represented by formula I has characteristic peaks at 4.7±0.2°, 6.4±0.2°, 9.4±0.2°, 13.8±0.2°, 15.0±0.2°, 16.8±0.2°, 19.7±0.2° and 20.4±0.2°; Preferably, the X-ray powder diffraction 2θ of the crystalline form C of the diethylamine salt of the compound represented by formula I has characteristic peaks at 4.7±0.2°, 6.4±0.2°, 9.4±0.2°, 13.1±0.2°, 13.8±0.2°, 14.1±0.2°, 15.0±0.2°, 16.8±0.2°, 18.0±0.2°, 18.9±0.2°, 19.4±0.2°, 19.7±0.2°, 20.4±0.2°, 23.0±0.2°, 23.3±0.2° and 24.9±0.2°; More preferably, the 2θ, d and peak intensity of the X-ray powder diffraction of the crystalline form C of the diethylamine salt of the compound represented by formula I are as shown in Table C; Most preferably, the crystalline form C of the diethylamine salt of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that of Figure 62; (16) The dimethylethanolamine salt of the compound represented by formula I is crystalline form A, wherein the crystalline form A of the dimethylethanolamine salt of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that of Figure 44; (17) The betaine salt of the compound represented by formula I is crystalline form A, wherein the X-ray powder diffraction 2θ of the crystalline form A of the betaine salt of the compound represented by formula I has characteristic peaks at 8.7±0.2°, 11.8±0.2°, 15.4±0.2°, 16.3±0.2°, 19.6±0.2°, 21.4±0.2° and 23.4±0.2°; Preferably, the X-ray powder diffraction 2θ of the crystalline form A of the betaine salt of the compound represented by formula I has characteristic peaks at 8.1±0.2°, 8.7±0.2°, 11.8±0.2°, 15.4±0.2°, 16.3±0.2°, 18.4±0.2°, 19.6±0.2°, 20.4±0.2°, 21.4±0.2°, 22.3±0.2°, 23.4±0.2°, 24.2±0.2° and 25.9±0.2°; More preferably, the 2θ, d and peak intensity of the X-ray powder diffraction of the crystalline form A of the betaine salt of the compound represented by formula I are as shown in Table A; Most preferably, the crystalline form A of the betaine salt of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that of Figure 48; (18) The ethylenediamine salt of the compound represented by formula I is crystalline form A, wherein the crystalline form A of the ethylenediamine salt of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that of Figure 52; (19) The ethylenediamine salt of the compound represented by formula I is crystalline form B, wherein the crystalline form B of the ethylenediamine salt of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that of Figure 54; The choline salt of the compound represented by formula I is crystalline form C, wherein the X-ray powder diffraction 2θ of the crystalline form C of the choline salt of the compound represented by formula I has characteristic peaks at 4.7±0.2°, 6.4±0.2°, 9.4±0.2°, 15.0±0.2°, 16.8±0.2°, 19.7±0.2° and 20.4±0.2°; Preferably, the X-ray powder diffraction 2θ of the crystalline form C of the choline salt of the compound represented by formula I has characteristic peaks at 4.7±0.2°, 6.4±0.2°, 9.4±0.2°, 13.1±0.2°, 13.8±0.2°, 15.0±0.2°, 16.8±0.2°, 18.9±0.2°, 19.7±0.2°, 20.4±0.2°, 22.4±0.2°, 22.9±0.2°, 23.2±0.2° and 24.9±0.2°; More preferably, the 2θ, d and peak intensity of the X-ray powder diffraction of the crystalline form C of the choline salt of the compound represented by formula I are as shown in Table E; Most preferably, the crystalline form C of the choline salt of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that of Figure 70.

7. The salt according to any one of claims 1-6, wherein, The choline salt of the compound represented by formula I is crystalline form A, wherein the X-ray powder diffraction 2θ of the crystalline form A of the choline salt of the compound represented by formula I has characteristic peaks at 6.9±0.2°, 14.0±0.2°, 19.7±0.2°, 21.2±0.2°, 22.8±0.2°, 26.5±0.2° and 27.0±0.2°; Preferably, the X-ray powder diffraction 2θ of the crystalline form A of the choline salt of the compound represented by formula I has characteristic peaks at 6.9±0.2°, 11.7±0.2°, 14.0±0.2°, 15.0±0.2°, 17.7±0.2°, 18.6±0.2°, 19.1±0.2°, 19.7±0.2°, 20.9±0.2°, 21.2±0.2°, 22.8±0.2°, 26.3±0.2°, 26.5±0.2°, 27.0±0.2° and 27.6±0.2°; More preferably, the 2θ, d and peak intensity of the X-ray powder diffraction of the crystalline form A of the choline salt of the compound represented by formula I are as shown in Table B; Most preferably, the crystalline form A of the choline salt of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that of Figure 28.

8. The salt according to any one of claims 1-7, wherein, The choline salt of the compound represented by formula I is crystalline form B, wherein the X-ray powder diffraction 2θ of the crystalline form B of the choline salt of the compound represented by formula I has characteristic peaks at 8.9±0.2°, 17.0±0.2°, 17.4±0.2°, 19.5±0.2°, 20.4±0.2°, 22.2±0.2°, 22.7±0.2° and 27.2±0.2°; Preferably, the X-ray powder diffraction 2θ of choline salt polymorph B of the compound represented by formula I has characteristic peaks at 8.9±0.2°, 16.2±0.2°, 17.0±0.2°, 17.4±0.2°, 19.5±0.2°, 19.9±0.2°, 20.4±0.2°, 20.9±0.2°, 21.8±0.2°, 22.2±0.2°, 22.5±0.2°, 22.7±0.2°, 26.5±0.2°, 27.2±0.2° and 27.5±0.2°; More preferably, the 2θ, d and peak intensity of the X-ray powder diffraction of choline salt polymorph B of the compound represented by formula I are as shown in Table D; Most preferably, choline salt polymorph B of the compound represented by formula I has an X-ray powder diffraction pattern substantially the same as that in Figure 66.

9. A pharmaceutical composition, which comprises the salt according to any one of claims 1-8; optionally, further comprises a pharmaceutically acceptable excipient.

10. Use of the salt according to any one of claims 1-8 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for the treatment and / or prevention of heart failure with preserved ejection fraction.

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