Application of salt-form crystal form of compound to treatment of heart failure with preserved ejection fraction

Abstract

The invention relates to application of a salt crystal form of a compound to treatment of heart failure with preserved ejection fraction, and particularly provides a salt of a compound shown in a formula I. The salt is a sodium salt, a potassium salt, a calcium salt, a tert-butylamine salt, an ammonium salt, a choline salt, an L-lysine salt, a diethylamine salt, a dimethylethanolamine salt, a betaine salt or an ethylenediamine salt of the compound shown in the formula I. # imgabs0 #

Description

Technical Field

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

[0002] Cardiovascular diseases represent one of the difficult problems in medical challenges. Heart failure (HF) is the main cause of death in patients with cardiovascular diseases and is also a major problem faced clinically. Heart failure includes 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 the increasing mortality rate of cardiovascular diseases. HFpEF is a complex syndrome involving multiple organ disorders, which is caused by the joint action of the heart, lungs, kidneys, bones, immune system, inflammation, metabolism and other components, and is often accompanied by symptoms such as obesity, hypertension, myocardial hypertrophy, diabetes or atrial fibrillation. HFpEF is a syndrome with high morbidity and high mortality. According to clinical statistics, the mortality rate due to HF is 35%, and the proportion of deaths caused by HFpEF accounts for 57% of them. However, so far, few drug therapies or medical devices have been proven to be able to change the disease progression and prognosis of HFpEF patients. At present, there is an urgent need in the art to develop a drug and / or treatment method that can effectively treat HFpEF.

[0004] Jun is a transcription factor and one of the members of the AP1 family. It has chromatin-binding activity and transcriptional cis-regulatory region-binding activity, and is involved in regulating processes such as animal organ development, protein phosphorylation and cell proliferation. A Jun inhibitor refers to an inhibitor that can inhibit the expression of the Jun gene, reduce the binding activity of Jun to DNA, reduce the level of the expression product of the Jun gene, or prevent or block the signal transduction of Jun. Currently, studies have shown that Jun inhibitors have therapeutic effects in animal models of endometriosis, breast cancer, sepsis, etc. Summary of the Invention

[0005] The inventors of the present invention have for the first time discovered that inhibiting the high expression of Jun using a Jun inhibitor (such as T5524) can have a preventive and therapeutic effect on HFpEF. Based on this, the inventors further screened the salt forms and crystal forms of T5524, and obtained salt forms and crystal forms with high stability, good solubility, high bioavailability, and excellent therapeutic effects on HFpEF, which have high application value.

[0006] Therefore, in the first aspect of the present invention, the present invention provides a salt of the compound shown in 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 shown in Formula I,

[0007]

[0008] 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 shown in Formula I.

[0009] In some embodiments, the salt is a choline salt, diethylamine salt or betaine salt of the compound shown in Formula I.

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

[0011] In some embodiments, the salt is a choline salt of the compound shown in Formula I.

[0012] In some embodiments, the sodium salt of the compound shown in Formula I is Crystal Form A, wherein the sodium salt Crystal Form A of the compound shown in Formula I has the same Figure 4 X-ray powder diffraction pattern.

[0013] In some embodiments, the sodium salt of the compound shown in Formula I is Crystal Form B, wherein the sodium salt Crystal Form B of the compound shown in Formula I has the same Figure 6 X-ray powder diffraction pattern.

[0014] In some embodiments, the potassium salt of the compound shown in Formula I is Crystal Form A, wherein the potassium salt Crystal Form A of the compound shown in Formula I has the same Figure 10 X-ray powder diffraction pattern.

[0015] In some embodiments, the potassium salt of the compound shown in Formula I is Crystal Form B, wherein the potassium salt Crystal Form B of the compound shown in Formula I has the same Figure 12 X-ray powder diffraction pattern.

[0016] In some embodiments, the potassium salt of the compound of formula I is crystalline form C, wherein the crystalline form C of the potassium salt of the compound of formula I has the same Figure 16 X-ray powder diffraction pattern.

[0017] In some embodiments, the potassium salt of the compound of formula I is crystalline form D, wherein the crystalline form D of the potassium salt of the compound of formula I has the same Figure 18 X-ray powder diffraction pattern.

[0018] In some embodiments, the calcium salt of the compound of formula I is crystalline form A, wherein the crystalline form A of the calcium salt of the compound of formula I has the same Figure 19 X-ray powder diffraction pattern.

[0019] In some embodiments, the tert-butylamine salt of the compound of formula I is crystalline form A, wherein the crystalline form A of the tert-butylamine salt of the compound of formula I has the same Figure 20 X-ray powder diffraction pattern.

[0020] In some embodiments, the tert-butylamine salt of the compound of formula I is crystalline form B, wherein the crystalline form B of the tert-butylamine salt of the compound of formula I has the same Figure 22 X-ray powder diffraction pattern.

[0021] In some embodiments, the ammonium salt of the compound of formula I is crystalline form A, wherein the crystalline form A of the ammonium salt of the compound of formula I has the same Figure 26 X-ray powder diffraction pattern.

[0022] In some embodiments, the ammonium salt of the compound of formula I is crystalline form B, wherein the crystalline form B of the ammonium salt of the compound of formula I has the same Figure 27 X-ray powder diffraction pattern.

[0023] In some embodiments, the L-lysine salt of the compound of formula I is crystalline form A, wherein the crystalline form A of the L-lysine salt of the compound of formula I has the same Figure 32 X-ray powder diffraction pattern.

[0024] In some embodiments, the diethylamine salt of the compound of formula I is crystalline form A, wherein the crystalline form A of the diethylamine salt of the compound of formula I has the same Figure 36 X-ray powder diffraction pattern.

[0025] In some embodiments, the diethylamine salt of the compound of formula I is crystalline form B, wherein the crystalline form B of the diethylamine salt of the compound of formula I has the same Figure 40 X-ray powder diffraction pattern.

[0026] In some embodiments, the diethylamine salt of the compound of 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 of 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°.

[0027] In some embodiments, the X-ray powder diffraction 2θ of the crystalline form C of the diethylamine salt of the compound of 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°.

[0028] In some embodiments, the 2θ, d and peak intensity of the X-ray powder diffraction of the crystalline form C of the diethylamine salt of the compound of formula I are shown in Table C.

[0029] In some embodiments, the crystalline form C of the diethylamine salt of the compound of formula I has Figure 62 substantially the same X-ray powder diffraction pattern.

[0030] In some embodiments, the dimethylethanolamine salt of the compound of formula I is crystalline form A, wherein the crystalline form A of the dimethylethanolamine salt of the compound of formula I has Figure 44 substantially the same X-ray powder diffraction pattern.

[0031] In some embodiments, the betaine salt of the compound of 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 of 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°.

[0032] In some embodiments, the X-ray powder diffraction 2θ of the crystalline form A of the betaine salt of the compound of 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°.

[0033] In some embodiments, the 2θ, d and peak intensity of the X-ray powder diffraction of the betaine salt crystalline form A of the compound represented by formula I are shown in Table A.

[0034] In some embodiments, the betaine salt crystalline form A of the compound represented by formula I has an X-ray powder diffraction pattern Figure 48 substantially the same as

[0035] In some embodiments, the ethylenediamine salt of the compound represented by formula I is crystalline form A, wherein the ethylenediamine salt crystalline form A of the compound represented by formula I has an X-ray powder diffraction pattern Figure 52 substantially the same as

[0036] In some embodiments, the ethylenediamine salt of the compound represented by formula I is crystalline form B, wherein the ethylenediamine salt crystalline form B of the compound represented by formula I has an X-ray powder diffraction pattern Figure 54 substantially the same as

[0037] In some embodiments, the choline salt of the compound represented by formula I is crystalline form C, wherein the X-ray powder diffraction 2θ of the choline 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°, 15.0 ± 0.2°, 16.8 ± 0.2°, 19.7 ± 0.2° and 20.4 ± 0.2°.

[0038] In some embodiments, the X-ray powder diffraction 2θ of the choline 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°, 13.1 ± 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°.

[0039] In some embodiments, the 2θ, d and peak intensity of the X-ray powder diffraction of the choline salt crystalline form C of the compound represented by formula I are shown in Table E.

[0040] In some embodiments, the choline salt crystalline form C of the compound represented by formula I has an X-ray powder diffraction pattern Figure 70 substantially the same as

[0041] In some embodiments, the choline salt of the compound represented by Formula I is Crystal Form A, wherein the X-ray powder diffraction 2θ of the choline salt Crystal Form A 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°.

[0042] In some embodiments, the X-ray powder diffraction 2θ of the choline salt Crystal Form A 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°.

[0043] 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.

[0044] In some embodiments, the choline salt Crystal Form A of the compound represented by Formula I has Figure 28 substantially the same X-ray powder diffraction pattern.

[0045] In some embodiments, the choline salt of the compound represented by Formula I is Crystal Form B, wherein the X-ray powder diffraction 2θ of the choline salt Crystal Form B 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°.

[0046] In some embodiments, the X-ray powder diffraction 2θ of the choline salt Crystal Form 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°.

[0047] In some embodiments, the 2θ, d and peak intensity of the X-ray powder diffraction of the choline salt Crystal Form B of the compound represented by Formula I are as shown in Table D.

[0048] In some embodiments, the choline salt crystal form B of the compound represented by Formula I has the same Figure 66 X-ray powder diffraction pattern.

[0049] In a second aspect of the present invention, there is provided a pharmaceutical composition comprising the aforementioned salt; optionally, further comprising a pharmaceutically acceptable excipient.

[0050] In a third aspect of the present invention, there is provided the use of the aforementioned salt in the preparation of a drug for the treatment and / or prevention of heart failure with preserved ejection fraction;

[0051] alternatively, there is provided the aforementioned salt for the treatment and / or prevention of heart failure with preserved ejection fraction;

[0052] alternatively, there is provided a method for the treatment and / or prevention of heart failure with preserved ejection fraction, comprising administering to a subject in need an effective amount of the aforementioned salt. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 : Construction of the HFpEF model. A: Schematic diagram of the experimental procedure; B: Detection results of systolic function after 5 weeks of mouse feeding; C: Detection results of diastolic function after 5 weeks of mouse feeding.

[0054] Figure 2 : Relative expression level of myocardial cell Jun after 15 weeks of HFD+L-NAME feeding, indicating high expression of Jun in HFpEF model mice.

[0055] Figure 3 : T-5224 can effectively alleviate the occurrence and development of HFpEF. A: Detection results of systolic function of mice at different time points; B: Detection results of diastolic function of mice at different time points; C: Changes in body weight of mice under different treatments; D: Changes in the expression level of myocardial cell Jun in mice under different treatments.

[0056] Figure 4 : XRPD spectrum of T-5224 sodium salt crystal form A.

[0057] Figure 5 : 1 H-NMR spectrum of T-5224 sodium salt crystal form A.

[0058] Figure 6 : XRPD spectrum of T-5224 sodium salt crystal form B.

[0059] Figure 7 : DSC spectrum of T-5224 sodium salt crystal form B.

[0060] Figure 8 : TGA spectrum of T-5224 sodium salt crystal form B.

[0061] Figure 9 : The 1 H-NMR spectrum of Form B of T-5224 sodium salt.

[0062] Figure 10 : The XRPD spectrum of Form A of T-5224 potassium salt.

[0063] Figure 11 : The 1 H-NMR spectrum of Form A of T-5224 potassium salt.

[0064] Figure 12 : The XRPD spectrum of Form B of T-5224 potassium salt.

[0065] Figure 13 : The DSC spectrum of Form B of T-5224 potassium salt.

[0066] Figure 14 : The TGA spectrum of Form B of T-5224 potassium salt.

[0067] Figure 15 : The 1 H-NMR spectrum of Form B of T-5224 potassium salt.

[0068] Figure 16 : The XRPD spectrum of Form C of T-5224 potassium salt.

[0069] Figure 17 : The 1 H-NMR spectrum of Form C of T-5224 potassium salt.

[0070] Figure 18 : The XRPD spectrum of Form D of T-5224 potassium salt.

[0071] Figure 19 : The XRPD spectrum of Form A of T-5224 calcium salt.

[0072] Figure 20 : The XRPD spectrum of Form A of T-5224 tert-butylamine salt.

[0073] Figure 21 : The 1 H-NMR spectrum of Form A of T-5224 tert-butylamine salt.

[0074] Figure 22 : The XRPD spectrum of Form B of T-5224 tert-butylamine salt.

[0075] Figure 23 : The DSC spectrum of Form B of T-5224 tert-butylamine salt.

[0076] Figure 24: TGA spectrum of crystalline form B of T-5224 tert-butylamine salt.

[0077] Figure 25 : of crystalline form B of T-5224 tert-butylamine salt 1 1H-NMR spectrum.

[0078] Figure 26 : XRPD spectrum of crystalline form A of T-5224 ammonium salt.

[0079] Figure 27 : XRPD spectrum of crystalline form B of T-5224 ammonium salt.

[0080] Figure 28 : XRPD spectrum of crystalline form A of T-5224 choline salt.

[0081] Figure 29 : DSC spectrum of crystalline form A of T-5224 choline salt.

[0082] Figure 30 : TGA spectrum of crystalline form A of T-5224 choline salt.

[0083] Figure 31 : of crystalline form A of T-5224 choline salt 1 1H-NMR spectrum.

[0084] Figure 32 : XRPD spectrum of crystalline form A of T-5224 L-lysine salt.

[0085] Figure 33 : DSC spectrum of crystalline form A of T-5224 L-lysine salt.

[0086] Figure 34 : TGA spectrum of crystalline form A of T-5224 L-lysine salt.

[0087] Figure 35 : of crystalline form A of T-5224 L-lysine salt 1 1H-NMR spectrum.

[0088] Figure 36 : XRPD spectrum of crystalline form A of T-5224 diethylamine salt.

[0089] Figure 37 : DSC spectrum of crystalline form A of T-5224 diethylamine salt.

[0090] Figure 38 : TGA spectrum of crystalline form A of T-5224 diethylamine salt.

[0091] Figure 39 : of crystalline form A of T-5224 diethylamine salt 1 1H-NMR spectrum.

[0092] Figure 40 : XRPD spectrum of crystalline form B of T-5224 diethylamine salt.

[0093] Figure 41 : DSC spectrum of crystalline form B of T-5224 diethylamine salt.

[0094] Figure 42 : TGA spectrum of crystalline form B of T-5224 diethylamine salt.

[0095] Figure 43 : 1 H-NMR spectrum of crystalline form B of T-5224 diethylamine salt.

[0096] Figure 44 : XRPD spectrum of crystalline form A of T-5224 dimethylethanolamine salt.

[0097] Figure 45 : DSC spectrum of crystalline form A of T-5224 dimethylethanolamine salt.

[0098] Figure 46 : TGA spectrum of crystalline form A of T-5224 dimethylethanolamine salt.

[0099] Figure 47 : 1 H-NMR spectrum of crystalline form A of T-5224 dimethylethanolamine salt.

[0100] Figure 48 : XRPD spectrum of crystalline form A of T-5224 betaine salt.

[0101] Figure 49 : DSC spectrum of crystalline form A of T-5224 betaine salt.

[0102] Figure 50 : TGA spectrum of crystalline form A of T-5224 betaine salt.

[0103] Figure 51 : 1 H-NMR spectrum of crystalline form A of T-5224 betaine salt.

[0104] Figure 52 : XRPD spectrum of crystalline form A of T-5224 ethylenediamine salt.

[0105] Figure 53 : 1 H-NMR spectrum of crystalline form A of T-5224 ethylenediamine salt.

[0106] Figure 54 : XRPD spectrum of crystalline form B of T-5224 ethylenediamine salt.

[0107] Figure 55: DSC spectrum of T-5224 ethylenediamine salt crystal form B.

[0108] Figure 56 : TGA spectrum of T-5224 ethylenediamine salt crystal form B.

[0109] Figure 57 : 1 H-NMR spectrum of T-5224 ethylenediamine salt crystal form B.

[0110] Figure 58 : XRPD spectrum of T-5224 crystal form A.

[0111] Figure 59 : DSC spectrum of T-5224 crystal form A.

[0112] Figure 60 : TGA spectrum of T-5224 crystal form A.

[0113] Figure 61 : 1 H-NMR spectrum of T-5224 crystal form A.

[0114] Figure 62 : XRPD spectrum of T-5224 diethylamine salt crystal form C.

[0115] Figure 63 : DSC spectrum of T-5224 diethylamine salt crystal form C.

[0116] Figure 64 : TGA spectrum of T-5224 diethylamine salt crystal form C.

[0117] Figure 65 : 1 H-NMR spectrum of T-5224 diethylamine salt crystal form C.

[0118] Figure 66 : XRPD spectrum of T-5224 choline salt crystal form B.

[0119] Figure 67 : DSC spectrum of T-5224 choline salt crystal form B.

[0120] Figure 68 : TGA spectrum of T-5224 choline salt crystal form B.

[0121] Figure 69 : 1 H-NMR spectrum of T-5224 choline salt crystal form B.

[0122] Figure 70 : XRPD spectrum of T-5224 choline salt crystal form C.

[0123] Figure 71: DSC spectrum of choline salt polymorph C of T-5224.

[0124] Figure 72 : of choline salt polymorph C of T-5224 1 1H-NMR spectrum.

[0125] Figure 73 : Choline salt polymorph B of T-5224 can effectively inhibit the occurrence and development of HFpEF. Detailed implementation manners

[0126] It should be understood that the terms used herein are intended to describe specific implementation manners and are not intended to be limiting. In addition, although any methods, devices, and materials similar or equivalent to those described herein can be used to implement or test the present invention, the methods, devices, and materials described now are preferred.

[0127] In the present invention, the term "treatment" generally refers to obtaining the desired pharmacological and / or physiological effects. This effect can be preventive according to completely or partially preventing a disease or its symptoms; and / or can be therapeutic according to partially or completely stabilizing or curing a disease and / or side effects caused by the disease. "Treatment" as used herein covers any treatment of a patient's disease, including: (a) preventing a disease or symptoms from occurring in a patient who is susceptible to the disease or symptoms but has not been diagnosed with the disease; (b) inhibiting the symptoms of the disease, that is, preventing its development; or (c) alleviating the symptoms of the disease, that is, causing the disease or symptoms to regress.

[0128] In the present invention, "subject" refers to a vertebrate. In certain implementation manners, 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 implementation manners, the mammal refers to a human.

[0129] In the present invention, "effective amount" refers to the amount that effectively achieves the desired therapeutic effect at the required dose and time. The "therapeutically effective amount" of the substance / molecule of the present invention can vary according to factors such as the disease state, age, gender, and weight of an individual and the ability of the substance / molecule to elicit the desired response in the individual. The therapeutically effective amount also covers the amount where the therapeutic beneficial effects of the substance / molecule outweigh any toxic or harmful consequences.

[0130] The pharmaceutical compositions involved in the present invention may contain pharmaceutically acceptable excipients, including but not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering 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, polyvinylpyrrolidone, cellulose substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin, and the like.

[0131] The pharmaceutical compositions of the present invention can be prepared into various forms according to different administration routes. For example, the pharmaceutical compositions can be administered in any of the following ways: oral, spray inhalation, rectal administration, nasal administration, buccal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intracardiac, intracranial injection or infusion, or administration by means of an implantable reservoir. Among them, oral or intravenous administration is preferred.

[0132] The compounds of the present invention can optionally also be used in combination with one or more other active ingredients, and their respective dosages and proportions can be adjusted by those skilled in the art according to specific diseases, specific conditions of patients, and clinical needs, etc.

[0133] 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:

[0134]

[0135]

[0136]

[0137] The present invention will be further explained and illustrated below in conjunction with specific embodiments.

[0138] Example A: Preliminary Research

[0139] 1. Materials and Reagents

[0140] In this embodiment, C57BL / 6N wild-type mice were purchased from Beijing Vital River. The sources of the reagents are shown in the following table.

[0141]

[0142] Except as described above, other materials and reagents used in this embodiment are also commercially available products.

[0143] 2. Animal Experiment Guidelines

[0144] In this example, all animal studies were conducted under the guidance of the Experimental Animal Center of the Animal Health and Use Committee of Fuwai Hospital, Chinese National Center for Cardiovascular Diseases. All mice were bred and raised in the same environment, and the mice were randomly grouped during the experiment. Echocardiogram analysis was performed by independent researchers who were unaware of the research objectives.

[0145] 3. Induction of heart failure with preserved ejection fraction model

[0146] Male C57BL / 6N wild-type mice aged 8 to 10 weeks were divided into three groups, namely the normal group (normal diet and water), the model control group (high-fat diet combined with N-nitro-L-arginine methyl ester), and the model treatment group (high-fat diet combined with N-nitro-L-arginine methyl ester and treated with T-5224). Among them, the model control group and the model treatment group were modeled by 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 (abbreviated as L-NAME, 0.5 g / L in drinking water) were used to induce heart failure with preserved ejection fraction to obtain an HFpEF animal model.

[0147] At the fifth week of model induction, the systolic function parameter LVEF of the mice did not change, while the diastolic function parameter (E / E’) increased significantly at the fifth week of model induction, indicating that the heart failure model with preserved ejection fraction described in the aforementioned literature had 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. Subsequent drug administration was carried out under the same baseline conditions, as Figure 1 shown.

[0148] 4. The expression of Jun is correlated with HFpEF

[0149] At the 15th week of model induction, myocardial cells of normal mice and the HFpEF animal model of mice were extracted and separated by perfusion method, and quantitative RCR detection was performed. The specific operations are as follows:

[0150] 4.1. Isolation of adult mouse cardiomyocytes:

[0151] To isolate cardiomyocytes from adult mouse hearts, we used a classical perfusion method to isolate cardiomyocytes. Specifically, 100 μl of sodium heparin (1000 units in 50 ml) was injected into the mice 20 minutes before sacrifice to prevent blood clotting in the heart during the operation and increase the difficulty of digestion. After that, the mice were anesthetized and sacrificed, and the hearts were removed and transferred to calcium-free solution for washing. Then, digestion was performed using the Langendorff method. The heart was perfused with calcium-free solution using the Langendorff apparatus for 5 minutes, and then digested with digestive enzyme solution (0.7 mg / ml type II collagenase and 0.7 mg / ml bovine serum albumin in calcium-free solution) for about 30 minutes. The heart was continuously touched at about 20 minutes. When the heart became soft and slippery, it indicated that the digestion was basically completed. Then, the tissue from the ventricle was collected, minced, and gently pipetted to dissociate into single cells. After standing and sedimentation, the supernatant was taken to remove undigested and adherent tissues. The cells were centrifuged at 100 g for 2 minutes at 4°C to obtain cardiomyocyte pellets. Most of the supernatant was non-cardiomyocytes. The cardiomyocytes were resuspended in calcium-free solution containing 10% FBS for subsequent experiments. The non-cardiomyocytes could be reselected with medium or PBS for subsequent experiments. If purer cardiomyocytes and non-cardiomyocytes were to be obtained, the cell suspension could be centrifuged (100 g, 2 minutes at room temperature) three times to separate cardiomyocytes from non-cardiomyocytes. The cardiomyocytes were collected for further experiments.

[0152] 4.2. Quantitative PCR detection:

[0153] Total RNA was extracted from the cells using the GeneJet RNA Purification Kit (Thermo Scientific, K0732), and 0.1 μg of total RNA was reverse transcribed to generate cDNA using the iScript TM cDNA Synthesis Kit (Bio-Rad, 1708890). qPCR was performed using the iTaq Universl SYBR Green supermix (1725121, Bio-Rad) on the ABI Vii7 real-time system (Life Technologies, Q6), with b-Actin used for normalization of quantitative analysis. As Figure 2 shown, compared with normal mice, a significantly higher expression of Jun was observed in the mouse HFpEF animal model. This indicates that there is a correlation between the expression level of Jun and HFpEF in mice, and Jun is highly expressed in HFpEF.

[0154] 5. Administration method of T-5224

[0155] After obtaining the animal model, starting from the fifth week of the self-induced HFpEF model, the model treatment group was treated with T-5224 in mice, and the model control group was treated with a drug-free solvent as a control. Mice with normal diet and water intake were used as negative controls throughout the induction process. When the mice were raised to 5 weeks old, drug administration was carried out. For the treatment group, T-5224 was administered at 250 mg / kg according to the body weight of the mice. Specifically, it was administered every other day. Each time, 0.8 mg of T-5224 was dissolved in 200 μL of 1% PVP solution. Administration started from the fifth week and ended at the thirteenth week (a total of 15 administrations), and the total dose reached 250 mg / kg; the control group was given an equal volume of 1% PVP solution, and other treatment methods were the same.

[0156]

[0157] 6. Conventional ultrasound detection

[0158] After all mice were fed under different conditions for five weeks, conventional ultrasound detection was started, and it was measured every two weeks until the detection ended at the fifteenth week. Specifically, a VisualSonics Vevo 2100 system equipped with an MS400 transducer (Visual Sonics) was used for transthoracic echocardiography. Left ventricular ejection fraction (LVEF) and other systolic function indices were obtained from short-axis M-mode scans at the mid-ventricular level, as indicated by the presence of the papillary muscles, in conscious, gently restrained mice. Apical four-chamber views were obtained in anesthetized mice for diastolic function measurements using pulsed-wave and tissue Doppler imaging at the mitral valve level. Anesthesia was induced with 2.5% isoflurane and confirmed by lack of response to firm pressure on one of the hind paws. During echocardiogram acquisition (under temperature-controlled conditions), isoflurane was reduced to 1.0 - 1.5% and adjusted to keep the heart rate within the range of 500 beats per minute. The 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, left ventricular fractional shortening, LVEF, peak Doppler blood flow velocity in early diastole through the mitral valve, peak Doppler blood flow velocity in late diastole through the mitral valve, isovolumic relaxation time, early diastolic and early filling deceleration times, and tissue Doppler peak velocity of myocardial relaxation at the mitral annulus. At the end of the procedure, all mice recovered from anesthesia without any abnormalities. All parameters were measured at least 3 times and the mean values were given. The ultrasound detection included systolic function and diastolic function detection.

[0159] 7. Experimental results and conclusions

[0160] First, at 5 weeks, the systolic and diastolic functions of the mice were measured. There was no significant change in the systolic function, but the diastolic function parameter E / E’ increased significantly, proving that there was a diastolic dysfunction, indicating that the model described in the aforementioned literature was successfully obtained. At the same time, taking the 5th week as the starting point for drug administration, there was no significant difference in the cardiac diastolic function between the model control group and the model treatment group before drug administration ( Figure 1 A-C). Based on this, drug administration treatment was carried out.

[0161] Secondly, comparing the expression of Jun in the normal group and the model control group showed an upregulation, indicating the correlation between the expression of Jun and HFpEF. In the HFpEF mouse model, Jun was highly expressed ( Figure 2 ). Based on this correlation, it can be judged that Jun inhibitors can be used for the prevention and treatment of HFpEF.

[0162] Furthermore, T-5224 was used to verify the effect of Jun inhibitors in preventing and treating HFpEF. Under the condition that the model construction was successfully determined and the baselines of the model control group and the model treatment group were consistent, the model treatment group was treated with T-5224. The cardiac function test results showed that the occurrence and development of HFpEF in the model treatment group (after T-5224 administration) were well inhibited. Specifically, after treatment with T-5224, the diastolic function of the mice treated with high-fat diet combined with L-NAME treatment (HFD + 0.5 g / L L-NAME) was significantly improved and could last until the 15th week; however, in the model control group mice without T-5224 treatment, a continuous deterioration of the diastolic function was observed ( Figure 3 A-B); at the same time, in the model treatment group, the expression of Jun was downregulated compared with the model control group ( Figure 3 D), and the obesity of the mice was improved ( Figure 3 C). This indicates that T-5224, as a Jun inhibitor, can play a role in preventing and treating HFpEF in the mouse HFpEF model.

[0163] Example 1: Screening and Preparation of T-5524 Salt

[0164] Based on the experimental results of Example A, the inventors further explored the new salt forms and crystal forms of T-5524.

[0165]

[0166] 1. Suspension method

[0167] Weigh approximately 30 - 50 mg of compound T-5224 polymorph A, place it in a 2 mL glass bottle together with 0.5, 1, or 2 equivalents of base, add screening solvents, and conduct screening experiments using the suspension method. The obtained sample is suspended at 50 °C for 2 hours, then cooled naturally to 25 °C and suspended at 25 °C for at least 48 hours.

[0168] Centrifuge the obtained suspension through a 0.45 μm nylon filter membrane at 14,000 rpm. The obtained solid is dried under vacuum at 50 °C for 2 hours and then characterized by XRPD.

[0169] Table 1-1: Preparation of salts by suspension method

[0170]

[0171]

[0172]

[0173] Note: " / / " indicates not carried out

[0174] 2. Cooling method

[0175] Cool the foggy suspension and clear solution obtained from the above suspension to 5 °C to prepare solid samples for characterization. Only gels or clear solutions are obtained.

[0176] Table 1-2: Cooling method

[0177]

[0178] Based on the above screening experiments, 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 compound T-5224 and their corresponding polymorphs were prepared. For the characterization of the crystal forms of each salt, see Figures 4 - 57 . Among them, the X-ray powder diffraction (XRPD) data of betaine salt polymorph A and choline salt polymorph A of T-5224 are shown in Table A and Table B.

[0179] Table A: X-ray powder diffraction data table of betaine salt polymorph A of T-5224

[0180]

[0181]

[0182]

[0183]

[0184] Table B:X-ray Powder Diffraction Data Sheet of T-5224 Choline Salt Polymorph A

[0185]

[0186]

[0187]

[0188] Example 2: Preliminary Study on the Stability and Solubility of T-5524 Salt and Its Crystal Forms

[0189] The sodium salt polymorph B, potassium salt polymorph B, tert-butylamine salt polymorph B, choline salt polymorph A, L-lysine salt polymorph A, diethylamine salt polymorph A, diethylamine salt polymorph B, dimethylethanolamine salt polymorph A, betaine salt polymorph A and ethylenediamine salt polymorph 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.

[0190] The above alternative salt forms were exposed at 25°C / 60% RH for 3 days and at 25°C / 80% RH for 2 days respectively to preliminarily investigate the physical stability of these 10 alternative salt forms. In addition, the approximate solubility experiments of these 10 alternative salt forms in pH 4.5 acetate buffer solution were also investigated. Thus, candidate salt forms were selected for further evaluation from the perspectives of physical stability and solubility. The results are shown in Tables 2-1 and 2-2 below.

[0191] Table 2-1

[0192]

[0193]

[0194] Table 2-2

[0195]

[0196]

[0197] Among the above alternative salt forms, choline salt polymorph A, diethylamine salt polymorph A and betaine salt polymorph A exhibit good physicochemical properties including good crystallinity, good physical stability, etc. In addition, in the approximate solubility experiment in pH 4.5 acetate buffer solution at 25°C, they also show potential advantages in solubility. Therefore, the above three alternative salt forms were selected as candidate salt forms for scale-up preparation, and the three candidate salt forms were systematically evaluated and compared with compound T-5224 polymorph A in terms of stability, solubility and hygroscopicity.

[0198] Example 3: Comparison of the Physicochemical Properties between the Dominant Salt Form and Its Crystal Forms and Crystal Form A of T-5524

[0199] 1. Preparation of Control Compounds and Candidate Salt Forms

[0200] In this part, the choline salt polymorph A and betaine salt polymorph A of the amplified batch were successfully obtained. The polymorphs of the candidate salts in the amplified batch were consistent with those of the candidate salts obtained from the previous screening. For the diethylamine salt, even under the condition of adding the seed crystal of diethylamine salt polymorph A, a new polymorph of diethylamine salt, polymorph C, was still obtained. This indicates that diethylamine salt polymorph C is more stable than diethylamine salt polymorph A. Therefore, a systematic characterization of diethylamine salt polymorph C was carried out subsequently.

[0201] 1.1 Preparation of control T5224 polymorph A

[0202] Weigh 500 mg of compound T5224 and place it in an 8 mL glass bottle. Add 4 mL of acetone and stir at 50 °C for 5 min to obtain a suspension. (Suspension)

[0203] Add approximately 3 mg of T-5524 polymorph A seed crystal to the above suspension. (Suspension)

[0204] After stirring at 50 °C for 2 h, cool the suspension to 25 °C and stir at 25 °C for about 3 days. (Suspension)

[0205] Collect the solid part by centrifugation and dry the obtained solid part under vacuum at 50 °C for about 2 h.

[0206] Approximately 253 mg of off-white powder of polymorph A was obtained, with a yield of 51%.

[0207] For the characterization of the control T-5224 polymorph A, see Figures 58 - 61 .

[0208] 1.2 Preparation of T-5224 choline salt polymorph A

[0209] Weigh 500 mg of compound T5224 and place it in an 8 mL glass bottle together with 256 mg (1.05 equivalents) of choline. Add 1 mL of acetone and stir at 50 °C for 5 min to obtain a clear solution. (Clear solution)

[0210] After stirring at 50 °C for 2 h, the solution becomes turbid and gradually turns into a suspension. (Clear solution → Suspension)

[0211] Add approximately 3 mg of choline salt polymorph A seed crystal to the above suspension. (Suspension)

[0212] Cool the suspension to 25 °C and stir at 25 °C for about 2 days. (Suspension)

[0213] Cool the suspension to 5 °C and continue to stir at 5 °C for about 1 day. (Suspension)

[0214] The solid part was collected by centrifugation and the obtained solid part was dried under vacuum at 50 °C for about 2 hours.

[0215] A total of 150 mg of white powder of choline salt crystal form A was obtained, and the yield was 20%.

[0216] Preparation of 1.3 T-5224 diethylamine salt crystal form C

[0217] Weigh 500 mg of compound T5224 and place it in an 8 mL glass bottle together with 71 mg (1.05 equivalents) of diethylamine. Add 5.1 mL of acetone and stir at 50 °C for 5 min to obtain a suspension. (Suspension)

[0218] Add about 3 mg of diethylamine salt crystal form A seed crystals to the above suspension. (Suspension)

[0219] After stirring at 50 °C for 2 h, cool the suspension to 25 °C and stir at 25 °C for about 2 days. (Suspension)

[0220] The solid part was collected by centrifugation and the obtained solid part was dried under vacuum at 50 °C for about 2 hours.

[0221] A total of 523 mg of white powder of the new crystal form of diethylamine salt crystal form C was obtained, and the yield was 92%.

[0222] For the characterization of T-5224 diethylamine salt crystal form C, see Figures 62 - 65 . Among them, the X-ray powder diffraction (XRPD) data are shown in Table C.

[0223] Table C: X-ray powder diffraction data table of T-5224 diethylamine salt crystal form C

[0224]

[0225]

[0226]

[0227]

[0228] 1.4 Preparation of T-5224 betaine salt crystal form A

[0229] Weigh 500 mg of compound T5224 and place it in an 8 mL glass bottle together with 122 mg (1.05 equivalents) of betaine. Add 4.4 mL of acetone and stir at 50 °C for 5 min to obtain a suspension. (Suspension)

[0230] Add about 3 mg of betaine salt crystal form A seed crystals to the above suspension. (Suspension)

[0231] 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)

[0232] The solid part was collected by centrifugation, and the obtained solid part was placed in a vacuum at 50 °C and dried for about 2 h.

[0233] A total of 473 mg of white powder of betaine salt crystal form A was obtained, and the yield was 76%.

[0234] 2. Stability comparison

[0235] 2.1 Initial chemical purity

[0236] The chemical purities of crystal form A of T5224, crystal form A of choline salt, crystal form C of diethylamine salt, and crystal form A of betaine salt were 98.7%, 99.4%, 98.4%, and 98.8% respectively.

[0237] 2.2 Solid stability

[0238] Open containers containing crystal form A of T5224 and 3 candidate salt form samples were placed at 25 °C / 92.5% RH for 1 week. Closed containers containing crystal form A of T5224 and 3 candidate salt forms were placed at 60 °C for 1 week. The samples after solid stability evaluation were characterized by XRPD and HPLC, and the color change of the samples was observed.

[0239] Table 3-1: Solid stability: purity and appearance (color, CL)

[0240]

[0241]

[0242] Note: A: No color change; B: Slight discoloration; C: Moderate discoloration; D: Severe discoloration. All the above physical forms showed good physical and chemical stability.

[0243] 2.3 Solubility

[0244] This part studied the solubility of crystal form A of T5224 and 3 candidate salt forms in water, 5 aqueous buffer solutions, and simulated biological fluids at two time points of 2 h and 24 h at 37 °C. The selected solvents included acetate buffer (50 mM) at pH 4.5 (only tested for solubility at 2 h at 37 °C), water, FaSSGF at pH 1.6, FaSSIF-v1 at pH 6.5, and FeSSIF-v1 at pH 5.0. The residual solids in the solubility experiment were characterized by XRPD.

[0245] Accurately weigh 10.0 mg of T5224 polymorph A, 12.6 mg of choline salt polymorph A, 11.3 mg of diethylamine salt polymorph C, or 12.3 mg of betaine salt polymorph A and place them separately in 8-mL glass bottles. Add 5 mL of solvent to each. The mass of the salt taken is equivalent to 10 mg of T5224 polymorph A. Stir the resulting suspension at 37 °C at a speed of 400 rpm, and take samples at 2 hours and 24 hours respectively. Centrifuge the samples at 14,000 rpm for 5 min at 37 °C. Determine the supernatant concentration by HPLC and the pH value of the supernatant by a pH meter. The results are shown in Table 3-2.

[0246] Table 3-2: Solubility

[0247]

[0248]

[0249] Remark: " / / ": Not carried out

[0250] The solubility and pH of T-5524 polymorph A are related to those of three candidate salt forms. The solubility of the three candidate salt forms is equivalent to that of T-5524 in pH 1.6 FaSSGF, pH 4.5 acetate buffer (50 mM), and pH 5.0 FeSSIF-v1. The solubility is close to or lower than the lowest detection limit in pH 1.6 FaSSGF and pH 4.5 acetate buffer (50 mM), and the solubility is 15 - 20 μg / mL in pH 5.0 FeSSIF-v1.

[0251] The solubility of the three candidate salt forms (15 - 35 μg / mL) in pH 6.5 FaSSIF-v1 is slightly higher than that of T-5524 polymorph A (about 7 μg / mL).

[0252] The solubility of choline salt polymorph A and diethylamine salt polymorph C in water (0.9 - 1 mg / mL) is much higher than that of T-5524 polymorph A and betaine salt polymorph A (1 - 5 μg / mL).

[0253] In summary, the three candidate salt forms exhibit good crystallinity, as well as good physical stability, chemical stability, etc. In addition, the three candidate salt forms have obvious advantages in terms of solubility. However, the diethylamine salt has no advantage compared with the other two salt forms because of its poor thermal stability (gradually decomposing starting from about 120 °C). The choline salt and the betaine salt are relatively close in terms of physical and chemical properties, but considering the safety of the base, the choline salt is selected as the preferred salt form for subsequent polymorph screening.

[0254] Example 4: Crystal Form Screening of Choline Salt and Comparison of the Physicochemical Properties with Crystal Form A of T-5524

[0255] 1. Preparation of choline salt polymorph

[0256] 1.1 Preparation of choline salt crystal form B

[0257] Weigh 5.0 g of T-5524 crystal form A and place it in an 8 mL glass bottle together with approximately 2.6 g (1.05 equivalents) of choline. Add 5 mL of ethanol and stir at 50 °C for 5 min to obtain a suspension. (Suspension)

[0258] Add approximately 10 mg of choline salt crystal form B seed crystals to the above suspension. (Suspension)

[0259] Cool the suspension to 5 °C and stir at 5 °C for approximately 3 days. (Suspension)

[0260] Take a portion of the solid for XRPD testing to obtain a physical mixture of choline salt crystal form A and free base crystal form A. Additionally, add approximately 0.3 g (0.1 equivalent) of choline and stir for approximately 1 day.

[0261] Take a portion of the solid for XRPD testing to obtain a physical mixture of choline salt crystal form B and free base crystal form A.

[0262] Evaporate the solvent to dryness, add 13 mL of ethyl acetate, and stir at 25 °C for 5 min to obtain a suspension. (Suspension)

[0263] Add approximately 3 mg of choline salt crystal form B seed crystals to the above suspension. (Suspension)

[0264] Stir the suspension at 25 °C for approximately 1 day. (Suspension)

[0265] Collect the solid portion by centrifugation and place the obtained solid portion in a vacuum at 50 °C for drying for approximately 2 hours.

[0266] A total of 5.2 g of off-white powder of choline salt crystal form B was obtained, with a yield of 66%. For the characterization of choline salt crystal form B, see Figures 66 - 69 .

[0267] Table D: X-ray powder diffraction data table of T-5224 choline salt crystal form B

[0268]

[0269]

[0270]

[0271]

[0272] 1.2 Preparation of choline salt crystal form A

[0273] See Table 1-1 in Example 1.

[0274] 1.3 Preparation of choline salt crystal form C

[0275] Weigh 10 - 20 mg of choline salt crystal form A and add 100 - 300 μL of the solvent ethyl acetate. The resulting suspension is suspended at 25 °C for 2 days. The solid part is collected by centrifugation to obtain choline salt crystal form C. For the characterization of choline salt crystal form C, see Figures 70 - 72 . Among them, the X - ray powder diffraction (XRPD) data is shown in Table E.

[0276] Table E: X - ray powder diffraction data table of choline salt crystal form C of T - 5224

[0277]

[0278]

[0279] 2. Polymorph characterization

[0280] Choline salt crystal form A is a hydrate. Choline salt crystal form A has high crystallinity. DSC shows that it starts to dehydrate at about 21 °C, the melting point T onset 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 about 0.1% of its weight between 80 °C and 130 °C. 1 1H - NMR shows that its stoichiometric ratio is free acid: choline = 1:1.0 and no solvent residue is detected in the sample. The water activity competition experiment shows that choline salt crystal form A transforms into choline salt crystal form B under the conditions of 5 °C a.w. ≤ 0.6 and 25 °C a.w. ≤ 0.3.

[0281] Choline salt crystal form B is an anhydrous crystal form. Choline salt crystal form B has high crystallinity. DSC shows that its melting point is 153.3 °C and the melting enthalpy is about 62 J / g. TGA shows that it loses about 0.7% of its weight at about 140 °C. 1 1H - NMR shows that its stoichiometric ratio is free acid: choline = 1:1.0 and no solvent residue is detected in the sample. Choline salt crystal form B is the most stable crystal form discovered so far. The water activity competition experiment shows that choline salt crystal form B is thermodynamically more stable than choline salt crystal form A under the conditions of 5 °C a.w. ≤ 0.6 and 25 °C a.w. ≤ 0.3.

[0282] 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 1H - NMR shows that its stoichiometric ratio is free acid: choline = 1:1.0 and no solvent residue is detected in the sample. Choline salt crystal form C can transform into choline salt crystal form B after 2 days under environmental conditions (about 20 - 25 °C, 60% - 75% RH).

[0283] Table 4-1: Summary of Polymorph Characterization

[0284]

[0285] Note: " / / ": Characterization not performed

[0286] Among them, in order to study the critical water activity between polymorph A and polymorph B of T-5224 choline salt, water activity experiments were carried out at 5 °C and 25 °C in an ethanol / water system. The method is as follows.

[0287] Weigh about 2 mg of polymorph A of choline salt, about 2 mg of polymorph B of choline salt and about 1 mg of polymorph B of choline salt with additional diffraction peaks and place them in the corresponding saturated solution of 0.1 mL ethanol / water system. The obtained suspension was suspended for 5 days at 5 °C and 25 °C. Then the obtained suspension was centrifuged, and the obtained solid was subjected to XRPD detection. The results are shown in Table 4-2 below.

[0288] Among them, the preparation method of polymorph B of choline salt with additional diffraction peaks is as follows:

[0289] Weigh about 10 - 20 mg of polymorph B of T-5224 choline salt and add the minimum volume of solvent (ethanol, 2.0 mL) and dissolve it thoroughly at ambient temperature (about 20 - 25 °C). The obtained solution or thin suspension was passed through a 0.45 μm nylon membrane syringe filter to obtain a clear solution. Slowly add 4 - 8 times the volume of anti-solvent (n-heptane, 16 mL) to the obtained clear solution. The obtained suspension was centrifugally filtered through a 0.45 μm nylon membrane centrifuge tube at 14,000 rpm, and the obtained solid was subjected to XRPD characterization.

[0290] Table 4-2: Water Activity Experiment

[0291]

[0292] Note: " / / ": Not performed; "*": Water activity (a.w.) calculated by UNIFAC method

[0293] 3. Evaluation of the properties of polymorph B of T5224 choline salt

[0294] Polymorph B of T5224 choline salt is the most stable polymorph of choline salt discovered so far. Therefore, the stability, solubility and mechanical properties of polymorph B of T5224 choline salt were systematically studied.

[0295] 3.1 Solid stability

[0296] In this study, the solid stability of polymorph B of choline salt was evaluated under open conditions at 25 °C / 92.5% RH and under accelerated conditions at 60 °C in a sealed container for 1 week.

[0297] An open container containing T-5224 choline salt polymorph B was placed at 25 °C / 92.5% RH for 1 week. A closed container containing T-5224 choline salt polymorph B was placed at 60 °C for 1 week. The samples under these conditions were subjected to XRPD and HPLC tests and observed for color changes.

[0298] Table 4-3: Solid stability

[0299]

[0300] Note: A: No color change; B: Slight discoloration; C: Moderate discoloration; D: Severe discoloration. T-5224 choline salt polymorph B showed good physical and chemical stability under the above accelerated stability conditions.

[0301] 3.2 Solubility

[0302] In this study, the solubility of the initial materials, T-5224 polymorph A and choline salt polymorph B, was evaluated in water, pH 1.6 FaSSGF solution, pH 6.5 FaSSIF solution, and pH 5.0 FeSSIF solution at 37 °C for 2 hours and 24 hours. The residual solids from the solubility experiments were characterized by XRPD.

[0303] Accurately weigh 10.0 mg of T-5224 polymorph A and 12.5 mg of choline salt polymorph B and place them separately in 8 mL glass bottles. Add 5 mL of solvent to each. The mass of the salt taken is equivalent to 10 mg of T-5224 polymorph A. The resulting suspension was stirred at 400 rpm at 37 °C and sampled at 2 hours and 24 hours respectively. The samples were centrifuged at 14,000 rpm for 5 min at 37 °C. The supernatant concentration was determined by HPLC and the pH value of the supernatant was measured by a pH meter.

[0304] Table 4-4: Solubility

[0305]

[0306] The solubility of T-5224 polymorph A and choline salt polymorph B is related to pH. The solubility of choline salt polymorph B in pH 6.5 FaSSIF-v1 and pH 5.0 FeSSIF-v1 at 24 hours (30 - 45 μg / mL) is slightly higher than that of T-5224 polymorph A (20 - 35 μg / mL). In pH 1.6 FaSSGF, the solubility of both T-5224 polymorph A and choline salt polymorph B is lower than the lowest detection limit.

[0307] The solubility of choline salt polymorph B in water (>2 mg / mL) is much higher than that of T-5224 polymorph A (8 - 9 μg / mL).

[0308] 3.3 Mechanical properties

[0309] In this study, the mechanical properties of choline salt polymorph B were evaluated by simulating tableting, grinding, and granulation experiments.

[0310] 3.3.1 Simulated tableting experiment

[0311] Weigh approximately 10 mg of the T-5224 choline salt polymorph B sample and press it for 5 min at pressures of 2 MPa, 5 MPa, and 10 MPa, respectively. The polymorphic transformation and changes in crystallinity were studied by XRPD characterization.

[0312] Table 4-5: Simulated tableting experiment

[0313]

[0314] 3.3.2 Simulated dry grinding experiment

[0315] Weigh approximately 10 mg of the T-5224 choline salt polymorph B sample and grind it manually for 5 min. The polymorphic transformation and changes in crystallinity were studied by XRPD characterization.

[0316] Table 4-6: Simulated dry grinding experiment

[0317]

[0318] 3.3.3 Simulated wet granulation experiment

[0319] Weigh approximately 10 mg of the T-5224 choline salt polymorph B sample and add ethanol dropwise until the sample is fully wetted. Gently grind the wetted sample with a pestle and then dry the sample in the external environmental conditions for 10 min. The polymorphic transformation and changes in crystallinity were studied by XRPD characterization.

[0320] Table 4-7: Simulated wet granulation experiment

[0321]

[0322] Choline salt polymorph B showed good tolerance to tableting (<2 MPa), dry grinding, and wet grinding, with no polymorphic transformation and no significant decrease in crystallinity. When the tableting pressure increased to 5 to 10 MPa, there was no polymorphic transformation of choline salt polymorph B, and its crystallinity only decreased slightly.

[0323] Combined with the above research results, choline salt crystal form B of T-5224 is the most stable crystal form obtained so far. It exhibits good chemical stability, physical stability, and mechanical force tolerance, etc. In terms of solubility, the solubility of choline salt crystal form B in water, pH 6.5 FaSSIF solution, and pH 5.0 FeSSIF after 24 hours is higher than that of the initial material of T-5224 crystal form A. Therefore, choline salt crystal form B of T-5224 is used as the preferred crystal form for subsequent pharmacokinetic studies.

[0324] Example 5: Pharmacokinetic Study and Comparison between Salt-Free Crystal Form A of T-5224 (i.e., the aforementioned Crystal Form A of T5224) and Choline Salt Crystal Form B of T-5224 Example 6: Comparison of the Efficacy Test in Animal Models between Salt-Free Crystal Form A of T-5224 (i.e., the aforementioned Crystal Form A of T5224) and Choline Salt Crystal Form B of T-5224 1. Administration method

[0325]

[0326] 2. Preparation configuration method

[0327]

[0328]

[0329] 3. Detection method

[0330]

[0331] 4. Experimental results

[0332]

[0333]

[0334]

[0335] 5. Experimental conclusion

[0336] As can be seen from the above results, compared with the control T-5524 crystal form A, the bioavailability of choline salt crystal form B of this application is significantly improved (55.4% vs 17.5%), which is increased by at least more than 2 times, showing significant advantages.

[0337] Figure 73 Figure 73

[0338] The materials and reagents, animal experiment guidelines, and induction of heart failure models with preserved ejection fraction are the same as in Example A.

[0339] The administration method is as follows:

[0340] After successfully inducing the animal model, the model treatment group was treated with T-5224 salt-free crystal form A and T-5224 choline salt crystal form B on mice, and the model control group was treated with a solvent without drugs for control. During the whole induction process, mice with normal diet and water were used as negative controls. When the mice were raised to 5-8 weeks old, after the model was successfully constructed by ultrasonic detection, drug administration was started. For the treatment group, T-5224 salt-free crystal form A was administered at a single dose of 12 mg / kg according to the body weight of the mice. Specifically, it was administered every other day. Each time, 0.54 mg of the compound was dissolved in 100 μL of 0.5% PVP solution (specifically, in the actual drug preparation process, it was prepared together according to the number of mice. For example, for 16 mice, 9 mg of the drug was dissolved in 1.7 mL of the solution, and each mouse was administered 100 μL); T-5224 choline salt crystal form B was administered at a single dose of 3 mg / kg according to the body weight of the mice. Specifically, it was administered every other day. Each time, 0.14 mg of the compound was dissolved in 100 μL of 0.5% PVP solution (specifically, in the actual drug preparation process, it was prepared together according to the number of mice. For example, for 16 mice, 2.2 mg of the drug was dissolved in 1.7 mL of the solution, and each mouse was administered 100 μL); Drug administration started 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, and other treatment methods were the same otherwise.

[0341] The experimental results are as ​ shown (where 0 week on the abscissa represents the day when drug administration started after the model was successfully constructed, that is, the starting point of drug administration). It can be ​ clearly seen that at 3 weeks after drug administration, the diastolic function of the mice administered with T-5224 choline salt crystal form B (3 mg / kg) was significantly improved (E / E’ was significantly reduced), but the diastolic function of the mice administered with the positive control T-5224 salt-free crystal form A (12 mg / kg) did not change significantly. Obviously, compared with 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, with obvious advantages and broad application prospects. Therefore, the salt crystal form of the present invention (especially T-5224 choline salt crystal form B) has an obvious therapeutic effect on HFpEF at a lower dose and has a rapid onset of action.

Claims

1. A salt of the compound represented by formula I, wherein, The salt is the 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 of the following technical features (1)-(20): (1) The sodium salt of the compound represented by Formula I is Crystal Form A, wherein the sodium salt Crystal 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 Crystal Form B, wherein the sodium salt Crystal 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 Crystal Form A, wherein the potassium salt Crystal 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 Crystal Form B, wherein the potassium salt Crystal 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 Crystal Form C, wherein the potassium salt Crystal 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 Crystal Form D, wherein the potassium salt Crystal 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 Crystal Form A, wherein the calcium salt Crystal 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 Crystal Form A, wherein the tert-butylamine salt Crystal 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 Crystal Form B, wherein the tert-butylamine salt Crystal 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 Crystal Form A, wherein the ammonium salt Crystal 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 Crystal Form B, wherein the ammonium salt Crystal 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 Crystal Form A, wherein the L-lysine salt Crystal 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 Crystal Form A, wherein the diethylamine salt Crystal Form A of the compound represented by Formula I has an X-ray powder diffraction pattern substantially the same as that in Figure 36; The diethylamine salt of the compound represented by formula (I) is crystalline form B, wherein the crystalline form B 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 40; (15) 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; 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; (20) 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 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 of Figure 66.

9. A pharmaceutical composition comprising the salt according to any one of claims 1-8; optionally, further comprising 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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