Use of compound for treating heart failure with preserved ejection fraction

Abstract

The present invention relates to a use of a compound for treating heart failure with preserved ejection fraction, and particularly provides a use of a compound or a stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, pharmaceutically acceptable ester, or pharmaceutically acceptable solvate thereof in preparation of a drug for treating and / or preventing heart failure with preserved ejection fraction, wherein the compound is selected from T3 and T5.

Description

Use of compounds 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 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 this invention have discovered for the first time that suppressing elevated Jun expression using a Jun inhibitor can prevent and treat HFpEF. Based on this discovery, the inventors further investigated and found that the compounds of this invention have the activity of inhibiting JUN binding to DNA and inhibiting JUN transcription, potentially preventing and treating HFpEF and showing broad potential for application.

[0006] To this end, in a first aspect of the present invention, the present invention provides use of a compound or its stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, pharmaceutically acceptable ester or pharmaceutically acceptable solvate in the preparation of a medicament for treating and / or preventing heart failure with preserved ejection fraction, wherein the compound is selected from T3, T5,

[0007] Alternatively, a compound or a stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, pharmaceutically acceptable ester or pharmaceutically acceptable solvate thereof is provided for treating and / or preventing heart failure with preserved ejection fraction, wherein the compound is selected from T3, T5,

[0008] Alternatively, a method for treating and / or preventing heart failure with preserved ejection fraction is provided, comprising: administering to a subject in need thereof an effective amount of a compound or its stereoisomer, prodrug, crystalline form, pharmaceutically acceptable salt, pharmaceutically acceptable ester or pharmaceutically acceptable solvate, wherein the compound is selected from T3, T5,

[0009] In a second aspect of the present invention, the present invention provides use of a pharmaceutical composition in the preparation of a medicament for treating and / or preventing heart failure with preserved ejection fraction, wherein the pharmaceutical composition comprises a compound or its stereoisomer, prodrug, crystalline form, pharmaceutically acceptable salt, pharmaceutically acceptable ester or pharmaceutically acceptable solvate, wherein the compound is selected from T3, T5,

[0010] Alternatively, a pharmaceutical composition is provided for treating and / or preventing heart failure with preserved ejection fraction, wherein the pharmaceutical composition comprises a compound or a stereoisomer, prodrug, crystalline form, pharmaceutically acceptable salt, pharmaceutically acceptable ester or pharmaceutically acceptable solvate thereof, wherein the compound is selected from T3, T5,

[0011] Alternatively, a method for treating and / or preventing heart failure with preserved ejection fraction is provided, comprising: administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a compound or a stereoisomer, prodrug, crystalline form, pharmaceutically acceptable salt, pharmaceutically acceptable ester or pharmaceutically acceptable solvate thereof, wherein the compound is selected from T3, T5, BRIEF DESCRIPTION OF THE DRAWINGS

[0012] 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.

[0013] 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.

[0014] 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.

[0015] Figure 4: Inhibitory effect test results of compounds at different concentrations on the binding activity of Jun to DNA at the molecular level.

[0016] Figure 5: Detection results of the inhibitory effect of compounds at different concentrations on cell Jun transcription activity.

[0017] Figure 6: The compounds of the present invention can effectively inhibit the occurrence and development of HFpEF. DETAILED DESCRIPTION

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] As used herein, unless otherwise indicated, the term "prodrug" refers to a derivative that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide a compound of the present invention. Prodrugs become active compounds only through this reaction under biological conditions, or they have no or only low activity in their unreactive form. Prodrugs can generally be prepared using known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff, 5th edition).

[0026] When the stereoisomers in the compounds described herein are specifically designated as (R)- or (S)-isomers in the chemical name, it should be understood that the predominant configuration is the (R)-isomer or the (S)-isomer, respectively. Any asymmetric carbon atom may be present in the (R)-, (S)-, or (R, S)-configuration, preferably in the (R)- or (S)-configuration.

[0027] Tautomerism refers to that a functional group in some compounds changes its structure into another functional group isomer, and these two isomers can rapidly convert to each other, and this rapid and reversible conversion process makes two isomers coexist with a certain ratio under given conditions, forming a dynamic equilibrium state. Taking following compound A and compound B as example, it will be appreciated by those skilled in the art that following compound A and compound B are tautomers (the difference of structure as shown in the box), and two isomers can rapidly convert to each other, to reach a certain balance, therefore, for those skilled in the art, what following compound A and compound B represent is identical compound, and it is just that expression mode is slightly different. Similarly, for other compounds with similar structure in the application, it will be appreciated by those skilled in the art equally that different tautomers represent identical compound in essence, and it is just that expression mode is slightly different.

[0028] "Solvate" or "solvate" are used interchangeably to refer to a compound that exists in combination with a solvent molecule. The combination may include a stoichiometric amount of a solvent, such as a monohydrate or dihydrate, or may include any amount of water; for example, methanol or ethanol may form an "alcoholate," which may also be stoichiometric or non-stoichiometric. As used herein, the term "solvate" refers to a solid form, i.e., a compound in solution in a solvent, which may be solvated but is not a solvate as the term is used herein.

[0029] As used herein, the term "pharmaceutically acceptable salt" refers to (i) a salt formed by an acidic functional group (e.g., -COOH) present in the compounds provided herein with a suitable inorganic or organic cation (base), and includes, but is not limited to, alkali metal salts, such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, etc.; other metal salts, such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts, such as ammonium salts; organic base salts, such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylamine salts, tris(hydroxymethyl)aminomethane salts. and (ii) salts formed by basic functional groups (e.g., -NH2) present in the compounds provided by the present invention and appropriate inorganic or organic anions (acids), including but not limited to hydrohalides, such as hydrofluorides, hydrochlorides, hydrobromides, hydroiodides, etc.; inorganic acid salts, such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkanesulfonates, such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; arylsulfonates, such as benzenesulfonates, p-toluenesulfonates, etc.; organic acid salts, such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates, etc.; amino acid salts, such as glycine, trimethylglycine, arginine, ornithine, glutamate, aspartate, etc.

[0030] As used herein, the term "pharmaceutically acceptable ester" refers to an ester formed between a -COOH group present in a compound provided herein and a suitable alcohol, or an ester formed between a -OH group present in a compound provided herein and a suitable acid (e.g., a carboxylic acid or an oxygen-containing inorganic acid). Suitable ester groups include, but are not limited to, formates, acetates, propionates, butyrates, acrylates, ethylsuccinates, stearic acid esters, or palmitates. Esters can undergo hydrolysis in the presence of an acid or base to produce the corresponding acid or alcohol.

[0031] As used herein, the term "crystalline form" refers to the crystal structure of a substance. During crystallization, various factors may alter the intramolecular or intermolecular bonding patterns, resulting in different arrangements of molecules or atoms within the crystal lattice space, forming different crystal structures. The compounds of the present invention may exist in a single crystal structure or in multiple crystal structures, i.e., they may exhibit "polymorphism." The compounds of the present invention may exist in different crystalline forms.

[0032] The present invention will be further explained below with reference to specific examples. Unless otherwise specified, all reagents and raw materials are commercially available, or can be prepared according to conventional techniques in the art with reference to existing technologies and common knowledge, and all instruments are those conventionally used by those skilled in the art.

[0033] Example 1

[0034] 1. Materials and Reagents

[0035] In this example, C57BL / 6N wild-type mice were purchased from Beijing Weitonglihua. The sources of the reagents are shown in the following table.

[0036] In addition to the above, other materials and reagents used in this example are also commercially available products.

[0037] 2. Animal Experiment Guidelines

[0038] 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.

[0039] 3. Induction of Heart Failure Model with Preserved Ejection Fraction

[0040] 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.

[0041] 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.

[0042] 4. Jun expression is correlated with HFpEF

[0043] 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 as follows:

[0044] 4.1. Isolation of adult mouse cardiomyocytes:

[0045] 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.

[0046] 4.2. Quantitative PCR detection:

[0047] 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.

[0048] 5. T-5224 administration method

[0049] 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.

[0050] 6. Conventional ultrasonic testing

[0051] 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.

[0052] 7. Experimental results and conclusions

[0053] 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.

[0054] 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.

[0055] 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.

[0056] Example 2

[0057] Based on the experimental results of Example 1, the inventors used T5524 as Yangshen to further explore the therapeutic effects of other compounds on HFpEF.

[0058] 1. Synthesis of compounds

[0059] 1.1 Synthesis of compound Target 3 (abbreviated as T3)

[0060] 1) Synthesis of compound 2B

[0061] Under nitrogen, potassium carbonate (311 g, 2.26 mol, 3.00 eq) was added to a solution of compound 2A (125 g, 752 mmol, 1.00 eq) in acetonitrile (1.25 L). Methyl iodide (266 g, 1.88 mol, 117 mL, 2.50 eq) was then added to the mixture. The mixture was stirred at 25°C under nitrogen for 12 hours. TLC (petroleum ether:ethyl acetate = 5:1) confirmed the complete reaction of compound 2A (Rf = 0.15) with the appearance of two new spots (Rf = 0.40, 0.60). The reaction mixture was diluted with water (3.00 L) and extracted with ethyl acetate (1.00 L x 3). All organic phases were combined, washed with water (1.00 L*3) and brine (1.00 L*2) in sequence, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a brown liquid compound 2B (138 g, 710 mmol, 94.4% yield).

[0062] Proton spectrum: EC6536-710-P1A1, 400 MHz, deuterated chloroform

[0063] δ:7.26-7.13(m,2H),6.98-6.81(m,2H),3.91-3.83(m,3H),3.76-3.67(m,3H),3.00(t,J=7.6Hz,2H),2.67(t,J=7.6Hz,2H).

[0064] 2) Synthesis of Compound 2

[0065] To a mixed solution of compound 1 (40.0 g, 219 mmol, 1.00 eq) in N,N-dimethylformamide (160 mg, 2.20 mmol, 168 μL, 0.01 eq) and dichloromethane (400 mL) was added dropwise oxalyl chloride (33.4 g, 263 mmol, 23.0 mL, 1.20 eq). The mixture was stirred at 25°C for 2 hours and then concentrated under reduced pressure to obtain a residue. After dissolving the residue in dichloromethane (400 mL), aluminum trichloride (AlCl3) (73.1 g, 548 mmol, 30.0 mL, 2.50 eq) and a solution of compound 2B (51.1 g, 263 mmol, 1.20 eq) in dichloromethane (50.0 mL) were added at -30°C. After stirring the mixture at 0°C for 1 hour, ethyl acetate (61.2 g, 694 mmol, 68.0 mL, 3.16 eq) was added dropwise, followed by aluminum chloride (161 g, 1.21 mol, 66.0 mL, 5.50 eq) at 0°C. The reaction mixture was stirred at 40°C for 12 hours (in duplicate). LCMS (EC6536-741-P1A1) confirmed the complete reaction of compound 1, with the appearance of a single major peak at the target molecular weight (Rt = 0.37 min, MS cal.: 316.09, MS observed: [M+H]+ = 317.0). The reaction mixture was cooled to 25°C and poured into ice-cold 6M hydrochloric acid. The aqueous phase was extracted with ethyl acetate (1.00 L x 3). All organic phases were combined, washed with brine (1.00 L x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to yield a residue. The crude product was purified by column chromatography (silica gel, petroleum ether:ethyl acetate = 100:1 to 0:1) and monitored by TLC plate (petroleum ether:ethyl acetate = 1:1, Rf = 0.30) to give compound 2 (121 g, 382 mmol, 87.1% yield) as a light yellow oil.

[0066] LCMS 1: EC6536-741-P1A1, Rt=0.37min, MS cal.: 316.09, MS observed: [M+H]+=317.0.

[0067] LCMS 2: EC6536-741-P1A2, Rt=0.37min, MS cal.: 316.09, MS observed: [M+H]+=317.0.

[0068] Proton spectrum: EC6536-741-P1A, 400 MHz, DMSO-d6.

[0069] δ:12.27(s,1H),10.58(br s,1H),10.37(br s,1H),7.49-7.35(m,3H),6.93(d,J=8.4Hz,1H),6.45-6.35(m,2H),3.59(s,3H),2.90-2.83(m,2H),2.61(t,J=7.6Hz,2H).

[0070] 3) Synthesis of Compound Int A

[0071] To a solution of compound 2 (101 g, 319 mmol, 1.00 eq) in toluene (1.00 L) was added p-toluenesulfonic acid monohydrate (3.04 g, 15.9 mmol, 0.05 eq), and the mixture was stirred at 120°C for 12 hours. LCMS (EC6536-760-P1A) confirmed the complete reaction of compound 2, with the appearance of a single main peak at the target molecular weight (Rt = 0.56 min, MS cal.: 284.07, MS observed: [M+H]+ = 285.0). Ethyl acetate (1.50 L) and saturated sodium bicarbonate solution (1.50 L) were then added, and the aqueous phase was extracted with ethyl acetate (1.00 L x 3). All organic phases were combined, washed with brine (1.50 L x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to yield a residue. The crude product was triturated with petroleum ether:ethyl acetate = 3:1 (100 mL) at 25°C for 30 minutes, filtered, and the filter cake was concentrated under reduced pressure to give a light brown solid compound Int A (63.1 g, 220 mmol, 69.1% yield, 99.4% purity).

[0072] LCMS 1: EC6536-760-P1A, Rt=0.56min, MS cal.: 284.07, MS observed: [M+H]+=285.0.

[0073] LCMS 2: EC6536-760-P1A1, Rt=0.56min, MS cal.: 284.07, MS observed: [M+H]+=285.0, 99.4% purity.

[0074] Proton spectrum: EC6536-760-P1A, 400 MHz, DMSO-d6

[0075] δ:12.05(br s,1H),11.13-10.42(m,1H),7.72-7.47(m,2H),7.40(br d,J=8.4Hz,1H),7.18(br d,J=8.4Hz,1H),6.50-6.28(m,2H),3.07(br t,J=7.2Hz,2H),2.84(br t,J=7.2Hz,2H)

[0076] 4) Synthesis of compound Int A_3

[0077] A tetrahydrofuran solution of compound Int A (25.1 g, 87.9 mmol, 1.00 eq) was cooled to 0°C, and diisopropyl azodicarboxylate (21.3 g, 105 mmol, 20.4 mL, 1.20 eq), Int-A_4 (7.95 g, 92.3 mmol, 8.38 mL, 1.05 eq) and triphenylphosphine (27.6 g, 105 mmol, 1.20 eq) were added dropwise, and the mixture was stirred at 25°C for 12 hours. After monitoring the complete reaction of compound Int A by LCMS (EC6536-768-P1A) and the appearance of a single main peak with the target molecular weight (Rt = 0.51 min, MS cal.: 352.13, MS observed: [M+H]+ = 353.0), the reaction solution was poured into a mixture of ethyl acetate (300 mL) and water (300 mL), and the aqueous phase was extracted with ethyl acetate (200 mL*3). All organic phases were combined, washed with brine (300 mL*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The crude product was purified by reverse-phase HPLC (neutral conditions) to obtain compound Int A_3 as a pale yellow solid (15.2 g, 43.1 mmol, 49.0% yield, 100% purity).

[0078] LCMS 1: EC6536-768-P1A, Rt=0.51min, MS cal.: 352.13, MS observed: [M+H]+=353.0.

[0079] LCMS 2: EC6536-768-P1B, Rt=0.51min, MS cal.: 352.13, MS observed: [M+H]+=353.1, 100% purity.

[0080] Proton spectrum: EC6536-768-P1A1, 400 MHz, DMSO-d6

[0081] δ:11.90(s,1H),7.63(s,1H),7.57(dd,J=2.0,8.4Hz,1H),7.43(d,J=8.8Hz,1H),7.19(d,J=8.4Hz,1H),6.54-6.44(m,2H),4.91(br t,J=6.0Hz,1H),3.12-3.05(m,2H),2.89-2.82(m,2H),2.01-1.88(m,2H),1.78-1.52(m,6H)

[0082] 5) Synthesis of Compound Int D

[0083] After a solution of compound Int-A_3 (5.00 g, 14.1 mmol, 1.00 eq) in methanol (50.0 mL) was cooled to 0°C, a solution of sodium methoxide (1.84 g, 34.0 mmol, 2.40 eq) in methanol (10.0 mL) was added, and the mixture was stirred at 0°C for 2 h. LCMS (EC6536-769-P1A1) monitored the complete reaction of compound Int-A_3 and the appearance of a single main peak with the target molecular weight (Rt = 0.49 min, MS cal.: 384.16, MS observed: [M+H]+ = 385.1). The reaction solution was poured into water (200 mL), the pH value was adjusted to 5 with 1 M hydrochloric acid solution, and then extracted with ethyl acetate (200 mL*3). All organic phases were combined, washed with brine (200 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound Int-D (4.70 g, 12.2 mmol, 86.1% yield, crude product) as a pale yellow oil.

[0084] LCMS 1: EC6536-769-P1A1, Rt=0.49min, MS cal.: 384.16, MS observed: [M+H]+=385.1.

[0085] LCMS 2: EC6536-769-P1A4, Rt=0.50min, MS cal.: 384.16, MS observed: [M+H]+=385.0.

[0086] 6) Synthesis of Compound 4

[0087] Potassium carbonate (1.44 g, 10.4 mmol, 2.00 eq) was added to a solution of compound Int D (2.00 g, 5.20 mmol, 1.00 eq) and compound 1C (1.19 g, 5.20 mmol, 1.00 eq) in N,N-dimethylformamide (20.0 mL). The mixture was stirred at 40°C for 1 hour. Upon completion of the reaction of compound Int D and the appearance of a new spot, a TLC plate (petroleum ether:ethyl acetate = 2:1) was used to monitor the reaction. The reaction solution was poured into water (50.0 mL), adjusted to pH 2 with 1 M hydrochloric acid, and extracted with ethyl acetate (30.0 mL x 3). All organic phases were combined, washed with brine (30.0 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford compound 4 (1.70 g, 3.14 mmol, 60.3% yield) as a pale yellow oil.

[0088] Proton spectrum: EC6536-773-P1A, 400 MHz, DMSO-d6

[0089] δ:12.01(s,1H),8.05-7.87(m,2H),7.69-7.49(m,4H),7.43(br d,J=8.8Hz,1H),7.21-7.08(m,1H),6.72-6.40(m,2H),5.42-5.26(m,2H),4.90(br s,1H),4.44-4.26(m,2H),3.62-3.47(m,3H),3.03-2.82(m,2H),2.77-2.56(m,2H),1.96-1.87(m,2H),1.72(br s,2H),1.60(br s,2H),1.37-1.29(m,3H).

[0090] 7) Synthesis of Compound T3

[0091] A solution of compound 4 (1.70 g, 3.14 mmol, 1.00 eq) in methanol (20.0 mL) was cooled to 0°C, followed by the addition of a solution of sodium hydroxide (627 mg, 15.6 mmol, 5.00 eq) in water (5.00 mL). The mixture was stirred at 25°C for 12 hours. LCMS (EC6536-776-P1A3) confirmed the complete reaction of compound 4 and the appearance of a single main peak at the target molecular weight (Rt = 0.51 min, MS cal.: 504.18, MS observed: [M+H]+ = 505.3). The reaction solution was then poured into water (50.0 mL), adjusted to pH 5 with 1 M hydrochloric acid, and extracted with ethyl acetate (30.0 mL x 3). All organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to yield a residue, which was purified by column chromatography (silica gel, dichloromethane:methanol = 1:0 to 10:1) with TLC monitoring (petroleum ether:ethyl acetate = 1:2, Rf = 0.65) to afford the crude product. The crude product was triturated with dichloromethane (20.0 mL) at 25°C for 30 minutes, filtered, and the filter cake concentrated under reduced pressure to afford Target 3 as a white solid (510 mg, 1.01 mmol, 32.1% yield, 100% purity).

[0092] LCMS: EC6536-776-P1A3, Rt=0.51min, MS cal.: 504.18, MS observed: [M+H]+=505.3.

[0093] HRMS:EC20403-1.

[0094] HPLC: EC20403-1-P1A2, Rt=3.52min, 100% purity.

[0095] Proton spectrum: EC20403-1-P1A7, 400 MHz, DMSO-d6

[0096] δ:13.79-12.12(m,2H),12.04(br s,1H),7.98(d,J=8.4Hz,2H),7.60(br d,J=8.0Hz,2H),7.57-7.50(m,2H),7.45(br d,J=8.8Hz,1H),7.17(br d,J=9.2Hz,1H),6.54-6.44(m,2H),5.35(s,2H),4.91(br t,J=5.2Hz,1H),2.92(br t,J=7.2Hz,2H),2.57(br t,J=7.2Hz,2H),2.01-1.88(m,2H),1.79-1.65(m,4H),1.59(br d, J = 2.4 Hz, 2H)

[0097] 1.2 Synthesis of compound Target 5 (abbreviated as T5)

[0098] 1) Synthesis of compound 2

[0099] To a solution of NH2OH·HCl (31.3 g, 451 mmol, 3.00 eq) in methanol, under nitrogen and in an ice bath, slowly add a solution of NaOMe (135 g, 752 mmol, 30.0% purity, 5.00 eq) in methanol dropwise over at least 10 minutes. The reaction mixture was allowed to warm to room temperature and continue to react for 10 minutes. The reaction mixture was then kept in the ice bath and 80 mL of a solution of compound 1 (25.0 g, 150 mmol, 1.00 eq) in methanol was slowly added dropwise. The reaction mixture was allowed to warm to room temperature for 1 hour, then heated to 70°C and continued to react for 4 hours. LCMS analysis (EC6536-775-P1A1) showed that the reaction of compound 1 was complete, with a yield of 91.7% (Rt = 0.19 min, MS calcd: 167.0, MS assay: [M+H]+ = 168.0). The reaction mixture was poured into 300 mL of ice water and the pH was adjusted to 5 with 1 M HCl solution. The mixture was filtered, and the filter cake was collected and washed sequentially with 500 mL of water, diisopropyl ether, and n-hexane. The filter cake was concentrated under reduced pressure to obtain compound 2 as a white solid (21.7 g, 129 mmol, 85.7% yield, 99.4% purity). The product was characterized by LCMS (EC6536-775-P1A2) and 1H NMR (EC6536-775-P1A1).

[0100] LCMS 1:EC6536-775-P1A1,R t =0.19min,MS cal.:167.0,MS observed:[M+H] + =168.0.

[0101] LCMS:EC6536-775-P1A2,R t =0.20min,MS cal.:167.0,MS observed:[M+H] + =168.2.

[0102] 1 H NMR:EC6536-775-P1A1,400MHz,DMSO-d6

[0103] δ:14.03-10.20(m,2H),10.09-8.59(m,1H),7.56(br d,J=8.0Hz,1H),6.94-6.41(m,2H),2.25(s,3H)

[0104] 2) Synthesis of compound 3

[0105] Compound 2 (21.7 g, 129 mmol, 1.00 eq) was dissolved in 400 mL of THF. TEA (78.3 g, 774 mmol, 107 mL, 6.00 eq) and SOCl2 (23.0 g, 193 mmol, 14.0 mL, 1.50 eq) were slowly added dropwise in an ice bath under nitrogen. The reaction mixture was allowed to react at room temperature for 30 minutes. LCMS 1 (EC20395-3-P1A2) showed that compound 2 was completely reacted, with a yield of 93.2% for compound 3 (Rt = 0.29 min, MS calcd: 149.0, MS assay: [M+H]+ = 150.0). The reaction system was then poured into 200 mL of ice water and the pH was adjusted to 1 with 1 M aqueous HCl. Compound 3 (14.6 g, 97.6 mmol, 75.6% yield, 99.7% purity) precipitated as a yellow solid. The structure was confirmed by LCMS (EC20395-3-P1B1) and 1H NMR (EC20395-3-P1A1).

[0106] LCMS 1:EC20395-3-P1A2,R t =0.29min,MS cal.:149.0,MS observed:[M+H] + =150.0.

[0107] LCMS:EC20395-3-P1B1,R t =0.29min,MS cal.:149.0,MS observed:[M+H] + =150.2.

[0108] 1 H NMR:EC20395-3-P1A1,400MHz,DMSO-d6

[0109] δ:7.60(d,J=8.0Hz,1H),7.34(s,1H),7.13(d,J=8.0Hz,1H),2.43(s,3H).

[0110] 3) Synthesis of Compounds 4 and 4A

[0111] Compound 3 (9.60 g, 64.1 mmol, 1.00 eq) was dissolved in 100 mL of DCM. Place in an ice bath under nitrogen, and DIEA (12.4 g, 96.2 mmol, 16.7 mL, 1.50 eq) and CHOCHCl (18.4 g, 228 mmol, 17.3 mL, 3.56 eq) were slowly added dropwise. The reaction was stirred at room temperature for 30 minutes. LCMS (EC6536-790-P1A2) analysis showed that compound 3 was completely reacted, with a yield of 41.5% (Rt = 0.67 min, MS calculated: 193.0, MS detected: [M+H]+ = 194.1). 200 mL of ice water was added to the reaction system, and the organic phase was washed with 200 mL of water and 200 mL of saturated brine, then dried over anhydrous sodium sulfate. Filtration and evaporation to dryness gave a solid, which was purified by reverse-phase preparative liquid chromatography (neutral conditions) to afford compound 4 (4.14 g, 20.0 mmol, 31.2% yield, 93.7% purity) as a yellow oil. 1H NMR 1 (EC6536-790-P1A1) and LCMS 1 (EC6536-790-P1D1) were used to obtain compound 4A (4.25 g, 21.6 mmol, 33.7% yield, 98.3% purity) as a yellow oil. The structure was confirmed by LCMS 2 (EC6536-790-P2A1) and 1H NMR 2 (EC6536-790-P1A2).

[0112] LCMS:EC6536-790-P1A2,R t =0.67min,MS cal.:193.0,MS observed:[M+H] + =194.1.

[0113] LCMS 1:EC6536-790-P1D1,R t =0.66min,MS cal.:193.0,MS observed:[M+H] + =194.0.

[0114] LCMS 2:EC6536-790-P2A1,R t =0.56min,MS cal.:193.0,MS observed:[M+H] + =194.0.

[0115] 1 H NMR 1:EC6536-790-P1A1,400MHz,CDCl3

[0116] δ:7.54(d,J=8.0Hz,1H),7.25(s,1H),7.11(d,J=8.0Hz,1H),5.55(s,2H),3.64(s,3H),2.51(s,3H)

[0117] 1 H NMR 2:EC6536-790-P1A2,400MHz,CDCl3

[0118] δ:7.72(br d,J=8.0Hz,1H),7.10(br d,J=7.8Hz,1H),7.04(br s,1H),5.31(s,2H),3.44(s,3H),2.48(s,3H)

[0119] 4) Synthesis of Compound 5

[0120] Compound 4 (2.07 g, 10.0 mmol, 1.00 eq) was dissolved in 30 mL of acetonitrile, and AIBN (164 mg, 1.00 mmol, 0.10 eq) and DBDMH (3.73 g, 13.0 mmol, 1.30 eq) were added. The reaction mixture was allowed to react at 80°C for 8 hours. LCMS (EC20395-14-P2C4) confirmed the complete reaction of compound 4, with a yield of 60.5% (Rt = 0.69 min, MS calculated: 270.9, MS detected: [M+H]+ = 271.9). The reaction mixture was treated with 50 mL of ice water, separated, and the organic phase was washed sequentially with aqueous Na2S2O3 (50.0 mL), aqueous NaHCO3 (50.0 mL), and saturated brine (50.0 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure to afford compound 5 (5.27 g, crude) as a yellow oil.

[0121] LCMS: EC20395-14-P2C4, Rt = 0.69 min, MS calculated value: 270.9, MS detected value: [M+H] + = 271.9.

[0122] LCMS:EC20395-14-P2C4,Rt =0.69min,MS cal.:270.9,MS observed:[M+H] + =271.9.

[0123] 5) Synthesis of Compound Int E

[0124] Int A_3 (6.00 g, 17.0 mmol, 1.00 eq) was dissolved in 50 mL of ethanol. NaOEt (0.29 M, 146 mL, 2.50 eq) was added at room temperature. The reaction mixture was allowed to react at 50°C for 2 hours. LCMS (EC6536-771-P1A1) showed that Int A_3 was completely reacted with a yield of 91.8% (Rt = 0.52 min, MS calculated: 398.1, MS measured: [M+H]+ = 399.2). The reaction mixture was poured into 200 mL of pH 5 hydrochloric acid and extracted with ethyl acetate (200 mL x 3). The organic phases were combined, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound Int E (6.78 g, 16.6 mmol, yield 97.8%, purity 97.9%) as a yellow oil, which was verified by LCMS 1 (EC6536-771-P1A3).

[0125] LCMS: EC6536-771-P1A1, Rt=0.52 min, MS calculated value: 398.1, MS detected value: [M+H]+=399.2.

[0126] LCMS 1: EC6536-771-P1A3, Rt = 0.53 min, MS calculated value: 398.1, MS detected value: [M+H] + = 399.2.

[0127] LCMS:EC6536-771-P1A1,R t =0.52min,MS cal.:398.1,MS observed:[M+H] + =399.2.

[0128] LCMS 1:EC6536-771-P1A3,R t =0.53min,MS cal.:398.1,MS observed:[M+H] + =399.2.

[0129] 6) Synthesis of Compound 6

[0130] Compound 5 (5.27 g, 19.3 mmol, 1.43 eq) was dissolved in 50 mL of DMF and stirred at room temperature. Compound Int E (5.50 g, 13.5 mmol, 1.00 eq) and KCO (3.74 g, 27.0 mmol, 2.00 eq) were added. The reaction mixture was heated to 50°C and stirred for 2 hours. LCMS (EC20395-15-P1A3) showed that compound 5 was completely reacted with a yield of 36.9% (Rt = 0.93 min, MS calculated: 589.2, MS detected: [M+H]+ = 590.4). The reaction system was treated with 100 mL of ethyl acetate and 100 mL of water. After separation, the organic phase was washed with 100 mL of water and 100 mL of saturated brine and dried over anhydrous sodium sulfate. Filtration and evaporation under reduced pressure gave the crude product, which was then purified by reverse-phase HPLC to yield compound 6 as a yellow oil (3.17 g, 5.06 mmol, 37.4% yield, 94.2% purity). The product was analyzed by LCMS (EC20395-15-P1D16) and HPLC (EC20395-15-P1A23).

[0131] LCMS:EC20395-15-P1A3,R t =0.93min,MS cal.:589.2,MS observed:[M+H] + =590.4.

[0132] LCMS 1:EC20395-15-P1D16,R t =2.10min,MS cal.:589.2,MS observed:[M+H] + =590.1.

[0133] HPLC:EC20395-15-P1A23,R t =4.01min,purity:94.2%

[0134] 7) Synthesis of Compound T5

[0135] Compound 6 (2.00 g, 3.20 mmol, 1.00 eq) was dissolved in a mixture of 10.0 mL of dioxane and 10.0 mL of ethanol. 6 M HCl (14.5 mL, 27.3 eq) was added at room temperature, and the reaction mixture was stirred at room temperature for 3 hours. LCMS (EC20395-17-P1A4) analysis showed that compound 6 had reacted completely, with a yield of 92.1% (Rt = 0.66 min, MS calculated: 545.2, MS detected: [M+H]+ = 546.4). The reaction mixture was treated with 100 mL of ethyl acetate and 100 mL of water. The phases were separated, and the organic phase was washed sequentially with 100 mL of water and 100 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by reverse-phase preparative liquid chromatography to yield the target compound T5 (750 mg, 1.37 mmol, 67.9% yield, 99.4% purity) as a white solid. The structure was confirmed by LCMS (EC20395-18-P1A1), HPLC (EC20395-18-P1A2), HRMS (EC20395-18-P1A1), 1H NMR (EC20395-18-P1A3) and 13C NMR (EC20395-18-P1A2).

[0136] LCMS:EC20395-17-P1A4,R t =0.66min,MS cal.:545.2,MS observed:[M+H] + =546.4.

[0137] LCMS 1:EC20395-18-P1A1,R t =0.67min,MS cal.:545.2,MS observed:[M+H] + =546.4.

[0138] HRMS:EC20395-18-P1A1

[0139] HPLC:EC20395-18-P1A2,R t =3.82min,purity:99.4%

[0140] 1 H NMR:EC20395-18-P1A3,400MHz,CDCl3

[0141] 13 C NMR:EC20395-18-P1A2

[0142] δ:12.69(br s,1H),7.83(d,J=8.0Hz,1H),7.58(d,J=2.2Hz,1H),7.57-7.53(m,2H),7.51(d,J=9.0Hz, 1H),7.39(d,J=8.4Hz,1H),6.96(d,J=8.4Hz,1H),6.49(d,J=2.4Hz,1H),6.38(dd,J=2.4,8 .9Hz,1H),5.35(s,2H),4.83(tt,J=2.8,5.8Hz,1H),4.15(q,J=7.2Hz,2H),3.11(t,J=7.8 Hz,2H),2.70(t,J=7.6Hz,2H),2.00-1.76(m,6H),1.71-1.60(m,2H),1.24(t,J=7.2Hz,3H)

[0143] 2. Inhibition of DNA Binding Activity Test

[0144] Test Method

[0145] c-jun transcription factor DNA binding activity test

[0146] DNA binding to transcription factors was tested using TransAM kits (Active Motif 46096). The transcription factor used was c-Jun / AP-1. Drugs at final concentrations of 500 μM, 250 μM, 125 μM, 62.5 μM, and 31.25 μM, along with 1 μL of cell extract containing the transcription factor, were sequentially added to a 96-well plate pre-coated with double-stranded DNA oligomers. The plates were incubated for 1 hour and then incubated with an antibody against the c-Jun transcription factor. The absorbance at 450 nm was measured, with the absorbance at 655 nm used as background. This assay measures the binding activity of the dsDNA sequence to the transcription factor. Lower binding activity indicates stronger inhibitory activity of the compound.

[0147] 2.2 Test Results

[0148] The results are shown in the following table and Figure 4.

[0149] DNA & JUN binding activity

[0150] The above results indicate that the compounds of the present invention have the activity of inhibiting the binding of JUN to DNA, which is better or equivalent to the positive control T-5224. Therefore, the compounds of the present invention have the activity of inhibiting the binding of JUN to DNA.

[0151] 3. Transcriptional Activity Inhibition Test

[0152] 3.1 Test Method

[0153] Dual luciferase reporter system to detect inhibition of DNA binding activity

[0154] 293T cells were plated at passage 1 and 3, incubated for 24 hours, and then replaced with antibiotic-free medium (Opti-MEM, Gibco, 11058021). 293T cells were transiently transfected with the luciferase reporter plasmid pGL4.44[luc2P / AP1 RE / Hygro] (Promega E411) and cultured for 24 hours. Cells were incubated for 1 hour in 1% PS / 10% FBS / DMEM (Gibco, 11965092) containing drugs at final concentrations of 50 μM, 25 μM, 12.5 μM, and 6.25 μM. They were then stimulated with PMA (final concentration 10 ng / ml) (Sigma, 79346-5MG) and cultured for 34 hours. Lysates were assayed using the Dual-Luciferase Reporter Gene Assay System (Promega). Drugs and PMA were stored in DMSO and diluted in culture medium before addition to the culture medium.

[0155] 3.2 Test Results

[0156] The results are shown in the following table and Figure 5.

[0157] The above results indicate that T3 and T5 further have the activity of inhibiting JUN transcription. Therefore, the compounds of the present invention have the activity of inhibiting JUN transcription.

[0158] 4. Animal Model Efficacy Testing

[0159] Materials and reagents, animal experiment guidelines, and induction of the heart failure model with preserved ejection fraction were the same as those in Example 1.

[0160] The dosage is as follows:

[0161] After successful model induction, mice in the treatment group were treated with compounds T3, T5, and T-5524, while the control group was treated with a drug-free solvent. Mice receiving normal diet and water throughout the induction period served as negative controls. When the mice were 5-8 weeks old, ultrasound confirmed successful model establishment and drug administration began. The treatment groups received a single dose of 12 mg / kg of T3, T5, and T-5524, based on body weight, every other day. Each dose consisted of 0.54 mg of compound dissolved in 100 μL of 0.5% PVP solution (the specific dosage was tailored to the number of mice; for example, for 14 mice, 8 mg of drug was dissolved in 1.5 mL of solution, with 100 μL administered to each mouse). Dosing began after successful model induction and continued for 7 weeks. The control group received an equal volume of 0.5% PVP solution. All other treatments were identical.

[0162] The experimental results are shown in Figure 6 (wherein week 0 on the abscissa represents the day dosing began after successful modeling, i.e., the dosing starting point). As can be clearly seen in Figure 6 , after 3 weeks of dosing, the positive control, T-5524, had not yet shown an inhibitory effect on disease progression. However, the compounds T3 and T5 of the present invention had already significantly inhibited disease progression after 3 weeks of dosing, demonstrating a clear advantage. This indicates that compared to the positive control, T-5524, the compounds T3 and T5 of the present invention can inhibit disease progression earlier, demonstrating a clear advantage. It can be seen that the compounds of the present invention have a significant therapeutic effect on HFpEF, and the effect is rapid.

[0163] In summary, the inventors of the present application have discovered that the compounds of the present invention have the activity of inhibiting the binding of JUN to DNA and the activity of inhibiting JUN transcription, and can produce a therapeutic effect on HFpEF. Compared with the positive control T-5524, they have obvious advantages in the treatment of HFpEF and have broad application prospects.

Claims

1. Use of a compound or its stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, pharmaceutically acceptable ester or pharmaceutically acceptable solvate in the preparation of a medicament for treating and / or preventing heart failure with preserved ejection fraction, wherein the compound is selected from T3, T5, 2. Use of a pharmaceutical composition in the preparation of a medicament for the treatment and / or prevention of heart failure with preserved ejection fraction, wherein, The pharmaceutical composition comprises a compound or its stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, pharmaceutically acceptable ester or pharmaceutically acceptable solvate, and the compound is selected from T3 and T5.

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