Use of small molecule compound in treatment of cardiovascular diseases

Abstract

The present invention relates to the use of a small molecule compound in the treatment of cardiovascular diseases. Specifically provided is the use of a compound as shown in formula I or a stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, pharmaceutically acceptable ester or pharmaceutically acceptable solvate thereof in the preparation of a drug for treating and / or preventing heart failure with preserved ejection fraction.

Description

Application of small molecule compounds in the treatment of cardiovascular diseases Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the application of small molecule compounds in the treatment of cardiovascular diseases. 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 represented by Formula I or its stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, pharmaceutically acceptable esters or pharmaceutically acceptable solvates in the preparation of a medicament for treating and / or preventing heart failure with preserved ejection fraction.

[0007] Alternatively, a compound of Formula I or a stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, pharmaceutically acceptable ester or pharmaceutically acceptable solvate thereof is provided for use in treating and / or preventing heart failure with preserved ejection fraction.

[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 of Formula I or its stereoisomer, prodrug, crystalline form, pharmaceutically acceptable salt, pharmaceutically acceptable ester or pharmaceutically acceptable solvate,

[0009] in:

[0010] R is selected from C6-C10 aryl, 6-10 membered heteroaryl, said C6-C10 aryl, 6-10 membered heteroaryl are each independently optionally replaced by R 1 and / or R 5 replaced by;

[0011] Preferably, R is selected from C6-C10 aryl, 6-10 membered heteroaryl, and the C6-C10 aryl and 6-10 membered heteroaryl are each independently optionally replaced by R 1 or R 1 and R 5 replaced by;

[0012] More preferably, R is selected from C6-C10 aryl, 6-10 membered heteroaryl, and the C6-C10 aryl and 6-10 membered heteroaryl are each independently optionally replaced by R 1 replaced by;

[0013] R 1 Selected from hydrogen, C1-C6 alkyl, -COOH, -C(=O)O-C1-C6 alkyl, -(CH2) m -O-C1-C6 alkyl, -NR c R d ;

[0014] R c 、R d Each independently selected from hydrogen, C1-C6 alkyl, -(CH2) m -O-C1-C6 alkyl;

[0015] m is selected from 0, 1, 2, 3, 4, 5, 6;

[0016] Preferably, m is selected from 0, 1;

[0017] More preferably, m is 1;

[0018] R 2 Selected from -O-(CH2) n -R 2’ ;

[0019] n is selected from 0, 1, 2, 3, 4, 5, 6;

[0020] Preferably, n is selected from 0, 1;

[0021] R 2’ Selected from C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, 5-6 membered heteroaryl;

[0022] R 3 is selected from hydroxy, -O-C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted by a 5-6 membered heterocyclic group;

[0023] Preferably, R 3 is selected from hydroxyl, -O-C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted by a 6-membered heterocyclic group;

[0024] More preferably, R 3 is selected from hydroxy, -O-C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with morpholinyl;

[0025] Most preferably, R 3 Selected from hydroxyl, -O-C1-C6 alkyl;

[0026] R 4 Selected from hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, -NR a R b 、-CONR a R b, C6-C10 aryl, -O-C6-C10 aryl, -C(=O)-C1-C6 alkyl, -CH2-C6-C10 aryl, C1-C6 alkoxy, C2-C6 alkenyl, C3-C8 cycloalkyl, -C(=O)O-C1-C6 alkyl, -C(=O)O-C6-C10 aryl, C1-C6 alkylthio, -S(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, 5-6 membered heteroaryl;

[0027] R a 、R b Each is independently selected from hydrogen, C1-C6 alkyl, -C(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, -S(=O)2-phenyl, wherein the phenyl group is optionally substituted with C1-C6 alkyl;

[0028] R 5 Selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio;

[0029] The compound shown in formula I does not contain

[0030] In some embodiments, R is selected from phenyl, benzimidazolyl, The phenyl group, benzimidazolyl group, Each independently optionally replaced by R 1 and / or R 5 replaced by;

[0031] Preferably, R is selected from phenyl, benzimidazolyl, The phenyl group, benzimidazolyl group, Each independently optionally replaced by R 1 or R 1 and R 5 replaced by;

[0032] More preferably, R is selected from phenyl, benzimidazolyl, The phenyl group, benzimidazolyl group, Each independently optionally replaced by R 1 replaced by;

[0033] Or preferably, R is selected from phenyl, The phenyl group, Each independently optionally replaced by R 1 and / or R 5 replaced by;

[0034] Or more preferably, R is selected from phenyl, The phenyl group, Each independently optionally replaced by R 1 or R 1 and R 5 replaced by;

[0035] Or further preferably, R is selected from phenyl, The phenyl group, Each independently optionally replaced by R 1 replaced by;

[0036] Or preferably, R is selected from

[0037] Or more preferably, R is selected from

[0038] Preferably, R 1 Selected from hydrogen, -COOH, -C(=O)O-C1-C6 alkyl, -(CH2) m -O-C1-C6 alkyl, -NR c R d ;

[0039] More preferably, R 1 Selected from hydrogen, -COOH, -C(=O)O-C1-C6 alkyl, -(CH2) m -O-C1-C6 alkyl;

[0040] Preferably, R c 、R d are each independently selected from hydrogen, -(CH2) m -O-C1-C6 alkyl;

[0041] More preferably, R c 、R d Any one of them is hydrogen, and the other is selected from -(CH2) m -O-C1-C6 alkyl;

[0042] More preferably, R 1 Selected from hydrogen, -COOH, -COOCH3, -COOCH2CH3, -COO(CH2)2CH3, -COO(CH2)3CH3, -COO(CH2)4CH3, -COO(CH2)5CH3,

[0043] More preferably, R 1 Selected from hydrogen, -COOH, -COOCH3, -COOCH2CH3,

[0044] Most preferably, R 1 Selected from hydrogen, -COOH, -COOCH2CH3,

[0045] Preferably, R 5 Selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy;

[0046] More preferably, R 5 Selected from C1-C6 alkoxy;

[0047] Most preferably, R 5 It is a methoxy group.

[0048] In some embodiments, R 2’ Selected from C1-C6 alkyl, C3-C8 cycloalkyl, phenyl, pyrazinyl (such as ), furanyl (such as );

[0049] Preferably, R 2’ Selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, Phenyl, pyrazinyl (such as ), furanyl (such as );

[0050] Or, R 2’ Selected from C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl;

[0051] Or preferably, R 2’ Selected from C1-C6 alkyl, C3-C8 cycloalkyl, phenyl;

[0052] Or more preferably, R 2’ Selected from cyclopentyl, cyclohexyl, phenyl;

[0053] Or more preferably, R 2’ Selected from cyclopentyl, phenyl;

[0054] Preferably, R 2 Selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-C6-C10 aryl, -O-CH2-5-6 membered heteroaryl;

[0055] More preferably, R 2 Selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-phenyl, -O-CH2-5-6 membered heteroaryl, wherein the 5-6 membered heteroaryl is selected from pyrazinyl (such as ), furanyl (such as );

[0056] Most preferably, R2 Selected from

[0057] Or preferably, R 2 Selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-C6-C10 aryl;

[0058] Or more preferably, R 2 Selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-phenyl;

[0059] Or more preferably, R 2 Selected from

[0060] Or most preferably, R 2 Selected from

[0061] In some embodiments, R 3 Selected from hydroxyl, -OCH3, -OCH2CH3, -OCH(CH3)CH3, -O(CH2)2CH3, -O(CH2)3CH3, -O(CH2)4CH3, -O(CH2)5CH3, Preferably, R 3 Selected from hydroxyl, -OCH3, -OCH2CH3, -OCH(CH3)CH3, -O(CH2)2CH3,

[0062] Or preferably, R 3 Selected from hydroxy, -OCH3, -OCH2CH3, -OCH(CH3)CH3, -O(CH2)2CH3, -O(CH2)3CH3, -O(CH2)4CH3, -O(CH2)5CH3;

[0063] Or more preferably, R 3 Selected from hydroxy, -OCH3, -OCH2CH3, -O(CH2)2CH3, -O(CH2)3CH3, -O(CH2)4CH3, -O(CH2)5CH3;

[0064] Or more preferably, R 3 Selected from hydroxy, -OCH3, -OCH2CH3, -OCH(CH3)CH3, -O(CH2)2CH3;

[0065] Or most preferably, R 3 Selected from hydroxyl, -OCH3, -OCH2CH3.

[0066] In some embodiments, R4 Selected from hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, -NR a R b 、-CONR a R b , phenyl, phenoxy, -C(=O)-C1-C6 alkyl, benzyl, C1-C6 alkoxy, C2-C6 alkenyl, C3-C8 cycloalkyl, -C(=O)O-C1-C6 alkyl, -C(=O)O-phenyl, C1-C6 alkylthio, -S(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, thienyl (such as 2-thienyl);

[0067] Preferably, R a 、R b Each independently selected from hydrogen, methyl,

[0068] Or preferably, R a 、R b wherein any one is selected from hydrogen, C1-C6 alkyl, and the other is selected from hydrogen, C1-C6 alkyl, -C(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, -S(=O)2-phenyl, and the phenyl group is optionally substituted with C1-C6 alkyl;

[0069] Or more preferably, R a 、R b Any one of them is selected from hydrogen, methyl, and the other is selected from hydrogen, methyl,

[0070] Preferably, R 4 Selected from hydroxy, cyano, nitro, carboxyl, -NH2, -SH, -CONH2, methyl, phenyl, benzyl, methoxy, phenoxy, acetyl, -F, -Cl, -Br, -CH=CH2, cyclopentyl, -COOMe, -COOPh, -SMe, -SOMe, -SO2Me, -NMe2, -NHAc, -NHMs, -NHTs, 2-thienyl;

[0071] More preferably, R 4 It is a hydroxyl group.

[0072] In some embodiments, the structural formula of the compound is as shown in Formula I-1 or Formula I-1'. Preferably, the structural formula of the compound is as shown in Formula I-1,

[0073] in:

[0074] R 1 Selected from -COOH, -C(=O)O-C1-C6 alkyl;

[0075] Preferably, R 1 Selected from -COOH, -COOCH3, -COOCH2CH3, -COO(CH2)2CH3, -COO(CH2)3CH3, -COO(CH2)4CH3, -COO(CH2)5CH3;

[0076] More preferably, R 1 Selected from -COOH, -COOCH3, -COOCH2CH3;

[0077] Most preferably, R 1 Selected from -COOH, -COOCH2CH3;

[0078] R 2 Selected from -O-(CH2) n -R 2’

[0079] n is selected from 0, 1, 2, 3, 4, 5, 6;

[0080] Preferably, n is selected from 0, 1;

[0081] R 2’ Selected from C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, 5-6 membered heteroaryl;

[0082] Preferably, R 2’ Selected from C1-C6 alkyl, C3-C8 cycloalkyl, phenyl, pyrazinyl (such as ), furanyl (such as );

[0083] More preferably, R 2’ Selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, Phenyl, pyrazinyl (such as ), furanyl (such as );

[0084] Or preferably, R 2’ Selected from C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl;

[0085] Or more preferably, R 2’ Selected from C1-C6 alkyl, C3-C8 cycloalkyl, phenyl;

[0086] Or most preferably, R 2’ Selected from cyclopentyl, phenyl;

[0087] Preferably, R 2Selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-C6-C10 aryl, -O-CH2-5-6 membered heteroaryl;

[0088] More preferably, R 2 Selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-phenyl, -O-CH2-5-6 membered heteroaryl, wherein the 5-6 membered heteroaryl is selected from pyrazinyl (such as ), furanyl (such as );

[0089] Most preferably, R 2 Selected from

[0090] Or preferably, R 2 Selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-C6-C10 aryl;

[0091] Or more preferably, R 2 Selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-phenyl;

[0092] Or most preferably, R 2 Selected from

[0093] R 3 Selected from hydroxyl, -O-C1-C6 alkyl;

[0094] Preferably, R 3 Selected from hydroxy, -OCH3, -OCH2CH3, -O(CH2)2CH3;

[0095] More preferably, R 3 is hydroxyl group;

[0096] R 4 Selected from hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, -NR a R b 、-CONR a R b , C6-C10 aryl, -O-C6-C10 aryl, -C(=O)-C1-C6 alkyl, -CH2-C6-C10 aryl, C1-C6 alkoxy, C2-C6 alkenyl, C3-C8 cycloalkyl, -C(=O)O-C1-C6 alkyl, -C(=O)O-C6-C10 aryl, C1-C6 alkylthio, -S(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, 5-6 membered heteroaryl;

[0097] Preferably, R 4 Selected from hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, -NR a R b 、-CONR a R b , phenyl, phenoxy, -C(=O)-C1-C6 alkyl, benzyl, C1-C6 alkoxy, C2-C6 alkenyl, C3-C8 cycloalkyl, -C(=O)O-C1-C6 alkyl, -C(=O)O-phenyl, C1-C6 alkylthio, -S(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, thienyl (such as 2-thienyl);

[0098] R a 、R b Each is independently selected from hydrogen, C1-C6 alkyl, -C(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, -S(=O)2-phenyl, wherein the phenyl group is optionally substituted with C1-C6 alkyl;

[0099] Preferably, R a 、R b Each independently selected from hydrogen, methyl,

[0100] Or preferably, R a 、R b wherein any one is selected from hydrogen, C1-C6 alkyl, and the other is selected from hydrogen, C1-C6 alkyl, -C(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, -S(=O)2-phenyl, and the phenyl group is optionally substituted with C1-C6 alkyl;

[0101] Or more preferably, R a 、R b Any one of them is selected from hydrogen, methyl, and the other is selected from hydrogen, methyl,

[0102] More preferably, R 4 Selected from hydroxy, cyano, nitro, carboxyl, -NH2, -SH, -CONH2, methyl, phenyl, benzyl, methoxy, phenoxy, acetyl, -F, -Cl, -Br, -CH=CH2, cyclopentyl, -COOMe, -COOPh, -SMe, -SOMe, -SO2Me, -NMe2, -NHAc, -NHMs, -NHTs, 2-thienyl;

[0103] Most preferably, R 4 is hydroxyl group;

[0104] R 5Selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio;

[0105] Preferably, R 5 Selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy;

[0106] More preferably, R 5 Selected from C1-C6 alkoxy;

[0107] Most preferably, R 5 It is a methoxy group.

[0108] In some embodiments, the compound has a structural formula as shown in Formula I-2,

[0109] in:

[0110] R 1 Selected from hydrogen, -(CH2) m -O-C1-C6 alkyl, -NR c R d ;

[0111] Preferably, R 1 Selected from hydrogen, -(CH2) m -O-C1-C6 alkyl;

[0112] R c 、R d Each independently selected from hydrogen, C1-C6 alkyl, -(CH2) m -O-C1-C6 alkyl;

[0113] Preferably, R c 、R d are each independently selected from hydrogen, -(CH2) m -O-C1-C6 alkyl;

[0114] More preferably, R c 、R d Any one of them is hydrogen, and the other is selected from -(CH2) m -O-C1-C6 alkyl;

[0115] m is selected from 0, 1, 2, 3, 4, 5, 6;

[0116] Preferably, m is selected from 0, 1;

[0117] More preferably, m is 1;

[0118] More preferably, R 1 Selected from hydrogen,

[0119] R 2Selected from -O-(CH2) n -R 2’ ;

[0120] n is selected from 0, 1, 2, 3, 4, 5, 6;

[0121] Preferably, n is selected from 0, 1;

[0122] R 2’ Selected from C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, 5-6 membered heteroaryl;

[0123] Preferably, R 2’ Selected from C1-C6 alkyl, C3-C8 cycloalkyl, phenyl, pyrazinyl (such as ), furanyl (such as );

[0124] More preferably, R 2’ Selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, Phenyl, pyrazinyl (such as ), furanyl (such as );

[0125] Or preferably, R 2’ Selected from C3-C8 cycloalkyl;

[0126] Or more preferably, R 2’ Selected from cyclopentyl and cyclohexyl;

[0127] Or most preferably, R 2’ selected from cyclopentyl;

[0128] Preferably, R 2 Selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-C6-C10 aryl, -O-CH2-5-6 membered heteroaryl;

[0129] More preferably, R 2 Selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-phenyl, -O-CH2-5-6 membered heteroaryl, wherein the 5-6 membered heteroaryl is selected from pyrazinyl (such as ), furanyl (such as );

[0130] Most preferably, R 2 Selected from

[0131] Or preferably, R 2 Selected from -O-C3-C8 cycloalkyl;

[0132] Or more preferably, R 2 Selected from

[0133] Or most preferably, R 2 Selected from

[0134] R 3 is selected from hydroxy, -O-C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted by a 5-6 membered heterocyclic group;

[0135] Preferably, R 3 is selected from hydroxyl, -O-C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted by a 6-membered heterocyclic group;

[0136] More preferably, R 3 is selected from hydroxy, -O-C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted by morpholinyl;

[0137] More preferably, R 3 Selected from hydroxyl, -OCH3, -OCH2CH3, -OCH(CH3)CH3, -O(CH2)2CH3, -O(CH2)3CH3, -O(CH2)4CH3, -O(CH2)5CH3,

[0138] More preferably, R 3 Selected from -OCH3, -OCH2CH3, -OCH(CH3)CH3, -O(CH2)2CH3,

[0139] Or preferably, R 3 Selected from hydroxyl, -O-C1-C6 alkyl;

[0140] Or more preferably, R 3 Selected from hydroxy, -OCH3, -OCH2CH3, -OCH(CH3)CH3, -O(CH2)2CH3, -O(CH2)3CH3, -O(CH2)4CH3, -O(CH2)5CH3;

[0141] Or more preferably, R 3 Selected from hydroxy, -OCH3, -OCH2CH3, -O(CH2)2CH3, -O(CH2)3CH3, -O(CH2)4CH3, -O(CH2)5CH3;

[0142] Or preferably, R 3 Selected from -O-C1-C6 alkyl;

[0143] Or more preferably, R3 Selected from -OCH3, -OCH2CH3, -OCH(CH3)CH3, -O(CH2)2CH3;

[0144] Or most preferably, R 3 Selected from -OCH3, -OCH2CH3;

[0145] R 4 Selected from hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, -NR a R b 、-CONR a R b , C6-C10 aryl, -O-C6-C10 aryl, -C(=O)-C1-C6 alkyl, -CH2-C6-C10 aryl, C1-C6 alkoxy, C2-C6 alkenyl, C3-C8 cycloalkyl, -C(=O)O-C1-C6 alkyl, -C(=O)O-C6-C10 aryl, C1-C6 alkylthio, -S(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, 5-6 membered heteroaryl;

[0146] Preferably, R 4 Selected from hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, -NR a R b 、-CONR a R b , phenyl, phenoxy, -C(=O)-C1-C6 alkyl, benzyl, C1-C6 alkoxy, C2-C6 alkenyl, C3-C8 cycloalkyl, -C(=O)O-C1-C6 alkyl, -C(=O)O-phenyl, C1-C6 alkylthio, -S(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, thienyl (such as 2-thienyl);

[0147] R a 、R b Each is independently selected from hydrogen, C1-C6 alkyl, -C(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, -S(=O)2-phenyl, wherein the phenyl group is optionally substituted with C1-C6 alkyl;

[0148] Preferably, R a 、R b Each independently selected from hydrogen, methyl,

[0149] Or preferably, R a 、R bwherein any one is selected from hydrogen, C1-C6 alkyl, and the other is selected from hydrogen, C1-C6 alkyl, -C(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, -S(=O)2-phenyl, and the phenyl group is optionally substituted with C1-C6 alkyl;

[0150] Or more preferably, R a 、R b Any one of them is selected from hydrogen, methyl, and the other is selected from hydrogen, methyl,

[0151] More preferably, R 4 Selected from hydroxy, cyano, nitro, carboxyl, -NH2, -SH, -CONH2, methyl, phenyl, benzyl, methoxy, phenoxy, acetyl, -F, -Cl, -Br, -CH=CH2, cyclopentyl, -COOMe, -COOPh, -SMe, -SOMe, -SO2Me, -NMe2, -NHAc, -NHMs, -NHTs, 2-thienyl;

[0152] Most preferably, R 4 is hydroxyl group;

[0153] The compound shown in formula I-2 does not contain

[0154] In some embodiments, the compound has a structural formula as shown in Formula I-3,

[0155] in:

[0156] R 1 Selected from hydrogen, C1-C6 alkyl;

[0157] Preferably, R 1 is hydrogen;

[0158] R 2 Selected from -O-C3-C8 cycloalkyl;

[0159] Preferably, R 2 for

[0160] R 3 Selected from -O-C1-C6 alkyl;

[0161] Preferably, R 3 is -OCH2CH3;

[0162] R 4 It is a hydroxyl group.

[0163] In some embodiments, the compound is selected from the group consisting of:

[0164] or,

[0165] or,

[0166] 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 represented by Formula I or a stereoisomer, prodrug, crystalline form, pharmaceutically acceptable salt, pharmaceutically acceptable ester or pharmaceutically acceptable solvate thereof,

[0167] Alternatively, a pharmaceutical composition is provided for treating and / or preventing heart failure with preserved ejection fraction, wherein the pharmaceutical composition comprises a compound represented by Formula I or a stereoisomer, prodrug, crystalline form, pharmaceutically acceptable salt, pharmaceutically acceptable ester or pharmaceutically acceptable solvate thereof,

[0168] 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 of Formula I or a stereoisomer, prodrug, crystalline form, pharmaceutically acceptable salt, pharmaceutically acceptable ester, or pharmaceutically acceptable solvate thereof,

[0169] Among them, R, R 2 、R 3 、R 4 Each independently as described in any technical solution of the first aspect;

[0170] Preferably, the structural formula of the compound is as shown in Formula I-1, Formula I-1', Formula I-2 or Formula I-3, wherein the compound represented by Formula I-1 or Formula I-1' is as described in the first aspect, the compound represented by Formula I-2 is as described in the first aspect, and the compound represented by Formula I-3 is as described in the first aspect;

[0171] More preferably, the structural formula of the compound is as shown in Formula I-1, Formula I-2 or Formula I-3, wherein the compound shown in Formula I-1 is as described in the first aspect, the compound shown in Formula I-2 is as described in the first aspect, and the compound shown in Formula I-3 is as described in the first aspect;

[0172] More preferably, the structural formula of the compound is as shown in Formula I-1, wherein the compound shown in Formula I-1 is as described in the first aspect;

[0173] More preferably, the structural formula of the compound is as shown in Formula I-1', wherein the compound shown in Formula I-1' is as described in the first aspect;

[0174] More preferably, the structural formula of the compound is as shown in Formula I-2, wherein the compound shown in Formula I-2 is as described in the first aspect;

[0175] More preferably, the structural formula of the compound is as shown in Formula I-3, wherein the compound shown in Formula I-3 is as described in the first aspect;

[0176] Most preferably, the compound is as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0177] 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.

[0178] 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.

[0179] 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.

[0180] Figure 4: Inhibitory effect test results of compounds at different concentrations on the binding activity of Jun to DNA at the molecular level.

[0181] Figure 5: Detection results of the inhibitory effect of compounds at different concentrations on cell Jun transcription activity.

[0182] Figures 6 to 9: The compounds of the present invention can effectively inhibit the occurrence and development of HFpEF.

[0183] Figures 10 to 18: NMR spectra of the compounds of the present invention. DETAILED DESCRIPTION

[0184] 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.

[0185] In the present invention, unless otherwise explicitly stated, the description method "... are independently selected from" used throughout this document can mean that in different groups, the specific options expressed by the same or different symbols do not affect each other, and can also mean that in the same group, the specific options expressed by the same or different symbols do not affect each other.

[0186] The substituents of the compounds of the present invention are disclosed by group class or range. It is specifically noted that the present invention includes each independent subcombination of the individual members of these group classes and ranges. For example, the term "C1-C6 alkyl" specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0187] The term "C1-C6 alkyl" refers to an alkyl group having 1 to 6 carbon atoms, preferably a "C1-C4 alkyl group", more preferably a "C1-C3 alkyl group", and most preferably a "C1-C2 alkyl group". Examples of "C1-C6 alkyl" include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. Examples of "C1-C4 alkyl" include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and the like. Examples of "C1-C3 alkyl" include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl, isopropyl), and the like. Examples of "C1-C2 alkyl" include, but are not limited to, methyl and ethyl.

[0188] The term "C1-C6 alkoxy" refers to any of the above C1-C6 alkyl groups linked to the rest of the molecule via an oxygen atom (-O-), and examples include methoxy, ethoxy, isopropoxy, and the like.

[0189] The term "C1-C6 alkylthio" refers to a group obtained by replacing the oxygen atom in any of the above C1-C6 alkoxy groups with a sulfur atom, such as C1-C6 alkylthio, C1-C4 alkylthio, C1-C3 alkylthio, and the like.

[0190] The term "C3-C8 cycloalkyl" refers to a saturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 8 carbon atoms, non-limiting examples of which include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc., preferably "C3-C6 cycloalkyl".

[0191] The term "C2-C6 alkenyl" refers to a straight-chain or branched aliphatic hydrocarbon group having 2 to 6 carbon atoms and containing one or more carbon-carbon double bonds, such as vinyl, allyl, and the like.

[0192] The term "aryl" refers to a group of a carbocyclic aromatic system. For example, "C6-C10 aryl" refers to a group of a carbocyclic aromatic system having 6 to 10 carbon atoms, non-limiting examples of which include but are not limited to phenyl, naphthyl, and the like.

[0193] The term "heteroaryl" refers to an aromatic monocyclic, bicyclic, tricyclic or more ring group having at least one heteroatom (N, O or S) in at least one ring, wherein the heteroatom-containing ring optionally further has 1, 2 or 3 heteroatoms selected from N, O or S, and is a bicyclic or tricyclic or more heteroaryl group, requiring each ring in the bicyclic or tricyclic or polycyclic ring to constitute an aromatic system. For example, a "5-6 membered heteroaryl" refers to an aromatic monocyclic, bicyclic or tricyclic or more ring group having at least one heteroatom (N, O or S) in at least one ring of a 5- or 6-membered ring, wherein the non-limiting examples thereof are pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, imidazolyl, thiazolyl, isothiazolyl, thioxazolyl, pyrrolyl, furyl, oxazolyl, isoxazolyl, pyrazolyl, thienyl. For another example, the term "6-10 membered heteroaryl" refers to a 6-, 7-, 8-, 9- or 10-membered aromatic monocyclic, bicyclic, tricyclic or higher ring group having at least one heteroatom (N, O or S) in at least one ring, non-limiting examples of which are pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, imidazolyl, phenyl-pyrrolyl, phenyl-furanyl, benzofuranyl, benzothiophenyl, benzimidazolyl, indazolyl, quinolinyl, isoquinolinyl and the like.

[0194] The term "heterocyclic radical" refers to a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-membered (preferably 3-7 or 3-6 membered) saturated or partially unsaturated carbocyclic ring, wherein one or more carbon atoms are replaced by heteroatoms such as nitrogen, oxygen and sulfur. Non-limiting examples of heterocyclic radicals include, for example, pyrans, pyrrolidines, pyrroline, imidazoline, imidazolidine, pyrazolidine, pyrazoline, thiazoline, thiazolidine, dihydrofuran, tetrahydrofuran, 1,3-dioxolane, piperidines, piperazine, morpholine, morpholinyl, tetrahydropyrrolyl, thiomorpholinyl, etc. For example, a "6-membered heterocyclic radical" refers to a 6-membered saturated or partially unsaturated carbocyclic ring, wherein one or more carbon atoms are replaced by heteroatoms such as nitrogen, oxygen and sulfur. Non-limiting examples of 6-membered heterocyclic radicals include, for example, pyrans, piperidines, piperazine, morpholine, morpholinyl, thiomorpholinyl, etc. For another example, "5-membered heterocyclyl" refers to a 5-membered saturated or partially unsaturated carbocyclic ring in which one or more carbon atoms are replaced by heteroatoms such as nitrogen, oxygen, and sulfur. Non-limiting examples of 5-membered heterocyclyls are, for example, pyrrolidine, pyrroline, imidazoline, imidazolidine, pyrazolidine, pyrazoline, thiazoline, thiazolidine, 1,3-dioxolane, and the like.

[0195] The term "heteroatom" refers to N, O or S.

[0196] The term "substituted" means that any hydrogen on the designated atom or group is replaced with the selection of the designated group, provided that the normal valence of the designated atom is not exceeded.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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).

[0204] 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.

[0205] 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.

[0206] "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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] Example 1

[0212] 1. Materials and Reagents

[0213] In this example, C57BL / 6N wild-type mice were purchased from Beijing Weitonglihua. The sources of the reagents are shown in the following table.

[0214] In addition to the above, other materials and reagents used in this example are also commercially available products.

[0215] 2. Animal Experiment Guidelines

[0216] 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.

[0217] 3. Induction of Heart Failure Model with Preserved Ejection Fraction

[0218] 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.

[0219] 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.

[0220] 4. Jun expression is correlated with HFpEF

[0221] At 15 weeks of model induction, myocardial cells from normal mice and the HFpEF model were extracted and separated using a perfusion method, and quantitative RCR detection was performed. The specific procedures are as follows:

[0222] 4.1. Isolation of adult mouse cardiomyocytes:

[0223] 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.

[0224] 4.2. Quantitative PCR detection:

[0225] 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.

[0226] 5. T-5224 administration method

[0227] 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.

[0228] 6. Conventional ultrasonic testing

[0229] 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.

[0230] 7. Experimental results and conclusions

[0231] 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.

[0232] 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.

[0233] 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.

[0234] Example 2

[0235] 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.

[0236] 1. Synthesis of compounds

[0237] 1.1 Synthesis of compound Target 1 (abbreviated as T1)

[0238] 1) Synthesis of compound 2

[0239] Under nitrogen, a solution of compound 1 (10.0 g, 34.9 mmol, 1.00 eq), compound 1A (2.85 g, 38.4 mmol, 3.55 mL, 1.10 eq), and triphenylphosphine (11.0 g, 41.9 mmol, 1.20 eq) in tetrahydrofuran (100 mL) was cooled to 0°C, and diisopropyl azodicarboxylate (8.48 g, 41.9 mmol, 8.14 mL, 1.20 eq) was added dropwise. The mixture was stirred at 25°C for 10 hours. LCMS (EC20183-4-P1A1) monitored the complete reaction of compound 1, and the appearance of a single main peak of the target molecular weight (R t =0.36min,MS cal.:340.13,MS observed:[M+H] + =341.0), water (300 mL) was added to the reaction system at 25°C to quench the reaction, followed by extraction with ethyl acetate (200 mL x 3). All organic phases were combined, washed with brine (300 mL), 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 yield Compound 2 (4.68 g, 13.3 mmol, 38.1% yield, 97.1% purity) as a light yellow solid.

[0240] LCMS:EC20183-4-P1A1,R t =0.36min,MS cal.:340.13,MS observed:[M+H] + =341.0.

[0241] LCMS:EC20183-4-P1A3,R t =0.39min,MS cal.:340.13,MS observed:[M+H] + =341.1.

[0242] HPLC:EC20183-4-P1A4,R t =2.34min,97.1%purity.

[0243] 1 H NMR:EC20183-4-P1B,400MHz,DMSO-d6

[0244] δ:11.84(s,1H),7.63(s,1H),7.57(d,J=8.4Hz,1H),7.44(d,J=9.6Hz,1H),7.19(d,J=8.4Hz,1H),6.54-6.53(m,2 H),3.83(d,J=8.8Hz,2H),3.07(t,J=6.8Hz,2H),2.84(t,J=8.0Hz,2H),2.07-1.98(m,1H),0.97(d,J=6.4Hz,6H).

[0245] 2) Synthesis of compound 3

[0246] Compound 2 (4.68 g, 13.3 mmol, 1.00 eq) was dissolved in methanol (20.0 mL), and a solution of sodium methoxide (1.73 g, 32.0 mmol, 2.40 eq) in methanol (30.0 mL) was added. The mixture was stirred at 0°C for 1 hour. LCMS (EC20183-6-P1A) monitored the complete reaction of compound 2, and a single main peak (R t =0.53min,MS cal.:372.16,MS observed:[M+H] + =373.2), the reaction solution was poured into 1N hydrochloric acid (50.0 mL) and diluted with water (200 mL). Extraction was performed with ethyl acetate (100 mL x 3). All organic phases were combined, washed with brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 3 (4.97 g, 13.0 mmol, 97.4% yield, 97.5% purity) as a light yellow solid.

[0247] LCMS:EC20183-6-P1A,R t =0.53min,MS cal.:372.16,MS observed:[M+H] + =373.2.

[0248] LCMS:EC20183-6-P1A1,R t =0.53min,MS cal.:372.16,MS observed:[M+H] + =373.2.

[0249] HPLC:EC20183-6-P1A2,R t =3.60min,97.5%purity.

[0250] 3) Synthesis of Compound 4-Notebook Page: EC20183-7

[0251] A solution of compound 3 (2.48 g, 6.49 mmol, 1.00 eq), compound 3A (1.86 g, 8.12 mmol, 1.25 eq) and potassium carbonate (1.79 g, 12.9 mmol, 2.00 eq) in N,N-methylformamide (15.0 mL) was stirred at 50°C for 2 hours. LCMS (EC20183-7-P1A1) monitored the complete reaction of compound 3 and the appearance of a single main peak of the target molecular weight (R t =0.47min,MS cal.:520.21,MS observed:[M+H] + =521.3), the reaction solution was poured into 1N HCl (50.0 mL) at 0°C, then diluted with water (150 mL) and extracted with ethyl acetate (100 mL*3). All organic phases were combined, washed with water (200 mL*2) and brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 4 (3.47 g, crude product) as a light yellow solid.

[0252] LCMS:EC20183-7-P1A1,R t =0.47min,MS cal.:520.21,MS observed:[M+H] + =521.3.

[0253] LCMS:EC20183-7-P1A2,R t =0.47min,MS cal.:520.21,MS observed:[M+H] + =521.3

[0254] 4) Synthesis of Compound T1

[0255] A solution of lithium hydroxide monohydrate (1.12 g, 26.6 mmol, 4.00 eq) in water (7.00 mL) was added to a solution of compound 4 (3.47 g, 6.67 mmol, 1.00 eq) in tetrahydrofuran (7.00 mL). The mixture was stirred at 50°C for 2 hours. LCMS (EC20183-8-P1A) monitored the complete reaction of compound 4 and the appearance of a single main peak (R t =0.39min,MS cal.:492.18,MS observed:[M+H] +=493.2), the reaction solution was poured into 1N HCl (50.0 mL), diluted with 150 mL of water, and extracted with ethyl acetate (100 mL*3). All organic phases were combined, washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. TLC (dichloromethane:methanol=10:1) was used to monitor 4 points (R f =0.25, 0.20, 0.10, 0.00). The residue was purified by column chromatography (silica gel, dichloromethane:methanol=100:1 to 1:1, R f =0.20) to obtain white solid compound T1 (0.90 g, 1.82 mmol, 27.3% yield, 99.6% purity).

[0256] LCMS:EC20183-8-P1A,R t =0.39min,MS cal.:492.18,MS observed:[M+H] + =493.2.

[0257] HRMS:EC20183-8-P1F4.

[0258] HPLC:EC20183-8-P1F,R t =3.62min,99.6%purity.

[0259] 1 H NMR:EC20183-8-P1B,400MHz,DMSO-d6

[0260] δ:12.57(br s,2H),12.00(s,1H),7.98(d,J=8.0Hz,2H),7.60(d,J=8.0Hz,2H),7.55-7.54(m,2H),7.46(d,J=9.6Hz,1H),7.17(br d,J=8.8Hz,1H),6.53-6.53(m,2H),5.34(s,2H),3.82(d,J=6.4Hz,2H),2.91(t ,J=7.2Hz,2H),2.57(t,J=7.6Hz,2H),2.07-1.97(m,1H),0.97(d,J=6.4Hz,6H)

[0261] 1.2 Synthesis of compound Target 2 (abbreviated as T2)

[0262] 1) Synthesis of compound 2

[0263] A solution of compound 1 (2.49 g, 6.52 mmol, 1.00 eq), compound 1A (1.98 g, 8.15 mmol, 1.25 eq) and potassium carbonate (1.80 g, 13.0 mmol, 2.00 eq) in N,N-dimethylformamide (15.0 mL) was stirred at 50°C for 2 hours. LCMS (EC20184-4-P1A) monitored the complete reaction of compound 1 and the appearance of a single main peak (R t =0.49min,MS cal.:534.23,MS observed:[M+H] + =535.3), the reaction solution was poured into 50.0 mL of 1N hydrochloric acid solution at 0°C, diluted with 150 mL of water, and extracted with ethyl acetate (100 mL*3). All organic phases were combined, washed with water (200 mL*2) and brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a light yellow solid compound 2 (3.39 g, crude product).

[0264] LCMS:EC20184-4-P1A,R t =0.49min,MS cal.:534.23,MS observed:[M+H] + =535.3

[0265] LCMS:EC20184-4-P1A1,R t =0.49min,MS cal.:534.23,MS observed:[M+H] + =535.3

[0266] 2) Synthesis of compound T2

[0267] Trimethyltin hydroxide (2.58 g, 14.3 mmol, 2.25 eq) was added to a solution of compound 2 (3.39 g, 6.34 mmol, 1.00 eq) in 1,2-dichloroethane (30.0 mL), and the mixture was stirred at 70°C for 9 hours. LCMS (EC20184-5-P1A) showed that the reaction of compound 2 was not complete, and the target molecular weight was the main peak (R t =0.44min,MS cal.:520.21,MS observed:[M+H] +=521.3), the reaction solution was adjusted to pH 6 with 1N hydrochloric acid solution at 25°C, then diluted with 150 mL of water and extracted with ethyl acetate (100 mL*3). All organic phases were combined, washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. TLC (dichloromethane: methanol = 20:1) monitored 4 points (R f =0.35, 0.30, 0.25, 0.00). The residue was purified by silica gel column chromatography (dichloromethane: methanol = 100:1 to 1:1, R f =0.30), and then the crude product was purified by preparative HPLC (TFA conditions) to give a white solid T2 (0.55 g, 1.06 mmol, 16.6% yield, and 100% purity).

[0268] LCMS:EC20184-5-P1A,R t =0.44min,MS cal.:520.21,MS observed:[M+H] + =521.3.

[0269] HRMS:EC20184-5-P1E4.

[0270] HPLC:EC20184-5-P1E3,R t =2.85min,100%purity.

[0271] 1 H NMR:EC20184-5-P1B,400MHz,DMSO-d6

[0272] δ:12.14(br s,1H),11.99(s,1H),8.00(d,J=8.4Hz,2H),7.63(d,J=8.4Hz,2H),7.55-7.53(m,2H),7.47–7.44(m,1H),7.17(br d,J=9.2Hz,1H),6.54-6.51(m,2H),5.36(s,2H),4.32(q,J=7.2Hz,2H),3.83(d,J=6.8Hz,2H),2.91(t ,J=7.6Hz,2H),2.57(t,J=7.2Hz,2H),2.07-1.98(m,1H),1.32(t,J=6.8Hz,3H),0.98(d,J=6.4Hz,6H)

[0273] 1.3 Synthesis of compound Target 3 (abbreviated as T3)

[0274] 1) Synthesis of compound 2B

[0275] 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).

[0276] Proton spectrum: EC6536-710-P1A1, 400 MHz, deuterated chloroform

[0277] 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).

[0278] 2) Synthesis of Compound 2

[0279] 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.

[0280] LCMS1: EC6536-741-P1A1, Rt=0.37min, MS cal.: 316.09, MS observed: [M+H]+=317.0.

[0281] LCMS2: EC6536-741-P1A2, Rt=0.37min, MS cal.: 316.09, MS observed: [M+H]+=317.0.

[0282] Proton spectrum: EC6536-741-P1A, 400 MHz, DMSO-d6.

[0283] 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).

[0284] 3) Synthesis of Compound Int A

[0285] 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).

[0286] LCMS1: EC6536-760-P1A, Rt=0.56min, MS cal.: 284.07, MS observed: [M+H]+=285.0.

[0287] LCMS2: EC6536-760-P1A1, Rt=0.56min, MS cal.: 284.07, MS observed: [M+H]+=285.0, 99.4% purity.

[0288] Proton spectrum: EC6536-760-P1A, 400 MHz, DMSO-d6

[0289] 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)

[0290] 4) Synthesis of compound Int A_3

[0291] 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).

[0292] LCMS1: EC6536-768-P1A, Rt=0.51min, MS cal.: 352.13, MS observed: [M+H]+=353.0.

[0293] LCMS2: EC6536-768-P1B, Rt=0.51min, MS cal.: 352.13, MS observed: [M+H]+=353.1, 100% purity.

[0294] Proton spectrum: EC6536-768-P1A1, 400 MHz, DMSO-d6

[0295] 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)

[0296] 5) Synthesis of Compound Int D

[0297] 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.

[0298] LCMS1: EC6536-769-P1A1, Rt=0.49min, MS cal.: 384.16, MS observed: [M+H]+=385.1.

[0299] LCMS2: EC6536-769-P1A4, Rt=0.50min, MS cal.: 384.16, MS observed: [M+H]+=385.0.

[0300] 6) Synthesis of Compound 4

[0301] 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.

[0302] Proton spectrum: EC6536-773-P1A, 400 MHz, DMSO-d6

[0303] 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).

[0304] 7) Synthesis of Compound T3

[0305] 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).

[0306] LCMS: EC6536-776-P1A3, Rt=0.51min, MS cal.: 504.18, MS observed: [M+H]+=505.3.

[0307] HRMS:EC20403-1.

[0308] HPLC: EC20403-1-P1A2, Rt=3.52min, 100% purity.

[0309] Proton spectrum: EC20403-1-P1A7, 400 MHz, DMSO-d6

[0310] 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)

[0311] 1.4 Synthesis of compound Target 4 (abbreviated as T4)

[0312] 1) Synthesis of compound 2B

[0313] A solution of compound 1 (5.00 g, 17.4 mmol, 1.00 eq), benzyl alcohol (2.08 g, 19.2 mmol, 1.99 mL, 1.10 eq), and triphenylphosphine (5.50 g, 20.9 mmol, 1.20 eq) in tetrahydrofuran (50.0 mL) was cooled to 0°C. Diisopropyl azodicarboxylate (4.24 g, 20.9 mmol, 4.07 mL, 1.20 eq) was added dropwise under nitrogen. The mixture was stirred at 25°C for 10 hours. LCMS (EC20185-4-P1A1) confirmed the complete reaction of compound 1 and the appearance of a single peak at the target molecular weight (Rt = 0.35 min, MS cal.: 374.12, MS observed: [M+H]+ = 375.0). The reaction was quenched with water (200 mL) at 25°C and extracted with ethyl acetate (150 mL x 3). All organic phases were combined, washed with brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The crude product was purified by reverse HPLC (neutral conditions) to give a light yellow solid compound 2A (2.10 g, 4.81 mmol, 27.4% yield, 85.7% purity) and a light yellow solid compound 2B (2.89 g, 4.27 mmol, 24.4% yield, 55.3% purity).

[0314] LCMS: EC20185-4-P1A1, Rt=0.35min, MS cal.: 374.12, MS observed: [M+H]+=375.0.

[0315] LCMS: EC20185-4-P1C1, Rt=0.51min, MS cal.: 374.12, MS observed: [M+H]+=375.0.

[0316] LCMS: EC20185-4-P1D1, Rt=0.50min, MS cal.: 374.12, MS observed: [M+H]+=375.0.

[0317] HPLC: EC20185-4-P1C2, Rt=4.31min, 85.7% purity.

[0318] HPLC: EC20185-4-P1C3, Rt=4.31min, 55.3% purity.

[0319] 2) Synthesis of compounds 3B and 3C

[0320] A solution of compound 2 (2.89 g, 4.27 mmol, 1.00 eq) in methanol (15.0 mL) was cooled to 0°C, and a solution of sodium methoxide (553 mg, 10.2 mmol, 2.40 eq) in methanol (20.0 mL) was added. The mixture was stirred at 0°C for 1 hour. When the reaction of compound 2 (Rf = 0.50) was complete and two new spots appeared as monitored by TLC (petroleum ether:ethyl acetate = 3:1), the reaction solution was poured into 1 M hydrochloric acid (50.0 mL), diluted with water (150 mL), and extracted with ethyl acetate (100 mL x 3). All organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (silica gel, petroleum ether:ethyl acetate = 100:1 to 1:1, Rf = 0.30) to give white solid compound 3B (0.88 g, 2.05 mmol, 48.1% yield, 94.9% purity) and white solid compound 3C (0.75 g, 1.55 mmol, 36.2% yield, 83.8% purity).

[0321] LCMS: EC20185-6-P1A3, Rt=0.49min, MS cal.: 406.14, MS observed: [M+H]+=407.1.

[0322] LCMS: EC20185-6-P1A5, Rt=0.48min, MS cal.: 406.14, MS observed: [M+H]+=407.2.

[0323] HPLC: EC20185-6-P1A4, Rt=3.49min, 94.9% purity.

[0324] HPLC: EC20185-6-P1A6, Rt=3.50min, 83.8% purity.

[0325] 3) Synthesis of compound 4

[0326] A solution of compound 3 (1.63 g, 3.60 mmol, 1.00 eq), compound 3A (1.03 g, 4.50 mmol, 1.25 eq), and potassium carbonate (995 mg, 7.20 mmol, 2.00 eq) in N,N-dimethylformamide (5.00 mL) was heated to 50°C and stirred for 2 hours. LCMS (EC20185-12-P1A) confirmed the complete reaction of compound 3 and the appearance of a single peak at the target molecular weight (Rt = 0.43 min, MS cal.: 554.19, MS observed: [M+H]+ = 555.2). The reaction mixture was then poured into 1 M HCl (20.0 mL) at 0°C, diluted with water (50.0 mL), and extracted with ethyl acetate (30.0 mL x 3). All organic phases were combined, washed sequentially with water (30.0 mL x 2) and brine (30.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. Four spots (Rf = 0.50, 0.30, 0.25, 0.00) were detected on a TLC plate (petroleum ether:ethyl acetate = 2:1). The residue was purified by column chromatography (silica gel, petroleum ether:ethyl acetate = 100:1 to 1:1, Rf = 0.30). The crude product was purified by reverse HPLC (neutral conditions) to obtain compound 4 as a white solid (0.68 g, 1.23 mmol, 34.0% yield, 100% purity).

[0327] LCMS: EC20185-12-P1A, Rt=0.43min, MS cal.: 554.19, MS observed: [M+H]+=555.2.

[0328] LCMS: EC20185-12-P1E, Rt=0.43min, MS cal.: 554.19, MS observed: [M+H]+=555.2.

[0329] HPLC: EC20185-12-P1E1, Rt=3.04min, 100% purity.

[0330] 1H NMR:EC20185-12-P1B,400MHz,DMSO-d6

[0331] δ:11.90(s,1H),8.00(d,J=8.0Hz,2H),7.62(d,J=8.4Hz,2H),7.56-7.54(m,2H),7.47–7.33(m,6H),7.17(d,J=8.4Hz,1 H),6.63-6.58(m,2H),5.35(s,2H),5.19(s,2H),3.86(s,3H),3.56(s,3H),2.94(t,J=7.2Hz,2H),2.65(t,J=7.6Hz,2H).

[0332] 4) Synthesis of Compound T4

[0333] To a solution of compound 4 (0.68 g, 1.23 mmol, 1.00 eq) in tetrahydrofuran (5.00 mL) was added a solution of lithium hydroxide monohydrate (205 mg, 4.90 mmol, 4.00 eq) in water (5.00 mL), and the mixture was stirred at 50°C for 4 hours. LCMS (EC20185-15-P1A4) confirmed the complete reaction of compound 4 and the appearance of a single peak at the target molecular weight (Rt = 0.27 min, MS cal.: 526.16, MS observed: [M+H]+ = 527.2). The reaction mixture was then adjusted to pH 1 with 1 M hydrochloric acid at 25°C and stirred at this temperature for 1 hour. The reaction mixture was filtered, the filter cake collected, dispersed in deionized water, and subsequently lyophilized to yield Target 4 as a white solid (0.51 g, 968 μmol, 79.0% yield, 100% purity).

[0334] LCMS: EC20185-15-P1A4, Rt=0.27min, MS cal.: 526.16, MS observed: [M+H]+=527.2.

[0335] HRMS:EC20185-15-P1E.

[0336] HPLC: EC20185-15-P1C3, Rt=3.53min, 100% purity.

[0337] Proton spectrum EC20185-15-P1B2, 400 MHz, DMSO-d6

[0338] δ:12.56(br s,2H),11.95(s,1H),7.98(d,J=8.4Hz,2H),7.61(d,J=8.0Hz,2H),7.56-7.54(m,2H),7.49-7.45(m,3H),7.41(t,J=6.8Hz,2H),7. 36-7.33(m,1H),7.17(d,J=8.8Hz,1H),6.64-6.59(m,2H),5.34(s,2H),5.19(s,2H),2.91(t,J=6.8Hz,2H),2.57(t,J=7.6Hz,2H).

[0339] 1.5 Synthesis of compound Target 5 (abbreviated as T5)

[0340] 1) Synthesis of compound 2

[0341] To a solution of NH2OH·HCl (31.3 g, 451 mmol, 3.00 eq) in methanol, under nitrogen and in an ice bath, was slowly added dropwise a solution of NaOMe (135 g, 752 mmol, 30.0% purity, 5.00 eq) in methanol 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. LCMS1 (EC6536-775-P1A1) analysis 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).

[0342] LCMS1:EC6536-775-P1A1,R t =0.19min,MS cal.:167.0,MS observed:[M+H] + =168.0.

[0343] LCMS:EC6536-775-P1A2,R t=0.20min,MS cal.:167.0,MS observed:[M+H] + =168.2.

[0344] 1 H NMR:EC6536-775-P1A1,400MHz,DMSO-d6

[0345] 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)

[0346] 2) Synthesis of compound 3

[0347] 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. LCMS1 (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 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).

[0348] LCMS1:EC20395-3-P1A2,R t =0.29min,MS cal.:149.0,MS observed:[M+H] + =150.0.

[0349] LCMS:EC20395-3-P1B1,R t =0.29min,MS cal.:149.0,MS observed:[M+H] + =150.2.

[0350] 1 H NMR:EC20395-3-P1A1,400MHz,DMSO-d6

[0351] δ:7.60(d,J=8.0Hz,1H),7.34(s,1H),7.13(d,J=8.0Hz,1H),2.43(s,3H).

[0352] 3) Synthesis of Compounds 4 and 4A

[0353] 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 afforded 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. Detection by 1H NMR 1 (EC6536-790-P1A1) and LCMS1 (EC6536-790-P1D1) revealed compound 4A (4.25 g, 21.6 mmol, 33.7% yield, 98.3% purity) as a yellow oil. The structure was confirmed by LCMS2 (EC6536-790-P2A1) and 1H NMR 2 (EC6536-790-P1A2).

[0354] LCMS:EC6536-790-P1A2,R t =0.67min,MS cal.:193.0,MS observed:[M+H] + =194.1.

[0355] LCMS1:EC6536-790-P1D1,R t =0.66min,MS cal.:193.0,MS observed:[M+H] + =194.0.

[0356] LCMS2:EC6536-790-P2A1,R t=0.56min,MS cal.:193.0,MS observed:[M+H] + =194.0.

[0357] 1 H NMR 1:EC6536-790-P1A1,400MHz,CDCl3

[0358] 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)

[0359] 1 H NMR 2:EC6536-790-P1A2,400MHz,CDCl3

[0360] 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)

[0361] 4) Synthesis of Compound 5

[0362] 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.

[0363] LCMS: EC20395-14-P2C4, Rt = 0.69 min, MS calculated value: 270.9, MS detected value: [M+H] + = 271.9.

[0364] LCMS:EC20395-14-P2C4,R t=0.69min,MS cal.:270.9,MS observed:[M+H] + =271.9.

[0365] 5) Synthesis of Compound Int E

[0366] 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 LCMS1 (EC6536-771-P1A3).

[0367] LCMS: EC6536-771-P1A1, Rt=0.52 min, MS calculated value: 398.1, MS detected value: [M+H]+=399.2.

[0368] LCMS1: EC6536-771-P1A3, Rt=0.53 min, MS calculated value: 398.1, MS detected value: [M+H]+=399.2.

[0369] LCMS:EC6536-771-P1A1,R t =0.52min,MS cal.:398.1,MS observed:[M+H] + =399.2.

[0370] LCMS1:EC6536-771-P1A3,R t =0.53min,MS cal.:398.1,MS observed:[M+H] + =399.2.

[0371] 6) Synthesis of Compound 6

[0372] 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 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).

[0373] LCMS:EC20395-15-P1A3,R t =0.93min,MS cal.:589.2,MS observed:[M+H] + =590.4.

[0374] LCMS1:EC20395-15-P1D16,R t =2.10min,MS cal.:589.2,MS observed:[M+H] + =590.1.

[0375] HPLC:EC20395-15-P1A23,R t =4.01min,purity:94.2%

[0376] 7) Synthesis of Compound T5

[0377] 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 LCMS1 (EC20395-18-P1A1), HPLC (EC20395-18-P1A2), HRMS (EC20395-18-P1A1), 1H NMR (EC20395-18-P1A3) and 13C NMR (EC20395-18-P1A2).

[0378] LCMS:EC20395-17-P1A4,R t =0.66min,MS cal.:545.2,MS observed:[M+H] + =546.4.

[0379] LCMS1:EC20395-18-P1A1,R t =0.67min,MS cal.:545.2,MS observed:[M+H] + =546.4.

[0380] HRMS:EC20395-18-P1A1

[0381] HPLC:EC20395-18-P1A2,R t =3.82min,purity:99.4%

[0382] 1 H NMR:EC20395-18-P1A3,400MHz,CDCl3

[0383] 13 C NMR:EC20395-18-P1A2

[0384] 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)

[0385] 1.6 Synthesis of compound Target 6 (abbreviated as T6)

[0386] 1) Synthesis of compound 7

[0387] Compound 4A (2.00 g, 10.3 mmol, 1.00 eq) was dissolved in 20 mL of acetonitrile and stirred at room temperature. AIBN (169 mg, 1.04 mmol, 0.10 eq) and DBDMH (3.85 g, 13.4 mmol, 1.30 eq) were added, and the reaction mixture was allowed to react at 80°C for 8 hours. LCMS (EC20419-5-P2A4) analysis showed that 4A was completely reacted with a yield of 32.6% (Rt = 0.58 min, MS cal.: 270.9, MS observed: [M+H]+ = 271.9). The reaction mixture was treated with 50 mL of ice water, separated, and the organic phase was washed with aqueous Na2S2O3 (50.0 mL), aqueous NaHCO3 (50.0 mL), and saturated brine (50.0 mL), then dried over anhydrous sodium sulfate. The mixture was filtered and evaporated to dryness under reduced pressure to yield 5.74 g of crude compound 7 as a yellow oil.

[0388] LCMS: EC20419-5-P2A4, Rt = 0.58 min, MS calculated value: 270.9, MS detected value: [M+H] + = 271.9.

[0389] 2) Synthesis of Compound T6

[0390] Compound 7 (5.74 g, 21.1 mmol, 1.35 eq) was dissolved in 50 mL of DMF and stirred at room temperature. Int D (6.41 g, 15.6 mmol, 1.00 eq) and KCO (4.31 g, 31.2 mmol, 2.00 eq) were added, and the reaction mixture was heated to 50°C for 1 hour. LCMS (EC20419-6-P1A) showed that compound 7 was completely reacted in a 17.5% yield (Rt = 0.87 min, MS calcd: 575.2, MS detected: [M+H]+ = 576.2). The reaction mixture was diluted with 50 mL of water and extracted with ethyl acetate (50.0 mL x 3). The combined organic phases were washed with water (50.0 mL x 5) and then with 100 mL of saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a solid. This solid was purified by reverse-phase preparative liquid chromatography to afford the target compound T6 (683 mg, 1.18 mmol, 7.56% yield, 99.5% purity) as a yellow solid. The structure was confirmed by HPLC (EC20419-6-P1D2), 1H NMR (EC20419-6-P1A1), 12C NMR (EC20419-6-P1A2), and HRMS (EC20419-6-P1A1).

[0391] LCMS:EC20419-6-P1A,R t =0.87min,MS cal.:575.2,MS observed:[M+H] + =576.2.

[0392] HRMS:EC20419-6-P1A1

[0393] HPLC:EC20419-6-P1D2,R t =4.19min,purity:99.5%

[0394] 13 C NMR 1:EC20419-6-P1A2

[0395] 1 H NMR 1:EC20419-6-P1A1,400MHz,CDCl3

[0396] δ:12.68(s,1H),7.90(d,J=8.0Hz,1H),7.57(d,J=2.0Hz,1H),7.56-7.53(m,1H),7.51(d,J=8. 8Hz,1H),7.40(s,1H),7.35(d,J=8.0Hz,1H),6.94(d,J=8.4Hz,1H),6.49(d,J=2.4Hz,1H),6.3 8(dd,J=2.4,8.8Hz,1H),5.36(s,2H),5.31(s,2H),4.83(td,J=2.8,5.6Hz,1H),3.69(s,3H),3 .47(s,3H),3.10(t,J=7.6Hz,2H),2.71(t,J=7.6Hz,2H),2.01-1.76(m,6H),1.71-1.64(m,2H)

[0397] 1.7 Synthesis of Target 7 (abbreviated as T7)

[0398] Synthesis of compound T7

[0399] Compound 2 (1.78 g, 12.0 mmol, 1.10 eq), compound 1 (4.45 g, 10.9 mmol, 1.00 eq) and triphenylphosphine (3.44 g, 13.1 mmol, 1.20 eq) were dissolved in tetrahydrofuran (40.0 mL) at 0°C. Diisopropyl azodicarboxylate (2.65 g, 13.1 mmol, 2.54 mL, 1.20 eq) was added dropwise to the reaction solution, and the mixture was stirred at 25°C for 10 hours. LCMS (EC19915-16-P1A) monitoring of compound 1 (Rt = 0.400 min) revealed incomplete reaction. Upon the appearance of a major peak at the target molecular weight (Rt = 0.32 min, MS cal.: 528.23, MS observed: [M+H]+ = 529.3), the reaction solution was concentrated under reduced pressure to yield a residue. Five TLC spots (Rf = 0.50, 0.30, 0.20, 0.15, 0.00) were observed on a TLC plate (dichloromethane:methanol = 15:1). The residue was purified by column chromatography (silica gel, dichloromethane:methanol = 100:1 to 1:1, Rf = 0.20). The crude product was then purified by preparative HPLC (trifluoroacetic acid) to yield Target 7 as a white solid (0.65 g, 1.23 mmol, 11.2% yield, 100% purity).

[0400] LCMS:EC19915-16-P1A,R t=0.32min,MS cal.:528.23,MS observed:[M+H] + =529.3.

[0401] HRMS:EC21255-1-P1A2.

[0402] HPLC:EC21255-1-P1A1,R t =3.03min,100%purity.

[0403] 1 H NMR:EC21255-1-P1B,400MHz,DMSO-d6

[0404] δ:11.98(s,1H),9.32(s,1H),7.93(s,1H),7.86(d,J=8.4Hz,1H),7.64(dd,J1=1.2Hz,J 2=8.4Hz,1H),7.57-7.53(m,2H),7.42(d,J=8.8Hz,1H),7.23(d,J=8.8Hz,1H),6.50-6. 45(m,2H),5.43(s,2H),4.90(t,J=5.6Hz,1H),4.00(q,J=7.2Hz,2H),2.92(t,J=7.6Hz, 2H), 2.62(t,J=7.2Hz,2H),1.98-1.90(m,2H),1.73–1.59(m,6H),1.09(t,J=7.2Hz,3H).

[0405] 1.8 Synthesis of compound Target 8 (abbreviated as T8)

[0406] Compound C230915014-C (1 g) was added to anhydrous methanol (5 mL), and 10 μL of concentrated sulfuric acid was added dropwise. The reaction was carried out at 60°C for 3 h. TLC (Plate 1:PE:EA = 1:1, 254 nm) showed a small amount of residual starting material. Saturated aqueous sodium bicarbonate was added to the system to adjust the pH to 8-9. The reaction was extracted three times with EA (20 mL x 3), washed with 20 mL of saturated brine, and the organic phase was collected and concentrated to yield 0.35 g of T8. The NMR spectrum of the product is shown in Figure 10.

[0407] 1.9 Synthesis of Target 9 (abbreviated as T9)

[0408] Compound C230915014-C (0.7 g) was added to isopropanol (30 ml), and 0.29 ml (4 eq) of concentrated sulfuric acid was added dropwise. The reaction was allowed to proceed at 85°C for 2 h. TLC (Plate 1:PE:EA = 1:1, 254 nm) indicated the formation of the desired product. The mixture was concentrated, extracted three times with 40 ml of EA and 30 ml of water, and washed with 20 ml of saturated brine. The organic phases were mixed and separated by column chromatography using a PE:EA ratio of 50:1 to 1:1. The desired fraction was concentrated, and the organic phase was concentrated to yield 0.52 g of T9. The NMR spectrum of the product is shown in Figure 11.

[0409] 1.10 Synthesis of Compound Target 10 (abbreviated as T10)

[0410] Compound C230915014-SM1E (8 g) was added to DMF (80 mL), potassium carbonate (15.6 g, 4 eq) and bromocyclohexane (36.9 g, 8 eq). The reaction was continued at 70°C for 12 h. LCMS indicated the formation of the desired product. The mixture was extracted with 240 mL of ethyl acetate and 300 mL of H₂O. The organic phase was washed with 200 mL of saturated sodium chloride solution and concentrated. The concentrate was filtered through silica gel (300-400 mesh) using a 500 mL mixture of ethyl acetate and n-heptane (1:4). The filtrate was then added with 500 mL of n-heptane and filtered through a 300-400 mesh silica gel pad to yield 1.95 g of the product fraction TM-10A.

[0411] Compound TM-10A (1.7 g) was added to THF (30 ml), wet Pd(OH)2 (0.5 g, 0.75 eq) and H2 (15 psi). The reaction was allowed to proceed at 25°C for 2 h. HPLC indicated the formation of the desired product. The reaction solution was concentrated to yield 1.6 g of TM-10B.

[0412] Compound TM-10B (0.8 g) was added to methanol (10 ml), followed by sodium methoxide (0.36 g, 3 eq). The reaction was allowed to proceed at 25°C for 3 h. LCMS indicated the formation of the desired product. The reaction solution was concentrated to remove MeOH, and 40 ml of EA and 40 ml of water were added. The mixture was separated by extraction, and the organic phase was collected. The aqueous phase was then separated by addition of 30 ml of EA and 20 ml of aqueous ammonium chloride. The organic phases were collected, combined, washed with 60 ml of saturated brine, and dried over anhydrous sodium sulfate to yield 0.9 g of TM-10C.

[0413] Compound TM-10C (0.9 g) was added to acetone (20 ml), along with potassium carbonate (0.3 g, 1 eq) and 1.06 g (1 eq) of C230915014-SM4. The reaction was allowed to proceed at 55°C for 16 h. LCMS indicated the formation of the desired product. The reaction solution was filtered and concentrated. 40 ml of EA and 30 ml of water were added to the reaction solution, followed by extraction. The organic phase was collected, washed with 30 ml of saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (TLC) (Plate 1:PE:EA = 3:1, 254 nm), with PE:EA = 50:1-5:1. The fraction was concentrated to yield 1.0 g, which was then lyophilized to yield 1 g of TM-10D.

[0414] Compound TM-10D (1 g) was added to DCM (20 ml), followed by TFA (10 ml). The reaction was allowed to react at 25°C for 16 h. LCMS indicated the formation of the desired product. 40 ml of water was added to the reaction solution, and the mixture was extracted with 40 ml of DCM. The organic phase was collected and washed with 40 ml of saturated brine. The organic phase was separated and collected, and purified by column chromatography (TLC) (Plate 1, PE:EA = 1:1, 254 nm) (PE:EA = 25:1-2:1). The fraction was concentrated to yield 0.1 g of T10. The NMR spectrum of the product is shown in Figure 12.

[0415] 1.11 Synthesis of Target 11 (abbreviated as T11)

[0416] Compound TM-10B (1.6 g) was added to ethanol (20 ml), followed by sodium ethoxide (0.6 g, 2 eq) and H2 (15 psi). The reaction was allowed to proceed at 25°C for 12 h. LCMS indicated the formation of the desired product. The reaction solution was concentrated to remove EtOH, and 40 ml of EA and 40 ml of water were added. The mixture was separated by extraction, and the organic phase was collected. 30 ml of EA was added to the aqueous phase, followed by extraction with 20 ml of aqueous ammonium chloride. The organic phases were collected, combined, washed with 60 ml of saturated brine, and dried over anhydrous sodium sulfate to yield 1.8 g of TM-11A.

[0417] Compound TM-11A (1.8 g) was added to acetone (30 ml), along with potassium carbonate (0.6 g, 1.1 eq) and 2.15 g (1.05 eq) of C230915014-SM4. The reaction was allowed to proceed at 55°C for 2 h. LCMS indicated the formation of the desired product. The reaction mixture was filtered and concentrated. 40 ml of EA and 30 ml of water were added to the reaction mixture, followed by extraction. The organic phase was collected, washed with 30 ml of saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (TLC) (Plate 1:PE:EA = 3:1, 254 nm), with PE:EA = 10:1-5:1 (10% DCM). The fraction was concentrated to yield 2 g of TM-11B.

[0418] Compound TM-11B (0.6 g) was added to D (10 ml), and 0.37 ml (2 M) HCl / Dioxane was added. The mixture was reacted at 25°C for 12 h. LCMS showed the formation of the target product. The reaction solution was concentrated and extracted with 40 ml of ethyl acetate and 30 ml of H2O. The organic phase was washed with 20 ml of saturated sodium chloride aqueous solution, concentrated, and slurried with 10 mL of ethanol to obtain 330 mg of T11. The NMR spectrum of the product is shown in Figure 13.

[0419] 1.12 Synthesis of Target 12 (abbreviated as T12)

[0420] Compound C230915014-SM2 (1 g) was added to anhydrous methanol (5 ml), and 0.03 ml (0.2 eq) of concentrated sulfuric acid was added dropwise. The reaction was allowed to proceed at 60°C for 3 h. LCMS indicated the formation of the desired product. Saturated sodium bicarbonate solution was added to adjust the pH to 8-9, and the mixture was extracted with EA (20 ml x 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to yield 0.75 g of TM-12A.

[0421] Compound TM-12A (1 g) was added to acetone (20 ml), and potassium carbonate (0.4 g, 1.1 eq) and 0.77 g (1.1 eq) of tert-butyl 4-bromomethylbenzoate were added. The mixture was reacted at 55°C for 10 h. LCMS showed that the target product was produced. The reaction solution was filtered and concentrated. 20 mL of water was added to the reaction solution, and 20 mL of EA was added for extraction. The organic phase was concentrated and mixed, and purified by column chromatography (TLC) (Patent 1:PE:EA=5:1, 254 nm), PE:EA=50:1-20:1. The fraction was concentrated to obtain 1.2 g of TM-12B.

[0422] Compound TM-12B (1.2 g) was added to DCM (20 ml), followed by TFA (6 ml, 39 eq). The mixture was reacted at 25°C for 3 h. LCMS indicated the formation of the desired product. 40 ml of water was added to the reaction solution, and the mixture was extracted with 20 ml of DCM. The organic phase was collected, washed with 30 ml of water, separated, and concentrated to yield 0.8 g of T12. The NMR spectrum of the product is shown in Figure 14.

[0423] 1.13 Synthesis of Target 13 (abbreviated as T13)

[0424] Compound TM-13 (1.3 g) was added to methanol (20 ml), followed by sulfuric acid (0.37 g, 1.5 eq). The mixture was reacted at 80°C for 0.5 h. TLC (Plate 1:PE:EA = 1:1, 254 nm) revealed the formation of new spots. The reaction solution was concentrated and extracted with 40 ml of ethyl acetate and 30 ml of H₂O. The organic phase was washed with 20 ml of saturated sodium chloride solution, concentrated, and slurried with ethyl acetate:n-heptane = 1:5 to yield 1.0 g of T13. The NMR spectrum of the product is shown in Figure 15.

[0425] 1.14 Synthesis of Target 14 (abbreviated as T14)

[0426] Compound C230915014-C (2 g) was added to 2-bromoethanol (30 ml), and 0.5 ml (2.5 eq) of concentrated sulfuric acid was added dropwise. The reaction was allowed to proceed at 85°C for 2 h. LCMS indicated the formation of the desired product. The mixture was concentrated, extracted three times with 40 ml of EA and 30 ml of water, and washed with 20 ml of saturated brine. The organic phase was concentrated and slurried with 50 ml of CAN to yield 1.6 g of TM-14A.

[0427] Compound TM-14A (1.4 g) was added to DMF (14 ml), along with potassium carbonate (0.25 g, 0.8 eq) and morpholine (0.2 ml, 1 eq). The reaction was carried out at 5°C for 0.5 h. LCMS indicated the formation of the desired product. 15 ml of H2O was added to the reaction solution, which was filtered. The filtrate was then subjected to preparative separation, and the fraction was lyophilized to afford 0.28 g of T14. The NMR spectrum of the product is shown in Figure 16.

[0428] 1.15 Synthesis of Target 15 (abbreviated as T15)

[0429] 2-Methoxy-4-methylbenzoic acid (4 g) was added to ethanol (20 mL), and 3.5 mL (2 eq) of SOCl2 was added dropwise. The reaction was allowed to react at 25°C for 14 h. TLC (Plate 1:PE:EA = 3:1, 254 nm) indicated the formation of the desired product. 12 mL of water was slowly added dropwise to the reaction solution, and the pH was adjusted to 7-8 with saturated aqueous NaHCO3. The mixture was extracted three times with 40 mL of 3% EA, and the organic phases were combined. The organic phases were washed twice with 20 mL of 2% saturated aqueous NaCl, dried over anhydrous Na2SO4, and filtered. The filtrate was then evaporated to dryness under reduced pressure at 45°C to yield 4 g of TM-15B.

[0430] Compound TM-15B (0.6 g) was added to chloroform (9 ml), along with 0.55 g (1 eq) of NBS and 0.04 g (0.05 eq) of benzoyl peroxide. The mixture was reacted at 35°C for 1 h. HPLC indicated the formation of the desired product. 20 mL of saturated aqueous NaHCO₃ was added to the reaction mixture, and the organic phase was collected. The aqueous phase was extracted twice with 15 mL of 2% DCM, and the combined organic phases were washed three times with 20 mL of 3% water. The organic phase was dried over 10 g of anhydrous Na₂SO₄, filtered, and concentrated to yield 0.7 g of TM-15C.

[0431] Compound TM-15C (0.44 g) was added to acetone (7 ml), along with potassium carbonate (0.22 g, 1 eq) and C230915014-A (0.7 g, 1 eq). The reaction was continued at 65°C for 14 h. LCMS indicated the formation of the desired product. 21 mL of water was added to the reaction mixture, and the aqueous phase was extracted three times with 10 mL of 3% EA. The organic phases were combined and washed twice with 20 mL of water. The organic phase was dried over 10 g of anhydrous Na2SO4 and filtered. The filtrate was concentrated to yield 1 g of TM-15A.

[0432] Compound TM-15A (1 g) was added to DCM (10 ml), followed by TFA (2.4 ml, 20 eq). The mixture was reacted at 25°C for 2 h. TLC (Plate 1:PE:EA = 3:1, 254 nm) indicated completion of the reaction. 30 ml of water was added to the reaction solution, and the mixture was extracted with 20 ml of DCM. The organic phase was collected and washed with 30 ml of water. The mixture was separated and concentrated, and then slurried with 6 mL of n-heptane:ethyl acetate at 15°C for 20 min. Filtering afforded 0.53 g of T15. The NMR spectrum of the product is shown in Figure 17.

[0433] 1.16 Synthesis of Target 16 (abbreviated as T16)

[0434] Compound C230915014-C (0.7 g) was added to n-propanol (30 ml), and 0.29 ml (4 eq) of concentrated sulfuric acid was added dropwise. The reaction was carried out at 85°C for 2 h. TLC (Plate 1:PE:EA = 1:1, 254 nm) indicated the formation of the desired product. The mixture was concentrated and extracted three times with 40 ml of EA and 30 ml of water. The mixture was washed with 20 ml of saturated brine. The organic phase was mixed and separated by column chromatography with PE:EA = 50:1 to 1:1. The desired fraction was concentrated, and the organic phase was collected to yield 0.6 g of T16. The NMR spectrum of the product is shown in Figure 18.

[0435] 2. Inhibition of DNA Binding Activity Test

[0436] Test Method

[0437] c-jun transcription factor DNA binding activity test

[0438] 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.

[0439] 2.2 Test Results

[0440] The results are shown in the following table and Figure 4.

[0441] DNA & JUN binding activity

[0442] The above results indicate that, with the exception of T7, the other six compounds all showed activity in inhibiting JUN-DNA binding. Among them, T3, T4, and T5 were superior to or comparable to the positive control, T-5224. This indicates that the compounds of the present invention (particularly those of Formula I-1, Formula I-1′, and Formula I-2) exhibit activity in inhibiting JUN-DNA binding.

[0443] 3. Transcriptional Activity Inhibition Test

[0444] 3.1 Test Method

[0445] Dual luciferase reporter system to detect inhibition of DNA binding activity

[0446] 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.

[0447] 3.2 Test Results

[0448] The results are shown in the following table and Figure 5.

[0449] The above results indicate that T3 and T5 further have the activity of inhibiting JUN transcription. It can be seen that the compounds of the present invention (especially the compounds of Formula I-1, Formula I-1′ and Formula I-2) have the activity of inhibiting JUN transcription.

[0450] 4. Animal Model Efficacy Testing

[0451] 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.

[0452] The dosage is as follows:

[0453] After successful induction of the animal model, mice in the treatment group were treated with compounds of the present invention (e.g., compounds T3, T5, T8-T16) and T-5524. A control group was treated with a drug-free solvent. Mice that received normal diet and water throughout the induction process served as negative controls. When the mice were 5-8 weeks old, ultrasound confirmed successful model establishment and began drug administration. The treatment groups were administered a single dose of 12 mg / kg of compounds T3, T5, T8-T16, and T-5524, based on mouse body weight, every other day. Each dose consisted of 0.54 mg of compound dissolved in 100 μL of 0.5% PVP solution (the actual 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). Drug administration began after successful model induction. The control group received an equal volume of 0.5% PVP solution. All other treatments were identical.

[0454] The experimental results are shown in Figures 6 to 9 (wherein week 0 on the horizontal axis represents the day when drug administration begins after successful modeling, i.e., the starting point of drug administration). It can be clearly seen from Figure 6 that at 3 weeks of administration, the positive control T-5524 has not yet shown an effect of inhibiting disease progression. However, the compounds T3 and T5 of the present invention have significantly inhibited disease progression at 3 weeks of administration, showing a clear advantage. This shows that compared to the positive control T-5524, the compounds T3 and T5 of the present invention can inhibit disease progression earlier, with a clear advantage. Similarly, it can be seen that the compounds T8 to T16 of the present invention also have a significant therapeutic effect on HFpEF, and the effect is very fast. It can be seen that the compounds of the present invention (especially the compounds of Formula I-1, Formula I-1' and Formula I-2 series) have a significant therapeutic effect on HFpEF, and the effect is very fast.

[0455] In summary, the inventors of the present application have found 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, can produce a therapeutic effect on HFpEF, have obvious advantages in the treatment of HFpEF, and have broad application prospects.

Claims

1. Use of a compound of formula I or its stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, pharmaceutically acceptable esters or pharmaceutically acceptable solvates in the preparation of a medicament for the treatment and / or prevention of heart failure with preserved ejection fraction, Wherein: R is selected from C6-C10 aryl, 6- to 10-membered heteroaryl, and each of the C6-C10 aryl and 6- to 10-membered heteroaryl is independently optionally substituted by R 1 and / or R 5 ; Preferably, R is selected from C6-C10 aryl and 6-10 membered heteroaryl, and each of the C6-C10 aryl and 6-10 membered heteroaryl is independently optionally substituted by R 1 or R 1 and R 5 substituted; More preferably, R is selected from C6-C10 aryl, 6-10 membered heteroaryl, and each of the C6-C10 aryl and 6-10 membered heteroaryl is independently optionally substituted by R 1 ; R 1 selected from hydrogen, C1-C6 alkyl, -COOH, -C(=O)O-C1-C6 alkyl, -(CH2) m -O-C1-C6 alkyl, -NR c R d ; R c 、R d each independently selected from hydrogen, C1-C6 alkyl, -(CH2) m -O-C1-C6 alkyl; m is selected from 0, 1, 2, 3, 4, 5, 6; Preferably, m is selected from 0, 1; More preferably, m is 1; R 2 selected from -O-(CH2) n -R 2’ ; n is selected from 0, 1, 2, 3, 4, 5, 6; Preferably, n is selected from 0, 1; R 2’ selected from C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, 5- or 6-membered heteroaryl; R 3 selected from a hydroxyl group, -O-C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted by a 5- or 6-membered heterocyclic group; Preferably, R 3 is selected from a hydroxyl group, -O-C1-C6 alkyl, and the C1-C6 alkyl is optionally substituted with a 6-membered heterocyclic group; More preferably, R 3 is selected from hydroxy, -O-C1-C6 alkyl, and the C1-C6 alkyl is optionally substituted by morpholinyl; Most preferably, R 3 is selected from hydroxy, -O-C1-C6 alkyl; R 4 Selected from hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, -NR a R b 、-CONR a R b 、C6-C10 aryl, -O-C6-C10 aryl, -C(=O)-C1-C6 alkyl, -CH2-C6-C10 aryl, C1-C6 alkoxy, C2-C6 alkenyl, C3-C8 cycloalkyl, -C(=O)O-C1-C6 alkyl, -C(=O)O-C6-C10 aryl, C1-C6 alkylthio, -S(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, 5-6 membered heteroaryl; R a and R b each independently selected from hydrogen, C1-C6 alkyl, -C(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, -S(=O)2-phenyl, said phenyl optionally substituted by C1-C6 alkyl; R 5 selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio; and the compound represented by Formula I does not contain 2. The use according to claim 1, wherein R is selected from phenyl, benzimidazolyl, said phenyl, benzimidazolyl, each is independently optionally substituted by R 1 and / or R 5 ; Preferably, R is selected from phenyl, benzimidazolyl, The phenyl group, benzimidazolyl group, each independently optionally substituted by R 1 or R 1 and R 5 ; More preferably, R is selected from phenyl, benzimidazolyl, The phenyl group, benzimidazolyl group, Each is independently optionally substituted by R 1 ; Alternatively, preferably, R is selected from phenyl, The phenyl group, each independently optionally substituted by R 1 and / or R 5 ; Alternatively, more preferably, R is selected from phenyl, The phenyl group, each independently and optionally substituted by R 1 or R 1 and R 5 ; Alternatively, more preferably, R is selected from phenyl, The phenyl group, each independently optionally substituted by R 1 ; Alternatively, preferably, R is selected from Alternatively, more preferably, R is selected from Preferably, R 1 is selected from hydrogen, -COOH, -C(=O)O-C1-C6 alkyl, -(CH2) m -O-C1-C6 alkyl, -NR c R d ; More preferably, R 1 is selected from hydrogen, -COOH, -C(=O)O-C1-C6 alkyl, -(CH2) m -O-C1-C6 alkyl; Preferably, R c , R d are each independently selected from hydrogen, -(CH2) m -O-C1-C6 alkyl; More preferably, R c , R d Among them, any one is hydrogen, and the other is selected from -(CH2) m -O-C1-C6 alkyl; Further preferably, R 1 is selected from hydrogen, -COOH, -COOCH3, -COOCH2CH3, -COO(CH2)2CH3, -COO(CH2)3CH3, -COO(CH2)4CH3, -COO(CH2)5CH3, More preferably, R 1 is selected from hydrogen, -COOH, -COOCH3, -COOCH2CH3, Most preferably, R 1 is selected from hydrogen, -COOH, -COOCH2CH3, Preferably, R 5 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy; More preferably, R 5 is selected from C1-C6 alkoxy groups; Most preferably, R 5 is a methoxy group.

3. Use according to any one of claims 1-2, wherein, R 2’ selected from C1-C6 alkyl, C3-C8 cycloalkyl, phenyl, pyrazinyl (such as ), furyl (such as ); Preferably, R 2’ is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, Phenyl, pyrazinyl (such as )、Furyl (such as )； Alternatively, R 2’ is selected from C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl; Alternatively, preferably, R 2’ is selected from C1-C6 alkyl, C3-C8 cycloalkyl, phenyl; Alternatively, more preferably, R 2’ is selected from cyclopentyl, cyclohexyl, phenyl; Or more preferably, R 2’ is selected from cyclopentyl, phenyl; Preferably, R 2 is selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-C6-C10 aryl, -O-CH2-5-6 membered heteroaryl; More preferably, R 2 is selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-phenyl, -O-CH2-5-6 membered heteroaryl, and the 5-6 membered heteroaryl is selected from pyrazinyl (such as )、furanyl (such as )； Most preferably, R 2 is selected from Alternatively, preferably, R 2 is selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-C6-C10 aryl; Or more preferably, R 2 is selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-phenyl; Or further preferably, R 2 is selected from Or most preferably, R 2 is selected from 4. Use according to any one of claims 1 - 3, wherein R 3 selected from hydroxy, -OCH3, -OCH2CH3, -OCH(CH3)CH3, -O(CH2)2CH3, -O(CH2)3CH3, -O(CH2)4CH3, -O(CH2)5CH3, Preferably, R 3 is selected from hydroxyl, -OCH3, -OCH2CH3, -OCH(CH3)CH3, -O(CH2)2CH3, Alternatively, preferably, R 3 is selected from hydroxy, -OCH3, -OCH2CH3, -OCH(CH3)CH3, -O(CH2)2CH3, -O(CH2)3CH3, -O(CH2)4CH3, -O(CH2)5CH3; Alternatively, more preferably, R 3 is selected from hydroxy, -OCH3, -OCH2CH3, -O(CH2)2CH3, -O(CH2)3CH3, -O(CH2)4CH3, -O(CH2)5CH3; Alternatively, more preferably, R 3 is selected from hydroxy, -OCH3, -OCH2CH3, -OCH(CH3)CH3, -O(CH2)2CH3; Alternatively, or most preferably, R 3 is selected from hydroxy, -OCH3, -OCH2CH3.

5. Use according to any one of claims 1 - 4, wherein, R 4 selected from hydroxy, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, -NR a R b , -CONR a R b , phenyl, phenoxy, -C(=O)-C1-C6 alkyl, benzyl, C1-C6 alkoxy, C2-C6 alkenyl, C3-C8 cycloalkyl, -C(=O)O-C1-C6 alkyl, -C(=O)O-phenyl, C1-C6 alkylthio, -S(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, thienyl (such as 2-thienyl); Preferably, R a , R b are each independently selected from hydrogen, methyl, Alternatively, preferably, R a , R b is independently selected from hydrogen, C1-C6 alkyl, and the other is selected from hydrogen, C1-C6 alkyl, -C(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, -S(=O)2-phenyl, and the phenyl is optionally substituted with C1-C6 alkyl; Alternatively, more preferably, R a , R b Among them, any one is selected from hydrogen and methyl, and the other is selected from hydrogen, methyl, Preferably, R 4 is selected from hydroxy, cyano, nitro, carboxyl, -NH2, -SH, -CONH2, methyl, phenyl, benzyl, methoxy, phenoxy, acetyl, -F, -Cl, -Br, -CH=CH2, cyclopentyl, -COOMe, -COOPh, -SMe, -SOMe, -SO2Me, -NMe2, -NHAc, -NHMs, -NHTs, 2-thienyl; More preferably, R 4 is a hydroxyl group.

6. Use according to any one of claims 1-5, wherein, The structural formula of the said compound is shown as Formula I-1 or Formula I-1', preferably, the structural formula of the said compound is shown as Formula I-1. Wherein: R 1 Selected from -COOH, -C(=O)O-C1-C6 alkyl; Preferably, R 1 is selected from -COOH, -COOCH3, -COOCH2CH3, -COO(CH2)2CH3, -COO(CH2)3CH3, -COO(CH2)4CH3, -COO(CH2)5CH3; More preferably, R 1 is selected from -COOH, -COOCH3, -COOCH2CH3; Most preferably, R 1 is selected from -COOH, -COOCH2CH3; R 2 Selected from -O-(CH2) n -R 2’ n is selected from 0, 1, 2, 3, 4, 5, 6; Preferably, n is selected from 0, 1; R 2’ selected from C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, 5- or 6-membered heteroaryl; Preferably, R 2’ is selected from C1-C6 alkyl, C3-C8 cycloalkyl, phenyl, pyrazinyl (such as )、furanyl (such as )； More preferably, R 2’ is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, phenyl, pyrazinyl (such as )、furanyl (such as )； Alternatively, preferably, R 2’ is selected from C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl; Or more preferably, R 2’ is selected from C1-C6 alkyl, C3-C8 cycloalkyl, phenyl; Or most preferably, R 2’ is selected from cyclopentyl, phenyl; Preferably, R 2 is selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-C6-C10 aryl, -O-CH2-5-6 membered heteroaryl; More preferably, R 2 is selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-phenyl, -O-CH2-5-6 membered heteroaryl, and the 5-6 membered heteroaryl is selected from pyrazinyl (such as )、Furyl (such as )； Most preferably, R 2 is selected from Alternatively, preferably, R 2 is selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-C6-C10 aryl; Or more preferably, R 2 is selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-phenyl; Or most preferably, R 2 is selected from R 3 selected from hydroxy, -O-C1-C6 alkyl; Preferably, R 3 is selected from hydroxy, -OCH3, -OCH2CH3, -O(CH2)2CH3; More preferably, R 3 is a hydroxyl group; R 4 selected from hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, -NR a R b , -CONR a R b , C6-C10 aryl, -O-C6-C10 aryl, -C(=O)-C1-C6 alkyl, -CH2-C6-C10 aryl, C1-C6 alkoxy, C2-C6 alkenyl, C3-C8 cycloalkyl, -C(=O)O-C1-C6 alkyl, -C(=O)O-C6-C10 aryl, C1-C6 alkylthio, -S(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, 5-6 membered heteroaryl; Preferably, R 4 is selected from the group consisting of hydroxy, cyano, nitro, carboxy, mercapto, halogen, C1-C6 alkyl, -NR a R b , -CONR a R b , phenyl, phenoxy, -C(=O)-C1-C6 alkyl, benzyl, C1-C6 alkoxy, C2-C6 alkenyl, C3-C8 cycloalkyl, -C(=O)O-C1-C6 alkyl, -C(=O)O-phenyl, C1-C6 alkylthio, -S(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, thienyl (such as 2-thienyl); R a 、R b are each independently selected from hydrogen, C1-C6 alkyl, -C(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, -S(=O)2-phenyl, and the phenyl is optionally substituted by C1-C6 alkyl; Preferably, R a , R b are each independently selected from hydrogen, methyl, Alternatively, preferably, R a , R b is independently selected from hydrogen, C1-C6 alkyl, and the other is selected from hydrogen, C1-C6 alkyl, -C(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, -S(=O)2-phenyl, and the phenyl is optionally substituted with C1-C6 alkyl; Alternatively, more preferably, R a , R b is selected from hydrogen and methyl, and the other is selected from hydrogen, methyl, More preferably, R 4 is selected from the group consisting of hydroxyl, cyano, nitro, carboxyl, -NH2, -SH, -CONH2, methyl, phenyl, benzyl, methoxy, phenoxy, acetyl, -F, -Cl, -Br, -CH=CH2, cyclopentyl, -COOMe, -COOPh, -SMe, -SOMe, -SO2Me, -NMe2, -NHAc, -NHMs, -NHTs, 2-thienyl; Most preferably, R 4 is a hydroxyl group; R 5 selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio; Preferably, R 5 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy; More preferably, R 5 is selected from C1-C6 alkoxy groups; Most preferably, R 5 is methoxy.

7. Use according to any one of claims 1-5, wherein The structural formula of the said compound is as shown in Formula I-2, Wherein: R 1 selected from hydrogen, -(CH2) m -O-C1-C6 alkyl, -NR c R d ; Preferably, R 1 is selected from hydrogen, -(CH2) m -O-C1-C6 alkyl; R c 、R d each independently selected from hydrogen, C1-C6 alkyl, -(CH2) m -O-C1-C6 alkyl; Preferably, R c , R d are each independently selected from hydrogen, -(CH2) m -O-C1-C6 alkyl; More preferably, R c , R d , any one of them is hydrogen, and the other is selected from -(CH2) m -O-C1-C6 alkyl; m is selected from 0, 1, 2, 3, 4, 5, 6; Preferably, m is selected from 0, 1; More preferably, m is 1; More preferably, R 1 is selected from hydrogen, R 2 Selected from -O-(CH2) n -R 2’ ; n is selected from 0, 1, 2, 3, 4, 5, 6; Preferably, n is selected from 0, 1; R 2’ selected from C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, 5- or 6-membered heteroaryl; Preferably, R 2’ is selected from C1-C6 alkyl, C3-C8 cycloalkyl, phenyl, pyrazinyl (such as )、furanyl (such as )； More preferably, R 2’ is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, Phenyl, pyrazinyl (such as )、Furyl (such as )； Alternatively, preferably, R 2’ is selected from C3-C8 cycloalkyl; Or more preferably, R 2’ is selected from cyclopentyl, cyclohexyl; Alternatively, or most preferably, R 2’ is selected from cyclopentyl; Preferably, R 2 is selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-C6-C10 aryl, -O-CH2-5-6 membered heteroaryl; More preferably, R 2 is selected from -O-C1-C6 alkyl, -O-C3-C8 cycloalkyl, -O-CH2-phenyl, -O-CH2-5- or 6-membered heteroaryl, and the 5- or 6-membered heteroaryl is selected from pyrazinyl (such as )、furanyl (such as )； Most preferably, R 2 is selected from Alternatively, preferably, R 2 is selected from -O-C3-C8 cycloalkyl; Or more preferably, R 2 is selected from Or most preferably, R 2 is R 3 selected from hydroxy, -O-C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with a 5-6 membered heterocyclic group; Preferably, R 3 is selected from a hydroxyl group, -O-C1-C6 alkyl, and the C1-C6 alkyl is optionally substituted by a 6-membered heterocyclic group; More preferably, R 3 is selected from hydroxy, -O-C1-C6 alkyl, and the C1-C6 alkyl is optionally substituted by morpholinyl; More preferably, R 3 is selected from hydroxy, -OCH3, -OCH2CH3, -OCH(CH3)CH3, -O(CH2)2CH3, -O(CH2)3CH3, -O(CH2)4CH3, -O(CH2)5CH3, More preferably, R 3 is selected from -OCH3, -OCH2CH3, -OCH(CH3)CH3, -O(CH2)2CH3, Alternatively, preferably, R 3 is selected from hydroxy, -O-C1-C6 alkyl; Or more preferably, R 3 is selected from hydroxy, -OCH3, -OCH2CH3, -OCH(CH3)CH3, -O(CH2)2CH3, -O(CH2)3CH3, -O(CH2)4CH3, -O(CH2)5CH3; Alternatively, more preferably, R 3 is selected from hydroxy, -OCH3, -OCH2CH3, -O(CH2)2CH3, -O(CH2)3CH3, -O(CH2)4CH3, -O(CH2)5CH3; Alternatively, preferably, R 3 is selected from -O-C1-C6 alkyl; Alternatively, more preferably, R 3 is selected from -OCH3, -OCH2CH3, -OCH(CH3)CH3, -O(CH2)2CH3; Or most preferably, R 3 is selected from -OCH3, -OCH2CH3; R 4 Selected from hydroxy, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, -NR a R b , -CONR a R b , C6-C10 aryl, -O-C6-C10 aryl, -C(=O)-C1-C6 alkyl, -CH2-C6-C10 aryl, C1-C6 alkoxy, C2-C6 alkenyl, C3-C8 cycloalkyl, -C(=O)O-C1-C6 alkyl, -C(=O)O-C6-C10 aryl, C1-C6 alkylthio, -S(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, 5-6 membered heteroaryl; Preferably, R 4 is selected from hydroxy, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, -NR a R b , -CONR a R b , phenyl, phenoxy, -C(=O)-C1-C6 alkyl, benzyl, C1-C6 alkoxy, C2-C6 alkenyl, C3-C8 cycloalkyl, -C(=O)O-C1-C6 alkyl, -C(=O)O-phenyl, C1-C6 alkylthio, -S(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, thienyl (such as 2-thienyl); R a 、R b each independently selected from hydrogen, C1-C6 alkyl, -C(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, -S(=O)2-phenyl, said phenyl optionally substituted by C1-C6 alkyl; Preferably, R a , R b are each independently selected from hydrogen, methyl, Alternatively, preferably, R a , R b is selected from the group consisting of hydrogen, C1-C6 alkyl, and the other is selected from the group consisting of hydrogen, C1-C6 alkyl, -C(=O)-C1-C6 alkyl, -S(=O)2-C1-C6 alkyl, -S(=O)2-phenyl, and the phenyl is optionally substituted with C1-C6 alkyl; Or more preferably, R a , R b Among them, any one is selected from hydrogen and methyl, and the other is selected from hydrogen, methyl, More preferably, R 4 is selected from the group consisting of hydroxy, cyano, nitro, carboxyl, -NH2, -SH, -CONH2, methyl, phenyl, benzyl, methoxy, phenoxy, acetyl, -F, -Cl, -Br, -CH=CH2, cyclopentyl, -COOMe, -COOPh, -SMe, -SOMe, -SO2Me, -NMe2, -NHAc, -NHMs, -NHTs, 2-thienyl; Most preferably, R 4 is a hydroxyl group; and the compound represented by Formula I-2 does not contain 8. Use according to any one of claims 1-5, wherein The structural formula of the said compound is as shown in Formula I-3, Wherein: R 1 selected from hydrogen, C1-C6 alkyl; Preferably, R 1 is hydrogen; R 2 selected from -O-C3-C8 cycloalkyl; Preferably, R 2 is R 3 selected from -O-C1-C6 alkyl; Preferably, R 3 is -OCH2CH3; R 4 is a hydroxyl group.

9. Use according to any one of claims 1 - 8, wherein, The compound is selected from the following: Or, Or, 10. 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 represented by formula I or its stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, pharmaceutically acceptable ester or pharmaceutically acceptable solvate, Among them, R, R 2 , R 3 , R 4 are each independently defined as in any one of claims 1-5; Preferably, the structural formula of the compound is as shown in Formula I-1, Formula I-1', Formula I-2 or Formula I-3, wherein the compound shown in Formula I-1 or Formula I-1' is defined as in Claim 6, the compound shown in Formula I-2 is defined as in Claim 7, and the compound shown in Formula I-3 is defined as in Claim 8; More preferably, the structural formula of the compound is as shown in Formula I-1, Formula I-2 or Formula I-3, wherein the compound shown in Formula I-1 is defined as in Claim 6, the compound shown in Formula I-2 is defined as in Claim 7, and the compound shown in Formula I-3 is defined as in Claim 8; Most preferably, the compound is defined as in Claim 9.

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