Compound, pharmaceutical composition comprising the compound and use thereof

Abstract

The present application relates to a compound represented by Formula 1, a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof, a pharmaceutical composition comprising the compound represented by Formula 1 and one or more pharmaceutically acceptable excipients, a transcription factor regulator comprising the compound represented by Formula 1 or a pharmaceutical composition thereof, and use thereof in the preparation of a drug for treating a disease related to the regulation of myocyte enhancer factor 2.

Description

Technical Field

[0001] The present disclosure relates to the field of medicine, and more specifically, the present disclosure relates to a compound represented by Formula 1, a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof, a pharmaceutical composition comprising the compound represented by Formula 1 and one or more pharmaceutically acceptable excipients, a transcription factor regulator comprising the compound represented by Formula 1 or a pharmaceutical composition thereof, and use thereof in the preparation of a medicament for treating a disease associated with the regulation of myocyte enhancer factor 2. Background Art

[0002] The transcription initiation process in eukaryotic organisms is very complex and often requires the assistance of multiple protein factors. Transcription factors are proteins that bind to specific DNA sequences and regulate gene expression directly or through related proteins such as coactivators and co-repressors, or by recruiting histone modifying enzymes such as histone acetyltransferases (HATs) and histone deacetylases (HDACs).

[0003] MyoCyte Enhancer Factor 2 (MEF2) is a specific transcription factor belonging to the MADS-Box family of transcriptional regulators. It is widely present in muscle cells, lymphocytes and neurons. It participates in various physiological processes such as muscle formation, bone development, nervous system development, and liver fibrosis by controlling gene transcription during muscle cell differentiation. In vertebrates, this family includes four members: MEF2A, MEF2B, MEF2C and MEF2D. They have a unit composed of 56 amino acids common to the MADS superfamily. Their function is to form dimers between proteins and recognize and bind to target DNA, regulating the expression of genes related to cell proliferation, differentiation, survival, and apoptosis. They regulate the survival and differentiation of various cells, involving different links of gene regulation and functional regulation mechanisms, and are receiving increasing attention. Their outstanding function is to control gene transcription during muscle cell differentiation and mediate cell differentiation in skeletal muscle, cardiac muscle and smooth muscle.

[0004] Although it is known in the art that MEF2 is involved in various cellular processes, it has not been rationally exploited so far due to the lack of suitable molecular tools. In particular, how to regulate the activity of MEF2 by small molecules has been a long-standing challenge. This is because MEF2 is a relatively small transcription factor without any obvious enzymatic activity; its main function is to bind to specific DNA and recruit transcriptional co-regulators such as Cabin1, histone deacetylase (HDAC) and histone acetyltransferase (HAT) CBP / lysine acetyltransferase (KAT) p300 to specific promoters. This functional mode is generally considered to be non-drugable, or at least very difficult to target.

[0005] In WO2010 / 028193, several classes of compounds targeting transcriptional co-regulator HDAC are disclosed, and the effect of inhibiting HDAC can be achieved by contacting the compound with HDAC. HDAC is generally divided into four categories, type I (HDAC1, 2, 3 and 8), type II (HDAC4, 5, 7, 9, 6 and 10), type III (deacetylase, Sirt1-Sirt7) and type IV (HDAC11). Type II HDAC can be further subdivided into type IIa (HDAC4, 5, 7 and 9) and type IIb (HDAC6 and 10). Generally, using active site inhibitors to broadly inhibit HDAC can cause complex cell reactions, and therefore, in WO2010 / 028193, mainly studied the effective inhibitors for type I HDAC, mainly HDAC3.

[0006] CN101851173A also discloses several types of compounds targeting transcriptional co-regulator HDAC, but the compounds mainly focus on type I HDAC, mainly effective inhibitors of HDAC1.

[0007] However, inhibitors for type I HDACs are not universal for diseases related to the regulation of MEF2. The inventors found that MEF2 binds to a short amphipathic helix with a ligand-like pocket to interact with related transcriptional co-regulators, which provides a new idea for using small molecules to regulate MEF2. By using small molecules to competitively bind to the ligand-like pocket of MEF2 with transcriptional co-regulators, the interaction between MEF2 and transcriptional regulators can be blocked. This compound that can regulate MEF2 will promote the development of this field and will promote the treatment of diseases related to the regulation of MEF2. Summary of the invention

[0008] Provided herein are compounds represented by Formula 1:

[0009]

[0010] or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof,

[0011] in:

[0012] Y can be a hydroxyl group, unsubstituted or substituted with at least one R 10a Substituted C6-C 20 Aryl groups, unsubstituted or substituted with at least one R 10a Substituted C1-C 20 The heterocycloalkyl group is either unsubstituted or substituted with at least one R 10a Substituted C1-C 20 heteroaryl groups;

[0013] X may be unsubstituted or substituted with at least one R 10a Substituted C1-C 10 an alkylene group;

[0014] R1 can be C3-C 10 Cycloalkyl groups, C1-C 10 Heterocycloalkyl groups, C6-C 10 Aryl group, C1-C 10 Heteroaryl groups, C3-C 10 Cycloalkyloxy groups, C1-C 10 Heterocycloalkyloxy group, C6-C 10 Aryloxy group or C1-C 10 heteroaryloxy groups;

[0015] R2 to R4 can each independently be hydrogen, halogen, hydroxyl group, cyano group, nitro group or C1-C 10 an alkyl group; and

[0016] R 10a Each may independently be deuterium, a hydroxyl group, an amino group, a cyano group, a nitro group, a halogen, an oxydene group, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a C1-C6 alkoxy group, a C3-C6 carbocyclic group or a C1-C6 heterocyclic group.

[0017] In an embodiment, Y can be a hydroxyl group, unsubstituted or substituted with at least one R 10a Substituted C6-C 10 Aryl groups, unsubstituted or substituted with at least one R 10a Substituted C1-C 10 The heterocycloalkyl group is either unsubstituted or substituted with at least one R 10a Substituted C1-C 10 Heteroaryl groups.

[0018] In another embodiment, Y can be a hydroxyl group, unsubstituted or substituted with at least one R 10a substituted phenyl group, unsubstituted or replaced by at least one R 10a substituted naphthyl group, unsubstituted or replaced by at least one R 10a substituted pyridine group, unsubstituted or replaced by at least one R 10a substituted pyrazine group, unsubstituted or replaced by at least one R 10a substituted indole group, unsubstituted or replaced by at least one R 10a substituted benzimidazole group, or unsubstituted or replaced by at least one R 10a Substituted benzotriazole groups.

[0019] In yet another embodiment, Y can be a hydroxyl group, a phenyl group optionally substituted by a hydroxyl or amino group, a naphthyl group optionally substituted by a hydroxyl or amino group, a pyridine group optionally substituted by a hydroxyl or amino group, a pyridine group optionally substituted by a hydroxyl or amino group, a pyrazine group optionally substituted by a hydroxyl or amino group, an indole group optionally substituted by a hydroxyl or amino group, a benzimidazole group optionally substituted by a hydroxyl or amino group, or a benzotriazole group optionally substituted by a hydroxyl or amino group.

[0020] In other embodiments, Y can be 2-aminophenyl.

[0021] In embodiments, X may be unsubstituted or substituted with at least one R 10a Substituted C1-C6 alkylene group.

[0022] In another embodiment, X can be an ethylene group optionally substituted with a halogen or oxyylidene group, a propylene group optionally substituted with a halogen or oxyylidene group, a butylene group optionally substituted with a halogen or oxyylidene group, a pentylene group optionally substituted with a halogen or oxyylidene group, or a hexylene group optionally substituted with a halogen or oxyylidene group.

[0023] In yet another embodiment, X can be ethylene, propylene, butylene, fluoroethylene, bromoethylene, oxyethylene, fluoropropylene, bromopropylene, oxypropylene, fluorobutylene, bromobutylene, or oxybutylene.

[0024] In other embodiments, X can be ethylene, propylene, difluoroethylene, difluoropropylene, oxyethylene, or oxypropylene.

[0025] In an embodiment, R1 can be a C3-C6 cycloalkyl group, a C1-C6 heterocycloalkyl group, a C6-C 10 Aryl group, C1-C 10Heteroaryl groups, C3-C6 cycloalkyloxy groups, C1-C6 heterocycloalkyloxy groups, C6-C 10 Aryloxy group or C1-C 10 Heteroaryloxy groups.

[0026] In another embodiment, R1 can be cyclohexyl, azetidinyl, oxetanyl, pyrrolidinyl, morpholinyl, pyridinyl, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, oxetanyloxy, or phenoxy.

[0027] In yet another embodiment, R1 can be azetidinyl, oxetanyl, pyrrolidinyl, morpholinyl, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, or oxetanyloxy.

[0028] In other embodiments, R1 can be cyclopropyloxy.

[0029] In an embodiment, one of R2 to R4 may be hydrogen.

[0030] In another embodiment, two of R2 to R4 may be hydrogen.

[0031] In yet another embodiment, R2 to R4 may all be hydrogen.

[0032] In an embodiment, the compound represented by Formula 1 may be selected from the following compounds 1 to 5:

[0033]

[0034] Also provided herein is a pharmaceutical composition, which may include the above-mentioned compound; and one or more pharmaceutically acceptable excipients.

[0035] In an embodiment, the pharmaceutically acceptable excipients may be selected from carriers, excipients, binders, suspending agents, glidants, flavoring agents, sweeteners, disintegrants, dispersants, surfactants, lubricants, colorants, diluents, solubilizers, wetting agents, plasticizers, stabilizers, penetration enhancers, wetting agents, defoaming agents, antioxidants, preservatives, solvents or a combination thereof.

[0036] The present invention also provides a transcription factor regulator, which may include the above compound or the above pharmaceutical composition.

[0037] In embodiments, the transcription factor regulator may be a myocyte enhancer factor 2 regulator.

[0038] The present invention further provides the use of the above compound or the above pharmaceutical composition in the preparation of a drug for treating a disease related to the regulation of myocyte enhancer factor 2.

[0039] In an embodiment, the disease associated with the regulation of myocyte enhancer factor 2 may be selected from the group consisting of cardiac hypertrophy, heart failure, coronary atherosclerosis, coronary heart disease and diabetic cardiomyopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Possible metabolites of Compound 2 according to an embodiment of the present disclosure in the plasma of SD rats and beagle dogs are shown.

[0041] Figure 2 Possible metabolic pathways of Compound 2 according to an embodiment of the present disclosure in liver microsomes of different species (mouse (MI), rat (R), beagle (D), cynomolgus monkey (MO), minipig (P) and human (H)) are shown.

[0042] Figure 3A The remaining percentage of Compound 2 according to an embodiment of the present disclosure in the plasma of humans (H), rhesus monkeys (Mo), beagles (D), mini pigs (P), and rats (R) is shown, and Figure 3B The remaining percentage of Compound 2 according to an embodiment of the present disclosure in the plasma of mice (Mi) is shown.

[0043] FIG. 4A to FIG. 4C The content changes of the metabolite M1 (m / z=323.1278) of compound 2 according to an embodiment of the present disclosure in the plasma of 6 species are shown, wherein Figure 4A The figure shows the changes in the content of metabolite M1 in the plasma of beagle dogs (D), mini pigs (P), rhesus monkeys (Mo) and mice (Mi) over time. Figure 4B The changes in the content of metabolite M1 in the plasma of rats (R) over time are shown, and Figure 4C The changes in the content of the metabolite M1 in the plasma of human (H) over time are shown.

[0044] Figure 5A shows the inhibition curve of compound 2 on HDAC1; and Figure 5B The inhibition curve of compound 2 on the interaction between MEF2 and HDAC4 is shown.

[0045] Fig. 6A The immunofluorescence images of cardiomyocytes before and after induction and after treatment with the same concentration of comparative compound 1, m3 and compound 1-2 are shown. Figure 6B Shown are the average areas of cardiomyocytes before induction, after induction, and after treatment with the same concentrations of comparative compound 1 (CC1H), m3 (m3H), and compounds 1 (C1 H) and 2 (C2 H).

[0046] Figure 7The average area of ​​cardiomyocytes before induction, after induction, and after treatment with different concentrations of compounds 3-5 of the present disclosure (C3 L, C3 M, C4L, C4 M, C4 H, C5 L, C5 M, C5 H) are shown.

[0047] Fig. 8A and Figure 8B The left ventricular end-diastolic diameter of rats is shown. Fig. 8A ) and left ventricular end-systolic diameter ( Figure 8B ) comparison chart.

[0048] Fig. 9A and Fig. 9B The left ventricular end-diastolic volume of rats is shown. Fig. 8A ) and left ventricular end-systolic volume ( Figure 8B ) comparison chart.

[0049] Fig. 10A and Fig. 10B The ejection fraction ( Fig. 9A ) and shortened fractions ( Fig. 9B ) comparison chart. DETAILED DESCRIPTION

[0050] The following detailed description will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the accompanying drawings show embodiments of the invention. However, the invention is not limited to the precise arrangements, examples and instrumentalities shown.

[0051] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0052] The terms used herein are for the purpose of describing specific embodiments and are not intended to be limiting. In addition, the terms "comprise" and / or "include" when used in this specification indicate the presence of specified features, integers, steps, operations, elements, components and / or groups thereof, but do not exclude the presence or addition of one or more than one other features, integers, steps, operations, elements, components and / or groups thereof.

[0053] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Terms (such as those defined in commonly used dictionaries) should be interpreted as having a meaning consistent with their meaning in the context of the relevant art, and should not be interpreted in an idealized or overly formal sense unless explicitly defined as such herein.

[0054] definition

[0055] As used herein, the term "substituted" when used with respect to a chemical substituent or moiety (e.g., a C1-C6 alkyl group) means that one or more hydrogen atoms of the substituent or moiety have been replaced with one or more non-hydrogen atoms or groups, provided that valence requirements are met and the substitution results in a chemically stable compound.

[0056] As used herein, the term "about" or "approximately" when used with a numerical variable refers to the indicated value of the variable and the variable is within the experimental error of the indicated value or within ±15%, ±20%, ±15% or ±10% of the indicated value.

[0057] All values ​​within ±10%, ±5%.

[0058] As used herein, the term "alkyl group" refers to straight-chain and branched saturated hydrocarbon groups generally having a specified number of carbon atoms (e.g., C1-C6 alkyl refers to an alkyl group having 1 to 6 (i.e., 1, 2, 3, 4, 5 or 6) carbon atoms, C1-C6 alkyl refers to an alkyl group having 1 to 6 (i.e., 1, 2, 3, 4, 5 or 6) carbon atoms, 10 Alkyl refers to an alkyl group having 1 to 10 carbon atoms). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentan-1-yl, pentan-2-yl, pentan-3-yl, 3-methylbutan-1-yl, 3-methylbutan-2-yl, 2-methylbutan-2-yl, 2,2,2-trimethyleth-1-yl, n-hexyl, and the like. An "alkylene group" refers to a divalent group having the same structure as the corresponding alkyl group.

[0059] As used herein, the term "alkenyl group" refers to a monovalent hydrocarbon group having at least one carbon-carbon double bond in the middle or at the end of an alkyl group (e.g., C2-C6 alkenyl refers to an alkenyl group having 2 to 6 (i.e., 2, 3, 4, 5 or 6) carbon atoms). Examples of alkenyl groups may include vinyl groups, propenyl groups, butenyl groups, and the like.

[0060] As used herein, the term "alkynyl group" refers to a monovalent hydrocarbon group having at least one carbon-carbon triple bond in the middle or at the end of an alkyl group (e.g., C2-C6 alkynyl refers to an alkynyl group having 2 to 6 (i.e., 2, 3, 4, 5 or 6) carbon atoms). Examples of alkenyl groups may include ethynyl groups, propynyl groups, butynyl groups, and the like.

[0061] As used herein, the term "cycloalkyl group" refers to a saturated cyclic hydrocarbon group (e.g., C 3-10 The cycloalkyl group refers to a cycloalkyl group having 3 to 10 carbon atoms as ring-constituting atoms). Examples of the cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

[0062] As used herein, the term "heterocycloalkyl group" refers to a cyclic group further comprising at least one heteroatom (wherein the number of heteroatoms may be 1 to 5 or 1 to 3, such as 1, 2, 3, 4 or 5) other than carbon atoms as a ring-forming atom (for example, a C1-C6 heterocycloalkyl group refers to a heterocycloalkyl group having at least one heteroatom and 1 to 6 carbon atoms as ring-forming atoms). Examples of heterocycloalkyl groups are oxetanyl, morpholinyl, and the like.

[0063] As used herein, the terms "oxyene" and "oxo" are used interchangeably and refer to a double-bonded oxygen (=0).

[0064] As used herein, the terms "halo" and "halogen" are used interchangeably and refer to fluoro, chloro, bromo, and iodo.

[0065] As used herein, the term "C6-C 20 The term "aryl group" refers to a monovalent group having a carbocyclic aromatic system containing 6 to 20 carbon atoms (wherein the number of carbon atoms may be 6 to 15, 6 to 12 or 6 to 10). 20 Examples of the aryl group may be a phenyl group, a naphthyl group, an indenyl group, an anthracenyl group, and the like.

[0066] As used herein, the term "C1-C 20 The term "heteroaryl group" refers to a monovalent group of a heterocyclic aromatic system further comprising at least one heteroatom (wherein the number of heteroatoms may be 1 to 5 or 1 to 3, such as 1, 2, 3, 4 or 5) other than carbon atoms and 1 to 20 carbon atoms as ring-forming atoms. Examples of heteroaryl groups are pyridyl, benzimidazolyl, benzotriazolyl, and the like.

[0067] As used herein, the term "C1-C 20 The term "heterocyclyl group" refers to a monovalent group of a heterocyclic ring system having 1 to 20 carbon atoms (wherein the number of carbon atoms may be 1 to 15, 1 to 12, 1 to 10 or 1 to 6) further containing at least one heteroatom (wherein the number of heteroatoms may be 1 to 5 or 1 to 3, such as 1, 2, 3, 4 or 5) other than carbon atoms as a ring-forming atom. The heterocyclyl group may include a heterocycloalkyl group and a heteroaryl group.

[0068] As used herein, the term "heteroatom" refers to any atom other than carbon and hydrogen atoms. Examples of heteroatoms are O, S, N, P, Si, B, Ge, Se, or any combination thereof.

[0069] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0070] As used herein, the term "pharmaceutically acceptable excipient" refers to other ingredients in a pharmaceutical formulation, other than the active ingredient, such as the compounds described herein, which are compatible with the active ingredient and suitable for contact with the tissues or organs of a biological subject without causing excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0071] As used herein, the term "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, salts derived from the following organic or inorganic acids: 1,2-ethanedisulfonic acid, 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonic acid, carbonic acid, citric acid, ethylenediaminetetraacetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hexylresorcinolic acid, hyaluronanic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxymaleic acid, hydroxynaphthoic acid, isethionic acid, lactic acid, lactobionic acid, laurylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, naphthalenesulfonic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfuric acid, tannic acid, tartaric acid, and toluenesulfonic acid.

[0072] As used herein, the term "solvate" refers to a complex of variable stoichiometry formed by a solute (e.g., a compound provided herein) and a solvent. These solvents do not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, methanol, ethanol, and acetic acid. In one embodiment, a solvate refers to a hydrate.

[0073] As used herein, the term "patient" or "individual" is used to refer to warm-blooded animals such as mammals, for example, cats, dogs, mice, guinea pigs, horses, cows, sheep, non-humans and humans.

[0074] As used herein, the term "therapeutically effective amount" means that the dose of active ingredient can prevent, improve, alleviate or cure the state of the disease, reduce one or more symptoms associated with the disease, or slow, curb or reverse the progression of the disease when compared to a corresponding individual who has not received the active ingredient. The therapeutically effective amount can be affected by a variety of factors, including but not limited to the properties of the active ingredient (e.g., activity, pharmacokinetics, pharmacodynamics, bioavailability, etc.), the condition of the individual to be treated (e.g., age, sex, type and stage of disease, general health, response to a given dose, etc.), the dosage form and route of administration, etc.

[0075] As used herein, the term "diseases associated with the regulation of myocyte enhancer factor 2" includes, but is not limited to, diseases caused by the disorder or abnormality of myocyte enhancer factor 2, and also includes diseases that can be prevented, improved, alleviated or cured by regulating myocyte enhancer factor 2. Diseases associated with the regulation of myocyte enhancer factor 2 include, but are not limited to, cardiovascular and cerebrovascular diseases, such as myocardial hypertrophy, heart failure, coronary atherosclerosis, coronary heart disease and diabetic cardiomyopathy, etc.; nervous system diseases, such as Alzheimer's disease, Parkinson's disease, cerebral stroke, etc.; leukemia, such as acute myeloid leukemia, acute promyelocytic leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, etc.

[0076] Compound

[0077] Provided herein are compounds represented by Formula 1:

[0078]

[0079] or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof,

[0080] in:

[0081] Y can be a hydroxyl group, unsubstituted or substituted with at least one R 10a Substituted C6-C 20 Aryl groups, unsubstituted or substituted with at least one R 10a Substituted C1-C 20 The heterocycloalkyl group is either unsubstituted or substituted with at least one R 10a Substituted C1-C 20 heteroaryl groups;

[0082] X may be unsubstituted or substituted with at least one R 10a Substituted C1-C 10 an alkylene group;

[0083] R1 can be C3-C 10 Cycloalkyl groups, C1-C 10 Heterocycloalkyl groups, C6-C 10 Aryl group, C1-C 10 Heteroaryl groups, C3-C 10 Cycloalkyloxy groups, C1-C 10 Heterocycloalkyloxy group, C6-C 10 Aryloxy group or C1-C 10 heteroaryloxy groups;

[0084] R2 to R4 can each independently be hydrogen, halogen, hydroxyl group, cyano group, nitro group or C1-C 10 an alkyl group; and

[0085] R 10a Each may independently be deuterium, a hydroxyl group, an amino group, a cyano group, a nitro group, a halogen, an oxydene group, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a C1-C6 alkoxy group, a C3-C6 carbocyclic group or a C1-C6 heterocyclic group.

[0086] In an embodiment, Y can be a hydroxyl group, unsubstituted or substituted with at least one R 10a Substituted C6-C 10 Aryl groups, unsubstituted or substituted with at least one R 10a Substituted C1-C 10 The heterocycloalkyl group is either unsubstituted or substituted with at least one R 10a Substituted C1-C 10 In a specific embodiment, Y can be a hydroxyl group, unsubstituted or substituted with at least one R 10a substituted phenyl group, unsubstituted or replaced by at least one R 10a substituted naphthyl group, unsubstituted or replaced by at least one R 10a substituted pyridine group, unsubstituted or replaced by at least one R 10a substituted pyrazine group, unsubstituted or replaced by at least one R 10a substituted indole group, unsubstituted or replaced by at least one R 10a substituted benzimidazole group, or unsubstituted or replaced by at least one R 10a substituted benzotriazole group. Y can be multiple R 10a Replaced by multiple R 10a When replacing, multiple R 10a They can be the same or different, and multiple R 10a Substitution may be at the same position or at a different position.

[0087] In other embodiments, Y can be a hydroxyl group, a phenyl group optionally substituted by a hydroxyl or amino group, a naphthyl group optionally substituted by a hydroxyl or amino group, a pyridine group optionally substituted by a hydroxyl or amino group, a pyridine group optionally substituted by a hydroxyl or amino group, a pyrazine group optionally substituted by a hydroxyl or amino group, an indole group optionally substituted by a hydroxyl or amino group, a benzimidazole group optionally substituted by a hydroxyl or amino group, or a benzotriazole group optionally substituted by a hydroxyl or amino group. In a preferred embodiment, Y can be a 2-aminophenyl group.

[0088] In embodiments, X may be unsubstituted or substituted with at least one R 10a substituted C1-C6 alkylene group. X can be multiple R10a When multiple R 10a When replacing, multiple R 10a They can be the same or different, and multiple R 10a Substitution may be at the same position or at a different position.

[0089] In other embodiments, X can be ethylene optionally substituted by halogen or oxyylidene group, propylene optionally substituted by halogen or oxyylidene group, butylene optionally substituted by halogen or oxyylidene group, pentylene group optionally substituted by halogen or oxyylidene group or hexylene optionally substituted by halogen or oxyylidene group. In specific embodiments, X can be ethylene, propylene, butylene, fluoroethylene, bromoethylene, oxyethylene, fluoropropylene, bromopropylene, oxypropylene, fluorobutylene, bromobutylene or oxybutylene. In other specific embodiments, X can be ethylene, propylene, difluoroethylene, difluoropropylene, oxyethylene or oxypropylene.

[0090] In an embodiment, R1 can be a C3-C6 cycloalkyl group, a C1-C6 heterocycloalkyl group, a C6-C 10 Aryl group, C1-C 10 Heteroaryl groups, C3-C6 cycloalkyloxy groups, C1-C6 heterocycloalkyloxy groups, C6-C 10 Aryloxy group or C1-C 10 Heteroaryloxy groups. In specific embodiments, R1 can be cyclohexyl, azetidinyl, oxetanyl, pyrrolidinyl, morpholinyl, pyridyl, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, oxetanyloxy or phenoxy. In other embodiments, R1 can be azetidinyl, oxetanyl, pyrrolidinyl, morpholinyl, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy or oxetanyloxy.

[0091] In a preferred embodiment, R1 may be cyclopropyloxy.

[0092] In an embodiment, one of R2 to R4 may be hydrogen. In another embodiment, two of R2 to R4 may be hydrogen. In yet another embodiment, R2 to R4 may all be hydrogen.

[0093] In an embodiment, the compound represented by Formula 1 may be selected from the following Compounds 1 to 5:

[0094]

[0095] Compounds provided herein include all diastereoisomers, enantiomers and epimeric forms and suitable mixtures thereof. Compounds and methods provided herein include all cis and trans isomers and suitable mixtures thereof. In certain embodiments, by reacting the racemic mixture of the compound with an optically active resolving agent to form a pair of diastereomeric compounds / salts, separating diastereomers and reclaiming optically pure enantiomers, the compounds described herein are prepared as their individual stereoisomers. In some embodiments, the covalent diastereomeric derivatives of the compounds described herein are used to resolve enantiomers. In another embodiment, diastereomers are separated by separation / resolving techniques based on solubility differences. In other embodiments, the separation of stereoisomers is carried out by chromatography or by forming diastereomeric salts and by recrystallization or chromatography or any combination thereof.

[0096] In some embodiments, the compounds described herein can be prepared as prodrugs. "Prodrug" refers to a medicament that is converted into a parent drug in vivo. Prodrugs are generally useful because, in some cases, they can be more easily administered than the parent drug. For example, when the parent drug is not suitable for oral administration, it is prepared as a prodrug and then has good bioavailability by oral administration. Compared with the parent drug, the prodrug can also have improved solubility in the pharmaceutical composition. In some embodiments, the design of the prodrug increases effective water solubility. Examples of prodrugs are (but not limited to) compounds described herein, which are administered as esters ("prodrugs") to promote the transfer of transcellular membranes where water solubility impairs mobility, but once in cells where water solubility is beneficial, it is subsequently metabolically hydrolyzed into carboxylic acid (active entity). Another example of a prodrug can be a short peptide (polyamino acid) bonded to an acid group, wherein the peptide is metabolized to show an active portion. In certain embodiments, when administered in vivo, the prodrug is chemically converted into a biological, pharmaceutical or therapeutically active form of the compound. In certain embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically, or therapeutically active form of the compound.

[0097] Pharmaceutical composition

[0098] Also provided herein is a pharmaceutical composition comprising at least one compound represented by Formula 1, and one or more than one pharmaceutically acceptable excipients.

[0099] In an embodiment, the pharmaceutical composition provided herein may further comprise an additional drug. The additional drug may play the same or similar biological role as the compound represented by Formula 1 for the disease or condition suffered by the individual, such as simultaneously alleviating a certain symptom of the disease or condition with the same mechanism of action; or may play a synergistic biological role with the compound represented by Formula 1 for the disease or condition suffered by the individual, such as synergistically alleviating a certain symptom of the disease or condition with different mechanisms of action; or may independently play a different biological role with the compound represented by Formula 1 but play a role in preventing, alleviating or curing the disease or condition suffered by the individual, such as preventing adverse reactions that may be caused by the compound represented by Formula 1 with different mechanisms of action; and the like.

[0100] As used herein, the term "excipient" can be selected from carriers, excipients, binders, suspending agents, glidants, flavoring agents, sweeteners, disintegrants, dispersants, surfactants, lubricants, colorants, diluents, solubilizers, wetting agents, plasticizers, stabilizers, penetration enhancers, wetting agents, defoaming agents, antioxidants, preservatives, solvents or a combination thereof commonly used in the art.

[0101] As used herein, the term "carrier" refers to a pharmaceutically acceptable liquid or solid carrier or combination of carriers that participates in carrying or transporting the active ingredients from one location, fluid, tissue, organ (internal or external) or body part to another location, fluid, tissue, organ, or body part in a formulation. Pharmaceutically acceptable carriers are well known in the art and include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; alcohols such as ethanol and propanol; phosphate buffered saline; and other nontoxic compatible substances used in pharmaceutical formulations.

[0102] As used herein, the term "excipient" generally refers to auxiliary materials used in tablets.

[0103] As used herein, the term "diluent" refers to a substance used to dilute the active pharmaceutical ingredient before delivery. Diluents can also be used as stabilizers. Non-limiting examples of diluents include starch and its processed and co-processed derivatives, sugars, disaccharides, sucrose, lactose, polysaccharides, cellulose, cellulose ethers, cellulose acetate, hydroxypropyl cellulose, sugar alcohols, xylitol, sorbitol, maltitol, lactitol; microcrystalline cellulose, magnesium carbonate or calcium carbonate or sodium carbonate, lactose, lactose monohydrate, dicalcium phosphate, compressible sugar, calcium hydrogen phosphate dihydrate, mannitol, anhydrous lactose, magnesium oxide, maltodextrin, maltose, pullulan, sodium alginate, sodium bicarbonate, calcium silicate, calcium sulfate, cells and tricalcium phosphate or a suitable combination thereof.

[0104] As used herein, the term "binder" refers to any pharmaceutically acceptable material that can be used to bind active and inert components together to keep cohesive and discrete parts. The limiting examples of binders are polyethylene glycol 12-hydroxy glycol (SOLUTOL HS-15), polyoxyethylene hydrogenated castor oil RH40, polyethylene glycol PEG400, chitosan, hydrogenated castor oil, sodium alginate, carbomer, cellulose acetate phthalate, polyvidone, sugar, hydroxypropyl methylcellulose, hydroxypropyl cellulose, starch, alginic acid, pregelatinized starch, gum arabic, tragacanth, ethylcellulose, acrylic acid and methacrylic acid copolymer or its suitable combination.

[0105] As used herein, the term "disintegrant" refers to a substance that, after being added to a solid formulation, promotes its decomposition or disintegration after administration and allows the active ingredient to be released as effectively as possible to allow it to dissolve rapidly. Non-limiting examples of disintegrants include corn starch, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone, microcrystalline cellulose, modified corn starch, sodium carboxymethyl starch, polyvinylpyrrolidone, pregelatinized starch, agar, calcium or sodium carboxymethyl cellulose, colloidal silicon dioxide, chitosan, sodium docusate, hydroxypropyl cellulose, magnesium aluminum silicate, maltose, methylcellulose, polacrilin potassium and alginic acid or a suitable combination thereof.

[0106] As used herein, the term "lubricant" refers to an excipient that is added to the powder blend to prevent the compacted powder material from sticking to the equipment during the tableting or encapsulation process. It helps the tablet to pop out of the die and can improve powder flow. Non-limiting examples of lubricants include magnesium stearate, stearic acid, silicon dioxide, fats, zinc stearate or sucrose stearate or sodium stearate or calcium stearate, castor oil, hydrogenated castor oil. Polyethylene glycol and its derivatives, sodium stearyl fumarate, talc or fatty acids, including lauric acid, oleic acid, glyceryl behenate, glyceryl monostearate and C1-C 10 Fatty acids or suitable combinations thereof.

[0107] As used herein, the term "glidant" is intended to mean an agent used in tablet and capsule formulations to improve flowability during tablet compression and produce an anti-caking effect. Non-limiting examples of glidants include colloidal silicon dioxide, talc, fumed silica, starch, starch derivatives and bentonite or a suitable combination thereof.

[0108] As used herein, the term "plasticizer" refers to, but is not limited to, polyols such as polyethylene glycol, propylene glycol, glycerol (glycerin), organic esters such as phthalates (diethyl, dibutyl), dibutyl sebacate, citrates (triethyl, acetyl triethyl, acetyl tributyl), triacetin, oils / glycerides such as castor oil; acetylated monoglycerides, fractionated coconut oil, or suitable combinations thereof.

[0109] In specific embodiments, the solvent may be selected from water, acetone, chloroform, dichloromethane, ethanol, ethyl acetate, methanol, isopropanol, N,N-dimethylformamide, combinations thereof, and other such substances known to those of ordinary skill in the art.

[0110] In a specific embodiment, the pharmaceutical composition provided herein can be prepared as an oral solution and administered to a mammal by gavage. In a specific embodiment, the pharmaceutical composition provided herein can be prepared as an injection solution and administered to a mammal by intravenous injection. However, the dosage form and administration route of the pharmaceutical composition provided herein are not limited thereto.

[0111] The pharmaceutical compositions provided herein can be prepared into a variety of pharmaceutical preparations as needed, which can be administered to mammals, such as humans, in a variety of ways through a variety of routes of administration, including but not limited to oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intralymphatic, intranasal injection), intranasal, oral, topical or transdermal routes of administration. The pharmaceutical preparations provided herein include but are not limited to aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate release preparations, controlled release preparations, fast-dissolving preparations, tablets, capsules, pills, delayed release preparations, extended release preparations, pulsed release preparations, multi-particulate preparations, and mixed immediate release and controlled release preparations.

[0112] For example, in specific embodiments, the pharmaceutical compositions provided herein can be administered in appropriate dosage forms and doses: (a) systemically to a mammal; and / or (b) orally to a mammal; and / or (c) intravenously to a mammal; and / or (d) by inhalation to a mammal; and / or (e) by nasal administration to a mammal; and / or (f) by injection to a mammal; and / or (g) topically to a mammal; and / or (h) ocularly to a mammal; and / or (i) rectally to a mammal; and the like.

[0113] The pharmaceutical compositions provided herein may also be administered in appropriate dosage forms and doses by the following regimens: (i) a single administration to a mammal; (ii) multiple administrations of the compound to a mammal over the span of a day; (iii) continuous administration to a mammal; or (iv) continuous administration to a mammal.

[0114] The pharmaceutical compositions provided herein can be administered in a therapeutically effective amount. Although the human dosage level is still to be optimized for the pharmaceutical compositions described herein, the therapeutically effective amount can generally be about 0.25 mg / kg body weight to about 120 mg / kg body weight or more, about 0.5 mg / kg body weight or less to about 70 mg / kg body weight, about 1.0 mg / kg body weight to about 50 mg / kg body weight, or about 1.5 mg / kg body weight to about 10 mg / kg body weight. For example, the therapeutically effective amount can be about 0.25 mg / kg body weight, about 0.5 mg / kg body weight, about 1.0 mg / kg body weight, about 5.0 mg / kg body weight, about 10.0 mg / kg body weight, about 50 mg / kg body weight, or about 100 mg / kg body weight. In a specific embodiment, the pharmaceutical compositions provided herein can be administered in the form of an oral solution with 5 mg / kg body weight. In another specific embodiment, the pharmaceutical compositions provided herein can be administered in the form of an injection with 1 mg / kg body weight. But the administration scheme of the pharmaceutical compositions provided herein is not limited thereto.

[0115] Myocyte enhancer factor 2 regulator

[0116] In this article, transcription factor regulators mainly refer to myocyte enhancer factor 2 (MEF2) regulators.

[0117] MEF2 plays an important role in the differentiation, proliferation, and survival / apoptosis of a wide range of cells, including myocytes, lymphocytes, and neurons. For example, MEF2 is one of the important transcription factors used to mediate calcium signaling in the development of the lymphatic system. MEF2 can regulate cytokine expression and immune response. MEF2 can also regulate transcriptional programs that are the basis of neuronal survival and synaptic remodeling. The main function of MEF2 is to bind to specific DNA and recruit transcriptional co-regulators such as Cabin1, HDAC, CBP / p300, and myocardin to specific promoters; among them, CBP / p300 and myocardin are common co-activator molecules that promote MEF2DNA binding and transcriptional activation; while type II HDAC and Cabin1 are common co-inhibitory molecules that maintain MEF2 in a transcriptionally inactive state.

[0118] In the art, it is generally believed that MEF2 is a relatively small transcription factor without any obvious enzymatic activity and is difficult to be targeted. Therefore, inhibitors in the prior art are usually inhibitors against co-regulators, such as inhibitors against HDAC, thereby indirectly regulating MEF2. In addition, in order to avoid the serious side effects that may be caused by broad-spectrum inhibition of co-regulators, co-regulator inhibitors usually only target a very narrow range of co-regulators.

[0119] The compounds provided herein can directly target MEF2 and exert their biological activity by blocking the interaction between MEF2 and transcriptional co-regulators. For example, the compounds provided herein can competitively bind to a highly conserved hydrophobic MAD-box domain of MEF2 with type IIa HDAC, thereby preventing type IIa HDAC from binding to the targeted gene, thereby inhibiting the function of type IIa HDAC. The binding ability of the compounds provided herein to MEF2 is usually several times that of HDAC4 to MEF2, so the inhibitory effect on HDAC4 can be indirectly provided by targeting MEF2.

[0120] The inventors determined the half-maximal inhibitory concentration (IC) of compound 2 provided herein on HDAC1 using an HDAC1 kit. 50 ) was 16.8 μM. The half inhibitory concentration (IC ) of compound 2 provided herein for the interaction between MEF2 and HDAC4 (type IIa HDAC) was determined by dual luciferase reporter gene assay. 50 ) is only 0.15 μM, which shows that the compound 2 provided herein has an inhibitory effect on the interaction between MEF2 and a co-regulator such as HDAC4, and produces a good indirect inhibitory effect on HDAC4. This is different from existing catalytic HDAC inhibitors because the compounds provided herein can retain the activity of HDAC.

[0121] Therefore, the compounds or pharmaceutical compositions provided herein can directly target MEF2, which can directly and effectively treat diseases related to the regulation of MEF2 on the one hand, and fundamentally avoid the side effects that may be caused by broad-spectrum coregulator inhibitors on the other hand.

[0122] For example, in an embodiment, the compounds provided herein can compete with p300 for binding to the hydrophobic MAD-box domain of MEF2, thereby preventing p300 from binding to a target gene, thereby inhibiting the function of p300.

[0123] For example, myocardial hypertrophy can be divided into physiological hypertrophy and pathological hypertrophy. Physiological hypertrophy refers to the normal mechanism of increase in heart size during growth and exercise training after birth; while pathological hypertrophy refers to the increase in heart size caused by the continuous increase in workload caused by diseases such as hypertension and valvular dysfunction. According to statistics, the global incidence of myocardial hypertrophy is as high as about 1 / 500. Myocardial hypertrophy may lead to cardiac systolic dysfunction, myocardial stiffness, fibrosis, circulatory failure and increased risk of death. Myocardial hypertrophy is also a common precursor to heart failure, especially heart failure with preserved ejection fraction, which is an extremely common and fatal disease with no effective treatment. 10% of patients with myocardial hypertrophy may die of heart failure or sudden cardiac death.

[0124] Recent studies have found that cardiac hypertrophy is driven by changes in myocardial gene expression that require the MEF2 family of DNA-binding transcription factors and the nuclear lysine acetyltransferase (KAT) p300. MEF2 acetylation is essential for the development and maintenance of pathological cardiac hypertrophy. In individuals with cardiac hypertrophy, the levels of both MEF2 and p300 are significantly increased, which may be due to the formation of the MEF2 transcription complex with p300 and the promotion of type II HDAC from the nucleus to the cytoplasm to separate from MEF2. In this case, HDAC inhibitors will no longer be applicable.

[0125] In an embodiment, in an individual with cardiac hypertrophy caused by aortic constriction, the compounds provided herein can block the interaction between MEF2 and p300, thereby inhibiting the abnormal activation of MEF2 and p300, thereby alleviating or even reversing cardiac hypertrophy without impairing physiological adaptation.

[0126] Example

[0127] The compounds of the present invention can be prepared using the reactions and techniques described below, as well as conventional techniques known to those skilled in the art of organic synthesis or variations thereof as understood by those skilled in the art. The reaction can be carried out in a solvent suitable for the reagents and materials used and suitable for being affected to undergo transformation. Preferred methods include, but are not limited to, those described below.

[0128] The present invention is further illustrated by the following non-limiting examples which describe preferred modes of carrying out the invention. These are not intended to limit the scope of the invention in any way.

[0129] The examples given in the 1 H NMR spectral data (see below) were recorded using a 400 MHz spectrometer (Bruker AVANCE-400) and are reported in delta scale. Unless otherwise stated, the solvent used for NMR was CDCl3 with TMS as internal standard.

[0130] The expression "B is used instead of A" used in describing a synthesis example means that B is used instead of A in an equimolar equivalent.

[0131] Synthesis Example 1: Synthesis of Compound 1

[0132]

[0133] 1.1 Synthesis of intermediate 2

[0134] Intermediate 1 (6.4 g, 38 mmol) was added to 50 mL of DMF, followed by cyclopropane bromide (9.18 g, 76 mmol), and finally Cs2CO3 (37 g, 114 mmol) and KI (631 mg, 3.8 mmol). The mixture was heated to 150 ° C in a sealed tube for 16 hours. The reaction mixture was cooled to room temperature, diluted with 150 mL of water, and extracted with EA (50 mL*3). The combined organic phases were washed with brine (100 mL*2), water (100 mL*2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography, eluted with EA / PE=1 / 50 to obtain intermediate 2 (6.1 g) as a colorless oil.

[0135] 1.2 Synthesis of intermediate 4

[0136] To a 200 mL DMF solution of intermediate 3 (21.5 g, 100 mmol) was added N, O-dimethylhydroxylamine hydrochloride (14.6 g, 150 mmol), followed by HATU (57 g, 150 mmol). The resulting mixture was cooled in an ice-water bath, DIEA (38.7 g, 300 mmol) was added dropwise, and after the addition was complete, the resulting mixture was stirred at 20-30 ° C for 16 hours. The mixture was diluted with 600 mL of water and extracted with EA (200 mL * 3). The combined organic phases were washed with brine (500 mL * 2), water (500 mL * 2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography, eluted with EA / PE = 1 / 3 to obtain intermediate 4 (20 g) as a light yellow oil.

[0137] 1.3 Synthesis of intermediate 5

[0138] n-BuLi (4.8 mL, 12.19 mmol) was added to a 20 mL anhydrous tetrahydrofuran solution of intermediate 2 (2 g, 9.38 mmol) cooled to -78 ° C under nitrogen, keeping the temperature below -75 ° C, and the mixture was stirred at -78 ° C for 1 hour. Then, a 10 mL THF solution of intermediate 4 (2.4 g, 9.38 mmol) was added dropwise, keeping the temperature below -75 ° C. After the addition was complete, the mixture was slowly warmed to room temperature, then quenched with saturated NH4Cl solution (50 mL), and extracted with EA (30 mL * 3). The combined organic phase was washed with brine (50 mL * 2) and water (50 mL * 2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography, eluting with EA / PE = 1 / 9 to obtain intermediate 5 (500 mg) as a light yellow oil.

[0139] 1.4 Synthesis of intermediate 6

[0140] At 0°C, NaBH4 (228 mg, 6.04 mmol) was added to a 30 mL MeOH solution of intermediate 5 (1 g, 3.02 mmol). The resulting mixture was stirred for 30 minutes at 0°C and then quenched with a saturated NH4Cl solution (50 mL). The reaction mixture was extracted with EA (30 mL*3). The combined organic phases were washed with water (50 mL*2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give intermediate 6 (1 g) as a brown oil, which was used in the next step without further purification.

[0141] 1.2 Synthesis of intermediate 7

[0142] To a 10 mL DCM solution of intermediate 6 (1 g, crude product), Et3SiH (3.4 g, 30 mmol) and BF3.Et2O (3.4 g, 24 mmol) were added. The resulting mixture was stirred at room temperature for 3 hours, the mixture was quenched with Na2CO3 solution until pH = 8, and then extracted with DCM (20 mL * 2). The combined organic phase was washed with water (50 mL * 2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain intermediate 7 (1.2 g) as a brown oil, which was used in the next step without further purification.

[0143] 1.3 Synthesis of intermediate 8

[0144] To a 10 mL DMF solution of intermediate 7 (1.2 g, crude product) was added methyl acrylate (1.51 g, 15.09 mmol), followed by PPh (791 mg, 3.02 mmol) and TEA (915 mg, 9.06 mmol), the mixture was replaced 3 times with N, then 300 mg Pd (OAc) was added, and the resulting mixture was heated to 100 ° C for 16 hours in a sealed tube under N. The reaction mixture was cooled to room temperature, diluted with 50 mL of water, and extracted with EA (20 mL * 3). The combined organic phases were washed with brine (50 mL * 2), water (50 mL * 2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography, eluted with EA / PE = 1 / 9 to give intermediate 8 (360 mg) as a yellow oil.

[0145] 1.4 Synthesis of intermediate 9

[0146] To the 10mL THF solution of intermediate 8 (360mg, crude product), 5mL water was added, and then 420mg LiOH.H2O (10mmol) was added. The resulting mixture was stirred at room temperature for 3 days. The reaction mixture was extracted with 10mL ether, and then the aqueous phase was acidified to pH=3 with 1N HCl, and then extracted with EA (10mL*3). The combined organic phase was washed with water (20mL*2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Intermediate 9 (180mg) was obtained as a yellow oil, which was used in the next step without further purification.

[0147] 1.5 Synthesis of compound 1

[0148] To a 10 mL DMF solution of intermediate 9 (180 mg, 0.62 mmol) was added benzene-1,2-diamine (80 mg, 0.75 mmol), followed by HATU (354 mg, 0.93 mmol). The resulting mixture was cooled in an ice-water bath, DIEA (240 mg, 1.86 mmol) was added dropwise, and after the addition was complete, the resulting mixture was stirred at 20-30°C for 16 hours. The mixture was diluted with 30 mL of water and extracted with EA (20 mL*3). The combined organic phases were washed with brine (50 mL*2), water (50 mL*2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography, eluted with EA / PE=1 / 1, and then further purified by Pre-HPLC to obtain compound 1 (51 mg, yield 21%) as a light yellow solid.

[0149] 1HNMR (400MHz, CDCl3) δ9.36 (s, -NH, 1H), 7.17~7.54 (m, ArH, -CH=CH, 7H), 6.58~6.88 (m, ArH, -CH =CH, 7H), 4.95 (s, -NH2, 2H), 3.76~3.78 (m, -CH, 1H), 2.90 (s, -CH2, 4H), 0.60~0.75 (m, -CH2, 4H). ESI:m / z 399.1[M+H] + .

[0150] Synthesis Example 2: Synthesis of Compounds 2 and 3

[0151]

[0152] 2.1 Synthesis of intermediate 10

[0153] Intermediate 2 was synthesized in the same manner as in Synthesis Example 1. Intermediate 2 (2.13 g, 10 mmoles) was added to 20 mL of anhydrous tetrahydrofuran and cooled to -78 ° C under nitrogen protection. n-BuLi (4.8 mL, 1.2 equivalents) was added dropwise. After the addition was complete, stirring continued at -78 ° C for 1 hour. 3.6 g of DMF was added dropwise. After the addition was complete, the temperature was gradually raised to room temperature and stirred for 1 hour. The reaction solution was quenched with 20 mL of saturated aqueous ammonium chloride solution and extracted with ethyl acetate (20 mL * 3). The ethyl acetate phase was backwashed twice with saturated brine (100 mL * 2), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (EA / PE = 1 / 50) to obtain intermediate 10 (1.3 g) as a yellow oil.

[0154] 2.2 Synthesis of intermediate 12

[0155] 12.5 g of intermediate 11 and 9.1 g of triethyl phosphite were uniformly mixed and heated to 140° C. for 5 hours. The reaction solution was cooled to room temperature to obtain intermediate 12, which was used in the next step without further purification.

[0156] 2.3 Synthesis of intermediate 13

[0157] Intermediate 10 (1.62 g) was added to 20 mL of anhydrous tetrahydrofuran, 480 mg of sodium hydride was added under nitrogen protection, and intermediate 12 (2.86 g) was added after stirring at room temperature for 15 minutes, and stirring was continued at room temperature for 16 hours. The reaction solution was quenched with 20 mL of saturated aqueous ammonium chloride solution and extracted with ethyl acetate (20 mL*3). The ethyl acetate phase was backwashed twice with saturated brine (100 mL*2), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (EA / PE=1 / 50) to obtain intermediate 13 (2.7 g) as a yellow oil.

[0158] 2.4 Synthesis of intermediates 14 and 15

[0159] Intermediate 13 (294 mg) was added to 2 mL of tetrahydrofuran, and 0.1 mL of 10 N BH3.DMS solution was added. After stirring at room temperature for two hours, 1.7 mL of 3N sodium hydroxide aqueous solution was added dropwise, followed by 1 mL of 30% hydrogen peroxide, and stirred at room temperature for 5 hours. The reaction solution was quenched with 20 mL of saturated ammonium chloride aqueous solution and extracted with ethyl acetate (20 mL*3). The ethyl acetate phase was backwashed twice with saturated brine (100 mL*2), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (EA / PE=1 / 2) to obtain a mixture of intermediates 14 and 15 (82 mg) as a yellow oil.

[0160] 2.5 Synthesis of intermediates 16 and 17

[0161] The mixture of intermediates 14 and 15 (333 mg) was dissolved in 10 mL DCM, 430 mg PCC was added, and stirred at room temperature for 2 hours. After the reaction was completed, 2 g of 200-300 mesh silica gel was added to the reaction solution, and the mixture was concentrated to dryness and directly subjected to column chromatography, eluted with EA / PE=1 / 3 to obtain a mixture of intermediates 16 and 17 (256 mg) as a yellow oil.

[0162] 2.6 Synthesis of intermediates 18 and 19

[0163] The mixture of intermediates 16 and 17 (1 g) was dissolved in 10 mL of DMF, methyl acrylate (1.94 g, 15.09 mmol) was added, and then Pd(OAc)2 (200 mg), Pd2(dba)3 (100 mg), PPh3 (786 mg, 3.02 mmol), TEA (604 mg, 6.04 mmol) were added at once, and heated to 100 ° C in a sealed tube for 16 hours. After the reaction was completed, 30 mL of water was added and extracted with ethyl acetate (20 mL*3). The ethyl acetate phase was backwashed twice with saturated brine (100 mL*2), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (EA / PE=1 / 10) to obtain a mixture of intermediates 18 and 19 (650 mg) as a yellow oil.

[0164] 2.7 Synthesis of intermediates 20 and 21

[0165] The mixture of intermediates 18 and 19 (650 mg) was added to 10 mL THF and 5 mL water. LiOH.H2O (509 mg, 12.1 mmol) was added and stirred at room temperature for 3 days. The reaction solution was extracted with 20 mL ether, the organic phase was discarded, the aqueous phase was adjusted to pH = 3 with 1N HCl, and then extracted with ethyl acetate (10 mL*3). The ethyl acetate phase was backwashed twice with saturated brine (100 mL*2), dried over anhydrous sodium sulfate, filtered and concentrated. A mixture of intermediates 20 and 21 (200 mg) was obtained.

[0166] 2.8 Synthesis of compounds 2 and 3

[0167] The mixture of intermediates 20 and 21 (200 mg) was added to 10 mL of DMF, and o-phenylenediamine (67 mg), HATU (283 mg), and DIEA (120 mg) were added in sequence, and stirred at room temperature for 16 hours. After the reaction was completed, 30 mL of water was added and extracted with ethyl acetate (20 mL*3). The ethyl acetate phase was backwashed twice with saturated brine (100 mL*2), backwashed twice with water (100 mL*2), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was first purified by Pre-HPLC to obtain a mixture of compounds 2 and 3 (150 mg). Separation was performed by Chiral-HPLC to obtain compound 2 (46 mg) and compound 3 (53 mg).

[0168] Compound 2:

[0169] 1HNMR (400MHz, CDCl3) δ9.39 (s, -NH, 1H), 7.32~7.73 (m, ArH, -CH=CH, 10H), 6.58~6.92 (m, ArH, -CH =CH, 4H), 4.96 (s, -NH2, 2H), 4.46 (s, -CH2, 2H), 3.93~3.94 (m, -CH, 1H), 0.67~0.83 (m, -CH2, 4H). ESI:m / z413.2[M+H] + .

[0170] Compound 3:

[0171] 1 HNMR (400MHz, CDCl3) δ9.42 (s, -NH, 1H), 7.23~8.30 (m, ArH, -CH=CH, 7H), 6.59~7.06 (m, ArH, -CH =CH, 7H), 4.97 (s, -NH2, 2H), 4.42 (s, -CH2, 2H), 3.79~3.80 (m, -CH, 1H), 0.63~0.78 (m, -CH2, 4H). ESI:m / z413.2[M+H] + .

[0172] Synthesis Example 3: Synthesis of Compounds 4 and 5

[0173]

[0174] 3.1 Synthesis of intermediates 22 and 23

[0175] Intermediates 18 and 19 were synthesized in the same manner as in Synthesis Example 2. A mixture of intermediates 18 and 19 (1 g) was added to 10 mL of dichloromethane, followed by 1,2-dithiol (426 mg) and boron trifluoride etherate (642 mg), and stirred at room temperature for 16 hours. After the reaction was completed, 30 mL of water was added, stirred for 30 minutes, the dichloromethane phase was separated, and concentrated to dryness to obtain a mixture of intermediates 22 and 23 (2.2 g), which was used directly in the next step without purification.

[0176] 3.2 Synthesis of intermediates 24 and 25

[0177] NIS (2.4g) is added to 20mL anhydrous DCM, cooled to -78°C under nitrogen protection, hydrofluoric acid pyridine solution (2.67g) is added dropwise, and stirred at -78°C for 10 minutes. The mixture (2.2g) of intermediates 22 and 23 is dissolved in 10mL DCM, and added dropwise to the above-mentioned reaction solution, the temperature is maintained at -78°C, and reacted at -78°C for 30 minutes. Then it is slowly warmed to room temperature and the reaction is continued for 2 hours. After the reaction is completed, water (30mL) is added to quench the reaction. Dichloromethane is separated, dried over anhydrous sodium sulfate, filtered and concentrated. A mixture (1g) of intermediates 24 and 25 is obtained, which is directly used in the next step without purification.

[0178] 3.3 Synthesis of intermediates 26 and 27

[0179] The mixture of intermediates 26 and 27 (1 g) was added to 10 mL of DMF, followed by tert-butyl acrylate (1.8 g), palladium acetate (200 mg), Pd2(dba)3 (100 mg), triphenylphosphine (741 mg), triethylamine (600 mg), and heated to 100 ° C in a sealed tube under nitrogen protection for 16 hours. After the reaction was completed, 30 mL of water was added and extracted with ethyl acetate (10 mL*3). The ethyl acetate phase was backwashed with saturated brine (30 mL*2), backwashed with water (30 mL*2), dried over anhydrous sodium sulfate, filtered and concentrated. Purification by column chromatography (EA / PE=1 / 5) gave a mixture of intermediates 26 and 27 (560 mg).

[0180] 3.4 Synthesis of intermediates 28 and 29

[0181] The mixture of intermediates 28 and 29 (560 mg) was dissolved in 10 mL DCM, 2 mL TFA was added, and stirred at room temperature for 16 hours. After the reaction was completed, it was concentrated directly to give a mixture of intermediates 28 and 29 (630 mg), which was used directly in the next step without purification.

[0182] 3.5 Synthesis of compounds 4 and 5

[0183] The mixture of intermediates 28 and 29 (630 mg) was added to 10 mL of DMF, followed by o-phenylenediamine (197 mg), HATU (695 mg) and DIEA (354 mg), and stirred at room temperature for 16 hours. After the reaction was completed, 30 mL of water was added and extracted with ethyl acetate (20 mL*3). The ethyl acetate phase was backwashed twice with saturated brine (100 mL*2), backwashed twice with water (100 mL*2), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was first purified by Pre-HPLC and then prepared and separated by Chiral-HPLC to obtain compound 4 (16 mg) and compound 5 (19 mg).

[0184] Compound 4:

[0185] 1 HNMR (400MHz, CDCl3) δ9.41 (s, -NH, 1H), 7.35~7.48 (m, ArH, -CH=CH, 7H), 6.56~7.21 (m, ArH, -CH=CH, 7H), 4.97 (s, -NH2, 2H), 3.83~3.86 (m, -CH, 1H), 3.56~3.65 (t, -CH2, 2H) (m, -CH, 1H), 0.62~0.78 (m, -CH2, 4H). ESI:m / z435.2[M+H] + .

[0186] Compound 5:

[0187] 1 HNMR (400MHz, CDCl3) δ9.40 (s, -NH, 1H), 7.18~7.57 (m, ArH, -CH=CH, 7H), 6.57~6.94 (m, ArH, -CH=CH, 7H), 4.98 (s, -NH2, 2H), 3.69 ~ 3.71 (m, -CH, 1H), 3.55 ~ 3.63 (t, -CH2, 2H) (m, -CH, 1H), 0.57 ~ 0.72 (m, -CH2, 4H). ESI:m / z435.2[M+H] + .

[0188] Example 1: Metabolic Stability

[0189] 1.1 Identification of in vivo metabolites

[0190] 3.12 mg of compound 2 was dissolved in 6.24 mL of methanol to prepare a stock solution (concentration of 500 μg / mL) of compound 2. 1 μL of the stock solution was diluted with a diluent (acetonitrile: water = 1:1, V / V) to obtain a test sample dilution solution with a concentration of 500 ng / mL.

[0191] The test sample dilution was administered to SD rats and beagle dogs by oral gavage. Blood was collected before and 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, and 24h after a single oral gavage. About 300μL of venous blood was collected from the rat eyeball, added to a centrifuge tube with sodium heparin precooled with ice water, and placed in an ice bath. After standing, centrifuge (4000rpm, 10min), divided into 50μL unit volumes, placed in sterile EP tubes, and stored at -80℃ for later use. 400μL of blood was collected from the radial vein of the upper limb of the beagle dog, added to a centrifuge tube with sodium heparin precooled with ice water, and placed in an ice bath. After standing, centrifuge (4000rpm, 10min), the upper plasma was taken, divided into 50μL unit volumes, placed in sterile EP tubes, and stored at -80℃ for later use.

[0192] The above sample was added with 200 μL of acetonitrile, centrifuged at 14000 rpm for 5 min, and the supernatant was collected and stored at -80°C for later use.

[0193] Take 200 μL of the sample before administration (blank control) and the sample at each time point after administration, mix them evenly, evaporate the solvent with nitrogen, add 100 μL of reconstitution solvent (water:acetonitrile 50:50), vortex for 5 minutes, centrifuge at 20000 rpm for 5 minutes, take the supernatant and measure.

[0194] A total of 17 metabolites of compound 2 were identified in SD rat and beagle dog plasma samples using LC-HRMS / MS technology, including 17 metabolites (M1, M2, M3, M4, M5, M6, M7, M8, M9, M10, M11, M12, M13, M14, M15, M16, and M17) in SD rat plasma; and 2 metabolites (M1 and M14) were identified in beagle dog plasma. Based on the inferred metabolite structures, the possible metabolic pathways of compound 2 in SD rats and beagle dogs are as follows: Figure 1 The relevant data of metabolites are summarized in Table 1 below.

[0195] Table 1

[0196]

[0197] 1.2 Identification of in vitro metabolites

[0198] 3.12 mg of compound 2 was dissolved in 6.24 mL of methanol to prepare a stock solution (concentration of 500 μg / mL) of compound 2. 1 μL of the stock solution was diluted with a diluent (acetonitrile: water = 1:1, V / V) to obtain a test sample dilution solution with a concentration of 500 ng / mL.

[0199] 1 μL of the test article dilution, 158.5 μL of liver microsome (mouse (MI), rat (R), beagle dog (D), cynomolgus monkey (MO), mini pig (P) and human (H)) solution (148.5 μL 1x PBS buffer (pH 7.4), 10 μL of liver microsomal enzyme), 20 μL UDPGA PBS solution, 20 μL NADPH PBS solution and 0.5 μL Lamethicin methanol solution were gently mixed and incubated in a 37°C water bath for 30 min and 60 min.

[0200] Add 400 μL of acetonitrile (4°C), vortex for 3 min, centrifuge at low temperature (14000 rpm, 4 min, 4°C), take 400 μL of the supernatant, blow off the solvent with nitrogen, reconstitute with 100 μL of 50% acetonitrile water, vortex for 5 min, centrifuge (24000 rpm, 5 min, 4°C), take the supernatant, and perform LC-MS / MS analysis.

[0201] A total of 7 metabolites of compound 2 were identified in the liver microsome incubation samples of 6 different species of animals using LC-HRMS / MS technology, including 6 metabolites (M10, M11, M13, M14, M15, M17) in mouse liver microsomes; 5 metabolites (M13, M14, M15, M16, M17) in rat liver microsomes; 1 metabolite (M18) in beagle liver microsomes; 2 metabolites (M13, M17) in mini pig liver microsomes; no metabolites were identified in cynomolgus monkey liver microsomes and human liver microsomes. Based on the inferred metabolite structure, the possible metabolic pathways of compound 2 in liver microsomes are as follows: Figure 2 The relevant data of metabolites are summarized in Table 2 below.

[0202] Table 2

[0203]

[0204]

[0205] 1.3 Metabolic stability

[0206] 2.34 mg of compound 2 was dissolved in 4.68 mL of methanol to prepare a stock solution of compound 2 (concentration of 500 μg / mL). 5 μL of the stock solution and 45 μL of blank plasma (1 μg / mL) of 6 species of human (H), rhesus monkey (Mo), beagle dog (D), mini pig (P), rat (R) and mouse (Mi) were placed in a centrifuge tube, vortexed for 1 min, and then incubated in a 37°C water bath for 2 min, 5 min, 15 min, 30 min, 60 min, and 90 min. Three parallel samples were set at each time point. Immediately thereafter, 150 μL of glacial acetonitrile solution (containing internal standard amiodarone) was added to terminate the reaction, vortexed for 5 min to precipitate the protein, centrifuged at low temperature (14000 rpm, 4 min, 4°C), and 150 μL of the supernatant was taken for LC-MS / MS analysis. The following shows the changes in the plasma content of compound 2 in 6 species (Table 3), as well as the half-life and clearance rate (Table 4). Figure 3A and Figure 3B The remaining percentage of compound 2 in the plasma of 6 species is shown in Figure 3A The remaining percentage of compound 2 in the plasma of human (H), rhesus monkey (Mo), beagle dog (D), mini pig (P), and rat (R) is shown. Figure 3B The remaining percentage of Compound 2 in the plasma of mice (Mi) is shown. Figures 4A to 4C The content of the metabolite M1 (m / z=323.1278) of compound 2 in plasma of 6 species is shown. Figure 4A The figure shows the changes in the content of metabolite M1 in the plasma of beagle dogs (D), mini pigs (P), rhesus monkeys (Mo) and mice (Mi) over time. Figure 4B The changes in the content of metabolite M1 in the plasma of rats (R) over time are shown, and Figure 4C The changes in the content of the metabolite M1 in the plasma of human (H) over time are shown.

[0207] Table 3

[0208]

[0209]

[0210] Table 4

[0211] K <![CDATA[t 1 / 2 (min)]]> <![CDATA[t 1 / 2 (h)]]> Beagle(D) 0.0230 30.1304 0.5022 Human (H) 0.0156 44.4231 0.7404 Mouse (Mi) 0.1605 4.3178 0.0720 Monkey(Mo) 0.0180 38.5000 0.6417 Pig (P) 0.0097 71.4433 1.1907 Rat (R) 0.0080 86.6250 1.4438

[0212] Example 2: In vivo toxicity experiment

[0213] 2.1 Animal husbandry

[0214] A total of 24 (12 females and 12 males) 6-7 week old SD rats (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were housed in polypropylene rat group cages. The control range of the main environmental indicators was: room temperature 20-26°C, relative humidity 40-70%. The minimum ventilation rate was 15 times / hour, artificial lighting, light: dark = 12h:12h. The cage specifications were: cage specifications: 545mm×395mm×200mm, no more than 5 rats per box. The animals were allowed to eat and move freely during the entire breeding process.

[0215] 2.2 Animal grouping and drug administration

[0216] 24 animals were divided into groups according to the following method, and the vehicle (control group), low dose (low dose group) and medium dose (medium dose group) of compound 2 were gavaged at 10 mg / kg to SD rats, respectively, and the administration was continued for 14 days, and the day of administration was D1. During this period, the cage was observed twice a day, and the observation content included the appearance of the animals, fur, physical signs, behavioral activities, respiratory status, glandular secretion, animal posture, fecal characteristics, death, etc. The body weight was measured once a week. The 24-hour feed consumption was measured once a week.

[0217]

[0218]

[0219] 2.3 Clinical pathological examination

[0220] Hematological indicators: red blood cell count, hemoglobin, hematocrit, mean red blood cell volume, mean hemoglobin content, mean hemoglobin concentration, red blood cell distribution width, white blood cell count, neutrophil percentage, lymphocyte percentage, monocyte percentage, eosinophil percentage, basophil percentage, platelet count, hematocrit, mean platelet volume, platelet distribution width, reticulocyte count, etc.

[0221] Serum biochemical indicators: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatine kinase, lactate dehydrogenase, urea, uric acid, creatinine, total protein, albumin, globulin, A / G, glucose, total bilirubin, total cholesterol, triglycerides, calcium ion concentration, potassium ion concentration, chloride ion concentration, and sodium ion concentration.

[0222] Coagulation indicators: prothrombin time, activated partial thromboplastin time.

[0223] Histopathological examination: D15 test.

[0224] Organs to be weighed: heart, liver, spleen, kidneys, brain, adrenal glands, thymus, testicles, epididymis, ovaries, and uterus.

[0225] Fixed and preserved tissues and organs: Gross observation of abnormal tissues and organs for histopathological examination.

[0226] According to general observation, during the experiment, there were no abnormalities in the appearance, behavior, respiratory status, fecal characteristics, etc. of the animals in each group. After administration, there was no significant difference in the body weight of male mice in each administration group compared with the control group (P>0.05). The food intake of female mice in the low-dose group and male mice in the medium-dose group was slightly lower than that of the control group in the first week, and returned to normal in the second week.

[0227] According to clinical pathological examination, after SD rats were gavaged with compound 2 (doses were 50 and 500 mg / kg, respectively) for 14 consecutive days, some clinical pathological indicators and organ weights fluctuated, but there was no clear toxicological significance.

[0228] Example 3: Mechanism studies

[0229] 3.1 Determination of the half-maximal inhibitory concentration of HDAC1

[0230] HDAC1 Kit: Abnova TM HDAC1 Inhibitor Screening Assay Kit. It includes: a. 1 bottle of HDAC assay buffer 10X, b. 1 bottle of HDAC1 (human recombinant), c. 1 bottle of HDAC Trichostatin A, d. 1 bottle of HDAC substrate, e. 1 plate of 96-well plate (black), f. 2 bottles of HDAC developer, g. 1 piece of 96-well cover sheet.

[0231] Test product: Compound 2

[0232] Determination process:

[0233] In a 96-well plate, 3 wells were added with 150 μL of assay buffer and 10 μL of DMSO as controls, 3 wells were added with 140 μL of assay buffer, 10 μL of diluted HDAC1 and 10 μL of DMSO as 100% initial activity wells, and 15 wells were added with 140 μL of assay buffer, 10 μL of diluted HDAC1 and 10 μL of test dilution (serial dilution of compound 2, solvent is DMSO, concentrations are 100 μM, 50 μM, 25 μM, 12.5 μM and 6.2 μM, 3 wells for each concentration). The reaction was started by adding 10 μL of HDAC substrate to all wells used. Cover the plate with a cover slip and incubate on a shaker at 37 ° C for 30 minutes. Remove the cover slip and add 40 μL of developer. Cover the plate again and incubate at room temperature for 15 minutes.

[0234] Fluorescence was read using an excitation wavelength of 340-360 nm and an emission wavelength of 440-465 nm, graphed, and the half inhibitory concentration (IC 50 ).

[0235] Figure 5A The inhibition curve of compound 2 on HDAC1 is shown. Figure 5A Calculation of IC of compound 2 against HDAC1 50 The inhibitory effect of compound 2 on HDAC1 was 16.8 μM.

[0236] 3.2 Determination of the half-maximal inhibitory concentration for the interaction between MEF2 and HDAC4

[0237] Test product: Compound 2

[0238] Determination process:

[0239] 293T cells were plated in 24-well plates with a cell density of 40-50%. After adherence, 1 μg pVP16-HDAC4 plasmid, 1 μg pM-MEF2 plasmid, 0.5 μg GK-luciferase plasmid, and 0.1 μg Renilla plasmid were transfected into each well using PEI transfection reagent.

[0240] 24 hours after transfection, the cells were treated with solvent (as a control) and test sample dilutions (serial dilutions of compound 2, with concentrations of 0.07 μM, 0.15 μM, 0.3 μM, 0.6 μM, 1.25 μM, 2.5 μM and 5 μM, respectively); 24 hours after treatment, the cells were harvested and prepared for subsequent measurements.

[0241] Mix the reporter gene cell lysate thoroughly, aspirate the cell culture medium, and add 100 μl of reporter gene cell lysate. After sufficient lysis, centrifuge at 10,000-15,000 g for 3-5 minutes, and take the supernatant for measurement.

[0242] Dissolve the Firefly Luciferase Assay Reagent and Renilla Luciferase Assay Buffer and bring to room temperature. Place the Renilla Luciferase Assay Substrate (100×) on ice or in an ice box until ready for use.

[0243] According to the amount of 100 μL required for each sample, take an appropriate amount of Renilla luciferase assay buffer and add Renilla luciferase assay substrate (100×) at a ratio of 1:100 to prepare the Renilla luciferase assay working solution.

[0244] Use a chemiluminometer or a multifunctional microplate reader with chemiluminescence detection function to measure each sample. Add 100 microliters of firefly luciferase detection reagent, mix well and measure RLU (relative light unit). Use reporter gene cell lysate as blank control.

[0245] After completing the above firefly luciferase assay steps, add 100 μl of Renilla luciferase assay working solution, mix well, and then measure RLU.

[0246] When Renilla luciferase was used as the internal reference, the RLU value obtained by the firefly luciferase assay was divided by the RLU value obtained by the Renilla luciferase assay. The activation degree of luciferase between different samples was compared based on the obtained ratio.

[0247] Figure 5B The inhibition curve of compound 2 on the interaction between MEF2 and HDAC4 is shown. Figure 5B Calculation of IC of compound 2 on the interaction between MEF2 and HDAC4 50 It was 0.15 μM, indicating that compound 2 had a significant inhibitory effect on the interaction between MEF2 and HDAC4.

[0248] Example 4: Experiment on reversing cardiomyocyte hypertrophy

[0249] 4.1. Primary cell culture:

[0250] SD rats and mice born 1-3 days old were selected for the experiment. Under normal circumstances, the cardiomyocytes obtained from every 7 mice can be inoculated into a T25 culture bottle. There are about 30 mice each time. Take out the mice, pinch off their cervical vertebrae to kill them, and put them in a jar filled with 75% alcohol to soak for 1 minute for sterilization, and then put them in the prepared culture dish. Use large tweezers to clamp the killed and sterilized mice, put them in a supine position in the culture dish and fix them. Use ophthalmic scissors to cut the sternum of the mice from the lower edge of the sternum to the neck along the midline of the sternum of the mice slightly to the left (when cutting the sternum, the front end of the ophthalmic scissors should be lifted up as much as possible to avoid cutting the heart) to expose its chest cavity. Use tweezers to squeeze the back of the mice from bottom to top, so that the beating heart can be squeezed out of the chest cavity. Cut off the front ventricular muscle part (apex, about 1 / 3 of the heart) directly, put the removed myocardial tissue into an ampoule filled with pre-cooled PBS, wash it twice, and set aside.

[0251] Cut the tissue into pieces of 1-3 mm in a small amount of PBS, wash it again with cold PBS, transfer the tissue to a 50 mL centrifuge tube, remove the residual PBS as much as possible, add 5 ml of digestion solution, and seal it with sealing film. Digest it in a 37°C water bath with uniform shaking (130 rpm / min) for 10 min, collect the supernatant with a sieve (to avoid sucking the tissue), terminate the digestion, centrifuge at 1200 rpm / min (200 g) at room temperature for 5 min, and wash the cells once with serum-free H-DMEM medium (except EDTA in trypsin).

[0252] Remove the supernatant, resuspend the cells in 10 ml H-DMEM medium containing 10% fetal bovine serum, and inoculate them in a culture dish. Incubate in a 37°C, 5% CO2 incubator for 4 hours for differential attachment to remove fibroblasts mixed with cardiomyocytes as much as possible, and aspirate the supernatant to inoculate in a 24-well plate. After culturing in a 37°C, 5% CO2 incubator for 24 hours, replace with fresh H-DMEM medium containing 10% fetal bovine serum and 0.05 mM BrdU.

[0253] 4.2 Purity Identification:

[0254] After culturing in a 37°C, 5% CO2 incubator for 48 hours, the beating of the cardiomyocytes was observed and the purity was identified by α-SMA immunofluorescence staining.

[0255] 4.3 Hypertrophy induction:

[0256] Identify cardiomyocytes with a purity greater than 90% for hypertrophy induction using 10 -7 mol / L angiotensin II was used to induce cardiomyocyte hypertrophy for 48 hours, and the size of the cells was observed.

[0257] 4.4 Drug treatment:

[0258] After 48 hours, A) the cells were treated with a high dose (H, 10 μM) of comparative compound 1 (structure shown below) and compound 1-2 (structure shown below); B) the cells were treated with different doses (high dose (H, 10 μM), medium dose (M, 1 μM), and low dose (L, 0.1 μM)) of compound 3-5. After 48 hours of treatment, the cells were stained to observe cell hypertrophy, including cell morphology, degree of fusion, and beating.

[0259]

[0260] Fig. 6A The immunofluorescence images of cardiomyocytes before and after induction and after treatment with the same concentration of comparative compound 1 and compound 1-2 are shown. Figure 6BThe average area of ​​cardiomyocytes before induction, after induction, and after treatment with the same concentration of comparative compound 1 (CC1 H) and compounds 1 (C1 H) and 2 (C2H) is shown. It can be seen that after the addition of Ang II, the size of cardiomyocytes increased significantly, while after treatment with high doses (10 μM) of comparative compound 1 and compound 1-2, the size of cardiomyocytes decreased significantly, even smaller than the size of cardiomyocytes before induction, and the effect of compound 1-2 in reversing cardiomyocyte hypertrophy was significantly better than that of comparative compound 1. Compared with comparative compound 1, compounds 1 and 2 have better binding affinity to MEF due to the introduction of lower chain alkyl between the two benzene rings, thereby showing a better effect of reversing cardiomyocyte hypertrophy.

[0261] Figure 7 The average area of ​​cardiomyocytes before and after induction and after treatment with different concentrations of compounds 3-5 (C3 L, C3M, C4 L, C4 M, C4 H, C5 L, C5 M, C5 H) is shown. The results show that the effect of low concentrations of compounds 3 and 4 in reversing cardiomyocyte hypertrophy is not obvious, but has a certain improvement effect; cardiomyocytes treated with medium concentrations of compounds 3 and 4 are already comparable to cardiomyocytes before induction, indicating that compounds 3 and 4 greater than 1μM can reverse cardiomyocyte hypertrophy; compound 5 greater than 0.1μM can already show a good effect of reversing cardiomyocyte hypertrophy.

[0262] Example 5: Experiment on alleviating myocardial hypertrophy and heart failure caused by TAC (aortic coarctation)

[0263] 5.1 TAC model preparation

[0264] Weigh the animal before surgery, anesthetize the animal with a small animal respiratory anesthesia machine, and fix it on the operating board in supine position after the animal shows anesthesia reactions such as loss of corneal reflex, decreased muscle strength, and flaccid paralysis. Prepare a stereo microscope, a small animal ventilator, and a pipeline. Ventilator parameter settings: respiratory rate 105-110 times / minute, tidal volume 2-3 ml / minute. Local skin disinfection. Cut the skin along the left edge of the sternum, separate the subcutaneous tissue layer by layer, cut the second rib along the left side of the sternum, separate the thymus tissue with toothless microtweezers, and peel and expose the ascending aorta. Between the first and second branches of the convex side branch of the aorta, pass the 6-0 silk thread through the aortic arch and tie the special smooth stainless steel round tube (diameter 0.75mm) along the direction of the aorta with the separated aortic arch. Then carefully remove the round tube, causing the ascending aorta to narrow, and then suture layer by layer. The diameter of the narrowed blood vessel is about 1 / 3 of the original blood vessel diameter. Postoperative analgesia, heat preservation, and close observation of the animal state.

[0265] 5.2 Experimental Animal Screening and Grouping

[0266] Within one week after surgery, all rats were subjected to ultrasound examination to calculate the blood flow velocity in the aortic arch, and animals with a uniform and stable blood flow velocity of 3 m / s were selected for the experiment. Among them, 12 animals were assigned to the control group, 10 animals were assigned to the positive drug (Betaloc) group, and 10 animals were assigned to the test drug (Compound 2) group.

[0267] 5.3 Drug treatment

[0268] One week after modeling, each group was treated according to the following scheme, once a day for 4 consecutive weeks.

[0269] Group dose Dosing volume Dosage regimen Control group —— 1ml / 100g Normal saline, intragastric administration Positive drug group 20 mg / kg / day 1ml / 100g Oral gavage Test drug group 50 mg / kg / day 1ml / 100g Oral gavage

[0270] 5.4 Efficacy evaluation

[0271] Rats were weighed twice a week;

[0272] Ultrasound examination: once one week after surgery (before medication), once two weeks after medication, and once before sampling four weeks after medication;

[0273] The heart was sampled for pathological experiments, HE staining and Masson staining.

[0274] The following Table 5 shows the left ventricular diastolic diameter (LVID; d) and end-systolic diameter (LVID; s) of the rat pressure overload type myocardial hypertrophy and heart failure model at each test time point. Fig. 8A and Figure 8B Also shown in the figure is a comparison of the left ventricular end-diastolic diameter and the left ventricular end-systolic diameter of rats. It can be seen that after modeling, the left ventricular internal diameter of each group of animals shows a trend of expansion with the modeling time, which shows that under the excessive volume load of the myocardium, the ventricular wall tension continues to increase, the myocardial sarcomeres proliferate in series, the myocardial cells grow, and the cardiac chamber volume increases. This indicator indicates the process of transition from myocardial hypertrophy to heart failure. After administration, the ventricular internal diameter of the control group is larger than that of other groups, and the increase trend is more obvious, while the ventricular internal diameter changes of the positive drug group and the test drug group are not obvious, especially the ventricular internal diameter changes of the test drug group are more minor, indicating that the compounds of the present application have a certain mitigating effect on the process of rat heart failure. From 2 weeks of administration to 4 weeks of administration, there are significant differences between the high-dose group and the control group (p<0.05p<0.01).

[0275] Table 5

[0276]

[0277] p<0.05*, p<0.01**

[0278] The following Table 6 shows the left ventricular end-diastolic volume (LV Vol; d) and end-systolic volume (LV Vol; s) of the rat pressure overload type myocardial hypertrophy heart failure model at each test time point. Fig. 9A and Fig. 9B The figure also shows the comparison of the left ventricular end-diastolic volume and left ventricular end-systolic volume of rats. It can be seen that after the aortic arch stenosis surgery, the heart will cause compensatory thickening of the ventricular wall due to pressure overload in the initial stage, and the heart function will also show compensatory enhancement. When entering the decompensation period, the heart contraction and diastolic tension will weaken, the ventricular volume will gradually expand, and the animals will gradually enter the heart failure stage. After 2 weeks of administration, the control group has entered the decompensation period, and the ventricular volume has increased significantly, while the ventricular volume of the test drug group has not increased significantly. It can be seen that the test drug group has a certain improvement on the cardiac function of the animals and slows down the development of heart failure. After 2 weeks of administration, compared with the control group, the left ventricular end-diastolic volume of the test drug group was significantly different (p<0.01). After 4 weeks of administration, compared with the control group, the left ventricular end-diastolic volume of the test drug group was significantly different (p<0.01).

[0279] Table 6

[0280]

[0281] p<0.05*, p<0.01**

[0282] The following Table 7 shows the ejection fraction (EF) and fractional shortening (FS) of the rat pressure overload type myocardial hypertrophy heart failure model at each test time point. Fig. 10A and Fig. 10B The comparison of the ejection fraction and shortening fraction of rats is also shown in the figure. It can be seen that after the aortic arch coarctation surgery, the heart will cause compensatory thickening of the ventricular wall due to pressure overload in the initial stage, and the cardiac function will also show compensatory enhancement. When entering the decompensation period, the cardiac function begins to decline. From 2 weeks after administration to 4 weeks after administration, the control group has entered the decompensation period, and the EF and FS values ​​have not continued to increase; the EF and FS values ​​of the positive drug group have declined to a certain extent; while the test drug group maintained a high level and did not decline. It can be seen that the test drug has a certain improvement effect on the cardiac function of animals and slows down the development of heart failure.

[0283] Table 7

[0284]

[0285]

[0286] In summary, the compounds provided herein have a certain improvement effect on the cardiac function of animals and slow down the development of ventricular remodeling and heart failure.

[0287] Example 6: Effects on stably overexpressed hERG channel currents

[0288] 6.1 Cell culture

[0289] HEK-293 cells stably expressing hERG potassium channel were used. The hERG potassium channel cells were purchased from Creacell (Cat. No. A-0320). The HEK293 cells stably expressing hERG potassium channel were cultured in DMEM medium containing 10% fetal bovine serum and 0.8 mg / mL G418 at 37°C and a carbon dioxide concentration of 5%.

[0290] Remove the old medium and wash once with PBS, then add 1 mL of TrypLE TM Express solution, incubate at 37℃ for about 0.5min. When the cells detach from the bottom of the dish, add about 5mL of complete medium preheated at 37℃. Use a pipette to gently blow the cell suspension to separate the aggregated cells. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 1000rpm for 5min to collect the cells. For expansion or maintenance culture, inoculate the cells in a 6cm cell culture dish, with 2.5×10 cells in each cell culture dish. 5 Cells (final volume: 5 mL).

[0291] 6.2 Patch clamp assay

[0292] Before the test, cells were treated with TrypLE TM Express separation, 4×10 3 The cells were plated onto coverslips and cultured in 24-well plates (final volume: 500 μL), and assayed after 18 hours.

[0293] The voltage stimulation scheme for whole-cell patch clamp recording of hERG potassium current is as follows: After the whole-cell seal is formed, the cell membrane voltage is clamped at -80mV. The clamping voltage is depolarized from -80mV to -50mV for 0.5s (as a leakage current detection), then stepped to 30mV for 2.5s, and then quickly restored to -50mV for 4s to stimulate the tail current of the hERG channel. Data is collected repeatedly every 10s to observe the effect of the drug on the hERG tail current. A 0.5s -50mV stimulation is used as a leakage current detection. The experimental data are collected by the EPC-10 amplifier (HEKA) and stored in the PatchMaster (HEKA) software.

[0294] Use a microelectrode puller to pull a capillary glass tube into a recording electrode. Under an inverted microscope, use the microelectrode manipulator to bring the recording electrode into contact with the cell, apply negative pressure and suction to form a GΩ seal. After the GΩ seal is formed, perform fast capacitance compensation, and then continue to apply negative pressure to break the cell membrane and form a whole-cell recording mode. Then perform slow capacitance compensation and record the membrane capacitance and series resistance. No leakage compensation is given.

[0295] When the hERG current recorded by the whole cell is stable, the drug administration (including positive drug cisapride and test drug compound 2) is started. The drug concentration shown below is applied to the cell for 5 minutes (or the current is stable) and then the next concentration is detected. Two concentrations are detected for each test compound. The coverslip with cells is placed in the recording bath in an inverted microscope. The test compound and the external solution without the compound are flowed through the recording bath from low concentration to high concentration in sequence by gravity perfusion to act on the cells. A vacuum pump is used for liquid exchange during recording. The current detected by each cell in the external solution without the compound serves as its own control group. At least two cells are used for each concentration and the test is repeated twice independently. All electrophysiological tests are performed at room temperature.

[0296] The results of the inhibition ratios of the positive drug cisapride and the test drug Compound 2 on hERG current are shown in Tables 8 and 9 below.

[0297] Table 8

[0298]

[0299] Table 9

[0300]

[0301] In this application, the general criteria are used to judge the inhibitory effect on hERG:

[0302] Very strong inhibition: IC 50 <0.1 μM

[0303] Strong inhibition: 0.1μM≤IC 50 ≤1μM

[0304] Moderate inhibition: 1 μM <IC 50 ≤10μM

[0305] Weak or no inhibition: IC 50 >10μM.

[0306] Inhibition of the rapidly activated potassium channels encoded by hERG will cause a prolongation of the repolarization time, which is manifested as a prolonged QT interval in the electrocardiogram, known as QT syndrome. Many drugs have withdrawn from the Chinese market due to their QT prolongation. Based on the above data, the positive drug cisapride IC50 The inhibition rate of the tested drug, compound 2 of the present application, at a concentration of 1 μM was 18.99% ± 5.36%, and at a concentration of 10 μM was 44.86% ± 1.78%, so IC 50 Above 10 μM, it has weak or no inhibitory effect on hERG channels.

[0307] Example 7: Myocardial infarction model drug efficacy experiment

[0308] 7.1 Test materials

[0309] Test drug: Compound 2. The oral solution of Compound 2 was prepared with physiological saline at a concentration of 10 mg / ml and used within 1.5 hours after preparation, and was administered orally at a concentration of 75 mg / kg.

[0310] Positive drug: Propranolol hydrochloride, Jiangsu Yabang Epson Pharmaceutical Co., Ltd. Batch number: E210630; Production date: 20210620; Validity period: 36 months; Specification 10 mg / tablet. Indications: As secondary prevention, reduce the mortality of myocardial infarction, hypertension, exertional angina pectoris, control supraventricular tachyarrhythmia, ventricular arrhythmia, hypertrophic cardiomyopathy, pheochromocytoma. Prepare the oral solution of the positive drug with normal saline at a concentration of 1.8 mg / ml, and administer it by gavage at 18 mg / kg.

[0311] Experimental animals: Animal strain: C57BL / 6j mice; Grade: SPF grade; Age: 8 weeks; Gender: 60 males; Animal production unit: Sibeifu (Beijing) Biotechnology Co., Ltd.

[0312] 7.2 Test methods

[0313] After 60 mice were fed with ordinary feed for one week, the left anterior descending coronary artery was ligated to construct a myocardial infarction model. After surgical ligation of the left anterior descending coronary artery, the electrocardiogram showed obvious ST segment elevation, proving that the model was successfully constructed. The animals with successful model construction were divided into a control group (20), a positive drug group (20), and a test drug group (20). The positive drug group was gavaged with an oral solution of propranolol hydrochloride at 18 mg / kg, once a day; the test drug group was gavaged with an oral solution of compound 2 at 75 mg / kg, once a day.

[0314] 7.3 Test indicators

[0315] ①Ultrasound examination: 3 days and 14 days after surgery

[0316] The left ventricular end-systolic diameter, left ventricular end-diastolic diameter, left ventricular posterior wall end-systolic thickness, left ventricular posterior wall end-diastolic thickness, left ventricular anterior wall end-systolic thickness, left ventricular anterior wall end-diastolic thickness, left ventricular ejection fraction, and shortening fraction were measured.

[0317] ② Myocardial enzyme test: 3 days and 14 days after surgery

[0318] CK-MB (creatine kinase isoenzyme) and ctnt (troponin) were measured.

[0319] ③After collecting materials:

[0320] 3 days after surgery: pathological staining and evaluation of myocardial fibrosis; detection of IL-6, TNF-α, and TGF-β in frozen tissues to evaluate inflammatory indicators.

[0321] 14 days after surgery: Pathological staining and evaluation of myocardial fibrosis, immunohistochemical CD31 staining to evaluate neovascularization.

[0322] In the following tables, *p<0.05, **p<0.01.

[0323] Table 10 Animal survival rate during the experiment

[0324]

[0325] Table 11 Ejection fraction (EF) and fractional shortening (FS) of mice in each group

[0326]

[0327] The results in Table 11 show that the ejection fraction (EF) and short-axis shortening rate (FS) of the test drug group 3 days and 14 days after surgery tended to be higher than those of the control group, but due to the large standard deviation, no difference was apparent; in terms of the course of disease, the ejection fraction and short-axis shortening rate of the control group were significantly reduced, while the ejection fraction and short-axis shortening rate of the positive drug group and the test drug group did not change much, and were basically the same 14 days after surgery and 3 days after surgery. Therefore, the test drug has a tendency to delay the progression of the disease.

[0328] Table 12 Diastolic thickness (LVAW; d) and systolic thickness (LVAW; s) of the left ventricular anterior wall of mice in each group

[0329]

[0330] The results in Table 12 show that 3 days after surgery, the diastolic thickness and systolic thickness of the left ventricular anterior wall in the test drug group were lower than those in the control group, but no significant difference was observed; 14 days after surgery, the diastolic thickness and systolic thickness of the left ventricular anterior wall in the test drug group were higher than those in the control group, but no significant difference was observed; from the perspective of the course of the disease, the thickness of the anterior wall in the test drug group had a tendency to gradually thicken.

[0331] Table 13 Diastolic thickness (LVPW; d) and systolic thickness (LVPW; s) of the left ventricular posterior wall of each group of mice

[0332]

[0333] The results in Table 13 show that 3 days after surgery, the diastolic thickness of the left ventricular posterior wall in the test drug group was lower than that in the control group, which was different (p<0.05), and the systolic thickness of the left ventricular posterior wall was also lower than that in the control group; but 14 days after surgery, compared with 3 days after surgery, the diastolic thickness and systolic thickness of the left ventricular posterior wall in the control group showed a significant thinning trend, the positive drug group also showed a thinning trend, but the test drug group did not show a thinning trend

[0334] Table 14 Left ventricular diastolic internal diameter (LVID; d) and systolic internal diameter (LVID; s) of mice in each group

[0335]

[0336] The results in Table 14 show that there was no significant difference in the trends of left ventricular diastolic internal diameter (LVID; d) and systolic internal diameter (LVID; s) between the test drug group, the positive drug group and the control group, whether 3 days or 14 days after surgery.

[0337] Table 15 Left ventricular end-diastolic volume (LV Vol; d) and end-systolic volume (LV Vol; s) of mice in each group

[0338]

[0339] The results in Table 15 show that there was no significant difference in the trend of left ventricular end-diastolic volume and end-systolic volume between the test drug group, the positive drug group and the control group, whether 3 days or 14 days after surgery.

[0340] Table 16 Left ventricular mass (LV Mass) and corrected left ventricular mass (LV Mass (corrected)) of mice in each group

[0341]

[0342] The results in Table 16 show that 3 days after surgery, the left ventricular weight of the test drug group was lower than that of the control group, which was significantly different (p<0.05); 14 days after surgery, the left ventricular weight of the test drug group was higher than that of the control group.

[0343] Table 17 ELISA test results of tissues 3 days after surgery

[0344]

[0345] The results in Table 17 show that none of the above inflammatory factors detected showed differences.

[0346] Table 18 Serum myocardial enzyme test results 3 days after surgery

[0347]

[0348]

[0349] The results in Table 18 show that compared with the control group, the ctnt in the serum of the test drug group increased significantly 3 days after surgery, while there was no difference in CK-MB.

[0350] Table 19 Collagen volume fraction of each group of mice

[0351]

[0352] The results in Table 19 show that there was no significant difference between the test drug group and the control group 3 days after surgery, but there was a trend of being lower than the control group 14 days after surgery, but no difference was shown.

[0353] Table 20 CD31 of each group of mice

[0354]

[0355] The results in Table 20 show that the number of new blood vessels in the test drug group tended to be higher than that in the control group 14 days after surgery, but no significant difference was observed.

[0356] Example 8: Ischemia-reperfusion model drug efficacy experiment

[0357] 8.1 Test materials

[0358] Test drug: Compound 2. The oral solution of Compound 2 was prepared with physiological saline at a concentration of 10 mg / ml and used within 1.5 hours after preparation, and was administered orally at a concentration of 75 mg / kg.

[0359] Positive drug: Edaravone, Nanjing Xiansheng Dongyuan Pharmaceutical Co., Ltd. Batch number: 80-190902; valid until: 20220907; specification 2ml: 10mg; dosage: 10min before reperfusion after coronary artery ligation, intravenous injection once, injection dose: 15mg / kg. Prepare the injection solution of the positive drug with physiological saline, and inject it into the tail vein at 15mg / kg.

[0360] Experimental animals: Animal strain: C57BL / 6j mice; Grade: SPF grade; Age: 8 weeks; Gender: 60 males; Animal production unit: Sibeifu (Beijing) Biotechnology Co., Ltd.

[0361] 8.2 Test methods

[0362] After 60 mice were adaptively fed with ordinary feed for one week, they were divided into a control group (20 mice), a positive drug group (20 mice), and a test drug group (20 mice) according to their body weight. The test drug group was pre-treated before the left anterior descending coronary artery ligation surgery, and the pre-treatment was administered by gavage at 75 mg / kg, once a day, for 7 consecutive days. After the end of the pre-treatment, all animals underwent left anterior descending coronary artery ligation surgery, and the positive drug group was injected with edaravone by tail vein 10 minutes before reperfusion.

[0363] 8.3 Test indicators

[0364] ①Ultrasonic testing was performed 24 hours after modeling;

[0365] ②After ultrasound, some animals underwent TTC+Evans Blue double staining to assess the myocardial infarction area;

[0366] ③ Some animals were sampled, the hearts were retained, and the fixed tissues were subjected to HE staining and fluorescent tunel+IF staining. The frozen tissues were used to detect IL-6, IL-1β, ROS, and TNF-α to evaluate inflammatory indicators.

[0367] Table 21 Animal survival rate during the experiment

[0368] Control group Positive drug group Test drug group Number of animals alive before sampling 18 16 18 Survival rate before sampling 90％ 80％ 90％

[0369] In the TTC+Evans Blue staining method, after collecting images, the Image-Pro Plus image analysis software was used to calculate the areas of non-infarcted myocardial tissue, risk zone tissue, and infarcted tissue, and the ratio of the infarcted tissue area to the risk zone tissue area was used to express the percentage of myocardial infarction area. Table 22 lists the percentage of myocardial infarction area in each group. The test drug group had a significantly lower percentage of infarction area compared with the control group, and was comparable to the positive drug group, with differences (P<0.05).

[0370] Table 22 Percentage of myocardial infarction area in each group

[0371]

[0372] The left ventricular end-systolic diameter, left ventricular end-diastolic diameter, left ventricular posterior wall end-systolic thickness, left ventricular posterior wall end-diastolic thickness, left ventricular anterior wall end-systolic thickness, left ventricular anterior wall end-diastolic thickness, left ventricular ejection fraction and shortening fraction of each group were measured by cardiac ultrasound.

[0373] Table 23 Ejection fraction (EF) and fractional shortening (FS) of mice in each group

[0374]

[0375] The results in Table 23 show that the ejection fraction (EF) and short-axis shortening rate (FS) of the test drug group and the positive drug group were significantly higher than those of the control group, and the ejection fraction (EF) and short-axis shortening rate (FS) of the test drug group were slightly higher than those of the positive drug group, but the test drug group had significant differences (p<0.01).

[0376] Table 24 Left ventricular diastolic (LVID; d) and end-systolic internal diameter (LVID; s) of mice in each group

[0377]

[0378]

[0379] The results in Table 24 show that the left ventricular diastolic (LVID; d) and end-systolic internal diameter (LVID; s) of the test drug group and the positive drug group were significantly lower than those of the control group, and the left ventricular diastolic (LVID; d) and end-systolic internal diameter (LVID; s) of the test drug group were also lower than those of the positive drug group, but the test drug group had significant differences (p<0.01).

[0380] Table 25 Left ventricular end-diastolic volume (LVVol; d) and end-systolic volume (LVVol; s) of each group of mice

[0381]

[0382] The results in Table 25 show that: the left ventricular end-diastolic volume of the test drug group and the positive drug group was significantly lower than that of the control group, and the left ventricular end-diastolic volume of the test drug group was also lower than that of the positive drug group, but the left ventricular end-diastolic volume of the test drug group was different (p<0.05); the end-systolic volume of the test drug group and the positive drug group was significantly lower than that of the control group, and the end-systolic volume of the test drug group was also lower than that of the positive drug group, but the end-systolic volume of the test drug group was significantly different (p<0.01).

[0383] Table 26 Left ventricular mass (LV Mass) and corrected left ventricular mass (LV Mass (corrected)) of mice in each group

[0384]

[0385] The results in Table 26 show that the left ventricular mass in the test drug group and the positive drug group was significantly lower than that in the control group, and the left ventricular mass in the test drug group was also lower than that in the positive drug group, but there was a difference between the test drug groups (p<0.05).

[0386] Table 27 Diastolic left ventricular anterior wall thickness (LVAW; d) and systolic left ventricular anterior wall thickness (LVAW; s) of mice in each group

[0387]

[0388] The results in Table 27 show that there was no significant change in the diastolic left ventricular anterior wall thickness and systolic left ventricular anterior wall thickness in the test drug group and the positive drug group compared with the control group.

[0389] Table 28 Diastolic left ventricular posterior wall thickness (LVPW; d) and systolic left ventricular posterior wall thickness (LVPW; s) of mice in each group

[0390]

[0391] The results in Table 28 show that compared with the control group, the left ventricular posterior wall thickness during diastole and the left ventricular posterior wall thickness during systole in the positive drug group tended to decrease, while compared with the control group, the left ventricular posterior wall thickness during diastole and the left ventricular posterior wall thickness during systole in the test drug group tended to increase, but no significant difference was observed.

[0392] Table 29 Inflammatory indicators (reactive oxygen species (ROS), IL-6, IL-1β and TNF-α) of mice in each group

[0393]

[0394] The results in Table 29 show that the inflammatory indexes of the test drug group and the positive drug group were significantly lower than those of the control group, and the inflammatory indexes of the test drug group were slightly higher than those of the positive drug group, but there were significant differences between the test drug group and the positive drug group (p<0.01).

[0395] In summary, the test drug can significantly reduce the myocardial infarction area ratio of mice, significantly improve the cardiac function of mice with myocardial ischemia-reperfusion, and significantly reduce the expression of verification factors during myocardial ischemia-reperfusion.

[0396] Example 9: Pharmacokinetic studies

[0397] 9.1 Pharmacokinetic studies in rhesus monkeys

[0398] 9.1.1 Preparation of test solution

[0399] Oral solution of compound 2: Weigh 50.00 mg of compound 2, add 1.00 mL DMSO to dissolve, and obtain 50.00 mg / mL liquid 1. Weigh polyethylene glycol PEG400 (8 g): polyoxyethylene hydrogenated castor oil RH40 (1 g) in a mass ratio of 8:1, mix well, and obtain liquid 2. Mix liquid 1 and liquid 2 in a volume ratio of 1:9 to obtain an oral solution of compound 2 with a drug concentration of 5.00 mg / mL. Prepare before use.

[0400] Compound 2 injection: Weigh 10.00 mg of compound 2, add 1.00 mL DMSO to dissolve, and obtain 10.00 mg / mL liquid 3. Mix liquid 3 and liquid 2 at a volume ratio of 4:1 to obtain a mixed solution of compound 2. Dilute the mixed solution of compound 2 4 times with water for injection to obtain an injection of compound 2 with a drug concentration of 0.50 mg / mL. Prepare before use.

[0401] 9.1.2 Route and method of administration

[0402] Administration route and frequency: oral administration / intravenous injection, single administration. There are two rhesus monkeys in each group, one female and one male, weighing about 5.5 kg. Before administration, the dosage of each animal is calculated according to the weight and dosage of the rhesus monkeys. The dosage is: 2.5 mg / kg by oral administration and 0.3 mg / kg by intravenous injection.

[0403] 9.1.3 Setting and basis of blood sampling time point

[0404] According to the Technical Guidelines for Nonclinical Pharmacokinetic Studies of Drugs, the blood sampling time points were set with reference to the previous pilot test results: 0 h before administration and 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after administration.

[0405] 9.1.4 Sample collection and storage

[0406] Whole blood collection and processing: 0.3-0.5 mL of blood was collected from the hind limb vein, added to a centrifuge tube with sodium heparin pre-cooled in ice water, and placed in an ice bath. It was divided into two portions with a unit volume of 50 μL, and then 3 times the volume (150 μL) of acetonitrile was immediately added and stored at -80°C for later use.

[0407] Plasma collection and processing: The remaining blood sample was added to a centrifuge tube pre-cooled with ice water and containing sodium heparin, and placed in an ice bath. After standing, it was centrifuged (4000 rpm, 10 min). The upper plasma was taken, placed in a sterile EP tube, and stored at -80°C for later use.

[0408] Table 30 Whole blood drug concentration after intravenous injection

[0409]

[0410] Table 31 Oral solution gavage whole blood drug concentration

[0411]

[0412] Table 32 Plasma drug concentration after intravenous injection

[0413]

[0414] Table 33 Plasma drug concentration after oral administration

[0415]

[0416] Absolute bioavailability:

[0417] F%=AUClastD 口服 / AUClast_D 静脉 ×100%=100.45 / 695.77×

[0418] 100% = 14.44%

[0419] 9.2 Pharmacokinetic studies in rats

[0420] 9.2.1 Preparation of test solution

[0421] Oral solution of compound 2: Weigh 50.00 mg of compound 2, add 1.00 mL DMSO to dissolve, and obtain 50.00 mg / mL liquid 1. Weigh polyethylene glycol PEG400 (8 g): polyoxyethylene hydrogenated castor oil RH40 (1 g) in a mass ratio of 8:1, mix well, and obtain liquid 2. Mix liquid 1 and liquid 2 in a volume ratio of 1:9 to obtain an oral solution of compound 2 with a drug concentration of 5.00 mg / mL. Prepare before use.

[0422] Injection of Compound 2: Dilute the oral solution of Compound 2 5 times with water for injection to obtain an injection of Compound 2 with a drug concentration of 1.00 mg / mL. Prepare before use.

[0423] 9.2.2 Route and method of administration

[0424] Administration route and frequency: oral administration / intravenous injection, single administration. SD rats were fasted for 12 hours before administration, but not water, and were fed uniformly 4 hours after administration, with free access to water. The dosage for each animal was calculated according to the rat's body weight and dosage before administration. The dosage was 5.0 mg / kg for oral administration and 1.0 mg / kg for intravenous injection.

[0425] 9.1.3 Setting and basis of blood sampling time point

[0426] According to the Technical Guidelines for Nonclinical Pharmacokinetic Studies of Drugs, the blood collection time points were set based on the results of the previous pilot experiment: the blood collection time points for animals given oral administration were 0 hours before administration and 0.25, 0.5, 1, 2, 3, 5, 7, 10, and 24 hours after administration. The blood collection time points for animals given tail vein administration were 0 hours before administration and 0.083, 0.25, 0.5, 1, 1.5, 2, 3, 5, 10, and 24 hours after administration.

[0427] 9.1.4 Sample collection and storage

[0428] At the planned blood collection point, about 0.2 mL of blood was collected from the jugular vein and placed in a sodium heparin anticoagulant tube with a temporary number. The plasma was separated by centrifugation at 12,000 rpm for 2 minutes. The supernatant was placed in a labeled EP tube and stored in a -20°C refrigerator.

[0429] Table 34 Related data of oral administration

[0430]

[0431] Table 35 Related data of intravenous administration

[0432]

[0433] Absolute bioavailability: F% = AUClast_D 口服 / AUClast_D 静脉 ×100%=723.34 / 1363.488×100%=53.05%

[0434] While the invention has been described with respect to specific embodiments thereof, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of this invention.

Claims

1. A compound represented by Formula 1: or a pharmaceutically acceptable salt thereof, in: Y is a C6-C 10 Aryl groups; X is unsubstituted or substituted with at least one R 10a Substituted C1-C4 alkylene groups; R1 is C3-C 10 Cycloalkyloxy groups; R2 to R4 are each independently hydrogen or C1-C 10 an alkyl group; and R 10a Each is independently a deuterium, a halogen or an oxydeoxy group.

2. The compound of claim 1, wherein Y is 2-aminophenyl.

3. The compound of claim 1, wherein X is ethylene, propylene, butylene, fluoroethylene, bromoethylene, oxyethylene, fluoropropylene, bromopropylene, oxypropylene, fluorobutylene, bromobutylene or oxybutylene.

4. The compound of claim 1, wherein X is ethylene, propylene, difluoroethylene, difluoropropylene, oxyethylene or oxypropylene.

5. The compound of claim 1, wherein R1 is a C3-C6 cycloalkyloxy group.

6. The compound of claim 1, wherein R1 is cyclopropyloxy, cyclobutyloxy or cyclopentyloxy.

7. The compound of claim 1, wherein R1 is cyclopropyloxy.

8. The compound of claim 1, wherein one of R2 to R4 is hydrogen.

9. The compound of claim 1, wherein two of R2 to R4 are hydrogen.

10. The compound of claim 1, wherein R2 to R4 are all hydrogen.

11. The compound according to any one of claims 1 to 10, wherein the compound is selected from the following compounds 1 to 5:

12. A pharmaceutical composition comprising the compound according to any one of claims 1 to 11, and one or more pharmaceutically acceptable excipients.

13. The pharmaceutical composition according to claim 12, wherein the pharmaceutically acceptable excipient is selected from a carrier, an excipient, a binder, a suspending agent, a glidant, a flavoring agent, a sweetener, a disintegrant, a dispersant, a surfactant, a lubricant, a colorant, a diluent, a solubilizer, a wetting agent, a plasticizer, a stabilizer, a penetration enhancer, a defoaming agent, an antioxidant, a preservative, a solvent or a combination thereof.

14. A transcription factor regulator, comprising the compound according to any one of claims 1 to 11 or the pharmaceutical composition according to claim 12 or 13.

15. The transcription factor regulator according to claim 14, which is a myocyte enhancer factor 2 regulator.

16. Use of a compound according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 12 or 13 in the preparation of a medicament for treating a disease associated with the regulation of myocyte enhancer factor 2.

17. The use according to claim 16, wherein the disease associated with the regulation of myocyte enhancer factor 2 is selected from the group consisting of cardiac hypertrophy, heart failure, coronary atherosclerosis, coronary heart disease and diabetic cardiomyopathy.

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