CN111094262B

CN111094262B - 1, 3-dioxane-4, 6-diketone compound, preparation method thereof, pharmaceutical composition and application thereof

Info

Publication number: CN111094262B
Application number: CN201980003854.6A
Authority: CN
Inventors: 刘东祥; 柳红; 王敏; 李淳朴; 杨利生; 陈凯先; 蒋华良
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2018-06-21
Filing date: 2019-06-20
Publication date: 2022-12-20
Anticipated expiration: 2039-06-20
Also published as: CN111094262A; CN108640898A; WO2019242687A1

Abstract

The invention discloses a 1, 3-dioxane-4, 6-diketone compound,The structure of the compound is shown as formula I, and the definition of each substituent group is described in the specification and the claims. The compound of the invention has the function of inhibiting the activity of SIRT1 protein deacetylase, and can be used in drugs for treating various diseases related to the abnormal expression of the SIRT1 protein deacetylase or the level of the enzyme activity thereof, such as neurodegenerative diseases, metabolic diseases, cancers and the like.

Description

1, 3-dioxane-4, 6-diketone compound, preparation method thereof, pharmaceutical composition and application thereof

Technical Field

The invention relates to 1, 3-dioxane-4, 6-diketone compounds, a preparation method thereof, a pharmaceutical composition and application thereof.

Background

Sirtuins are a class of NAD + -dependent protein deacetylases that share a high degree of homology in amino acid sequence and structure. The Sirtuin protein is widely present in many organisms such as archaea, nematodes, drosophila, yeast and mammals, and regulates many important biological processes including cell senescence, transcription, apoptosis, inflammation, stress, mitochondrial synthesis and body's biological clock.

The yeast Sir2 gene was the first sirtuin protein to be found. As early as the seventies of the 20 th century, people found that Sir2 gene can maintain the length of yeast telomere and regulate the generation of DNA repetitive sequence coded by rDNA. Later, sir2 gene was found to prolong the lifespan of yeast by suppressing genomic instability, knockout of the Sir2 gene in yeast significantly shortens the lifespan of yeast, and overexpression of Sir2 gene extended the lifespan of yeast by about 40%. Similarly, overexpression of Sir2.1 (a homologous gene to Sir 2) in nematodes prolongs nematode life by about 50%, and is similarly observed in Drosophila. These findings have made the research into sirtuin family proteins much more intense.

The mammalian genome encodes seven sirtuin proteins, designated SIRT1-7, which contain a highly conserved core region composed of NAD + binding domain and enzyme-active catalytic domain, and variable-length and sequence N-and C-termini. Differences in the N-and C-termini of Sirtuin proteins may affect binding of the proteins to ligands, mediate interactions of the proteins with other Sirtuin isoforms, or affect their subcellular localization. The results of the existing research show that SIRT1, SIRT6 and SIRT7 are nuclear proteins, but SIRT1 can also enter cytoplasm from nucleus through nucleoplasm transportation, thereby regulating target proteins in cytoplasmic stress response. SIRT2 is primarily located in the cell matrix, although SIRT2 may be transported into the nucleus through the nucleoplasm, while SIRT3, SIRT4 and SIRT5 are primarily located in the mitochondria. Mammalian sirtuins are distributed in different sub-cellular layers, which are closely related to the substrates and biological functions they act upon.

SIRT1 is the closest in sequence to yeast Sir2 in the mammalian sirtuin family of proteins and was the earliest studied member of the mammalian sirtuin family. SIRT1 regulates heterochromatin formation by deacetylating H1K26, H3K9 and H4K16, and in addition, SIRT1 is also involved in deacetylation of non-histones. Non-histone substrates of SIRT1 can be divided into three classes: (1) transcription factor: such as p53, FOX03a, E2F2, BCL6, etc.; (2) DNA repair proteins: such as Ku70 and MRE11-RAD50-NBS1 (MRN), etc.; (3) signal factor: smad7, and the like. SIRT1 is involved in regulating a variety of physiological functions including gene expression, energy metabolism, and aging through deacetylation of histone substrates as well as non-histone substrates.

SIRT1 is closely related to the occurrence and development of various diseases, and can inhibit A beta aggregation in microglia cells by deacetylating P65/RelA subunit of NF-kB, thereby controlling the development of Alzheimer's Disease (AD). SIRT1 may also protect nerve cells in models of Huntington's Disease (HD) by deacetylating PGC-1 α and increasing PGC-1 α activity. The involvement of the p53 protein, a tumor suppressor, in a variety of physiological processes including DNA repair, cell growth arrest, senescence and apoptosis has become one of the important targets for cancer therapy. Whereas SIRT1 can deacetylate lysine residue 382 of p53, deacetylation of p53 leads to tumorigenesis. SIRT1 can also enhance cell DNA repair effect and inhibit cell apoptosis caused by DNA damage through deacetylation of DNA repair factor Ku70 and forkhead transcription factor FOXOs. Research shows that the SIRT1 activity inhibition can induce the growth arrest of tumor cells and promote the apoptosis of the tumor cells. In addition, SIRT1 can regulate transcription of tumor suppressor genes by deacetylating lysines at positions 26, 9 and 16 of histones H1, H3 and H4, thereby participating in regulation of tumor cell cycle. The research shows that the overexpression of the SIRT1 protein is detected in most solid tumors and hematological malignancies including breast cancer, colon cancer, prostate cancer, liver cancer and leukemia. Since overexpression of SIRT1 is associated with the development of cancer, inhibition of SIRT1 activity can effectively inhibit cancer cell proliferation while inducing cancer cell apoptosis. Therefore, SIRT1 is possible to become a new target point for tumor treatment, and the SIRT1 inhibitor can become a potential anticancer candidate drug.

Disclosure of Invention

The invention aims at providing a 1, 3-dioxane-4, 6-diketone compound shown in a general formula (I), and a pharmaceutically acceptable salt, an enantiomer, a diastereoisomer or a racemate thereof.

Another object of the present invention is to provide a process for producing the compound represented by the above general formula (I).

It is a further object of the present invention to provide a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds of formula (I) above or a pharmaceutically acceptable salt thereof.

The invention also aims to provide application of the compound shown in the general formula (I) in preparing medicines for treating diseases related to SIRT1 deacetylase activity level, such as cancer, immune disorders and inflammation.

In a first aspect of the present invention, there is provided a compound of formula I, or a pharmaceutically acceptable salt, hydrate, solvate, enantiomer, diastereomer or racemate thereof, or a mixture thereof:

wherein, the first and the second end of the pipe are connected with each other,

R ¹ and R ⁷ Each independently hydrogen, C1-C6 alkyl or C2-C12 unsaturated hydrocarbyl;

R ² 、R ³ 、R ⁴ 、R ⁵ 、R ⁶ each independently hydrogen, halogen, cyano, nitro, amino, hydroxyl, carboxyl, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 unsaturated hydrocarbyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 acyl, substituted or unsubstituted C3-C12 cycloalkyl, -L1- (CH) ₂ ) m-substitutionOr unsubstituted C6-C12 aryl, -L1- (CH) ₂ ) m-substituted or unsubstituted 3-12 membered heterocyclyl, -L1- (CH) ₂ )m-C(＝O)-N(R ⁸ )(R ⁹ ) Wherein, L1 is none, -O-or-S-; m is 0, 1, 2,3,4 or 5; r ⁸ And R ⁹ Each independently selected from: hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-12 membered heterocyclyl, or substituted or unsubstituted C6-C12 aryl;

the substitution means that one or more substituents selected from the group consisting of: halogen, hydroxyl, phenyl, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 unsaturated alkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C12 cycloalkyl, 3-12 member heterocyclyl, cyano, nitro, hydroxymethyl, carboxyl, mercapto;

and R is ⁶ The number of (2) is 1-3.

In another preferred embodiment, R ¹ And R ⁷ Each independently hydrogen, C1-C4 alkyl or C2-C4 unsaturated hydrocarbyl.

In another preferred embodiment, R ² 、R ³ 、R ⁴ 、R ⁵ Each independently of the others is hydrogen, halogen, cyano, nitro, amino, hydroxyl, carboxyl, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C2-C6 unsaturated hydrocarbyl, substituted or unsubstituted C1-C4 alkoxy, substituted or unsubstituted C1-C4 acyl, substituted or unsubstituted C3-C6 cycloalkyl, -L1- (CH) ₂ ) m-substituted or unsubstituted C6-C12 aryl, -L1- (CH) ₂ ) m-substituted or unsubstituted 4-10 membered heterocyclyl, -L1- (CH) ₂ )m-C(＝O)-N(R ⁸ )(R ⁹ ) Wherein, L1 is none, -O-or-S-; m is 0, 1, 2,3,4 or 5; r ⁸ And R ⁹ Each independently selected from: hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 3-6 membered heterocyclyl, or substituted or unsubstituted C6-C12 aryl;

the substitution means that one or more substituents selected from the group consisting of: halogen, hydroxyl, phenyl, C1-C4 alkyl, C1-C4 haloalkyl, C2-C4 unsaturated alkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, C3-C6 cycloalkyl, 3-6 member heterocyclic radical, cyano, nitro, hydroxymethyl, carboxyl and sulfhydryl.

In another preferred embodiment, R ³ Is hydrogen, halogen, hydroxy, -L1- (CH) ₂ ) m-3-10 membered heterocyclyl, -L1- (CH) ₂ ) m-C6-C10 aryl, -L1- (CH) ₂ ) m-C6-C10 aryl-carboxy, C1-C4 alkoxy, 3-6 membered heterocyclyl, -L1- (CH) ₂ )m-C(＝O)-N(R ⁸ )(R ⁹ ) Wherein, L1 is none, -O-or-S-; m is 0, 1, 2 or 3; r ⁸ And R ⁹ Each independently selected from: hydrogen, C1-C4 alkyl or phenyl.

In another preferred embodiment, R ² 、R ⁴ Each independently of the other is hydrogen, halogen, C1-C4 alkyl, C1-C4 alkoxy or-L1- (CH) ₂ ) m-C6-C10 aryl, wherein L1 is none, -O-or-S-; m is 0, 1, 2 or 3.

In another preferred embodiment, R ⁵ Is hydrogen or C1-C4 alkoxy.

In another preferred embodiment, the compound is:

in a second aspect of the present invention, there is provided a process for the preparation of a compound according to the second aspect, comprising the steps of:

a) Reacting malonic acid with substituted benzaldehyde or ketone to obtain compound I _a ；

b) Compound I _a Reacting with substituted benzaldehyde or ketone to obtain a compound shown in a formula I,

wherein R is ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ 、R ⁶ 、R ⁷ Is as defined inIn one aspect.

In another preferred embodiment, the preparation method of the compound comprises the following steps:

a) Dissolving malonic acid and substituted benzaldehyde or ketone in a solvent, and heating, stirring and reacting to obtain a compound I _a The solvent is acetic anhydride;

b) The compound I _a Dissolving the substituted benzaldehyde or ketone in methanol, and stirring at room temperature to react to obtain the final product.

In a third aspect of the invention, there is provided a pharmaceutical composition comprising a compound of the first aspect, or a pharmaceutically acceptable salt, hydrate, solvate, enantiomer, diastereomer or racemate thereof, or a mixture thereof; and a pharmaceutically acceptable carrier.

"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and between the active ingredients of the present invention without significantly diminishing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (e.g. tween, etc.)

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

In a fourth aspect of the invention, there is provided the use of a compound of the first aspect, or a pharmaceutically acceptable salt, hydrate, solvate, enantiomer, diastereomer or racemate thereof, or a mixture thereof, (a) for the preparation of an inhibitor of SIRT1 deacetylase; or (b) is used for preparing a medicament for treating diseases related to the abnormal expression of the SIRT1 protein or the enzyme activity level thereof.

In another preferred embodiment, the diseases related to the abnormal expression of the SIRT1 protein or the enzyme activity level thereof are selected from the group consisting of: neurodegenerative diseases, cancer, metabolic diseases, immune disorders and inflammation.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. For reasons of space, they will not be described in detail.

Drawings

FIG. 1 is a diagram of the results of an in vitro SIRT1 enzyme activity experiment, illustrating that compound S3 inhibits SIRT1 activity in a concentration-dependent manner

FIG. 2 is a diagram of the inhibition type of a compound S3 on SIRT1 protein, A) and B) are respectively fixed NAD ⁺ Concentration, michaelis constant curve and double reciprocal curve of compound S3 versus substrate Abz polypeptide were measured while varying the substrate polypeptide concentration. C) D) respectively, varying NAD for fixing substrate Abz polypeptide concentration ⁺ At concentration, the detection compound S3 is on NAD ⁺ The mie constant curve and the double reciprocal curve. Indicating that Compound S3 is a competitive inhibitor of substrate polypeptide Abz, is NAD ⁺ A non-competitive inhibitor of (a).

FIG. 3 is a graph of binding of compound S3 to protein detected by microcalorimetric electrophoresis (MST) experiments. Graph illustrating the results of compound S3 binding competitively to the binding site of substrate polypeptide Abz.

FIG. 4 is a graph of the results of molecular modeling of the binding of compound S3 to SIRT 1; b) SIRT1 mutant enzyme dynamic experiment result chart.

FIG. 5 is a graph showing the effect of compounds on the level of p53 acetylation in SH-SY5Y human neuroblastoma cells. The control group (Con, control) was DMSO (10. Mu.M) added, and the literature-reported SIRT1 inhibitor Ex527 was used as a positive control, and beta-actin was used as an internal control. anti-p53 (acetyl K381) antibody (cat # ab 61241) and anti-p53 antibody (cat # ab 26) were purchased from IRDye,680RD Goat anti-Rabbit (cat # 926-68071), 800CW Goat anti-Mouse (cat # 926-32210) antibody was purchased from LICOR.

Detailed Description

Based on long-term and intensive research, the invention prepares a compound with a structure shown in formula I and finds that the compound has SIRT1 inhibition activity. The compound has an inhibiting effect on SIRT1 at low concentration, and the inhibiting activity is quite excellent, so that the compound can be used for treating diseases related to SIRT1 activity or expression quantity, such as neurodegenerative diseases, metabolic diseases and tumors. On the basis of this, the present invention has been completed.

Term(s)

As used herein, the term "C1-C12 alkyl" refers to a straight or branched chain alkyl group having 1 to 12 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, and isohexyl, or the like. "C1-C6 alkyl" refers to a straight or branched chain alkyl group having 1, 2,3,4, 5, or 6 carbon atoms, "C1-C4 alkyl" refers to a straight or branched chain alkyl group having 1, 2,3, or 4 carbon atoms, and so on. The term "C1-C12 haloalkyl" refers to a straight or branched chain alkyl group having 1-12 carbon atoms, such as trifluoromethyl and the like, substituted with 1, 2,3 or more halogens.

The term "C2-C12 unsaturated hydrocarbon group" means a straight or branched chain alkenyl or alkynyl group having 2 to 12 carbon atoms, such as vinyl, propynyl, etc.

The term "C1-C6 alkoxy" means a straight or branched chain alkoxy group having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy and tert-butoxy, or the like. Included within the definition of "C1-C6 alkoxy" are "C1-C4 alkoxy".

The term "C3-C12 cycloalkyl" refers to a saturated or unsaturated hydrocarbon group having 3 to 12 carbon atoms in the ring, such as cyclopropyl, cyclobutyl, cyclohexyl, cyclohexenyl and the like. The term "C3-C8 cycloalkyl" refers to a saturated hydrocarbon group having 3 to 8 carbon atoms in the ring, such as cyclopropyl, cyclobutyl, cyclohexyl and the like.

The term "C6-C12 aryl" refers to a monocyclic or fused bicyclic ring having 6-12 carbon atoms, substituents having a conjugated pi-electron system, such as phenyl and naphthyl, or similar groups. Included within the definition of "C6-C12 aryl" are "C6-C10 aryl".

The term "3-12 membered heterocyclyl" refers to a monocyclic or fused bicyclic ring having 3-12 ring atoms with one or more (preferably 1-5) heteroatoms selected from O, S, N or P in the ring system, for example piperidinyl, pyrrolidinyl, piperazinyl, tetrahydrofuranyl, morpholinyl, benzodioxolanyl, tetrahydropyrrolyl or the like. The definition of "3-12 membered heterocyclyl" includes "4-10 membered heterocyclyl".

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

Herein, unless otherwise specified, the term "substituted" means that one or more hydrogen atoms on a group are replaced with a substituent selected from the group consisting of: halogen, carboxyl, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C2-C6 acyl, unsubstituted or halogenated C1-C6 alkyl-hydroxyl.

Unless otherwise specified, all occurrences of a compound in the present invention are intended to include all possible optical isomers, such as a single chiral compound, or a mixture of various chiral compounds (i.e., a racemate). In all compounds of the invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.

As used herein, the term "compounds of the invention" refers to compounds of formula I. The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of formula I.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention with an acid or base that is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, phenylmethanesulfonic acid, benzenesulfonic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid.

A compound of formula I

The structure of the compound of formula I of the invention is shown as follows:

the substituents are as defined above.

Preferred compounds are shown in the following table:

TABLE 1 Compound Numbers, names and chemical structures

SIRT1 activity inhibitor and application thereof

The compounds of the invention can inhibit the activity of one or more sirtuin proteins. For example, the compounds of the invention can be used to inhibit the activity of a sirtuin enzyme in a cell or in a patient in which it is desired to inhibit the activity of the enzyme by administering to the cell, subject or patient an inhibitory amount of a compound of the invention such that inhibition of the function of the sirtuin protein is achieved.

Analysis of the data on the inhibitory activity of the compounds of the invention on SIRT1, SIRT2, SIRT3 and SIRT5 shows that the compounds of the invention are selective inhibitors of SIRT1 and are more selective than the positive compound EX 527.

As SIRT1 inhibitors, the compounds of the invention are useful in the treatment of various diseases associated with aberrant SIRT1 expression or activity. The abnormal proliferation diseases related to SIRT1 activity or expression quantity include but are not limited to the following diseases: histiocytic lymphoma, ovarian cancer, head and neck squamous cell carcinoma, gastric cancer, breast cancer, childhood hepatocellular carcinoma, colorectal cancer, cervical cancer, lung cancer, sarcoma, nasopharyngeal carcinoma, pancreatic cancer, glioblastoma, prostate cancer, small cell lung cancer, non-small cell lung cancer, multiple myeloma, thyroid cancer, testicular cancer, cervical cancer, lung adenocarcinoma, colon cancer, papillary renal cell carcinoma, glioblastoma, endometrial cancer, esophageal cancer, leukemia, renal cell carcinoma, bladder cancer, liver cancer, and astrocytoma, glioma, non-malignant skin cancer, and the like. More preferably for the treatment of hematological malignancies, including breast cancer, colon cancer, prostate cancer, liver cancer and leukemia.

The compounds and compositions of the invention are useful for treating, preventing or modulating metabolic-related disorders, including diabetes, hyperlipidemia, obesity, hyperglycemia, and hyperosmolar syndrome.

The compounds and compositions of the invention are useful for treating, preventing or modulating neurodegenerative diseases, including alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, spinocerebellar ataxia, and spinal muscular atrophy.

The invention has the advantages that:

the 1, 3-dioxane-4, 6-diketone compound has low toxicity and good solubility.

The preparation method of the 1, 3-dioxane-4, 6-diketone compound and the derivative thereof has the advantages of mild reaction conditions, abundant and easily available raw materials, simple operation and post-treatment, good corresponding selectivity and the like.

The 1, 3-dioxane-4, 6-diketone compound and the derivative thereof have good inhibitory activity and excellent selectivity on SIRT deacetylase.

Therefore, the compound of the invention can be used for treating various diseases related to the abnormal expression or activity of SIRT1 protein, such as neurodegenerative diseases, diabetes, tumors and the like.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally according to conventional conditions or according to conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are exemplary only.

EXAMPLE 1 preparation of the compound 5- (4-chlorobenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S1)

Reaction scheme 1.1

Step 1: preparation of 2-phenyl-1, 3-dioxane-4, 6-dione

Malonic acid (10.4 g, 100mmol) was placed in a round bottom flask, acetic anhydride (28.4 mL, 300mmol) was added, stirring at room temperature, and 0.1mL of concentrated H was added ₂ SO ₄ And stirred overnight. Benzaldehyde (10.2mL, 100mmol) was slowly added thereto at room temperature, and the reaction was carried out overnight at 5 ℃ after completion of the reaction. About 30mL of toluene was added, the acetic anhydride was removed by concentration under reduced pressure, a white solid was precipitated, and the filter cake was washed with 200mL of water after suction filtration to give a crude product. Dissolving the crude product in acetone, adding appropriate amount of water to precipitate solid, stirring for 15min, vacuum filtering, and washing the filter cake to obtain 2-phenyl-1, 3-dioxo-4, 6-dione (10.2 g, yield 53)％)。 ¹ H NMR(400MHz，DMSO-d ₆ )δ7.56(dtd，J＝10.2，5.1，2.0Hz，5H)，7.13(s，1H)，4.56(d，J＝18.4Hz，1H)，3.61(d，J＝18.4Hz，1H).MS(ESI，m/z)：191(M-H) ^- 。

Reaction scheme 1.2

And 2, step: preparation S1

2-phenyl-1, 3-dioxo-4, 6-dione (0.3 g, 1.56mmol) was dissolved in anhydrous DMSO, anhydrous sodium acetate (90mg, 1.09mmol) was added and stirred at room temperature to dissolve it, 4-chlorobenzaldehyde (0.22g, 1.56mmol) was added and stirred overnight, about 30mL of water was added to the solution and stirred to precipitate a solid, suction filtration was carried out, the cake was washed with water and dried to give a crude product, which was recrystallized from methanol/petroleum ether to give 310mg pure product in 63% yield. ¹ H NMR(400MHz，CDCl ₃ )δ8.32(s，1H)，8.08-7.94(m，2H)，7.65-7.58(m，2H)，7.56-7.40(m，5H)，6.76(s，1H).

EXAMPLE 2 preparation of the compound 5- (4-hydroxy-3-iodo-5-methoxybenzylidene) -2-methyl-2-phenyl-1, 3-dioxane-4, 6-dione (S2)

Reaction scheme 2.1

Step 1: preparation of 2-methyl-2-phenyl-1, 3-dioxane-4, 6-dione

Malonic acid (15.6 g, 150mmol) was placed in a round bottom flask, acetic anhydride (55.5 mL,585 mmol) was added, stirring was done at room temperature, 0.2mL of concentrated H was added ₂ SO ₄ And stirred overnight. After acetophenone (35ml, 300mmol) was slowly added at room temperature and reacted for 1 hour, acetic anhydride was removed by concentration under reduced pressure and the product, 2-methyl-2-phenyl-1, 3-dioxo-4, 6-dione (9.3 g, 30% yield) was isolated and purified by flash column chromatography (petroleum ether/ethyl acetate =15/1, v/v). MS (ESI, m/z): 205 (M-H) ^- 。

Reaction scheme 2.2

And 2, step: preparation S2

2-methyl-2-phenyl-1, 3-dioxo-4, 6-dione (0.3g, 1.45mmol) was dissolved in anhydrous DMSO, anhydrous sodium acetate (84mg, 1.02mmol) was added and stirred at room temperature to dissolve it, 4-hydroxy-3-iodo-5-methoxybenzaldehyde (0.41g, 1.45mmol) was added and stirred overnight, about 30mL of water was added to the solution and stirred to precipitate a solid, which was suction filtered, washed with water and dried to give a crude product, which was recrystallized from methanol/petroleum ether to give 340mg of pure product in 50% yield. ¹ H NMR(400MHz，DMSO-d ₆ )δ10.59(s，1H)，7.78-7.68(m，4H)，7.42-7.22(m，4H)，3.78(s，3H)，1.86(s，3H).

EXAMPLE 3 preparation of the Compound 5- (3-bromo-4-hydroxy-5-methoxybenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S3)

Compound S3 was prepared in the same manner as in example 1, except that 3-bromo-4-hydroxy-5-methoxybenzaldehyde was used instead of 4-chlorobenzaldehyde, resulting in a final reaction yield of 56%. ¹ H NMR(400MHz，DMSO-d ₆ )δ8.17(d，J＝6.8Hz，2H)，7.91(s，1H)，7.64-7.40(m，5H)，7.08(s，1H)，3.82(s，3H).MS(ESI，m/z)：404(M-H) ^- 。

EXAMPLE 4 preparation of the compound 5- [4- (1, 3-Benzodioxane-5-ylmethoxy) -3-methoxybenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S4)

Compound S4 was prepared in the same manner as in example 1 except that 4- (benzo-1, 3-dioxolan-5-ylmethoxy) -3-methoxybenzaldehyde was used instead of 4-chlorobenzaldehyde, and the final reaction yield was 68%. ¹ H NMR(400MHz，CDCl ₃ )δ8.34(s，1H)，8.22-8.18(m，1H)，7.64-7.42(m，6H)，6.99-6.76(m，4H)，6.70(s，1H)，5.96(s，2H)，5.18(s，2H)，3.90(s，3H)。

EXAMPLE 5 preparation of the compound 3- ({ 4- [ (4, 6-dioxo-2-phenyl-1, 3-dioxan-5-ylidene) methyl ] phenoxy } methyl) benzoic acid (S5)

Compound S5 was prepared in the same manner as in example 1, except that 3- ((4-formylphenoxy) methyl) benzoic acid was used instead of 4-chlorobenzaldehyde, resulting in a final reaction yield of 23%. ¹ H NMR(400MHz，DMSO-d ₆ )δ8.24(s，1H)，8.08-7.92(m，2H)，8.05-8.01(m，1H)，7.95-7.85(m，1H)，7.62-7.50(m，3H)，7.48-7.38(m，4H)，7.10-7.01(m，2H)，6.88(s，1H)，5.20(s，2H)。

EXAMPLE 6 preparation of 5- (4-hydroxy-3-iodo-5-methoxybenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S6)

Compound S6 was prepared in the same manner as in example 1, except that 4-hydroxy-3-iodo-5-methoxybenzaldehyde was used instead of 4-chlorobenzaldehyde, resulting in a final reaction yield of 70%. ¹ H NMR(400MHz，DMSO-d ₆ )δ10.65(s，1H)，8.14(s，1H)，8.08-7.92(m，2H)，7.56-7.48(m，2H)，7.42-7.38(m，3H)，6.82(s，1H)，3.83(s，3H)。

Example 7 preparation of 5- (3-ethoxy-4-hydroxybenzylidene) -2-methyl-2-phenyl-1, 3-dioxane-4, 6-dione (S7)

Compound S7 was prepared in the same manner as in example 2, except that 3-ethoxy-4-hydroxybenzaldehyde was used instead of 4-hydroxy-3-iodo-5-methoxybenzaldehyde, resulting in a final reaction yield of 46%. ¹ H NMR(400MHz，CDCl ₃ )δ8.08-8.01(m，2H)，7.54-7.25(m，5H)，6.91-6.84(m，1H)，6.35(s，1H)，4.22-4.10(m，2H)，1.56(s，3H)，1.50-1.42(m，3H)。

Example 8 preparation of 5-benzylidene-2-methyl-2-phenyl-1, 3-dioxane-4, 6-dione (S8)

Compound S8 was prepared in the same manner as in example 2, except that benzaldehyde was used instead of 4-hydroxy-3-iodo-5-methoxybenzaldehyde, resulting in a final reaction yield of 62%. ¹ H NMR(400MHz，CDCl ₃ )δ8.06(s，1H)，7.74-7.68(m，2H)，7.58-7.28(m，8H)，1.98(s，3H)。

EXAMPLE 9 preparation of (S9) 3- ({ 2-methoxy-4- [ (2-methyl-4, 6-dioxo-2-phenyl-1, 3-dioxan-5-ylidene) methyl ] phenoxy } methyl) benzoic acid

Compound S9 was prepared in the same manner as in example 2, except that 3- ((4-formyl-2-methoxyphenoxy) methoxy) benzoic acid was used instead of 4-hydroxy-3-iodo-5-methoxybenzaldehyde, resulting in a final reaction yield of 45%. ¹ H NMR(400MHz，CDCl ₃ )δ8.18-8.02(m，3H)，7.88(s，1H)，7.70-7.65(m，1H)，7.55-7.46(m，3H)，7.40-7.28(m，4H)，6.88-6.82(m，1H)，5.22(s，2H)，3.86(s，3H)，1.90(s，3H)。

EXAMPLE 10 preparation of 5- (3-chloro-4-hydroxy-5-methoxybenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S10)

Compound S10 was prepared in the same manner as in example 1, except that 4-hydroxy-3-chloro-5-methoxybenzaldehyde was used instead of 4-chlorobenzaldehyde, resulting in a final reaction yield of 72%. ¹ H NMR(400MHz，DMSO-d ₆ )δ8.15(s，1H)，7.90-7.82(m，1H)，7.80-7.74(m，1H)，7.58-7.45(m，2H)，7.42-7.36(m，3H)，6.82(s，1H)，3.84(s，3H)。

EXAMPLE 11 preparation of 5- (2, 5-dimethoxybenzylidene) -2-methyl-2-phenyl-1, 3-dioxane-4, 6-dione (S11)

Compound S11 was prepared in the same manner as in example 2 except that 2, 5-dimethoxybenzaldehyde was used instead of 4-hydroxy-3-iodo-5-methoxybenzaldehyde, and the final reaction yield was 54%. ¹ H NMR(400MHz，CDCl ₃ )δ8.50(s，1H)，7.58-7.50(m，2H)，7.43-7.30(m，3H)，7.25-7.20(m，1H)，7.05-7.02(m，1H)，6.80-6.75(m，1H)，3.78(s，3H)，3.76(s，3H)，1.96(s，3H)。

Example 12 preparation of 5- (3-ethoxy-4-hydroxybenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S12)

Compound S12 was prepared in the same manner as in example 1, except that 3-ethoxy-4-hydroxybenzaldehyde was used instead of 4-chlorobenzaldehyde, resulting in a final reaction yield of 43%. ¹ H NMR(400MHz，DMSO-d ₆ )δ10.22(s，1H)，8.20-8.04(m，2H)，7.60-7.38(m，6H)，6.90-6.82(m，2H)，4.14-4.02(m，2H)，1.43-1.38(m，3H)。

Example 13 preparation of 5- (2-methoxybenzylidene) -2-methyl-2-phenyl-1, 3-dioxane-4, 6-dione (S13)

Compound S13 was prepared in the same manner as in example 2, except that 2-methoxybenzaldehyde was used instead of 4-hydroxy-3-iodo-5-methoxybenzaldehyde, resulting in a final reaction yield of 76%. ¹ H NMR(400MHz，CDCl ₃ )δ8.51(s，1H)，7.58-7.52(m，3H)，7.44-7.30(m，4H)，6.91-6.82(m，2H)，3.80(s，3H)，1.98(s，3H)。

Example 14 preparation of 5- (2-Isopropoxybenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S14)

Compound S12 was prepared in the same manner as in example 1, except that 2-isopropoxybenzaldehyde was used instead of 4-chlorobenzaldehyde, and the final reaction yield was 43%. ¹ H NMR(400MHz，DMSO-d ₆ )δ10.22(s，1H)，8.20-8.04(m，2H)，7.60-7.38(m，6H)，6.90-6.82(m，2H)，4.14-4.02(m，2H)，1.43-1.38(m，3H)。

Example 15 preparation of 5- (3, 5-dichloro-4-hydroxybenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S15)

Compound S15 was prepared in the same manner as in example 1, except that 3, 5-dichloro-4-hydroxybenzaldehyde was used instead of 4-chlorobenzaldehyde, resulting in a final reaction yield of 68%. ¹ H NMR(400MHz，DMSO-d ₆ )δ8.15-8.07(m，3H)，7.58-7.39(m，6H)，6.93(s，1H)。

EXAMPLE 16 preparation of 5- (4-hydroxybenzylidene) -2-methyl-2-phenyl-1, 3-dioxane-4, 6-dione (S16)

Compound S16 was prepared in the same manner as in example 2, except that 4-hydroxybenzaldehyde was used instead of 4-hydroxy-3-iodo-5-methoxybenzaldehyde, resulting in a final reaction yield of 76%. ¹ H NMR(400MHz，CDCl ₃ )δ8.07(s，1H)，7.93-7.82(m，2H)，7.56-7.44(m，2H)，7.40-7.28(m，3H)，6.87-6.80(m，2H)，5.93(s，1H)，1.94(s，3H)。

EXAMPLE 17 preparation of 5- [4- (1, 3-Benzodioxane-5-ylmethoxy) benzylidene ] -2-phenyl-1, 3-dioxane-4, 6-dione (S17)

Compound S17 was prepared in the same manner as in example 1, except that 4- (benzo-1, 3-dioxolan-5-ylmethoxy) benzaldehyde was used instead of 4-chlorobenzaldehyde, resulting in a reaction yield of 41% in the last step. ¹ H NMR(400MHz，CDCl ₃ )δ8.37(s，1H)，8.23-8.16(m，2H)，7.64-7.58(m，2H)，7.51-7.42(m，3H)，7.08-6.80(m，5H)，6.70(s，1H)，5.96(s，2H)，5.06(s，2H)。

EXAMPLE 18 preparation of 5- (4-hydroxybenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S18)

Compound S18 was prepared in the same manner as in example 1, except that 4-hydroxybenzaldehyde was used instead of 4-chlorobenzaldehyde, and the final reaction yield was 55%. ¹ H NMR(400MHz，DMSO-d ₆ )δ10.56(s，1H)，8.19(s，1H)，8.10-8.02(m，2H)，7.55-7.43(m，2H)，7.42-7.35(m，3H)，6.85-6.75(m，3H)。

Example 19 preparation of 2-phenyl-5- (2, 3, 4-trimethoxybenzylidene) -1, 3-dioxane-4, 6-dione (S19)

Compound S19 was prepared in the same manner as in example 1, except that 2,3,4-trimethoxybenzaldehyde was used instead of 4-chlorobenzaldehyde, and the final reaction yield was 58%. ¹ H NMR(400MHz，CDCl ₃ )δ8.78(s，1H)，8.28-8.20(m，1H)，7.63-7.40(m，5H)，6.78-6.68(m，2H)，4.01(s，3H)，3.95(s，3H)，3.84(s，3H)。

EXAMPLE 20 preparation of 2-phenyl-5- [4- (1-tetrahydropyrrolyl) benzylidene ] -1, 3-dioxane-4, 6-dione (S20)

Compound S20 was prepared in the same manner as in example 1, except that 4- (1-tetrahydropyrrolyl) benzaldehyde was used instead of 4-chlorobenzaldehyde, and the final reaction yield was 22%. ¹ H NMR(400MHz，CDCl ₃ )δ8.32(s，1H)，8.28-8.19(m，2H)，7.68-7.58(m，2H)，7.52-7.40(m，3H)，6.64-6.52(m，3H)，3.58-3.40(m，4H)，2.18-1.97(m，4H)。

Example 21 preparation of 2- {4- [ (4, 6-dioxo-2-phenyl-1, 3-dioxan-5-ylbenzylidene) methyl ] phenoxy } -N-phenylacetamide (S21)

Except that 2- (4-formylbenzeneCompound S21 was prepared in the same manner as in example 1 except that oxy) -N-phenylacetamide was used instead of 4-chlorobenzaldehyde, and the final reaction yield was 36%. ¹ H NMR(400MHz，DMSO-d ₆ )δ9.98(s，1H)，8.25(s，1H)，8.20-8.01(m，2H)，7.62-7.50(m，4H)，7.49-7.40(m，3H)，7.28-7.20(m，2H)，7.18-6.97(m，4H)，4.78(s，2H)。

Example 22 preparation of 5- (5-bromo-2, 4-dimethoxybenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S22)

Compound S22 was prepared in the same manner as in example 1 except that 5-bromo-2, 4-dimethoxybenzaldehyde was used instead of 4-chlorobenzaldehyde, and the final reaction yield was 65%. ¹ H NMR(400MHz，CDCl ₃ )δ8.66(s，1H)，8.01(s，1H)，7.59(ddd，J＝7.3，2.0，1.0Hz，2H)，7.44(s，1H)，7.42-7.34(m，2H)，7.37-7.29(m，1H)，6.75(s，1H)，3.89(d，J＝10.1Hz，6H).

EXAMPLE 23 preparation of 5- (3-benzyl-4-hydroxy-5-methoxybenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S23)

Compound S23 was prepared in the same manner as in example 1, except that 3-benzyl-4-hydroxy-5-methoxybenzaldehyde was used instead of 4-chlorobenzaldehyde, and the final reaction yield was 78%. ¹ H NMR(400MHz，DMSO-d ₆ )δ10.42(s，1H)，8.22(s，1H)，8.04(s，1H)，7.75(s，1H)，7.69-7.45(m，5H)，7.32-7.05(m，6H)，3.92(s，2H)，3.85(s，3H).MS(ESI，m/z)：415(M-H) ^- 。

EXAMPLE 24 preparation of 5- (4-hydroxy-3, 5-dimethoxybenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S24)

Compound S24 was prepared in the same manner as in example 1 except that 4-hydroxy-3, 5-dimethoxybenzaldehyde was used instead of 4-chlorobenzaldehyde, and the final reaction yield was 82%. ¹ H NMR(400MHz，DMSO-d ₆ )δ10.22(s，1H)，8.28(s，1H)，7.79(s，2H)，7.65-7.60(m，2H)，7.57-7.49(m，3H)，7.13(s，1H)，3.82(s，6H).MS(ESI，m/z)：355(M-H) ^- 。

EXAMPLE 25 preparation of 5- (3-fluoro-4-hydroxybenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S25)

Compound S25 was prepared in the same manner as in example 1, except that 3-fluoro-4-hydroxybenzaldehyde was used instead of 4-chlorobenzaldehyde, resulting in a final reaction yield of 74%. ¹ H NMR(400MHz，DMSO-d ₆ )δ11.48(s，1H)，8.27(dd，J＝13.4，1.8Hz，2H)，7.89(dd，J＝8.6，2.1Hz，1H)，7.62(dd，J＝6.8，3.0Hz，2H)，7.54(dd，J＝5.1，1.9Hz，3H)，7.14(s，1H)，7.08(t，J＝8.8Hz，1H).MS(ESI，m/z)：313(M-H) ^- 。

Example 26 preparation of 5- (3-bromo-4-hydroxybenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S26)

Compound S26 was prepared in the same manner as in example 1, except that 3-bromo-4-hydroxybenzaldehyde was used instead of 4-chlorobenzaldehyde, resulting in a final reaction yield of 75%. ¹ H NMR(400MHz，DMSO-d ₆ )δ11.81(s，1H)，8.60(d，J＝2.2Hz，1H)，8.23(s，1H)，8.04(dd，J＝8.7，2.2Hz，1H)，7.65-7.59(m，2H)，7.57-7.50(m，3H)，7.15(s，1H)，7.06(d，J＝8.6Hz，1H).MS(ESI，m/z)：374(M-H) ^- 。

Example 27 preparation of 5- (3, 5-dibromo-4-hydroxybenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S27)

Compound S27 was prepared in the same manner as in example 1 except that 3, 5-dibromo-4-hydroxybenzaldehyde was used instead of 4-chlorobenzaldehyde, and the final reaction yield was 75%. ¹ H NMR(400MHz，DMSO-d ₆ )δ8.48(s，2H)，8.18(s，1H)，7.66-7.58(m，2H)，7.57-7.49(m，3H)，7.11(s，1H).MS(ESI，m/z)：453(M-H) ^- 。

Example 28 preparation of 5- (3, 5-difluoro-4-hydroxybenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S28)

Compound S28 was prepared in the same manner as in example 1, except that 3, 5-difluoro-4-hydroxybenzaldehyde was used instead of 4-chlorobenzaldehyde, resulting in a final reaction yield of 73%. ¹ H NMR(400MHz，DMSO-d ₆ )δ8.24(s，1H)，8.08-8.03(m，2H)，7.64-7.59(m，2H)，7.57-7.51(m，3H)，7.15(s，1H).MS(ESI，m/z)：331(M-H) ^- 。

Example 29 preparation of 5- (4-hydroxy-3, 5-diiodobenzylidene) -2-phenyl-1, 3-dioxane-4, 6-dione (S29)

Compound S29 was prepared in the same manner as in example 1, except that 4-hydroxy-3, 5-diiodobenzaldehyde was used instead of 4-chlorobenzaldehyde, and the final reaction yield was 80%. ¹ H NMR(400MHz，DMSO-d ₆ )δ7.75(s，1H)，7.62-7.55(m，2H)，7.52-7.45(m，3H)，7.08(s，3H)，6.81(s，1H).MS(ESI，m/z)：547(M-H) ^- 。

Pharmacological experiments

The invention measures the inhibitory activity of 1, 3-dioxane-4, 6-diketone compounds on SIRT deacetylase activity, and experimental materials used in pharmacological experiments are all purchased commercially except special instructions.

1. SIRT1 enzyme activity detection

The polypeptide Abz-GCLK is treated with DMSO _(Ac) AY _(NO2) GV-NH2 is prepared into 10mM storage liquid, and the storage liquid is frozen and stored in a refrigerator at the temperature of minus 80 ℃ after being subpackaged; NAD (nicotinamide adenine dinucleotide) ⁺ Preparing 50mM stock solution with enzyme activity reaction buffer (25mM Tris, pH 8.0, 137mM sodium chloride, 2.7mM potassium chloride, 1mM magnesium chloride); small molecule compounds were formulated in DMSO as 10mM stock solutions.

Determination of Compound IC ₅₀ When the concentration of the compound is high, the compound is diluted in a gradient. The reaction system is 100 mu L, and the system contains 1 mu M SIRT1 and 500 mu M NAD ⁺ 10 μ M substrate polypeptide and corresponding concentration of compound, 3 secondary wells per reaction condition, 3 replicates per experiment. After the reaction was shaken at 37 ℃ for 30min, 50. Mu.L of nicotinamide at a final concentration of 10mM and trypsin at a concentration of 0.01mg/mL were added to each well to terminate the reaction and perform the cleavage. After reacting for 15min at 37 ℃, reading the fluorescence value by using a microplate reader, wherein the excitation wavelength and the emission wavelength are respectively 320nm and 420nm.

As shown in Table 1, the compounds are 1, 3-dioxane-4, 6-dione, in which a phenyl group is connected to the C2 position and a benzylidene structure is connected to the C5 position. By applying SIRT1 enzyme activity detection experiment, the IC of the batch of compounds for inhibiting the SIRT1 deacetylase activity is detected ₅₀ (Table 2) shows that the compounds have SIRT1The deacetylase activity has an inhibitory effect.

Since each subtype of sirtuin family plays a very important role in life, several active compounds were selected and tested for their inhibitory activity against SIRT2, SIRT3 and SIRT5 in designing small molecule SIRT1 inhibitors, taking into account the inhibitory effects of the inhibitors on other subtypes of sirtuin. In this experiment, the SIRT1 inhibitor EX527 was selected as a positive compound. According to the determination of IC of SIRT1 inhibition by small molecule compounds ₅₀ The detection method and the concentration ratio of each component are used for detecting the inhibition effect of the compounds on SIRT1 homologous protein. As shown in Table 3, the compounds are selective inhibitors of SIRT1, and have better selectivity than the positive compound EX 527. Therefore, the compound has good inhibitory activity on SIRT1 and good selectivity.

Table 2: inhibition of SIRT1 deacetylase activity by compounds

Compound (I)	IC ₅₀ (μM)	Compound (I)	IC ₅₀ (μM)
				S1	18.81±0.42	S15	1.20±0.09
S2	2.45±0.37	S16	7.06±0.10
				S3	1.31±0.26	S17	3.41±0.19
S4	8.14±0.97	S18	6.33±0.21
				S5	10.04±0.57	S19	15.79±1.36
S6	0.89±0.01	S21	4.51±1.17
				S7	7.92±1.79	S22	4.89±0.35
S8	＞50	S23	2.47±0.10
				S9	12.88±2.09	S24	11.90±2.62
S10	1.90±0.53	S25	4.65±0.05
				S11	16.75±0.71	S26	1.40±0.43
S12	5.49±0.95	S27	0.70±0.03
				S13	14.38±1.86	S28	4.54±0.03
S14	14.01±2.01	S29	0.46±0.09

Table 3: inhibition of SIRT1, SIRT2, SIRT3, SIRT5 deacylase activity by partial compounds

Compound (I)	SITR1	SIRT2	SIRT3	SIRT5
					S3	1.31±0.26	54.04±6.35	136.7±14.3	19.84±1.24
S6	0.89±0.01	49.45±5.95	135.8±14.7	22.45±0.78
					S10	1.90±0.53	64.39±7.09	204.4±16.2	32.53±1.15
S15	1.20±0.09	56.44±0.44	171.8±13.6	37.42±1.47
					EX527	0.99±0.10	8.69±0.08	29.18±1.60	/

2. Enzyme kinetic reaction test

In vitro SIRT1 enzyme activity experiments show that the compound S3 inhibits the SIRT1 enzyme activity in a concentration-dependent manner (see figure 1), and the S3 inhibits the IC of SIRT1 ₅₀ 1.31. + -. 0.26. Mu.M, followed by the use of compound S3, and the determination of the type of inhibition of this type of inhibitor using the Michaelis-Menten plot and the double reciprocal plot (Lineweaver-Burk plot).

Acetylation of the polypeptide, NAD, is required for the SIRT1 deacetylase reaction ⁺ As double substrates, when performing enzyme kinetic reaction, it is necessary to fix the concentration of one reaction substrate and change the concentration of the other reaction substrate to determine the substrate acetylation polypeptide and NAD of SIRT1 by the inhibitor respectively ⁺ The type of inhibition of (c):

1) Immobilization of NAD ⁺ The type of inhibition of the inhibitor on the acetylated polypeptide was determined at a concentration of 1 mM: the fixed SIRT1 protein concentration is 0.5 mu M, the concentration of acetylated polypeptide is 50 mu M, 25 mu M, 12.5 mu M, 6.25 mu M, 3.125 mu M and 1.5625 mu M respectively, and the concentration of inhibitor S3 is 1.875 mu M, 0.9375 mu M, 0.46875 mu M and 0 mu M respectively. After shaking reaction at 37 ℃ for 15min, the enzyme was cleaved for 15min and detected with a microplate reader.

2) Determination of inhibitor vs NAD with fixed acetylated polypeptide concentration of 30. Mu.M ⁺ The inhibition type of (c): fixed SIRT1 protein concentration of 0.5. Mu.M, NAD ⁺ Respectively at a concentration of 1000. Mu.M, 500. Mu.M, 250. Mu.M, 125. Mu.M, 62.5. Mu.M and 31.25. Mu.M, and at a concentration of 1.875. Mu.M, 0.9375. Mu.M, 0.46875. Mu.M and 0. Mu.M, respectively, of inhibitor S3. After shaking reaction at 37 ℃ for 15min, the enzyme was digested for 15min and detected with a microplate reader.

The results of the experiment are shown in FIG. 2.

In FIG. 2, A and B are fixed NAD, respectively ⁺ Concentration, change in substrate Abz polypeptide concentration, michaelis constant curve and double reciprocal curve of compound S3 versus Abz polypeptide. In FIG. 2, C and D are respectively the fixed Abz polypeptide concentration and the changed NAD ⁺ At concentration, compound S3 is coupled to NAD ⁺ The mie constant curve and the double reciprocal curve. As can be seen from B in FIG. 2, when mapping the substrate Abz polypeptide, concentrations at different compounds were observedThe curve at intensity crosses the third quadrant, indicating that as the concentration of compound S3 increases, the initial velocity V ₀ Decrease of apparent Michaelis constant K _m ' reduced, so compound S3 is a mixed type of inhibition of the substrate polypeptide, and binding of compound S3 to SIRT1 is likely at the binding site of the substrate polypeptide. To NAD ⁺ When plotted (D in FIG. 2), the curves are plotted on the horizontal axis at different compound concentrations, indicating that the initial velocity V increases with increasing compound S3 concentration ₀ Decrease of apparent Michaelis constant K _m ' invariant, therefore Compound S3 is to NAD ⁺ Non-competitive inhibition, S3 binds SIRT1 at a site other than the NAD + binding site.

3. Microcalorie Thermophoresis (MST) experiment

The mini-hSIRT1 protein was centrifuged at high speed (12,000rpm, 10 min) to remove air, and then allowed to stand at room temperature for 30min. To 1mL of mini-hSIRT1 protein, 100. Mu.L of TCEP (PB buffer solution) with 100 times of protein molar concentration was added, the centrifuge tube containing the protein was flushed with nitrogen gas, quickly covered, thoroughly mixed and left for 10min. Then 50. Mu.L of 10 times molar concentration Cy5 was added to the centrifuge tube ^TM And (4) dissolving the dye solution (DMSO), flushing the centrifuge tube filled with the protein by using nitrogen, quickly covering the centrifuge tube, thoroughly mixing, and standing. Incubate at room temperature for 2 hours and mix well every half hour before standing at 4 ℃ overnight. 2g of SM-2 adsorbent (Bio-Rad) was weighed, the packing was activated with 1 to 2 column volumes of methanol, and washed with water and a buffer (20mM HEPES, pH 7.2, 200mM sodium chloride, 5% glycerol). And (3) after the protein liquid flows through the filler, washing the filler by using a buffer solution, collecting the flow-through liquid, and measuring the SIRT1 protein concentration.

Before MST experiments, the SIRT1 to be labeled ^Cy5 The protein was centrifuged at high speed (13,000rpm, 5 min) to remove aggregates. Labelled SIRT1 ^Cy5 The protein was diluted to 200nM in MST optimized buffer (50mM Tris, pH 7.4, 150mM NaCl, 10mM magnesium chloride, 0.05% Tween-20) for use. In the reaction system, fixing SIRT1 ^Cy5 The concentration was 100nM, the compound was set to an initial concentration of 500. Mu.M, diluted in 16 steps in two-fold, and the DMSO content in the system was kept at 10%. Three conditions were determined using Monolith NT115 (Nano tester Technologies)The following MST curves (Monolith NT115 parameter set to 20% Red, MST Power 40.0%, excitation Power 20%):

1)SIRT1 ^Cy5 interaction profile with Compound S3

2) At 500. Mu.M polypeptide Abz-GVL _(Ac) AY _(NO2) GV-NH ₂ In the presence of SIRT1 ^Cy5 Interaction curves with Compound S3

3) At 5mM NAD ⁺ In the presence of SIRT1 ^Cy5 Interaction profile with compound S3.

Experimental data were analyzed by mo.affinity Analysis software to obtain MST binding curves. As shown in FIG. 3, in the presence of the Abz polypeptide, the binding curve of compound S3 to SIRT1 protein shifts to the right, K _d The value increased, indicating that compound S3 is a competitive inhibitor for the Abz polypeptide. While in NAD ⁺ When the compound S3 participates, the binding curve of the compound S3 and the SIRT1 protein is not obviously changed, which indicates that the compound S3 is applied to NAD ⁺ Is a non-competitive inhibitor.

4. Molecular simulation and mutant experiments

The complex crystal complex structure (PDB ID:4I 5I) of SIRT1/EX527 is downloaded from RCSB-PDB database (www.rcsb.org), the EX527 molecule in the structure is removed, and the coordinates of a SIRT1 protein molecule are extracted and stored as PDB file. Compound S3 was docked into SIRT1 protein structure and binding models were analyzed according to structure-activity relationships. As shown in A in FIG. 4, compound S3 binds to phenylalanine 273, asparagine 346, isoleucine 347, 348 and 414 of SIRT1, therefore, site-directed mutagenesis was performed on these 5 amino acid residues and the inhibition constants (K) of compound S3 for these SIRT1 mutant proteins were examined _i ). As can be seen in FIG. 4B, compound S3 is active against K of SIRT1 wild type _i The value was 0.16. Mu.M, compound S3 vs mutant SIRT1 ^F273L 、SIRT1 ^N346A 、SIRT1 ^1347A 、SIRT1 ^D348A 、SIRT1 ^F414A Inhibition rate constant K of _i Respectively 0.85. Mu.M, 2.47. Mu.M, 0.67. Mu.M, 4.33. Mu.M and 25.5. Mu.M, thereby showing that the inhibition constants of the compounds on mutant proteins are all improved, wherein the mutant SIRT1 ^F414A The influence on the inhibitory activity of the compound is the largest, and based on a binding model, a hydrophobic effect is found between the phenyl at the C2 position and the phenylalanine at the 414 position of the compound, which all indicate that the phenylalanine at the 414 position of SIRT1 is a key amino acid residue for binding protein/S3 small molecule compound, and the fact that the hydrophobicity of the phenyl group of the compound can be properly increased in the next structural modification is suggested to further improve the activity of the compound. In addition, according to a binding model, the hydroxyl at the 3-position of the C5 benzylidene R of the compound forms a hydrogen bond with the 346 th aspartic acid residue of the SIRT1, and after mutation of the 346 th aspartic acid residue, the sensitivity of the compound S3 to mutant protein is reduced, which indicates that the site is also a key site for compound binding.

5. Effect of Compounds on intracellular levels of p53 acetylation

SH-SY5Y human neuroblastoma cells are inoculated in a 12-well plate, are put in a culture solution overnight, ATRA (all-trans-retinoic acid) and a compound (10 mu M) are added for acting for 2 hours, then the cells are collected, washed once by precooled PBS, and cell lysate is added. After heating the cell lysate in a boiling water bath for 5 minutes. Then, the mixture was centrifuged at 4 ℃ for 10 minutes in a high-speed centrifuge (12000 rpm), and the supernatant was collected.

And (3) carrying out SDS-PAGE electrophoresis on the supernatant, cutting the gel after electrophoresis, placing the gel in a glass dish containing electrotransfer liquid, measuring the length of the gel, shearing a membrane and filter paper according to the size of the gel, preparing a membrane transfer layer, placing the membrane in an anode, placing the gel in a cathode, and placing the filter paper at the outermost layer to wrap the membrane and the gel. The film transfer layer is arranged in an electric transfer tank filled with electric transfer liquid for film transfer. After washing the membrane with TBST detergent, the membrane was placed in blocking solution (5% nonfat dry milk) and blocked at 37 ℃ for 2 hours. After incubation overnight at 4 ℃ with the addition of primary antibody, washed three times with TBST for 10 minutes each, and then incubated with secondary antibody, incubated at 37 ℃ for 2 hours, washed three times with TBST and developed with a developer.

As can be seen from the experimental results (FIG. 5), each band of beta-actin has the same color, indicating that the concentrations of the loading proteins are consistent; the p53 bands were identical, indicating consistent levels of p53 background. Under the experimental condition, when the acetylated p53 (ac-p 53) bands in SH-SY5Y cells treated by ATRA and the compounds are compared, the p53 acetylation level is reduced when the compounds S3, S6, S10 and S15 inhibit ATRA-induced SH-SY5Y cell differentiation, and the compounds are proved to have good SIRT1 acetylation inhibition activity at the cell level.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. The application of the compound shown in the formula I or the pharmaceutically acceptable salt thereof is characterized in that (a) the compound is used for preparing an inhibitor of SIRT1 deacetylase; or (b) is used for preparing a medicine for treating diseases related to the abnormal expression of SIRT1 protein deacetylase or the enzyme activity level thereof,

R ¹ and R ⁷ Each independently hydrogen or C1-C6 alkyl;

R ² 、R ³ 、R ⁴ 、R ⁵ 、R ⁶ each independently of the others is hydrogen, halogen, hydroxy, carboxy, C1-C4 alkyl, C1-C6 alkoxy, -L1- (CH) ₂ ) m-substituted or unsubstituted C6-C12 aryl, -L1- (CH) ₂ ) m-substituted or unsubstituted 3-12 membered heterocyclyl, -L1- (CH) ₂ )m-C(＝O)-N(R ⁸ )(R ⁹ ) Wherein, L1 is none, -O-or-S-; m is 0, 1 or 2; r is ⁸ And R ⁹ Each independently selected from: hydrogen, C1-C6 alkyl, C6-C12 aryl;

the substitution refers to the inclusion of one or more substituents selected from the group consisting of: halogen, hydroxy, phenyl, hydroxymethyl, carboxy;

and R is ⁶ The number of (2) is 1-3.

2. The use according to claim 1, wherein R is ¹ And R ⁷ Each independently hydrogen or C1-C4 alkyl.

3. The use according to claim 1, wherein R is ² 、R ³ 、R ⁴ 、R ⁵ Each independently of the others is hydrogen, halogen, hydroxy, carboxy, C1-C4 alkyl, C1-C4 alkoxy, -L1- (CH) ₂ ) m-substituted or unsubstituted C6-C12 aryl, -L1- (CH) ₂ ) m-substituted or unsubstituted 4-10 membered heterocyclyl, -L1- (CH) ₂ )m-C(＝O)-N(R ⁸ )(R ⁹ ) Wherein, L1 is none, -O-or-S-; m is 1 or 2; r is ⁸ And R ⁹ Each independently selected from: hydrogen, C6 aryl;

the substitution refers to the inclusion of one or more substituents selected from the group consisting of: halogen, hydroxy, phenyl, hydroxymethyl, carboxy.

4. The use according to claim 1, wherein R is ³ Is hydrogen, halogen, hydroxy, -L1- (CH) ₂ ) m-3-10 membered heterocyclyl, -L1- (CH) ₂ ) m-phenyl, -L1- (CH) ₂ ) m-phenyl-carboxy, C1-C4 alkoxy, -L1- (CH) ₂ )m-C(＝O)-N(R ⁸ )(R ⁹ ) Wherein, L1 is none, -O-or-S-; m is 1 or 2; r is ⁸ And R ⁹ Each independently selected from: hydrogen, C1-C4 alkyl or phenyl.

5. The use according to claim 1, wherein R is ² 、R ⁴ Each independently of the others is hydrogen, halogen, C1-C4 alkyl, C1-C4 alkoxy or-L1- (CH) ₂ ) m-phenyl, wherein L1 is absent; m is 1 or 2.

6. The use according to claim 1, wherein R is ⁵ Is hydrogen or C1-C4 alkoxy.

7. The use according to claim 1, wherein the compound is:

8. the use of claim 1, wherein the disease associated with aberrant SIRT1 protein expression or its enzymatic activity level is selected from the group consisting of: neurodegenerative diseases, cancer, metabolic diseases, immune disorders and inflammation.

9. The use according to claim 8, wherein said cancer is selected from the group consisting of: histiocytic lymphoma, ovarian cancer, head and neck squamous cell carcinoma, gastric cancer, breast cancer, childhood hepatocellular carcinoma, colorectal cancer, cervical cancer, lung cancer, sarcoma, nasopharyngeal carcinoma, pancreatic cancer, glioblastoma, prostate cancer, multiple myeloma, thyroid cancer, testicular cancer, lung adenocarcinoma, glioblastoma, endometrial cancer, esophageal cancer, leukemia, renal cell carcinoma, bladder cancer, astrocytoma.

10. The use according to claim 8, wherein the metabolic disease is selected from the group consisting of: diabetes, hyperlipidemia, obesity, and hyperglycemia hyperostosis syndrome.

11. The use of claim 8, wherein the neurodegenerative disease is selected from the group consisting of: alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, spinocerebellar ataxia, and spinal muscular atrophy.

12. The use of claim 8, wherein the cancer is selected from the group consisting of: small cell lung cancer, non-small cell lung cancer, liver cancer, colon cancer, papillary renal cell carcinoma, glioma, non-malignant skin cancer.

13. A pharmaceutical composition comprising a compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof; and

a pharmaceutically acceptable carrier.

14. The use of a pharmaceutical composition according to claim 13, wherein (a) the use for the preparation of an inhibitor of SIRT1 deacetylase; or (b) is used for preparing a medicine for treating diseases related to the abnormal expression of SIRT1 protein deacetylase or the enzyme activity level thereof.

15. The use of claim 14, wherein the disease associated with aberrant expression of a SIRT1 protein or its enzymatic activity level is selected from the group consisting of: neurodegenerative diseases, cancer, metabolic diseases, immune disorders and inflammation.

16. The use according to claim 15, wherein the cancer is selected from the group consisting of: histiocytic lymphoma, ovarian cancer, head and neck squamous cell carcinoma, gastric cancer, breast cancer, childhood hepatocellular carcinoma, colorectal cancer, cervical cancer, lung cancer, sarcoma, nasopharyngeal carcinoma, pancreatic cancer, glioblastoma, prostate cancer, multiple myeloma, thyroid cancer, testicular cancer, lung adenocarcinoma, glioblastoma, endometrial cancer, esophageal cancer, leukemia, renal cell carcinoma, bladder cancer, astrocytoma.

17. The use according to claim 15, wherein the cancer is selected from the group consisting of: small cell lung cancer, non-small cell lung cancer, liver cancer, colon cancer, papillary renal cell carcinoma, glioma, and non-malignant skin cancer.

18. The use according to claim 15, wherein the metabolic disease is selected from the group consisting of: diabetes, hyperlipidemia, obesity, and hyperglycemia hyperostosis syndrome.

19. The use of claim 15, wherein the neurodegenerative disease is selected from the group consisting of: alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, spinocerebellar ataxia, and spinal muscular atrophy.