WO2019242687A1 - 1,3-dioxane-4,6-dione compound, and preparation method, pharmaceutical composition, and application thereof - Google Patents

Abstract

The present invention discloses a 1,3-dioxane-4,6-dione compound, and a preparation method, a pharmaceutical composition, and an application thereof. The structure of the compound is represented by formula I, and the definition of each substitution group is described in the specification and claims. The compound of the present invention inhibits the activity of SIRT1 protein deacetylase and can be used in drugs for treating various diseases associated with abnormal expression of SIRT1 protein deacetylase and enzyme activity thereof, such as neurodegenerative diseases, metabolic diseases, cancer, and the like.

Description

1,3-dioxane-4,6-dione compounds, preparation method, pharmaceutical composition and application thereof Technical field

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

Background technique

Sirtuin is a class of NAD ⁺ -dependent protein deacetylases, which have a high degree of homology in amino acid sequence and structure. Sirtuin protein is widely present in various organisms such as archaea, nematodes, fruit flies, yeast, and mammals, and regulates a variety of cells including cell senescence, transcription, apoptosis, inflammation, stress, mitochondrial synthesis, and the body's biological clock. Important biological processes.

The yeast Sir2 gene was the first sirtuin protein discovered. As early as the 1970s, it was discovered that the Sir2 gene can maintain the length of yeast telomeres and regulate the generation of DNA repeats encoded by rDNA. It was later discovered that the Sir2 gene can prolong the lifespan of yeast by suppressing genomic instability. Knockout of the Sir2 gene can significantly shorten the yeast's lifespan, while overexpression of the Sir2 gene can extend the yeast's lifespan by about 40%. Similarly, overexpression of Sir2.1 (a homologous gene of Sir2) in nematodes can extend the lifespan of nematodes by about 50%, and a similar phenomenon occurs in fruit flies. These findings have made research on sirtuin family proteins more popular.

The mammalian genome encodes seven sirtuin proteins, named SIRT1-7, which contain a highly conserved core region consisting of a NAD ⁺ binding region and an enzyme catalytic region, as well as a variable length N-terminus and a C-terminus. end. Differences in the N- and C-termini of Sirtuin proteins can affect protein-ligand binding, mediate protein interactions with other sirtuin isoforms, or affect their subcellular localization. Existing research results show that SIRT1, SIRT6, and SIRT7 are nuclear proteins, but SIRT1 can also pass from the nucleus into the cytoplasm through nuclear transport, thereby regulating the target proteins in the cytoplasmic stress response. SIRT2 is mainly located in the cell matrix, but SIRT2 can be transported into the nucleus through nuclear transport, while SIRT3, SIRT4, and SIRT5 are mainly located in the mitochondria. Mammalian sirtuins are distributed in different subcellular layers, which is closely related to the substrates and biological functions they act on.

SIRT1 is the mammalian sirtuin family protein that is closest in sequence to yeast Sir2 and is the earliest member of the mammalian sirtuin family to be studied. SIRT1 regulates heterochromatin formation by deacetylating H1K26, H3K9, and H4K16. In addition, SIRT1 is also involved in the deacetylation of non-histones. The non-histone substrates of SIRT1 can be divided into three categories: (1) transcription factors: such as p53, FOXO3a, E2F2, BCL6, etc .; (2) DNA repair proteins: such as Ku70 and MRE11-RAD50-NBS1 (MRN); 3) Signal factor: Smad7 and so on. SIRT1 participates in regulating a variety of physiological functions including gene expression, energy metabolism, and aging by deacetylating histone substrates and non-histone substrates.

SIRT1 is closely related to the occurrence and development of various diseases. It can control the development of Alzheimer's disease (AD) by deacetylating the P65 / RelA subunit of NF-κB and inhibiting the accumulation of Aβ in microglia. . SIRT1 can also protect nerve cells in Huntington's disease (HD) disease models by deacetylating PGC-1α and increasing PGC-1α activity. It is known that the tumor suppressor p53 protein is involved in many physiological processes including DNA repair, cell growth arrest, aging and apoptosis, and has become one of the important targets for cancer treatment. SIRT1 can deacetylate the lysine residue at position 382 of p53. Deacetylation of p53 can lead to tumorigenesis. SIRT1 can also deacetylate DNA repair factor Ku70 and forkhead transcription factor FOXOs to enhance cell DNA repair and inhibit apoptosis caused by DNA damage. Studies have shown that inhibition of SIRT1 activity can induce tumor cell growth arrest and promote tumor cell apoptosis. In addition, SIRT1 can regulate the transcription of tumor suppressor genes by deacetylating histone H1 at position 26, H3 at position 9, and H4 at position 16 to participate in the regulation of tumor cell cycle. The study found that overexpression of SIRT1 protein was detected in most solid tumors and hematological malignancies including breast, colon, prostate, liver and leukemia. Since the overexpression of SIRT1 is related to the occurrence of cancer, inhibiting the activity of SIRT1 can effectively inhibit the proliferation of cancer cells and induce apoptosis of cancer cells at the same time. Therefore, SIRT1 may become a new target for tumor therapy, and SIRT1 inhibitors may become potential anticancer drug candidates.

Summary of the Invention

An object of the present invention is to provide a 1,3-dioxane-4,6-dione compound represented by the general formula (I), a pharmaceutically acceptable salt thereof, an enantiomer, Enantiomers or racemates.

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

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

Yet another object of the present invention is to provide the use of the compound represented by the general formula (I) in the preparation of a medicament for treating diseases related to the activity level of SIRT1 deacetylase, such as cancer, immune disorders and inflammation.

According to a first aspect of the present invention, there is provided a compound represented by Formula I, or a pharmaceutically acceptable salt, hydrate, solvate, enantiomer, diastereomer or racemate thereof, or mixture:

among them,

R ¹ and R ⁷ are each independently hydrogen, C1-C6 alkyl or C2-C12 unsaturated hydrocarbon group;

R ² , R ³ , R ⁴ , R ⁵ , R ⁶ are each independently hydrogen, halogen, cyano, nitro, amino, hydroxyl, carboxyl, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 unsaturated hydrocarbon, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 acyl, substituted or unsubstituted C3-C12 cycloalkyl, -L1- (CH ₂ ) m-substituted Or unsubstituted C6-C12 aryl, -L1- (CH ₂ ) m-substituted or unsubstituted 3-12 membered heterocyclyl, -L1- (CH ₂ ) mC (= O) -N (R ⁸ ) (R ⁹ ), wherein L 1 is none, -O- or -S-; m is 0, 1, 2, 3, 4 or 5; R ⁸ and R ⁹ are 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 refers to including one or more substituents selected from the group consisting of halogen, hydroxy, phenyl, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 unsaturated hydrocarbon, C1-C6 alkoxy , C1-C6 haloalkoxy, C3-C12 cycloalkyl, 3-12 membered heterocyclic group, cyano, nitro, methylol, carboxyl, mercapto;

And the number of R ⁶ is 1-3.

In another preferred example, R ¹ and R ⁷ are each independently hydrogen, C1-C4 alkyl, or C2-C4 unsaturated hydrocarbon group.

In another preferred example, R ² , R ³ , R ⁴ , and R ⁵ are each independently hydrogen, halogen, cyano, nitro, amino, hydroxyl, carboxyl, substituted or unsubstituted C1-C4 alkyl, substituted Or unsubstituted C2-C6 unsaturated hydrocarbon group, substituted or unsubstituted C1-C4 alkoxy group, substituted or unsubstituted C1-C4 acyl group, substituted or unsubstituted C3-C6 cycloalkyl group, -L1- (CH ₂ ) m-substituted or unsubstituted C6-C12 aryl, -L1- (CH ₂ ) m-substituted or unsubstituted 4-10 membered heterocyclic group, -L1- (CH ₂ ) mC (= 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 are 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 refers to including one or more substituents selected from the group consisting of halogen, hydroxy, phenyl, C1-C4 alkyl, C1-C4 haloalkyl, C2-C4 unsaturated hydrocarbon, C1-C4 alkoxy , C1-C4 haloalkoxy, C3-C6 cycloalkyl, 3-6 membered heterocyclic group, cyano, nitro, methylol, carboxyl, mercapto.

In another preferred example, R ³ is hydrogen, halogen, hydroxyl, -L1- (CH ₂ ) m-3-10 membered heterocyclic group, -L1- (CH ₂ ) m-C6-C10 aryl group, -L1 -(CH ₂ ) m-C6-C10aryl-carboxyl, C1-C4 alkoxy, 3-6 membered heterocyclyl, -L1- (CH ₂ ) mC (= O) -N (R ⁸ ) (R ^9), wherein, Ll is no, -O- or -S-; m is 0, 1 or 3; R ⁸ and R ⁹ are each independently selected from: hydrogen, C1-C4 alkyl or phenyl.

In another preferred example, R ² and R ⁴ are each independently 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 example, the compound is:

According to a second aspect of the present invention, a method for preparing a compound according to the second aspect is provided, including the following steps:

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

b) Compound I _{a is} reacted with a substituted benzaldehyde or ketone to obtain a compound represented by Formula I,

The definitions of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ are as described in the first aspect.

In another preferred embodiment, the method for preparing the compound includes the following steps:

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

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

According to a third aspect of the present invention, there is provided a pharmaceutical composition comprising the compound described in the first aspect, or a pharmaceutically acceptable salt, hydrate, solvate, enantiomer, diastereomer Enantiomers or racemates, or mixtures thereof; and pharmaceutically acceptable carriers.

)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。 "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and low enough toxicity. "Compatibility" here means that each component in the composition can blend with the active ingredient of the present invention and each other without significantly reducing the medicinal effect of the active ingredient. Examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, and solid lubricants (such as stearic acid). , Magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween

), Wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

According to a fourth aspect of the present invention, there is provided the compound described in the first aspect, or a pharmaceutically acceptable salt, hydrate, solvate, enantiomer, diastereomer or racemate thereof, or The use of their mixture, (a) for the preparation of inhibitors of SIRT1 deacetylase; or (b) for the preparation of drugs for treating diseases related to abnormal expression of SIRT1 protein or its enzyme activity level.

In another preferred example, the abnormal expression of SIRT1 protein or a disease associated with its enzyme activity level is selected from the group consisting of: neurodegenerative disease, cancer, metabolic disease, immune disorder, and inflammation.

It should be understood that, within the scope of the present invention, the above technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Each feature disclosed in the description may be replaced by any alternative feature serving the same, equivalent, or similar purpose. Due to space limitations, I will not repeat them here.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of SIRT1 enzyme activity in vitro experiments, illustrating that compound S3 inhibits SIRT1 activity in a concentration-dependent manner.

Figure 2 shows the inhibition type of compound S3 on SIRT1 protein. A) and B) are the fixed NAD ⁺ concentration. When the substrate polypeptide concentration is changed, the Mie constant curve and double reciprocal curve of the compound S3 on the substrate Abz polypeptide are detected. C) and D) are the concentrations of the fixed substrate Abz polypeptide, respectively. When the NAD ⁺ concentration is changed, the Mie constant curve and the double reciprocal curve of the compound S3 against NAD ⁺ are detected. This shows that compound S3 is a competitive inhibitor of the substrate polypeptide Abz and a non-competitive inhibitor of NAD ⁺ .

Figure 3 is a graph showing the binding curve of compound S3 and protein detected by a microthermophoresis test (MST). Result chart illustrating that compound S3 competitively binds to the binding site of the substrate polypeptide Abz.

In Fig. 4 A) the molecular simulation results of compound S3 and SIRT1 binding; B) the results of SIRT1 mutant enzyme kinetic experiment.

Figure 5 shows the effect of compounds on p53 acetylation levels in SH-SY5Y human neuroblastoma cells. The control group (Con, control) was added with DMSO (10 μM), the SIRT1 inhibitor Ex527 reported in the literature was used as a positive control, and β-actin was used as an internal reference. anti-p53 (acetyl K381) antibody (article number: ab61241) and anti-p53 antibody (article number: ab26) were purchased from IRDye, 680RD Goat anti-Rabbit (article number 926-68071), 800CW Goat anti-Mouse (article number 926-32210) Antibodies were purchased from LICOR.

detailed description

Based on long-term and in-depth research, the present inventors have prepared a class of compounds having a structure represented by Formula I and found that they have SIRT1 inhibitory activity. In addition, the compound has an inhibitory effect on SIRT1 at a low concentration, and the inhibitory activity is quite excellent. Therefore, it can be used to treat diseases related to SIRT1 activity or expression, such as neurodegenerative diseases, metabolic diseases, and tumors. Based on this, the present invention has been completed.

the term

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, S-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl and isohexyl, or similar groups. "C1-C6 alkyl" refers to a straight or branched chain alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms, and "C1-C4 alkyl" refers to a group having 1, 2, 3, or 4 Straight or branched chain alkyl groups of one carbon atom, and so on. The term "C1-C12 haloalkyl" refers to a straight or branched chain alkyl group having 1 to 12 carbon atoms, such as trifluoromethyl, etc., which is substituted with 1, 2, 3 or more halogens.

The term "C2-C12 unsaturated hydrocarbon group" refers to a linear or branched alkenyl or alkynyl group having 2 to 12 carbon atoms, such as vinyl, propynyl, and the like.

The term "C1-C6 alkoxy" refers to a straight or branched chain alkoxy group having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso Butoxy, sec-butoxy, and tert-butoxy, or similar groups. The definition of "C1-C6 alkoxy" includes "C1-C4 alkoxy".

The term "C3-C12 cycloalkyl" refers to a saturated or unsaturated hydrocarbon group having 3-12 carbon atoms on 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 on 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 to 12 carbon atoms, a substituent having a conjugated pi electron system, such as phenyl and naphthyl, or the like. The definition of "C6-C12 aryl" includes "C6-C10 aryl".

The term "3-12 membered heterocyclyl" refers to a monocyclic or fused bicyclic ring having 3-12 ring atoms and having one or more (preferably 1-5) ring systems selected from O, S, N or Heteroatoms of P, such as piperidinyl, pyrrolidinyl, piperazinyl, tetrahydrofuranyl, morpholinyl, benzodioxolyl, tetrahydropyrrolyl or the like. The definition of "3-12 membered heterocyclic group" includes "4-10 membered heterocyclic group".

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

As used herein, unless specifically stated, the term "substituted" refers to the replacement of one or more hydrogen atoms on a group 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-hydroxy.

Unless otherwise specified, all compounds present in the present invention are intended to include all possible optical isomers, such as a single chiral compound, or a mixture (ie, a racemate) of various chiral compounds. Among all the compounds of the present 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 "compound of the invention" refers to a compound of formula I. The term also includes various crystalline forms of the compounds of Formula I, pharmaceutically acceptable salts, hydrates or solvates.

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

Compound of formula I

The structure of the compound of formula I of the present invention is shown below:

The definition of each substituent is as described above.

The preferred compounds are shown in the following table:

Table 1. Compound number, name and chemical structural formula

SIRT1 activity inhibitor and its application

The compounds of the invention inhibit the activity of one or more sirtuin proteins. For example, the compound of the present invention can be used to inhibit the activity of the sirtuin enzyme in a cell or a patient, and the inhibitory function of the sirtuin protein can be achieved by applying an inhibitory amount of the compound of the present invention to the cell, individual or patient. .

Analysis of the inhibitory activity data of the compounds of the present invention on SIRT1, SIRT2, SIRT3, and SIRT5 shows that the compounds of the present invention are selective inhibitors of SIRT1 and have better selectivity than the positive compound EX527.

As SIRT1 inhibitors, the compounds of the present invention are suitable for treating various diseases associated with abnormal expression or activity of SIRT1. The abnormal proliferation diseases related to the activity or expression of SIRT1 include but are not limited to the following diseases: histiocytic lymphoma, ovarian cancer, head and neck phosphorous epithelial cell cancer, gastric cancer, breast cancer, childhood hepatocellular carcinoma, colorectal cancer , Cervical cancer, lung cancer, sarcoma, nasopharyngeal cancer, 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, etc. It is more preferably used for treating hematological malignancies including breast cancer, colon cancer, prostate cancer, liver cancer and leukemia.

The compounds and compositions of the present invention are used to treat, prevent or regulate metabolic related diseases, including diabetes, hyperlipidemia, obesity, hyperglycemia and hypertonic syndrome.

The compounds and compositions of the present invention are used to treat, prevent or regulate neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar ataxia and spinal sex Muscular atrophy.

The benefits of the present invention are:

The 1,3-dioxane-4,6-dione compounds of the present invention have low toxicity and good solubility.

The preparation method of the 1,3-dioxane-4,6-dione compound and its derivatives has the advantages of mild reaction conditions, abundant raw materials, easy operation and post-treatment, and good corresponding selectivity.

The 1,3-dioxane-4,6-dione compounds and derivatives thereof according to the present invention have very good inhibitory activity and excellent selectivity on SIRT deacetylase.

Therefore, the compounds of the present invention can be used to treat various diseases related to abnormal expression or activity of SIRT1 protein, such as neurodegenerative diseases, diabetes, tumors and other diseases.

The present invention will be further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are usually performed according to conventional conditions or conditions recommended by the manufacturer. Unless stated otherwise, percentages and parts are percentages by weight and parts by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equal to those described can be used in the method of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

Example 1 Preparation of Compound 5- (4-chlorobenzylidene) -2-phenyl-1,3-dioxane-4,6-dione (S1)

Reaction Formula 1.1

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

Place malonic acid (10.4 g, 100 mmol) in a round bottom flask, add acetic anhydride (28.4 mL, 300 mmol), stir at room temperature, add 0.1 mL of concentrated H ₂ SO ₄ , and stir overnight. At room temperature, benzaldehyde (10.2 mL, 100 mmol) was slowly added and reacted overnight at 5 ° C. After the reaction was completed. About 30 mL of toluene was added, and the acetic anhydride was removed by concentration under reduced pressure. A white solid precipitated, and the filter cake was washed with 200 mL of water after suction filtration to obtain a crude product. The crude product was dissolved in acetone, a solid was precipitated after adding an appropriate amount of water, stirred for 15 minutes, and then filtered with suction, and the filter cake was washed with water to obtain 2-phenyl-1,3-dioxo-4,6-dione (10.2 g, yield 53%). ¹ H NMR (400MHz, DMSO-d ₆ ) δ 7.56 (dtd, J = 10.2, 5.1, 2.0 Hz, 5H), 7.13 (s, 1H), 4.56 (d, J = 18.4 Hz, 1H), 3.61 ( d, J = 18.4 Hz, 1H). MS (ESI, m / z): 191 (MH) ^- .

Reaction 1.2

Step 2: preparing S1

Dissolve 2-phenyl-1,3-dioxo-4,6-dione (0.3 g, 1.56 mmol) in anhydrous DMSO, add anhydrous sodium acetate (90 mg, 1.09 mmol) and stir to dissolve at room temperature. Add 4-chlorobenzaldehyde (0.22g, 1.56mmol), stir overnight, add about 30mL of water to the solution, stir to precipitate the solid, suction filter, wash the filter cake with water, and obtain the crude product after drying, methanol / petroleum ether Recrystallization gave 310 mg of pure product with a yield of 63%. 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 Compound 5- (4-hydroxy-3-iodo-5-methoxybenzylidene) -2-methyl-2-phenyl-1,3-dioxane-4,6-dione Preparation of (S2)

Reaction 2.1

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

Put malonic acid (15.6 g, 150 mmol) in a round bottom flask, add acetic anhydride (55.5 mL, 585 mmol), stir at room temperature, add 0.2 mL of concentrated H ₂ SO ₄ , and stir overnight. At room temperature, acetophenone (35mL, 300mmol) was slowly added, and after reacting for 1 hour, acetic anhydride was removed by concentration under reduced pressure and separated by flash column chromatography (petroleum ether / ethyl acetate = 15/1, v / v). The product was purified to obtain 2-methyl-2-phenyl-1,3-dioxo-4,6-dione (9.3 g, yield 30%). MS (ESI, m / z): 205 (MH) ^- .

Reaction 2.2

Step 2: preparing S2

Dissolve 2-methyl-2-phenyl-1,3-dioxo-4,6-dione (0.3 g, 1.45 mmol) in anhydrous DMSO and add anhydrous sodium acetate (84 mg, 1.02 mmol) at room temperature Stir to dissolve, add 4-hydroxy-3-iodo-5-methoxybenzaldehyde (0.41g, 1.45mmol), stir overnight, add about 30mL of water to the solution, stir to precipitate the solid, suction filter, water The filter cake was washed and dried to obtain the crude product. The methanol / petroleum ether was recrystallized to obtain 340 mg of pure product with a yield of 50%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 10.59 (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 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 4-chlorobenzaldehyde was replaced with 3-bromo-4-hydroxy-5-methoxybenzaldehyde, and the reaction yield in the last step was 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 (MH) ^- .

Example 4 Compound 5- [4- (1,3-benzodioxolane-5-alkylmethoxy) -3-methoxybenzylidene) -2-phenyl-1,3-di Preparation of oxane-4,6-dione (S4)

Except for replacing 4-chlorobenzaldehyde with 4- (benzo-1,3-dioxolane-5-alkylmethoxy) -3-methoxybenzaldehyde, in the same manner as in Example 1 Compound S4 was prepared with a final reaction yield of 68%. ¹ H NMR (400MHz, CDCl ₃ ) δ 8.34 (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 Compound 3-({4-[(4,6-dioxo-2-phenyl-1,3-dioxane-5-ylidene) methyl] phenoxy} methyl) benzoic acid Preparation of (S5)

The compound S5 was prepared in the same manner as in Example 1 except that 4- (benzylphenoxy) methyl) benzoic acid was used instead of 4-chlorobenzaldehyde, and the final reaction yield was 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-chlorobenzaldehyde was replaced with 4-hydroxy-3-iodo-5-methoxybenzaldehyde, and the reaction yield in the last step was 70%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 10.65 (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 4-hydroxy-3-iodo-5-methoxybenzaldehyde was replaced with 3-ethoxy-4-hydroxybenzaldehyde. The yield of the final step was 46. %. ¹ H NMR (400MHz, CDCl ₃ ) δ 8.08-8.01 (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 4-hydroxy-3-iodo-5-methoxybenzaldehyde was replaced with benzaldehyde, and the reaction yield in the last step was 62%. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.06 (s, 1H), 7.74-7.68 (m, 2H), 7.58-7.28 (m, 8H), 1.98 (s, 3H).

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

Except for replacing 4-hydroxy-3-iodo-5-methoxybenzaldehyde with 3-((4-formyl-2-methoxyphenoxy) methoxy) benzoic acid, the same procedure as in Example 2 was used. Compound S9 was prepared in the same manner, with a final reaction yield of 45%. ¹ H NMR (400MHz, CDCl ₃ ) δ 8.18-8.02 (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-chloro-3-chlorobenzaldehyde was replaced with 4-hydroxy-3-chloro-5-methoxybenzaldehyde, and the reaction yield in the last step was 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 4-hydroxy-3-iodo-5-methoxybenzaldehyde was replaced with 2,5-dimethoxybenzaldehyde. The yield of the final reaction was 54%. . ¹ H NMR (400MHz, CDCl ₃ ) δ 8.50 (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 4-chlorobenzaldehyde was replaced with 3-ethoxy-4-hydroxybenzaldehyde, and the reaction yield in the final step was 43%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 10.22 (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 4-methoxy-3-iodo-5-methoxybenzaldehyde was replaced with 2-methoxybenzaldehyde, and the reaction yield in the last step was 76%. ¹ H NMR (400MHz, CDCl ₃ ) δ 8.51 (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-chlorobenzaldehyde was replaced with 2-isopropoxybenzaldehyde, and the final reaction yield was 43%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 10.22 (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 4-chlorobenzaldehyde was replaced with 3,5-dichloro-4-hydroxybenzaldehyde. The reaction yield in the last step was 68%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.15-8.07 (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-hydroxy-3-iodo-5-methoxybenzaldehyde was replaced with 4-hydroxybenzaldehyde, and the reaction yield in the last step was 76%. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.07 (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 5- [4- (1,3-Benzodioxolane-5-alkylmethoxy) benzylidene] -2-phenyl-1,3-dioxane-4,6 -Preparation of dione (S17)

Compound S17 was prepared in the same manner as in Example 1 except that 4- (benzo-1,3-dioxolane-5-ylmethoxy) benzaldehyde was used instead of 4-chlorobenzaldehyde. The final reaction was Yield 41%. ¹ H NMR (400MHz, CDCl ₃ ) δ 8.37 (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-chlorobenzaldehyde was replaced with 4-hydroxybenzaldehyde, and the final step yield was 55%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 10.56 (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 reaction yield in the last step was 58%. ¹ H NMR (400MHz, CDCl ₃ ) δ 8.78 (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-tetrahydropyrrolidinyl) benzylidene] -1,3-dioxane-4,6-dione (S20)

The 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 step yield was 22%. ¹ H NMR (400MHz, CDCl ₃ ) δ 8.32 (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 2- {4-[(4,6-Dioxo-2-phenyl-1,3-dioxane-5-ylbenzylidene) methyl] phenoxy} -N-phenylacetamide Preparation of (S21)

Compound S21 was prepared in the same manner as in Example 1 except that 4- (chloroformylphenoxy) -N-phenylacetamide was used in place 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 reaction yield in the last step 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 = 10.1 Hz, 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 4-benzaldehyde was replaced with 3-benzyl-4-hydroxy-5-methoxybenzaldehyde, and the yield of the reaction in the last step was 78%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 10.42 (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 (MH) ^- .

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-chloro-3,5-dimethoxybenzaldehyde was used instead of 4-chlorobenzaldehyde. The reaction yield in the last step was 82%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 10.22 (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 (MH) ^- .

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 4-chlorobenzaldehyde was replaced with 3-fluoro-4-hydroxybenzaldehyde. The reaction yield in the last step was 74%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.48 (s, 1H), 8.27 (dd, J = 13.4, 1.8 Hz, 2H), 7.89 (dd, J = 8.6, 2.1 Hz, 1H), 7.62 ( dd, J = 6.8, 3.0 Hz, 2H), 7.54 (dd, J = 5.1, 1.9 Hz, 3H), 7.14 (s, 1H), 7.08 (t, J = 8.8 Hz, 1H) .MS (ESI, m / z): 313 (MH) ^- .

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 4-chlorobenzaldehyde was replaced with 3-bromo-4-hydroxybenzaldehyde, and the reaction yield in the last step was 75%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 11.81 (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 (MH) ^- .

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 4-chlorobenzaldehyde was replaced with 3,5-dibromo-4-hydroxybenzaldehyde, and the reaction yield in the last step was 75%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.48 (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 (MH) ^- .

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 4-chlorobenzaldehyde was replaced with 3,5-difluoro-4-hydroxybenzaldehyde, and the reaction yield in the last step was 73%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.24 (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 (MH) ^- .

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-chlorobenzaldehyde was replaced with 4-hydroxy-3,5-diiodobenzaldehyde, and the reaction yield in the last step was 80%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 7.75 (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 (MH) ^- .

Pharmacological experiment

In the present invention, the inhibitory activity of 1,3-dioxane-4,6-dione compounds on SIRT deacetylase activity is determined. The experimental materials used in pharmacological experiments are commercially purchased except for special instructions.

First, SIRT1 enzyme activity test

The peptide Abz-GVLK _(Ac) AY _(NO2) GV-NH2 was prepared into a 10 mM storage solution with DMSO, and then frozen in a -80 ° C refrigerator after being dispensed; NAD ⁺ enzyme-activated reaction buffer (25 mM Tris, pH 8.0) (137 mM sodium chloride, 2.7 mM potassium chloride, 1 mM magnesium chloride) was prepared into a 50 mM stock solution; small molecule compounds were prepared into a 10 mM stock solution with DMSO.

To determine the IC ₅₀ of a compound, the compound was subjected to gradient dilution. The reaction system was 100 μL. The system contained 1 μM SIRT1, 500 μM NAD ⁺ , 10 μM substrate peptide, and compounds at corresponding concentrations. Each reaction condition had 3 auxiliary wells, and each experiment was repeated 3 times. After shaking the reaction at 37 ° C for 30 minutes, 50 μL of 10 mM nicotinamide and 0.01 mg / mL trypsin were added to each well to terminate the reaction and perform digestion. After a reaction at 37 ° C for 15 minutes, the fluorescence value was read with a microplate reader, and the excitation and emission wavelengths were 320 nm and 420 nm, respectively.

As shown in Table 1, these compounds are all 1,3-dioxane-4,6-dione with a phenyl group at the C2 position and a benzylidene structure at the C5 position. The SIRT1 enzyme activity test was used to test the IC ₅₀ (Table 2) of SIRT1 deacetylase activity inhibition by these batches of compounds, indicating that these compounds have inhibitory effects on SIRT1 deacetylase activity.

Since each subtype of the sirtuin family has a very important role in life, when designing SIRT1 small molecule inhibitors, the inhibitory effect of the inhibitor on other sirtuin subtypes should be considered. Several active compounds were selected and tested Inhibitory activity on SIRT2, SIRT3 and SIRT5. In this experiment, SI527 inhibitor EX527 was selected as the positive compound. According to the IC ₅₀ detection method for determining the inhibitory effect of small molecule compounds on SIRT1, and the concentration ratio of each component, the inhibitory effect of these compounds on SIRT1 homologous proteins was detected. As shown in Table 3, these compounds are selective inhibitors of SIRT1 and have better selectivity than the positive compound EX527. It can be seen that the compounds of the present invention have very good inhibitory activity on SIRT1 and have good selectivity.

Table 2: Inhibitory effects of compounds on SIRT1 deacetylase activity

Compound IC ₅₀ (μM) Compound 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 some compounds on SIRT1, SIRT2, SIRT3, SIRT5 deacylation

Compound 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 /

Second, enzyme kinetic reaction test

In vitro SIRT1 enzyme activity experiments showed that compound S3 inhibited SIRT1 enzyme activity in a concentration-dependent manner (see Figure 1). The IC ₅₀ of S3 inhibited SIRT1 was 1.31 ± 0.26 μM. Next, compound S3 was selected and the Mie constant constant curve (Michaelis- Menten plot and Lineweaver-Burk plot to detect the type of inhibition of this type of inhibitor.

Since the deacetylase reaction of SIRT1 requires acetylated peptide and NAD ⁺ as dual substrates, it is necessary to fix the concentration of one reaction substrate and change the concentration of the other reaction substrate in the enzymatic kinetic reaction, and judge the inhibitors separately. Types of inhibition of SIRT1 substrate acetylated peptides and NAD ⁺ :

1) Determine the type of inhibition of acetylated polypeptide by the fixed NAD ⁺ concentration of 1 mM: fixed SIRT1 protein concentration is 0.5 μM, the concentration of acetylated polypeptide is 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM The inhibitor S3 concentration was 1.875 μM, 0.9375 μM, 0.46875 μM, and 0 μM, respectively. After shaking reaction at 37 ° C for 15min, digestion was performed for 15min, and detection was performed using a microplate reader.

2) Determine the type of inhibition of NAD ⁺ by the inhibitor with fixed acetylated peptide concentration of 30 μM: fixed SIRT1 protein concentration of 0.5 μM, NAD ⁺ concentration of 1000 μM, 500 μM, 250 μM, 125 μM, 62.5 μM, and 31.25 μM, inhibitor The concentrations of S3 were 1.875 μM, 0.9375 μM, 0.46875 μM, and 0 μM, respectively. After shaking reaction at 37 ° C for 15min, digestion was performed for 15min, and detection was performed using a microplate reader.

The experimental results are shown in Figure 2.

In FIG. 2, A and B are respectively the Mie constant curve and the double reciprocal curve of compound S3 to the Abz polypeptide when the concentration of NAD ⁺ is fixed and the substrate Abz polypeptide concentration is changed. C and D in FIG. 2 are respectively the Mie constant curve and the double reciprocal curve of compound S3 versus NAD ⁺ when the concentration of Abz polypeptide is fixed and the NAD ⁺ concentration is changed. It can be seen from Figure B that when plotting the substrate Abz polypeptide, the curve intersects in the third quadrant at different compound concentrations, indicating that as the concentration of compound S3 increases, the initial velocity V ₀ decreases and the apparent Mie The constant K _m ′ decreases, so compound S3 inhibits the substrate polypeptide mixedly, and the binding of compound S3 to SIRT1 may be at the binding site of the substrate polypeptide. When plotting NAD ⁺ (D in Figure 2), the curve intersects the horizontal axis at different compound concentrations, indicating that as the concentration of compound S3 increases, the initial velocity V ₀ decreases and the apparent Mie constant K _m ′ does not Therefore, the compound S3 is a non-competitive inhibitor of NAD ⁺ , and the binding of S3 to SIRT1 is not at the NAD ⁺ binding site.

3. Microscale Thermophoresis (MST) experiment

After mini-hSIRT1 protein was centrifuged at high speed (12,000 rpm, 10 min) to remove gas, it was left at room temperature for 30 min. Add 100 μL of 100-fold protein molar TCEP (PB buffer dissolution) to 1 mL of mini-hSIRT1 protein, flush the centrifuge tube containing the protein with nitrogen, quickly cover the centrifuge tube, mix thoroughly and let stand for 10 min. Then add 50 μL of 10 times protein molar Cy5 ^TM dye solution (dissolved in DMSO) to the centrifuge tube. After flushing the centrifuge tube containing the protein with nitrogen, quickly cover the centrifuge tube, mix thoroughly and place. Incubate at room temperature for 2 hours and mix every half an hour, and then leave at 4 ° C overnight. Weigh 2 g of SM-2 adsorbent (Bio-Rad), activate the packing with 1 to 2 column volumes of methanol, and rinse with water and buffer (20 mM HEPES, pH 7.2, 200 mM sodium chloride, 5% glycerol). After the protein solution was flowed through the filler, the filler was washed with a buffer solution, the flow-through solution was collected, and the SIRT1 protein concentration was measured.

Prior to the MST experiment, the labeled SIRT1 ^Cy5 protein was centrifuged at high speed (13,000 rpm, 5 min) to remove aggregates. The labeled SIRT1 ^Cy5 protein was diluted to 200 nM with MST optimized buffer (50 mM Tris, pH 7.4, 150 mM sodium chloride, 10 mM magnesium chloride, 0.05% Tween-20). In the reaction system, the concentration of SIRT1 ^{Cy5 was} fixed at 100 nM, the compound was set to an initial concentration of 500 μM, the dilution ratio was 16 gradients, and the DMSO content in the system was maintained at 10%. Monolith NT115 (Nano Temper Technologies) was used to determine the MST curve under three conditions (Monolith NT115 parameters were set to 20% Red, MST Power 40.0%, excitation power 20%):

1) Interaction curve of SIRT1 ^Cy5 and compound S3

2) Interaction curve of SIRT1 ^Cy5 and compound S3 with the participation of 500 μM peptide Abz-GVLK _(Ac) AY _(NO2) GV-NH ₂

3) The interaction curve of SIRT1 ^Cy5 with compound S3 with the participation of 5mM NAD ⁺ .

The experimental data was analyzed by MO.Affinity Analysis software to obtain the MST binding curve. As shown in Figure 3, with the participation of Abz polypeptide, the binding curve of compound S3 and SIRT1 protein shifted to the right, and the K _d value increased, indicating that compound S3 is a competitive inhibitor of Abz polypeptide. However, when NAD ^{+ was} involved, the binding curve of compound S3 and SIRT1 protein did not change significantly, indicating that compound S3 is a non-competitive inhibitor of NAD ⁺ .

4. Molecular simulation and mutant experiments

Download the SIRT1 / EX527 complex crystal complex structure (PDB ID: 4I5I) from the RCSB-PDB database (www.rcsb.org), remove the EX527 molecules in the structure, extract the coordinates of a SIRT1 protein molecule, and save it as a pdb file . Compound S3 was docked into the SIRT1 protein structure and the binding model was analyzed based on structure-activity relationship. As shown in Figure 4A, compound S3 binds to phenylalanine at position 273, asparagine at position 346, isoleucine at position 347, aspartic acid at position 348, and phenylalanine at position 414. These five amino acid residues were subjected to site-directed mutagenesis, and the inhibitory constant (K _i ) of these SIRT1 mutant proteins by compound S3 was examined. It can be seen from FIG. 4B that the K _i value of compound S3 on SIRT1 wild type is 0.16 μM, and the inhibitory rate constants K _{i of} compound S3 on mutants SIRT1 ^F273L , SIRT1 ^N346A , SIRT1 ^I347A , SIRT1 ^D348A , and SIRT1 ^F414A are respectively It is 0.85μM, 2.47μM, 0.67μM, 4.33μM, and 25.5μM. It can be seen that the inhibitory constant of the compound on the mutant protein has been improved. Among them, the mutant SIRT1 ^F414A has the largest effect on the inhibitory activity of the compound. The compound has a hydrophobic effect between the phenyl group at the C2 position and the phenylalanine at the 414th position, which indicates that the phenylalanine at the 414th position of the SIRT1 is a key amino acid residue bound by the "protein / S3 small molecule compound", suggesting that in the next step When the structure is modified, the hydrophobicity of the phenyl compound can be appropriately increased to further improve the activity of the compound. In addition, according to the binding model, it was found that the hydroxyl group at the benzylidene R3 position at the C5 position of the compound formed a hydrogen bond with the aspartic acid residue at the 346th position in SIRT1. The reduced sensitivity of the mutant protein indicates that this site is also a key site for compound binding.

V. Effects of compounds on the level of p53 acetylation in cells

SH-SY5Y human neuroblastoma cells were seeded in a 12-well plate, overnight in the culture solution, ATRA (all-trans-retinoic acid) and compound (10 μM) were added for 2 hours, and then collected Cells were washed once with pre-chilled PBS and cell lysate was added. After the cell lysate was heated in a boiling water bath for 5 minutes. After centrifugation at 4 ° C for 10 minutes using a high-speed centrifuge (12000 rpm), the supernatant was collected.

The supernatant was subjected to SDS-PAGE electrophoresis. The gel after electrophoresis was cut and placed in a glass dish containing electrotransformation solution. After measuring the length, the membrane and filter paper were cut according to the size of the gel, and then a transfer membrane layer was prepared. It is placed on the anode, the gel is placed on the cathode, and the filter paper is on the outermost layer, which encloses the membrane and the gel. The transfer film layer is placed in an electrorotation tank filled with an electro-transfer liquid to transfer the film. The membrane was washed with a TBST washing solution, put in a blocking solution (5% skimmed milk powder), and blocked at 37 ° C for 2 hours. Add the primary antibody and incubate at 4 ° C overnight, wash three times with TBST for 10 minutes, and then add secondary antibody to incubate. After 2 hours of incubation at 37 ° C, wash TBST three times and develop color with a color developer.

From the experimental results (Figure 5), it can be seen that the color depth of each band of β-actin is the same, indicating that the loading protein concentration is the same; the p53 bands are the same, indicating that the p53 background level is the same. Under this experimental condition, comparing the acetylated p53 (ac-p53) bands in SH-SY5Y cells treated with ATRA and compounds, it can be seen that compounds S3, S6, S10, and S15 inhibit ATRA-induced SH-SY5Y The level of p53 acetylation decreases during cell differentiation, which proves that the compound has a good SIRT1 acetylation inhibitory activity at the cell level.

All documents mentioned in the present invention are cited in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

A compound of formula I, or a pharmaceutically acceptable salt, hydrate, solvate, enantiomer, diastereomer or racemate thereof, or a mixture thereof:

among them, R ¹ and R ⁷ are each independently hydrogen, C1-C6 alkyl or C2-C12 unsaturated hydrocarbon group; R ² , R ³ , R ⁴ , R ⁵ , R ⁶ are each independently hydrogen, halogen, cyano, nitro, amino, hydroxyl, carboxyl, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 unsaturated hydrocarbon, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 acyl, substituted or unsubstituted C3-C12 cycloalkyl, -L1- (CH ₂ ) m-substituted Or unsubstituted C6-C12 aryl, -L1- (CH ₂ ) m-substituted or unsubstituted 3-12 membered heterocyclyl, -L1- (CH ₂ ) mC (= O) -N (R ⁸ ) (R ⁹ ), wherein L 1 is none, -O- or -S-; m is 0, 1, 2, 3, 4 or 5; R ⁸ and R ⁹ are 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 refers to including one or more substituents selected from the group consisting of halogen, hydroxy, phenyl, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 unsaturated hydrocarbon, C1-C6 alkoxy , C1-C6 haloalkoxy, C3-C12 cycloalkyl, 3-12 membered heterocyclic group, cyano, nitro, methylol, carboxyl, mercapto; And the number of R ⁶ is 1-3. The compound of claim 1, wherein R ¹ and R ⁷ are each independently hydrogen, C1-C4 alkyl, or C2-C4 unsaturated hydrocarbon group. The compound of claim 1, wherein R ² , R ³ , R ⁴ , and R ⁵ are each independently hydrogen, halogen, cyano, nitro, amino, hydroxyl, carboxyl, substituted or unsubstituted C1 -C4 alkyl, substituted or unsubstituted C2-C6 unsaturated hydrocarbon, 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 heterocyclic group, -L1- (CH ₂ ) mC (= O) -N (R ⁸ ) (R ⁹ ), where L1 is none, -O- or -S-; m is 0, 1, 2, 3, 4 or 5; R ⁸ and R ⁹ are independent Is selected from: hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 3-6 membered heterocyclic group, or substituted or unsubstituted C6- C12 aryl; The substitution refers to including one or more substituents selected from the group consisting of halogen, hydroxy, phenyl, C1-C4 alkyl, C1-C4 haloalkyl, C2-C4 unsaturated hydrocarbon, C1-C4 alkoxy , C1-C4 haloalkoxy, C3-C6 cycloalkyl, 3-6 membered heterocyclic group, cyano, nitro, methylol, carboxyl, mercapto. The compound according to claim 1, wherein R ³ is hydrogen, halogen, hydroxyl, -L1- (CH ₂ ) m-3-10 membered heterocyclic group, -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 ) mC (= 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 are each independently selected from: hydrogen, C1-C4 alkane Or phenyl. The compound according to claim 1, wherein R ² and R ⁴ are each independently 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. The compound of claim 1, wherein R ⁵ is hydrogen or C1-C4 alkoxy. The compound of claim 1, wherein the compound is:

The method for preparing a compound according to claim 1, comprising the following steps:

a) reacting malonic acid with a substituted benzaldehyde or ketone to obtain compound I _a ; b) Compound I _{a is} reacted with a substituted benzaldehyde or ketone to obtain a compound represented by Formula I, The definitions of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ are as described in claim 1. A pharmaceutical composition, characterized in that the composition comprises the compound of claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, enantiomer, diastereomer thereof Or racemates, or mixtures thereof; and Pharmaceutically acceptable carrier. Use of a compound according to claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, enantiomer, diastereomer or racemate thereof, or a mixture thereof, It is characterized in that (a) is used for preparing inhibitors of SIRT1 deacetylase; or (b) is used for preparing drugs for treating diseases related to abnormal expression of SIRT1 protein deacetylase or its enzyme activity level.

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