WO2016206138A1 - Histone deacetylase inhibitor and preparation method and use thereof - Google Patents

Abstract

Provided are a compound for inhibiting a histone deacetylase (HDAC) and preparation method and use thereof. Provided is a compound represented by the following general formula, wherein a substituent group is defined as in the specification. The compound has an inhibitory effect on colorectal and breast cancer cells.

Description

Histone deacetylase inhibitor and preparation method and application thereof Technical field

The invention relates to the field of anticancer compound histone deacetylase inhibitors, in particular to histone deacetylase inhibitors and preparation methods and applications thereof.

Background technique

Histone deacetylase (HDAC) is a series of zinc-dependent metalloproteinases involved in the modification and modification of chromosome structure, regulating gene transcription and some important factors such as tumor suppressor p53 heat shock protein 90 and α-tubulin. The activity of proteins and cytokines plays a key role in tumorigenesis and is a new target for antitumor drug design in recent years.

Histone deacetylase inhibitor (HDACI), abbreviated as HDACIs, is a series of compounds that interfere with the function of histone deacetylase and can effectively inhibit the proliferation of cancer cells and induce apoptosis. Histone deacetylase inhibitors inhibit the proliferation of tumor cells and induce cell differentiation and/or apoptosis by increasing the degree of acetylation of intracellular histones and increasing the expression levels of p21 and other genes. Histone deacetylase inhibitors have become a new research hotspot in tumor-targeted therapy. Their inhibitory effects on tumor cell migration, invasion, metastasis and anti-tumor angiogenesis have also been confirmed. Some of them have better activity in vivo and in vitro. HDACI has entered clinical trials.

According to incomplete statistics, the sales of colorectal cancer treatment drugs in the seven traditional major pharmaceutical markets (English, French, American, German, Italian, Spanish, and Japanese) in 2000 were about 630 million US dollars (the number of cases was 438,500). People), sales in 2010 were $1.7 billion (onset) The number of people is 532,900). According to the 3rd Roche Cancer Forum (2012), the number of new cases of colorectal cancer worldwide is 940,000 per year, and nearly 500,000 people die of colorectal cancer each year. The estimated colorectal cancer treatment market is at least It is $3 billion. The incidence of colorectal cancer is now significantly increased, with the most obvious in the elderly and female groups. One of the reasons is the advancement of diagnostic techniques, and more and more early patients are diagnosed.

Oxaliplatin, irinotecan, 5-FU/leucovorin or capecitabine, which are used to treat colorectal cancer, are traditional cytotoxic drugs, and new targeted drugs are urgently needed to further improve the efficacy and reduce side effects.

Breast cancer is a common malignant tumor in women. According to statistics published by CA: A Cancer Journal for Clinicians (influence factor 94.262 in 2010), there were 232,670 women with breast cancer in the United States in 2014, accounting for 29% of women's new malignant tumors, ranking the incidence of female malignancies. first. Statistics in major cities such as Beijing, Shanghai, and Tianjin show that breast cancer is also the most common malignant tumor in women in China, and the incidence rate is increasing year by year. The age distribution of breast cancer is different in Eastern and Western countries. In high-incidence areas such as Northern Europe and North America, breast cancer begins to appear around the age of 20, and maintains a rapid upward trend before the menopause, 45-50 years old, about every age. The incidence of growth increased by 10 times in the age of 10-20 years, and the rise after menopause was relatively slow, reaching the highest in 75-85 years old. In low-incidence areas such as Asia, the incidence of breast cancer will decline slightly after menopause. The peak incidence of breast cancer is between 45 and 55 years old. Asians still maintain this age distribution after moving to Western countries.

Cytotoxic chemicals currently used clinically to treat breast cancer, such as docetaxel and methotrexate, have large side effects; while targeted new drugs, such as the monoclonal antibodies Herceptin and Avastin, are very expensive.

A novel inhibitor currently clinically studied is the histone deacetylase inhibitor FK228 (also known as depsipeptide, romidepsin; under the trade name Istodax), which exhibits antitumor properties in both human and in vitro human cell lines. However, many studies have confirmed that treatment of tumor cells by FK228 leads to inhibition of angiogenesis and cell growth, while inducing apoptosis, cell necrosis and cell differentiation. In 2009, FK228 was approved by the FDA for the treatment of cutaneous T-cell lymphoma and peripheral T-cell lymphoma.

Chinese Patent 201310130579.9 discloses a histone deacetylase inhibitor having the structural formula:

Wherein the R1 group is a methyl group, an ethyl group or an isopropyl group, the R2 group is a methyl group or an n-octyl group; and the R3 group is a methyl group, an ethyl group or an isopropyl group. The compound is mainly used for the preparation of a medicament for preventing or treating a mammalian disease associated with dysregulation of histone deacetylase, including cancer, neurodegenerative disease, malaria and diabetes; lymphoma, lung cancer, stomach cancer, pancreatic cancer Breast cancer, prostate cancer, leukemia and cervical cancer.

Chinese Patent No. 201110364545.7 discloses a histone deacetylase inhibitor, a synthesis method thereof and a pharmaceutical use, and the structural formula is:

Wherein the R ₅ group is hydrogen, C _1-12 alkyl, C _3-12 cycloalkyl, -O-(C _1-12 alkyl), -NH-(C _1-12 alkyl) or -S -(C _1-12 alkyl), R ₁ - R ₈ As defined in the specification, these compounds are mainly used in the preparation of a medicament for the prevention or treatment of a mammalian disease associated with histone deacetylase dysregulation.

Summary of the invention

The object of the present invention is to provide a novel structure of histone deacetylase inhibitor which has less toxicity and restores the normal expression of tumor suppressor genes and promotes cancer by acting on the epigenetic process of cancerous cells. Apoptosis, in order to achieve cancer-causing carcinogenesis, they act on specific molecular targets, high selectivity, low side effects, and clear mechanism of action.

Another object of the present invention is to provide a process for the preparation of the histone deacetylase inhibitor.

A third object of the present invention is to provide a use of the novel structure of a histone deacetylase inhibitor for the preparation of a pharmaceutical composition for treating colorectal cancer and breast cancer.

The object of the invention is achieved in this way:

The histone deacetylase inhibitor has the following structural formula or a pharmaceutically acceptable salt thereof,

Wherein R1 and R2 are each selected from hydrogen, hydroxy, siloxy or alkoxy, or R1=R2=O;

R3 is selected from the group consisting of hydrogen, triphenylmethylthio, p-methoxybenzylthio, 2-(trimethylsilyl)ethylthio, 9-fluorenylthio, n-pentylthio, n-hexylthio , n-heptylthio or n-octanoylthio;

R4 is selected from the group consisting of triphenylmethyl, p-methoxybenzyl, 2-(trimethylsilyl)ethyl, 9-fluorenylmethyl, n-pentanoyl, n-hexanoyl, n-heptanoyl or n-octanoyl.

The histone deacetylase inhibitor provided by the invention has less toxicity, high selectivity, low side effects, clear mechanism of action and less objection.

Further, R1 is H, R2 is OH, or R1=R2=O.

Further, R3 is n-octanoylthio or H.

Further, R4 is n-octanoyl.

Further, the structural formula of the histone deacetylase inhibitor Qing An 101 (hereinafter referred to as CA101) is:

Further, the structural formula of the histone deacetylase inhibitor Qing An 102 (hereinafter referred to as CA102) is:

Further, the structural formula of the histone deacetylase inhibitor Qing An 103 (hereinafter referred to as CA103) is:

The above three compounds CA101-CA103 belong to cyclic peptide high-efficiency histone deacetylase inhibitors, which act on specific molecular targets with high selectivity, low side effects, and clear mechanism of action.

Meanwhile, the present invention also provides a synthesis method for preparing the above-mentioned histone deacetylase inhibitor, and the reaction process is as follows:

[Correct according to Rule 26 16.09.2015]

Among them, the structural formula of Qing'an 101-3 (hereinafter referred to as CA101-3) added in the second step reaction is

Preferably, in the last two steps of the above synthetic method:

In the reaction of the sixth step, the peptide bond condensing agent is: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytris(dimethylamino) Phosphonium hexafluorophosphate, 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4-one, O-benzotriazole-N,N,N' , N'-tetramethylurea tetrafluoroboric acid, hexafluorophosphate Any one of acid benzotriazol-1-yl-oxytripyrrolidinyl or bis(2-oxo-3-oxazolidinyl)phosphoryl chloride.

The base is a basic inorganic salt or an organic base. Wherein the basic inorganic salt is any one of sodium hydrogencarbonate, potassium hydrogencarbonate, sodium carbonate, potassium carbonate or potassium phosphate; the organic base is triethylamine, diisopropylethylamine or pyridine. Any of them.

The organic solvent is N,N-dimethylformamide, dichloromethane or acetonitrile.

In the sixth step reaction, the molar ratio of the n-octanoic acid to the reactant is 1-10:1;

Wherein the reactant: n-octanoic acid: peptide bond condensing agent: 1-hydroxybenzotriazole or 1-hydroxy-7-azobenzotriazole: base molar ratio: 1:1-2:1- 3:1-3:1-5.

In the reaction of the seventh step, preferably, the organic solvent is any one of dichloromethane, tetrahydrofuran, acetonitrile, dimethyl sulfoxide or acetone.

The oxidizing agent is any one of pyridinium chlorochromate, pyridine dichromate, 2-iodobenzoic acid, and Dess-Martin oxidant.

The molar ratio of the oxidizing agent to the reactants is from 1 to 20:1.

This series of synthesis methods is the first proposed new method, which uses known small molecular materials and known intermediates for synthesis, and chemical synthesis improves the yield of the compound and reduces the production cost.

The invention also provides the use of the histone deacetylase inhibitor in the preparation of a medicament for treating colorectal cancer and breast cancer.

The invention also provides the histone deacetylase inhibitor for treating cancer including but not limited to colorectal, breast; skin; lymph node; cervical; uterus; Pancreas, lung; ovary; prostate; mouth; brain; head and neck; throat; testis; kidney; pancreas; bone; spleen; liver; bladder; larynx; or nasal cancer and relapsed or refractory cancer. Also includes cardiac hypertrophy, chronic heart failure, inflammation, cardiovascular disease, hemoglobinopathy, thalassemia, sickle cell disease, CNS disease, autoimmune disease, diabetes, osteoporosis, MDS, benign prostatic hypertrophy, oral mucosa White spot, genetically related metabolic disorders, infection, Rubens-Taybi, Fragile X syndrome or alpha-1 antitrypsin deficiency, or used to accelerate wound healing, or to protect hair follicles or as an immunosuppressive agent.

The medicament may also be used to alleviate the effects of chronic lymphocytic leukemia, T-cell lymphoma or skin inflammation, particularly psoriasis, acne or eczema.

A pharmaceutical composition comprising a histone deacetylation inhibitor of the invention and a pharmaceutically acceptable carrier or diluent. Preferably, the drug is a pharmaceutically acceptable carrier or diluent, and the carrier or diluent is a tablet, a capsule, a tablet, a lozenge, a water, an oil suspension, a dispersible powder, a granule, a sublingual tablet. Agent or injection.

The medicament is in a form suitable for oral, rectal, parenteral, intranasal or transdermal administration or administration by inhalation or by suppository or injection.

The compounds are capable of simultaneously or sequentially using other HDAC inhibitors, chemotherapy or anti-tumor agents, respectively, in the treatment or prevention of HDAC-mediated disorders.

Compared with the prior art:

1. Has a similar main structure to FK228, but contains new structural functional groups.

2. Less toxic than FK228.

3. It acts on specific molecular targets with high selectivity, low side effects, clear mechanism of action, and less objection, especially for colorectal cancer cells and breast cancer cells.

Preclinical test data indicates that the inhibitors provided by the present invention can be used independently, especially low doses of CA101, CA102, CA103 and the current mainstream treatment of colon cancer drug 5-fluorouracil (5-FU) to the colorectal cancer HCT-116 The activity of SW480 cell line is very strong;

Low-dose CA101, CA102, CA103 and the current mainstream treatment of breast cancer drug paclitaxel (TAX) have strong activity against breast cancer MCF-7 cell line, and the prospect is quite promising, which obviously has great development value.

DRAWINGS

Figure 1 is a graph showing the anti-proliferative effect of the compounds CA101, CA102, CA103 and 5-FU on the tumor cell line HCT116;

2 is a graph showing the anti-proliferative effect of the compounds CA101, CA102, CA103 and 5-FU on the tumor cell line SW480;

Figure 3 is a graph showing the anti-proliferative effect of the compounds CA101, CA102, CA103 and TAX on the human breast cancer cell line MCF-7.

detailed description

The present invention will be further described in detail below by way of specific examples.

The synthetic routes of the following examples of histone deacetylase inhibitors CA101, CA102, CA103 are for better explanation of the contents of the present invention, and those skilled in the art The present invention can be better understood and understood by the embodiments. However, the scope of the protection and claims of the invention includes and is not limited to the examples provided.

The synthetic route of the histone deacetylase inhibitors CA101, CA102, CA103 in the examples of the present invention is as follows:

Example 1: A specific preparation method for synthesizing CA101 includes the following steps:

Step one, prepare CA101-2:

The CA101-2 structure is:

The raw material CA101-1 (2.88 g, 4.54 mmol) was weighed, and the structural formula of CA101-1 was

The mixture was placed in a 50 mL flask, and 25 mL of dichloromethane was added, and the mixture was stirred to dissolve all the solid, and then 5.76 mL of trifluoroacetic acid was slowly added dropwise. After the completion of the dropwise addition, the reaction was stirred at room temperature for 2 hours, and the progress of the reaction was observed by TLC dot plate, and there was no remaining material. The reaction solution was spun dry, dissolved in toluene (5 mL×2) and then dried. It was then drained with a low pressure oil pump for 1 hour to give a light brown, clear, semi-solid, which was used directly in the next step without further purification.

Step 2, prepare CA101-4:

The CA101-4 structure is:

The product obtained from the upper reaction was dissolved in 25 mL of anhydrous N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added sequentially under ice bath. (1.73g, 9.08mmol), 1-hydroxybenzotriazole (1.23g, 9.08mmol), hydrogencarbonate (2.29g, 27.22mmol) and CA101-3 (2.89g, 5.45mmol), structural formula of CA101-3 for

After stirring for 10 minutes, the ice bath was removed, and the reaction was stirred at room temperature for 16 hours. The reaction progress was observed by TLC dot plate. After the raw material was not left, the reaction solution was poured into 100 mL of distilled water, stirred for 10 minutes, and extracted with ethyl acetate (40 mL × 4). The organic phase was combined, washed with saturated brine and dried over anhydrous sodium sulfate. A white solid (CA101-4) of 3.54 g was obtained in a two-step total yield of 74.4%.

CA101-4 nuclear magnetic data:

_{^{1 H NMR (400MHz, CDCl 3}} ) δ7.49-7.37 (m, 12H), 7.35-7.26 (m, 13H), 7.23 (dt, J = 14.6,7.3Hz, 6H), 7.00 (d, J = 7.5 Hz, 1H), 6.11 (t, J = 7.8 Hz, 2H), 5.54-5.42 (m, 1H), 5.36 (dd, J = 15.4, 6.3 Hz, 1H), 4.36 (s, 1H), 4.23 (dd , J=11.9, 8.4 Hz, 1H), 4.07 (dd, J=13.4, 6.9 Hz, 1H), 4.02-3.91 (m, 2H), 3.66 (s, 3H), 3.34 (s, 1H), 3.15- 2.99 (m, 1H), 2.65 (ddd, J = 22.3, 14.6, 4.7 Hz, 2H), 2.53-2.32 (m, 3H), 2.29-2.21 (m, 3H), 2.09 (dd, J = 13.7, 6.3 Hz, 2H), 1.91 (d, J = 3.4 Hz, 1H), 1.73 (s, 3H), 1.29 (dd, J = 14.1, 6.6 Hz, 7H), 1.20-1.11 (m, 1H), 0.90 (tt , J = ¹ 7.2, 3.4 Hz, 10H).

Step 3, prepare CA101-5:

The structural formula of CA101-5 is:

The raw material CA101-4 (3.50 g, 3.33 mmol) was weighed and dissolved in tetrahydrofuran (25 mL), and a lithium hydroxide solution (0.5 mol/L, 33.3 mL, 16.7 mmol) was slowly added dropwise with stirring under ice bath, and added dropwise. After stirring, the mixture was stirred for 10 minutes, then the ice bath was removed, and the reaction was stirred at room temperature for 2 hours. The reaction progress was checked by TLC dot plate. After confirming that there was no remaining material, the reaction solution was spun dry, toluene was added and then spun twice, and the oil pump was drained for 1 hour. , a light brown transparent semi-solid is obtained. It was used directly in the next reaction without further purification.

Step 4, prepare CA101-6:

The structural formula of CA101-6 is:

The product CA101-5 obtained in the previous step was dissolved in re-distilled dichloromethane (300 mL), placed in a 500 mL constant pressure dropping funnel; another 5000 mL round bottom flask was placed, and a magnetic stirrer was added to add 2-methyl- 6-Nitrobenzoic anhydride (2.27 g, 6.6 mmol), 4-dimethylaminopyridine (1.6 g, 13.32 mmol), dissolved in 3000 mL of re-distilled dichloromethane, and slowly mixed with CA101-5 dichloromethane solution with stirring. Add to the above solution, when added 8 hours between. After the completion of the dropwise addition, the reaction was stirred at room temperature for 16 h, and the reaction mixture was evaporated to dryness. The product-containing eluate was collected, and concentrated to give 421 mg of white solid (yield: EtOAc) (yield: 16%).

CA101-6 nuclear magnetic data:

_{^{1 H NMR (400MHz, CDCl 3}} ) δ7.45-7.34 (m, 13H), 7.31-7.27 (m, 10H), 7.24-7.16 (m, 7H), 7.03-6.91 (m, 2H), 5.99 (d , J=6.6 Hz, 1H), 5.67-5.56 (m, 1H), 5.55-5.45 (m, 1H), 5.34 (dd, J = 15.4, 6.7 Hz, 1H), 4.36-4.24 (m, 1H), 4.21 (dd, J=13.7, 7.3 Hz, 1H), 3.48 (d, J=33.1 Hz, 2H), 3.25-3.07 (m, 2H), 2.62-2.31 (m, 5H), 2.24-2.14 (m, 2H), 2.11-1.97 (m, 2H), 1.86-1.70 (m, 2H), 1.60-1.49 (m, 1H), 1.39-1.28 (m, 5H), 1.19-1.07 (m, 1H), 0.89- 0.91 (m, 9H).

Step 5, prepare CA101-7:

The structural formula of CA101-7 is:

The raw material CA101-6 (406 mg, 0.398 mmol) was weighed and dissolved in dichloromethane (5 mL), and triethylsilane (334 mg, 1.99 mmol, 459 μL) and trifluoroacetic acid (453 mg) were added with stirring under ice bath. 3.98mmol, 296μL), stirring for 10 minutes, removing the ice bath, stirring the reaction at room temperature for 2 hours, the reaction of the starting material was completed, the reaction solution was spun dry, and then added with an appropriate amount of toluene, and then the solution was dissolved twice. The crude silica gel was slurried and separated by column chromatography. The first fraction was eluted with PE/EA=2/1 eluent and the eluent was changed to DCM/MeOH = 10/1 to give a white solid (CA101-7). 143 mg, yield 67.5%, due to product instability, directly to the next step.

Step six, prepare CA101:

The starting material CA101-7 (141 mg, 0.26 mmol) was dissolved in 5 mL of anhydrous N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl) carbon was added in sequence under ice bath. Diimine hydrochloride (200 mg, 1.04 mmol), 1-hydroxybenzotriazole (141 mg, 1.04 mmol) and sodium hydrogencarbonate (175 mg, 2.08 mmol). After stirring for 10 minutes, n-octanoic acid (93.6 mg, 103 μL, 0.65 mmol), after the completion of the addition, the mixture was stirred for 10 minutes in an ice bath, and then the reaction was stirred at room temperature for 16 hours. Then, 10 mL of distilled water was added to the ice bath, and the mixture was extracted with EA (5 mL×5). The organic phase was combined, washed with a saturated aqueous solution of sodium chloride, dried over anhydrous magnesium sulfate, filtered, and then evaporated. Elution with =2/1 gave a colorless oily product (CA101), 75 mg, yield 36.8%.

CA101 nuclear magnetic data:

_{^{1 H NMR (400MHz, CDCl 3}} ) δ7.48 (d, J = 7.4Hz, 1H), 7.11 (d, J = 8.1Hz, 1H), 6.21 (d, J = 6.1Hz, 1H), 5.85-5.65 (m, 2H), 5.48 (dd, J = 15.5, 6.7 Hz, 1H), 4.55 (s, 1H), 4.36 (s, 1H), 4.24 (dd, J = 13.8, 5.7 Hz, 1H), 3.45 ( t, J = 6.3 Hz, 2H), 3.27 (s, 1H), 2.91 (dd, J = 15.4, 8.2 Hz, 2H), 2.67-2.48 (m, 7H), 2.31 (dd, J = 12.8, 5.9 Hz) , 2H), 2.00 (d, J = 5.9 Hz, 1H), 1.66 (dd, J = 14.5, 7.3 Hz, 5H), 1.32 (dd, J = 40.3, 8.4 Hz, 25H), 1.04-0.83 (m, 15H).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 199.52, 172.72, 172.23, 170.25, 170.02, 132.38, 128.29, 70.87, 67.57, 58.83, 54.74, 44.13, 44.04, 40.61,37.91,35.29,32.29,31.59,31.57,31.52 , 29.67, 28.94, 28.89, 28.87, 27.87, 27.79, 27.00, 25.68, 25.65, 22.56, 22.30, 15.25, 14.02, 13.83, 11.41.

Example 2: A specific preparation method for synthesizing CA102 includes the following steps:

Step one, prepare CA102-2:

The CA102-2 structure is:

Take CA102-1 (2.4g, 8.2mmol), the structural formula is

Dissolved in 24 mL of dichloromethane, then 4.8 mL of trifluoroacetic acid was added dropwise, and the addition was completed within 10 minutes. After stirring at room temperature for 2 hours, it was spun dry and taken twice with toluene (5 mL x 2). The oil pump is directly pumped to the next step after being drained for 1 hour.

Step 2: Prepare CA102-3:

The structural formula of CA102-3 is:

CA102-2 (1.57 g, 8.2 mmol) was dissolved in 30 mL of anhydrous N,N-dimethylformamide. At 0 ° C, 1-ethyl-(3-dimethylaminopropyl)carbon was added in sequence. Imine hydrochloride (3.1 g, 16.4 mmol), 1-hydroxybenzotriazole (2.1 g, 16.4 mmol) and sodium bicarbonate (4.2 g, 49.2 mmol), then N-Boc-alanine (1.87) g, 9.8 mmol), stirred at 0 ° C for 10 minutes and stirred at room temperature for 12 hours. Then add 100mL The mixture was stirred for 10 minutes at rt. The organic phase was dried over anhydrous sodium sulfate. Spinning through the column (PE/EA = 4/1-2/1) gave product (CA102-3) 3 g, pale yellow oil, yield 80%.

CA102-3 nuclear magnetic data:

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.41 (s, 1H), 5.06 (d, J = 6.8 Hz, 1H), 4.09 (tt, J = 11.3, 5.7 Hz, 1H), 4.00 - 3.89 (m, 2H) ), 3.67 (s, 3H), 3.44 (d, J = 4.6 Hz, 1H), 2.89 (d, J = 31.2 Hz, 1H), 2.54 (dd, J = 16.5, 2.2 Hz, 1H), 2.44 (dd) , J = 16.5, 8.5 Hz, 1H), 2.10 (d, J = 35.5 Hz, 1H), 1.92 (dt, J = 6.7, 4.6 Hz, 1H), 1.41 (s, 9H), 1.33 (dd, J = 15.8, 7.1 Hz, 3H), 1.28-1.21 (m, 1H), 1.12 (tt, J = 14.7, 7.4 Hz, 1H), 0.95-0.76 (m, 6H).

Step 3: Preparation of CA102-4: The preparation method of this step is the same as that of CA101-2, and the same reaction solvent is added to obtain CA102-4.

The structural formula of CA102-4 is:

Step 4: Preparation of CA102-5: The preparation method of this step was the same as that of CA101-4, and the same reaction solvent was added to obtain CA102-5.

The structural formula of CA102-5 is:

CA102-5 nuclear magnetic data:

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.42 (d, J = 7.6 Hz, 6H), 7.37-7.26 (m, 6H), 7.22 (dd, J = 14.2, 7.0 Hz, 3H), 6.45 (d, J=9.8 Hz, 1H), 6.28 (d, J=7.5 Hz, 1H), 5.68-5.56 (m, 1H), 5.45 (dd, J=15.0, 5.9 Hz, 1H), 4.39 (ddd, J=20.9) , 16.0, 10.1 Hz, 3H), 4.01 (dd, J = 13.6, 9.9 Hz, 2H), 3.72 (s, 1H), 2.94 (d, J = 29.0 Hz, 2H), 2.67-2.40 (m, 5H) , 2.32-2.18 (m, 2H), 2.17-2.07 (m, 3H), 1.36 (d, J = 7.0 Hz, 9H), 0.91 (t, J = 6.9 Hz, 10H).

Step 5: Preparation of CA102-6: The preparation method of this step is the same as that of CA101-5, and the same reaction solvent is added to obtain CA102-6.

The structural formula of CA102-6 is:

Step 6: Preparation of CA102-7: The preparation method of this step was the same as that of CA101-6, and the same reaction solvent was added to obtain CA102-7.

The structural formula of CA102-7 is:

CA102-7 nuclear magnetic data:

_{^{1 H NMR (400MHz, CDCl 3}} ) δ7.42 (d, J = 7.4Hz, 6H), 7.31 (d, J = 7.3Hz, 6H), 7.23 (t, J = 7.2Hz, 3H), 5.75-5.58 (m, 1H), 5.50 (dd, J = 13.4, 6.6 Hz, 1H), 5.40 (dd, J = 15.4, 6.6 Hz, 1H), 4.38 (dd, J = 14.5, 7.5 Hz, 1H), 4.26- 4.10 (m, 1H), 3.96 (s, 1H), 3.88 (s, 1H), 3.77 (t, J = 6.3 Hz, 1H), 2.59-2.32 (m, 3H), 2.29-2.15 (m, 2H) , 2.07 (d, J = 3.8 Hz, 2H), 1.92-1.85 (m, 1H), 1.78 (d, J = 6.1 Hz, 1H), 1.65 (dd, J = 18.2, 7.3 Hz, 3H), 1.48- 1.22 (m, 10H), 0.92 (t, J = 6.0 Hz, 9H).

Step 7: Preparation of CA102-8: The preparation method of this step was the same as that of CA101-7, and the same reaction solvent was added to obtain CA102-8.

The structural formula of CA102-8 is:

CA102-8 nuclear magnetic data:

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.49 (d, J = 9.0 Hz, 1H), 5.76 (dd, J = 13.8, 7.1 Hz, 1H), 5.64 - 5.47 (m, 2H), 4.48 - 4.28 (m) , 1H), 4.14 (d, J = 6.8 Hz, 1H), 3.97 (d, J = 26.3 Hz, 2H), 2.64 - 2.51 (m, 4H), 2.45 - 2.32 (m, 3H), 1.91 - 1.72 ( m, 3H), 1.63 (d, J = 7.1 Hz, 3H), 1.46-1.20 (m, 12H), 1.04-0.82 (m, 9H).

Step eight, prepare CA102:

CA102-8 (190 mg, 0.38 mmol) was dissolved in 5 mL of anhydrous N,N-dimethylformamide. At 0 ° C, 1-ethyl-(3-dimethylaminopropyl)carbodiene was added in sequence. Amine hydrochloride (146 mg, 0.72 mmol), 1-hydroxybenzotriazole (103 mg, 0.72 mmol), sodium hydrogencarbonate (191.5 mg, 2.28 mmol), then octanoic acid (67 μL, 0.42 mmol), stirred at 0 ° C for 10 min and stirred at room temperature for 12 h. Then, 20 mL of water and 5 mL of ethyl acetate were added, and the mixture was stirred at room temperature for 10 minutes, and the mixture was separated. The aqueous phase was extracted with ethyl acetate (10 mL×3), and the organic phase was combined and washed with 10 mL of water and 10 mL of saturated brine. Sodium is dry.

The product was dried (DCM / MeOH = 100 / 1-40/1)

CA102 nuclear magnetic data:

_{^{1 H NMR (400MHz, CDCl 3}} ) δ8.17 (s, 1H), 7.64 (d, J = 10.1Hz, 1H), 6.52 (s, 1H), 5.86-5.68 (m, 1H), 5.54 (dt, J=9.5, 6.2 Hz, 2H), 4.42 (dd, J = 14.5, 7.8 Hz, 1H), 4.16 (s, 1H), 3.97-3.76 (m, 2H), 3.49 (s, 1H), 2.92 (t , J = 7.4 Hz, 2H), 2.62 - 2.53 (m, 2H), 2.52 - 2.37 (m, 3H), 2.33 (dt, J = 7.3, 6.7 Hz, 3H), 1.75 (dd, J = 12.2, 6.0 Hz, 2H), 1.66 (t, J = 8.2 Hz, 5H), 1.45-1.17 (m, 15H), 1.04-0.77 (m, 12H).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 199.74, 174.20, 174.02, 171.09, 169.68, 131.71, 128.92, 71.92, 67.79, 56.47, 53.30, 44.15, 42.24, 37.93, 34.50, 32.92, 32.31, 31.62, 28.91, 28.90 , 27.89, 27.68, 26.64, 25.68, 22.58, 22.32, 16.01, 14.47, 14.05, 13.91, 11.26.

Example 3:

The specific preparation method of the synthetic CA103 includes the following steps:

CA102 (20 mg, 0.03 mmol) was dissolved in 1 mL of EtOAc (MeOH). Dess-Martin oxidant (DMP) (34 mg, 0.075 mmol) was added to an ice bath at 0 ° C, stirred at 0 ° C for 10 minutes and stirred at room temperature for 12 hours. After the TLC monitoring reaction was completed, the temperature was lowered to 0 ° C, diluted with 1 mL of dichloromethane, and then 1 mL of saturated sodium hydrogencarbonate and 1 mL of 1 M sodium thiosulfate were added in sequence, followed by stirring. After 10 minutes, the mixture was separated and evaporated, evaporated, evaporated, evaporated

The column was dried (DCM / MeOH = 50/1).

CA103 nuclear magnetic data:

_{^{1 H NMR (400MHz, CDCl 3}} ) δ7.78 (d, J = 8.4Hz, 1H), 7.60 (d, J = 7.1Hz, 1H), 7.01 (d, J = 6.7Hz, 1H), 5.76-5.57 (m, 2H), 5.42 (dd, J = 15.4, 6.5 Hz, 1H), 4.37-4.10 (m, 3H), 3.45 (dd, J = 67.4, 16.6 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H), 2.47 (dd, J = 14.8, 7.7 Hz, 2H), 2.22 (dd, J = 13.6, 6.7 Hz, 4H), 1.61-1.54 (m, 3H), 1.44 (t, J = 12.9 Hz) , 3H), 1.20 (d, J = 9.2 Hz, 15H), 0.92 - 0.73 (m, 12H).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 201.80, 199.40, 173.05, 172.22, 170.52, 166.75, 132.44, 128.35, 72.35, 62.18, 54.81, 51.40, 45.46, 44.12, 41.55, 34.13, 32.19, 31.59, 30.72, 29.67 , 28.88, 28.05, 27.78, 27.03, 25.63, 22.55, 22.34, 16.52, 14.98, 14.02, 13.89, 11.52.

In vitro activity verification test:

1. In vitro activity test of colorectal cancer cell lines (HCT-116, SW480):

1) Reagent preparation:

2) Preparation of the medium:

Cell line	Medium
		SW480	DMEM+10%FBS

HCT116

DMEM+10%FBS

3) Preparation of the compound:

CA101, CA102, CA103 and 5-FU were diluted with DMSO to a final concentration of 10 mM, of which 5-FU (5-fluorouracil) was the first clinical drug for colorectal cancer.

CA101, CA102, CA103 and 5-FU, 4 candidate compounds were applied at a final concentration ranging from 100 μM to 0 μM, 4 fold dilutions, for a total of 10 or 12 concentration points.

4) Cell culture:

Collect logarithmic growth phase cells, count, resuspend the cells with complete medium, adjust the cell concentration to the appropriate concentration, inoculate 96-well plates, inoculate 100 μl of cell suspension per well, incubate at 37 ° C, 100% relative humidity, 5% CO 2 Incubate in the box for 24 hours.

5) IC50 experiment:

The logarithmic growth phase cells were collected, counted, and the cells were resuspended in complete medium, and the cell concentration was adjusted to an appropriate concentration (determined according to the cell density optimization test results), and 96-well plates were seeded, and 100 μl of the cell suspension was added to each well. After incubating the cells for 24 hours at 37 ° C, 100% relative humidity, 5% CO 2 incubator, dilute the test compound with DMSO, 4 times gradient dilution 8 or 10 times; then dilute the diluted compound 20 times with medium. A total of 9 or 11 points were added to the cells at 25 μl/well. The final concentration of the compound was from 100 μM to 0 μM, 4 fold dilutions, for a total of 10 or 12 concentration points.

The cells were incubated for 72 hours at 37 ° C in 100% relative humidity in a 5% CO 2 incubator.

The medium was aspirated, and the complete medium containing 10% CCK-8 was added and incubated in a 37 ° C incubator for 2-6 hours.

The absorbance at a wavelength of 450 nm was measured on a Spectra Max M5 Microplate Reader with gentle shaking, and the absorbance at 650 nm was used as a reference to calculate the inhibition rate.

6) Data processing:

The inhibition rate of the drug on tumor cell growth was calculated as follows: tumor cell growth inhibition rate % = [(Ac-As) / (Ac-Ab)] × 100%;

As: OA of the sample (cell + CCK-8 + test compound);

Ac: negative control OA (cell + CCK-8 + DMSO);

Ab: positive control OA (medium + CCK-8 + DMSO);

IC50 curves were fitted using software Graphpad Prism 5 and IC50 values were calculated.

7) Experimental results:

This experiment tested the anti-value-adding effects of four candidate compounds (CA101, CA102, CA103 and 5-FU) on two human-derived tumor cell lines (HCT116 and SW480). The final concentration of the compound was diluted from 100 μM to 0 μM in 4 fold gradients for 10 or 12 points. The experimental results are shown in Tables 1 and 2 below:

Tables 1 and 2 show the IC50 values of the respective compounds in different cell lines, respectively, and the graphs are shown in Figures 1 and 2, respectively.

RESULTS: Three of the compounds, CA101, CA102 and CA103, had strong inhibitory effects on the proliferation of these two tumor cell lines (HCT-116 and SW480) with IC50 values between 1 and 50 nM.

Table 1 IC50 values of compounds in tumor cell line HCT11

Compound	5-FU	CA101	CA102	CA103
					IC50 value (nM)	1300	4.947	2.739	45.34

Table 2 IC50 values of compounds in tumor cell line SW480

Compound	5-FU	CA101	CA102	CA103
					IC50 value (nM)	9590	6.631	10.42	43.63

2. In vitro activity test of breast cancer cell line (MCF-7):

1) Reagent preparation:

2) Preparation of the medium:

Cell line	Medium
		MCF-7	1640 medium + 10% FBS

3) Preparation of the compound:

CA101, CA102, CA103 and TAX were diluted in DMSO to a final concentration of 10 mM. Among them, TAX (Paclitaxel) is a first-line drug for treating breast cancer.

CA101, CA102, CA103 and TAX, 4 candidate compounds were applied at a final concentration ranging from 10 μM to 1 nM, 10-fold gradient dilution, for a total of 5 concentration points.

4) Cell culture:

Collect logarithmic growth phase cells, count, resuspend the cells with complete medium, adjust the cell concentration to the appropriate concentration, inoculate 96-well plates, inoculate 100 μl of cell suspension per well, incubate at 37 ° C, 100% relative humidity, 5% CO 2 Incubate in the box for 24 hours.

5) IC50 experiment:

The logarithmic growth phase cells were collected, counted, and the cells were resuspended in complete medium, and the cell concentration was adjusted to an appropriate concentration (determined according to the cell density optimization test results), and 96-well plates were seeded, and 100 μl of the cell suspension was added to each well. After incubating the cells for 24 hours at 37 ° C, 100% relative humidity, 5% CO 2 incubator, dilute the test compound with DMSO, 4 times gradient dilution 8 or 10 times; then dilute the diluted compound 20 times with medium. A total of 5 points were added to the cells at 25 μl/well. The final concentration of the compound was from 10 μM to 1 nM, 10-fold dilution, for a total of 5 concentration points.

The cells were incubated for 72 hours at 37 ° C in 100% relative humidity in a 5% CO 2 incubator.

The medium was aspirated, and the complete medium containing 10% CCK-8 was added and incubated in a 37 ° C incubator for 2-6 hours.

The absorbance at a wavelength of 450 nm was measured on a SpectraMax M5 Microplate Reader with gentle shaking, and the absorbance at 650 nm was used as a reference to calculate the inhibition rate.

6) Data processing:

The inhibition rate of the drug on tumor cell growth was calculated by the following formula: tumor cell growth inhibition rate % = [(Ac-As) / (Ac-Ab)] × 100%.

As: OA of the sample (cell + CCK-8 + test compound);

Ac: negative control OA (cell + CCK-8 + DMSO);

Ab: positive control OA (medium + CCK-8 + DMSO);

IC50 curves were fitted using software Graphpad Prism 5 and IC50 values were calculated.

7) Experimental results: This experiment tested the anti-value-adding effects of four candidate compounds (CA101, CA102, CA103 and TAX) on human breast cancer cell line MCF-7. The final concentration of the compound was from 10 μM to 1 nM, and the 10-fold gradient was diluted by 5 points. The experimental results are shown in Table 3 below:

Table 3 shows the IC50 values of the above respective compounds in different cell lines, and the graph is shown in Fig. 3.

RESULTS: The literature reported that the IC50 of paclitaxel to MCF-7 was 0.4 μmol/L after 48 h of drug treatment, which was similar to the 0.211 μmol/L measured in this experiment. Three of the compounds, CA101, CA102 and CA103, had a strong inhibitory effect on the proliferation of this tumor cell line MCF-7, with IC50 values between 10-500 nM.

Table 3 IC50 values of compounds in breast cancer cell line MCF-7

Compound	TAX	CA101	CA102	CA103
					IC50 value (nM)	211.13	35.272	335.999	19.643

The above description is only the preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes can be made to the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and scope of the present invention are intended to be included within the scope of the present invention.

Claims (10)

1. The histone deacetylase inhibitor has the following structural formula or a pharmaceutically acceptable salt thereof,

   

   Wherein R1 and R2 are each selected from hydrogen, hydroxy, siloxy or alkoxy, or R1=R2=O;

   R3 is selected from the group consisting of hydrogen, triphenylmethylthio, p-methoxybenzylthio, 2-(trimethylsilyl)ethylthio, 9-fluorenylthio, n-pentylthio, n-hexylthio , n-heptylthio or n-octanoylthio;

   R4 is selected from the group consisting of triphenylmethyl, p-methoxybenzyl, 2-(trimethylsilyl)ethyl, 9-fluorenylmethyl, n-pentanoyl, n-hexanoyl, n-heptanoyl or n-octanoyl.
2. The histone deacetylase inhibitor according to claim 1, wherein R1 is H, R2 is OH, or R1=R2=O.
3. The histone deacetylase inhibitor according to claim 1 or 2, wherein R3 is n-octanoylthio or H.
4. The histone deacetylase inhibitor according to claim 1, wherein R4 is n-octanoyl.
5. The histone deacetylase inhibitor according to claim 1, wherein the structural formula of the histone deacetylase inhibitor CA101 is:

   
6. The histone deacetylase inhibitor according to claim 1, wherein the structural formula of the histone deacetylase inhibitor CA102 is:

   
7. The histone deacetylase inhibitor according to claim 1, wherein the structural formula of the histone deacetylase inhibitor CA103 is:

   
8. [Correct according to Rule 26 16.09.2015]
   The method for synthesizing the histone deacetylase inhibitor according to any one of claims 1 to 7, wherein the reaction process is as follows:
   

   

   
   Wherein, the structural formula of CA101-3 added in the second step reaction is
   

   
9. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 7 and a pharmaceutically acceptable carrier or diluent.
10. A histone deacetylase inhibitor according to any one of claims 1 to 7 and a compound synthesized according to the method of claim 8, wherein the histone deacetylase inhibitor is used in the preparation of a therapeutic knot Rectal, breast, skin, lymph node, cervical, uterine, gastrointestinal, pancreatic, lung, ovarian, prostate, oral, brain, head, neck, throat , testicular cancer, kidney cancer, pancreatic cancer, bone cancer, spleen cancer, liver cancer, bladder cancer, throat cancer, nasal cancer, recurrent or refractory cancer, cardiac hypertrophy, chronic heart failure, inflammation, cardiovascular disease, hemoglobinopathy, Thalassemia, sickle cell disease, CNS disease, autoimmune disease, diabetes, osteoporosis, MDS, benign prostatic hyperplasia, oral leukoplakia, genetically related metabolic disorders, infection, Rubens-Taybi, fragile X syndrome The use of α-1 antitrypsin deficiency, accelerated wound healing, protection of hair follicles, and use as a drug for immunosuppressants.

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