CN112920242B

CN112920242B - Benzimidazole derivative BI292 and preparation method and application thereof

Info

Publication number: CN112920242B
Application number: CN202110118455.3A
Authority: CN
Inventors: 卢光明; 潘璟; 马龙
Original assignee: Eastern Theater General Hospital of PLA
Current assignee: Eastern Theater General Hospital of PLA
Priority date: 2021-01-28
Filing date: 2021-01-28
Publication date: 2021-12-21
Anticipated expiration: 2041-01-28
Also published as: CN112920242A

Abstract

The invention discloses a benzimidazole derivative BI292 and a preparation method thereof, wherein the chemical name of the benzimidazole derivative BI292 is 1- [ (2R,4S,5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl ] -1H-benzo [ d ] imidazole-4-carboxylic acid methyl ester. The benzimidazole derivative and the pharmaceutically acceptable salt, solvate and hydrate thereof have excellent antitumor in-vitro and in-vivo activities on MCF-7, SK-BR-3, HCT116, U-118MG, U-87MG and MDA-MB-468, and have good application prospects in preparation of antitumor drugs.

Description

Benzimidazole derivative BI292 and preparation method and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a benzimidazole derivative BI292 as well as a preparation method and application thereof.

Background

Malignant tumor is a common disease and frequently encountered disease seriously threatening human health, and the death rate of the tumor in China has increased 29.42% in recent 20 years. In the middle-aged and senior population, between the ages of 35 and 59, tumors have been the first cause of death of various types. And (3) displaying data: the disease rate of tumors in China is about 200/10 ten thousands of people, more than 220 thousands of new cases are generated every year, and more than 600 thousands of patients are treated. The tumor treatment method includes operation treatment, radiotherapy and chemotherapy. Currently, chemotherapy remains the primary means of clinical treatment of tumors. The search for anti-tumor drugs is one of the hot spots in the research of new drugs. In recent years, benzimidazole compounds have attracted much attention because of their excellent biological activity, and have become a focus of research for researchers in biology and chemistry. The indications include: peptic ulcer, parasitic infection, bacterial infection, viral infection, tumor, etc., and particularly, has the effect of selectively inhibiting glioblastoma and other tumors. The inventor unexpectedly finds that methyl 1- [ (2R,4S,5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl ] -1H-benzo [ d ] imidazole-4-carboxylate has certain antitumor activity, and proposes the invention related to methyl 1- [ (2R,4S,5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl ] -1H-benzo [ d ] imidazole-4-carboxylate or a pharmaceutically acceptable salt, solvate or prodrug or stereoisomer or tautomer or metabolite of the compound.

Disclosure of Invention

The purpose of the invention is as follows: in order to solve the problems in the prior art, the invention provides a benzimidazole derivative BI292 which is chemically named as 1- [ (2R,4S,5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl ] -1H-benzo [ d ] imidazole-4-carboxylic acid methyl ester and has inhibitory activity on proliferation of MCF-7, SK-BR-3, HCT116, U-118MG, U-87MG and MDA-MB-468 six tumor cells.

The technical scheme is as follows: in order to achieve the technical purpose, the invention provides a benzimidazole derivative BI292, which is chemically named as 1- [ (2R,4S,5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl ] -1H-benzo [ d ] imidazole-4-carboxylic acid methyl ester and has the following structural formula:

the invention further provides a preparation method of the benzimidazole derivative BI292, which comprises the following steps:

s1: under the protection of nitrogen, methanol solution, 5- (hydroxymethyl) oxapentane-2, 4-diol and CH₃COCl is mixed and stirred for 20-45 minutes, pyridine is diluted and concentrated, Py, 4-dimethylaminopyridine and 4-methylbenzoyl chloride are added, stirring is carried out for 1-1.5 hours at the temperature of 0 ℃, and stirring is carried out at the room temperature overnight; adding water to stop the reaction, extracting with dichloromethane, combining organic layers, and sequentially using saturated NaHCO₃Solution, 15% H₂SO₄Washing, drying over anhydrous sodium sulfate, concentrating in vacuo and triturating with ether, filtering off the solid, concentrating the filtrate in vacuo, adding ether acetic acid to the resulting residue at 0 deg.C, introducing hydrochloric acid gas for 10-15 minutes, collecting the solid by filtration and washing with ether/petroleum ether to give compound (1), i.e. (2R,3S,5S) -5-chloro-2- { [ (4-methylbenzoyl) oxy)]Methyl } tetrahydrofuran-3-yl-4-benzoic acid methyl ester.

S2：1H-1, 3-benzodiazole-4-carboxylic ester and CH₃CN and BSA are put into a round-bottom flask and stirred for 15 to 20 minutes at the temperature of 80 ℃; to CH at room temperature₃CN TMSOTf and compound (1) are added; mixing the above solutions at 85 deg.C, stirring for 2.5-3h, cooling to room temperature, diluting with water, extracting with ethyl acetate for several times, combining organic layers, drying over anhydrous sodium sulfate, vacuum concentrating, eluting the obtained residue with ethyl acetate/petroleum ether on silica gel column, and purifying the crude product by flash preparative high performance liquid chromatography to obtain compound (2), i.e. methyl- { (2R,4S,5R) -4- [ (4-methylbenzoyl) oxy ] carbonyl]-5- { [ (4-methylbenzoyl) oxy]Methyl } tetrahydrofuran-2-yl } -1H-benzo [ d]Imidazole-4-carboxylic acid esters;

s3: placing compound (2) and methanol, tetrahydrofuran, sodium methoxide in a round bottom flask, stirring at room temperature for 1.5-2H, adjusting the solution to neutrality with hydrochloric acid, concentrating the reaction mixture in vacuum, and purifying the obtained residue by flash preparative high performance liquid chromatography to obtain compound (3), i.e. methyl 1- [ (2R,4S,5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl ] -1H-benzo [ d ] imidazole-4-carboxylate.

Preferably, in step S1, the methanol solution, 5- (hydroxymethyl) oxapentane-2, 4-diol, and CH₃The dosage ratio of COCl, pyridine, 4-dimethylaminopyridine and 4-methylbenzoyl chloride is 60-120 mL: 6-12 g: 0.1-0.5 mL: 20-60 mL: 0.5-5 g: 10-30 mL.

In step S2, 1H-1, 3-benzodiazole-4-carboxylate, CH₃CN, BSA, TMSOTf and compound (1) were used in the following ratios: 0.5-5 g: 60-100 mL: 0.5-5 g: 0.5-5 g: 3-10 g, the reaction temperature is 60-100 ℃, and the reaction time is 90-180 minutes. The mobile phase gradient elution conditions for purifying the crude product by flash preparative high performance liquid chromatography are H₂O(0.1％HCOOH)：CH₃CN＝1:10～10:1。

In step S3, the compound (2), methanol, THF, and sodium methoxide are used in the following amounts: 1-5 g: 60-100 mL: 60-100 mL: 0.1-0.5 g, and the mobile phase gradient elution conditions for purification by preparative chromatography are as follows: h₂O(0.1％NH₄HCO₃)：CH₃CN＝20:1～1:5。

The invention further provides application of the benzimidazole derivative in preparing an antitumor agent.

The present invention also provides a pharmaceutical composition comprising a compound as set forth in claim 1 above and a pharmaceutically acceptable carrier.

More particularly, the invention provides the use of the compound or the pharmaceutical composition in the preparation of medicaments.

Meanwhile, the invention also provides application of the benzimidazole derivative BI292 or pharmaceutically acceptable salt, solvate or prodrug or stereoisomer or tautomer or metabolite of the compound in preparation of antitumor agents.

Finally, the invention provides the application of the benzimidazole derivative BI292 or the pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, tautomer or metabolite of the compound in preparing the medicine for treating tumors by combining with one or more anticancer agents.

Has the advantages that: the invention discloses a benzimidazole derivative BI292, which is used for evaluating the proliferation activities of 6 tumor cells of MCF-7, SK-BR-3, HCT116, U-118MG, U-87MG and MDA-MB-468 inhibition by adopting an MTT method, and calculating the IC for inhibiting the proliferation of the six tumor cells₅₀The value and the result show that the prepared benzimidazole derivative BI292 has an inhibition effect on the tumor cells and can be used for preparing an anti-tumor preparation.

Drawings

Fig. 1 is a scheme for the synthesis of benzimidazole derivatives, wherein: i) acetyl chloride, methanol, pyridine, 4-dimethylaminopyridine, 4-methylbenzoyl chloride; ii) BSA, TMSOTf, acetonitrile; iii) methanol, sodium methoxide.

Detailed Description

To further illustrate the invention, a series of examples are given below. These examples are purely illustrative and are intended to be a detailed description of the invention only and should not be taken as limiting the invention.

EXAMPLE 1 preparation of methyl (2R,3S,5S) -5-chloro-2- { [ (4-methylbenzoyl) oxy ] methyl } tetrahydrofuran-3-yl-4-benzoate (Compound 1)

To a 250mL round bottom flask maintained under nitrogen at room temperature were added 100mL of methanol solution, 6.8g of 5- (hydroxymethyl) oxapentane-2, 4-diol, and 0.4mL of CH₃COCl. The mixture was stirred for 45 minutes, diluted with 10mL pyridine and concentrated to a yellow oil. 20mL of Py, 0.5g of 4-dimethylaminopyridine and 14mL of 4-methylbenzoyl chloride were added, and the mixture was stirred at 0 ℃ for 1.5 hours and at room temperature overnight. The reaction was then quenched by the addition of 100mL of water and extracted several times with 100mL of dichloromethane. The organic layers were combined and washed with 100mL saturated NaHCO₃Solution, 100mL 15% H₂SO₄Washed several times, dried over anhydrous sodium sulfate, concentrated in vacuo and triturated with 20mL of diethyl ether. The resulting solid was filtered off and the filtrate was concentrated in vacuo. The residue was added with 100mL of diethyl ether and 50mL of acetic acid at 0 ℃. Hydrochloric acid gas was introduced into the above solution for 10 minutes. The solid was collected by filtration and washed with 100mL diethyl ether/petroleum ether (1: 1). 12.5g (yield 63%) of Compound 1 are obtained as a white solid.

Compound 1 was characterized by LC-MS as follows:

LC-MS:(ES,m/z):353[M-Cl^-]+

EXAMPLE 2 preparation of methyl- { (2R,4S,5R) -4- [ (4-methylbenzoyl) oxy ] -5- { [ (4-methylbenzoyl) oxy ] methyl } tetrahydrofuran-2-yl } -1H-benzo [ d ] imidazole-4-carboxylate (Compound 2)

2.1g of 1H-1, 3-benzodiazole-4-carboxylate, 80mL of CH₃CN was charged into a 250mL round-bottom flask, and 2.68g BSA was added at 80 ℃ and stirred for 15 minutes. Followed by addition of the solution in 10mLCH at room temperature₃CN 3.97g of TMSOTf and 6.94g of Compound (1). The above solutions were mixed and stirred at 85 ℃ for 2.5 h. Cooled to room temperature, diluted with 100mL of water and extracted several times with 100mL of ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was eluted with ethyl acetate/petroleum ether (10:1) on a silica gel column. The crude product was purified by flash preparative high performance liquid chromatography under the following conditions: c₁₈Mobile phase, H₂O(0.1％HCOOH)：CH₃CN is 3:1, inDetector, UV254nm, within 25 minutes. 2.4g (38% yield) of Compound 2 are obtained as a pale yellow solid.

Compound 2 was characterized by LC-MS as follows:

LC-MS:(ES,m/z):529.1[M+H]+

H-NMR:(300MHz,DMSO-d6,ppm):δ8.623(s,1H),7.93-8.04(m,3H),7.85-7.79(m,3H),7.40-7.19(m,5H),6.66(dd,J＝6Hz,J＝1.8Hz,1H),5.76(d,J＝6.3Hz,1H),4.65-4.55(m,3H),3.95(s,3H),3.16-3.06(m,1H),2.86-2.74(m,1H),2.42(s,3H),2.38(s,3H).

EXAMPLE 3 preparation of methyl 1- [ (2R,4S,5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl ] -1H-benzo [ d ] imidazole-4-carboxylate (Compound 3)

After 2.4g of compound 2 and 80mL of methanol, 80mL of THF and 245mg of sodium methoxide have been stirred at room temperature for 1.5h, the solution is adjusted to neutrality with hydrochloric acid. The reaction mixture was concentrated under vacuum. The resulting residue was purified by flash preparative high performance liquid chromatography under the following conditions: chromatography column, C₁₈(ii) a Mobile phase, water (0.1% NH)₄HCO₃)：CH₃CN 20:1, added to water (0.1% NH)₄HCO₃)：CH₃CN 1:2 in 20 minutes; detector, UV254 nm. 0.6498g (yield 49%) of compound 3 were obtained as a white solid.

ESI-MS and the like were performed on the prepared Compound 3, and the results were as follows:

LC-MS:(ES,m/z):293.20[M+H]+

H-NMR:(300MHz,DMSO-d6,ppm):δ8.59(s,1H),8.01(d,J＝7.5Hz,1H),7.79(d,J＝6.9Hz,1H),7.36(t,J＝7.8Hz,1H),6.42(t,J＝6.8Hz,1H),5.36(s,1H),4.99(s,1H),4.42-4.40(m,1H),3.89(s,4H),3.56(s,2H),2.63-2.57(m,1H),2.37-2.30(m,1H).

experimental example 4 evaluation of antitumor cell proliferating activity by Compound 3.

(1) And (3) testing a sample:

the compounds 3 of the invention were all formulated to the desired concentration in media containing 0.1% DMSO.

(2) Cell lines:

MCF-7 (human breast cancer cells, ATCC: HTB-22), SK-BR-3 (human breast cancer cells, ATCC: HTB-30), HCT116 (human colon cancer cells, ATCC: CCL-247), U-118MG (human brain astrocytoma, ATCC: HTB-15), U-87MG (human brain astrocytoma, ATCC: HTB-14), and MDA-MB-468 (human breast cancer cells, ATCC: HTB-132)6 tumor cells were purchased from American Standard culture Collection (ATCC).

(3) Main instruments and materials

Ultra-pure water instrument: MILLIPORE Direct-Q3;

an autoclave: HVE-50, Hirayama corporation;

digital display constant temperature water bath: HH-4, Kyowa electric appliances, Inc.;

an ultra-clean bench: VS-1300-U clean bench, Suzhou Antai air technology, Inc.;

a cell incubator: HF151UVCO2 incubator, shanghai li cheng;

low-temperature centrifuger: shanghai' an pavilion scientific instrument factory

An enzyme-labeling instrument: ELx800 Biotek Inc

A flat plate oscillator: ZD-9556, Taicang scientific and educational facilities;

96-well cell culture plate, 25cm²Culture bottles: corning Costar corporation;

2mL of frozen tubes: corning Costar corporation;

(4) primary reagent

RPMI-1640 medium: gibco corporation;

DMEM medium: gibco corporation;

l-15 Medium: gibco corporation;

McCoy's 5A medium: gibco corporation;

MEM medium: gibco corporation;

PBS buffer: gibco corporation;

fetal bovine serum: gibco corporation;

0.25% pancreatin solution: hyclone company;

MTT (thiazolidine blue): sigma, dissolving in PBS solution to make 5mg/mL solution, filtering, sterilizing, and storing in dark place;

doxorubicin (ADR): beijing Huafeng Union technologies, Inc.

DMSO, DMSO: dimethyl sulfoxide, Sigma corporation;

(5) test method

The MCF-7 and U-118MG cells adopt DMEM culture medium, the U-87MG cells adopt MEM culture medium, the MDA-MB-468 cells adopt L-15 culture medium, the HCT116 cells adopt McCoy's 5A culture medium, and other cells adopt RPMI-1640 culture medium. The culture medium contains 10% of extinguishant fetal calf serum and 80 U.mL^-1Penicillin and 0.08 mg/mL^-1Streptomycin.

MCF-7, SK-BR-3, HCT116, U-118MG, U-87MG and MDA-MB-468 cells in logarithmic growth phase with good growth state are divided into 1 × 10⁴The cells were plated at a density of 100. mu.l/well in 96-well plates. Placing at 37 ℃ and 5% CO₂Culturing in an incubator for 12 hours to adhere to the wall. Adding a compound 3 dissolved in a culture medium to be detected and subjected to sterilization treatment into a drug-added cell hole according to a preset concentration gradient, wherein each hole is 200 mu l, adding an equal volume of the culture medium into a blank cell hole, adding an equal volume of Adriamycin (ADR) dissolved in the culture medium into a control cell hole according to a preset concentration gradient, and paralleling 6 holes. At 37 deg.C, 5% CO₂After 48 hours of incubation in an incubator, 10. mu.l of 5mg/mL MTT solution was added to each well, and the mixture was further incubated at 37 ℃ with 5% CO₂The culture was carried out in an incubator for 4 hours. The supernatant was carefully aspirated, 150. mu.l of DMSO was added to each well to dissolve the purple residue (formazan), the plate was shaken for 10 minutes to dissolve the precipitate completely, and the O.D. value (absorbance) was measured on a microplate reader at a wavelength of 570 nm.

The inhibition rate of the sample on tumor cells at each sample concentration was calculated according to the formula "relative survival rate ═ D blank containing drug (D)/(D control-D blank) × 100%".

The experiment was repeated 3 times in parallel and the inhibition rate was plotted against the compound concentration to calculate the IC of compound 3 of the invention₅₀(median effective inhibitory concentration) value. While doxorubicin (ADR) was used as a positive control drug.

(6) Results of the experiment

TABLE 1 Compound 3BI292 antitumor cell proliferation Activity (IC)₅₀±SDμM)

As shown in Table 1, the test results of the compound 3 on the proliferation activity of the tumor cells are shown, and the results show that the prepared benzimidazole derivative has an inhibition effect on the tumor cells and can be used for preparing an anti-tumor preparation.

Claims

1. A benzimidazole derivative BI292, its chemical name is 1- [ (2R,4S,5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl ] -1H-benzo [ d ] imidazole-4-carboxylic acid methyl ester, and its pharmaceutically acceptable salt.

2. The process for preparing the benzimidazole derivative BI292 according to claim 1, comprising the steps of:

s1: under the protection of nitrogen, methanol solution, 5- (hydroxymethyl) oxapentane-2, 4-diol and CH₃COCl is mixed and stirred for 20-45 minutes, pyridine is diluted and concentrated, Py, 4-dimethylaminopyridine and 4-methylbenzoyl chloride are added, stirring is carried out for 1-1.5 hours at the temperature of 0 ℃, and stirring is carried out at the room temperature overnight; adding water to stop the reaction, extracting with dichloromethane, combining organic layers, and sequentially using saturated NaHCO₃Solution, 15% H₂SO₄Washing, drying over anhydrous sodium sulfate, concentrating in vacuo and triturating with ether, filtering off the solid, concentrating the filtrate in vacuo, adding ether acetic acid to the resulting residue at 0 deg.C, introducing hydrochloric acid gas for 10-15 minutes, collecting the solid by filtration and washing with ether/petroleum ether to give compound (1), i.e. (2R,3S,5S) -5-chloro-2- { [ (4-methylbenzoyl) oxy)]Methyl } tetrahydrofuran-3-yl-4-benzoic acid methyl ester;

s2: 1H-1, 3-benzodiazole-4-carboxylic ester and CH₃CN and BSA are put into a round-bottom flask and stirred for 15 to 20 minutes at the temperature of 80 ℃; to CH at room temperature₃CN TMSOTf and compound (1) are added; mixing the above solutions at 85 deg.C, stirring for 2.5-3h, cooling to room temperature, diluting with water, extracting with ethyl acetate for several times, mixing organic layers, drying with anhydrous sodium sulfate, vacuum concentrating, eluting the obtained residue with ethyl acetate/petroleum ether on silica gel column, and purifying with silica gel columnFlash preparative high performance liquid chromatography purification of the crude product to yield compound (2), methyl- { (2R,4S,5R) -4- [ (4-methylbenzoyl) oxy]-5- { [ (4-methylbenzoyl) oxy]Methyl } tetrahydrofuran-2-yl } -1H-benzo [ d]Imidazole-4-carboxylic acid esters;

3. The method of claim 2, wherein in step S1, the mixture of the benzimidazole derivative BI292 comprises methanol solution, 5- (hydroxymethyl) oxapentane-2, 4-diol, and CH₃The dosage ratio of COCl, pyridine, 4-dimethylaminopyridine and 4-methylbenzoyl chloride is 60-120 mL: 6-12 g: 0.1-0.5 mL: 20-60 mL: 0.5-5 g: 10-30 mL.

4. The method of claim 2, wherein in step S2, 1H-1, 3-benzodiazole-4-carboxylate, CH₃CN, BSA, TMSOTf and compound (1) were used in the following ratios: 0.5-5 g: 60-100 mL: 0.5-5 g: 0.5-5 g: 3-10 g, and the mobile phase gradient elution conditions for purifying the crude product by adopting the rapid preparative high performance liquid chromatography are as follows: h containing 0.1% HCOOH₂O：CH₃CN=3:1。

5. The method for preparing the benzimidazole derivative BI292 according to claim 2, wherein the compound (2), methanol, THF, and sodium methoxide are used in the following ratio in step S3: 1-5 g: 60-100 mL: 60-100 mL: 0.1-0.5 g, and the mobile phase gradient elution conditions for purification by preparative chromatography are as follows: in 20 minutes, the mobile phase is composed of a mixture containing 0.1% NH₄HCO₃H of (A) to (B)₂O：CH₃CN =20:1 increase to 0.1% NH₄HCO₃H of (A) to (B)₂O：CH₃CN=1:2。

6. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier.

7. Use of a compound of claim 1, or a pharmaceutical composition of claim 6, in the manufacture of a medicament.

8. Use of the benzimidazole derivative BI292 or a pharmaceutically acceptable salt thereof as claimed in claim 1 for the preparation of an antitumor agent.

9. Use of the benzimidazole derivative BI292 or a pharmaceutically acceptable salt of said compound of claim 1 in combination with one or more anticancer agents for the preparation of a medicament for the treatment of tumors.