CN112300235A

CN112300235A - Benzimidazole derivative BI321 and preparation method and application thereof

Info

Publication number: CN112300235A
Application number: CN202011312389.5A
Authority: CN
Inventors: 潘璟; 卢光明; 许静; 马龙
Original assignee: Eastern Theater General Hospital of PLA
Current assignee: Eastern Theater General Hospital of PLA
Priority date: 2020-11-20
Filing date: 2020-11-20
Publication date: 2021-02-02
Anticipated expiration: 2040-11-20
Also published as: CN112300235B

Abstract

The invention discloses a benzimidazole derivative BI321 and a preparation method thereof, wherein the chemical name of the benzimidazole derivative BI321 is 1- [ tetrahydro-3, 4-dihydroxy-5- (hydroxymethyl) furan-2-yl ] -N, N-dimethyl-1H-benzo [ d ] imidazole-4-formamide. 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 BI321 and preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a benzimidazole derivative BI321 and 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 inventors have unexpectedly found that 1- [ tetrahydro-3, 4-dihydroxy-5- (hydroxymethyl) furan-2-yl ] -N, N-dimethyl-1H-benzo [ d ] imidazole-4-carboxamide has some anti-tumor activity and have proposed inventions relating to 1- [ tetrahydro-3, 4-dihydroxy-5- (hydroxymethyl) furan-2-yl ] -N, N-dimethyl-1H-benzo [ d ] imidazole-4-carboxamide or a pharmaceutically acceptable salt, solvate or prodrug or stereoisomer or tautomer or metabolite of such a 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 BI321 which is chemically named as 1- [ tetrahydro-3, 4-dihydroxy-5- (hydroxymethyl) furan-2-yl ] -N, N-dimethyl-1H-benzo [ d ] imidazole-4-formamide 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 objects, the present invention provides a benzimidazole derivative BI321, which is chemically named as 1- [ tetrahydro-3, 4-dihydroxy-5- (hydroxymethyl) furan-2-yl ] -N, N-dimethyl-1H-benzo [ d ] imidazole-4-carboxamide, and has the following structural formula:

the invention further provides a preparation method of the benzimidazole derivative BI321, which comprises the following steps:

s1: putting 1H-1, 3-benzobisoxazole-4-carboxylic acid, methanol and sulfuric acid into a round bottom flask, refluxing in an oil bath, stirring overnight, adjusting the pH value to be neutral by using a saturated solution of sodium bicarbonate, filtering, collecting a solid, and drying to obtain a compound (1), namely 1H-1, 3-benzobisoxazole-4-carboxylic acid methyl ester.

S2: placing compound (1), ACN and BSA in a 250mL occlusion round bottom flask, stirring at 80-85 deg.C for 10-15 min, and adding CH at room temperature₃CN to which TMSOTf and [3,4, 5-tris (acetoxy) oxopentan-2-yl group were added]Mixing the above solutions at 80-85 deg.C for 2.5-3 hr, vacuum concentrating, and adding NaHCO₃Dissolving the solution, and extracting with 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 (1:1) on a silica gel column to give the compound (2), methyl 1- [3, 4-bis (acetoxy) -5- [ (acetoxy) methyl]Oxazol-2-yl]-1H-1, 3-benzodiazole-4-carboxylic acid methyl ester;

s3: in a round-bottomed flask, Me is placed under ice bath₃Al addition to Me₂Adding solution of NH and THF into compound (2) solution dissolved in THF, refluxing in oil bath, stirring overnight, filtering off solid, and vacuum concentrating the filtrate to obtain compound (3) {3, 4-bis (acetoxy) -5- [4- (dimethylcarbamoyl) -1H-1, 3-benzodiazol-1-yl]Oxazol-2-yl } acetic acid methyl ester;

s4: placing compound (3) and NH 3-methanol solution in a round-bottom flask, stirring overnight at room temperature, and then concentrating the reaction mixture in vacuo; the resulting residue was purified by flash preparative high performance liquid chromatography to give compound (4), 1- [ tetrahydro-3, 4-dihydroxy-5- (hydroxymethyl) furan-2-yl ] -N, N-dimethyl-1H-benzo [ d ] imidazole-4-carboxamide.

Preferably, in step S1, the ratio of the 1H-1, 3-benzodiazole-4-carboxylic acid, methanol and sulfuric acid is 10 to 50 g: 20-100 mL: 5-50 mL.

In step S2, compound 1, ACN, BSA, TMSOTf, and methyl [3,4, 5-tris (acetoxy) oxopentan-2-yl ] acetate were used in the following ratios: 3-9 g: 100-350 mL: 3-20 g: 3-20 g: 2-30 g. The reaction temperature is 60-100 ℃, and the reaction time is 60-180 minutes.

In the step S3, the oil bath temperature is 60-120 ℃, and dimethylamine, THF and Me are used₃The dosage ratio of Al to the compound (2) is 1-10 mL: 30-100 mL: 3-20 mL: 1-5 g.

In step S4, Compound (3), NH₃-the ratio of the amount of methanol: 1-5 g: 10-60 mL; the mobile phase gradient elution conditions for purification by preparative chromatography are as follows: h₂O(0.5％NH₃.H₂O)：CH₃CN＝1:2～5:1。

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 BI321 or pharmaceutically acceptable salt, solvate or prodrug or stereoisomer or tautomer or metabolite of the compound in preparation of an antitumor agent.

Finally, the invention provides the application of the benzimidazole derivative BI321 or the pharmaceutically acceptable salt, solvate or prodrug or stereoisomer or tautomer or metabolite of the compound in preparing the drugs for treating tumors by combining with one or more anticancer drugs.

Has the advantages that: the invention discloses a benzimidazole derivative BI321, and adopts an MTT method to evaluate the proliferation activities of 6 tumor cells of MCF-7, SK-BR-3, HCT116, U-118MG, U-87MG and MDA-MB-468, and calculates IC for inhibiting the proliferation of the six tumor cells₅₀The value and the result show that the prepared benzimidazole derivative BI321 has an inhibiting 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) sulfuric acid, methanol(ii) a ii) BSA, TMSOTf, acetonitrile; iii) Me₃Al, dimethylamine, THF; iv) ammonia-methanol.

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 1H-1, 3-benzobisoxazole-4-carboxylate (Compound 1)

30g of 1H-1, 3-benzodiazole-4-carboxylic acid, 80mL of methanol and 20mL of sulfuric acid were placed in a 250mL round-bottom flask, and the resulting solution was refluxed in an oil bath and stirred overnight. Adjusting the pH value to be neutral by using a saturated solution of sodium bicarbonate. The solid was collected by filtration and dried. 27.5g (84% yield) of Compound 1 are obtained as a yellow solid. Compound 1 was characterized by LC-MS as follows:

LC-MS:(ES,m/z):177.2[M+H]+

EXAMPLE 2 preparation of methyl 1- [3, 4-bis (acetoxy) -5- [ (acetoxy) methyl ] oxazol-2-yl ] -1H-1, 3-benzobisoxazole-4-carboxylic acid methyl ester (Compound 2)

5g of Compound 1, 150mL of ACN and 5.77g of BSA were placed in a 250mL closed round bottom flask and stirred at 85 ℃ for 15 minutes. Then 20mL CH at room temperature₃CN was added 8.20g of TMSOTf and 9.94g of [3,4, 5-tris (acetoxy) oxopentan-2-yl group]Methyl acetate solution. The above solutions were mixed and stirred at 85 ℃ for 2.5 h. Concentrated in vacuo using 100mL NaHCO₃The solution was dissolved and extracted with 100mL 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 (1:1) on a silica gel column. 8.4g (68% yield) of Compound 2 are obtained as a yellow oil.

Compound 2 was characterized by LC-MS as follows:

LC-MS:(ES,m/z):435.1[M+H]+

EXAMPLE 3 preparation of methyl {3, 4-bis (acetoxy) -5- [4- (dimethylcarbamoyl) -1H-1, 3-benzoxadiazol-1-yl ] oxazol-2-yl } acetate (Compound 3).

13 was placed in a 100mL round-bottom flask in an ice bath over 5 minutes.8mL Me₃Al was added to 9.2mL Me₂To a solution of NH in 40mL of THF was then added 5mL of THF with 4g of Compound 2 dissolved in 5mL of THF, the above solutions were mixed, and the resulting solution was stirred in an oil bath at reflux overnight. The solids were filtered off. The filtrate was concentrated in vacuo to give 4g of compound 3 as a yellow oil. Compound 3 was characterized by LC-MS as follows:

LC-MS:(ES,m/z):448.2[M+H]+

EXAMPLE 4 preparation of 1- [ tetrahydro-3, 4-dihydroxy-5- (hydroxymethyl) furan-2-yl ] -N, N-dimethyl-1H-benzo [ d ] imidazole-4-carboxamide (Compound 4)

4g of Compound 3 and 50mL of NH₃The methanol solution was placed in a 100mL round bottom flask. After stirring at room temperature overnight, 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, H₂O(0.5％NH₃·H₂O)：CH₃CN 1:0, increasing to H₂O(0.5％NH₃·H₂O)：CH₃CN 2:1 in 30 minutes; detector, UV 254 nm. 867.4mg (yield 30%) of Compound 4 are obtained as a white solid.

ESI-MS and the like were performed on the prepared Compound 4, and the results were as follows:

LC-MS:(ES,m/z):322.15[M+H]⁺

1H-NMR:(300MHz,DMSO-d₆,ppm):δ8.51(s,1H),7.81(d,J＝7.8Hz,1H),7.30(t,J＝7.5Hz,1H),7.18(d,J＝6.9Hz,1H),5.89(d,J＝6.3Hz,1H),5.49(d,J＝6.6Hz,1H),5.22(d,J＝4.8Hz,1H),5.12(t,J＝5.1Hz,1H),4.42-4.36(q,1H),4.14-4.10(q,1H),3.99-3.98(q,1H),3.71-3.58(m,2H),3.06(s,3H),2.78(s,3H).

[α]＝-33.737(C＝0.41g/100mL,T＝24.9℃,MeOH)

experimental example 5 evaluation of antitumor cell proliferation Activity of Compound 4.

(1) And (3) testing a sample:

the compounds 4 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 4 which is to be detected and is dissolved in a culture medium after 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 the 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 4 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 4BI321 antitumor cell proliferation Activity (IC)₅₀±SDμM)

As shown in Table 1, the test results of the compound 4 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 BI321, chemically designated as 1- [ tetrahydro-3, 4-dihydroxy-5- (hydroxymethyl) furan-2-yl ] -N, N-dimethyl-1H-benzo [ d ] imidazole-4-carboxamide, and pharmaceutically acceptable salts, solvates or prodrugs or stereoisomers or tautomers or metabolites thereof.

2. The process for preparing the benzimidazole derivative BI321 of claim 1, comprising the steps of:

s1: putting 1H-1, 3-benzobisoxazole-4-carboxylic acid, methanol and sulfuric acid into a flask, refluxing in an oil bath, and stirring overnight; adjusting the pH value to be neutral by using a saturated solution of sodium bicarbonate; the solid was collected by filtration and dried; to obtain a compound (1), namely 1H-1, 3-benzobisoxazole-4-carboxylic acid methyl ester;

s2: placing the compound 1, ACN and BSA into a blocking reaction bottle, and stirring for 10-15 minutes at 80-85 ℃; to CH at room temperature₃CN to which TMSOTf and [3,4, 5-tris (acetoxy) oxopentan-2-yl group were added]A methyl acetate solution; mixing the above solutions at 80-85 deg.C, stirring for 2.5-3h, vacuum concentrating, and adding NaHCO₃Dissolving the solution, and extracting with ethyl acetate; the organic layers were combined, dried over anhydrous sodium sulfate and concentrated in vacuo; eluting the residue with ethyl acetate/petroleum ether on silica gel column to obtain compound (2), i.e. methyl 1- [3, 4-bis (acetoxy) -5- [ (acetoxy) methyl]Oxazol-2-yl]-1H-1, 3-benzodiazole-4-carboxylic acid methyl ester;

s3: in a round-bottomed flask, Me is placed under ice bath₃Al addition to Me₂NH with THF, thenAdding compound (2) dissolved in THF, refluxing in oil bath, stirring overnight, filtering off solid, and vacuum concentrating the filtrate to obtain compound (3) {3, 4-bis (acetoxy) -5- [4- (dimethylcarbamoyl) -1H-1, 3-benzodiazol-1-yl]Oxazol-2-yl } acetic acid methyl ester;

s4: after placing compound (3) and NH 3-methanol solution in a round bottom flask and stirring at room temperature overnight, the reaction mixture was concentrated in vacuo and the resulting residue was purified by flash preparative high performance liquid chromatography to give compound (4), i.e., 1- [ tetrahydro-3, 4-dihydroxy-5- (hydroxymethyl) furan-2-yl ] -N, N-dimethyl-1H-benzo [ d ] imidazole-4-carboxamide.

3. The method for preparing the benzimidazole derivative BI321 of claim 2, wherein in step S1, the use amount ratio of 1H-1, 3-benzodiazole-4-carboxylic acid, methanol and sulfuric acid is 10 to 50 g: 20-100 mL: 5-50 mL.

4. The method of claim 2, wherein in step S2, compound 1, ACN, BSA, TMSOTf, and methyl [3,4, 5-tris (acetoxy) oxopentan-2-yl ] acetate are used in the following ratios: 3-9 g: 100-350 mL: 3-20 g: 3-20 g: 2-30 g, the reaction temperature is 60-100 ℃, and the reaction time is 60-180 minutes.

5. The method of claim 2, wherein in step S3, the oil bath temperature is 60-120 ℃, dimethylamine, THF, Me₃The dosage ratio of Al to the compound (2) is 1-10 mL: 30-100 mL: 3-20 mL: 1-5 g.

6. The method of claim 2, wherein in step S4, compound (3), NH₃-the ratio of the amount of methanol: 1-5 g: 10-60 mL, and the mobile phase gradient elution conditions for purification by preparative chromatography are as follows: h₂O（0.5%NH₃.H₂O）：CH₃CN=1:2~5:1。

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

8. Use of a compound according to claim 1, or a pharmaceutical composition according to claim 10, in the manufacture of a medicament.

9. Use of the benzimidazole derivative BI321 or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, tautomer, or metabolite of the compound of claim 1 for the preparation of an antitumor agent.

10. Use of the benzimidazole derivative BI321 or a pharmaceutically acceptable salt, solvate, or prodrug or stereoisomer or tautomer or metabolite of the compound of claim 1 in combination with one or more anti-cancer agents for the preparation of a medicament for the treatment of tumors.