CN103288803B - Benzimidazole amides compound and its preparation method and application - Google Patents

Abstract

The invention discloses a kind of benzimidazole amides compound or its pharmaceutically acceptable salt and preparation method thereof, it, which is used for the tumour related to Hh signal path sustained activations, includes rodent ulcer, brain tumor, medulloblastoma, lung cancer, tumor in digestive tract, breast cancer or cancer of pancreas and the disease relevant with Hh signal paths.

Description

Benzimidazole amide compound and its preparation method and application

technical field

The invention relates to the technical field of medicine, in particular to a benzimidazole amide compound, a preparation method and an application thereof.

Background technique

The Hedgehog gene was first discovered in Drosophila in the early 1980s by Wieschaus and Nusslein-Vollhard. Hedgehog (Hh) signaling pathway is mainly composed of secreted glycoprotein ligand Hedgehog, transmembrane protein receptor Ptched (Ptch), transmembrane protein Smoothened (Smo), nuclear transcription factor Gli protein and downstream target genes. In normal body, the expression of Hh ligands in tissues is closed, and the combination of Ptch and Smo inhibits the activity of Smo, and the pathway is closed. When Hh binds to Ptch, it releases the inhibitory effect of Ptch on Smo, and Smo transmits the signal to the cytoplasm, activates the downstream Gli transcription factor and activates the entire signaling pathway.

In recent years, the relationship between Hh signaling pathway and tumor has been paid more and more attention. Literature has shown that under normal conditions, the Hedgehog (Hh) signaling pathway regulates the growth and differentiation of tissue cells during the embryonic period. After the embryo matures, the pathway goes into a closed state. Many other studies have shown that the abnormal activation of the Hh signaling pathway is closely related to a variety of tumors, such as skin basal cell carcinoma, medulloblastoma, lung cancer, digestive tract tumors, breast cancer, and pancreatic cancer. Therefore, blocking the Hh signaling pathway in tumor cells will be a new and effective means for human treatment of tumors. With the progress of research, the role of Hh signaling pathway in the process of tumorigenesis and development has become more and more clear. In a large number of human tumor cells, the Hh signaling pathway is continuously activated and regulates downstream gene transcription, thereby participating in tumor proliferation and differentiation, cell apoptosis, angiogenesis, and invasion and metastasis. Therefore, targeted inhibition of the Hh signaling pathway has also become a hot spot in anticancer therapy. It has been documented that the inhibitors of the Hh signaling pathway are mainly divided into three categories: Shh inhibitors, Smo inhibitors and Gli transcription inhibitors. Among them, the Smo inhibitor Cyclopamine can inhibit the activity of Smo and prevent the activation of the Hh signaling pathway, thereby exerting an anti-tumor effect. In recent years, the research on Cyclopamine and its derivatives as antitumor drugs has developed rapidly abroad. Some have entered the clinical stage, among which GDC-0449 was approved by the FDA for the treatment of basal cell carcinoma (BCC) in early 2012, and the research of GDC-0449 in the treatment of various other solid tumors has also entered clinical phase II/I. Taken together, the Hh signaling pathway provides a promising target for the development of anticancer drugs.

However, GDC-0449, the only Hh signaling pathway inhibitor currently on the market, also has some problems in its application. If there are mutations in some Smo sites, the efficacy of GDC-0449 will be reduced or even ineffective.

Contents of the invention

The purpose of the present invention is to provide a benzimidazole amide compound, which is used for tumors related to the continuous activation of the Hh signaling pathway, including skin basal cell carcinoma, brain tumor, medulloblastoma, lung cancer, digestive tract tumors, breast cancer Or pancreatic cancer and diseases related to the Hh signaling pathway.

Another object of the present invention is to provide a preparation method of the above-mentioned compound and its pharmaceutically acceptable salt.

A further object of the present invention is to provide the application of the above compound in the preparation of antitumor drugs.

A further object of the present invention is to provide a pharmaceutical composition containing the above-mentioned compound and its pharmaceutically acceptable salt as active ingredients.

To achieve the above object, the technical solution of the present invention is:

A compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

Wherein, R ¹ is H, halogen, C _1-6 straight chain, branched chain alkyl or C _1-6 straight chain or branched chain alkyl substituted by halogen; Ar is substituted or unsubstituted aryl, and the substituent Including halogen, C _1-6 straight chain, branched chain alkyl, halogen substituted C _1-6 straight chain or branched chain alkyl substituted benzene; R ² is: the heteroatom is nitrogen or oxygen, and the number of heteroatoms is one or Two, unsubstituted or 6-membered heterocyclic rings substituted with alkyl, hydroxyl or amino groups.

^The R2 is

The compound or a pharmaceutically acceptable salt thereof is selected from one of the following compounds:

The preparation method of formula (I) compound, comprises the following steps:

a. The nitro group of compound 1 is reduced to obtain compound 2;

b. Compound 2 was protected with p-toluenesulfonyl chloride to obtain compound 3;

c. Nitrating compound 3 with nitric acid mixed acid to obtain compound 4;

d. deprotecting compound 4 to obtain compound 5;

e. Compound 6 was obtained by reacting compound 5 with 6-chloronicotinyl chloride;

f. Compound 6 is heated and ring-closed in a solvent to obtain benzimidazole compound 7;

g. Prepare the compound of formula (I) from benzimidazole compound 7.

The g step is preparation method I or preparation method II,

Wherein, preparation method I comprises the following steps:

① reduction of benzimidazole compound 7 to obtain compound 14;

② acylation of compound 14 to obtain amide compound 16;

③ Substituting the chlorine on the pyridine with the amide compound 16 to obtain the compound of formula (I).

Preparation method II comprises the following steps:

① Substituting chlorine on the pyridine in benzimidazole compound 7 to obtain compound 17;

② Re-reduction of the nitro group in compound 17 to obtain compound 18;

③ acylation of the amino group of compound 18 to obtain the compound of formula (I);

The reaction formula is as follows:

Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of an antitumor drug, wherein the tumor is a disease related to the Hh signaling pathway.

The tumor is a tumor related to the continuous activation of the Hh signaling pathway; the tumor includes skin basal cell carcinoma, brain tumor, medulloblastoma, lung cancer, digestive tract tumor, breast cancer or pancreatic cancer.

In another aspect, the present invention provides a pharmaceutical composition, which comprises the compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient, and one or more pharmaceutically acceptable excipients.

The pharmaceutical combination provided by the present invention contains the above-mentioned compound or its pharmaceutically acceptable salt, a pharmaceutically acceptable carrier, or a combination with the compound as an active ingredient mixed with an excipient or diluent.

The pharmaceutically acceptable carrier in the drug combination for treating tumors of the present invention refers to the conventional drug carrier in the field of pharmacy, for example: diluents such as water, etc.; excipients such as starch, sucrose, etc., binders such as Cellulose derivatives, alginate, gelatin, etc.; wetting agents such as glycerin; disintegrants such as agar, calcium carbonate, calcium bicarbonate, etc.; absorbents such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as Kaolin, etc.; lubricants such as talc, calcium stearate, magnesium, etc. In addition, other adjuvants such as flavoring agents and sweetening agents can also be added to the composition.

The compounds of the present invention may be administered in the form of compositions to patients in need of such treatment by oral, nasal inhalation, rectal or parenteral administration. For oral administration, it can be made into conventional solid preparations such as tablets, powders, granules, capsules, etc., into liquid preparations such as water or oil suspensions or other liquid preparations such as syrups, tinctures, etc.; for parenteral administration When used as a medicine, it can be made into a solution for injection, water or oily suspension, etc., and the preferred forms are tablets, capsules and injections.

Various dosage forms of the pharmaceutical combination of the present invention can be prepared according to conventional production methods in the field of pharmacy. For example, the active ingredient is mixed with one or more carriers and brought into the desired dosage form.

The compounds of the present invention and their pharmaceutically acceptable salts have an inhibitory effect on cancer cell growth of tumors related to the continuous activation of the Hh signaling pathway, which is equivalent to or even better than GDC-0449, thus providing a new option for the treatment of related tumors.

detailed description

The present invention will be described in further detail below in conjunction with specific examples. It should be understood that these examples are only to illustrate the present invention and not to limit the scope of the invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. The reagents used were of commercially available analytical or chemical grade without indicating their sources and specifications.

The structure of the compound was confirmed by nuclear magnetic resonance spectrum and mass spectrum.

Example 1

Synthesis of N-{5-chloro-2-[6-(4-hydroxypiperidinyl)(3-pyridyl]benzimidazol-6-yl}-3-chlorobenzamide (I-1)

Experimental procedure

Compound 1 (20.0g) was dissolved in 600ml mixed solution (EtOH:H ₂ O=5:1), ammonium chloride (20g) and acetic acid (20ml) were added, the system was heated to 60°C, and iron powder (32.4g) was added in batches join in. Keep at 60°C for 1 hour. The temperature was lowered, extracted with ethyl acetate, and spin-dried to obtain compound 2 (14 g, yield 85%).

Compound 2 (14.0 g) was dissolved in pyridine (315 ml), p-toluene chloride (39.3 g) was added in batches, and the temperature was raised to 75° C. for 1.5 hours. The reaction solution was spin-dried, dissolved in ethyl acetate, washed three times with 0.1N HCl aqueous solution, dried, and spin-dried to obtain compound 3 (29 g, yield 65.6%).

Compound 3 (21g) was dissolved in acetic acid (170ml), the reaction was heated to 70°C, the mixed solution (7ml sulfuric acid/4.9ml fuming nitric acid) was added dropwise, and the reaction was completed at 70°C for 2 hours. The reaction solution was cooled to room temperature, filtered, the filter cake was washed three times with water, and dried to obtain compound 4 (14.6 g, yield 63%)

Compound 4 (12g) was dissolved in 121ml of sulfuric acid (H ₂ SO ₄ :H ₂ O=10:1), the system was heated to 85°C and reacted for 0.5 hours. The reaction solution was poured into ice water, the pH was adjusted to 9 with ammonia water, and compound 5 (3.0 g, yield 67%) was obtained by filtration.

Compound 5 (3.0g), N,N-diisopropylethylamine (6.2g), was dissolved in tetrahydrofuran (75ml), the reaction system was lowered to 0°C, and the mixture (3.4g 6-chloronicotinoyl chloride/ 10ml tetrahydrofuran), react at room temperature for 0.5 hours. Spin-dried, washed with EA dissolved in water, dried, and spin-dried to obtain compound 6 (3.3 g, yield 63.6%).

Compound 6 (3.0 g) was dissolved in acetic acid (120 ml), and the reaction system was raised to 100° C. for 0.5 hour. The reaction system was poured into ice water and filtered to obtain compound 7 (2.4 g, yield 85%).

Compound 7 (1.2g), 4-hydroxypiperidine (0.79g), N,N-diisopropylethylamine (3.0g) dissolved in N-methylpyrrolidone (50ml), added to the sealed tube under nitrogen protection overnight at 130°C. The reaction solution was poured into ice water, extracted with ethyl acetate, washed five times with water, dried and spin-dried to obtain compound 8 (0.8 g, yield 55%).

Compound 8 (0.5g), imidazole (0.2g) was dissolved in N,N-dimethylformamide (60ml), tert-butyldimethylsilyl chloride (0.4g) was added at 0°C, and the system was raised to room temperature for overnight reaction . The reaction solution was poured into water, extracted with ethyl acetate, dried, and spin-dried to obtain compound 9 (0.45 g, yield 69%).

Compound 9 (450mg) was dissolved in EtOH:H2O=5:1 (96ml), ammonium chloride (450mg) and acetic acid (0.45ml) were added, the system was heated to 60°C, and iron powder (258mg) was added in batches. Keep at 60°C for 1 hour. The temperature was lowered, extracted with ethyl acetate, and spin-dried to obtain compound 10 (350 mg, yield 83%). LC/MS=458(M+1)

Compound 10 (100mg), N,N-diisopropylethylamine (84mg), was dissolved in (15ml) tetrahydrofuran, the reaction system was lowered to 0°C, and the mixed solution (46mg, m-chlorobenzoyl chloride/5ml , tetrahydrofuran), reacted at room temperature for 2 hours. Spin-dried, dissolved in ethyl acetate, washed with water, and spin-dried to obtain compound 11 (92 mg, yield 71%).

Compound 11 (90mg), tetrabutylammonium fluoride (197mg) was dissolved in tetrahydrofuran (50ml), heated to 50°C for 3 hours. It was quenched with water, extracted with ethyl acetate, washed with water, and spin-dried to prepare and isolate compound I-1 (10 mg, yield 13.8%).

¹ H-NMR (400MHz, CD3OD) δ: 1.46-1.51 (m, 2H), 1.88-1.92 (m, 2H), 3.19-3.23 (m, 2H), 3.83-3.85 (m, 1H), 4.12-4.18 (m,2H),6.88-6.91(d,1H),7.46-7.50(m,1H),7.56-7.61(m,2H),7.82(s,1H),7.87-7.89(d,1H),7.96 (s,1H),8.06-8.09(m,1H),8.72-8.73(d,1H)LC/MS=482(M+1)

Example 2

synthesis

N-{5-chloro-2-[6-(4-methylpiperazinyl)(3-pyridyl]benzimidazol-6-yl}-3-chlorobenzamide (I-2)

Experimental procedure

Compound 7 (500mg), N-methylpiperazine (488mg), N,N-diisopropylethylamine (1.26g) dissolved in N-methylpyrrolidone (20ml), added to a sealed tube under nitrogen protection React overnight at 130°C. The reaction solution was poured into ice water, extracted with ethyl acetate, washed with water five times, dried and spin-dried to obtain compound 12 (450 mg, yield 75%).

Compound 12 (450mg) was dissolved in EtOH/H2O=5/1 (96ml), ammonium chloride (450mg) was added, the system was heated to 60°C, and iron powder (408mg) was added in batches. Keep at 60°C for 1 hour. The temperature was lowered, extracted with ethyl acetate, and spin-dried to obtain compound 13 (200 mg, yield 48%).

Compound 13 (100mg), N,N-diisopropylethylamine (113mg), was dissolved in (15ml) tetrahydrofuran, the reaction system was lowered to 0°C, and the mixed solution (61mg p-chlorobenzoyl chloride/5ml tetrahydrofuran ), reacted at room temperature for 2 hours. Spin-dry, dissolve in ethyl acetate, wash with water, spin-dry, and then separate by column chromatography to obtain compound I-2 (90 mg). LCMS=481.4(M+1)

Example 3

synthesis

N-{5-chloro-2-[6-(4-methylpiperazinyl)(3-pyridyl]benzimidazol-6-yl}-3-trifluoromethylbenzamide (I-3)

Experimental procedure

Compound 13 (100mg), N,N-diisopropylethylamine (113mg), was dissolved in (15ml) tetrahydrofuran, the reaction system was lowered to 0°C, and the mixed solution (72.6mg m-trifluoromethylbenzyl acid chloride/5ml tetrahydrofuran), react at room temperature for 2 hours. Spin to dry, dissolve in ethyl acetate, wash with water, spin dry and separate by column chromatography to obtain compound I-3 (65mg). LCM S=515.9(M+1)

Example 4

synthesis

N-{5-chloro-2-[6-(2,6-dimethylmorpholin-4-yl)(3-pyridyl]benzimidazol-6-yl}-3-chlorobenzamide (I- 4)

Experimental procedure

Compound 7 (1.0g) was dissolved in EtOH/H ₂ O=5/1 (96ml), ammonium chloride (1.0g) was added, the system was heated to 60°C, and iron powder (0.9g) was added in batches. Keep at 60°C for 1 hour. Cool down, spin dry, add water and ethyl acetate for extraction, and spin dry to obtain compound 14 (0.5 g, 56%).

Compound 14 (500mg), N,N-diisopropylethylamine (696mg), was dissolved in tetrahydrofuran (90ml), the reaction system was lowered to 0°C, and the mixed solution (378mg m-chlorobenzoyl chloride/10ml tetrahydrofuran ), reacted at room temperature for 2 hours. Spin dry and pass through a column (PE/EA=3:1) to obtain compound 15 (200 mg, yield 27%). LCMS=417(M+1)

Compound 15 (50mg), 2,6-dimethylmorpholine (69mg), N,N-diisopropylethylamine (93mg) dissolved in N-methylpyrrolidone (1ml), added to a sealed tube under nitrogen overnight at 130°C under protection. The reaction solution was poured into ice water, filtered and washed with water to obtain compound I-4 (14 mg, yield 24%).

¹ H-NMR(400MHz,DMSO_d6)δ:1.18-1.20(d,6H),3.61-3.64(m,2H),4.29-4.33(d,2H),7.03-7.05(d,1H),7.58-7.62 (m,1H),7.69-7.71(m,3H),7.98-8.00(d,1H),8.07(s,1H),8.24-8.26(d,1H),8.90(s,1H),7.96(s ,1H), 10.23(s,1H), 12.88-12.96(m,1H); LC/MS=497.3(M+1)

Example 5

Synthesis of N-{5-chloro-2-[6-(3,5-dimethylpiperazinyl)(3-pyridyl]benzimidazol-6-yl}-3-chlorobenzamide (I-5)

Experimental procedure

Compound 15 (50mg), 2,6-dimethylpiperazine (68mg), N,N-diisopropylethylamine (93mg) dissolved in N-methylpyrrolidone (1ml), added to the sealed tube under nitrogen overnight at 130°C under protection. The reaction solution was poured into ice water, filtered, and washed with water to obtain compound I-5 (13 mg, yield 22%).

¹ H-NMR(400MHz,DMSO_d6)δ:1.04-1.05(d,6H),2.32-2.38(m,2H),2.75(s,1H),4.28-4.31(d,2H),6.99-7.01(d ,1H),7.57-7.99(m,4H),7.98-7.99(d,1H),8.07(s,1H),8.19-8.21(d,1H),8.86(s,1H),10.22(s,1H ), 12.89-12.92(s,1H); LC/MS=496.3(M+1)

Test Example 6

The compounds of the present invention having the structure of formula (I) and pharmaceutically acceptable salts thereof have significant antitumor effects, which are now illustrated by the following pharmacological experiments:

MTS cell proliferation assay

1. Test material

1.1 Compounds and solvents

Test sample

Formula (I) series compounds I-1, I-4, and I-5 prepared in the foregoing examples. ;

Positive control substance: GDC-0449 was purchased from Shanghai Hanxiang Fragrance Co., Ltd.

DMSO was used as the solvent in this experiment and was purchased from Sigma Company, item number: D8418.

1.2 Cell lines

Two types of cell lines were used in this experiment. 1) Human bladder cancer cell line T24 was derived from the Cell Bank of the Chinese Academy of Sciences. 2) The human chronic myeloid leukemia cell line K562 was obtained from the Cell Bank of the Chinese Academy of Sciences.

1.3 Reagents

MTS detection of cell proliferation reagent powder, purchased from Promega, product number: G1111. PMS was purchased from sigma company, article number: P9625. Cell culture media RPMI-1640 and DMEM were purchased from Gibco. Fetal bovine serum was purchased from HyClone Company, item number: SV30087 02

2. Test method

2.1 Drug treatment dosage and preparation method

Blank vehicle composition: DMSO

The preparation method of the test product formula (I) series compound and the positive reference substance GDC-0449: Weigh an appropriate amount of sample, add an appropriate amount of DMSO to make the drug storage concentration 20mmol/L, and vortex to mix evenly. According to the solubility of drugs in DMSO and the safe concentration of DMSO in cell culture (DMSO is below 1%), the following drug treatment concentrations μmol/L were set: 60, 20, 6.67, 2.22, 0.74, 0.25, 0.082, 0.027.

2.2 Cell culture

T24 human bladder cancer cell line was cultured in complete DMEM medium (containing 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin). Human chronic myeloid leukemia cell line K562 (suspension cells) was cultured in RPMI-1640 complete medium (containing 10% fetal bovine serum, 100U/ml penicillin, 100μg/ml streptomycin).

2.3 MTS method to detect the inhibitory effect of HB series compounds on the growth of cancer cells in vitro

Digest the T24 cells in the logarithmic growth phase with 0.25% trypsin to make a cell suspension, add 5×10 ³ /well into a 96-well cell culture plate (150 μL/well), and place three replicate wells at 37 Cultivate in an incubator with 5% _CO at ℃, and after sticking to the wall the next day, according to the experimental design, add 50ul series concentrations of the test compound or the culture medium of the control to each well, and set up the cell control group (only containing cells and culture medium but not Wells without drug), blank control group (only medium without cells). K562 suspension cells were directly counted, 1X10 ⁴ /well was added to a 96-well cell culture plate, and other treatments were the same as T24. Both human bladder cancer cell T24 and human chronic myeloid leukemia cell line K562 were cultured for 3 days after administration. After the culture was completed, the cell proliferation was detected by the MTS method and the cell viability of the cells was calculated relative to the cell control group.

Relative cell viability%=(cell absorbance value of drug-dosed group-average absorbance value of blank control group)/(average absorbance value of cell control group-average absorbance value of blank control group)×100%

3. Test results

Inhibitory Effects of HB Series Compounds on the Growth of Cancer Cells Cultured in Vitro

After 3 days of drug addition, MTS method was used to detect the inhibitory effect of the series of compounds of the present invention on the growth of T24 and K562 cells. The results showed that the inhibitory effects of several compounds were comparable to or even better than GDC-0449, and the IC50 results are shown in Table 1.

Table 1: The inhibitory effect of compound of the present invention on cancer cell growth

Claims (8)

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, Wherein, R ¹ is H, halogen, C _1-6 straight chain, branched chain alkyl or C _1-6 straight chain or branched chain alkyl substituted by halogen; Ar is substituted or unsubstituted aryl, and the substituent Including halogen, C _1-6 straight chain, branched chain alkyl, halogen substituted C _1-6 straight chain or branched chain alkyl substituted benzene; R ² is heteroatom nitrogen or oxygen, the number of heteroatoms is one or two 6-membered heterocyclic rings that are unsubstituted or substituted with alkyl, hydroxyl or amino groups. ^2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein said R is 3. The compound or pharmaceutically acceptable salt thereof according to claim 2, characterized in that it is selected from one of the following compounds: 4. the preparation method of formula (I) compound described in claim 1 is characterized in that comprising the steps: a. The nitro group of compound 1 is reduced to obtain compound 2; b. Compound 2 was protected with p-toluenesulfonyl chloride to obtain compound 3; c. Nitrating compound 3 with nitric acid mixed acid to obtain compound 4; d. deprotecting compound 4 to obtain compound 5; e. Compound 6 was obtained by reacting compound 5 with 6-chloronicotinyl chloride; f. Compound 6 is heated and ring-closed in a solvent to obtain benzimidazole compound 7; g. Prepare the compound of formula (I) from benzimidazole compound 7. The g step is preparation method I or preparation method II, Wherein, preparation method I comprises the following steps: ① reduction of benzimidazole compound 7 to obtain compound 14; ② acylation of compound 14 to obtain amide compound 16; ③ Substituting the chlorine on the pyridine with the amide compound 16 to obtain the compound of formula (I). Preparation method II comprises the following steps: ① Substituting chlorine on the pyridine in benzimidazole compound 7 to obtain compound 17; ② Re-reduction of the nitro group in compound 17 to obtain compound 18; ③ acylation of the amino group of compound 18 to obtain the compound of formula (I); The reaction formula is as follows: 5. The use of the compound or its pharmaceutically acceptable salt according to claim 1, 2 or 3 in the preparation of antitumor drugs, wherein the tumor is a disease related to Hh signaling pathway. 6. The application according to claim 5, characterized in that the tumor is a tumor associated with the continuous activation of the Hh signaling pathway. 7. The application according to claim 6, wherein the tumor is skin basal cell carcinoma, brain tumor, medulloblastoma, lung cancer, digestive tract tumor, breast cancer or pancreatic cancer. 8. The pharmaceutical composition, characterized in that it contains the compound of claim 1, 2 or 3 or a pharmaceutically acceptable salt thereof as an active ingredient, and one or more pharmaceutically acceptable excipients.