CN103214456B - Benzimidazole compound with antitumour activity as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a benzimidazole compound with antitumour activity as well as a preparation method and an application thereof. The benzimidazole compound is shown in a formula (I), wherein R1, R2, R3, R4 and R5 are described in the specification. The related tumour cancer cell growth inhibiting effect continuously activated by an Hh signal channel of the benzimidazole compound and medicinal salt thereof is equivalent to and even better than that of GDC-0449, so that a new choice is provided for treating the related tumours.

Description

Benzimidazole compound with antitumor activity, preparation method and application thereof

technical field

The invention relates to the technical field of medicine, in particular to a benzimidazole compound with antitumor activity, 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 object of the present invention is to provide compounds with anti-tumor activity and pharmaceutically acceptable salts thereof that inhibit the Hh signaling pathway against the Smo target for use in the preparation of anti-tumor drugs.

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 , R ² , R ³ , R ⁴ are independently H, halogen, C1-6 straight chain, branched chain alkyl, cycloalkyl or halogen substituted C1-6 straight chain or branched chain alkyl; Y =C or N; R ⁵ is:

The heteroatom is nitrogen or oxygen, the number of heteroatoms is one or two, unsubstituted or substituted by alkyl, hydroxyl or amino, 4-8 membered heterocyclic rings, or Among them, R ⁶ is independently C1-4 straight chain alkyl, branched chain alkyl or cycloalkyl; R ⁷ and R ⁸ are independently H, C1-6 straight chain alkyl, cycloalkyl, amino or hydroxyl substitution Alkyl, alkylamino substituted alkyl R ⁹ R ¹⁰ NR, or alkoxy substituted alkyl R ¹¹ OR; wherein, R ⁹ and R ¹⁰ can be independently H, C1-6 straight chain alkyl, branched chain alkane Group, cycloalkyl or R ⁹ , R ¹⁰ are closed to form a ring; R ¹¹ is C1-4 straight chain alkyl, branched chain alkyl or cycloalkyl.

Preferably, R ⁵ is a 4, 6, 8-membered monoheterocyclic ring whose heteroatom is nitrogen or oxygen, the number of heteroatoms is one or two, unsubstituted or substituted with alkyl or hydroxyl.

More preferably R ⁵ is

or

More preferably, the compound of the present invention or a pharmaceutically acceptable salt thereof is selected from one of the following compounds:

Most preferably, the compound of the present invention or a pharmaceutically acceptable salt thereof is selected from one of the following compounds:

In the preparation method of the compound of the present invention, the final product is prepared according to the following steps:

a. Compound 1 was reacted with acetic anhydride in a solvent (CH ₂ Cl ₂ , THF, etc.) to prepare Compound 2;

b. Compound 2 reacts with bis(pinacolate) diboron to prepare compound 3;

c. Compound 3 was reacted with 2-bromopyridine to obtain compound 4;

d. Compound 4 was nitrated in concentrated sulfuric acid to prepare compound 5;

e. reducing compound 5 to obtain compound 6;

f. Deprotecting compound 6 to remove the acetyl group to obtain compound 7a

g. Alkylation of 7a to obtain compound 7;

h. Then reaction 7 or 7a with substituted aryl carboxylic acid gives the desired compound

Specifically, it can be carried out with reference to the following reaction formula (II):

target compound (Ⅱ),

Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and Y are as defined above.

In another aspect, the present invention provides the compound or a pharmaceutically acceptable salt thereof for use in the preparation of antitumor drugs. The tumor is a tumor associated with continuous activation of the Hh signaling pathway. More specifically, the tumor is: skin basal cell carcinoma, medulloblastoma, lung cancer, digestive tract tumor, breast cancer or pancreatic cancer, etc.

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 ways

The present invention will be described in further detail below in conjunction with specific embodiments. 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 5-chloro-2-(2-chloro-4-methylsulfonyl)phenyl-6-(pyridin-2-yl)benzimidazole (I-1):

Compound 1 (10g) was dissolved in dichloromethane, acetic anhydride (5.93g) was added, and the system was heated to 40°C for 2 hours. The system was cooled down to room temperature, washed twice with NaHCO3 aqueous solution, and the organic phase was spin-dried to obtain compound 2 (11 g, yield 92%).

Compound 2 (11g), potassium acetate (8.8g) was dissolved in dioxane, and bisboron (20.5g) was added. The system was replaced with nitrogen for 15 minutes, Pd(dppf)Cl2 (1 g) was added, and heated to 80° C. to react overnight (12 hours). The temperature was lowered, spin-dried and passed through the column (PE/EA=2/1) to obtain compound 3 (8 g, yield 61%).

Compound 3 (2.1 g), cesium carbonate (4 g) was dissolved in dioxane, and 2-bromopyridine (1.27 g) was added. The system was replaced with nitrogen for 15 minutes, and Pd(dppf)Cl2 (0.2 g) was added, heated to 80° C. and reacted overnight (12 hours). Spin-dried and passed through the column (PE/EA=1/1) to obtain compound 4 (1.2 g, yield 61%).

Compound 4 (10 g) was dissolved in concentrated sulfuric acid (10 ml), cooled to 5° C., potassium nitrate (15 g) was added in batches, and the TLC plate was spotted until the reaction of the raw materials was complete. The system was poured into ice water, and the pH was adjusted to 7 with 15% NaOH aqueous solution. Solids slowly precipitated out, filtered by suction, and dried to obtain compound 5 (8 g, yield 92%).

Compound 5 (2g) was dissolved in EtOH/H2O=5/1 (60ml), ammonium chloride (1.8g) and acetic acid (5ml) were added, the system was heated to 60°C, and iron powder (1.9g) was added in batches. Keep at 60°C for 1 hour. The temperature was lowered, extracted with ethyl acetate, spin-dried and passed through the column (PE/EA=1/1) to obtain compound 6 (1.2 g, yield 67%).

Compound 6 (3g) was dissolved in 15% NaOH, heated to 70°C, and spotted on a TLC plate until the reaction of the raw material was complete. The temperature was lowered, a solid was precipitated, filtered, and the solid was dried to obtain compound 7 as a red solid (1.5 g, yield 60%).

Compound 7 (500, mg), 2-chloro-4-thiamphenicol benzoic acid (1 g) was dissolved in polyphosphoric acid, heated to 60° C. for 5 hours, and spotted on a TLC plate until the reaction of the raw materials was complete. The system was poured into ice water, and the pH was adjusted to 7 with 15% NaOH aqueous solution. A solid slowly precipitated out. The solid was filtered with suction and dried to give Compound Ⅰ-1 (510 mg, yield 54%) as a gray solid. ¹ HNMR CDCl ₃ δ: 3.12(s,3H),7.36(m,1H),7.54-7.89(m,5H),8.02(s,1H),8.37-8.47(dd,1H),8.73(s,1H) ), 11.64(s,1H); LC-MS: m/z=417.8(M+1)

Example 2

Synthesis of 5-chloro-2-(6-(4-methylpiperazin-1-yl)pyridin-3-yl)-6-(pyridin-2-yl)benzimidazole (I-2):

Compound 1 (10g) was dissolved in dichloromethane, acetic anhydride (5.93g) was added, and the system was heated to 40°C for 2 hours. The system was cooled down to room temperature, washed twice with NaHCO3 aqueous solution, and the organic phase was spin-dried to obtain compound 2 (11 g, 92%).

Compound 2 (11g), potassium acetate (8.8g) was dissolved in dioxane, and bisboron (20.5g) was added. The system was replaced with nitrogen for 15 minutes, then Pd(dppf)Cl2 (1g) was added, and heated to 80°C to react overnight. Cool down, spin dry and pass through the column (PE/EA=2/1) to obtain compound 3 (8g, 61%).

Compound 3 (2.1 g), cesium carbonate (4 g) was dissolved in dioxane, and 2-bromopyridine (1.27 g) was added. The system was replaced with nitrogen for 15 minutes, then Pd(dppf)Cl2 (0.2g) was added, and heated to 80°C to react overnight. Spin-dried and passed through the column (PE/EA=1/1) to obtain compound 4 (1.2 g, 61%).

Compound 4 (10 g) was dissolved in concentrated sulfuric acid (10 ml), cooled to 5° C., potassium nitrate (15 g) was added in batches, and the TLC plate was spotted until the reaction of the raw materials was complete. The system was poured into ice water, and the pH was adjusted to 7 with 15% NaOH aqueous solution. A solid slowly precipitated out. The solid was filtered and dried to obtain compound 5 (8 g, 92%).

Compound 5 (2g) was dissolved in EtOH/H2O=5/1 (60ml), ammonium chloride (1.8g) and acetic acid (5ml) were added, the system was heated to 60°C, and iron powder (1.9g) was added in batches. Keep at 60°C for 1 hour. The temperature was lowered, extracted with ethyl acetate, spin-dried and passed through the column (PE/EA=1/1) to obtain compound 6 (1.2 g, 67%).

Compound 6 (3g) was dissolved in 15% NaOH, heated to 70°C, and spotted on a TLC plate until the reaction of the raw material was complete. After cooling down, a solid was precipitated, filtered, and the solid was dried to obtain compound 7 (1.5 g, 60%) as a red solid.

Compound 7 (5g) was dissolved in dichloromethane (200ml), added with 6-chloronicotinic acid (3.6g), HOBT (4g), Et3N (5.7g), EDCI (5.2g), and stirred overnight at room temperature. The system was poured into water, extracted with dichloromethane, and concentrated through a column (PE/EA=1/1-EA) to obtain compound 8 (5.2 g, 63%).

Compound 8 (2g) was dissolved in acetic acid, the system was heated to 100°C, and the TLC plate was spotted until the reaction of the raw materials was complete. The system was cooled down, poured into water, adjusted the pH to 7 with 15% NaOH aqueous solution, a solid slowly precipitated out, filtered with suction, and dried the solid to obtain compound 9 (1.5 g, 79%).

Compound 9 (500mg) was dissolved in N-methylpyrrolidone (10ml), N-methylpiperazine (147mg) and DIEA (114mg) were added, and the system was heated to 130°C for overnight reaction. The temperature was lowered, poured into water, extracted with ethyl acetate, spin-dried and passed through the column (MeOH/DCM=1/10) to obtain compound Ⅰ-2 (204 mg, yield 34%) as a red solid. ¹ HNMR CDCl ₃ δ:2.304(s,3H),2.445-2.456(d,4H),3.569-3.579(d,4H),6.491-6.514(d,1H),7.264-7.607(m,4H),7.733 -7.775(m,1H),7.943-7.965(d,1H),8.635-8.646(d,1H),8.75-8.756(d,1H); LC-MS: m/z=405.9(M+1)

Example 3

Synthesis of 5-chloro-2-(6-(2,6-dimethylmorpholinyl)pyridin-3-yl)-6-(pyridin-2-yl)benzimidazole (I-3):

Compound 9 was dissolved in N-methylpyrrolidone (10ml), (2S,6R)-2,6-dimethylmorpholine (147mg) and DIEA (455mg) were added, and the system was heated to 130°C for overnight reaction. The temperature was lowered, poured into water, extracted with ethyl acetate, spin-dried and passed through the column (MeOH/DCM=1/10) to obtain compound Ⅰ-3 (32 mg, yield 13%) as a yellow solid. ¹ HNMR CD ₃ ODδ:1.269-1.304(t,6H),2.582-2.642(q,2H),3.702-3.749(m,2H),4.301-4.332(d,2H),6.976-6.999(d,1H) ,7.471-7.502(q,1H),7.678-7.721(t,3H),7.949-7.992(m,1H),8.176-8.232(m,1H),8.654-8.666(d,1H),8.852-8.858( d,1H); LC-MS: m/z=420(M+1)

Example 4

Synthesis of 5-chloro-2-(6-(3,5-dimethylpiperazin-1-yl)pyridin-3-yl)-6-(pyridin-2-yl)benzimidazole (I-4) :

Compound 9 (200 mg) was dissolved in N-methylpyrrolidone (10 ml), 2,6-dimethylpiperazine (134 mg) and DIEA (455 mg) were added, and the system was heated to 130° C. to react overnight. The temperature was lowered, poured into water, extracted with ethyl acetate, spin-dried and passed through the column (MeOH/DCM=1/10) to obtain compound Ⅰ-4 (18 mg, yield 7%) as a yellow solid. ¹ HNMR CD ₃ ODδ:1.427-1.443(d,6H),2.898-2.962(q,2H),3.368-3.410(t,2H),4.679-4.711(d,2H),7.086-7.109(d,1H) ,7.474-7.505(q,1H),7.686-7.722(t,3H),8.246-8.274(q,1H),8.422(br.s,1H),8.660-8.672(d,1H),8.887-8.892( d,1H); LC-MS: m/z=420(M+1)

Example 5

Synthesis of 5-chloro-2-(6-(4-hydroxypiperidinyl)pyridin-3-yl)-6-(pyridin-2-yl)benzimidazole (I-5):

Compound 9 (200 mg) was dissolved in N-methylpyrrolidone (10 ml), 4-hydroxypiperidine (120 mg) and DIEA (455 mg) were added, and the system was heated to 130° C. to react overnight. The temperature was lowered, poured into water, extracted with ethyl acetate, spin-dried and passed through the column (MeOH/DCM=1/10) to obtain compound Ⅰ-5 (23 mg, yield 10%) as a yellow solid. ¹ HNMR CD ₃ ODδ:1.124-1.588(m,2H),1.943-2(m,2H),3.248-3.337(m,2H),3.882-3.926(m,1H),4.191-4.245(m,2H) ,6.949-6.972(d,1H),7.457-7.488(m,1H),7.650-7.707(q,3H),7.931-7.974(m,1H),8.136-8.164(m,1H),8.648-8.659( d,1H), 8.804-8.810(d,1H); LC-MS: m/z=406.9(M+1)

Example 6

Synthesis of 5-chloro-2-(6-(4-morpholinopiperidinyl)pyridin-3-yl)-6-(pyridin-2-yl)benzimidazole (I-6)

Compound 9 (200 mg) was dissolved in N-methylpyrrolidone (10 ml), 4-piperidinylmorpholine (200 mg) and DIEA (455 mg) were added, and the system was heated to 130° C. to react overnight. The temperature was lowered, poured into water, extracted with ethyl acetate, spin-dried and passed through the column (MeOH/DCM=1/10) to obtain compound Ⅰ-6 (41 mg, purity 95%, yield 15%) as a yellow solid. ¹ HNMR CDCl ₃ δ:1.401-1.50(m,2H),1.855-1.884(d,2H),2.372-2.4(m,1H),2.519-2.54(t,4H),2.805-2.865(t,2H) ,3.691-3.713(t,4H),4.293-4.326(d,2H),6.516-6.539(d,1H),7.269-7.305(m,1H),7.434-7.486(d,2H),7.606-7.626( d,1H),7.741-7.784(m,1H),7.933-7.962(q,1H),8.654-8.665(d,1H),8.728-8.733(d,1H); LC-MS: m/z=476.1 (M+1)

Example 7

Synthesis of 5-chloro-2-(6-(ethoxyethylamino)pyridin-3-yl)-6-(pyridin-2-yl)benzimidazole (I-7):

Compound 9 (100 mg) was dissolved in N-methylpyrrolidone (10 ml), 4-hydroxypiperidine (120 mg) and DIEA (400 g) were added, and the system was heated to 130° C. to react overnight. The temperature was lowered, poured into water, extracted with ethyl acetate, spin-dried and passed through the column (MeOH/DCM=1/10) to obtain compound I-7 (16 mg, yield 13.9%) as a yellow oil. ¹ HNMR CD ₃ ODδ:1.181-1.241(m,3H),3.528-3.668(m,6H),6.688-6.708(d,1H),7.452-7.482(m,1H),7.643-7.701(m,3H) ,7.930-7.967(q,1H),8.061-8.080(m,1H),8.645-8.718(m,2H); LC-MS: m/z=394.9(M+1)

Example 8

Synthesis of 5-chloro-2-(6-(3-N,N-dimethylaminopropylamino)pyridin-3-yl)-6-(pyridin-2-yl)benzimidazole (I-8) :

Compound 9 (100 mg) was dissolved in N-methylpyrrolidone (10 ml), 4-hydroxypiperidine (120 mg) and DIEA (400 g) were added, and the system was heated to 130° C. to react overnight. The temperature was lowered, poured into water, extracted with ethyl acetate, spin-dried and passed through the column (MeOH/DCM=1/10) to obtain compound Ⅰ-8 (25 mg, yield 21%) as a yellow solid. ¹ HNMR CD ₃ ODδ:1.490-1.508(d,2H),2.938(s,6H),3.189-3.239(m,2H),3.533-3.564(m,2H),6.709-6.731(d,1H),6.466 -7.501(m,1H),7.673-7.720(m,3H),7.945-7.988(m,1H),8.089-8.116(m,1H),8.3(s,2H),8.655-8.666(d,1H) ,8.787-8.792(d,1H); LC-MS: m/z=407.9(M+1)

Example 9

hydrochloride

Take compound Ⅰ-2 (50 mg), dissolve it in methanol (1-2 ml), and then add 2 ml of methanolic hydrochloric acid. Then the solvent was evaporated to obtain 52 mg of solid. LC-MS:m/z=405.9(M+1)

Example 10

The compounds of the present invention having the structure of formula (I) and their pharmaceutically acceptable salts have obvious anti-tumor 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-2, I-3, I-4, I-5, I-6; I-7, I-8 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) The human pancreatic cancer cell line PANC-1 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: SV3008702

2. Test method

2.1 Drug treatment dosage and preparation method

Blank vehicle composition: DMSO

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

2.2 Cell Culture

Human pancreatic cancer cell line PANC-1 was cultured in complete DMEM medium (containing 10% fetal bovine serum, 100U/ml penicillin, 100μg/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 PANC-1 cells in the logarithmic growth phase with 0.25% trypsin to make a cell suspension, add 5X10 ³ /well to a 96-well cell culture plate (150μL/well), and place three replicate wells at 37 After culturing in a 5% CO2 incubator at ℃ and adhered to the wall the next day, add 50ul series concentrations of the test compound or control medium to each well according to the experimental design, and set up a cell control group (only containing cells and medium without drugs) wells), 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 PANC-1. Both PANC-1 and K562 were cultured for 3 days after administration. After the culture was completed, the MTS method was used to detect the cell proliferation and calculate the cell viability of the cells 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 PANC-1 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: Inhibitory effect of compounds of the present invention on cancer cell growth (IC50: Mean ± SD)

IC50(μM) K562 PANC-1 Ⅰ-1 6.003±0.646 28.214±9.491 Ⅰ-2 5.858±0.795 17.348±3.320 Ⅰ-3 3.325±1.252 12.863±4.644 Ⅰ-4 6.205±0.999 9.072±0.394 Ⅰ-5 3.779±1.027 8.667±1.892 Ⅰ-6 2.598±0.447 8.915±2.495 I-7 12.48±0.603 I-8 19.245±0.808

RT-PCR Gli1 mRNA detection

1. Test material

1.1 Compounds and solvents

Test sample

Formula (I) series compounds (I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8); Positive control substance: GDC-0449

DMSO was used as the vehicle in this experiment.

1.2 Cell lines

This experiment uses a type of cell line, human chronic myeloid leukemia cell line K562.

1.3 Reagents and instruments

Reagent: SuperfecTRI RNA extraction reagent, purchased from Shanghai Pufei Biotechnology Co., Ltd., item number: 3101-100. RevertAid First Strand cDNA Synthesis Kit was purchased from FERMENTAS company, item number: K1622. FastStartUniversal SYBR Green Master (Rox) reagent was purchased from Roche, catalog number: 04913914001.

Instruments: Bio-Rad MJ Mini 48-Well Personal Thermal Cycler PCR instrument, model: PTC-1148; Bio-Rad company iQ5 real-time fluorescent quantitative PCR instrument, model: iQ5.

2. Test method

Add K562 cells into a 12-well plate, ^2X105 cells/well. According to the experimental design, add the test compound and the positive control substance (30, 10, 3.3μM) with a preset concentration to each well, two pairs of wells. After 24 hours, the SuperfecTRI RNA extraction reagent was used to extract total RNA according to the standard protocol, and the mRNA was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit. Then, qPCR experiments were performed on the iQ5 Real-Time PCR Detection System using FastStart Universal SYBR Green Master (Rox) reagents according to standard protocols. The primer sequences are as follows: Gli1 upstream primer: AGCGTGAGCCTGAATCTGTG, downstream primer: CAGCATGTACTGGGCTTTGAA; GADPH upstream primer: TTCACCACCATGGAGAAGGC, downstream primer: GGCATGGACTGTGGTCATGA. All values were analyzed relative quantitatively by 2ΔCt1 ^-ΔCt2 method.

3. Test results

Effects of Formula (I) Series Compounds on Gli1 mRNA Expression Level

After 24 hours of dosing, RNA was extracted, and the effects of HB series compounds on the expression level of Gli1 mRNA were detected by RT-PCR. The results showed that I-1 and I-2 had the same inhibitory effect as GDC-0449 under the conditions of 30, 10, and 3.3 μM treatment for 24 hours. The results are shown in Table 2.

Table 2: the compound of the present invention is on the influence of Gli1 mRNA expression level

Claims (10)

1. A benzimidazole compound represented by formula (I) or a pharmaceutically acceptable salt thereof, Wherein, R ¹ , R ² , R ⁴ are independently H, halogen, C _1-6 straight chain, branched chain alkyl; R ³ is independently H, C _1-6 straight chain, branched chain alkyl; Y= C or N; ^R5 is: the heteroatom is nitrogen or oxygen, the number of heteroatoms is one or two, unsubstituted or substituted with alkyl, hydroxyl or amino group, 4-8 membered heterocyclic ring, Wherein, R ⁶ is independently a C _1-4 straight-chain alkyl group or a branched-chain alkyl group; R ⁷ and R ⁸ are independently H, a C _1-6 straight-chain alkyl group. 2. The compound shown in formula (I) according to claim 1 or its pharmaceutically acceptable salt, it is characterized in that ^R is nitrogen or oxygen, the number of heteroatoms is one or two, unsubstituted Or a 6-membered monoheterocycle substituted with an alkyl group or a hydroxyl group. 3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein R ^is 4. Selected from the following compounds or pharmaceutically acceptable salts thereof: Wherein, the definitions of R ¹ , R ² , R ³ , R ⁴ and Y are the same as those in claim 1. 5. Selected from the following compounds or pharmaceutically acceptable salts thereof: 6. The preparation method of the compound or pharmaceutically acceptable salt thereof according to one of claims 1, 2, 3, 4 or 5, characterized in that, the compound shown in formula (I) is prepared from compound 7, the compound 7 The structure is: The reaction formula is: 7. The use of the compound according to any one of claims 1, 2, 3, 4 or 5 or a pharmaceutically acceptable salt thereof in the preparation of antitumor drugs. 8. The application according to claim 7, characterized in that the tumor is a tumor associated with the continuous activation of the Hh signaling pathway. 9. The application according to claim 8, characterized in that the tumor associated with the continuous activation of the Hh signaling pathway is skin basal cell carcinoma, medulloblastoma, lung cancer, digestive tract tumors, breast cancer or pancreatic cancer 10. The pharmaceutical composition, characterized in that it contains the compound or pharmaceutically acceptable salt thereof according to one of claims 1, 2, 3, 4 or 5 as an active ingredient, and one or more pharmaceutically acceptable accessories.