Use of dabigatran chloride derivatives for the preparation of medicaments for the inhibition and treatment of colon cancer

Abstract

The application discloses application of a dabigatran chlorine-containing derivative in preparation of a medicine for inhibiting and treating colon cancer, and a structure of the compound is: the compound is an artificial synthetic compound, and experiments by MTT colorimetry prove that the compound with an amount greater than 12.5 mu g / mL can effectively inhibit HCT116 colon cancer cell activity, and the greater the amount of the compound, the more significant the inhibiting effect on HCT116 cells is, and the compound can be used for research on human colon cancer as a medicine for inhibiting proliferation of human colon cancer cells or an antitumor medicine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the application of dabigatran chloride derivatives in the preparation of drugs for inhibiting and treating colon cancer. BACKGROUND

[0002] Colon cancer is a cancer originating from the colon or rectum, and is one of the common malignant tumors in the digestive tract. It is the third most common cancer in men and the second most common cancer in women in the world, and has the pathological characteristics of rapid onset, strong metastasis ability and high recurrence rate. With the development of social economy, the improvement of people's living standards, the change of eating habits and structure, and the problem of population aging, colon cancer has gradually become a major disease threatening human health.

[0003] In 2018, colon cancer ranked third and second in the global incidence and mortality rates of new cancers, respectively. In addition, in the past 10 years, the incidence of colon cancer in China has shown a significant upward trend, and it is predicted that by 2035, the incidence and mortality of colon cancer in China will increase significantly, seriously threatening the health and life of patients.

[0004] For the treatment of colon cancer, the current main method is to combine surgical resection and chemotherapy. Commonly used chemotherapy drugs include 5-fluorouracil (5-FU), irinotecan, oxaliplatin, etc. When used alone, the drug efficacy is extremely limited, and in most cases, two or more of these drugs are used in combination to improve efficacy, but have strong side effects. Therefore, it is urgent to discover new drugs with high activity against colon cancer cells to improve the efficacy of chemotherapy and enhance the overall quality of life of patients, and to alleviate the pain of more cancer patients.

[0005] MTT method, also known as MTT colorimetric method, is a method for detecting cell survival and growth. Its detection principle is that succinate dehydrogenase in the mitochondria of living cells can reduce the exogenous MTT in the culture medium to water-insoluble blue-purple crystals-melanin and deposit in the cells, while dead cells do not produce succinate dehydrogenase and have no such function. By establishing the relationship between melanin production and the number of living cells, a method for measuring the number of living cells is obtained.

[0006] In the specific measurement process, dimethyl sulfoxide (DMSO) is used to dissolve the melanin in the cells, and an enzyme-linked immunodetection instrument is used to measure the light absorption value at 492 nm wavelength, to establish the relationship between the amount of melanin production and the number of living cells, and thus obtain the cell survival rate. Studies have shown that within a certain range of cell concentration, the light absorption value is proportional to the number of living cells.

[0007] MTT method has the advantages of small workload, simple operation, rapidness, good repeatability, etc., and the method has been widely applied to activity detection of eukaryotic cells (such as tumor cells, fungal cells), viable bacterial counting of prokaryotes (such as bacteria), detection of biological active factors, tumor cell drug sensitivity detection, large-scale screening of anti-tumor drugs, fungal drug sensitivity experiment, cytotoxicity experiment, etc. SUMMARY

[0008] The application aims to provide application of the dabigatran chloride derivative in preparation of a drug for inhibiting and treating colon cancer, so as to solve the problems of few types of chemotherapeutic drugs for colon cancer, strong cytotoxicity, poor drug efficacy of single drug, and large toxic and side effects of mixed drugs, and provide a new chemotherapeutic drug for colon cancer treatment.

[0009] To achieve the above-mentioned object, the application provides application of the dabigatran chloride derivative in preparation of a drug for inhibiting and treating colon cancer.

[0010]

[0011] The synthesis steps of the compound are as follows:

[0012] S1, 3-chloroaniline (compound 1) and ethyl acrylate are used as reactants, 10% mol trifluoromethanesulfonic acid is used as a catalyst, the mixture is heated to 100 DEG C under the protection of nitrogen atmosphere, and refluxed for 16 h to prepare 3-((3-chlorophenyl)amino)propionic acid ethyl ester (compound 2);

[0013] S2, 4-chloro-3-nitrobenzoic acid (compound 3) is reacted with a corresponding amine solution in the presence of acetic acid under the condition that the pH is 4-5 for 5 h to generate 4-methylamino-3-nitrobenzoic acid (compound 4);

[0014] S3, compound (4) is reacted with dichloromethane and thionyl chloride at room temperature for 2 h to generate 3-nitro-4-methylamino-benzoyl chloride (compound 5);

[0015] S4, compound 5 and 2 are reacted at room temperature in the presence of dichloromethane and triethylamine for 3 h to generate a ring-opening reaction to obtain 3-(N-(3-chlorophenyl)-4-(methylamino)-3-nitrobenzamide) propionic acid ethyl ester (compound 6);

[0016] S5, at room temperature, zinc / acetic acid is used as a reducing agent to occur a reduction reaction in a tetrahydrofuran aqueous solution to reduce nitro compound 6 to obtain 3-(3-amino-N-(3-chlorophenyl)-4-(methylamino) benzamide) propionic acid ethyl ester (compound 7);

[0017] S6, mixing bromoacetic acid and p-amino benzonitrile (compound 8), and stirring at 100℃ for 10h to obtain N-(4-cyanophenyl) glycine (compound 9);

[0018] S7, reacting compound 7 with compound 9 in the presence of dimethylformamide / tetrahydrofuran, acetic acid and ammonium hydroxide, and using acetic acid carbodiimide hydrochloride and 1-hydroxybenzotriazole as catalysts to obtain ethyl 3-(N-(3-chlorophenyl)-2-(((4-cyanophenyl)amino)methyl)-1-methyl-1H-benzo[d]imidazole-5-carboxamido)propanoate (compound 10);

[0019] S8, reacting hydroxylamine hydrochloride-15N with compound 10 in the presence of triethylamine, anhydrous ethanol, palladium carbon, ammonium formate, acetic acid and nitrogen to obtain ethyl 3-(2-(((4-carbamoylphenyl)amino)methyl)-N-(3-chlorophenyl)-1-methyl-1H-benzo[d]imidazole-5-carboxamido)propanoate intermediate (compound 11);

[0020] S9, hydrolyzing compound 11 with sodium hydroxide in an ethanol aqueous solution at room temperature, and hydrolyzing compound 11 into the target compound (compound 12), i.e. the dabigatran chloride derivative in the present application, after 2h.

[0021] The reaction formula of the compound is as follows:

[0022]

[0023] Therefore, the dabigatran chloride derivative provided by the present application is used for the preparation of a drug for inhibiting and treating colon cancer. The compound with a concentration greater than 12.5μg / mL has a strong inhibitory effect on the activity of HCT116 colon cancer cells in vitro, and the greater the amount of the compound added, the more significant the inhibitory effect on HCT116 cells. The compound can be used for the research of human colon cancer as a drug for inhibiting the proliferation of human colon cancer cells or an antitumor drug.

[0024] The technical solutions of the present application are further described in detail below by means of the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is the result of inhibiting HCT116 cells in vitro by different concentrations of compounds in the present application. DETAILED DESCRIPTION

[0026] The technical solutions of the present application are further described in detail below by means of the accompanying drawings and examples.

[0027] In order to make the purposes, technical solutions and advantages of the present application more clear, thorough and complete, the technical solutions in the embodiments of the present application are described clearly and completely below by means of the drawings and examples. The following detailed description is a description of the embodiments, which aims to provide further detailed description of the present application. Unless otherwise specified, all technical terms employed by the present application have the same meanings as those generally understood by the general technical personnel in the field to which the present application belongs.

[0028] The complete culture medium used in the embodiments is RPMI-1640 culture medium (purchased from Hyclone Company, with a product number of R8758) containing 10% fetal bovine serum (purchased from Biyun Tian Company, with a product number of C0234), 1% double antibody (purchased from Biyun Tian Company, with a product number of C0222) and no Hepes.

[0029] The HCT116 colon cancer cells are purchased from Shanghai Zetyng Biotech Co., Ltd., with a batch number of CC-Y1194.

[0030] The digestive juice is 0.25% trypsin-0.53 mM EDTA, purchased from Beijing Solarbio Science and Technology Co., Ltd., with a brand of Solarbio and a product number of T1300.

[0031] Embodiment one

[0032] The synthesis of the dabigatran chlorine-containing derivative is as follows:

[0033] S1, 3-chloroaniline (compound 1) and ethyl acrylate are used as reactants, 10% mol trifluoromethanesulfonic acid is used as a catalyst, the mixture is heated to 100°C under the protection of nitrogen atmosphere, and refluxed for 16 h to prepare ethyl 3-((3-chlorophenyl)amino)propanoate (compound 2);

[0034] S2, 4-chloro-3-nitrobenzoic acid (compound 3) is reacted with a corresponding amine solution in the presence of acetic acid at a pH of 4-5 for 5 h to generate 4-methylamino-3-nitrobenzoic acid (compound 4);

[0035] S3, compound (4) is reacted with dichloromethane and thionyl chloride at room temperature for 2 h to generate 3-nitro-4-methylamino-benzoyl chloride (compound 5);

[0036] S4, compound 5 and 2 are reacted in the presence of dichloromethane and triethylamine at room temperature for 3 h to generate a ring-opening reaction to obtain ethyl 3-(N-(3-chlorophenyl)-4-(methylamino)-3-nitrobenzamide)propanoate (compound 6);

[0037] S5, at room temperature, zinc / acetic acid is used as a reducing agent to reduce the nitro compound 6 in a tetrahydrofuran aqueous solution to obtain ethyl 3-(3-amino-N-(3-chlorophenyl)-4-(methylamino)benzamido)propanoate (compound 7);

[0038] S6, bromoacetic acid and p-aminophenyl nitrile (compound 8) are mixed and stirred at 100℃ for 10h to obtain N-(4-cyanophenyl)glycine (compound 9);

[0039] S7, compound 7 is reacted with compound 9 in the presence of dimethylformamide / tetrahydrofuran, acetic acid and ammonium hydroxide, with ethyl carbodiimide acetate and 1-hydroxybenzotriazole as catalysts to obtain ethyl 3-(N-(3-chlorophenyl)-2-(((4-cyanophenyl)amino)methyl)-1-methyl-1H-benzo[d]imidazole-5-carboxamido)propanoate (compound 10);

[0040] S8, hydroxylamine-15N and compound 10 are reacted in the presence of triethylamine, anhydrous ethanol, palladium carbon, ammonium formate, acetic acid and nitrogen to obtain ethyl 3-(2-(((4-carbamoylphenyl)amino)methyl)-N-(3-chlorophenyl)-1-methyl-1H-benzo[d]imidazole-5-carboxamido)propanoate intermediate (compound 11);

[0041] S9, at room temperature, sodium hydroxide and compound 11 are hydrolyzed in an ethanol aqueous solution, and compound 11 is hydrolyzed into the target compound (compound 12), i.e., the dabigatran chloride derivative in the present application, after 2h.

[0042] The reaction formula is as follows:

[0043]

[0044] Example Two

[0045] Cultivation of HCT116 colon cancer cells (adherent cells)

[0046] After the purchased HCT116 colon cancer cells are taken back to the laboratory, the outer package is opened, the entire bottle is disinfected with 75% alcohol and then placed in a clean bench, and strict aseptic operation is performed.

[0047] The bottle cap is not opened, and the cells are placed in a 37℃, 5% CO2 incubator for 2-3h to stabilize the cell state. Under an inverted microscope, it is found that the cell confluence does not exceed 80%, the complete culture solution in the bottle is collected into a centrifuge tube, 5ml of complete culture medium is added again, and it is placed in a 37℃, 5% CO2 incubator for cultivation;

[0048] After 8h culture, the cells were found to have grown to fullness (confluence reached 80-90%), and the cells were subcultured according to the following steps:

[0049] A. Discard the culture supernatant, and rinse the cells twice with PBS not containing calcium ions and magnesium ions;

[0050] B. Add 2ml of the digestion solution to the culture bottle, and place it in a 37°C incubator for 2min of digestion. Then observe the cell digestion under a microscope. If most of the cells are rounded and detached, quickly take the culture bottle back to the operation table, tap the culture bottle a few times, and then add 7ml of complete culture medium containing 10% serum to terminate the digestion.

[0051] C. Transfer the suspension to a 15ml centrifuge tube, centrifuge at 750rpm for 5min, discard the supernatant, and resuspend the sediment at the bottom of the centrifuge tube with fresh complete culture medium. Subculture at 1:2, and continue to culture in a 37°C, 5% CO2 cell incubator.

[0052] Example Three

[0053] Culture of HCT116 colon cancer cells (adherent cells)

[0054] After taking the purchased HCT116 colon cancer cells back to the laboratory, first open the outer packaging, spray the entire bottle with 75% alcohol for sterilization, and then place it in a clean bench for strict aseptic operation.

[0055] Without opening the bottle cap, place it in a 37°C, 5% CO2 incubator for 2-3h to stabilize the cell state. Under an inverted microscope, the cells were found to have grown to fullness (confluence reached 80-90%), and the cells were subcultured according to the following steps:

[0056] A. Discard the culture supernatant, and rinse the cells twice with PBS not containing calcium ions and magnesium ions;

[0057] B. Add 2ml of the digestion solution to the culture bottle, and place it in a 37°C incubator for 2min of digestion. Then observe the cell digestion under a microscope. If most of the cells are rounded and detached, quickly take the culture bottle back to the operation table, tap the culture bottle a few times, and then add 7ml of complete culture medium containing 10% serum to terminate the digestion.

[0058] C. Transfer the suspension to a 15ml centrifuge tube, centrifuge at 750rpm for 5min, discard the supernatant, and resuspend the sediment at the bottom of the centrifuge tube with fresh complete culture medium. Subculture at 1:2, and continue to culture in a 37°C, 5% CO2 cell incubator.

[0059] Example Four

[0060] Culture of HCT116 colon cancer cells (adherent cells)

[0061] After taking the purchased HCT116 colon cancer cells back to your own laboratory, first open the outer packaging, spray the entire bottle with 75% alcohol to disinfect, and then place it in a clean bench. Strict aseptic operation.

[0062] Without opening the bottle cap, place it in a 37°C, 5% CO2 incubator for 2-3 hours to stabilize the cell state. Under an inverted microscope, the results show that the cell confluence does not exceed 80%. Collect the complete culture medium from the bottle into a centrifuge tube, add 5ml of complete culture medium, and place it in a 37°C, 5% CO2 incubator.

[0063] After 8 hours of culture, it is found that the cells have grown (confluence reaches 80-90%). The cells are subcultured, and the specific operation steps are as follows:

[0064] A. Discard the culture supernatant and rinse the cells once with PBS without calcium and magnesium ions.

[0065] B. Add 1ml of digestion solution to the culture bottle and place it in a 37°C incubator for 1 minute. Then observe the cell digestion under a microscope. If most of the cells are rounded and detached, quickly return to the operation platform, gently tap the culture bottle, and then add 8ml of complete culture medium containing 10% serum to terminate digestion.

[0066] C. Transfer the suspension to a 15ml centrifuge tube, centrifuge at 750rpm for 5 minutes, discard the supernatant. Add fresh complete culture medium to resuspend the sediment at the bottom of the centrifuge tube, subculture at a ratio of 1:3, and continue to culture in the cell culture incubator.

[0067] Example Five

[0068] The MTT colorimetric method is used to determine the viability of HCT116 colon cancer cells, as follows:

[0069] (1) Under an inverted microscope, observe the cells when they have grown (confluence reaches 80-90%), and subculture the cells according to the method of Example Two. Resuspend with fresh complete culture medium to prepare a cell suspension.

[0070] (2) Count the cells, take 10μL of cell suspension, slowly add the cell suspension along one side of the cover glass, and count the cells. Then dilute the HCT116 cells in the logarithmic growth phase to 2x10 5 cell / mL according to the counting results, inoculate in a 96-well culture plate, 0.1mL per well, and culture for 24 hours.

[0071] (3) The dabigatran chlorine-containing derivative prepared in Example One was dissolved in normal saline and diluted to 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL and 100 μg / mL, respectively, and then added to RPMI-1640 medium containing 10% fetal bovine serum, 1% double antibody and no Hepes to prepare a culture medium containing different concentrations of the dabigatran chlorine-containing derivative;

[0072] (4) The supernatant in the culture cells was removed, 200 μL of the culture medium containing different concentrations of the dabigatran chlorine-containing derivative prepared in step (3) was added to each well, 6 parallel wells were set in each group, and the plate was incubated in a 37°C, 5% CO2 and saturated humidity incubator for 24 h;

[0073] (5) An appropriate amount of PBS was added around the hole plate to prevent the cell liquid in the 96-well plate from being dried; the plate was incubated in a 37°C, 5% CO2 and saturated humidity incubator for 24 h to allow the cells to adhere to the wall;

[0074] (6) The supernatant was removed, 20 μL of MTT with a concentration of 5 mg / mL was added to each well, and after 4 h of culture, 100 μL of DMSO was added to dissolve the formazan, and the mixture was shaken for 5 min. The MTT reduction product was completely dissolved, and the optical density (OD) value was detected on an enzyme-labeled instrument (dual-wavelength, 492 nm and 630 nm, rapid shaking for 60 s), and the cell activity, inhibition rate and P value were calculated.

[0075] (7) Data processing, two extreme values were removed and calculated according to the following formula:

[0076] Cell activity = (experimental well-blank well) / (control well-blank well) x 100%,

[0077] Cell growth inhibition rate = (control well-experimental well) / (control well-blank well) x 100%.

[0078] Comparative Example One

[0079] The blank group was treated in the same way as the HCT116 colon cancer cells in Example Five, except that no dabigatran chlorine-containing derivative and HCT116 colon cancer cells were added, and an equal amount of the culture medium in Example Five was added to make up.

[0080] Comparative Example Two

[0081] The negative control group was treated in the same way as the HCT116 colon cancer cells in Example Five, except that no dabigatran chlorine-containing derivative was added, i.e. the concentration of the dabigatran chlorine-containing derivative was 0 μg / mL, and an equal amount of the culture medium in Example Five was added to make up.

[0082] Result Analysis

[0083] The cell activity, inhibition rate and P value data obtained in Example 3 and Comparative Examples 1 and 2 are shown in Table 1, and a line graph is shown in Figure 1 .

[0084] Table 1

[0085]

[0086] As can be seen from Table 1 and Figure 1 the 12.5 μg / mL dabigatran chlorine-containing derivative has no obvious inhibitory effect on the activity of HCT116 colon cancer cells, the 6.25 μg / mL, 50 μg / mL and 100 μg / mL compounds all have inhibitory effects on the activity of HCT116 colon cancer cells, and when the concentration of the compound is greater than 12.5 μg / mL, the higher the concentration, the more significant the inhibitory effect on HCT116 cells, and the inhibitory rate of the 100 μg / mL compound on HCT116 cells reaches 50.4%.

[0087] Therefore, the dabigatran chlorine-containing derivative provided by the present application can be used in the preparation of a drug for inhibiting and treating colon cancer, and the compound greater than 12.5 μg / mL can effectively inhibit the activity of HCT116 colon cancer cells, and the greater the amount of the compound added, the more significant the inhibitory effect on HCT116 cells, and the compound can be used in the research of human colon cancer as a drug for inhibiting the proliferation of human colon cancer cells or an antitumor drug.

[0088] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application but not to limit it, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: it can still modify or equivalently replace the technical solutions of the present application, and these modifications or equivalent replacements also cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. Use of a chloro derivative of dabigatran for the preparation of a medicament for the inhibition and treatment of colon cancer, characterized in that, The structural formula of a chloro-containing derivative of dabigatran is:

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