CN114751841B - N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide as well as preparation method and application thereof - Google Patents
N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of compound synthesis, in particular to N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide, a preparation method and application thereof, wherein m-phenylenediamine, triethylamine and m-chlorobenzoyl chloride are used as main raw materials for reaction to obtain an intermediate product; dissolving 3-nitro-4-methoxybenzoic acid in dichloromethane, sequentially adding N, N-diisopropylethylamine and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, dissolving an intermediate product in dichloromethane under stirring, adding the intermediate product, stirring at room temperature for reaction, washing the obtained reactant with dilute hydrochloric acid and clear water, drying the washed organic phase with anhydrous sodium sulfate, spin-drying, and passing the organic phase through a column with silica gel to obtain N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide. The N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide can obviously inhibit the activity and proliferation growth of HCT116 and HT29 cells, and can be applied to the preparation of antitumor drugs.
Description
Technical Field
The invention relates to the technical field of compound synthesis, in particular to N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide and a preparation method and application thereof.
Background
Colorectal cancer (Colorectal cancer) is one of common tumors in China, including rectal cancer and colon cancer, and is one of tumors with the highest incidence rate in the last two decades, and at present, colorectal cancer already occupies the 4 th, 5 th or 6 th position of the cause of malignant tumor death, and the incidence rate in China is about twenty-four parts per million, and men are slightly higher. With the development of economy, the improvement of living standard and the change of living style, the incidence rate of the Chinese herbal medicine is also in a continuous rising trend.
Many epidemiological studies of colorectal cancer have shown that there are many factors that cause colorectal cancer, such as dietary factors, occupational factors, physical activities, genetic factors, disease factors, and other carcinogenic factors. The colon cancer occurrence and development process can be divided into five stages: 0. cancer cells appear in the mucosa of the colon or the inner wall of the colon; cancer cells reach submucosa; colon cancer penetrates the colon wall; III, cancer cells begin to spread to lymph nodes; IV. tumor nodules form in tissues surrounding the colon and the cancer begins to metastasize to distant tissues such as liver, lung, etc.
The early symptoms of colon cancer are mostly unobvious, and most patients find that lymphatic metastasis, blood circulation metastasis, intestinal cavity metastasis, abdominal cavity metastasis and the like can occur in the middle and late stage of colon cancer, so that the treatment is difficult. The colon cancer is hidden, and the proportion of early diagnosis of intestinal cancer in China is only 5-10%. Early colon cancer can be cured through surgery, and for patients with advanced colon cancer, methods such as chemical drug treatment, radiotherapy and the like are generally needed to control tumors, so that the survival time of the patients is prolonged. The chemical drug treatment mainly adopts general chemotherapeutics such as fluorouracil (5-fluorouracil and capecitabine), mitomycin, doxorubicin and the like, and lacks of high-efficiency specific clinical treatment drugs for colon cancer.
Disclosure of Invention
In view of the above, the present invention aims to provide N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide, and a preparation method and application thereof, wherein the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide can obviously inhibit the activity and growth proliferation of HCT116 and HT29 cells, and can be applied to the preparation of antitumor drugs.
The invention solves the technical problems by the following technical means:
in one aspect, the invention provides a compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide, wherein the N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide has the following structural formula:
in another aspect of the present invention, there is provided a process for preparing the above compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide, which comprises the steps of:
preferably, the preparation method comprises the following steps:
dissolving m-phenylenediamine in dichloromethane, and adding triethylamine under ice bath to obtain a mixed solution I; dissolving m-chlorobenzoyl chloride in dichloromethane under stirring, slowly dropwise adding the obtained m-chlorobenzoyl chloride solution into the mixed solution I, removing the ice bath after dropwise adding, continuously stirring at room temperature for reaction for 2 hours, washing the obtained reaction solution with saturated saline water and clear water in sequence after the reaction is finished, drying an organic phase with anhydrous sodium sulfate, and spin-drying to obtain an intermediate product;
dissolving 3-nitro-4-methoxybenzoic acid in dichloromethane, sequentially adding N, N-diisopropylethylamine and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to obtain a mixed solution II, dissolving an intermediate product in dichloromethane under stirring, adding the mixed solution II into the mixed solution II, stirring at room temperature for reaction, washing the obtained reactant with dilute hydrochloric acid and clear water successively after the reaction is finished, drying the washed organic phase with anhydrous sodium sulfate, spin-drying, and passing the organic phase through a column with silica gel to obtain N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide.
Preferably, the molar ratio of the m-phenylenediamine to the triethylamine to the m-chlorobenzoyl chloride is 1:2:1.
Preferably, the molar ratio of the 3-nitro-4-methoxybenzoic acid, the N, N-diisopropylethylamine and the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 1:2:2.
In another aspect, the invention provides application of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide in preparing antitumor drugs.
Preferably, the tumor is colon cancer.
Preferably, the N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide is added with pharmaceutically acceptable auxiliary materials and carriers to prepare an anti-tumor preparation.
Preferably, the preparation is any one of granules, tablets, pills, capsules, injections or dispersing agents.
The compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide has simple preparation method and easily obtained preparation raw materials. The compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide has obvious inhibition effect on the activity and growth proliferation of cells HCT116 and HT29, and in vivo experiments show that the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide has obvious inhibition effect on the proliferation of HCT116 cells in vivo, has no great toxicity, and is safe and reliable. Therefore, the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide can inhibit proliferation and growth of colorectal cancer cells, and can be used for preparing antitumor drugs.
Drawings
FIG. 1 is a structural formula of N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide according to the invention;
FIG. 2 is a nuclear magnetic resonance spectrum of N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide according to the invention;
FIG. 3 is a nuclear magnetic resonance spectrum of N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide according to the invention;
FIG. 4 is a graph showing the activity of HCT116 cells, HT29 cells, FHC cells treated with different concentrations of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide at different times, wherein FIG. 4A shows the activity of HCT116 cells, FIG. 4B shows the activity of HT29 cells, and FIG. 4C shows the activity of FHC cells from normal colorectal;
FIG. 5 is a graph showing the results of EdU staining experiments of cells treated with different concentrations of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide, HCT116 and HT 29;
FIG. 6 is a graph showing colony formation experiments of HCT116 and HT29 cells treated with different concentrations of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide;
FIG. 7 is a graph showing the effect of tumor size and weight on nude mice bearing HCT116 xenograft tumors with or without the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide;
FIG. 8 is a graph showing the effect of tumor volume on nude mice bearing HCT116 xenograft tumors with or without the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide;
FIG. 9 is a graph showing the weight effect of nude mice with or without the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide on HCT116 xenograft tumor bearing nude mice;
FIG. 10 is a graph of flow cytometry experimental results;
FIG. 11 is a graph showing the effect of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide on S phase CyclinB, cyclinD, CDK and CDK2 protein expression levels;
FIG. 12 is a graph showing the effect of N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide on the levels of SKP2, p21, p27 and Tubulin protein expression during S phase;
FIG. 13 is a graph showing the effect of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide on the level of SKP2, tubulin protein expression over time;
FIG. 14 is a graph showing the results of EdU staining experiments of cells treated with different concentrations of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide for HCT116 and HCT116-SKP 2;
FIG. 15 is a graph showing the change in tumor volume size of experiments using xenograft tumor models formed from HCT116 or HCT116-T7-SKP2 colorectal cancer cells;
FIG. 16 is a graph showing tumor size and weight changes in experiments using xenograft tumor models formed from HCT116 or HCT116-T7-SKP2 colorectal cancer cells;
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that the letter abbreviations used in the following description have the following meanings:
MTT:3- (4, 5-dimethyl-2-thiazole) -2, 5-diphenyl tetrazolium bromide thiazole blue; DAPI:4', 6-diamino-2-phenylindole; PI: propidium iodide; CQ: an autophagy inhibitor; edU: 5-bromo-2-deoxyuracil; 202#: n- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide.
Example 1
This example describes N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide and a preparation method thereof, wherein the structural formula of the N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide is shown in figure 1, and the preparation method of the N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide is as follows:
wherein, the mol ratio of m-phenylenediamine, triethylamine and m-chlorobenzoyl chloride is 1:2:1, 3-nitro-4-methoxybenzoic acid, N-diisopropylethylamine and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 1:2:2.
Specifically, the following is an illustration of the preparation of N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide:
2.16g of m-phenylenediamine (20 mmol) is dissolved in 100mL of dichloromethane, 5.5mL of triethylamine is added under ice bath to obtain a mixed solution I, 3.5g of m-chlorobenzoyl chloride (20 mmol) is dissolved in 20mL of dichloromethane under stirring, the obtained m-chlorobenzoyl chloride solution is slowly added into the mixed solution I in a dropwise manner, the ice bath is removed after the dropwise addition, the stirring reaction is continued for 2h at room temperature, after the reaction is finished, the obtained reaction solution is washed with 100mL of saturated saline and 100mL of clear water in sequence, and the organic phase is dried by anhydrous sodium sulfate and then dried by spin to obtain an intermediate product. The structural formula of the intermediate product is as follows:
3.94g of 3-nitro-4-methoxybenzoic acid (20 mmol) was dissolved in 100mL of methylene chloride, 7mL of N, N-diisopropylethylamine (40 mmol) and 7.68g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride EDCI (40 mmol) were sequentially added to obtain a mixed solution II, the intermediate amide obtained in the previous step was dissolved in 20mL of methylene chloride with stirring, and the mixture was added to the reaction system, and the reaction was stirred at room temperature overnight. After the completion of the reaction, the resultant reaction product was washed with 1M diluted hydrochloric acid (100 mL) once, and after the organic phase was dried over anhydrous sodium sulfate and spin-dried, it was passed through a column with silica gel to obtain 6.2g of a white solid, and the yield was calculated to be 73%. The nuclear magnetic patterns of the N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide are shown in figures 2 and 3.
The nuclear magnetic data detection conditions were as follows:
on a 400MHz solid state nuclear magnetic resonance spectrometer (Bruker AVANCE III 400 MHz), tetramethyl silicon (TMS) is used as an internal marker for recording 1 H and 13 C NMR。 1 h NMR data are reported below: chemical shift, in ppm (δ), multiplicity (s=singlet, d=doublet, t=triplet, m=multiplet), coupling constant (Hz), relative intensity; 13 the C NMR data are reported below: chemical shift (ppm).
HPLC-MS analysis was performed on a Shimadzu-2020LC-MS instrument using the following conditions: a Shim-pack VPODS C18 column (reversed phase, 150X 2.0 mm); 80% acetonitrile and 20% water were used over 10.0 minutes; the flow rate is 0.4ml/min; the detection range of the ultraviolet photodiode array is 200 to 300nm. The product was purified by Biotage Isolera TM The Spektra system and hexane/ethyl acetate solvent system. All reagents and solvents were purchased commercially and used without further purification.
Example 2 related cell assays
(1) Cell culture
HCT116, HT29, SW620, HEK293T and FHC used in this example were all purchased from american-type culture collection (ATCC, virginia). HCT116 was cultured in McCoy's 5A medium (SH 30200.01, hyclone), HT29, SW620, HEK293T and FHC in high sugar DMEM medium (SH 30022.01, hyclone), both of which were placed at 37℃with 5% CO 2 And 10% fetal bovine serum (FBS, 10100147, gibco) and 1% penicillin streptomycin were added.
(2) Plasmid transfection and lentiviral infection
Plasmid constructs expressing T7-SKP2 and Myc-SKP2 were obtained from Shandong Uygur autonomous Biotechnology Co. According to the manufacturer's protocol, lipo8000 is used TM The transfection reagent (Beyotime) transfects cells with the indicated plasmids. For stable transfection, lipo8000 was used TM Transfection reagents targeting plasmid, pspax2 and pMD2G vectors were co-transfected into HEK293T cells. Collection of disease 72h after transfectionToxic particles, filtered through a 0.22 μm membrane, are added to a medium containing 10. Mu.g/mL polybrene. After 24 hours of culture, the medium was changed, and cells were selected with 10. Mu.g/mL puromycin to obtain transgenic cells.
(3) Cell viability assay
HCT116, HT29 and FHC cells were seeded in 96-well plates at a density of 2 x10 3 After treatment with different concentrations of N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide at 37℃per well, 20. Mu. LMTT working solution (5 mg/ml) was added to each well and incubated at 37℃for 4h, followed by complete dissolution of insoluble crystals by addition of DMSO, and absorbance was measured at 570nm with a microplate reader (Bio-Tek) and data analyzed by GraphPad Prism 9.0. All experiments were performed independently in triplicate.
(4) Colony formation assay
HCT116 and HT29 cells were seeded in 6-well dishes at a density of 1000 cells per well, incubated with different concentrations of N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide for 14 days, and the colonies formed were washed three times with cold phosphate buffered saline, fixed with 4% paraformaldehyde for 25 minutes at room temperature, and stained with 0.5% crystal violet for 30 minutes. All statistical measurements were three times, as observed and visualized using an Epson scanner (Epson) scan.
(5) EdU detection assay
Using BeyoClick according to manufacturer's instructions TM The EdU cell proliferation kit analyzes cell proliferation. Cells were grown at 2X 10 cells per well 3 Density of individual cells were seeded into CellCarrierTM-96 well plates (Perkinemer) and incubated for 24 hours, treated with the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide for 24 hours. Subsequently, 10. Mu.M EdU working solution was added at 37℃for 4 hours, then fixed with 4% paraformaldehyde, infiltrated with 0.3% Triton X-100, and stained with 1 Xhoechst nuclear dye for 30 minutes. Images were observed using a fluorescence microscope (Olympus) and the percentage of EdU positive cells was calculated using GraphPad Prism 9.0.
(6) Flow cytometry analysis
For cell cycle analysis, cells were seeded in 100mm dishes for 24 hours and then treated with different concentrations of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide, respectively, for 24 hours. Cells were collected by trypsinization and fixed overnight with 70% ethanol at low temperature of 4 ℃. After washing with cold PBS, cells were incubated with 50 μg/mL propidium iodide (PI, sigma) and rnase a (Beyotime) supplemented PBS for 30 min at 37 ℃ and detected with BD Accuri C6 flow cytometry (BD Biosciences) connected to Cell Quest software (BD Biosciences) and Cell cycle distribution was analyzed by FlowJo 7.6.1 software.
(7) Construction of tumor xenogeneic animal transplanting model
Will be 1x10 6 HCT116 cells or HCT116 cells which stably overexpress T7-SKP2 were suspended in 100. Mu.L of serum-free McCoy's 5a and inoculated subcutaneously on both sides of female Nude Balb/c mice of 4-6 weeks of age and body weight of 16-20 g. Mice were randomly grouped (n=6 per group) when the average tumor volume was about 100mm 3 Dosing was started at that time. The mice were orally administered every 3 days at a dose of 10mg/kg or 30mg/kg of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide, which also contained 5% dimethyl sulfoxide, 30% polyethylene glycol 300, 10% tween-80 and 55% physiological saline. Tumor length (L) and width (W) were measured every 3 days with vernier calipers and tumor volume was calculated using standard formula (l×w2)/2. Mice were sacrificed 36 days after drug treatment, xenograft tumors were isolated and weighed. Tumor tissue was then harvested and divided into two parts, one fixed in 4% pfa, hematoxylin-eosin (HE) and immunohistochemical staining with paraffin embedding and the other immunoblotted.
Statistical analysis
All data were subjected to more than three independent experiments. Statistical analysis was performed using GraphPad Prism 9.0. Significance between groups was calculated using a one-way anova test. Data are shown as mean ± standard deviation, significance set to p <0.05.
Result determination
FIG. 4 is a graph showing the change in cell viability as a function of treatment time for cells HCT116, HT29, FHC, using MTT assay with varying concentrations (3.125. Mu.M, 6.25. Mu.M, 12.5. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M) of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide, FIG. 4A shows the change in cell HCT116, FIG. 4B shows the change in cell HT29, FIG. 4C shows the change in normal colorectal cell FHC, and the data of FIG. 4 shows that the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide has significant inhibition on the activities of both cell HCT116 and HT29, but little inhibition on normal colorectal cell FHC, i.e. no toxicity on normal cells.
To further determine the growth and proliferation inhibition of colorectal cancer cells by the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide, edU and colony formation experiments were performed, as shown in fig. 5 and 6, fig. 5 is a graph of EdU experiments with different concentrations of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide to treat cells HCT116 and HT29, fig. 6 is a graph of colony formation experiments with different concentrations of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide to treat cells HCT116 and HT29, and fig. 5 shows that the inhibitor can significantly reduce DNA synthesis of cells HCT116 and HT29 as the concentration of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide increases, and that the colony formation experiments shown in fig. 6 show that the indicated concentration (10 μm) of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide can significantly reduce the number of colorectal cancer cells and colonies. As can be seen from fig. 5 and 6, the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide can significantly inhibit the proliferation of cell HCT116 and HT29, indicating that it has an inhibitory effect on the growth of cell HCT116 and HT 29.
To further evaluate the tumor-inhibiting activity of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide in vivo, xenograft model experiments were performed, and the experimental results are shown in fig. 7, 8 and 9, fig. 7 is a graph showing the effect of tumor cell size and weight of nude mice carrying HCT116 xenograft tumors with or without the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide, and the experimental data of fig. 7 show that the use of 30mg/Kg of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide can significantly reduce the size and weight of HCT116 tumors compared to the control group; FIG. 8 is a graph showing the effect of tumor volume on nude mice bearing HCT116 xenograft tumors with or without the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide, and the experimental data of FIG. 8 show that the use of 30mg/Kg of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide significantly reduced the volume of HCT116 tumors in the mice compared to the control group; fig. 9 is a graph showing the weight effect of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide on the weight of nude mice bearing HCT116 xenograft tumors, and the experimental data of fig. 9 shows that the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide has no significant effect on the weight of the mice, indicating no significant toxicity. Taken together, the experimental data of fig. 7, 8 and 9 show that the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide can significantly inhibit the growth of HCT116 tumor cells in vivo, and no significant toxicity is found during use.
To assess the mechanism of antiproliferative effect of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide in colorectal cancer cells, analyses were performed using flow cytometry and western immunoblotting. The results of flow cytometry evaluation are shown in FIG. 10, and the data in FIG. 10 indicate that the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide can reduce the distribution of HCT116 and HT29 cells in the G0/G1 and G2/M phases, and significantly induce S phase cell cycle arrest. The results of western blot analysis are shown in fig. 11, and the data of fig. 11 demonstrate that the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide significantly reduced the levels of CyclinB, cyclinD, CDK and CDK2 in a dose-dependent manner, meaning that the antiproliferative effect of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide is caused by cell cycle arrest in S phase.
To explore the molecular mechanism of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide to inhibit cell cycle arrest, SKP2 expression level detection was performed, and the results are shown in fig. 12, SKP2 protein levels were significantly down-regulated after treatment with the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide, while p21 and p27 protein levels were significantly up-regulated in a dose-dependent manner, and SKP2 protein reduction by the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide was time-dependent, as shown in fig. 13. To investigate whether SKP2 overexpression was able to reduce the anti-proliferative effect induced by the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide, the proliferation of HCT116 cells overexpressing SKP2 treated with the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide was examined, as shown in fig. 14, the data of fig. 14 indicate that overexpression of SKP2 attenuated the proliferation inhibition induced by the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide, as SKP2 transfected cells grew better than wild-type HCT116 cells.
To examine whether overexpression of SKP2 could attenuate the in vivo growth inhibitory effect of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide on colorectal cancer, experiments were performed using xenograft tumor models formed from HCT116 or HCT116-T7-SKP2 colorectal cancer cells, the results of which are shown in fig. 15 and 16, fig. 15 is a graph of the change in the size of the relevant tumor volume, fig. 16 is a graph of the change in the size and weight of the relevant tumor, and the data shown in fig. 15 and 16 indicate that SKP2 overexpression can significantly attenuate the growth inhibitory effect of the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide on colorectal cancer tumors.
The above results indicate that the mechanism of inhibition of colorectal cancer proliferation and growth by the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide is achieved by inhibiting cell cycle progression.
In conclusion, the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide can obviously inhibit proliferation and growth of colorectal cancer HCT116 and HT29 cells, can be used for preparing antitumor drugs, especially medicines for resisting colon cancer, and can be used for preparing antitumor preparations after adding pharmaceutically acceptable auxiliary materials and carriers into the compound N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide, wherein the preparations are any one of granules, tablets, pills, capsules, injections or dispersing agents.
The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention. The technology, shape, and construction parts of the present invention, which are not described in detail, are known in the art.
Claims (3)
- Use of n- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide for the preparation of an anti-tumor drug, wherein the tumor is colon cancer.
- 2. The use of N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide for preparing antitumor drugs according to claim 1, wherein the N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide is used for preparing antitumor preparations after adding pharmaceutically acceptable auxiliary materials and carriers.
- 3. Use of N- (3- (3-chlorobenzamide) phenyl) -4-methoxy-3-nitrobenzamide for preparing antitumor drugs according to claim 2, wherein the formulation is any one of granules, tablets, pills, capsules or injections.
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| CN101362718A (en) * | 2008-09-28 | 2009-02-11 | 四川大学 | 4-(4-benzamido phenoxy)-2-(methylcarbamoyl) pyridine derivatives, preparation method and application thereof |
| KR20180137248A (en) * | 2017-06-16 | 2018-12-27 | 연세대학교 산학협력단 | Pharmaceutical composition for inhibiting invasion and metastasis of cancer tumorsphere |
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| US8067608B2 (en) * | 2003-09-29 | 2011-11-29 | The Johns Hopkins University | Hedgehog pathway antagonists |
| US7994222B2 (en) * | 2006-09-05 | 2011-08-09 | Bipar Sciences, Inc. | Monitoring of the inhibition of fatty acid synthesis by iodo-nitrobenzamide compounds |
| US8466180B2 (en) * | 2006-09-11 | 2013-06-18 | Syngenta Crop Protection Llc | Insecticidal compounds |
| HUE062956T2 (en) * | 2017-08-14 | 2023-12-28 | Teva Pharmaceuticals Int Gmbh | Processes for the preparation of niraparib and intermediates thereof |
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| CN101362718A (en) * | 2008-09-28 | 2009-02-11 | 四川大学 | 4-(4-benzamido phenoxy)-2-(methylcarbamoyl) pyridine derivatives, preparation method and application thereof |
| KR20180137248A (en) * | 2017-06-16 | 2018-12-27 | 연세대학교 산학협력단 | Pharmaceutical composition for inhibiting invasion and metastasis of cancer tumorsphere |
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