1, 5-benzodiazepine derivative as well as preparation method and application thereof

Abstract

The invention relates to the field of medicinal chemistry, in particular to a novel heterocyclic system, namely a 1, 5-benzodiazepine # imgabs0 # derivative as well as a preparation method and application of the 1, 5-benzodiazepine # imgabs0 # derivative. The benzodiazepine # imgabs 1 # derivative has activity of resisting solid tumors such as colon cancer, pancreatic cancer, cervical cancer, gastric cancer, liver cancer and the like; the benzodiazepine # imgabs2 # derivative has good selectivity on colon cancer cells, and can inhibit proliferation and migration of the colon cancer cells and promote apoptosis of the colon cancer cells; pharmacodynamic experiments show that the benzodiazepine # imgabs3 # derivative has better colon cancer treatment effect than a clinical drug 5-Fu, and has low toxicity to normal intestinal epithelial cells HIEC; in addition, the benzodiazepine # imgabs4 # derivative T0 also has good cytotoxicity to pancreatic cancer cells PANC-1, cervical cancer cells Hela, gastric cancer cells AGS and liver cancer cells HepG-2, can also be used as a potential broad-spectrum anti-cancer drug, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular to a new heterocyclic system, namely 1,5-benzodiazepine Derivatives and preparation methods and uses thereof. Background Art

[0002] Heterocyclic compounds are a collection of compounds that contain heterocyclic rings in their molecules, and are large in number and complex in types. Due to their special biological activity properties, heterocyclic compounds are considered to be key structural motifs for pharmaceuticals and agricultural chemicals, and can be used as various active lead compounds. Statistics show that heterocyclic compounds account for more than 67% of the total compounds included in the Comprehensive Medicinal Chemistry database. As an important branch of heterocyclic compounds, N-heterocyclic compounds are widely distributed in nature, have a variety of physiological and pharmacological properties, and are components of many important biological molecules, including vitamins, nucleic acids, antibiotics, dyes and pesticides. Due to their unique properties and important applications in the medical field, chemists are committed to developing efficient, economical and selective nitrogen heterocyclic transformations. The lone pair of electrons on the nitrogen atom of N-heterocyclic compounds can easily accept protons and can also establish various weak interactions. Some of these intermolecular interactions, such as hydrogen bond formation, hydrophobicity, pyramidal stacking, dipole-dipole interactions, and van der Waals forces, increase the importance of N-heterocyclic compounds in the field of medicinal chemistry and enable them to bind to a variety of enzymes and receptors in biological targets with high affinity. Therefore, the design and synthesis of physiologically active nitrogen-containing heterocyclic compounds has always been a research hotspot in the field of synthesis.

[0003] According to relevant statistics, among the seven-membered heterocyclic molecules reported in the published literature, 55% of them have biological activity in anti-tumor. The seven-membered ring of benzazepine is a parent structure composed of a benzene ring and a nitrogen-containing seven-membered ring fused together. It is a potential anti-tumor biologically active structural unit and has been widely used in the research and development of new drugs in recent years. The derivatives have antiviral, antianxiety, anticonvulsant, sedative, analgesic, antidepressant, hypnotic, anti-inflammatory, anti-HIV, anti-obesity, anticoagulant, anti-ulcer and other activities. In addition, they can also be used as BET protease inhibitors, non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTI), calcium channel blockers, cholecystokinin antagonists, endothelin antagonists, thrombopoietin receptor agonists and vasopressin receptor antagonists, and semi-creatine protease FP-2 inhibitors.

[0004] Therefore, it is of great practical significance to carry out the synthesis and activity research of this type of derivatives. Summary of the invention

[0005] In view of the above technical problems, the present invention aims to provide a 1,5-benzodiazepine having anti-tumor activity. Derivatives. Specifically include the following:

[0006] In a first aspect, the present invention provides a 1,5-benzodiazepine The derivative has the structural formula shown in the following formula (I):

[0007]

[0008] In a second aspect, the present invention provides the 1,5-benzodiazepine described in the first aspect. Application of derivatives in the preparation of anti-tumor drugs.

[0009] Preferably, the tumor includes colon cancer, pancreatic cancer, ampullary cancer, liver cancer, gastric cancer, and cervical cancer.

[0010] Preferably, the cancer is colon cancer.

[0011] In a third aspect, the present invention provides a pharmaceutical preparation, wherein the pharmaceutical preparation is the 1,5-benzodiazepine described in the first aspect. The derivatives are added with pharmaceutically acceptable carriers and / or excipients to prepare any pharmaceutically acceptable dosage form.

[0012] Preferably, the dosage form includes any one of capsules, granules, tablets, sprays, oral solutions, suspensions, and injections.

[0013] In a fourth aspect, the present invention provides a 1,5-benzodiazepine as described in the first aspect. A method for preparing a derivative, the method comprising:

[0014] The product is prepared by mixing o-phenylenediamine and (1E,4E)-1,5-di-p-toluene-1,4-pentadien-3-one, heating under reflux with anhydrous ethanol and triethylamine as solvent, column chromatography, and recrystallization from anhydrous ethanol.

[0015] Preferably, the mass ratio of o-phenylenediamine to (1E,4E)-1,5-di-p-toluene-1,4-pentadien-3-one is 1-2:2-3.

[0016] Preferably, the triethylamine and anhydrous ethanol are added in a volume ratio of 1:4, with a total of 25 ml.

[0017] Preferably, the heating reflux time is 48 hours.

[0018] Preferably, column chromatography is performed using a 93:7 mixture of n-hexane and ethyl acetate as eluent.

[0019] The beneficial effects of the present invention are: the present invention provides a benzodiazepine Derivatives, the benzodiazepine The derivatives have anti-colon cancer, pancreatic cancer, cervical cancer, gastric cancer, and liver cancer activities; the benzodiazepine The derivatives have good selectivity for colon cancer cells, can inhibit the proliferation and migration of colon cancer cells, and promote apoptosis of colon cancer cells; pharmacodynamic experiments show that benzodiazepines The derivatives are more effective than the clinical drug 5-Fu in treating colon cancer and have less toxicity to normal intestinal epithelial cells (HIEC). The derivatives also have good cytotoxicity to pancreatic cancer cells PANC-1, cervical cancer cells Hela, gastric cancer cells AGS, and liver cancer cells HepG-2, and can also be used as potential broad-spectrum anticancer drugs with good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Benzodiazepines Derivatives T1-T16 structural formula;

[0021] Figure 2 Benzodiazepines Synthetic route of derivative T0;

[0022] Figure 3 Benzodiazepines Synthetic routes of derivatives 3a-d and 4a-d;

[0023] Figure 4 Benzodiazepines Synthetic routes of derivatives 6a-d and 7a-d;

[0024] Figure 5 MTT assay for benzodiazepines The survival rate of derivative T0 on colorectal cancer cell SW480;

[0025] Figure 6 The MTT method was used to detect the survival rate of colorectal cancer cell SW480 by 5-Fu;

[0026] Figure 7 Benzodiazepines Effect of derivative T0 on the migration of colon cancer cell SW480;

[0027] Figure 8 Benzodiazepines The inhibitory effect of derivative T0 on colon cancer cell SW480 clone formation;

[0028] Fig. 9 Hoechst 33258 staining assay for analysis of benzodiazepines Effect of derivative T0 on apoptosis of colorectal cancer cell line SW480. DETAILED DESCRIPTION

[0029] In order to make the present invention easier to understand, the present invention is further described below in conjunction with specific embodiments. However, it should be understood that these embodiments are for illustration and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the contents involved in the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the present invention.

[0030] The human colon cancer cell lines SW480, LOVO cells and HCT116, ampullary cancer cells DPC-X1, pancreatic cancer cells PANC-1, liver cancer cells HepG-2, gastric cancer cells AGS, cervical cancer cells Hela, and intestinal epithelial cells HIEC described in the following examples are all commercially available.

[0031] Example 1 Benzodiazepine Synthesis of derivatives

[0032] Benzodiazepines The synthetic route of derivative T0 is as follows Figure 2 As shown, the specific synthesis method is:

[0033] Add 1-2g of o-phenylenediamine and 2-3g of (1E, 4E)-1,5-di-p-toluene-1,4-pentadiene-3-one to a 100mL round-bottom flask with a branch tube; mix triethylamine and anhydrous ethanol at a volume ratio of 1:4, add 25ml in total to dissolve o-phenylenediamine and (1E, 4E)-1,5-di-p-toluene-1,4-pentadiene-3-one, add zeolite and heat under reflux for 48h. After the reaction is complete, the solution turns dark red, and then the mixture is subjected to column chromatography, using a mixture of n-hexane / ethyl acetate (93:7) as the eluent. The product is recrystallized in anhydrous ethanol to obtain a yellow solid with a yield of 84%.

[0034] 1H NMR(300MHz, CDCl3), δppm:2.84(Ha),3.06(Hb)(m,2H,-CH2-3),2.38(s,6H,Ar-CH3),3.77( s,1H,-NH-),5.15(m,1H,-CH-2),6.76-7.37(m,12H,H-Ar,1H,Ph-CH=CH-and1H,Ph-CH=CH-).

[0035] 13C NMR (75MHz, CDCl3), δppm: 21.16, 21.42 (2C, CH3-Ar), 36.39 (1C, -CH2-), 70.02 (1C, -CH-), 133.41, 137.79, 138.51, 139.10, 139. 29,141.97(6C,Ar-Cquaternary),120.64,121.34,125.94,126.52,127.39,128.82,129.49,129.55,130.15,136.45(10C,CH-Ar and 2C,Ph-CH=CH-),168.06(1C,-C=N).

[0036] The structural formula is shown in the following formula (I):

[0037]

[0038] At the same time, this application synthesized 16 other benzodiazepines Derivatives, named T1-T16, with structural formula Figure 1 shown.

[0039] The synthetic route of T1-T8 is as follows Figure 3 As shown, specifically: synthesized benzodiazepine The derivative T0 is mixed with the precursor compound 2 (N-aryl-C-ethoxycarbonyl nitrile imine, formula (II)), and heated to reflux in tetrahydrofuran solvent containing triethylamine for 12-48h to obtain benzodiazepine Derivatives 3a-d (T1, T3, T5, T7) and 4a-d (T2, T4, T6, T8).

[0040]

[0041] The synthetic route of T9-T16 is as follows Figure 4 As shown, specifically: synthesized benzodiazepine The derivative T0 is mixed with the precursor compound and the corresponding precursor compound 5 (α-chloroarylene hydrazone, formula (III)), and refluxed at room temperature for 12-48h in tetrahydrofuran solvent containing triethylamine to obtain benzodiazepine Derivatives 6a-d (T9, T11, T13, T15) and 7a-d (T10, T12, T14, T16).

[0042]

[0043] Example 2 Benzodiazepine Inhibitory effects of derivatives on different tumor cells

[0044] The benzodiazepine prepared in Example 1 Derivatives T0 and T1-T16 were used to treat colon cancer cells SW480, ampullary cancer cells DPC-X1, pancreatic cancer cells PANC-1, liver cancer cells HepG-2, gastric cancer cells AGS, cervical cancer cells Hela and intestinal epithelial cells HIEC, and then 10 μL of 5 mg / mL MTT solution was added and incubated in an incubator at 37°C and 5% CO2 for 4 hours. The old culture medium and MTT solution were carefully discarded, and 100 μL of DMSO solution was added. The mixture was shaken on a shaker at 120 r / min for 30 minutes to allow the reacted formazan to fully dissolve in DMSO. The absorbance value was then measured at 490 nm using an enzyme reader to calculate the concentration of different benzodiazepines. The inhibition rate of the derivatives on the above cells was calculated by SPSS software. 50 .

[0045] Benzodiazepines The cytotoxicity IC values ​​of derivatives T0-T16 against colon cancer cells SW480, ampullary cancer cells DPC-X1, pancreatic cancer cells PANC-1, liver cancer cells HepG-2, gastric cancer cells AGS, cervical cancer cells Hela and intestinal epithelial cells HIEC 50 The test results are shown in Table 1 below. The derivative T0 showed significant anticancer activity against colon cancer, pancreatic cancer, liver cancer cells, gastric cancer, and cervical cancer, and had the best inhibitory effect on colon cancer cell SW480. The inhibitory effect of the derivatives on the above tumor cells is not obvious. The derivative T0 showed strong cytotoxicity to SW480 cells, and its IC 50 The IC values ​​for human pancreatic cancer cells PANC-1 were 3.54 μM and 50 The IC value of human hepatoma cell HepG-2 is 5.76 μM. 50 The IC value of human gastric cancer cell AGS is 12.66 μM. 50 The IC value of Hela human cervical cancer cells is 7.61 μM. 50 The IC value for human ampullary carcinoma cell DPC-X1 was 17.54 μM. 50 >30μM, IC of intestinal epithelial cells HIEC 50 The above experimental results show that the benzodiazepines described in this application The derivative T0 not only has significant cytotoxicity to colon cancer cells, but also has obvious cytotoxicity to pancreatic cancer, gastric cancer, cervical cancer, and liver cancer.

[0046] Table 1 Benzodiazepines IC of derivatives against tumor cells 50 Test results (μM)

[0047]

[0048] Example 3 Benzodiazepine Inhibitory effect of derivative T0 on other colon cancer cells

[0049] 1. MTT method for detecting benzodiazepines Derivatives' inhibitory effects on other colon cancer cells

[0050] LOVO cells and HCT116 cells were cultured in cell culture dishes. When the cells grew to about 80%, they were taken out of the incubator at 37°C and 5% CO2. Trypsin containing phenol red and EDTA was added to the culture dishes in a clean bench for digestion. After digestion, 1640 culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added to resuspend the cells. The suspended cells were counted using a hemocytometer and the cell concentration was adjusted to 1×10 5 / 100μL, then use a spray gun to add the cells of this concentration to a 96-well plate at 100μL / well for plating, then put the 96-well culture plate into a cell culture incubator and continue to culture for 24h, then discard the old culture medium, add 100μL of culture medium containing different concentrations of T0 and continue to culture for 48h. Then add 10μL of 5mg / mL MTT solution to each well and continue to incubate in the cell culture incubator for 4h. After the incubation, carefully aspirate the liquid in the 96-well plate, and add 100μL of DMSO solution to each well, and shake on a shaker at 120r / min for 30min to fully dissolve the formazan in DMSO. Then use an enzyme reader to measure the absorbance at 490nm to calculate the benzodiazepine described in this application The inhibition rate of the derivatives on the above cells was calculated by SPSS software. 50 .

[0051] The benzodiazepines described in this application The derivative T0 showed strong cytotoxicity against LOVO cells and HCT116 cells, and its IC 50 4.38 μM and 5.87 μM respectively.

[0052] 2. MTT method for detecting the benzodiazepines described in this application The experimental methods for the inhibitory effects of derivatives and 5-Fu on colon cancer cell SW480 were the same as described above.

[0053] The benzodiazepines described in this application The results of MTT experiments of derivatives T0 and 5-Fu on colon cancer cell SW480 are shown as follows: Figure 5 and Figure 6 As shown. Experimental verification shows that the benzodiazepines described in this application IC of derivative T0 against colon cancer cell SW480 50 is 3.54 μM, while the IC 50 is 10.73 μM, indicating that the benzodiazepine The inhibitory rate of derivative T0 on SW480 cells was significantly better than that of 5-Fu.

[0054] In summary, the benzodiazepines of the present invention The derivative T0 can inhibit the proliferation of solid tumors such as colorectal cancer, pancreatic cancer, gastric cancer, and cervical cancer. Its anti-colon cancer effect is better than the positive drug 5-Fu, and it has less toxicity to normal intestinal epithelial cells, showing the characteristics of high efficiency and low toxicity. It can selectively treat colon cancer and has broad-spectrum anti-tumor properties.

[0055] 3. Cell scratch test

[0056] First, use a ruler to draw horizontal lines evenly on the back of the 6-well plate with a marker pen, approximately every 0.5 to 1 cm, across the wells, and at least 5 lines in each well; then take out the colon cancer SW480 cells in the logarithmic growth phase, discard the old culture medium, wash the cells twice with 1× PBS, and then digest them with trypsin to make a single cell suspension, and then use 2×10 5 The density of cell / well is inoculated in a 6-well culture plate. The inoculation principle is that the fusion rate reaches 100% after overnight, and the final total amount of culture medium in each well is 2mL; then culture in a 37℃, 5% CO2 incubator for 24h. On the second day, use a 10μL pipette tip to vertically scratch the 6-well plate cover perpendicular to the horizontal line on the back. The pipette tip cannot be tilted during the entire operation. After the scratch, wash the cells 3 times with PBS to remove the scratched cell fragments. Wipe off the marker horizontal scratches on the back of the 6-well plate. Then add serum-free culture medium and different concentrations of benzodiazepines The serum-free medium of derivative T0 was photographed under a 10x microscope. Finally, Image J software was used for image processing and result analysis.

[0057] The results are as follows Figure 7 As shown, the benzodiazepines described in the present invention The derivative T0 can significantly inhibit the migration of colon cancer SW480 cells.

[0058] 4. Cell cloning experiment

[0059] SW480 cells were cultured in a medium cell culture dish. When the cells grew to 80%, the cells were digested as described above to prepare a cell suspension; then the cells were inoculated in a 24-well plate at a density of 800 cells / well, with a total culture medium of 500 μL, and then the inoculated culture plate was placed in a cell culture incubator for continued culture for about 24 hours. Then the old culture medium was discarded, and the culture medium containing different concentrations of T0 was added to continue incubation for 7 to 10 days, and the culture medium containing different concentrations of T0 was replaced every 48 hours. When the cells grew to a suitable density, the old culture medium was discarded, and the cells were washed twice with 1×PBS solution, and 300 to 400 μL of 4% paraformaldehyde solution was added to fix the cells for about 40 minutes, and then the fixative was aspirated, and the 24-well plate was continued to be washed with 1×PBS, and then 0.1% crystal violet staining solution was added for staining for 20 minutes. After the crystal violet solution was recovered, it was washed twice with 1×PBS, and photographed after drying.

[0060] The results are as follows Figure 8 As shown, the benzodiazepines described in the present invention The derivative T0 can significantly inhibit the cloning of colorectal cancer cells.

[0061] 5. Hoechst33258 staining experiment

[0062] SW480 cells were cultured in medium-sized cell culture dishes. When the cells grew to 80%, the cells were digested as described above to prepare a cell suspension. Then, 2 × 10 4 The cells were inoculated in a 12-well plate at a density of 1000 μL / well, and the total culture medium was 1000 μL. The inoculated culture plate was then placed in a cell culture incubator for continued culture for about 24 hours. The old culture medium was then discarded, and the culture medium containing different concentrations of T0 was added for continued incubation for 48 hours. The old culture medium was discarded, and the cells were washed twice with 1×PBS solution. A 1 mg / mL Hoechst 33258 solution was prepared with 1×PBS solution, and then diluted to a concentration of 50 μg / mL. 400 μL of diluted Hoechst 33258 staining solution was added to each well of the 12-well plate, and the cells were continued to be incubated in a cell culture incubator for 30 minutes. The Hoechst 33258 staining solution was then recovered, and the cells were washed twice with 1×PBS solution. A little PBS was left in the cell culture plate for the last wash, and the cells were immediately observed with an inverted fluorescence microscope. The nuclear staining of apoptotic cells was enhanced, the fluorescence was brighter, and the cells were round, condensed, or lumpy.

[0063] The results are as follows Fig. 9 As shown, after SW480 cells were treated with different concentrations of benzazepine derivatives T0 for 48 hours, the cell morphology of SW480 cells changed significantly with the increase of compound concentration, the cell nucleus became round or condensed, and the number of cells with strong blue fluorescence increased in a concentration-dependent manner.

[0064] In summary, the benzodiazepines described in this application The derivatives have anti-colon cancer, pancreatic cancer, cervical cancer, gastric cancer, and liver cancer activities, and have low toxicity to normal intestinal epithelial cells HIEC, and can be used as potential broad-spectrum anticancer drugs; and the benzodiazepine #imgpt55# derivatives have good selectivity for colon cancer cells, can inhibit the proliferation and migration of colon cancer cells, and promote the apoptosis of colon cancer cells. The effect of treating colon cancer is better than the clinical drug 5-Fu, and has good application prospects.

[0065] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features thereof may be replaced by equivalents. However, such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A 1,5-benzodiazepine A derivative, characterized in that The structural formula of the derivative is shown in the following formula (I):

2. 1,5-benzodiazepine as claimed in claim 1 Application of derivatives in the preparation of anti-tumor drugs.

3. The use according to claim 2, characterized in that The tumors include colon cancer, pancreatic cancer, liver cancer, stomach cancer, and cervical cancer.

4. The use according to claim 3, characterized in that The cancer is colon cancer.

5. A pharmaceutical preparation, characterized in that The pharmaceutical preparation is the 1,5-benzodiazepine according to claim 1 The derivatives are added with pharmaceutically acceptable carriers and / or excipients to prepare any pharmaceutically acceptable dosage form.

6. The pharmaceutical preparation according to claim 5, characterized in that The dosage form includes any one of capsules, granules, tablets, sprays, oral liquids, suspensions and injections.

7. 1,5-benzodiazepine as claimed in claim 1 A method for preparing a derivative, characterized in that: The method comprises: The compound is prepared by mixing o-phenylenediamine and (1E,4E)-1,5-di-p-toluene-1,4-pentadien-3-one, heating under reflux with anhydrous ethanol and triethylamine as solvent, column chromatography, and recrystallization from anhydrous ethanol.

8. The preparation method according to claim 7, characterized in that: The mass ratio of the o-phenylenediamine to (1E, 4E)-1,5-di-p-toluene-1,4-pentadiene-3-one is 1-2:2-3.

9. The preparation method according to claim 7, characterized in that: The triethylamine and anhydrous ethanol were added in a volume ratio of 1:4, with a total of 25 ml; the heating reflux time was 48 hours.

10. The preparation method according to claim 7, characterized in that: Column chromatography was performed using a mixture of n-hexane and ethyl acetate in a volume ratio of 93:7 as the eluent.

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