1,5-benzodiazepine derivatives, processes for their preparation and their use

Abstract

The present application relates to the field of medicinal chemistry, in particular to a new heterocyclic system, namely 1,5-benzodiazepine derivatives and preparation method and use thereof.The benzodiazepine derivative has the activity of anti-solid tumor such as colon cancer, pancreatic cancer, cervical cancer, gastric cancer, liver cancer, etc.; the benzodiazepine derivative has good selectivity to colon cancer cells, can inhibit the proliferation and migration of colon cancer cells, and promote the apoptosis of colon cancer cells; pharmacodynamic experiments show that the effect of the benzodiazepine derivative in treating colon cancer is better than that of the clinical drug 5-Fu, and the toxicity to normal intestinal epithelial cells HIEC is smaller; in addition, the benzodiazepine 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, and can also be used as a potential broad-spectrum anticancer drug, and has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a novel heterocyclic system, namely 1,5-benzodiazepine. Derivatives, their preparation methods, and applications. Background Technology

[0002] Heterocyclic compounds are a large and complex collection of compounds containing heterocycles in their molecules. Due to their unique biological activities, heterocyclic compounds are considered key structural motifs in pharmaceuticals and agrochemicals, serving as lead compounds for various activities. Statistics show that heterocyclic compounds account for over 67% of all compounds listed in the Comprehensive Medicinal Chemistry database. N-heterocyclic compounds, as an important branch of heterocyclic compounds, are widely distributed in nature, possessing diverse physiological and pharmacological properties, and are components of many important biomolecules, including vitamins, nucleic acids, antibiotics, dyes, and pesticides. Their unique properties and important applications in the pharmaceutical field have prompted chemists to dedicate themselves to developing efficient, economical, and selective nitrogen heterocycle transformations. The lone pair of electrons on the nitrogen atom in N-heterocyclic compounds can easily accept protons and establish various weak interactions. Some of these intermolecular interactions, such as hydrogen bond formation, hydrophobicity, π-stacking, dipole-dipole interactions, and van der Waals forces, have increased the importance of N-heterocyclic compounds in medicinal chemistry and enabled them to bind with high affinity to a variety of enzymes and receptors in biological targets. 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 published literature, 55% exhibit anti-tumor biological activity. The benzodiazepine seven-membered ring, a parent structure consisting of a benzene ring and a nitrogen-containing seven-membered ring fused together, is a potential anti-tumor bioactive structural unit and has been widely used in the development of novel drugs in recent years. Benzodiazepines... The derivatives possess antiviral, anti-anxiety, anticonvulsant, sedative, analgesic, antidepressant, hypnotic, anti-inflammatory, anti-HIV, anti-obesity, anticoagulant, and anti-ulcer 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 angiotensin receptor antagonists, and cysteine ​​protease FP-2 inhibitors.

[0004] Therefore, conducting research on the synthesis and activity of such derivatives is of great practical significance. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a 1,5-benzodiazepine with antitumor activity. Derivatives. Specifically, they include the following:

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

[0007]

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

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

[0010] Preferably, the cancer is colon cancer.

[0011] Thirdly, the present invention provides a pharmaceutical formulation, wherein the pharmaceutical formulation is the 1,5-benzodiazepine described in the first aspect above. The derivative is added to a pharmaceutically acceptable carrier and / or excipients to form any pharmaceutically acceptable dosage form.

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

[0013] Fourthly, the present invention provides a 1,5-benzodiazepine described in the first aspect above. A method for preparing derivatives, the method comprising:

[0014] o-Phenylenediamine was mixed with (1E,4E)-1,5-di-p-toluene-1,4-pentadien-3-one, and obtained by heating under reflux, column chromatography, and recrystallization in anhydrous ethanol and triethylamine as solvents.

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

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

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

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

[0019] The beneficial effects of this invention are: this invention provides a benzodiazepine Derivatives, the benzodiazepines The derivatives exhibit anti-colon cancer, anti-pancreatic cancer, anti-cervical cancer, anti-gastric cancer, and anti-liver cancer activity; the benzodiazepines mentioned above... The derivatives exhibit good selectivity for colon cancer cells, inhibiting their proliferation and migration while promoting apoptosis. Pharmacodynamic experiments show that benzodiazepines... The derivative is more effective than the clinical drug 5-Fu in treating colon cancer and has less toxicity to normal intestinal epithelial cells (HIEC); in addition, benzodiazepines... The derivative also exhibits good cytotoxicity against pancreatic cancer cells PANC-1, cervical cancer cells HeLa, gastric cancer cells AGS, and liver cancer cells HepG-2, and can also serve as a potential broad-spectrum anticancer drug with promising application prospects. Attached Figure Description

[0020] Figure 1 benzodiazepines Derivative T1-T16 structural formulas;

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

[0022] Figure 3 benzodiazepines Synthetic routes for derivatives 3a-d and 4a-d;

[0023] Figure 4 benzodiazepines Synthetic routes for derivatives 6a-d and 7a-d;

[0024] Figure 5 MTT assay for benzodiazepines Survival rate of derivative T0 against colorectal cancer cells SW480;

[0025] Figure 6 MTT assay for the survival rate of 5-FU against SW480 colorectal cancer cells;

[0026] Figure 7 benzodiazepines Effects of derivative T0 on the migration of colon cancer cells SW480;

[0027] Figure 8 benzodiazepines The inhibitory effect of derivative T0 on the clonogenesis of colon cancer cells SW480;

[0028] Figure 9 hoechst33258 staining experiment analysis of benzodiazepines Effect of derivative T0 on apoptosis of colorectal cancer cells SW480. Detailed Implementation

[0029] To make the present invention more readily understood, specific embodiments are described below to further illustrate the invention. However, it should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various modifications or alterations to the invention, and these equivalent forms also fall within the scope defined by this invention.

[0030] The human colon cancer cell lines SW480, LOVO, 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 as follows:

[0033] In a 100 mL round-bottom flask equipped with a side arm, add 1–2 g of o-phenylenediamine and 2–3 g of (1E,4E)-1,5-di(p-toluene-1,4-pentadien-3-one. Mix triethylamine and anhydrous ethanol at a volume ratio of 1:4, then add 25 mL of the mixture to dissolve the o-phenylenediamine and (1E,4E)-1,5-di(p-toluene-1,4-pentadien-3-one. Add zeolite and heat under reflux for 48 h. After the reaction is complete, the solution turns deep red. Then, perform column chromatography using a hexane / ethyl acetate (93:7) mixture as eluent. Recrystallize the product from anhydrous ethanol to give a yellow solid in 84% yield.

[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 equation (I) below:

[0037]

[0038] This application also synthesized 16 other benzodiazepines. Derivatives, named T1-T16, have the following structural formulas: Figure 1 As shown.

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

[0040]

[0041] The synthetic routes for T9-T16 are as follows: Figure 4 As shown, specifically: the synthesized benzodiazepines... Derivative T0 is mixed with the precursor compound and the corresponding precursor compound 5 (α-chloroarylhydrazone, formula (III)), and refluxed at room temperature for 12-48 h in a 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, respectively. Then, 10 μL of 5 mg / mL MTT solution was added, and the mixture was incubated at 37°C with 5% CO2 for 4 h. The old culture medium and MTT solution were carefully discarded, and 100 μL of DMSO solution was added. The mixture was shaken at 120 rpm for 30 min to ensure complete dissolution of the formazan in the DMSO. The absorbance was then measured at 490 nm using a microplate reader, and the values ​​of different benzodiazepines were calculated. The inhibitory rate of the derivatives on the above-mentioned cells was finally calculated using SPSS software for all IC50 values. 50 .

[0045] benzodiazepines The cytotoxic IC50 values ​​of derivative 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 were measured. 50 The test results are shown in Table 1 below. The benzodiazepine described in this application... Derivative T0 showed significant anticancer activity against colon cancer, pancreatic cancer, liver cancer cells, gastric cancer, and cervical cancer, with the best inhibitory effect on colon cancer cells SW480. However, other benzodiazepines... However, the derivatives did not show significant inhibitory effects on the aforementioned tumor cells. Among them, the benzodiazepines described in this application... Derivative T0 exhibits strong cytotoxicity against SW480 cells, with an IC50 value of [missing information]. 50 The IC50 values ​​for 3.54 μM and PANC-1 human pancreatic cancer cells were respectively... 50 The IC50 value was 5.76 μM, which was the IC50 value for HepG-2 human liver cancer cells. 50 The IC50 of human gastric cancer cells AGS was 12.66 μM. 50 The IC50 value was 7.61 μM, which was the IC50 value of HeLa cervical cancer cells. 50 The concentration was 17.54 μM, while the IC50 against human ampullary cancer cells DPC-X1 was 17.54 μM. 50 >30 μM, IC50 of HIEC in intestinal epithelial cells 50 Greater than 30 μM. The above experimental results indicate that the benzodiazepine described in this application... The derivative T0 not only exhibits significant toxicity to colon cancer cells, but also shows marked cytotoxicity to pancreatic cancer, gastric cancer, cervical cancer, and liver cancer.

[0046] Table 1 Benzodiazepines Derivatives on IC50 of tumor cells 50 Detection results (μM)

[0047]

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

[0049] 1. MTT assay for the detection of benzodiazepines Inhibitory effect of derivatives on other colon cancer cells

[0050] LOVO cells and HCT116 cells were cultured separately in cell culture dishes. When the cells reached approximately 80% confluence, they were removed from the 37°C, 5% CO2 incubator and digested with trypsin containing phenol red and EDTA in a clean bench. After digestion, the cells were resuspended in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The resuspended cells were counted using a hemocytometer, and the cell concentration was adjusted to 1×10⁻⁶. 5 100 μL of the cell culture medium was added to each well of a 96-well plate using a microparticle pipette. The plates were then incubated for 24 hours. The old culture medium was discarded, and 100 μL of medium containing different concentrations of T0 was added, followed by incubation for another 48 hours. Then, 10 μL of 5 mg / mL MTT solution was added to each well, and the plates were incubated for another 4 hours. After incubation, the liquid in the 96-well plates was carefully aspirated, and 100 μL of DMSO solution was added to each well. The plates were shaken at 120 rpm for 30 minutes to ensure complete dissolution of the formazan in the DMSO. The absorbance was then measured at 490 nm using a microplate reader to calculate the benzodiazepines described in this application. The inhibitory rate of the derivatives on the above-mentioned cells was finally calculated using SPSS software for all IC50 values. 50 .

[0051] The benzodiazepines described in this application Derivative T0 exhibits strong cytotoxicity against LOVO and HCT116 cells, with an IC50 value of [missing information]. 50 The values ​​are 4.38 μM and 5.87 μM, respectively.

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

[0053] The benzodiazepines described in this application The MTT assay results of derivatives T0 and 5-Fu against SW480 colon cancer cells are as follows: Figure 5 and Figure 6 As shown. Experimental verification confirms that the benzodiazepine described in this application... Derivative T0 IC50 against SW480 colon cancer cells 50 The concentration was 3.54 μM, while the IC50 of 5-FU against SW480 colon cancer cells was 3.54 μM. 50 The value was 10.73 μM, indicating that the benzodiazepine described in this application... The derivative T0 showed a significantly better inhibitory effect on SW480 cells than 5-Fu.

[0054] In summary, the benzodiazepines described in this 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 that of the positive control drug 5-Fu, and it has less toxicity to normal intestinal epithelial cells, showing the characteristics of high efficiency and low toxicity. It can be used to selectively treat colon cancer and has broad-spectrum anti-tumor activity.

[0055] 3. Cell scratch assay

[0056] First, using a ruler as a guide, draw evenly spaced horizontal lines on the back of a 6-well plate with a marker pen, approximately every 0.5–1 cm, passing through each well with at least 5 lines. Then, remove SW480 colon cancer cells in the logarithmic growth phase, discard the old culture medium, wash the cells twice with 1× PBS, and then digest them into a single-cell suspension using trypsin. (The text then repeats the process of adding cells at 2×10⁻⁶ ppm.) 5 Cells were seeded at a density of 100% per well in 6-well plates. The seeding principle was to achieve 100% confluence overnight, with a final total culture medium volume of 2 mL per well. The plates were then incubated at 37°C with 5% CO2 for 24 hours. The next day, a 10 μL pipette tip was used to make a vertical mark along the marker line on the back of the 6-well plate, ensuring the tip remained straight throughout the process. After marking, the cells were washed three times with PBS to remove cell debris. The marker line marks on the back of the 6-well plate were wiped away. Serum-free culture medium and different concentrations of benzodiazepines were then added. The serum-free culture medium containing derivative T0 was photographed under a 10x microscope. Image processing and result analysis were then performed using ImageJ software.

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

[0058] 4. Cell cloning experiments

[0059] SW480 cells were cultured in medium-sized cell culture dishes. When the cells reached 80% confluence, they were digested as described previously to prepare a cell suspension. The suspension was then seeded at a density of 800 cells / well in 24-well plates with a total culture medium of 500 μL. The seeded plates were then placed in a cell culture incubator and cultured for approximately 24 hours. The old culture medium was then discarded, and medium containing different concentrations of T0 was added for incubation for 7–10 days, with the T0 medium being changed every 48 hours. Once the cells reached a suitable density, the old culture medium was discarded, and the cells were washed twice with 1×PBS. The cells were then fixed with 300–400 μL of 4% paraformaldehyde solution for approximately 40 minutes. The fixative was then aspirated, and the 24-well plates were washed again with 1×PBS. The plates were then stained with 0.1% crystal violet solution for 20 minutes. After recovering the crystal violet solution, the plates were washed twice with 1×PBS, air-dried, and photographed.

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

[0061] 5. Hoechst 33258 staining experiment

[0062] SW480 cells were cultured in medium-sized cell culture dishes. When the cells reached 80% confluence, they were digested as described earlier to prepare a cell suspension; then, they were cultured in 2×10⁻⁶ cells / mL solution. 4 Cells were seeded at a density of 1 / well in 12-well plates with a total culture medium of 1000 μL. The seeded plates were then incubated in a cell culture incubator for approximately 24 hours. The old culture medium was then discarded, and medium containing different concentrations of T0 was added, followed by incubation for another 48 hours. The old culture medium was discarded, and the cells were washed twice with 1×PBS. A 1 mg / mL Hoechst 33258 solution was prepared using 1×PBS and then diluted to a concentration of 50 μg / mL. 400 μL of the diluted Hoechst 33258 staining solution was added to each well of the 12-well plate, and the plates were incubated for another 30 minutes. The Hoechst 33258 staining solution was then recovered, and the cells were washed twice with 1×PBS. After the final wash, a small amount of PBS was left in the cell culture plate, and the cells were immediately observed using an inverted fluorescence microscope. Apoptotic cells showed enhanced nuclear staining, brighter fluorescence, and exhibited round, condensed, or clumped structures.

[0063] The results are as follows Figure 9 As shown, after treating SW480 cells with different concentrations of benzo[a]aza derivative T0 for 48 hours, the cell morphology of SW480 cells changed significantly with the increase of compound concentration. The cell nuclei became round or shrunken, and the number of cells with strong blue fluorescence increased, showing a concentration-dependent effect.

[0064] In summary, the benzodiazepines described in this application... The derivatives exhibit anti-colon cancer, pancreatic cancer, cervical cancer, gastric cancer, and liver cancer activities, and show low toxicity to normal intestinal epithelial cells (HIEC), making them potential broad-spectrum anticancer drugs. Furthermore, the benzodiazepines #imgpt55# derivatives described above have good selectivity for colon cancer cells, inhibiting their proliferation and migration, and promoting apoptosis. Their therapeutic effect on colon cancer is superior to that of the clinical drug 5-Fu, demonstrating promising application prospects.

[0065] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. However, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

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

2. The 1,5-benzodiazepine derivative of claim 1 for use in the preparation of a medicament for the treatment of a neoplasm selected from the group consisting of colon cancer, pancreatic cancer, liver cancer, gastric cancer, cervical cancer. for use in the preparation of a medicament for the treatment of a neoplasm selected from the group consisting of colon cancer, pancreatic cancer, liver cancer, gastric cancer, cervical cancer.

3. Use according to claim 2, wherein the compound is ###0002### The tumor is colon cancer.

4. A pharmaceutical preparation, characterized by, The pharmaceutical preparation is 1,5-benzodiazepine according to claim 1 The derivatives are incorporated into pharmaceutically acceptable carriers and / or adjuvants to make any pharmaceutically acceptable dosage form.

5. The pharmaceutical preparation according to claim 4, characterized in that The dosage form includes any dosage form of capsule, granule, tablet, spray, oral liquid, suspension, and injection.

6. The 1,5-benzodiazepine derivative of claim 1, wherein A process for the preparation of a 1,5-benzodiazepine derivative, characterized in that, The method comprises: The o-phenylenediamine is mixed with (1E, 4E)-1, 5-di-p-toluene-1, 4-pentadiene-3-ketone, heated to reflux in anhydrous ethanol and triethylamine as a solvent, column chromatography, and recrystallized in anhydrous ethanol to obtain.

7. The production method according to claim 6, wherein The mass ratio of the o-phenylenediamine to (1E, 4E)-1, 5-di-p-toluene-1, 4-pentadiene-3-ketone is 1-2: 2-3.

8. The production method according to claim 6, wherein The triethylamine and anhydrous ethanol are jointly added in a volume ratio of 1:4, and the total amount is 25ml; the heating reflux time is 48h.

9. The production method according to claim 6, wherein A mixture of n-hexane and ethyl acetate in a volume ratio of 93:7 is used as an eluent for column chromatography.

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