6-Azophenylcoumarin-3-carboxamide compound and its preparation method and application

Abstract

The present invention belongs to the field of medical technology and drug synthesis technology, and is particularly related to a 6-azophenylcoumarin-3-formamide compound and its preparation method and application. First, 5-azophenyl salicylaldehyde and diethyl malonate are used to produce a Claisen ester condensation reaction to prepare 6-azophenylcoumarin-3-ethyl formate, and then 6-azophenylcoumarin-3-ethyl formate and halogenated benzylamine or amine substituted alkyl pyridine are reacted through aminolysis to prepare 6-azophenylcoumarin-3-formamide compound. The present invention expands the types of amide coumarin compounds and lays the foundation for studying the relationship between the structure and performance of coumarin amide compounds. At the same time, this type of compound has good anticancer activity against lung cancer cells and breast cancer cells, and is less toxic, providing a new idea for the development of anticancer drugs. The preparation method of the compound is simple and easy to operate, the required items are less toxic, and natural products are combined with good practicality.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology and drug synthesis technology, and in particular relates to a 6-azophenylcoumarin-3-carboxamide compound, a preparation method and an application thereof. Background Art

[0002] Cancer, caused by the uncontrolled proliferation of cancer cells, is a disease with a high mortality rate and a serious threat to human health. While chemotherapy is a common treatment for cancer, it can also cause adverse reactions such as inflammation, nausea and vomiting, hair loss, anemia, and thrombocytopenia. Therefore, the development of new, more effective, and less harmful anticancer drugs is crucial. Natural products are a treasure trove left to us by nature. Millions of years of evolution have ensured that most natural products possess certain biological activities. Natural products are structurally rich and have diverse effects. For example, kaempferol extract and paclitaxel extract can both inhibit the proliferation of liver cancer cells and exhibit significant antiproliferative activity. Therefore, the search for active ingredients from natural products for cancer prevention and treatment has long been a hot research topic among pharmacologists and chemists.

[0003] Coumarin is a class of natural products with benzopyrone structures extracted from legumes. Coumarin has a variety of pharmacological properties, such as antibacterial, anticoagulant, anticancer and antiviral activities. In addition, there are multiple modification sites on the coumarin parent structure, which can be modified to obtain coumarin derivatives with better biological activity. For example, coumarin acylhydrazone derivatives (CHHD) were found to have certain anticancer activity against human pancreatic cancer cells (Panc-1) (IC 50 =0.129 μM), flow cytometry showed that CHHD induced apoptosis by arresting the Panc-1 cell cycle at the G2 / M phase. It has been reported that 4-hydroxyphenol-6-aminobutanol coumarin has certain anticancer activity against human breast cancer cells (MCF-7) (IC 50 =2.9 μM), and has low toxicity to normal human hepatocytes (selectivity index SI; SI>20; SI = normal cell IC 50 Value / liver cancer cell IC 50 Its mechanism of action is to induce apoptosis by arresting cells in the G2 / M phase. In vitro cell viability assays demonstrated that the furanocoumarin derivative aflatoxin (AME) exhibited antiproliferative effects on human hepatocellular carcinoma (HepG-2) cells (IC50 = 6.9 μg / mL). Treatment of HepG-2 cells with 6.9 μg / mL AME induced significant changes in DNA and cell cycle activity (increases in the pre-apoptotic G0 / G1 and G2 / M phases and a decrease in the S phase). Furthermore, AME significantly increased Annexin V responses and the number of HepG-2 cells at both the early and late stages of apoptosis compared to control cells.

[0004] To date, many bioactive coumarins have been designed and synthesized. However, these reported coumarins still suffer from poor anticancer activity and high toxicity. Therefore, the design and synthesis of highly effective and low-toxic coumarins is of great significance. Summary of the Invention

[0005] In view of the above problems existing in the prior art, the object of the present invention is to provide a 6-azophenylcoumarin-3-carboxamide compound and a preparation method and application thereof.

[0006] In order to solve the above problems, the technical solutions adopted by the present invention are as follows:

[0007] A method for preparing a 6-azophenylcoumarin-3-carboxamide compound is characterized in that the compound is prepared by aminolysis reaction of 6-azophenylcoumarin-3-ethylcarboxylate and halogenated benzylamine or aminoalkylpyridine.

[0008] The 6-azophenylcoumarin-3-carboxamide compound obtained by the above preparation method.

[0009] Optionally, its structural formula is:

[0010] ,

[0011] Wherein R is 2-pyridyl or m-substituted phenyl or p-substituted phenyl; and the substituent is halogen.

[0012] Optionally, the halogenated benzylamine is m-fluorobenzylamine or p-fluorobenzylamine; and the aminoalkylpyridine is 2-(aminomethyl)pyridine.

[0013] Optionally, the aminolysis reaction comprises the following specific steps: dissolving 6-azophenylcoumarin-3-carboxylic acid ethyl ester in anhydrous ethanol, heating under reflux, adding halogenated benzylamine or 2-aminomethylpyridine thereto for reaction, filtering, recrystallizing with anhydrous ethanol, and filtering and drying.

[0014] Optionally, the ratio of ethyl 6-azophenylcoumarin-3-carboxylate to the halogenated benzylamine or 2-aminomethylpyridine is 1:1.

[0015] Optionally, the 6-azophenylcoumarin-3-ethylcarboxylate is obtained by a Claisen ester condensation reaction of 5-azophenylsalicylaldehyde and diethyl malonate.

[0016] Optionally, the specific steps for preparing 6-azophenylcoumarin-3-ethyl carboxylate are: dissolving 5-azophenylsalicylaldehyde in anhydrous ethanol, heating under reflux, adding diethyl malonate, piperidine and glacial acetic acid thereto to react, cooling after the reaction to precipitate a solid, and purifying and drying the solid.

[0017] Optionally, the amount ratio of the 5-azophenyl salicylaldehyde to the diethyl malonate is 1:(1.2-7).

[0018] Application of the above 6-azophenylcoumarin-3-carboxamide compound in anticancer aspects.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The 6-azophenylcoumarin-3-carboxamide compound with heteroatoms designed and synthesized by the present invention has good anticancer activity and low toxicity;

[0021] (2) The 6-azophenylcoumarin-3-carboxamide compound of the present invention has good anticancer activity against lung cancer cells and breast cancer cells, and has low toxicity, providing new ideas for the development of anticancer drugs;

[0022] (3) The preparation method of the 6-azophenylcoumarin-3-carboxamide compound designed and synthesized by the present invention is easy to operate, the substances required in the method are low in toxicity, and the method is combined with natural products, which has good practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The synthetic route of the 6-azophenylcoumarin-3-carboxamide compound provided by the present invention is

[0024] Figure 2 This is an analysis chart of the apoptosis test results of the 6-azophenylcoumarin-3-carboxamide compound provided by the present invention;

[0025] Figure 3 This is an analysis chart of the cell cycle detection results of the 6-azophenylcoumarin-3-carboxamide compound provided by the present invention;

[0026] Figure 4 The diagram shows ultraviolet and fluorescence spectra of the interaction between the 6-azophenylcoumarin-3-carboxamide compound provided by the present invention and DNA. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to specific embodiments.

[0028] The preparation method of 5-azophenyl salicylaldehyde is as follows:

[0029] Add 1.68 g of aniline to a hydrochloric acid dilution solution of 15 ml of concentrated hydrochloric acid and 9 ml of distilled water, and stir to mix. Control the temperature at 0-5°C, and add dropwise a mixed solution of 1.242 g of sodium nitrite and 6 ml of water while stirring, and react for 1.5 hours to obtain a diazonium salt solution. Add 13.35 g of Na2CO3 to 90 ml of water, and then add dropwise 1.89 ml of salicylaldehyde to obtain a salicylaldehyde solution. Add the prepared diazonium salt solution dropwise to the salicylaldehyde solution while stirring at 0-5°C, react in an ice bath for 2 hours, and filter to obtain 5-azobenzene salicylaldehyde.

[0030] The preparation method of 6-azophenylcoumarin-3-ethylcarboxylate is as follows:

[0031] Place 3.5 mmol (0.791 g) of 5-azophenyl salicylaldehyde in a 100 ml single-necked flask. Add 15 ml of anhydrous ethanol and heat under reflux until the solution is completely dissolved and turns yellow. Add 17.5 mmol (2.8 g, 2.65 ml) of diethyl malonate, 5 drops of piperidine, and 5 drops of glacial acetic acid. Allow to react for 6 hours, then cool to room temperature. A large amount of solid precipitates. Filter, rinse with glacial ethanol, and dry to obtain orange-red needle-shaped crystals (0.7 g, 62%).

[0032] Example 1

[0033] In a 100 ml three-necked flask, 2.00 mmol (0.644 g) of 6-azophenylcoumarin-3-carboxylic acid ethyl ester was added, followed by 40 ml of anhydrous ethanol. The mixture was heated to reflux, and then 2.00 mmol of m-fluorobenzylamine was added. After reacting for 6 h, the mixture was filtered off with suction. The crude product was recrystallized from anhydrous ethanol, filtered, dried, and weighed to obtain 0.5584 g of an orange solid, which was recorded as compound 1 (6-azophenylcoumarin-3-formyl-(3-fluorobenzylamine)). The synthetic route of 6-azophenylcoumarin-3-carboxamide compounds can be found in Figure 1 The yield is 70%, and the melting point is 240.1℃~241.8℃. H NMR spectrum analysis 1 H NMR (CDCl3, 600 MHz, δ / ppm, TMS as internal standard): δ 9.208-9.189 (t, J = 5.8 Hz, 1H), 9.100 (s, 1H), 8.303-8.287 (m, J = 4.8 Hz, 2H), 7.993 (m, J = 4.5 Hz, 2H), 7.596-7.540 (dd, J = 10.1 Hz, 4H),7.363 (td, J= 8.0 Hz, 1H), 7.179-7.166 (d, J= 7.7 Hz, 1H), 7.112-7.095 (m,1H), 7.023-6.991 (td, J = 8.3, 2.5 Hz, 1H), 4.712-4.702 (d, J = 6.0 Hz, 2H); C-NMR analysis 13 C NMR (CDCl3, 151 MHz, δ / ppm, TMS as internal standard): δ 161.36, 161.09, 155.63,152.25, 149.48, 140.44, 140.40, 131.83, 130.29, 130.24, 129.27, 127.57,125.10, 123.21, 123.14, 118.95, 118.89, 117.61, 114.69, 114.54, 114.53,114.39, 43.38; Infrared spectroscopy analysis IR (KBr) v max / cm -1 : 3331(NH), 1720(C=O), 1655(C=O),1612(N=N); mass spectrometry analysis HRMS m / z [M+Na] + Calculated value: 424.1068, Measured value: 424.1064.

[0034] Example 2

[0035] In a 100ml three-necked flask, 2.00 mmol (0.644g) of ethyl 6-azophenylcoumarin-3-carboxylate was added, followed by 40ml of anhydrous ethanol. The mixture was heated to reflux, followed by 2.00 mmol of p-fluorobenzylamine. The reaction was allowed to proceed for 6h and then filtered. The crude product was recrystallized from anhydrous ethanol, filtered, dried, and weighed to yield 0.4590g of an orange solid, designated as compound 2 (6-azophenylcoumarin-3-formyl-(4-fluorobenzylamine)). The yield was 57%, and the melting point was 240.1-241.8°C. H NMR analysis 1 H NMR (CDCl3, 600 MHz, δ / ppm, TMS as internal standard): δ 9.162 (d, J = 6.3 Hz, 1H), 9.093 (s,1H), 8.297 (d, J = 7.6 Hz, 2H), 7.989 (d, J = 7.5 Hz, 2H), 7.593 (m, J = 10.1 Hz,4H), 7.384 (dd,J = 8.3, 5.4 Hz, 2H), 7.081 (t, J = 8.5 Hz, 2H), 4.676 (d, J = 5.9Hz, 2H); C-NMR analysis 13 C NMR (CDCl3, 151 MHz, δ / ppm, TMS as internal standard): δ 161.24,161.07, 155.61, 152.25, 149.47, 148.71, 148.50, 131.83, 129.52, 129.46,129.27, 129.09, 127.53, 125.07, 123.14, 123.08, 122.56, 119.02, 118.89,117.60, 115.68, 115.53, 43.23; Infrared spectroscopy analysis IR (KBr) v max / cm -1 : 3341(NH), 1721(C=O), 1649(C=O), 1618(N=N); mass spectrometry analysis HRMS m / z [M+Na] + Calculated: 424.1068, Measured: 424.1059.

[0036] Example 3

[0037] In a 100 ml three-necked flask, 2.00 mmol (0.644 g) of ethyl 6-azophenylcoumarin-3-carboxylate was added, followed by 40 ml of anhydrous ethanol. The mixture was heated to reflux, followed by 2.00 mmol (0.324 g) of 2-(aminomethyl)pyridine. The reaction was allowed to proceed for 6 h, followed by filtration. The crude product was recrystallized from anhydrous ethanol, filtered, dried, and weighed to yield 0.585 g of a yellow solid, designated as compound 3 (6-azophenylcoumarin-3-formyl-(2-aminomethylpyridine)). The yield was 76%, and the melting point was 216.0°C-217.4°C. H NMR analysis 1 H NMR (CDCl3., 600 MHz, δ / ppm, TMS as internal standard): δ 9.618-9.602 (t, J =4.8 Hz, 1H), 9.070 (s, 1H), 8.640 (d, J = 4.7 Hz, 1H), 8.273 (d, J = 8.2 Hz, 2H),7.969 (d, J= 7.3 Hz, 2H), 7.705 (t, J = 8.1 Hz, 1H),7.572 (dd, J = 10.5 Hz, 4H),7.360 (d, J = 7.8 Hz, 1H), 7.236 (m, J = 6.3 Hz, 1H), 4.843 (d, J = 5.4 Hz, 2H); C NMR analysis 13 C NMR (CDCl3., 151 MHz, δ / ppm, TMS as internal standard): δ 161.36, 160.85, 156.58, 155.69, 152.24, 149.51, 149.39, 148.44, 136.78, 131.78, 129.25, 127.40, 125.06, 123.12, 122.40, 121.78, 119.20, 118.90, 117.59, 45.44; Infrared spectroscopy analysis IR (KBr) v max / cm -1 : 3331(NH), 1729(C=O), 1656(C=O), 1616(N=N); mass spectrometry analysis HRMS m / z [M+Na] + Calculated value: 407.1115, Measured value: 407.1118.

[0038] Comparative Example 1

[0039] In a 100ml three-necked flask, 2.00 mmol (0.644g) of ethyl 6-azophenylcoumarin-3-carboxylate was added, followed by 40ml of anhydrous ethanol. The mixture was heated to reflux, and then 2.00 mmol of o-fluorobenzylamine was added. After reacting for 6h, the mixture was filtered and the crude product was recrystallized from anhydrous ethanol, filtered, dried, and weighed to obtain 0.5019g of an orange solid, designated as compound 4 (6-azophenylcoumarin-3-formyl-(2-fluorobenzylamine)). The yield was 63%, and the melting point was 208.2-208.9°C. H NMR analysis 1 H NMR (CDCl3, 600 MHz, δ / ppm, TMS as internal standard) δ 9.183 (d, J = 6.1 Hz, 1H), 9.079 (s,1H,), 8.292 (m, J = 5.7 Hz, 2H), 7.987 (m, J= 6.6 Hz, 2H,), 7.591 (m, J = 6.6 Hz,4H), 7.457 (td, J = 7.5, 1.8 Hz, 1H), 7.325 (td, J = 7.8 Hz, 1H,), 7.169 (m, J =7.5 Hz, 1H), 7.121 (m, J = 8.4Hz, 1H), 4.766 (d, J = 5.9 Hz, 2H); C-NMR analysis 13 C NMR (CDCl3, 151 MHz, δ / ppm, TMS as internal standard) δ 161.83, 161.28, 160.99, 160.20,155.62, 152.24, 149.43, 148.64, 131.80, 130.02, 129.26, 127.44, 125.10,124.77, 123.13, 119.05, 118.88, 117.58, 115.58, 115.44, 37.91; Infrared spectrum analysis IR (KBr) v max / cm -1 : 3331(NH), 1720(C=O), 1655(C=O), 1612(N=N); mass spectrometry analysis HRMS m / z [M+Na] + Calculated value: 424.1068, Measured value: 424.1060.

[0040] Comparative Example 2

[0041] Under the same reaction conditions as in Example 1, compound 5 (coumarin-3-formyl-(3-fluorobenzylamine)) was obtained from m-fluorobenzylamine and coumarin-3-carboxylic acid ethyl ester with a yield of 60.6% and a melting point of 170.8-171.6°C. H NMR analysis 1 H NMR (DMSO- d 6., 600 MHz, δ / ppm, TMS as internal standard): δ 9.18 (t, J =12.6Hz, 1H), 8.86 (s, 1H), 7.96 (d, J =7.8 Hz, 1H) , 7.75~7.72 (m, 1H) , 7.50 (d, J =7.8 Hz, 1H), 7.44~7.41 (t,J =15.0 Hz, 1H) , 7.39~7.35 (m, 1H) , 7.20~7.16 (m, 2H) , 7.08 (t, J =10.8 Hz ,1H), and 4.57 (d, J =6.0 Hz, 2H); C-NMR analysis 13 C NMR (DMSO- d 6, 151 MHz, δ / ppm, TMS as internal standard) δ 163.51, 161.91, 160.73, 154.36, 148.00, 142.55, 142.51,134.54, 130.75, 130.71, 130.70, 125.57, 123.80, 123.79, 119.55, 118.92,116.58, 114.57, 114.43, 114.16, 114.03, 42.73, 42.72; Infrared spectrum analysis IR (KBr) v max / cm -1 :3321(-CONH-), 3053(=CH), 1705(C=O), 1655(C=O); Mass spectrometry analysis HRMS m / z [M+H] + Calculated: 298.0874, Found: 298.0885. (Shi JZ, Lu W, Chen J C. Synthesis, antiproliferative activities and DNA binding of coumarin-3-formamidoderivatives[J]. Arch Pharm. 2021: 354.)

[0042] Comparative Example 3

[0043] Under the same reaction conditions as in Example 2, compound 6 (coumarin-3-formyl-(4-fluorobenzylamine)) was obtained from p-fluorobenzylamine and coumarin-3-carboxylic acid ethyl ester with a yield of 64.6% and a melting point of 174.4-175.6°C. H NMR analysis 1 H NMR (DMSO- d 6., 600 MHz, δ / ppm, TMS as internal standard): δ 9.09 (t, J=12.6Hz, 1H), 8.85 (s, 1H),7.97 (m, 1H), 7.76 (m, 1H), 7.49 (m, 4H), 7.17~ 7.11 (m ,2H), and 4.54 (d, J =12.0 Hz, 2H); C-NMR analysis 13 C NMR (DMSO- d 6, 151 MHz, δ / ppm, TMS as internal standard) δ 163.06,162.52, 161.92, 160.74, 156.44, 155.00, 154.35, 149.11, 148.02, 135.68,135.66, 134.93, 134.55, 130.74, 130.71, 129.95, 129.90, 125.58, 125.30,119.51, 118.92, 118.27, 118.16, 116.61, 116.59, 115.60, 115.46, 42.51;Infrared spectrum analysis IR (KBr) v max / cm -1 : 3332(-CONH-), 3049(=CH, Ar-H), 1703(C=O), 1655(C=O); Mass spectrometry analysis HRMS m / z [M+H] + Calculated: 320.0693, Found: 320.0708. (Shi JZ, Lu W, Chen J C. Synthesis, antiproliferative activities and DNA binding of coumarin-3-formamido derivatives[J]. Arch Pharm. 2021: 354.).

[0044] Comparative Example 4

[0045] Compound 7 (7-(3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide)-oxyethoxy-coumarin-3-formyl-(4-fluorobenzylamine)) was obtained by the reaction of 7-(2-hydroxyethoxy)coumarin-3-formyl-(4-fluorobenzyl) and 3,4-diphenylsulfonyl-1,2,5-oxadiazole-2-oxide with a yield of 57% and a melting point of 169-172°C. H NMR analysis 1 H NMR (DMSO- d6., 400 MHz, δ / ppm, TMS as internal standard): δ 9.10 (t, J = 6.2 Hz, 1H), 8.87 (s, 1H), 7.96 (dd, J 1= 8.4 Hz, J 2= 2.9 Hz, 3H), 7.82 (t, J = 7.6 Hz, 1H), 7.65 (t, J = 7.6Hz, 2H), 7.39 (dd, J 1= 8.4 Hz, J 2= 5.8 Hz, 2H), 7.21 (d, J = 0.8 Hz, 1H), 7.16 (t, J = 8.7 Hz, 2H), 7.10 (dd, J 1= 8.7, J 2= 1.5 Hz, 1H), 4.77 (t, J = 4.0 Hz, 2H), 4.53(s, 2H), 4.51 (t, J = 4.0 Hz, 2H); C NMR analysis 13 C NMR (DMSO- d 6, 151 MHz, δ / ppm, TMS as internal standard) δ 37.02, 135.94, 134.25, 129.78, 129.70, 129.65, 128.13, 126.66,121.12, 115.10, 114.86, 114.74, 113.90, 112.44, 110.38, 101.10, 68.56, 65.93,32.21, 15.04; HRMS m / z [M+H] +Calculated: 583.1, Found: 582.5. (Yu Z, LiM, Wang K, et al. Novel Hybrids of 3-Substituted Coumarin and Phenylsulfonylfuroxan as Potent Antitumor Agents with Collateral Sensitivity against MCF-7 / ADR[J]. J. Med. Chem. 2022, 65, 13: 9328-9349.)

[0046] Comparative Example 5

[0047] Compound 8 (coumarin-3-formyl-(3-aminomethylpyridine)) was obtained from 3-aminomethylpyridine and coumarin-3-carboxylic acid ethyl ester with a yield of 75% and a melting point of 188.4~189.7℃. H NMR analysis 1 H NMR (CDCl3, 600 MHz, δ / ppm, TMS as internal standard): δ 4.663-4.673 (d, J =6Hz, 2H), 7.267-7.280 (t, J =7.8Hz, 1H),7.367-7.407 (m, 2H), 7.653-7.706 (m, 3H), 8.522-8.532 (q, J =1.2Hz, 1H), 8.620-8.623 (d, J =1.8Hz, 1H),8.931 (s, 1H), 9.233 (s, 1H); C NMR analysis 13 C NMR (CDCl3, 151 MHz, δ / ppm, TMS as internal standard) δ 41.12, 116.49, 117.91, 118.35, 123.38,125.20, 129.68, 133.47, 134.10, 135.32, 148.63, 148.66, 149.05, 154.27,161.23, 161.60; Infrared spectrum analysis IR (KBr) v max / cm -1 : 3316(-NH), 1706(-C=O), 1650(NHC=O); mass spectrometry analysis HRMS m / z [M+H] + Calculated: 281.09124, Measured: 281.09244.

[0048] Experimental Example Study on the Anticancer Activity of Coumarin Carboxamide Compounds

[0049] 1. Anticancer Activity Testing of Compounds

[0050] The specific operation of the MTT method is as follows: HepG-2, MCF-7, A549, HeLa cancer cells and HUVEC normal cells were cultured in MEM medium, α-MEM medium, F12k medium and DMEM medium (the medium contains D-glucose, L-glutamine, penicillin and streptomycin) containing 10% fetal bovine serum, respectively, and cultured under humid conditions of 37°C and 5% CO2. MTT (5 mg / mL) was dissolved in phosphate-buffered saline (PBS, pH=7.4), filtered and stored at −20°C in the dark. First, the cultured cells in the logarithmic phase were digested, centrifuged and counted. 100 μL of 5-8×10 3 Cells were added to each well of the 96-well plate and cultured at 37°C, 5% CO2, and 90% humidity for 24 hours. Different concentrations of target compounds were added to the 96-well plate. After 48 hours, the old culture medium was aspirated, 100 μL of MTT (1 mg / mL) was added to each well and incubated for 4 hours. The MTT solution was completely removed, and 100 μL of DMSO was added to each well and shaken for 10 minutes to dissolve the purple formazan. DMSO solution was used as a negative control, and the absorbance was read at 595 nm using a microplate reader, and the half-inhibitory concentration IC for cancer cells was calculated. 50 values (see Table 1 for the results).

[0051] II. Apoptosis Test of Compounds 1, 2, and 3

[0052] The effects of compounds 1, 2, and 3 on apoptosis of A549 and MCF-7 cells were analyzed by Annexin V-FITC / PI staining. First, 2 mL of 2 × 10 6 Cells were plated at 4% RI / well and incubated for 24 hours. Compounds 1, 2, and 3 were diluted in culture medium at various concentrations and incubated with the cells for 24 hours. The culture medium was discarded, and the cells were washed twice with PBS, digested with EDTA-free trypsin, harvested (2000 rpm / 5 minutes), and rinsed twice again with PBS. After centrifugation, the supernatant was discarded, and 500 μL of binding buffer, 5 μL of Annexin V-FITC, and 5 μL of PI were added. The cells were incubated in the dark at room temperature for 10 minutes and immediately assayed.

[0053] 3. Cell cycle detection of compound 1 and compound 3

[0054] Plating and cell incubation times for cell cycle experiments were consistent with those for apoptosis experiments. After incubating the samples with cells for 24 hours, the medium was discarded, the cells were washed with PBS, digested with EDTA-free trypsin, harvested (2000 rpm / 5 minutes), and washed again with PBS. The supernatant was discarded, and the cells were fixed with 75% anhydrous ethanol for at least 2 hours at 4°C, washed with PBS, and centrifuged. After discarding the supernatant, 500 μL of RNase A was added, and the cells were resuspended and incubated at room temperature in the dark for 30 minutes before assay.

[0055] IV. Interactions between Compounds 1, 2, and 3 and DNA

[0056] HS-DNA was diluted to 0.2 × 10 −3 M (pH = 7.2; A 260 / A 280 Compounds 1 and 2 were diluted to 1 × 10 −3 M, 5 µL of HS-DNA stock solution (1 × 10 –3 M) were added with 3 mL of compound solution (1 × 10 −5 M) and the UV absorption intensity was detected at 298 nm. Ethidium bromide (EB) was added to the HS-DNA solution (1 × 10 −4 M) to make the concentration of EB 1×10 –5 M and incubate in the dark for 2 hours. 5 μL of compound solution (1 × 10 −3 M), and the fluorescence intensity was measured at an excitation wavelength of 420 nm. The quenching intensity of the fluorescence intensity of the EB-HS-DNA system was calculated using the Stern–Volmer equation as follows:

[0057] I0 / I = 1 + K q [Q],

[0058] Where I0 is the fluorescence intensity of the HS-DNA–EB system at 615 nm, I is the fluorescence intensity of the EB–HS-DNA system after the addition of the compound; [Q] is the concentration of the compound, K q is the quenching constant.

[0059] The experimental results are as follows:

[0060] As can be seen from Table 1, fluorobenzene-modified compounds 1, 2, and 4 have no anti-proliferative effect on MCF-7 cells, but compound 1 with meta-fluorine substitution and compound 2 with para-fluorine substitution have strong inhibitory effects on HeLa, A549, and HepG-2 cells. Compounds 1 and 2 have particularly strong anti-proliferative effects on A549 cells, with IC 50 The values were 0.51µmol / L and 0.29µmol / L, respectively, and the SI values were 9.38 and 13.69, respectively, which were higher than those of DOX (SI = 1.36). Compared with m- and p-fluorine-substituted compounds, o-fluorine-substituted compound 4 exhibited poor antiproliferative activity against cancer cells and was also highly toxic to normal HUVEC cells. Pyridine-modified compound 3 exhibited excellent antiproliferative activity against MCF-7 cells, with an antiproliferative capacity 49-fold higher than that of the positive control DOX. Its safety factor was nearly 3000-fold higher than that of DOX, indicating its reduced toxicity to normal cells and its potential as an anticancer drug. The anti-proliferative activity of compound 7 with para-fluorine substitution of the sulfonyl group in the comparative example against MCF-7 cells is also far inferior to that of compounds 1, 2, and 3. In the comparative example, compound 5 with meta-fluorine substitution without azophenyl substitution, compound 6 with para-fluorine substitution without azophenyl substitution, and compound 8 with 3-aminomethylpyridine substitution without azophenyl have no obvious anti-proliferative activity against cancer cells.

[0061] Table 1 Half inhibitory concentration IC of compounds against cancer cells 50 (μM)

[0062]

[0063] The apoptosis results of compound 1, compound 2 and compound 3 are shown in Figure 2. Figure 2 As shown, compared with the control group, after co-incubation of A549 cells with compound 1, the number of cancer cells undergoing apoptosis increased significantly with increasing compound 1 concentration. At a 20 μM concentration, the number of apoptotic cells increased from 2.43% to 50.40% compared with the control group, demonstrating that compound 1 can induce apoptosis in A549 cells. After co-incubation of A549 cells with compound 2 for 24 hours, the number of cancer cells undergoing apoptosis increased with increasing compound 2 concentration, but the apoptotic effect was not significant. At a 20 μM concentration, the number of cells undergoing apoptosis was only 3.39%, indicating that compound 2 has a weak ability to induce apoptosis in A549 cells. When compound 3 was co-incubated with MCF-7 cells for 24 hours, the number of cancer cells undergoing apoptosis increased significantly with increasing compound 3 concentration. At a 20 μM concentration, the proportion of cells undergoing apoptosis increased from 7.36% in the control group to 28.24%, demonstrating that compound 3 can induce apoptosis in MCF-7 cells.

[0064] The cell cycle results of compound 1 and compound 3 are as follows Figure 3 As shown, after 24 hours of incubation of A549 cells with compound 1 at various concentrations (0, 0.5, 5, and 20 μM), the number of cells in the G0 / G1 phase decreased from 53.93% (0 μM) to 47.81% (20 μM) compared to the control group, while the number of cells in the S phase significantly increased from 18.55% to 39.96%. Therefore, compound 1 can block A549 cells in the S phase, thereby inhibiting their proliferation. When MCF-7 cells were incubated with compound 3 at various concentrations (0, 0.5, 5, and 20 μM) for 24 hours, the number of cells in the G0 / G1 phase decreased from 56.32% to 36.55%, a significant decrease of 19.77% compared to the control group. Furthermore, the number of cells in the G2 / M phase showed a downward trend, while the number of cells in the S phase increased from 16.29% to 41.69%. Therefore, compound 3 can block MCF-7 cells in the S phase, thereby inhibiting their proliferation. In conclusion, compound 1 and compound 3 arrested A549 and MCF-7 cells at S phase, respectively, in a concentration-dependent manner, and inhibited cell proliferation by blocking the DNA synthesis process.

[0065] UV and fluorescence tests of the interaction of compound 1, compound 2, and compound 3 with DNA Figure 4 As shown, UV-visible absorption spectroscopy experiments showed that the addition of HS-DNA caused the characteristic absorption peaks of compounds 1, 2, and 3 to red-shift, and the hypochromic effect increased with the increase of HS-DNA concentration, indicating that there was an intercalation pattern between compounds 1, 2, and 3 and HS-DNA. EB is a highly conjugated molecule that emits strong fluorescence when bound to DNA. The addition of compounds will cause changes in the fluorescence intensity of the EB–DNA solution, which can indirectly study the interaction between DNA and compounds that can replace EB from the EB–DNA adduct. The test results show that after the addition of compounds 1, 2, and 3, the fluorescence intensity of EB–HS-DNA gradually decreased, indicating that compounds 1, 2, and 3 all replaced the EB bound to HS-DNA and interacted with DNA. According to the Stern-Volmer equation, the quenching constants of compounds 1, 2, and 3 are 3.75×10 5 M, 4.9×10 6 M and 5.17×10 5 The larger the quenching constant of a compound, the stronger its ability to bind to HS-DNA.

Claims

1. A method for preparing a 6-azophenylcoumarin-3-carboxamide compound, characterized in that: The product is prepared by aminolysis reaction of 6-azophenylcoumarin-3-carboxylic acid ethyl ester with halogenated benzylamine or alkylamine substituted pyridine. The halogenated benzylamine is m-fluorobenzylamine or p-fluorobenzylamine, and the alkylamine substituted pyridine is 2-(aminomethyl)pyridine.

2. A 6-azophenylcoumarin-3-carboxamide compound, characterized in that Prepared according to the method of claim 1, its structural formula is: , Wherein R is 2-pyridyl or m-substituted phenyl or p-substituted phenyl; and the substituent is fluoro.

3. The method for preparing a 6-azophenylcoumarin-3-carboxamide compound according to claim 1, wherein The aminolysis reaction specifically comprises the following steps: dissolving 6-azophenylcoumarin-3-ethylcarboxylate in anhydrous ethanol, heating under reflux, adding halogenated benzylamine or 2-aminomethylpyridine to the solution for reaction, filtering, recrystallizing with anhydrous ethanol, and filtering and drying.

4. The method for preparing a 6-azophenylcoumarin-3-carboxamide compound according to claim 1, wherein The molar ratio of 6-azophenylcoumarin-3-carboxylic acid ethyl ester to halogenated benzylamine or 2-aminomethylpyridine is 1:

1.

5. The method for preparing a 6-azophenylcoumarin-3-carboxamide compound according to claim 1, wherein: The 6-azophenylcoumarin-3-ethyl carboxylate is obtained by a Claisen ester condensation reaction between 5-azophenylsalicylaldehyde and diethyl malonate.

6. The method for preparing a 6-azophenylcoumarin-3-carboxamide compound according to claim 5, wherein: The specific steps for preparing ethyl 6-azophenylcoumarin-3-carboxylate are as follows: dissolving 5-azophenylsalicylaldehyde in anhydrous ethanol, heating under reflux, adding diethyl malonate, piperidine and glacial acetic acid thereto to carry out a reaction, cooling after the reaction to precipitate a solid, and purifying and drying the solid.

7. The method for preparing a 6-azophenylcoumarin-3-carboxamide compound according to claim 5, wherein: The amount ratio of the 5-azophenyl salicylaldehyde to the diethyl malonate is 1:(1.2-7).

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