Coumarin-thiosemicarbazide compound and application thereof

Abstract

The invention discloses a coumarin-thiosemicarbazide compound and application of the coumarin-thiosemicarbazide compound. The preparation method comprises the following steps: firstly, synthesizing a coumarin-thiosemicarbazide derivative, and carrying out in-vitro antioxidant activity screening to obtain preferable derivatives 4g and 4i; analyzing an antioxidant action target based on network pharmacology and molecular docking, and performing intersection mapping on the action target and the antioxidant target of the compounds 4g and 4i to obtain a common target; constructing a PPI interaction network, and screening to obtain potential core targets; carrying out GO function and KEGG pathway enrichment analysis on the core target spot to obtain a related biological process and signal pathway; and carrying out molecular docking operation on the key target spot and compounds 4g and 4i to verify the binding capacity of the compounds and the target spot. Potential antioxidant targets and modern biological mechanisms of the derivatives 4g and 4i are preliminarily revealed by using a network pharmacology method, and important guidance and basis are provided for development of antioxidant drugs.

Description

Technical Field

[0001] The present invention belongs to the fields of pharmacology and bioinformatics, and particularly relates to a coumarin-thiosemicarbazide compound and its application. Background Art

[0002] In a healthy human body, normal metabolic processes produce free radicals and other highly reactive substances, such as ions, molecules with unpaired electrons, reactive oxygen species, carbon, nitrogen or sulfur species (ROS, RCS, RNS or RSS). Although these free radicals and highly reactive substances are crucial for certain physiological processes (such as cell signaling and immune defense), their excessive production can disrupt the delicate balance between oxidants and antioxidants, thereby causing oxidative stress. In cardiovascular diseases, oxidative stress can lead to endothelial dysfunction, inflammation and atherosclerosis. In neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, oxidative damage to neurons can lead to impaired function and cell death. Chronic inflammation, such as rheumatoid arthritis and inflammatory bowel disease, is both a cause and a result of persistent oxidative stress. Therefore, it is particularly important to develop novel antioxidants.

[0003] Natural products have diverse chemical structures and broad biological activities, and are an important source for drug discovery. Coumarin, also known as 1,2-benzopyrone, is a secondary metabolite widely distributed in plants such as Rutaceae, Asteraceae and Oleaceae, and has characteristics such as high solubility, high bioavailability and low toxicity. Numerous studies have shown that coumarin has pharmacological activities such as free radical scavenging, anti-inflammatory, anti-cancer, antibacterial and anti-cholinesterase. Substitution at different positions (C-3, C-4 or C-5 positions) on the coumarin skeleton can obtain pharmacologically active molecules with significantly improved activities. These characteristics make coumarin an ideal skeleton with scientific value in drug development and research.

[0004] Network pharmacology and molecular docking are important tools for analyzing the potential action targets and mechanisms of compounds. Based on the theoretical knowledge and research methods of systems biology and pharmacology, network pharmacology reveals the complex interactions between biological systems and drugs from an overall and systematic perspective. The main contents of its research revolve around multiple aspects such as drug efficacy, toxicity, metabolic characteristics, target prediction, biological network construction, mechanism analysis and clinical application. The advantages of network pharmacology research are, on the one hand, to break through the bottleneck of the traditional "single drug, single target" R & D model and provide a new model and idea of "multiple drugs, multiple targets" interaction relationship, which is more in line with the complexity of biological systems. On the other hand, by constructing a drug-target-disease network, comprehensively analyze the regulatory effect of drugs on the disease network.

[0005] Taking coumarin as the basic skeleton, constructing novel coumarin-thiosemicarbazide compounds, and predicting the biological processes, potential targets and signaling pathways involved in the antioxidant activity of the compounds by means of network pharmacology and molecular docking is a feasible strategy. Summary of the Invention

[0006] The present invention aims to provide a coumarin-thiosemicarbazide compound and its applications. Taking coumarin as the basic skeleton, 10 novel coumarin-thiosemicarbazide compounds were prepared, and the anti-free radical activity was screened to obtain the preferred compounds. By means of network pharmacology and molecular docking, the biological processes, potential targets and signaling pathways involved in the antioxidant activity of the compounds were predicted.

[0007] One of the objects of the present invention is to provide a coumarin-thiosemicarbazide compound.

[0008] Another object of the present invention is to provide a preparation method of the coumarin-thiosemicarbazide compound.

[0009] A further object of the present invention is to provide an application of the coumarin-thiosemicarbazide compound in antioxidant.

[0010] Another object of the present invention is to provide a pharmaceutical use of the coumarin-thiosemicarbazide compound.

[0011] In the first aspect of the present invention, a coumarin-thiosemicarbazide compound is provided, and its structural formula is shown as formula (I):

[0012]

[0013] Wherein, R is selected from any one of the following:

[0014]

[0015] In the second aspect of the present invention, a preparation method of the above-mentioned coumarin-thiosemicarbazide compound is provided.

[0016] Specifically, the preparation method of the coumarin-thiosemicarbazide compound comprises the following steps:

[0017] The thiosemicarbazide compound was dissolved in anhydrous ethanol and acetic acid, stirred at room temperature for 30 minutes, and then the 6-formyl coumarin dissolved in ethanol was slowly added dropwise, the temperature was raised to 85°C, and the reaction was continued for 8 hours. TLC thin layer detection was performed until the reaction was complete. Filtered to obtain a solid crude product, which was recrystallized from a mixed solvent of n-hexane and ethanol, and the pure product obtained by recrystallization was further filtered, and the filter cake was vacuum dried to obtain the target compound. The molar ratio of the 6-formyl coumarin, thiosemicarbazide compound, and acetic acid was 1:1.5:0.5. The 6-formyl coumarin was synthesized according to the method disclosed in CN119707900A.

[0018] The synthesis route of coumarin-thiosemicarbazide compounds is as follows:

[0019]

[0020] In one or more specific embodiments of the present invention, compounds 4a-4j were synthesized, and their specific structures are as follows:

[0021]

[0022]

[0023] The third aspect of the present invention provides the use of coumarin-thiosemicarbazide compounds of formula (I) in anti-oxidation.

[0024] In one embodiment of the present invention, the in vitro antioxidant activity of the compound of formula (I) was evaluated.

[0025] Specifically, vitamin C (Vc) was used as a positive control, and DPPH free radicals and hydroxyl free radicals were used as indicators to evaluate the antioxidant activity.

[0026] The DPPH method was used to detect the scavenging ability of the derivatives on DPPH free radicals. At the maximum final concentration of 200 μM, the derivatives obtained by the present invention showed different degrees of scavenging effect on DPPH free radicals (20.58-78.36%). Among them, derivatives 4g and 4i had strong scavenging ability on DPPH free radicals, and their scavenging rates were above 70%.

[0027] With the help of Fe 2+ -H2O2-salicylic acid system method was used to determine the scavenging effect of the derivatives on ·OH free radicals. At the maximum final concentration of 150μM, the scavenging rates of derivatives 4a-4j on ·OH free radicals ranged from 6.93% to 76.38%. Among them, the scavenging ability of compounds 4g and 4i on ·OH free radicals was significantly better than that of Vc.

[0028] Based on the above evaluation results of in vitro antioxidant activity, the present invention provides the application of the coumarin-thiosemicarbazide compounds of formula (I) as antioxidants in daily chemical products such as food, cosmetics and washing and care products.

[0029] In the fourth aspect of the present invention, there is provided the pharmaceutical use of the coumarin-thiosemicarbazide compounds of formula (I).

[0030] In a specific embodiment of the present invention, the in vitro antioxidant activity of the compound of formula (I) was evaluated.

[0031] In a specific embodiment of the present invention, the network pharmacology research method was adopted to analyze the potential targets of compounds 4g and 4i for antioxidant stress.

[0032] The prediction results of the drug-likeness and toxicity parameters of compounds 4g and 4i show that both derivatives 4g and 4i follow the Lipinski rules, indicating that they have a high potential for oral drugability, that is, they may exhibit good absorption, distribution, metabolism and excretion characteristics in the human body and are suitable for the development of oral small molecule drugs. At the same time, they show low toxicity.

[0033] The targets of the compounds were intersected with the oxidative stress-related targets, and 90 intersection targets of 4g and 4i for antioxidant effects were screened out. Sorted according to the DC (degree centrality) value, the top 18 core targets were obtained. That is, the key targets of derivatives 4g and 4i for antioxidant effects involve AKT1 (protein kinase B), BCL2 (B-cell lymphoma-2 protein), EGFR (epidermal growth factor receptor), SRC (sarcoma virus protein), HIF1A (hypoxia-inducible factor-1α), MTOR (mammalian target of rapamycin), MMP9 (matrix metalloproteinase-9), PIK3CA (phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit α), PTGS2 (prostaglandin-endoperoxide synthase 2), GSK3β (glycogen synthase kinase 3β), MAPK1 (mitogen-activated protein kinase 1), MCL1 (myeloid cell leukemia sequence 1), JAK2 (Janus kinase 2), KDR (kinase insert domain receptor), PTK2 (protein tyrosine kinase 2), MAPK8 (mitogen-activated protein kinase 8), APP (amyloid precursor protein) and PRKACA (protein kinase cAMP-dependent catalytic subunit α), etc. GO and KEGG enrichment analysis shows that its antioxidant activity is closely related to the PI3K / Akt and MAPK signaling pathways.

[0034] In a specific embodiment of the present invention, a molecular docking research method was used to verify the binding affinity of derivatives 4g and 4i with key targets. Specifically, the preferred compounds 4g and 4i were used as ligands, and the core targets AKT1, BCL2, EGFR, SRC, HIF1A, and MTOR were used as receptors for molecular docking simulation operations. The results showed that derivatives 4g and 4i had good binding properties with the above six target proteins. Among them, derivatives 4g and 4i had the best binding free energy with SRC protein, showing strong binding affinity.

[0035] In view of the above results of network pharmacology and molecular docking studies, coumarin-thiosemicarbazide derivatives have antioxidant stress activity and can be used to prepare drugs for preventing and treating oxidative stress-related diseases. The antioxidant stress described in the present invention includes scavenging DPPH free radicals and / or scavenging hydroxyl free radicals.

[0036] Preferably, the application of coumarin-thiosemicarbazide compounds in the preparation of drugs for scavenging DPPH and hydroxyl free radicals, and the structural formula of coumarin-thiosemicarbazide compounds is:

[0037]

[0038] The oxidative stress-related diseases described in the present invention include, but are not limited to, nervous system diseases, cardiovascular system diseases, respiratory system diseases, metabolic diseases, liver diseases, kidney diseases, cancers, inflammatory and immune diseases, skin diseases, etc. The nervous system diseases include, but are not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), cerebral ischemia / reperfusion injury, etc.; the cardiovascular diseases include, but are not limited to, atherosclerosis, hypertension, myocardial ischemia-reperfusion injury, etc.; the respiratory system diseases include, but are not limited to, chronic obstructive pulmonary disease (COPD), asthma, etc.; the metabolic diseases include, but are not limited to, diabetes and its complications, non-alcoholic fatty liver disease (NAFLD), etc.; the liver diseases include, but are not limited to, alcoholic liver disease, drug-induced liver injury, etc.; the kidney diseases include, but are not limited to, diabetic nephropathy, chronic kidney disease (CKD), etc., the cancers include, but are not limited to, liver cancer, breast cancer, colorectal cancer, etc.; the inflammatory and immune diseases include, but are not limited to, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), etc.; the skin diseases include, but are not limited to, ultraviolet-induced photoaging, psoriasis, etc.

[0039] The present invention also provides a multi-target antioxidant stress drug composition, which contains the compound of formula (1).

[0040] The present invention further provides a pharmaceutical preparation, comprising a compound of formula (I) or the above-mentioned multi-target antioxidant stress pharmaceutical composition, and a pharmaceutically acceptable carrier.

[0041] Preferably, the administration form of the pharmaceutical preparation is oral administration.

[0042] The present invention also provides a method for predicting and optimizing antioxidant targets of a compound based on network pharmacology, comprising the following steps:

[0043] (1) Evaluating the drug-likeness and toxicity of the optimized compound;

[0044] (2) Screening and correcting the action targets of the compound;

[0045] (3) Obtaining potential targets for antioxidant activity;

[0046] (4) Intersecting the targets described in steps (2) and (3) to obtain potential action targets for the antioxidant activity of the compound;

[0047] (5) Importing the common targets described in step (4) into the STRING database, constructing a target protein interaction network, and performing analysis to obtain key targets;

[0048] (6) Performing GO biological process enrichment analysis and KEGG metabolic pathway analysis on the key targets described in step (5), and visualizing the analysis results;

[0049] The present invention also provides a method for verifying antioxidant targets of an optimized compound using molecular docking technology, comprising the following steps:

[0050] Draw the structure of the compound in ChemDraw software and convert it into a pdb format file; download the protein structure from the PDB database and process the crystal structure with software to prepare a protein receptor file. The molecular docking software is selected from AutoDock1.5.6. The molecular docking results are scored by the obtained binding energy to verify whether the compound has good binding affinity with the target. The visualization analysis software is selected from PyMOL 3.1.3.1.

[0051] The present invention synthesized a class of coumarin-thiosemicarbazide compounds by structurally modifying coumarin, and evaluated their applications in oxidative stress; analyzed the action targets and signal pathways of such compounds in improving oxidative stress through network pharmacology and molecular docking, and speculated their potential action mechanisms, providing certain guidance and basis for the future development of coumarin antioxidants. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1Schematic diagram of the method for analyzing the antioxidant targets of coumarin-thiosemicarbazide compounds based on network pharmacology and molecular docking of the present invention.

[0053] Figure 2 Intersection targets of derivatives 4g and 4i with antioxidant.

[0054] Figure 3 PPI network diagram of the antioxidant effects of derivatives 4g and 4i; (a) Screening process of the PPI interaction network, 18 targets were obtained by screening with DC, BC, and CC thresholds; (b) PPI interaction network, the node color is proportional to the degree value in the network; (c) Diagram of the top 18 core targets.

[0055] Figure 4 GO enrichment analysis diagram.

[0056] Figure 5 KEGG enrichment analysis diagram.

[0057] Figure 6 Molecular docking heat map of derivatives 4g and 4i with potential targets.

[0058] Figure 7 Docking visualization diagram of derivatives 4g and 4i with target protein SRC; (a) 4g-SRC; (b) 4i-SRC. Specific implementation mode

[0059] The present invention is described in detail below through specific examples. It should be understood that the following examples are only for explanation and illustration, and do not limit the scope of the present invention in any form. In the following embodiments, biochemical reagents not specifically described are conventional reagents in the art, and can be prepared according to conventional methods in the art or obtained through commercial purchase, and the specification is laboratory pure grade.

[0060] Example 1

[0061] 1. Preparation of compounds 4a-4j, the overall synthesis route is as follows:

[0062]

[0063] Reagents and conditions: (i) Different thiosemicarbazide compounds, acetic acid, ethanol, 85 °C.

[0064] General method for the synthesis of coumarin-thiosemicarbazide derivatives:

[0065] Synthesize the intermediate 6-formylcoumarin according to the method disclosed in CN119707900A. Weigh the arylaminothiourea compound (0.69 mmol) and place it in a 20 mL Schlenk tube. Add 2 mL of absolute ethanol and acetic acid (0.23 mmol) respectively. After stirring at room temperature for 30 min, slowly add dropwise 6-formylcoumarin (0.46 mmol) dissolved in ethanol (0.5 mL). Raise the temperature to 85 °C and continue the reaction for 8 h. Detect by TLC thin layer until the reaction is complete, then filter by suction to obtain the solid crude product. The crude product is recrystallized with a mixed solvent of n-hexane and ethanol, and the pure product obtained by recrystallization is filtered by suction again. The filter cake is dried under vacuum to obtain the target products 4a - 4j.

[0066] 2. Physical and chemical properties, chemical structures and NMR data of compounds 4a - 4j

[0067] 2.1 Structure confirmation of compound 4a

[0068] 4a:

[0069] The arylaminothiourea compound is 4-phenylaminothiourea, and compound 4a is prepared. Its property is a white solid, and the yield is 89.3%. 1 H NMR (400 MHz, DMSO-d6): δ (ppm) 11.93 (s, 1H), 10.18 (s, 1H), 8.31 (d, J = 8.8 Hz, 1H), 8.20 (s, 1H), 8.14 (s, 1H), 8.05 (d, J = 9.6 Hz, 1H), 7.55 (d, J = 7.6 Hz, 2H), 7.46 (d, J = 8.4 Hz, 1H), 7.39 (t, J = 7.6 Hz, 2H), 7.23 (t, J = 7.4 Hz, 1H), 6.56 (d, J = 9.6 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6): δ (ppm) 176.1, 159.8, 154.5, 144.0, 141.2, 139.1, 130.6, 130.4, 128.2 (2C), 128.1, 126.2 (2C), 125.6, 119.0, 116.9, 116.9.

[0070] 2.2 Structure confirmation of compound 4b

[0071] 4b:

[0072] The arylaminothiourea compound is 4-(4-methylphenyl)-3-aminothiourea, and compound 4b is prepared. Its property is a white solid, and the yield is 88.5%. 11H NMR (400 MHz, DMSO-d6): δ (ppm) 11.88 (s, 1H), 10.11 (s, 1H), 8.30 (dd, J = 8.7, 2.1 Hz, 1H), 8.19 (s, 1H), 8.14 (d, J = 2.1 Hz, 1H), 8.05 (d, J = 9.6 Hz, 1H), 7.46 (d, J = 8.7 Hz, 1H), 7.40 (d, J = 8.3 Hz, 2H), 7.18 (d, J = 8.1 Hz, 2H), 6.55 (d, J = 9.6 Hz, 1H), 2.31 (s, 3H). 13 13C NMR (101 MHz, DMSO-d6): δ (ppm) 176.2, 159.8, 154.4, 144.1, 141.1, 136.5, 134.7, 130.7, 130.4, 128.6 (2C), 128.1, 126.1 (2C), 119.0, 116.9, 116.9, 20.7.

[0073] 2.3 Structural confirmation of compound 4c

[0074] 4c:

[0075] The arylaminothiourea compound is 4-(4-methoxyphenyl)-3-thiosemicarbazide. Compound 4c was prepared, which is a yellow solid with a yield of 89.7%. 1 1H NMR (400 MHz, DMSO-d6): δ (ppm) 11.85 (s, 1H), 10.08 (s, 1H), 8.29 (dd, J = 8.7, 2.1 Hz, 1H), 8.18 (s, 1H), 8.13 (d, J = 2.0 Hz, 1H), 8.04 (d, J = 9.6 Hz, 1H), 7.45 (d, J = 8.7 Hz, 1H), 7.38 (d, J = 8.9 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 6.55 (d, J = 9.6 Hz, 1H), 3.77 (s, 3H). 13 13C NMR (101 MHz, DMSO-d6): δ (ppm) 176.5, 159.8, 157.1, 154.4, 144.0, 141.0, 131.9, 130.7, 130.4, 128.0, 127.8 (2C), 119.0, 116.9, 116.8, 113.4 (2C), 55.3.

[0076] 2.4 Structural confirmation of compound 4d

[0077] 4d:

[0078] The arylaminothiourea compound is 4-(4-tert-butylphenyl)-3-aminothiourea. Compound 4d was prepared, which is a yellow solid with a yield of 85.0%. 1 H NMR(400MHz,DMSO-d6):δ(ppm)11.88(s,1H),10.09(s,1H),8.29(dd,J=8.7,2.1Hz,1H),8.19(s,1H),8.14(d,J=2.1Hz,1H),8.05(d,J=9.6Hz,1H),7.45(dd,J=8.8,2.4Hz,2H),7.44(s,1H),7.39(dt,J=8.8,2.2Hz,2H),6.56(d,J=9.6Hz,1H),1.30(s,9H). 13 C NMR(101MHz,DMSO-d6):δ(ppm)176.1,159.7,154.4,148.0,144.0,141.1,136.4,130.6,130.4,128.0,125.7(2C),124.9(2C),119.0,116.9,116.8,34.3,31.2(3C).

[0079] 2.5 Structure confirmation of compound 4e

[0080] 4e:

[0081] The arylaminothiourea compound is 4-(4-fluorophenyl)-3-aminothiourea. Compound 4e was prepared, which is a yellow solid with a yield of 97.7%. 1 H NMR(400MHz,DMSO-d6):δ(ppm)11.94(s,1H),10.17(s,1H),8.30(dd,J=8.7,2.0Hz,1H),8.20(s,1H),8.13(d,J=2.4Hz,1H),8.05(d,J=9.6Hz,1H),7.54(m,2H),7.46(d,J=8.8Hz,1H),7.22(t,J=8.8Hz,2H),6.55(d,J=9.6Hz,1H). 13 C NMR(101MHz,DMSO-d6):δ(ppm)176.5,159.8(d,J=242.9Hz),159.7,154.5,144.0,141.4,135.4(d,J=2.8Hz),130.6,130.4,128.4(d,J=8.3Hz,2C),128.1,119.0,116.9,116.9,114.8(d,J=22.5Hz,2C). 19F NMR (282 MHz, DMSO-d6): δ (ppm) -116.9.

[0082] 2.6 Structural confirmation of compound 4f

[0083] 4f:

[0084] The arylaminothiourea compound is 4-(4-chlorophenyl)-3-thiosemicarbazide. Compound 4f was prepared, which is a yellow solid with a yield of 95.6%. 1 H NMR (400 MHz, DMSO-d6): δ (ppm) 12.01 (s, 1H), 10.21 (s, 1H), 8.30 (dd, J = 8.8, 2.1 Hz, 1H), 8.20 (s, 1H), 8.13 (d, J = 2.1 Hz, 1H), 8.06 (d, J = 9.6 Hz, 1H), 7.61 (dt, J = 8.8, 2.6 Hz, 2H), 7.47 (d, J = 8.8 Hz, 1H), 7.44 (dt, J = 8.8, 2.6 Hz, 2H), 6.55 (d, J = 9.6 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6): δ (ppm) 176.1, 159.7, 154.5, 144.0, 141.6, 138.1, 130.5, 130.4, 129.5, 128.2, 128.1 (2C), 127.8 (2C), 119.0, 117.0, 116.9.

[0085] 2.7 Structural confirmation of compound 4g

[0086] 4g:

[0087] The arylaminothiourea compound is 4-(4-bromophenyl)-3-thiosemicarbazide. Compound 4g was prepared, which is a yellow solid with a yield of 98.3%. 1 H NMR (400 MHz, DMSO-d6): δ (ppm) 12.01 (s, 1H), 10.20 (s, 1H), 8.29 (td, J = 9.2, 2.4 Hz, 1H), 8.20 (s, 1H), 8.14 (s, 1H), 8.03 (d, J = 9.2 Hz, 1H), 7.56 (s, 1H), 7.48 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 8.8 Hz, 2H), 6.56 (dd, J = 9.6, 1.6 Hz, 1H). 1313C NMR (101 MHz, DMSO-d6): δ (ppm) 176.1, 159.8, 154.5, 144.0, 141.7, 138.5, 131.0 (2C), 130.7, 130.5, 130.4, 130.3, 128.1 (2C), 119.0, 117.0.

[0088] 2.8 Structural confirmation of compound 4h

[0089] 4h:

[0090] The arylaminothiourea compound is 4-(4-nitrophenyl)-3-aminothiourea. Compound 4h was prepared, which is a yellow solid with a yield of 98.1%. 1 1H NMR (400 MHz, DMSO-d6): δ (ppm) 12.28 (s, 1H), 10.48 (s, 1H), 8.32 (d, J = 8.8 Hz, 1H), 8.26 (d, J = 9.2 Hz, 2H), 8.25 (s, 1H), 8.14 (s, 1H), 8.08 (d, J = 8.4 Hz, 1H), 8.06 (d, J = 9.2 Hz, 2H), 7.49 (d, J = 8.8 Hz, 1H), 6.56 (d, J = 9.6 Hz, 1H). 13 13C NMR (101 MHz, DMSO-d6): δ (ppm) 175.4, 159.7, 154.7, 145.4, 144.0, 143.6, 142.6, 130.5, 130.3, 128.5, 124.7 (2C), 123.8 (2C), 119.0, 117.0, 116.9.

[0091] 2.9 Structural confirmation of compound 4i

[0092] 4i:

[0093] The arylaminothiourea compound is 4-(4-trifluoromethylphenyl)-3-aminothiourea. Compound 4i was prepared, which is a brown solid with a yield of 90.1%. 1HNMR(400MHz, DMSO-d6): δ(ppm) 11.95 (s, 1H), 10.51 (s, 1H), 8.28 (dd, J=8.8, 2.0Hz, 1H), 8.15 (brs, 2H), 8.11 (d, J=9.6Hz, 1H), 8.04 (d, J=9.7Hz, 1H), 7.93 - 7.87 (m, 1H), 7.69 (m, 1H), 7.48 (d, J=8.4Hz, 1H), 7.46 (d, J=8.4Hz, 1H), 6.56 (d, J=9.6Hz, 1H). 13 C NMR(151MHz, DMSO-d6): δ(ppm) 178.9, 160.2, 154.9, 144.4, 141.8, 131.1, 131.1 (q, J=75.9Hz), 128.5, 127.2 (q, J=2.1Hz, 2C), 126.8, 126.1 (q, J=19.2Hz, 2C), 119.4, 118.5 (q, J=295.4Hz), 117.4, 117.3, 117.0. 19 F NMR(282MHz, DMSO-d6): δ(ppm) -59.8.

[0094] 2.10 Structural confirmation of compound 4j

[0095] 4j:

[0096] The arylaminothiourea compound is 4-(4-trifluoromethoxyphenyl)-3-aminothiourea. Compound 4j was prepared, which is a white solid with a yield of 87.1%. 1 H NMR(400MHz, DMSO-d6): δ(ppm) 12.02 (s, 1H), 10.24 (s, 1H), 8.30 (dd, J=8.8, 2.0Hz, 1H), 8.21 (s, 1H), 8.14 (d, J=2.0Hz, 1H), 8.06 (d, J=9.6Hz, 1H), 7.69 (dt, J=8.8, 2.4Hz, 2H), 7.47 (d, J=8.4Hz, 1H), 7.39 (d, J=8.5Hz, 2H), 6.56 (d, J=9.6Hz, 1H). 13 C NMR(101MHz, DMSO-d6): δ(ppm) 176.2, 159.7, 154.5, 145.5, 145.5, 144.0, 141.7, 138.3, 130.5, 129.3 (q, J=221.6Hz), 127.8 (2C), 124.6, 120.9 (2C), 119.0, 117.0, 116.9.19 F NMR (282 MHz, DMSO-d6): δ (ppm) -56.9.

[0097] Example 2

[0098] In vitro antioxidant activity evaluation: According to the thiosemicarbazide-coumarin compounds described in Example 1, the present invention used vitamin C (Vc) as a positive control and DPPH free radical and hydroxyl free radical as indicators to evaluate its antioxidant activity.

[0099] 1. Determination of DPPH free radical scavenging ability

[0100] The DPPH method was used to detect the scavenging ability of the derivatives on DPPH free radicals. As shown in Table 1, at the maximum final concentration of 200 μM, the derivatives obtained in the present invention showed different degrees of scavenging effects on DPPH free radicals (20.58 - 78.36%). Among them, derivatives 4g and 4i had strong scavenging abilities on DPPH free radicals, and their scavenging rates were 73.69% and 78.36% respectively.

[0101] Table 1. Scavenging rates of derivatives 4a - 4j on DPPH free radicals

[0102]

[0103] 2. Determination of hydroxyl free radical scavenging ability

[0104] The present invention used the Fe 2+ -H2O2-salicylic acid system method to determine the scavenging effect of the derivatives on ·OH free radicals. As shown in Table 2, at the maximum final concentration of 150 μM, the scavenging rates of derivatives 4a - 4j on ·OH free radicals were between 6.93 - 76.38%. Among them, the scavenging abilities of compound 4g (32.04%) and 4i (76.38%) on ·OH free radicals were significantly better than that of Vc (25.07%).

[0105] Table 2. Scavenging rates of derivatives 4a - 4j on hydroxyl free radicals

[0106]

[0107] Example 3

[0108] According to Example 2, compounds 4g and 4i, which had strong or better abilities to capture DPPH and hydroxyl free radicals than Vc, were screened out, and their potential targets for antioxidant effects were analyzed based on network pharmacology and molecular docking.

[0109] I. Prediction of drug-likeness and toxicity parameters of compounds 4g and 4i

[0110] 1. Method

[0111] By using the SwissADME website (http: / / www.swissadme.ch / index.php), according to Lipinski's rules: molecular weight (Mw, < 500 g / mol), topological polar surface area lipid-water partition coefficient (MLogP, ≤ 5), hydrogen bond acceptor (HBA, < 10), and hydrogen bond donor (HBD, ≤ 5). The compound molecule should not violate more than 2 of the 5 parameters. Using the ProTox 3.0 website (https: / / tox.charite.de / protox3 / index.php), screen out organ toxicities (hepatotoxicity, neurotoxicity, nephrotoxicity, respiratory toxicity, and cardiotoxicity) and toxicity endpoints (carcinogenicity, immunotoxicity, mutagenicity, cytotoxicity, BBB barrier, ecotoxicity, clinical toxicity, and nutritional toxicity), and score the toxicity level of the compound to preliminarily predict toxicity.

[0112] 2. Result Analysis

[0113] As shown in Table 3, both derivatives 4g and 4i follow Lipinski's rules, indicating that they have a high potential for oral drugability, that is, they may exhibit good absorption, distribution, metabolism, and excretion characteristics in the human body and are suitable for the development of oral small molecule drugs. At the same time, their toxicity levels are both 5, showing low toxicity.

[0114] Table 3. Druglikeness and Toxicity of Derivatives 4g and 4i

[0115]

[0116] II. Acquisition and Integration of Intersection Targets

[0117] 1. Method

[0118] Use ChemDraw software to obtain the molecular structures of compounds 4g and 4i, and then import them into the Swiss TargetPrediction online data website (http: / / www.swisstargetprediction.ch) to predict the potential targets of 4g and 4i. Use the GeneCards database (https: / / www.genecards.org) to determine the potential targets of antioxidant. Match and integrate the targets of the compound with the antioxidant-related targets through Microbial Information (https: / / www.bioinformatics.com.cn) to obtain the common targets of the preferred compounds 4g and 4i and antioxidant effects, that is, the common targets, and draw a Venn diagram. The intersection targets in the diagram are the related targets of the antioxidant effects of 4g and 4i.

[0119] 2. Result Analysis

[0120] A total of 167 action targets of 4g and 4i were retrieved from the SwissTargetPrediction database. A total of 5,098 antioxidant targets were obtained from the GeneCards database. The top 2,000 antioxidant targets were intersected with the compound targets, and a Venny diagram was drawn. As Figure 2 shown, a total of 90 intersection targets of 4g and 4i with antioxidant effects were screened out.

[0121] III. Construction of a visualization graph of the network relationship between the action targets of 4g and 4i and antioxidant targets and screening of core targets

[0122] 1. Method

[0123] The obtained intersection targets were uploaded to the online protein interaction platform STRING (https: / / cn.string-db.org / ). "Organism:Homo sapiens" was retrieved in the Multiple Proteins item, and scattered nodes were removed to obtain the target protein interaction network and the tsv data file. The data was imported into the NetworkAnalyzer module in Cytoscape 3.10 software to analyze the topological parameters of all nodes in the interaction network. Using the thresholds of degree centrality (DC), betweenness centrality (BC), and closeness centrality (CC) as the screening conditions for core targets, different colors were formed, and different circle sizes represented different gradients to construct a PPI network diagram.

[0124] 2. Result Analysis

[0125] Figure 3 (a) The screening process of the PPI interaction network. 18 targets were obtained by screening with DC, BC, and CC thresholds; Figure 3 (b) The PPI interaction network, where the node color is proportional to the degree value in the network; Figure 3 (c) The diagram of the top 18 core targets. As Figure 3 can be seen, under the conditions of DC≥22.956, BC≥78.800, and CC≥0.006, 18 nodes and 138 edges were obtained. Sorting by the DC value, the top 18 core targets were obtained (as shown in Table 4). The key targets for the antioxidant effects of derivatives 4g and 4i involve AKT1, BCL2, EGFR, SRC, HIF1A, MTOR, and MMP9, etc.

[0126] Table 4. Core targets of derivatives 4g and 4i for antioxidant

[0127]

[0128]

[0129] IV. GO Biological Process Analysis

[0130] 1. Method

[0131] The 18 obtained core targets were imported into the David website (https: / / david.ncifcrf.gov / ) for Gene Ontology (GO) enrichment analysis. The identifier was set to "OFFICE_GENE_SYMBOL", the species was set to "Homo sapiens", and the list type was set to "Gene List". Then, "GOTERM_BP_DIRECT", "GOTERM_CC_DIRECT", and "GOTERM_MF_DIRECT" were selected in Gene_Ontology, and the results were subjected to visual analysis.

[0132] 2. Result Analysis

[0133] To clarify the biological processes of the core targets, GO enrichment analysis was performed. According to the screening criteria, a total of 692 entries were obtained from the GO functional enrichment analysis, including 486 in Biological process (BP), accounting for approximately 70.23%; 79 in Molecular function (MF), accounting for approximately 11.42%; and 127 in Cellular component (CC), accounting for approximately 18.35%. The top 10 entries were selected for visual analysis. As Figure 4 shown, the core targets are involved in multiple biological processes, including chromatin remodeling, EGFR signaling pathway, tyrosine phosphorylation, insulin receptor, and PI3K / Akt signaling pathway, etc. The CC process mainly involves cytoplasm, mitochondria, nucleus, and postsynaptic membrane, etc. The MF process mainly involves ATP binding, kinase, and receptor activities, such as H3Y41 protein kinase, tyrosine protein kinase, Ephrin receptor, and insulin receptor, etc. These results indicate that the anti-free radical process of derivatives 4g and 4i may be related to the signal pathways regulated by kinases.

[0134] V. KEGG Pathway Enrichment Analysis

[0135] 1. Method

[0136] Similarly, the 18 obtained core targets were imported into the David website (https: / / david.ncifcrf.gov / ), with the identifier set to "OFFICE_GENE_SYMBOL", the species set to "Homo sapiens", and the list type set to "Gene List". Then, "KEGG_PATHWAY" was selected in Pathways for KEGG pathway analysis and visualization analysis.

[0137] 2. Result Analysis

[0138] According to the results of KEGG enrichment analysis, a total of 162 significantly enriched signaling pathways were screened out. The top 10 entries were obtained according to the P-value for visualization analysis. As Figure 5 shown, the pathways with relatively high correlations with the core targets mainly involve signaling pathways such as PI3K / Akt, EGFR, MAPK, and Ras. Among them, the PI3K / Akt and MAPK signaling pathways are the core regulators of the antioxidant response, enhancing the antioxidant capacity of cells by upregulating the expression of antioxidant enzymes and inhibiting apoptosis. The EGFR, ErbB, and Ras signaling pathways regulate the antioxidant response of cells by activating downstream signaling pathways (such as PI3K / Akt, MAPK). The core of the signaling pathways all points to the PI3K / Akt and MAPK signaling pathways.

[0139] VI. Molecular Docking Analysis

[0140] 1. Method

[0141] Molecular docking was performed between the top 5 key targets and coumarin derivatives using AutoDock 1.5.6 software. The docking process is as follows:

[0142] (1) Receptor preparation: Download the protein from the PDB database, import the protein into the PyMOL 3.1.3.1 software to remove water and ligands, and save it in pdb format.

[0143] (2) Ligand preparation: Draw the compound molecule using Chemdraw, import the saved file into Chem3D, minimize the energy by adjusting the conformation of the substance through MM2, and save it in pdb format.

[0144] (3) Molecular docking: Set the protein with all hydrogens added as the receptor and save it in pdbqt format. Set the compound with all hydrogens added as the ligand, detect the torsion bonds, select the torsion bonds, and save it in pdbqt format. Set the docking Box, save and run Autogrid4, set the docking parameters and operation methods, run Autodock4, save and view the results.

[0145] 2. Result Analysis

[0146] To verify the binding affinity of derivatives 4g and 4i with key targets, the present invention uses the preferred compounds 4g and 4i as ligands, and respectively uses the core targets AKT1, BCL2, EGFR, SRC, HIF1A, and MTOR as receptors for molecular docking simulation operations. The results are shown in Table 5. The binding free energies of derivatives 4g and 4i with the above six target proteins are all negative values, and are all ≤ -5.0 kcal / mol, indicating that both have good binding properties. At the same time, as Figure 6 can be seen, derivatives 4g and 4i have the best binding free energy with SRC protein, showing strong binding affinity. As Figure 7 shown in a, the ester bond in the structure of derivative 4g can form two hydrogen bonds with the NH2 on the Lys300 residue, and the NH on the thiosemicarbazide can respectively have hydrogen bond interactions with the Ser283 residue. Derivative 4i establishes a hydrogen bond interaction through the carbonyl group on the coumarin mother nucleus and the NH on the imidazole ring in the His6 residue. At the same time, the Glu332 residue in the protein receptor targets the amino group in the thiosemicarbazide in derivative 4i, generating a hydrogen bond interaction ( Figure 7 b).

[0147] Table 5. Binding energy data of derivatives 4g and 4i with potential targets

[0148]

[0149] The present invention prepared 10 coumarin-thiosemicarbazide derivatives by a drug synthesis method, and detected the anti-free radical activity of the obtained derivatives using an enzyme-labeled instrument, and preferably selected derivatives 4g and 4i therefrom. Using the integrated strategy of network pharmacology and molecular docking, the antioxidant candidate targets, biological functions, and molecular action pathways of derivatives 4g and 4i were respectively analyzed. Among them, the main core targets include AKT1, BCL2, EGFR, SRC, HIF1A, and MTOR, etc. The changes in these targets involve biological processes regulated by the activation of various protein kinases and receptors (tyrosine, protein, H3Y41 protein, insulin receptor), and the transduction of signals involved in PI3K / Akt, EGFR, MAPK, and Ras and other signal pathways.

[0150] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A class of coumarin-thiosemicarbazide compounds, the general chemical structure formula of which is shown in formula (I): , In the formula, R is one of the following structural formulas: 。 2. Use of the coumarin-thiosemicarbazide compound according to claim 1 as an antioxidant in food and daily chemical products.

3. Use of the coumarin-thiosemicarbazide compound according to claim 1 in the preparation of a drug for preventing and treating oxidative stress-related diseases.

4. The application according to claim 3, characterized in that The structural formula of the coumarin-thiosemicarbazide compound is: or 。 5. The application according to claim 3, characterized in that The drug for preventing and treating oxidative stress-related diseases targets multiple oxidative stress-related targets.

6. The application according to claim 5, characterized in that, The oxidative stress-related targets include: AKT1, BCL2, EGFR, SRC, HIF1A, MTOR, MMP9, PIK3CA, PTGS2, GSK3B, MAPK1, MCL1, JAK2, KDR, PTK2, MAPK8, APP and PRKACA.

7. The application according to claim 3, wherein The drug for preventing and treating oxidative stress-related diseases targets the PI3K / Akt and / or MAPK signaling pathways.

8. The application according to claim 3, wherein The oxidative stress-related diseases include neurological diseases, cardiovascular diseases, respiratory diseases, metabolic diseases, liver diseases, kidney diseases, cancers, inflammatory and immune diseases, and skin diseases.

9. A pharmaceutical composition, characterized in that, It includes the coumarin-thiosemicarbazide compound according to claim 1.

10. A pharmaceutical preparation, characterized in that, It includes the coumarin-thiosemicarbazide compound according to claim 1 or the pharmaceutical composition according to claim 9, and a pharmaceutically acceptable carrier.

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