Coumarin-aminothiourea compound and application thereof

Abstract

This invention discloses a class of coumarin-aminothiourea compounds and their applications. First, a class of coumarin-aminothiourea derivatives were synthesized and screened for in vitro antioxidant activity, yielding preferred derivatives 4g and 4i. Then, based on network pharmacology and molecular docking, the antioxidant targets were analyzed. The intersection mapping of the target sites of compounds 4g and 4i with antioxidant targets revealed common targets. A PPI interaction network was constructed to screen for potential core targets. GO function and KEGG pathway enrichment analysis was performed on the core targets to obtain the involved biological processes and signaling pathways. Molecular docking operations were performed between the key targets and compounds 4g and 4i to verify the binding ability of the compounds to the targets. This invention, using network pharmacology methods, preliminarily reveals the potential antioxidant targets and modern biological mechanisms of derivatives 4g and 4i, providing important guidance and basis for the development of antioxidant drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmacology and bioinformatics, specifically relating to a coumarin-aminothiourea compound and its applications. Background Technology

[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, and carbon, nitrogen, or sulfur compounds (ROS, RCS, RNS, or RSS). While these free radicals and highly reactive substances are essential for certain physiological processes, such as cell signaling and immune defense, their excessive production disrupts the delicate balance between oxidants and antioxidants, leading to oxidative stress. In cardiovascular diseases, oxidative stress can cause endothelial dysfunction, inflammation, and atherosclerosis. In neurodegenerative diseases such as Alzheimer's and Parkinson's, oxidative damage to neurons leads to impaired function and cell death. Chronic inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease, are both causes and consequences of persistent oxidative stress. Therefore, the development of novel antioxidants is of paramount importance.

[0003] Natural products, with their diverse chemical structures and broad biological activities, are an important source for drug discovery. Coumarin, also known as 1,2-benzopyranone, is a secondary metabolite widely distributed in plants of the Rutaceae, Asteraceae, and Oleaceae families, characterized by high solubility, high bioavailability, and low toxicity. Numerous studies have shown that coumarins possess pharmacological activities such as free radical scavenging, anti-inflammatory, anticancer, antibacterial, and anticholinesterase effects. Substitution at different positions on the coumarin skeleton (C-3, C-4, or C-5) can yield molecules with significantly enhanced pharmacological activity. These properties make coumarins an ideal skeleton with scientific value for drug development and research.

[0004] Network pharmacology and molecular docking are important tools for analyzing the potential targets and mechanisms of action 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 a holistic and systemic perspective. Its main research content revolves around multiple aspects such as drug efficacy, toxicity, metabolic characteristics, target prediction, biological network construction, mechanism analysis, and clinical applications. The advantages of network pharmacology research lie in two aspects: firstly, it breaks through the bottleneck of the traditional "single drug, single target" development model, providing a new model and approach for "multi-drug, multi-target" interaction relationships, which is more in line with the complexity of biological systems; secondly, by constructing drug-target-disease networks, it comprehensively analyzes the regulatory effects of drugs on disease networks.

[0005] Constructing novel coumarin-aminothiourea compounds using coumarin as the basic framework, and then using network pharmacology and molecular docking techniques to predict the biological processes, potential targets, and signaling pathways involved in the antioxidant effects of these compounds, is a feasible strategy. Summary of the Invention

[0006] This invention aims to provide a coumarin-aminothiourea compound and its applications. Using coumarin as the basic framework, this invention prepared 10 novel coumarin-aminothiourea compounds and screened them for their anti-free radical activity, obtaining preferred compounds. Using network pharmacology and molecular docking techniques, the biological processes, potential targets, and signaling pathways involved in the antioxidant effects of these compounds were predicted.

[0007] One of the objectives of this invention is to provide a coumarin-aminothiourea compound.

[0008] The second objective of this invention is to provide a method for preparing coumarin-aminothiourea compounds.

[0009] A third objective of this invention is to provide an application of coumarin-aminothiourea compounds in antioxidant applications.

[0010] The fourth objective of this invention is to provide a pharmaceutical use for a coumarin-aminothiourea compound.

[0011] In a first aspect, the present invention provides a coumarin-aminothiourea compound, the structural formula of which is shown in formula (I):

[0012]

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

[0014]

[0015] In a second aspect, the present invention provides a method for preparing the above-mentioned coumarin-aminothiourea compounds.

[0016] Specifically, the preparation method of coumarin-aminothiourea compounds includes the following steps:

[0017] The aminothiourea compound was dissolved in anhydrous ethanol and acetic acid. After stirring at room temperature for 30 min, 6-aldehyde coumarin dissolved in ethanol was slowly added dropwise. The temperature was raised to 85 °C, and the reaction was continued for 8 h. TLC thin-layer chromatography was performed until the reaction was complete. The mixture was filtered to obtain a crude solid. The crude solid was recrystallized from the crude solid using a mixed solvent of n-hexane and ethanol. The purified product obtained from the recrystallization was further filtered, and the filter cake was dried under vacuum to obtain the target compound. The molar ratio of 6-aldehyde coumarin, the aminothiourea compound, and acetic acid was 1:1.5:0.5. The 6-aldehyde coumarin was synthesized according to the method disclosed in CN119707900A.

[0018] The synthetic route for coumarin-aminothiourea compounds is shown below:

[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 application of coumarin-aminothiourea compounds of formula (I) in antioxidant properties.

[0024] In one specific 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 its antioxidant activity was evaluated using DPPH free radicals and hydroxyl free radicals as indicators.

[0026] The scavenging ability of the derivatives against DPPH free radicals was detected using the DPPH method. At a maximum final concentration of 200 μM, the derivatives obtained in this invention exhibited varying degrees of scavenging effect against DPPH free radicals (20.58-78.36%). Among them, derivatives 4g and 4i showed strong scavenging ability against DPPH free radicals, with scavenging rates exceeding 70%.

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

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

[0029] In a fourth aspect, the present invention provides the pharmaceutical use of a coumarin-thiourea compound 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 one specific embodiment of the present invention, a network pharmacology research method was used to analyze the potential targets of the antioxidant stress effects of compounds 4g and 4i.

[0032] The predicted drug-likeness and toxicity parameters of compounds 4g and 4i show that both derivatives 4g and 4i follow Lipinski's rule, indicating that they have high potential for oral drug development, meaning they may exhibit good absorption, distribution, metabolism, and excretion in the human body, making them suitable for development as oral small molecule drugs. They also show low toxicity.

[0033] By intersecting the targets of the compounds with targets related to oxidative stress, 90 overlapping targets of 4g and 4i with antioxidant effects were identified. These were then sorted according to their degree centrality (DC) values, resulting in the top 18 core targets. The key targets for the antioxidant effects of derivatives 4g and 4i 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 intraperoxide synthase 2), GSK3β (glycogen synthase kinase 3β), MAPK1 (mitogen-activated protein kinase 1), MCL1 (myeloid leukemia sequence 1), JAK2 (Janus kinase 2), KDR (kinase insertion 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 showed that its antioxidant activity was closely related to the PI3K / Akt and MAPK signaling pathways.

[0034] In one specific embodiment of the present invention, molecular docking was used to verify the binding affinity of derivatives 4g and 4i to key targets. Specifically, molecular docking simulations were performed using preferred compounds 4g and 4i as ligands and the core targets AKT1, BCL2, EGFR, SRC, HIF1A, and mTOR as acceptors. The results showed that derivatives 4g and 4i exhibited good binding properties to all six target proteins, with derivatives 4g and 4i showing the best binding free energy and strong binding affinity to the SRC protein.

[0035] Based on the above network pharmacology and molecular docking research results, coumarin-aminothiourea derivatives have antioxidant stress activating properties and can be used to prepare drugs for the prevention and treatment of oxidative stress-related diseases. The antioxidant stress described in this invention includes scavenging DPPH free radicals and / or scavenging hydroxyl free radicals.

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

[0037]

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

[0039] The present invention also provides a multi-target anti-oxidative stress pharmaceutical composition comprising a compound of formula (1).

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

[0041] Preferably, the pharmaceutical preparation is administered orally.

[0042] This invention also provides a method for predicting and selecting antioxidant targets for compounds based on network pharmacology, comprising the following steps:

[0043] (1) Evaluate the drug-likeness and toxicity of the preferred compounds;

[0044] (2) Screening and correction of compound targets;

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

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

[0047] (5) Import the common target points described in step (4) into the STRING database, create a target protein interaction network, and analyze it to obtain key targets.

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

[0049] This invention also provides a method for verifying the antioxidant targets of preferred compounds using molecular docking technology, comprising the following steps:

[0050] The compound structure was drawn in ChemDraw software and converted into a PDB file. The protein structure was downloaded from the PDB database, and the crystal structure was processed using software to prepare a protein receptor file. AutoDock 1.5.6 was selected as the molecular docking software. The obtained binding energies were used to score the molecular docking results, verifying whether the compound had a good binding affinity to the target site. PyMOL 3.1.3.1 was selected as the visualization analysis software.

[0051] This invention synthesizes a class of coumarin-aminothiourea compounds by structural modification of coumarin and evaluates their application in oxidative stress. Through network pharmacology and molecular docking analysis, the target sites and signaling pathways of these compounds in improving oxidative stress are identified, and their potential mechanisms of action are speculated, providing guidance and basis for the future development of coumarin antioxidants. Attached Figure Description

[0052] Figure 1This is a schematic diagram of the method for analyzing the antioxidant targets of coumarin-aminothiourea compounds based on network pharmacology and molecular docking according to the present invention.

[0053] Figure 2 The intersection of derivatives 4g and 4i with antioxidants is the target.

[0054] Figure 3 PPI network diagrams for the antioxidant effects of derivatives 4g and 4i; (a) Screening process of PPI interaction network, 18 targets were obtained by screening through 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 first 18 core targets.

[0055] Figure 4 A graph showing GO enrichment analysis.

[0056] Figure 5 This is a graph showing KEGG enrichment analysis.

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

[0058] Figure 7 Visualization of the docking of derivatives 4g and 4i with the target protein SRC; (a) 4g-SRC; (b) 4i-SRC. Detailed Implementation

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

[0060] Example 1

[0061] 1. The overall synthetic route for the preparation of compounds 4a-4j is as follows:

[0062]

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

[0064] General methods for synthesizing coumarin-aminothiourea derivatives:

[0065] The intermediate 6-aldehyde coumarin was synthesized according to the method disclosed in CN119707900A. 0.69 mmol of an arylaminothiourea compound was weighed and placed in a 20 mL Shrek tube. 2 mL of anhydrous ethanol and 0.23 mmol of acetic acid were added. After stirring at room temperature for 30 min, 0.46 mmol of 6-aldehyde coumarin dissolved in 0.5 mL of ethanol was slowly added dropwise. The temperature was raised to 85 °C, and the reaction was continued for 8 h. TLC analysis was performed until the reaction was complete. The mixture was filtered to obtain a crude solid. The crude solid was recrystallized from the crude solid using a mixed solvent of hexane and ethanol. The purified product obtained from the recrystallization was further filtered, and the filter cake was dried under vacuum to obtain the target products 4a-4j.

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

[0067] 2.1 Structural confirmation of compound 4a

[0068] 4a:

[0069] The aryl aminothiourea compound is 4-phenylaminothiourea. Compound 4a was prepared as a white solid with a yield of 89.3%. 1 H NMR (400MHz, DMSO-d6): δ (ppm) 11.93 (s, 1H), 10.18 (s, 1H), 8.31 (d, J = 8.8Hz, 1H), 8.20 (s, 1H), 8.14 (s, 1H), 8.05 (d, J = 9. 6Hz, 1H), 7.55 (d, J = 7.6Hz, 2H), 7.46 (d, J = 8.4Hz, 1H), 7.39 (t, J = 7.6Hz, 2H), 7.23 (t, J = 7.4Hz, 1H), 6.56 (d, J = 9.6Hz, 1H). 13 C NMR (101MHz, 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 Structural confirmation of compound 4b

[0071] 4b:

[0072] The aryl aminothiourea compound is 4-(4-tolyl)-3-aminothiourea. Compound 4b was prepared as a white solid with a yield of 88.5%. 1H NMR (400MHz, DMSO-d6): δ (ppm) 11.88 (s, 1H), 10.11 (s, 1H), 8.30 (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),7.46(d,J=8.7Hz,1H),7.40(d,J=8.3Hz,2H),7.18(d,J=8.1Hz,2H),6.55(d,J=9.6Hz,1H),2.31(s,3H). 13 C NMR (101MHz, DMSO-d6): δ (ppm) 176.2, 159.8, 154.4, 144.1, 141.1, 136.5, 13 4.7,130.7,130.4,128.6(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 aryl aminothiourea compound is 4-(4-methoxyphenyl)-3-aminothiourea. Compound 4c was prepared as a yellow solid with a yield of 89.7%. 1 H NMR (400MHz, DMSO-d6): δ (ppm) 11.85 (s, 1H), 10.08 (s, 1H), 8.29 (dd, J = 8.7, 2.1Hz, 1H), 8.18 (s, 1H), 8.13 (d, J = 2.0Hz, 1H), 8 .04(d,J=9.6Hz,1H),7.45(d,J=8.7Hz,1H),7.38(d,J=8.9Hz,2H),6.94(d,J=9.0Hz,2H),6.55(d,J=9.6Hz,1H),3.77(s,3H). 13 C NMR (101MHz, DMSO-d6): δ (ppm) 176.5, 159.8, 157.1, 154.4, 144.0, 141.0, 13 1.9,130.7,130.4,128.0,127.8(2C),119.0,116.9,116.8,113.4(2C),55.3.

[0076] 2.4 Structural confirmation of compound 4d

[0077] 4d:

[0078] The aryl aminothiourea compound is 4-(4-tert-butylphenyl)-3-aminothiourea. Compound 4d was prepared as 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, 1 30.6,130.4,128.0,125.7(2C),124.9(2C),119.0,116.9,116.8,34.3,31.2(3C).

[0079] 2.5 Structural confirmation of compound 4e

[0080] 4e:

[0081] The aryl aminothiourea compound is 4-(4-fluorophenyl)-3-aminothiourea. Compound 4e was prepared as 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 (282MHz, DMSO-d6): δ (ppm)-116.9.

[0082] 2.6 Structural confirmation of compound 4f

[0083] 4f:

[0084] The aryl aminothiourea compound is 4-(4-chlorophenyl)-3-aminothiourea. Compound 4f was prepared as a yellow solid with a yield of 95.6%. 1 H NMR (400MHz, DMSO-d6): δ (ppm) 12.01 (s, 1H), 10.21 (s, 1H), 8.30 (dd, J = 8.8, 2.1Hz, 1H), 8.20 (s, 1H), 8.13 (d, J = 2.1Hz, 1H), 8.06(d,J=9.6Hz,1H),7.61(dt,J=8.8,2.6Hz,2H),7.47(d,J=8.8Hz,1H),7.44(dt,J=8.8,2.6Hz,2H),6.55(d,J=9.6Hz,1H). 13 C NMR (101MHz, 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 aryl aminothiourea compound is 4-(4-bromophenyl)-3-aminothiourea. 4 g of the compound was prepared as a yellow solid with a yield of 98.3%. 1 H NMR (400MHz, DMSO-d6): δ (ppm) 12.01 (s, 1H), 10.20 (s, 1H), 8.29 (td, J = 9.2, 2.4Hz, 1H), 8.20 (s, 1H), 8.14 (s, 1H) ),8.03(d,J=9.2Hz,1H),7.56(s,1H),7.48(d,J=8.4Hz,2H),7.47(d,J=8.8Hz,2H),6.56(dd,J=9.6,1.6Hz,1H). 13C NMR (101MHz, 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 aryl aminothiourea compound is 4-(4-nitrophenyl)-3-aminothiourea. The compound 4h was prepared as a yellow solid with a yield of 98.1%. 1 H NMR (400MHz, DMSO-d6): δ (ppm) 12.28 (s, 1H), 10.48 (s, 1H), 8.32 (d, J = 8.8Hz, 1H), 8.26 (d, J = 9.2Hz, 2H), 8.25 ( s,1H),8.14(s,1H),8.08(d,J=8.4Hz,1H),8.06(d,J=9.2Hz,2H),7.49(d,J=8.8Hz,1H),6.56(d,J=9.6Hz,1H). 13 C NMR (101MHz, 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 aryl aminothiourea compound is 4-(4-trifluoromethylphenyl)-3-aminothiourea. Compound 4i was prepared as 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, 12 7.2(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 aryl aminothiourea compound is 4-(4-trifluoromethoxyphenyl)-3-aminothiourea. Compound 4j was prepared as 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 (282MHz, DMSO-d6): δ (ppm)-56.9.

[0097] Example 2

[0098] In vitro antioxidant activity evaluation: Based on the aminothiourea coumarin compounds described in Example 1, the antioxidant activity of these compounds was evaluated using vitamin C (Vc) as a positive control and DPPH free radicals and hydroxyl free radicals as indicators.

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

[0100] The scavenging ability of the derivatives against DPPH radicals was detected using the DPPH method. As shown in Table 1, at a maximum final concentration of 200 μM, the derivatives obtained in this invention exhibited varying degrees of scavenging effect against DPPH radicals (20.58-78.36%). Among them, derivatives 4g and 4i showed strong scavenging ability against DPPH radicals, with scavenging rates of 73.69% and 78.36%, respectively.

[0101] Table 1. Scavenging rate of derivatives 4a-4j against DPPH radicals

[0102]

[0103] 2. Determination of hydroxyl radical scavenging ability

[0104] This invention utilizes Fe 2+ The scavenging effect of the derivatives on ·OH radicals was determined using the -H2O2-salicylic acid system method. As shown in Table 2, at a maximum final concentration of 150 μM, the scavenging rates of derivatives 4a-4j on ·OH radicals ranged from 6.93% to 76.38%. Among them, compounds 4g (32.04%) and 4i (76.38%) showed significantly better scavenging abilities on ·OH radicals than Vc (25.07%).

[0105] Table 2. Scavenging rate of hydroxyl radicals by derivatives 4a-4j

[0106]

[0107] Example 3

[0108] Based on Example 2, compounds 4g and 4i, which have strong or better ability to capture both DPPH and hydroxyl radicals than vitamin C, were screened out. Their potential targets for antioxidant activity were analyzed based on network pharmacology and molecular docking.

[0109] I. Drug-likeness and toxicity parameter prediction of compounds 4g and 4i

[0110] 1. Method

[0111] Using the SwissADME website (http: / / www.swissadme.ch / index.php), according to Lipinski's rule: molar mass (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) were used. The compound molecule could not violate more than two of the five parameters. Using the ProTox 3.0 website (https: / / tox.charite.de / protox3 / index.php), organ toxicity (hepatotoxicity, neurotoxicity, nephrotoxicity, respiratory toxicity, and cardiotoxicity) and toxicity endpoints (carcinogenicity, immunotoxicity, mutagenicity, cytotoxicity, BBB barrier, ecotoxicity, clinical toxicity, and nutritional toxicity) were screened, and the compounds were scored to preliminarily predict toxicity.

[0112] 2. Results Analysis

[0113] As shown in Table 3, derivatives 4g and 4i both follow Lipinski's rule, indicating that they have high potential for oral drug development, meaning they may exhibit good absorption, distribution, metabolism, and excretion properties in the human body, making them suitable for development as oral small molecule drugs. Meanwhile, their toxicity rating is 5, indicating low toxicity.

[0114] Table 3. Drug-like properties and toxicity of derivatives 4g and 4i

[0115]

[0116] II. Acquisition and Integration of Intersecting Targets

[0117] 1. Method

[0118] The molecular structures of compounds 4g and 4i were obtained using ChemDraw software and then imported into the Swiss TargetPrediction (http: / / www.swisstargetprediction.ch) online database to predict potential targets for 4g and 4i. The GeneCards database (https: / / www.genecards.org) was used to identify potential antioxidant targets. The targets of the compounds were matched and integrated with antioxidant-related targets using bioinformatics (https: / / www.bioinformatics.com.cn) to obtain the common targets of the preferred compounds 4g and 4i with antioxidant activity, i.e., common targets. A Venn diagram was drawn, and the intersection points in the diagram represent the relevant targets for the antioxidant activity of 4g and 4i.

[0119] 2. Results Analysis

[0120] A total of 167 target sites for 4g and 4i were retrieved from the SwissTargetPrediction database. A total of 5098 antioxidant targets were obtained from the GeneCards database. The intersection of the first 2000 antioxidant targets with the compound targets was analyzed, and a Venny plot was plotted. Figure 2 As shown, 90 overlapping targets of 4g and 4i with antioxidant effects were screened.

[0121] III. Constructing a visualization of the network relationship between 4G and 4I targets and antioxidant targets, and screening core targets.

[0122] 1. Method

[0123] The obtained intersection target points were uploaded to the online protein-protein interaction platform STRING (https: / / cn.string-db.org / ). The search term "Organism:Homo sapiens" was used in the Multiple Proteins category to remove scattered nodes, resulting in the target protein interaction network and TSV data file. The data was imported into the NetworkAnalyzer module of Cytoscape 3.10 software to analyze the topological parameters of all nodes in the interaction network. Using thresholds for degree centrality (DC), betweenness centrality (BC), and proximity centrality (CC) as the selection criteria for core targets, different colors and circle sizes were used to represent different gradients, constructing a PPI network graph.

[0124] 2. Results Analysis

[0125] Figure 3 The screening process of the PPI interaction network in (a) was carried out by screening through DC, BC and CC thresholds to obtain 18 targets; Figure 3 In the PPI interaction network (b), the node color is proportional to the degree value in the network; Figure 3 Image of the first 18 core targets in (c). From Figure 3 It can be seen that under the conditions of DC≥22.956, BC≥78.800, and CC≥0.006, 18 nodes and 138 edges were obtained. Sorted according to DC values, 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 antioxidant targets of derivatives 4g and 4i

[0127]

[0128]

[0129] IV. GO biological process analysis

[0130] 1. Method

[0131] The 18 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 to "Homo sapiens", and the list type to "Gene List". Then, in Gene_Ontology, "GOTERM_BP_DIRECT", "GOTERM_CC_DIRECT", and "GOTERM_MF_DIRECT" were selected, and the results were visualized.

[0132] 2. Results Analysis

[0133] To clarify the biological processes of the core targets, GO enrichment analysis was performed. Based on the screening criteria, GO enrichment analysis yielded 692 items, including 486 biological processes (BP), accounting for approximately 70.23%; 79 molecular functions (MF), accounting for approximately 11.42%; and 127 cellular components (CC), accounting for approximately 18.35%. The top 10 items were used for visualization analysis, such as... Figure 4 As shown, the core targets involve multiple biological processes, including chromatin remodeling, the EGFR signaling pathway, tyrosine phosphorylation, the insulin receptor, and the PI3K / Akt signaling pathway. The CC process mainly involves the cytoplasm, mitochondria, nucleus, and postsynaptic membrane. The MF process mainly involves ATP binding, kinase and receptor activity, such as H3Y41 protein kinase, tyrosine protein kinase, Ephrin receptor, and insulin receptor. These results suggest that the free radical scavenging processes of derivatives 4g and 4i may be related to kinase-regulated signaling pathways.

[0134] V. KEGG Pathway Enrichment Analysis

[0135] 1. Method

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

[0137] 2. Results Analysis

[0138] Based on the KEGG enrichment analysis results, a total of 162 significantly enriched signaling pathways were identified. The top 10 pathways were then ranked by p-value for visualization analysis. Figure 5 As shown, the pathways highly correlated with the core targets mainly involve the PI3K / Akt, EGFR, MAPK, and Ras signaling pathways. Among them, the PI3K / Akt and MAPK signaling pathways are the core regulators of antioxidant responses, enhancing cellular antioxidant capacity by upregulating the expression of antioxidant enzymes and inhibiting apoptosis. The EGFR, ErbB, and Ras signaling pathways regulate cellular antioxidant responses by activating downstream signaling pathways (such as PI3K / Akt and MAPK). The core of all these signaling pathways points to the PI3K / Akt and MAPK signaling pathways.

[0139] VI. Molecular docking analysis

[0140] 1. Method

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

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

[0143] (2) Ligand preparation: Use Chemdraw to draw compound molecules, import the saved file into Chem3D, adjust the conformation of the substance through MM2 to achieve energy minimization, and save it as pdb format.

[0144] (3) Molecular docking: Add all hydrogen to the protein to set it as the acceptor and save it as a pdbqt file. Add all hydrogen to the compound to set it as the ligand, detect the torsion bond, select the torsion bond, and save it as a pdbqt file. Set the docking box, save and run Autogrid4, set the docking parameters and calculation method, run Autodock4, save and view the results.

[0145] 2. Results Analysis

[0146] To verify the binding affinity of derivatives 4g and 4i to key targets, this invention used preferred compounds 4g and 4i as ligands and performed molecular docking simulations with the core targets AKT1, BCL2, EGFR, SRC, HIF1A, and mTOR as acceptors. The results are shown in Table 5. The binding free energies of derivatives 4g and 4i to the above six target proteins were all negative and ≤-5.0 kcal / mol, indicating good binding performance. Meanwhile, from... Figure 6 It can be seen that derivatives 4g and 4i have the best binding free energy with SRC protein, exhibiting strong binding affinity. For example... Figure 7 As 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 aminothiourea can interact with the Ser283 residue through hydrogen bonding. Derivative 4i establishes a hydrogen bond with the NH on the imidazole ring in the His6 residue through the carbonyl group on the coumarin core. Simultaneously, the Glu332 residue in the protein receptor targets the amino group in the aminothiourea of ​​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] This invention prepared 10 coumarin-thiourea derivatives through drug synthesis and detected their anti-free radical activity using an enzyme-linked immunosorbent assay (ELISA) reader. Derivatives 4g and 4i were selected from these. Using network pharmacology and molecular docking integration strategies, the candidate targets, biological functions, and molecular pathways of action of derivatives 4g and 4i were elucidated. The main core targets include AKT1, BCL2, EGFR, SRC, HIF1A, and mTOR. Changes at these targets involve biological processes regulated by various protein kinases and receptor activations (tyrosine, protein, H3Y41 protein, insulin receptor), as well as participation in the transduction of signaling pathways such as PI3K / Akt, EGFR, MAPK, and Ras.

[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 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. Such 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 class of coumarin-aminothiourea compounds, whose general chemical structure is shown in formula (I): ， In the formula, R is one of the following structural formulas: 。 2. The use of the coumarin-aminothiourea compound according to claim 1 in the preparation of drugs for anti-oxidative stress.

3. The application according to claim 2, characterized in that, The antioxidant stress mentioned includes scavenging DPPH free radicals and / or scavenging hydroxyl free radicals.

4. A pharmaceutical composition, characterized in that, Includes the coumarin-aminothiourea compounds as described in claim 1.

5. A pharmaceutical preparation, characterized in that, It includes the coumarin-thiourea compound of claim 1 or the pharmaceutical composition of claim 4, and a pharmaceutically acceptable carrier.

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