Isoflavone compound, preparation method thereof and application of isoflavone compound in preparation of medicine for treating porcine epidemic diarrhea

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an isoflavone compound, a preparation method thereof and application of the isoflavone compound in preparation of a medicine for treating porcine epidemic diarrhea. The isoflavone compound is prepared by the following method: carrying out Friedel-Crafts acylation reaction on substituted phenylacetic acid and resorcinol under the catalytic action of boron trifluoride diethyl etherate to generate an acylated product; reacting DMF (Dimethyl Formamide) with phosphorus pentachloride to generate a Vilsmeier reagent; and carrying out condensation reaction on the acylation product and a Vilsmeier reagent to obtain the compound. In-vitro experiments prove that the isoflavone compound not only has good anti-PEDV (porcine epidemic diarrhea virus) activity, but also can remarkably relieve inflammatory response induced by PEDV. In addition, a fluorescence resonance energy transfer experiment is adopted, and it is verified that the isoflavone compound can inhibit PEDV 3C sample protease activity. The invention provides a candidate medicine for treating porcine epidemic diarrhea, and has important significance on development and innovation of isoflavone compounds.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an isoflavone compound, its preparation method, and its application in the preparation of a drug for treating swine epidemic diarrhea. Background Technology

[0002] Porcine epidemic diarrhea (PED) is an acute, highly contagious intestinal disease in pigs caused by porcine epidemic diarrhea virus (PEDV). Affected pigs exhibit clinical symptoms such as vomiting, watery diarrhea, and dehydration, with suckling piglets showing the most severe symptoms. Since 2010, the emergence of highly pathogenic PEDV variants has led to a surge in PED infection rates in my country, making PEDV one of the leading viruses causing diarrhea in piglets and posing a significant challenge to the pig farming industry. Currently, vaccination is the most common and effective way to prevent PED; however, the high variability of PEDV means that existing vaccines are insufficient to provide effective immune protection against circulating strains. Therefore, developing highly effective, low-toxicity, and low-residue PEDV treatments is of great importance.

[0003] The coronavirus 3CL protease belongs to the cysteine ​​protease family. Its molecular structure consists of approximately 300 amino acid residues and contains three functional regions. This protease exists in a homodimeric active form, with each monomer forming a functional complex through specific interactions. A unique Cys-His catalytic duplex (His41 and Cys145) exists at the junction of domains I and II. This atypical active site can precisely recognize 11 cleavage sites on the polyprotein, enabling self-cleavage. The non-structural proteins (nsp4-nsp16) released after protease cleavage not only participate in viral genome replication and transcription but also play important roles in key biological processes such as protein translation and nucleic acid synthesis. Therefore, inhibitors targeting this protease can interfere with the replication process of viral genetic material, thereby effectively inhibiting the pathogen's proliferation ability.

[0004] Studies have shown that porcine epidemic diarrhea virus 3C-like protease (PEDV 3CL) pro This enzyme plays a crucial role in viral particle assembly, intracellular transport, and systemic transmission. Notably, it exhibits high sequence conservation among different PEDV strains, with an amino acid sequence variation rate of less than 1.2%. This molecular stability provides an ideal target for developing broad-spectrum antiviral agents. Drug design strategies targeting this enzyme hold promise for inhibiting all currently prevalent PEDV genotypes (including genotype I, genotype II, and recombinant genotypes), thus providing a more universal solution for controlling swine diarrhea caused by this virus.

[0005] Isoflavones are an important class of plant secondary metabolites, with a core structure of the benzochrome ring system. Their biosynthesis begins with the phenylalanine metabolic pathway in plants, proceeding through the extension and cyclization of the cinnamoyl-CoA side chain, ultimately forming a 3-phenyl-substituted chromenet skeleton. Compared to traditional flavonoids, these compounds possess unique molecular characteristics: higher toxicity thresholds, better biomembrane permeability, and structural modifications significantly impacting solubility. In recent years, with the deepening of natural product research, the pharmacological activities of isoflavones have been gradually revealed. Studies have shown that some isoflavones can participate in anti-stress responses as phytodefensins, and also demonstrate multi-target therapeutic potential in the pharmaceutical field, including tumor suppression, antibacterial and anti-inflammatory effects, and cardiovascular disease intervention. However, isoflavones also suffer from poor water and lipid solubility and low bioavailability, limiting their application in the pharmaceutical field. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention aims to provide a novel class of isoflavone compounds. Through molecular docking and ADMET prediction, and following cytotoxicity tests, in vitro efficacy verification tests, and fluorescence cotransfer assays, ideal compounds with good anti-PEDV activity were screened, providing a target for PEDV 3CL. pro Inhibitors provide new drug candidates.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] An isoflavone compound, with the structural formula shown in Formula I below:

[0009]

[0010] In this system, R1 is one of hydrogen, methyl, or halogen; R2 is hydrogen or nitro; R3 is one of hydrogen, methoxy, benzyloxy, cyano, hydroxyl, halogen, amino, bromomethyl, trifluoromethyl, or -NHSO2CH3; R4 is hydrogen or halogen; and R5 is hydrogen or fluorine.

[0011] Preferably, the isoflavone compound is one of the following three compounds:

[0012]

[0013] More preferably, the isoflavone compound is:

[0014] The isoflavone compounds can be prepared by the following method: substituted phenylacetic acid and resorcinol undergo Friedel-Crafts acylation reaction under the catalysis of boron trifluoride diethyl ether to generate an acylated product; DMF and phosphorus pentachloride react to generate a Vilsmeier reagent; the acylated product undergoes a condensation reaction with the Vilsmeier reagent to obtain the isoflavone compounds. Specifically, the method includes the following steps:

[0015] (1) Add resorcinol, substituted phenylacetic acid and boron trifluoride diethyl ether to the reaction vessel in sequence, heat in an oil bath to 80°C, stir magnetically, and after the reaction is complete, obtain mixture I;

[0016] (2) Add PCl5 and DMF to another reaction vessel, heat to 55°C, stir and react for 20 min to obtain mixture II;

[0017] (3) Add mixture II to mixture I in batches, keep the reaction temperature not exceeding 40°C, and react with stirring. After the reaction is complete, pour into 0.1N hydrochloric acid, heat to 70°C, stir for 10 min, and let stand overnight at room temperature. Filter the product, and recrystallize the filter cake with methanol to obtain the isoflavone compounds.

[0018] The substituted phenylacetic acid is:

[0019] Preferably, in step (1), the ratio of resorcinol, substituted phenylacetic acid and boron trifluoride ether is 1 mmol: 1 mmol: 1 mL.

[0020] Preferably, in step (2), the ratio of PCl5 to DMF is 5 mmol: 9 mL.

[0021] Preferably, the molar ratio of resorcinol to PCl5 is 2:3.

[0022] The use of the isoflavone compounds in the preparation of drugs or feed additives for the prevention or treatment of swine epidemic diarrhea is also within the scope of protection of this invention.

[0023] Another object of the present invention is to provide the use of the said isoflavone compounds in the preparation of pharmaceuticals or feed additives against swine epidemic diarrhea virus.

[0024] The isoflavone compounds can significantly downregulate the transcription levels of N and M genes of porcine epidemic diarrhea virus (PEDV) and the inflammatory factors IL-1β, IL-6, and TNF-α genes, and can significantly reduce the expression of N, IL-1β, IL-6, and TNF-α proteins in PEDV.

[0025] Another object of the present invention is to provide the use of the isoflavone compounds in the preparation of swine epidemic diarrhea virus 3C-like protease inhibitors.

[0026] The present invention also provides a drug for treating porcine epidemic diarrhea virus infection, which is made alone from the isoflavone compound or from the isoflavone compound mixed with a pharmaceutically acceptable carrier; the drug includes various acceptable dosage forms, preferably one of tablets, injections, capsules, powders, granules, syrups, and oral liquids.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0028] This invention first screened novel isoflavone compounds targeting PEDV 3C-like protease using molecular docking and ADMET prediction. Then, cytotoxicity and in vitro anti-PEDV efficacy assays verified the good anti-PEDV activity of these novel isoflavone compounds. Finally, fluorescence resonance energy transfer assays confirmed that these novel isoflavone compounds can inhibit PEDV 3C-like protease activity. This invention provides suitable drug candidates for PEDV treatment, new drug development, and further research, and is of great significance to the development of the pharmaceutical field. Attached Figure Description

[0029] Figure 1 The hydrogen spectrum of compound 2 in Example 2;

[0030] Figure 2 The carbon spectrum of compound 2 in Example 2;

[0031] Figure 3 The mass spectrum of compound 2 in Example 2;

[0032] Figure 4 The hydrogen spectrum of compound 8 in Example 2;

[0033] Figure 5 The carbon spectrum of compound 8 in Example 2;

[0034] Figure 6 The mass spectrum of compound 8 in Example 2;

[0035] Figure 7 The hydrogen spectrum of compound 3 in Example 2;

[0036] Figure 8 The carbon spectrum of compound 3 in Example 2;

[0037] Figure 9 The mass spectrum of compound 3 in Example 2;

[0038] Figure 10 The cytotoxicity of different concentrations of the compound in Example 3 on Vero cells and LLC-PK1 cells was detected.

[0039] Figure 11 The effect of compound 2 in Example 3 on the expression of PEDV-N and PEDV-M genes after PEDV infection of Vero cells; Note: Compared with the PEDV group, **p<0.01, *p<0.05;

[0040] Figure 12 The effect of compound 8 in Example 3 on the expression of PEDV-N and PEDV-M genes after PEDV infection of Vero cells; Note: Compared with the PEDV group, **p<0.01, *p<0.05;

[0041] Figure 13 The effect of compound 3 in Example 3 on the expression of PEDV-N and PEDV-M genes after PEDV infection of Vero cells; Note: Compared with the PEDV group, **p<0.01, *p<0.05;

[0042] Figure 14 The effect of compound 2 in Example 3 on PEDV-N protein expression after PEDV infection of Vero cells; Note: **p<0.01 compared with the PEDV group;

[0043] Figure 15 The effect of compound 8 in Example 3 on PEDV-N protein expression after PEDV infection of Vero cells; Note: **p<0.01 compared with the PEDV group;

[0044] Figure 16 The effect of compound 3 in Example 3 on PEDV-N protein expression after PEDV infection of Vero cells; Note: Compared with the PEDV group, **p<0.01, *p<0.05;

[0045] Figure 17 The effect of compound 2 in Example 3 on the expression of PEDV-N, PEDV-M, IL-1β, IL-6 and TNF-α genes after PEDV infection of LLC-PK1 cells; Note: Compared with the PEDV group, **p<0.01, *p<0.05;

[0046] Figure 18 The effect of compound 8 in Example 3 on the expression of PEDV-N, PEDV-M, IL-1β, IL-6 and TNF-α genes after PEDV infection of LLC-PK1 cells; Note: Compared with the PEDV group, **p<0.01, *p<0.05;

[0047] Figure 19The effect of compound 3 in Example 3 on the expression of PEDV-N, PEDV-M, IL-1β, IL-6 and TNF-α genes after PEDV infection of LLC-PK1 cells; Note: Compared with the PEDV group, **p<0.01, *p<0.05;

[0048] Figure 20 The effect of compound 2 in Example 3 on the expression of PEDV-N, IL-1β, IL-6 and TNF-α proteins after PEDV infection of LLC-PK1 cells; Note: Compared with the PEDV group, **p<0.01, *p<0.05;

[0049] Figure 21 The effect of compound 8 in Example 3 on the expression of PEDV-N, IL-1β, IL-6 and TNF-α proteins after PEDV infection of LLC-PK1 cells; Note: Compared with the PEDV group, **p<0.01, *p<0.05;

[0050] Figure 22 The effect of compound 3 in Example 3 on the expression of PEDV-N, IL-1β, IL-6 and TNF-α proteins after PEDV infection of LLC-PK1 cells; Note: Compared with the PEDV group, **p<0.01, *p<0.05;

[0051] Figure 23 The expression induced by SDS-PAGE was detected in Example 4; where M: protein marker; empty: empty vector; whole: whole protein;

[0052] Figure 24 For example, in Example 4, SDS-PAGE was used to detect 3CL. pro Protein purification; where M: protein marker; lane 1: whole protein induced for expression; lane 2: precipitate induced for expression; lane 3: supernatant induced for expression; lane 4: protein supernatant not eluted; lanes 5–10: elution results of 25 mmol / L imidazole for 1–6 times; lanes 11–16: elution results of 50 mmol / L imidazole for 1–6 times; lanes 17–21: elution results of 100 mmol / L imidazole for 1–5 times; lanes 22–28: elution results of 150 mmol / L imidazole for 1–7 times;

[0053] Figure 25 The concentrated protein in Example 4 was detected by Coomassie Brilliant Blue incubation; where M: protein marker; A: ultrafiltration tube 1; B: ultrafiltration tube 2;

[0054] Figure 26 The concentrated protein in Example 4 was detected by His antibody incubation; where M: protein marker; A: ultrafiltration tube 1; B: ultrafiltration tube 2;

[0055] Figure 27 PEDV 3CL in Example 4 pro Activity detection;

[0056] Figure 28 Compound 2 in Example 4 inhibits PEDV 3CL pro active;

[0057] Figure 29 Compound 8 in Example 4 inhibits PEDV 3CL pro active;

[0058] Figure 30 Compound 3 in Example 4 inhibits PEDV 3CL pro active. Detailed Implementation

[0059] To make the purpose and content of this invention clearer, the applicant will now provide a clear and complete description of the technical solution of this invention in conjunction with specific embodiments.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0061] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0062] Example 1: Design, molecular docking and ADMET prediction of novel isoflavone compounds

[0063] Considering that poor solubility and low bioavailability limit the development of isoflavones in the pharmaceutical field, this embodiment focuses on chemical modification and structural alteration to increase solubility, improve bioavailability, and enhance biological activity. Nitro, hydroxyl, halogen, and sulfonyl groups were introduced into the molecular structure to design novel isoflavone compounds. These compounds were then molecularly docked with the PEDV non-structural protein 3C-like protease. ADMET prediction and solubility prediction were performed on the novel isoflavone compounds, and ideal compounds were screened.

[0064] 1. Databases and software

[0065] PDB database (https: / / www.rcsb.org / ),

[0066] The admetSAR 3.0 database (https: / / lmmd.ecust.edu.cn / admetsar3)

[0067] pkcsm database (https: / / biosig.lab.uq.edu.au / pkcsm / prediction),

[0068] Swissadme database (https: / / www.swissadme.ch / ),

[0069] Chem3D software, ChemDraw software, and SYBYL-X 2.0 software.

[0070] 2. Test methods

[0071] 2.1 Design of novel isoflavone compounds

[0072] The substitution pattern of the C7 hydroxyl group and the C4' methoxy group in isoflavones significantly affects the interaction between isoflavones and 3CL. pro The binding affinity suggests that structural modification can optimize inhibitory activity. This embodiment, based on the fundamental structure-activity relationship of isoflavones and following three design principles—activation, detoxification, and drug formulation—introduces nitro, hydroxyl, halogen atoms, and sulfonamide groups (as shown in Formula II below) at key active sites to design a series of novel isoflavone compounds. The aim is to improve their low solubility, enhance their biological activity and pharmacological effects, and further advance their development in the pharmaceutical field.

[0073]

[0074] 2.2 Compounds and 3CL pro Molecular docking

[0075] To investigate the relationship between novel small molecule ligands and PEDV 3CL pro To explore the potential binding possibilities, this study employed computer-aided molecular docking simulations. The experimental procedure can be divided into four main stages: first, a two-dimensional structural model of the compound was constructed using the chemical drawing tool ChemDraw, and then optimized using the three-dimensional conformation (Chem3D) to generate a standard molecular file. 3CLs from the PDB database were selected. proUsing the crystal structure (ID: 4XFQ) as the acceptor model, the following analysis workflow was established in the SYBYL-X 2.0 computing platform: First, a compound database was created in the molecular modeling module, and preprocessed .mol ​​format small molecule files were imported. The docking module was accessed via the function menu, and the docking program based on the surface matching algorithm (SFXC mode) was selected. Key operations were performed in the acceptor processing interface: identifying and separating co-crystallized ligands in the original crystal structure; removing solvent molecules such as the hydration layer; optimizing the protein moleculeization state using an automatic hydrogenation algorithm; and performing charge balance using default parameters. The preprocessed protein file, validated by the system, will be used as the fixed acceptor model in subsequent calculations. In the ligand processing stage, the optimized compound file was converted to Mol2 format and input into the system. In the docking parameter settings, the complex scoring calculation function was disabled to improve computational efficiency, while the original crystal ligand was specified as a spatial positioning reference. Finally, the docking conformation was obtained through batch computation, and the binding mode was systematically evaluated using the module's built-in molecular interaction analysis tools, focusing on key binding parameters such as hydrogen bond networks, hydrophobic interactions, and spatial matching.

[0076] 2.3 Compound ADMET Prediction

[0077] To further predict the druggability of the compounds, they were imported into the AdmetSAR 3.0 and PKCSM databases in "smile" format. Their druggability was evaluated from aspects such as genotoxicity, carcinogenicity, oral toxicity, hepatotoxicity, and nephrotoxicity. Soy isoflavones (CAS No.: 574-12-9) were used as a control, and the solubility of the screened compounds was predicted in the SwissadME database.

[0078] 3. Test Results

[0079] 3.1 Design of novel isoflavone compounds

[0080] Twenty-three novel isoflavone compounds were designed and plotted in ChemDraw software. The results are shown in Table 1.

[0081] Table 1: Structural and molecular formulas of novel isoflavone compounds

[0082]

[0083]

[0084]

[0085] 3.2 Compounds and 3CL pro Molecular docking

[0086] Using SYBYL software, small molecules of compounds are combined with 3CL. proThe molecular docking scoring results are shown in Table 2. The results show that among the 23 novel isoflavone compounds designed, 14 had molecular docking scores above 4, with compound 2 having the highest score of 6.2933. Generally, scores between 3 and 5 indicate moderate binding activity between the ligand and receptor; scores between 5 and 7 indicate high binding activity; and scores above 7 indicate extremely high binding activity. This indicates that most of the 23 designed compounds interact with the receptor protein, with compound 2 exhibiting a strong interaction with the receptor protein.

[0087] Table 2: Molecular docking scoring results

[0088]

[0089]

[0090] 3.3 Compound ADMET Prediction

[0091] The ADMET prediction results are shown in Table 3. The results indicate that, considering genotoxicity, carcinogenicity, oral toxicity, hepatotoxicity, and nephrotoxicity, compounds 2, 8, and 3 all showed normal or good toxicity predictions, indicating no significant toxicity. The solubility predictions for compounds 2, 8, and 3 in the Swissadme database are shown in Table 4. The results show that, compared to soy isoflavones, the solubility of all three compounds was significantly improved, indicating potential for drug development and further validation of their antiviral effects.

[0092] Table 3: ADMET Prediction Results

[0093]

[0094]

[0095] Note: In the admetSAR3.0 database, Ames toxicity, carcinogenicity, oral toxicity, and nephrotoxicity are: 0-0.3 good; 0.3-0.7 normal; 0.7-1 relatively toxic.

[0096] Table 4: Swissadme Prediction Results

[0097]

[0098] (Note: Solubility class: Log S scale: Insoluble<-10 <Poorly soluble<-6<Moderately

[0099] soluble<-4 <Soluble<-2<Very soluble<0<Highly soluble)

[0100] This embodiment employs a molecular docking method to combine 23 novel isoflavone compounds with PEDV 3CL. pro Interaction simulations were conducted. Docking results showed that all compounds achieved a "Total_Score" above 2.9, with 14 compounds scoring above 4 and compound 2 scoring above 6. Most compounds had an absolute "Crash" value below 1; half of the compounds had a "CSCORE" value of 5; and nearly half had a "GLOBAL_CSCORE" value of 4 or 5. The "Total_Score" parameter integrates the energy contributions of various interactions between ligands and acceptors, such as van der Waals forces, electrostatic interactions, hydrogen bonding, and hydrophobic interactions, as well as potential ligand conformational changes and solvation effects. A higher score indicates better binding between the ligand and acceptor, providing important reference for docking results. "Crash" indicates whether there is unreasonable spatial overlap or collision between certain atoms or groups of the ligand and acceptor; a higher value suggests potential spatial irregularities between the ligand and acceptor. "Polar" generally relates to polar interactions, primarily reflecting the strength or contribution of the polar interaction between the ligand and receptor. A higher value indicates a stronger polar interaction between the ligand and receptor, and it is one of the important parameters for assessing ligand-receptor binding. "CSCORE" refers to a score designed for a specific molecular characteristic or interaction, assessing ligand-receptor binding from a specific perspective. "GLOBAL_CSCORE" is similar to "Total_Score," but it considers not only local interactions but also the overall molecular conformation, long-range intermolecular interactions, and the thermodynamic properties of the entire system, providing a more comprehensive assessment of the stability and rationality of ligand-receptor binding. Therefore, overall, compound 2 showed the best docking results. In a study based on network pharmacology and molecular docking technology to explore the target, signaling pathway, and biological function of Lianhua Qingwen capsules in improving the clinical cure rate of COVID-19 (Journal of Southern Medical University, 2021, 41(01):20-30), the authors screened compounds with a Total Score ≥ 5.0, which could form a stable conformation with the target protein and had good binding activity. This indicates that a Total Score of around 5 has the potential to explore the binding of compounds to target proteins. Based on the molecular docking scoring results of this example, to ensure the rigor of the experiment and prevent missed compounds, all compounds with a Total Score greater than 4 were further screened.

[0101] ADMET is a virtual method for predicting the properties of compounds in terms of absorption, distribution, metabolism, excretion, and toxicology. In a study on the screening of NLRP3 inhibitors (Dong Shuang. Virtual Screening and In Vitro Bioactivity Evaluation of NLRP3 Inflammasome Natural Product Inhibitors Based on Molecular Docking [D]. Huazhong Agricultural University, 2023), the authors used ADMET to predict the properties and perform drug-like analysis of compounds, screening out nine candidate compounds. Subsequent experiments verified that the screened candidate compounds did indeed have inhibitory effects on the target. Therefore, ADMET prediction is a very effective predictive and analytical tool. The purpose of this experiment is to predict the toxicology of compounds and assess the potential hazards of drugs in vivo to ensure drug safety. Genotoxicity, carcinogenicity, teratogenicity, acute toxicity, chronic toxicity, hepatotoxicity, and nephrotoxicity were used as the most important indicators for evaluation. The results showed that the toxicity prediction results of compounds 2, 8, and 3 were all normal or good. The compounds were also predicted for solubility in the Swissadme database, using soy isoflavones as a control. The results showed that the solubility of compounds 2, 8, and 3 was significantly improved, indicating their potential for drug development. Candidate compounds were selected in this way.

[0102] In summary, this embodiment, combining molecular docking results and ADMET prediction results, virtually screened compounds 2, 8, and 3 as targets for PEDV 3CL. pro The candidate compounds will be further validated.

[0103] Example 2: Synthesis and structural confirmation of novel isoflavone compounds

[0104] The results of Example 1 show that, after molecular docking and ADMET virtual screening, compounds 2, 8, 3, and 3CL were among the 23 novel isoflavone compounds designed. pro Compounds 2, 8, and 3 exhibit strong interactions and all possess drug-like potential; therefore, this embodiment employs a chemical method to synthesize compounds 2, 8, and 3, and utilizes... 1 H-NMR, 13 The structure of the compound was confirmed by C-NMR and MS.

[0105] 1. Test reagents and instruments

[0106] 1.1 Test Reagents

[0107] Table 5: Reagents used in synthesis and structural confirmation

[0108]

[0109]

[0110] 1.2 Test Instruments

[0111] Table 6: Instruments used for synthesis and structural confirmation

[0112] Instrument Name model Manufacturer Rotary evaporator RE-52AA Shanghai Yarong Biochemical Instrument Factory Thermostatic magnetic stirrer DF-101S Gongyi Yuhua Instrument Co., Ltd. Electronic balance AUY220 SHIMADZU Vacuum drying oven DZF-2060 Shanghai Precision Experimental Equipment Co., Ltd. Circulating multi-purpose vacuum pump SHB-III Shanghai Cangmao Industrial Co., Ltd. Nuclear magnetic resonance spectrometer Bruke-400M Swiss Bruker Company Box-type ultraviolet analyzer ZF-20D Shanghai Baoshan Gu Ke Electro-Optical Instrument Factory Digital melting point apparatus WRS-1C Shanghai Instrument & Electronics Physical Optical Instrument Co., Ltd.

[0113] 2. Test methods

[0114] 2.1 Synthesis of Novel Isoflavones

[0115] References (Li Wei, Li Xing, Wang Qiuan. Synthesis and antiproliferative activity of isoflavone Mannich base derivatives [J]. Chemical Research and Application, 2023, 35(04):753-758; Dai Liyan, Wang Xiaozhong, Chen Yingqi. A new method for the synthesis of isoflavone compounds [J]. Organic Chemistry, 2008, 28(12):2126-2131), the "one-pot method" was used, first acylation and then cyclization, and the crude product was purified by recrystallization. The synthetic routes of compounds 2, 8 and 3 are shown below:

[0116]

[0117] Compound 2 introduces NO2 at the R2 position; compound 8 introduces CH3SO2NH at the R3 position; and compound 3 introduces CH3O at the R3 position.

[0118] The specific synthesis process of compound 2 is as follows: (1) Add 1.1 g (10 mmol) of resorcinol, 1.81 g (10 mmol) of 3-nitrophenylacetic acid and 10 mL of boron trifluoride diethyl ether to a three-necked flask in sequence, heat to 80 °C in an oil bath, stir magnetically, monitor the reaction progress by TLC, and obtain mixture I after the reaction is completed; (2) Add 3.13 g (15 mmol) of PCl5 and 27 mL of DMF to another reaction flask, heat to 55 °C, stir for 20 min, and obtain red mixture II; (3) Add mixture II to mixture I in two batches, keep the reaction temperature not exceeding 40 °C, stir and react, monitor the reaction progress by TLC, and after the reaction is completed, pour into 0.1 N hydrochloric acid (the molar amount of HCl is 100 mmol), heat to 70 °C, stir for 10 min, and let stand overnight at room temperature; filter the product under reduced pressure, and recrystallize the filter cake with methanol to obtain compound 2.

[0119] Compounds 8 and 3 were synthesized using the same method, except that the reactant 3-nitrophenylacetic acid was replaced with 4-(methylsulfonamide)phenylacetic acid and 4-methoxyphenylacetic acid.

[0120] 2.2 Confirmation of the structure of novel isoflavone compounds

[0121] The product prepared under section 2.1 was analyzed by nuclear magnetic resonance (NMR).1 H-NMR and 13 Its structure was confirmed by C-NMR and mass spectrometry (MS).

[0122] 3. Test Results

[0123] Compounds 2, 8, and 3 were subjected to 1 H-NMR, 13 The structure was confirmed by C-NMR and MS, and its physicochemical properties and related spectral data are as follows:

[0124] Compound 2: pale yellow powder, 1.84 g, yield 65.0%, mp: 304.6-307.1 °C.

[0125] 1 H NMR (400MHz, DMSO) δ: 8.60 (s, 1H, 2′-H), 8.50~8.49 (t, J=4.0Hz, 1H, 4′-H), 8.24~8.20 (m, J=12.0Hz, 1H, 2-H), 8.05~8.01 (m, J=16.0Hz, 1H, 6′- H), 8.00~7.97 (d, J=12.0Hz, 1H, 5-H), 7.75~7.69 (t, J=24.0Hz, 1H, 5′-H), 6.99~6.95 (dd, J=12.0Hz, 1H, 6-H), 6.91~6.90 (d, J=4.0Hz, 1H, 8-H).

[0126] 13 C NMR(400MHz, DMSO)δ:179.24,168.18,162.72,160.33,152.83,140.52,139.08,134.88,132.58,128.71,127.76,126.58,121.65,120.76,107.53.

[0127] MS(ESI) m / z: 284.16 [M+H].

[0128] Compound 8: pale yellow powder, 1.56 g, yield 47.1%, mp: 264.8.6-266.4 °C.

[0129] 1H NMR (400MHz, DMSO) δ: 8.36 (s, 1H, 2-H), 7.98~7.96 (d, J=8.0Hz, 1H, 5-H), 7.55~7.53 (d, J=8.0Hz, 2H, 2′-H, 6′-H), 7.27~7.2 5(d,J=8.0Hz,2H,3′-H,5′-H),6.96~6.93(dd,J=12.0Hz,1H,8-H),6.88~6.87(d,J=4.0Hz,1H,6-H),3.02(s,3H,CH3SO2-).

[0130] 13 C NMR (400MHz, DMSO) δ: 179.68, 167.89, 162.67, 158.78, 143.20, 135.06, 132.77, 132.53, 128.16, 124.51, 121.79, 120.50, 107.40.

[0131] MS(ESI) m / z: 332.14 [M+H].

[0132] Compound 3: pale yellow powder, 1.58 g, yield 58.9%, mp: 256.2-258.9 °C.

[0133] 1 H NMR (400MHz, DMSO) δ: 10.80 (s, 1H, 7-OH), 8.30 (s, 1H, 2-H), 7.98~7.96 (d, J=8.0Hz, 1H, 5-H), 7.5 0~7.48(d,J=8.0Hz,2H,2′-H,6′-H),6.98~6.86(m,4H,3′-H,5′-H,6-H,8-H),3.77(s,3H,CH3O-).

[0134] 13 C NMR (400MHz, DMSO) δ: 179.83, 167.79, 164.16, 162.67, 158.32, 135.30, 132.52, 129.47, 128.38, 121.85, 120.38, 118.79, 107.35, 60.33.

[0135] MS(ESI) m / z: 269.12 [M+H].

[0136] Compounds 2, 8, and 3 are structurally similar to genistein. Genistein, also known as genistein, is an isoflavone widely found in legumes and other plants. It is poorly soluble in water but soluble in organic solvents such as ethanol, methanol, and acetone. It is relatively stable and possesses multiple effects including antiviral, antioxidant, estrogen-like, anti-inflammatory, and cardiovascular protective properties. The results of Example 1 indicate that compounds 2, 8, and 3 are similar to PEDV 3CL. pro The strong interactions suggest that it also possesses strong antiviral capabilities. Referring to the synthesis method of genistein, this embodiment employs a "one-pot" method, integrating multiple reactions into a single reaction system, first acylation followed by cyclization. Compared to literature reports, this embodiment optimizes the reaction process. While literature requires repeated distillation of the raw materials and recrystallization of the first-step crude product, this embodiment simplifies the related operations, eliminating the need for purification of the first-step crude product. Furthermore, this embodiment changes the purification method from column chromatography to methanol recrystallization, further simplifying the operation.

[0137] In this experiment, the acylation method was Friedel-Crafts acylation. Catalyzed by boron trifluoride diethyl ether, the carboxyl group in phenylacetic acid reacted with resorcinol, introducing a phenylacetyl group onto the benzene ring of resorcinol, generating the corresponding acylated product. Boron trifluoride diethyl ether, as a Lewis acid catalyst, enhanced the reaction activity. The specific mechanism is as follows: BF3 in boron trifluoride diethyl ether has an empty p orbital and is a strong Lewis acid. It coordinates with the oxygen atom of the carboxyl group in phenylacetic acid, increasing the positive charge of the carbonyl carbon in the carboxyl group, thereby improving its electrophilicity. Under the action of BF3, the carboxyl group undergoes proton transfer and structural rearrangement, forming an acyl cation intermediate with stronger electrophilicity. The benzene ring in resorcinol has abundant electron clouds and is a good nucleophile. The phenylacetyl cation, as an electrophile, attacks the position with higher electron cloud density on the benzene ring of resorcinol, forming a positively charged σ-complex intermediate. The σ-complex intermediate is unstable and rapidly loses a proton (H). + The aromaticity of the benzene ring is restored, thus generating the final acylated product. DMF and phosphorus pentachloride then form a Vilsmeier reagent, which undergoes a condensation reaction with the acylated product to finally obtain the target product.

[0138] Example 3: Compound cytotoxicity test and anti-PEDV efficacy test

[0139] Novel isoflavone compounds 2, 8, and 3, designed, screened, and synthesized in Examples 1 and 2, were further validated for their toxicity and efficacy in vitro. First, different concentrations of the compounds (0, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM) were used to perform cell proliferation and cytotoxicity tests (CCK8) on Vero and LLC-PK1 cells. Then, a PEDV infection model was established on Vero and LLC-PK1 cells, divided into five groups: a blank control group (Control group), a control group (PEDV group), a low-dose treatment group (PEDV + compound 25 μM), a medium-dose treatment group (PEDV + compound 50 μM), and a high-dose treatment group (PEDV + compound 100 μM). In addition to directly inhibiting proteases, isoflavones also exhibit good anti-inflammatory effects; therefore, the expression of related genes and proteins was detected by Q-RT PCR and Western blotting to verify the compounds' anti-PEDV ability.

[0140] 1. Experimental materials, reagent preparation and instruments

[0141] 1.1 Test Materials

[0142] Experimental cells and strains: African green monkey kidney cell (Vero) and porcine kidney cell (LLC-PK1) were provided by Professor Li Zhonghua of the Animal Nutrition and Gut Health Innovation Team of Wuhan University of Light Industry;

[0143] The PEDV-HB strain was provided by Professor Li Zhonghua of the Animal Nutrition and Gut Health Innovation Team at Wuhan University of Light Industry.

[0144] Table 7: Test Reagents

[0145]

[0146]

[0147] 1.2 Preparation of test reagents

[0148] Vero / LLC-PK1 cell culture medium: 91% DMEM High Glucose (cytiva, SH30243.01) basal medium supplemented with 8% FBS (ExCell, FSP500) and 1% penicillin-streptomycin (Solepro, P1400) in total system.

[0149] Compounds: Using DMSO as solvent, the compounds obtained in Example 2 were prepared into a series of sample solutions with different concentration gradients and stored in a refrigerator at 4°C for later use.

[0150] 75% DEPC alcohol: anhydrous ethanol: DEPC water = 75:25, v / v.

[0151] Electrotransfer buffer: Trans-Blot Turbo 5×Transfer Buffer: Anhydrous ethanol: Deionized water, mixed in a volume ratio of 1:1:3, and used immediately.

[0152] 5× Electrophoresis Buffer: Weigh 72g glycine, 15.15g Tris, and 5g SDS, dissolve them in deionized water, and bring the volume to 1L. Dilute with deionized water to 1× before use.

[0153] TBS buffer: Dissolve one packet of TBS buffer (ready-to-use dry powder) in deionized water and bring the volume up to 2L to obtain 1×TBS buffer.

[0154] TBST buffer: Prepare 1×TBST buffer by adding 0.1% (v / v) Tween 20 to 1×TBS buffer.

[0155] 1.3 Test Instruments

[0156] Table 8: Instruments Used in the Experiment

[0157] Instrument Name model Manufacturer Mini multi-amplitude track shaking bed ZHWY-103B Shanghai Smart City Vertical high-pressure steam sterilizer LDZX-75L Shanghai Shenan Inverted fluorescence microscope Moticam3000 MOTIC Electric thermostatic incubator DRP-9082 Shanghai Senxin CO2 incubator BPN-80CH Shanghai Yiheng Miniature Vortex Mixer WH-2 Shanghai Huxi ELISA reader EnSpire Shanghai Platinum Elmer Chemiluminescence imaging instrument DXI 600 Beckmann, Germany Electrophoresis apparatus DYY-6C type Beijing June 1st Gene Amplification Instrument mastercycle Eppendorf, Germany Real-time PCR instrument <![CDATA[ViiA TM 7]]> Thermo Fisher Scientific, USA Electrophoresis tank DYCZ-40S Beijing June 1st Rapid transfer system Trans-blot Turbo American talent scout

[0158] 2. Test methods

[0159] 2.1 Vero Cell and LLC-PK1 Cell Culture Methods

[0160] (1) Culture and passage of African green monkey kidney cells (Vero)

[0161] When the confluence of Vero cell monolayers reaches 80%-90%, standardized passage procedures are performed: First, the old culture medium is discarded, and the cell layer is gently washed twice along the flask wall with PBS pretreated at 37°C. Then, trypsin is added to cover the cell surface, and after a few seconds of contact, excess digestion solution is immediately aspirated with a sterile pipette. The culture flask is transferred to a 5% CO2 incubator (37°C) for precise digestion for 3 minutes. When the intercellular spaces are observed to widen and the cell shape becomes rounded under a microscope, DMEM complete medium containing 10% fetal bovine serum is quickly added to terminate the enzymatic digestion reaction. After repeated pipetting to form a homogenized cell suspension, the cells are aliquoted into fresh culture containers at a 1:2 amplification ratio.

[0162] (2) Culture and passage of porcine kidney cells (LLC-PK1)

[0163] When the culture system reaches a confluence of over 80%: First, remove the old culture medium. Gently wash the cell layer twice along the flask wall with PBS pretreated at 37°C to remove metabolic products. Add trypsin digestion solution, covering the cell layer, and let stand at room temperature for 2 minutes. Then remove excess digestion solution. Place the culture vessel in a humidified incubator for 3 minutes for digestion, observing under a microscope. After digestion, add DMEM complete medium containing 10% fetal bovine serum to terminate the enzymatic digestion reaction. After repeatedly pipetting to form a homogeneous cell suspension, aliquot into fresh culture containers at a 1:2 expansion ratio.

[0164] 2.2 Cell cryopreservation and thawing

[0165] (1) Cell cryopreservation: A mixture of 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) was used as the cell cryopreservation medium. After trypsin digestion and separation, the cells were added to serum-containing medium to terminate the digestion reaction. The supernatant metabolites were removed by gradient density centrifugation (2000 rpm, 5 min, 4℃), and the cell pellet was resuspended in pre-cooled cryopreservation medium. After aliquoting into sterile cryovials, a programmed cooling process was performed: first, incubation at 4℃ for 30 min, then at -20℃ for 60 min, followed by ultra-low temperature storage at -80℃ for 12 h, and finally, long-term storage in liquid nitrogen.

[0166] (2) Cell resuscitation: After removing the cryovials from the liquid nitrogen tank, immediately immerse them in a 37°C constant temperature water bath for thawing. Aseptically transfer the thawed cell suspension to a centrifuge tube containing pre-warmed basal medium and centrifuge at 2000 rpm for 5 min to remove the cryoprotectant. Gently resuspend the precipitate in 5 mL of fresh complete culture medium (containing 10% FBS + 1% antibiotics), inoculate it into a new cell culture flask, and then place it in a 5% CO2, 37°C constant temperature incubator to start the resuscitation culture.

[0167] 2.3 CCK-8 assay for the cytotoxicity of compounds

[0168] Vero cells in good growth condition were subjected to 10 4Cells were seeded per well in 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator. After the cells grew into a monolayer, they were treated with compounds at final concentrations of 0, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively, with 5 replicates per group. Cells were cultured for another 24 h, then washed twice with PBS solution preheated to 37°C. The supernatant in each well was then replaced with 100 μL of DMEM basal medium containing 1% enhanced CCK-8 solution, and incubated in the dark for 2 h. After incubation, the cells were gently shaken, and the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated using the formula: Cell viability = (Aa - A) ÷ (Ab - A) × 100%, where Aa: OD of the well treated with the compound after incubation with CCK-8 solution. 450 Value; A: OD of wells that are cell-free and untreated with compounds after incubation with CCK-8 solution. 450 Value; Ab: OD of the well without compound treatment after incubation with CCK-8 solution. 450 value.

[0169] The cell viability of LLC-PK1 cells was detected using the same method, with the difference being: LLC-PK1 cells were viable at a concentration of 2 × 10⁻⁶ cells / cells. 4 Cells / well were seeded in 96-well cell culture plates.

[0170] 2.4 Establishing a viral infection model

[0171] Vero cells in good growth condition were seeded into 24-well plates and cultured in a 37°C, 5% CO2 incubator. After the cells reached confluence, they were washed three times with PBS containing trypsin (final trypsin concentration of 8 μg / mL) and then seeded with PEDV (MOI = 0.001).

[0172] LLC-PK1 cells in good growth condition were seeded into 24-well plates and cultured in a 37°C, 5% CO2 incubator. After the cells reached confluence, they were washed three times with PBS containing trypsin (final trypsin concentration of 7 μg / mL) and then seeded with PEDV (MOI = 0.01).

[0173] The samples were divided into five groups: Control group (no compound added, no inoculation), PEDV group (inoculation only), low-dose group (compound at a final concentration of 25 μM, inoculated), medium-dose group (compound at a final concentration of 50 μM, inoculated), and high-dose group (compound at a final concentration of 100 μM, inoculated). Each group was incubated at 37°C and 5% CO2 for 24 hours before being collected.

[0174] 2.5Q-RT PCR

[0175] Take the 24-well plate incubated for 24 hours as described in section 2.4, add 300 μL of total RNA extraction reagent to each well, incubate at room temperature for 10 min, and pipette the mixture into a 1.5 mL enzyme-free centrifuge tube. Add 1 / 5 volume of chloroform to the centrifuge tube, vortex vigorously for 30 s to completely emulsify, and incubate on ice for 5 min. Centrifuge at 12000 g for 15 min at 4 °C. Pipe as much supernatant as possible into a new 1.5 mL enzyme-free centrifuge tube, add an equal volume of isopropanol, gently invert to mix, and incubate on ice for 10 min. Centrifuge at 12000 g for 10 min at 4 °C and discard the supernatant. Add 1 mL of 75% DEPC ethanol to the centrifuge tube, gently invert the tube to wash the precipitate, and then centrifuge at 12000 g for 5 min at 4 °C and discard the supernatant. Dry at room temperature for 5 min, add 20 μL of DEPC water to dissolve the precipitate, determine the RNA concentration, and dilute with DEPC water to a uniform concentration.

[0176] Following the instructions of the reverse transcription kit, reverse the RNA into cDNA and store at -20°C.

[0177] Q-RT-PCR was performed according to the instructions of the Taq Pro Universal SYBR qPCR Master MIX kit. HRPT1 was used as an internal control in the Vero cell model, and RPL19 was used as an internal control in the LLC-PK1 cell model. Experimental data were analyzed according to 2... -ΔΔCT Process it.

[0178] Primers were designed by NCBI and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequences of each primer are shown in Table 9.

[0179] Table 9: Primer Sequence List

[0180]

[0181] 2.6 Total Protein Extraction and Quantification

[0182] Vero cells in good growth condition were seeded into six-well plates and cultured in a 37°C, 5% CO2 incubator. After the cells reached confluence, they were washed three times with PBS containing trypsin (final trypsin concentration of 8 μg / mL) and then seeded with PEDV (MOI = 0.001).

[0183] LLC-PK1 cells in good growth condition were seeded into six-well plates and cultured in a 37°C, 5% CO2 incubator. After the cells reached confluence, they were washed three times with PBS containing trypsin (final trypsin concentration of 7 μg / mL) and then seeded with PEDV (MOI = 0.01).

[0184] The samples were divided into five groups: Control group (no compound added, no inoculation), PEDV group (inoculation only), low-dose group (compound at a final concentration of 25 μM, inoculated), medium-dose group (compound at a final concentration of 50 μM, inoculated), and high-dose group (compound at a final concentration of 100 μM, inoculated). Each group was incubated at 37°C and 5% CO2 for 24 hours before samples were collected.

[0185] Cellular total protein extraction assay steps:

[0186] (1) Sample pretreatment: After the above cell co-culture system is terminated, discard the residual culture medium, and wash the cell layer twice with pre-cooled phosphate-buffered saline (PBS) to remove impurities. The entire operation should be carried out at a low temperature of 4°C to prevent protein degradation.

[0187] (2) Construction of lysis system: Prepare high-efficiency lysis buffer: Use ice-pre-cooled basic lysis buffer (Lysis Buffer) as solvent, add compound phosphatase inhibitor, broad-spectrum protease inhibitor and benzyl sulfonyl fluoride (PMSF), vortex mix well and stand on ice for later use.

[0188] (3) Low-temperature lysis procedure: Add 200-300 μL of pre-cooled lysis buffer to each well to ensure complete coverage of the cell monolayer. Perform lysis for 20 minutes under ice bath conditions.

[0189] (4) Protein sample collection: The cells were mechanically disrupted using a cell scraper, and the lysate was transferred to a pre-cooled 1.5 mL centrifuge tube. After a brief centrifugation (12000×g, 30s), the supernatant was collected, aliquoted, and immediately stored in an ultra-low temperature freezer at -80℃ for later use.

[0190] BCA protein quantification assay steps:

[0191] (1) Redissolve the lyophilized standard protein in deionized water to prepare a 25 mg / mL stock solution, aliquot it and store it at -20°C.

[0192] (2) Take 25 mg / mL stock solution and dilute it in a gradient with PBS to prepare a standard solution with a working concentration of 0.5 mg / mL.

[0193] (3) Preparation of BCA working solution: Mix BCA reagent A and reagent B precisely at a volume ratio of 50:1 to prepare sufficient working solution. Vortex for 30 seconds until the solution is homogenized. The working solution should be stored away from light and used within 2 hours.

[0194] (4) Using an eight-gradient concentration standard system (0-0.5 mg / mL), add 0, 1, 2, 4, 8, 12, 16 and 20 μL of 0.5 mg / mL working concentration standard solution to the standard wells of a 96-well plate, respectively, and then add PBS to a final volume of 20 μL.

[0195] (5) Take 2 μL of the above-mentioned protein sample to be tested and mix it with 18 μL of PBS in the sample wells of a 96-well plate to maintain the uniformity of detection between samples.

[0196] (6) Add 200 μL of BCA working solution to each well and incubate at 37℃ for 25 ± 5 minutes to ensure the colorimetric reaction proceeds fully. Measure the absorbance at 562 nm using a microplate spectrophotometer, with three technical replicates for each sample. Establish a standard curve and calculate the protein concentration based on the sample OD value.

[0197] (7) Mix the quantified protein sample with 5× loading buffer at a volume ratio of 4:1, heat in a boiling water bath for 10 minutes to achieve full denaturation of the protein, centrifuge to remove heat-induced precipitates, and freeze at -80℃ for later use.

[0198] 2.7 Western blot

[0199] Experimental steps:

[0200] (1) Preparation of colloidal matrix: Select a suitable concentration of separating gel system based on the molecular weight range of the target protein. During preparation, 4.6 mL of separating gel matrix is ​​used, and 2 mL of gel solution is required for the preparation of the stacking gel system. Strict control of ambient temperature and coagulation time is necessary during colloidal polymerization.

[0201] (2) Sample loading: In the electrophoresis tank, accurately transfer 3 μL of protein marker to the first well, and add 10 μL of the denatured protein sample solution from section 2.6 to each of the remaining wells. The sample loading operation must be performed in an ice bath to ensure sample stability.

[0202] (3) Electrophoresis parameters: In the initial stage, 60V constant voltage electrophoresis was used for 40 minutes to allow the sample to migrate to the separation interface. In the second stage, the voltage was switched to 120V for continuous electrophoresis for 50 minutes to complete fine separation. The electrophoresis buffer was kept circulating and cooled throughout the process.

[0203] (4) Transfer: The PVDF transfer membrane was activated by pretreatment with methanol for 10 seconds, and then equilibrated with the transfer buffer. After the matching filter paper was fully soaked in the transfer buffer, the transfer sandwich was constructed in the order of cathode filter paper-transfer membrane-separating gel-anodic filter paper, and air bubbles between the layers were eliminated using a glass rod. The transfer device was set to 25V voltage and 25mA current, and the transfer was carried out continuously for 7 minutes.

[0204] (5) Blocking: Immediately after the transfer, immerse the membrane medium in blocking buffer containing 5% skim milk powder or 5% bovine serum albumin, and continuously block on a horizontal shaker at room temperature for 180 min at a speed of 60 rpm.

[0205] (6) Primary antibody incubation: After discarding the blocking buffer, rinse the membrane three times with TBST buffer (10 min each time). Add the specific primary antibody working solution (dilution ratio according to the antibody instructions) and incubate at 4°C on a shaker for 12-16 h.

[0206] (7) Secondary antibody incubation: Recover the primary antibody, repeat the TBST rinsing step (rinse 3 times, 10 min each time), then add HRP-labeled secondary antibody working solution (dilution ratio according to antibody instructions), and incubate at room temperature with shaking for 60 min. After incubation, rinse 3 times with TBST, 10 min each time.

[0207] (8) Color development: Prepare the color development working solution by mixing equal volumes of color development substrate A and solution B. After removing the residual buffer solution from the membrane surface, completely immerse the transfer membrane in the color development working solution, observe the band development under dark conditions, terminate the reaction in a timely manner, and record the imaging data.

[0208] 2.8 Statistical Analysis

[0209] One-way ANOVA was performed using SPSS software. A p-value < 0.01 and p-value < 0.05 were considered statistically significant, and a p-value < 0.001 was considered extremely statistically significant. "***" indicates that p < 0.001, meaning the difference between the two groups is extremely significant; "**" indicates that p < 0.01, meaning the difference between the two groups is significant; and "*" indicates that p < 0.05, meaning the difference between the two groups is significant.

[0210] 3. Test Results

[0211] 3.1 Cytotoxicity of the compound to African green monkey kidney cells and porcine kidney cells

[0212] In this embodiment, African green monkey kidney cells (Vero) and porcine kidney cells (LLC-PK1) were used as in vitro virus-infected cell models. First, the toxicity of the compound to the cells was verified. The CCK-8 results are as follows: Figure 10 As shown, treatment with compounds at concentrations of 12.5, 25, 50, 100, and 200 μM showed no significant toxicity to either type of cell. Therefore, compounds at 25, 50, and 100 μM were selected for subsequent cell experiments.

[0213] 3.2 Effects of the compound on the expression of related genes and proteins in PEDV-infected Vero cells

[0214] PEDV M protein is a transmembrane glycoprotein that determines and influences viral budding and is one of the main proteins in PEDV serological diagnostic antigens. PEDV N protein is the viral nucleocapsid protein, playing an important role in viral genome transcription, RNA packaging, and viral assembly. In this embodiment, the expression of N and M genes and proteins was detected in a PEDV-infected Vero cell model to verify the anti-PEDV ability of the compound.

[0215] 3.2.1 Effects of the compound on the expression of N and M genes after PEDV infection of Vero cells

[0216] (1) Effects of compound 2 on the expression of N and M genes after PEDV infection of Vero cells. The results are as follows: Figure 11 As shown in the figure. The experimental results showed that in the PEDV-infected Vero cell model, compound 2 significantly reduced the transcription levels of the key viral genes N and M. 25 μM compound 2 downregulated the transcription levels of N and M genes in cells to some extent (p < 0.05); 100 μM compound 2 significantly downregulated the transcription levels of N and M genes in cells (p < 0.01). This indicates that compound 2 has good anti-PEDV ability.

[0217] (2) Effects of compound 8 on the expression of N and M genes after PEDV infection of Vero cells. The results are as follows: Figure 12 As shown in the figure. The experimental results showed that in the PEDV-infected Vero cell model, compound 8 significantly reduced the transcription levels of the key viral genes N and M. 25 μM and 50 μM of compound 8 downregulated the transcription levels of N and M genes in cells to some extent (p < 0.05); 100 μM of compound 8 significantly downregulated the transcription levels of N and M genes in cells (p < 0.01). This indicates that compound 8 has good anti-PEDV activity at high doses.

[0218] (3) Effects of compound 3 on the expression of N and M genes after PEDV infection of Vero cells. The results are as follows: Figure 13 As shown in the figure. The experimental results showed that in the PEDV-infected Vero cell model, compound 3 significantly reduced the transcription levels of the key viral genes N and M. 25 μM and 50 μM of compound 3 downregulated the transcription levels of N and M genes in cells to some extent; 100 μM of compound 3 significantly downregulated the transcription levels of N and M genes in cells (p < 0.01). Compound 3 exhibited good anti-PEDV activity at high doses.

[0219] 3.2.2 Effects of the compound on PEDV-N protein expression after PEDV infection of Vero cells

[0220] (1) The effect of compound 2 on PEDV-N protein expression after PEDV infection of Vero cells. The results are as follows: Figure 14 As shown in the figure. The experimental results showed that in the PEDV-infected Vero cell model, compound 2 significantly reduced the expression level of the key viral protein PEDV-N. 25 μM, 50 μM and 100 μM of compound 2 all significantly downregulated the expression level of PEDV-N protein in cells (p < 0.01); compound 2 has good anti-PEDV ability.

[0221] (2) Effect of compound 8 on PEDV-N protein expression after PEDV infection of Vero cells. The results are as follows: Figure 15 As shown in the figure. The experimental results showed that in the PEDV-infected Vero cell model, compound 8 significantly reduced the expression level of the key viral protein PEDV-N. 50 μM and 100 μM of compound 8 significantly downregulated the expression level of PEDV-N protein in cells (p < 0.01); compound 8 showed good anti-PEDV ability at medium and high doses.

[0222] (3) Effect of compound 3 on PEDV-N protein expression after PEDV infection of Vero cells. The results are as follows: Figure 16 As shown in the figure. The experimental results showed that in the PEDV-infected Vero cell model, compound 3 significantly reduced the expression level of the key viral protein PEDV-N. 25 μM compound 3 downregulated the expression level of PEDV-N protein in cells to some extent (p < 0.05); 50 μM and 100 μM compound 3 significantly downregulated the expression level of PEDV-N protein in cells (p < 0.01); compound 3 exhibited good anti-PEDV activity at medium and high doses.

[0223] 3.3 Effects of the compound on the expression of related genes and proteins in PEDV-infected LLC-PK1 cells

[0224] PEDV has been reported to cause severe atrophic enteritis in newborn pigs and to lead to increased pro-inflammatory and innate immune responses. Pro-inflammatory factors such as IL-1β, IL-6, and TNF-α are key mediators of most inflammatory responses. To verify the compound's ability to combat PEDV, this example examined the expression of PEDV-N, PEDV-M, IL-1β, IL-6, and TNF-α genes and proteins when PEDV infected LLC-PK1 cells.

[0225] 3.3.1 Effects of the compound on the expression of PEDV-N, PEDV-M, IL-1β, IL-6 and TNF-α genes after PEDV infection of LLC-PK1 cells

[0226] (1) Effects of compound 2 on the expression of PEDV-N, PEDV-M, IL-1β, IL-6 and TNF-α genes after PEDV infection of LLC-PK1 cells. The results are as follows: Figure 17 As shown in the figure. The experimental results showed that in the PEDV-infected LLC-PK1 cell model, compound 2 significantly reduced the transcription levels of the key viral genes N and M, as well as the key inflammatory mediators IL-1β, IL-6, and TNF-α. 100 μM of compound 2 significantly downregulated the transcription levels of PEDV-N, PEDV-M genes, and the key inflammatory mediators IL-1β, IL-6, and TNF-α genes in cells (p < 0.01); compound 2 exhibited good anti-PEDV activity.

[0227] (2) Effects of compound 8 on the expression of PEDV-N, PEDV-M, IL-1β, IL-6 and TNF-α genes after PEDV infection of LLC-PK1 cells. The results are as follows: Figure 18 As shown in the figure. The experimental results showed that in the PEDV-infected LLC-PK1 cell model, compound 8 significantly reduced the transcriptional levels of key viral genes PEDV-N and PEDV-M, as well as key inflammatory mediators IL-1β, IL-6, and TNF-α. 25 μM and 50 μM of compound 8 downregulated the expression levels of PEDV-N, PEDV-M, IL-1β, IL-6, and TNF-α genes to some extent; 100 μM of compound 8 significantly downregulated the transcriptional levels of PEDV-N, PEDV-M, and key inflammatory mediators IL-1β, IL-6, and TNF-α genes in cells; compound 8 exhibited good anti-PEDV activity at high doses.

[0228] (3) Effects of compound 3 on the expression of PEDV-N, PEDV-M, IL-1β, IL-6 and TNF-α genes after PEDV infection of LLC-PK1 cells. The results are as follows: Figure 19 As shown in the figure. The experimental results showed that in the PEDV-infected LLC-PK1 cell model, compound 3 significantly reduced the transcriptional levels of key viral genes PEDV-N and PEDV-M, as well as key inflammatory mediators IL-1β, IL-6, and TNF-α. 25 μM and 50 μM of compound 3 downregulated the expression levels of PEDV-N, PEDV-M, IL-1β, IL-6, and TNF-α genes to some extent; 100 μM of compound 3 significantly downregulated the transcriptional levels of PEDV-N, PEDV-M, and key inflammatory mediators IL-1β, IL-6, and TNF-α genes in cells; compound 3 exhibited good anti-PEDV activity at high doses.

[0229] 3.3.2 Effects of the compound on the expression of PEDV-N, IL-1β, IL-6 and TNF-α proteins after PEDV infection of LLC-PK1 cells

[0230] (1) Effects of compound 2 on the expression of PEDV-N, IL-1β, IL-6 and TNF-α proteins after PEDV infection of LLC-PK1 cells. The results are as follows: Figure 20 As shown in the figure. The experimental results showed that in the PEDV-infected LLC-PK1 cell model, compound 2 significantly reduced the expression levels of the key viral protein PEDV-N and the key inflammatory mediators IL-1β, IL-6, and TNF-α. 25 μM, 50 μM, and 100 μM of compound 2 all significantly downregulated the expression level of PEDV-N protein in cells (p < 0.01); 100 μM of compound 2 significantly downregulated the expression levels of the key inflammatory mediators IL-1β, IL-6, and TNF-α (p < 0.01); compound 2 exhibited good anti-PEDV activity.

[0231] (2) Effects of compound 8 on the expression of PEDV-N, IL-1β, IL-6 and TNF-α proteins after PEDV infection of LLC-PK1 cells. The results are as follows: Figure 21 As shown in the figure. The experimental results showed that in the PEDV-infected LLC-PK1 cell model, compound 8 significantly reduced the expression levels of the key viral protein PEDV-N and the key inflammatory mediators IL-1β, IL-6, and TNF-α. Compound 8 at concentrations of 25 μM, 50 μM, and 100 μM downregulated the expression levels of PEDV-N and IL-1β, IL-6, and TNF-α proteins in cells to some extent. The effect was most pronounced at the 100 μM concentration. Compound 8 exhibited good anti-PEDV activity at high doses.

[0232] (3) Effects of compound 3 on the expression of PEDV-N, IL-1β, IL-6 and TNF-α proteins after PEDV infection of LLC-PK1 cells. The results are as follows: Figure 22 As shown in the figure. The experimental results showed that in the PEDV-infected LLC-PK1 cell model, compound 3 significantly reduced the expression levels of the key viral protein PEDV-N and the key inflammatory mediators IL-1β, IL-6, and TNF-α. Compound 3 at concentrations of 25 μM, 50 μM, and 100 μM downregulated the expression levels of PEDV-N and IL-1β, IL-6, and TNF-α proteins in cells to some extent. The effect was most pronounced at the 100 μM concentration. Compound 3 exhibited good anti-PEDV activity at high doses.

[0233] During viral infection, the release of pro-inflammatory cytokines and chemokines often triggers inflammatory responses, thereby promoting disease progression. Previous studies have shown that PEDV infection of Vero cells significantly increases the mRNA levels of pro-inflammatory factors such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). In this example, after PEDV infection of LLC-PK1 cells, a significant upregulation of IL-1β, IL-6, and TNF-α expression was observed, further confirming the conclusion that PEDV can induce inflammatory responses. Adding the screened compounds to the cell culture system significantly reduced the expression levels of inflammatory cytokines, indicating that the three compounds can effectively alleviate the PEDV-induced inflammatory response. Therefore, in the PEDV-infected LLC-PK1 cell model, the three compounds not only demonstrated anti-PEDV potential but also significantly alleviated the PEDV-induced inflammatory response in LLC-PK1 cells. In a study on the effects of baicalin and baicalein on PEDV-infected Vero and LLC-PK1 cell models (Frontiers in Microbiology, 2024, 15:1357470-1357470), the authors also used these two in vitro models to explore the anti-PEDV potential of baicalin and baicalein, consistent with the in vitro models used in this experiment. Among the three compounds, considering the results of PEDV-N, PEDV-M, and IL-1β, IL-6, and TNF-α genes and proteins, especially at high concentrations, compound 2 showed superior antiviral activity compared to compounds 8 and 3. Therefore, whether compound 2 has the same effect on PEDV-induced enteritis warrants further in vivo validation.

[0234] Example 4: Compound effect on PEDV 3CL pro Activity inhibition test

[0235] Example 3 has already verified the compounds' ability to fight PEDV; this example further explores the antiviral mechanism of the compounds. Among the many antiviral mechanisms, inhibition of viral protease activity is one of them. In Example 1, the three compounds were based on their interaction with PEDV 3CL. pro The results were obtained through virtual molecular docking screening, suggesting that the three compounds are related to PEDV 3CL. pro There is an interaction between the two, inhibiting the activity of viral proteases and exhibiting antiviral activity. To further verify the interaction between the two, this embodiment uses a fluorescence resonance energy transfer (FRET) assay to verify the interaction between the compound and PEDV 3CL. proTo investigate the interaction between the compounds and their antiviral mechanisms.

[0236] 1. Test materials

[0237] 1.1 Preparation of experimental materials and reagents

[0238] 3CL pro Recombinant plasmid: provided by Professor Li Zhonghua of the Animal Nutrition and Gut Health Innovation Team at Wuhan University of Light Industry;

[0239] Ni-NTA NUPharose FF, product number: NRPB57S-Z15, purchased from Hangzhou Newlong Biotechnology Co., Ltd.

[0240] Ultrafiltration centrifuge tubes (10kDa), item number: UFC2010, purchased from Mililpore;

[0241] Preparation of Tris-HCl: Dissolve Tris (121.14g) in deionized water (800mL), then adjust the pH to 7.4 with HCl;

[0242] Preparation of Buffer A: Add NaCl (29.22g) and Tris-HCl (120mL) to deionized water to a final volume of 1L, then adjust the pH to 7.4 using HCl.

[0243] Preparation of Buffer B: Add NaCl (29.22g), Tris-HCl (20mL), and imidazole (34.04g) to deionized water to a final volume of 1L, then adjust the pH to 7.4 using HCl;

[0244] LB liquid medium: LB broth (20.0 g) is dissolved in distilled water (800 mL), autoclaved, cooled, and stored at 4°C for later use.

[0245] LB solid medium: LB agar (20.0g) is dissolved in distilled water (800mL), autoclaved, poured into bacterial culture dishes, and sealed and stored in a 4℃ refrigerator for later use after cooling and solidification.

[0246] IPTG stock solution: Isopropyl-β-D-thiogalactoside (IPTG, 0.238 g) was dissolved in deionized water (10 mL), filtered through a 0.22 μm filter membrane, and stored in a -20°C refrigerator away from light for later use.

[0247] 2. Test methods

[0248] 2.1 Induced expression of PEDV 3C-like protease

[0249] 3CL proThe expression vector was provided by the Animal Nutrition and Gut Health Innovation Team of Wuhan University of Light Industry. The specific process was as follows: First, the RNA of the PEDV strain was extracted and reverse transcribed into cDNA. Using this as a template, the PEDV Nsp5 gene (expressing PEDV 3CL) was added. pro The amplification was performed using primers (as shown below), followed by gel recovery and enzyme digestion, and then ligated into the Pet-28a(+) prokaryotic expression vector. Finally, the recombinant plasmid was obtained through transformation. After sequencing identification, the recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells, and after shaking, 3CL was performed. pro The expression.

[0250] Primer sequences for the PEDV Nsp5 gene:

[0251] F: 5'-CGCGGATCCGCTGGCTTGCGTAAGATG-3';

[0252] R: 5'-CCGCTCGAGCTGAAGATTAACGCCATACAT-3'.

[0253] PEDV 3CL proThe specific process of induction expression is as follows: (1) First, the bacterial culture (the product obtained by transforming the above recombinant plasmid into Escherichia coli BL21 competent cells) was transferred to LB liquid medium supplemented with kanamycin at an inoculation volume of 1%, and amplified incubated at 37°C for 4 hours. (2) Then, 0.8 mM IPTG was added to the culture system to start protein induction, and cultured continuously at a constant temperature of 37°C and a shaking speed of 120 rpm for 24 hours. (3) After induction, the cells were centrifuged at 12000 rpm for 20 minutes at 4°C, and the supernatant was removed, leaving the bacterial precipitate. (4) The collected bacterial cells were washed twice with phosphate-buffered saline (PBS), and then the bacterial clumps were resuspended in PBS solution. (5) Transfer the prepared bacterial suspension to a pre-cooled centrifuge tube and place it in an ice bath for cell lysis: Adjust the probe of the ultrasonic disruptor to maintain a distance of about 1 mm from the bottom of the tube (ensure contact with the liquid surface but not touching the bottom of the tube); set the ultrasonic treatment program to a cyclic mode of 10 seconds on and 15 seconds off, control the power at 40% of the total output, and continue the treatment for 25 minutes. (6) After ultrasonic treatment, centrifuge again at 12,000 rpm for 20 minutes at 4°C, separate the supernatant and transfer it to a new centrifuge tube, and resuspend the precipitate with pre-cooled PBS buffer. (7) Take 10 μL of the bacterial suspension after ultrasonic disruption in step (5), the supernatant in step (6) and the precipitate suspension in step (6), respectively, mix each with 2.5 μL of 5× protein loading buffer, and heat-treat in a 98°C metal bath for 10 minutes to denature the protein. (8) Finally, perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on the whole bacterial protein sample, soluble supernatant and precipitate.

[0254] 2.2PEDV 3CL pro Purification and concentration

[0255] (1) Pretreatment of the chromatography column: First, the metal chelate affinity chromatography column (Ni-NTANUPharose FF) was activated with 20% ethanol solution, and then equilibrated with 10 column volumes of binding buffer (Buffer A) to ensure that the column bed was fully wetted.

[0256] (2) Construction of gradient elution system: Three gradient elution systems were pre-configured: Buffer A and Buffer B were mixed at volume ratios of 9.5:0.5, 9:1, 8:2, and 7:3, respectively, corresponding to final imidazole concentrations of 25, 50, 100, and 150 mmol / L, to construct a linear gradient elution environment.

[0257] (3) Sample loading and washing: The crude protein extract solution containing the His tag (the supernatant of step (6) in section 2.1) was loaded three times by circulating through the column, and then washed with Buffer A to remove non-specifically bound impurity proteins.

[0258] (4) Gradient elution collection: Elution buffer was loaded sequentially in order of increasing imidazole concentration, with 3 mL elution volume loaded for each concentration gradient. Protein fractions corresponding to each gradient were collected simultaneously during elution, labeled and stored.

[0259] (5) Elution effect verification: 16 μL of each gradient collection solution was mixed with 4 μL of 5× loading buffer, heat-denatured at 98℃ for 10 minutes, and then detected by SDS-PAGE. The characteristic elution concentration of the target protein LI0004 was determined by Coomassie brilliant blue staining.

[0260] (6) Target protein enrichment: Select the eluted fraction of the target protein enrichment and concentrate it using a 10kDa molecular weight cutoff ultrafiltration tube. Centrifuge at 2500 rpm at 4℃ until the protein solution volume is reduced to 1-2 mL.

[0261] (7) Purity identification: Finally, take 15 μL of the concentrated purified protein sample and perform immunological verification by Western Blot technology. Use specific primary antibody to detect the molecular weight of the target protein and the purification effect.

[0262] 2.3 PEDV 3C-like protease activity assay

[0263] To assess the enzymatic activity of the purified protease, this assay system used a dual-labeled fluorescent peptide as the specific substrate. The peptide's N-terminus was modified with an Edans donor fluorescent group (excitation wavelength 340 nm, emission wavelength 490 nm), and its C-terminus was bound to a Dabcyl acceptor quenching group. The intact substrate molecule maintained the donor-acceptor relationship via covalent bonds. The nanometer-scale spacing falls precisely within the effective radius of fluorescence resonance energy transfer (FRET). When the substrate is intact, the fluorescence energy generated by the excited donor group is transferred to the acceptor group non-radiatively, achieving an efficiency exceeding 90%. This results in the absence of Edans' characteristic blue fluorescence, creating a low-background detection environment. However, when a specific protease acts on the substrate peptide chain, the covalently linked fluorescent groups undergo spatial dissociation. At this point, the donor-acceptor distance exceeds the effective FRET range. The fluorescence quantum yield of the Edans group was restored to over 85% of the theoretical value, and a fluorescence signal with intensity positively correlated with enzyme activity could be detected at 490 nm. This detection strategy achieves background suppression through intramolecular self-quenching, and its signal-to-noise ratio (SNR) is 5-8 times higher than that of traditional single-labeled probes. The dynamic detection range covers enzyme concentration gradients from 0.1 to 100 μM, and the detection sensitivity reaches the pmol level.

[0264] Used to measure PEDV 3CL pro The active enzyme reaction system (100 μL) included FRET substrate at a final concentration of 10 μM, Tris / HCl buffer (pH 7.5) at a final concentration of 20 mM, and purified 3C-like protease at different final concentrations (0, 0.25, 0.5, 1, and 2 μM). Fluorescence emitted during the reaction was continuously monitored over 90 minutes at a sampling interval of 60 s, with an excitation wavelength of 340 nm and an emission wavelength of 490 nm.

[0265] 2.4 Inhibition test of compound on PEDV 3C-like protease activity

[0266] A 100 μL reaction system was constructed in a 96-well optical detection plate, with the following final concentrations of each component: 0.5 μM 3CL pro 10 μM MFRET fluorescent substrate and different concentrations of the compound (0, 25, 50, 100 μM) were used, and the ionic strength was maintained using a Tris-HCl buffer system (20 mM, pH = 7.5). The experimental group and the control group were set up synchronously, with the latter using an equal volume of buffer solution instead of the compound solution.

[0267] The specific process of the reaction system is as follows: the compound is reacted with 3Cl... pro The enzyme was pre-incubated at 37°C in a shaker for 30 min to ensure full formation of the inhibitor-enzyme complex. FRET fluorescent substrate was added to initiate the enzyme digestion reaction, maintaining a reaction environment of 37°C. Fluorescence signals were continuously acquired for 90 min using a multi-mode microplate reader (excitation 340 nm / emission 490 nm), with a sampling interval of 60 s. Three independent replicate experiments were used to reduce systematic error.

[0268] 3. Test Results

[0269] 3.1 Inducible expression of PEDV 3C-like protease

[0270] Induction at 37℃, 0.8mM IPTG, and 120rpm for 24h resulted in the collection of total protein, supernatant, and precipitate. An empty vector was used as a control. Coomassie Brilliant Blue gel electrophoresis results are as follows: Figure 23 As shown in the figure. The results showed that the induced protein size was around 36 kDa, and the target protein was also expressed in the supernatant.

[0271] 3.2 Purification and Concentration of PEDV 3C-like Protease

[0272] 3CL via Ni pillar pro The protein was purified, and the eluent obtained by eluting with imidazole at concentrations ranging from low to high was collected and identified by SDS-PAGE. Figure 24 As shown. The results indicated that the target protein could not be successfully eluted using solutions containing 25 mM and 50 mM imidazole; while elution was achieved using a solution containing 100 mM imidazole, the amount of target protein in the eluent was low and contained impurities; elution with a solution containing 150 mM imidazole successfully yielded purified target protein, and the amount of target protein obtained gradually decreased with increasing elution time. Therefore, the eluent obtained from elution with a solution containing 150 mM imidazole was collected and concentrated using two ultrafiltration tubes for easy centrifugation and balancing during concentration. Detection was performed by SDS-PAGE incubation with Coomassie Brilliant Blue and specific His antibody incubation, as shown... Figure 25 and Figure 26 As shown, the results indicate that the concentrated protein size is approximately 36 kDa. The protein concentrations after concentration in the two tubes were measured to be 27.8 μM and 16.7 μM, respectively.

[0273] 3.3 PEDV 3C-like protease activity assay

[0274] 10 μM FRET substrate, 20 mM Tris / HCl buffer (pH 7.5), and purified 3CL at different concentrations (0, 0.25, 0.5, 1, 2 μM) were used. pro The cleavage activity of the purified protein was verified and the optimal reaction concentration was determined using an enzymatic reaction system. The experimental results are as follows: Figure 27 As shown, the results indicate that the purified protein possesses cleavage activity, and the optimal 3CL was determined for subsequent experiments. pro The concentration is 0.5 μM.

[0275] 3.4 Inhibition test of compound on PEDV 3C-like protease activity

[0276] At 0.5μM 3CL pro Compounds 2, 8, and 3 were tested in a 100 μL reaction system consisting of 10 μM FRET fluorescent substrate, different concentrations of compounds (0, 25, 50, 100 μM), and Tris-HCl buffer. pro The interaction between them. Experimental results are as follows: Figures 28-30 As shown, the results indicate that the inhibition of PEDV 3C-like protease activity by the three compounds is dose-dependent, and at a concentration of 100 μM, compound 2 has a more significant inhibitory effect, consistent with the results of Example 3.

[0277] The results of Example 4 demonstrate that the compounds screened by molecular docking in Example 1 are compatible with PEDV 3CL. pro The interaction between them suggests that the compound may exhibit antiviral activity by inhibiting viral 3C-like proteases. pro The Cys-His catalytic dual at the active site is a core target for inhibitor design, acting as a 3CL for coronaviruses. pro The core unit of catalytic function, the active site composed of Cys145 and His41, plays a crucial role in enzymatic reactions. These two amino acid residues are present in the 3CL of SARS-CoV-2. pro Precisely positioned within the three-dimensional conformation, they collectively constitute the catalytic center at the bottom of the enzyme's active pocket. Specifically, the thiol group (-SH) of Cys145 and the imidazole ring of His41 form a dynamic synergistic effect through electrostatic attraction and a hydrogen bond network, constructing the molecular basis of the catalytic reaction. The mechanism of action can be summarized as follows: His41, acting as a proton transfer mediator, first captures a proton from the thiol group of Cys145, inducing the formation of a thiolate anion (-S-). Subsequently, this highly active thiolate initiates a nucleophilic attack on the carbonyl carbon of the substrate peptide bond, forming a transition tetrahedral intermediate. During this process, neighboring residues such as Gly143 and Ser144 stabilize the intermediate conformation through hydrogen bonds. Finally, the proton is transferred from His41 to the amino terminus of the substrate, leading to peptide bond cleavage and the release of hydrolysis products.

[0278] Significant progress has been made in the development of antiviral drugs using covalent binding strategies targeting this catalytic dual. Representative drugs such as nirmatrelvir and simnotrelvir are designed based on the selective covalent modification of the Cys145 thiol group with electrophilic groups (such as α-ketoamides or aldehydes), achieving irreversible inhibition of enzyme activity by forming stable thiohemiacetals or Michael adducts. In contrast, the natural product baicalein employs a non-covalent binding mode. Its molecular skeleton binds to the His41-Cys145 neighboring region through hydrophobic interactions and a hydrogen bond network, thereby competitively blocking substrate entry into the active site. In a FRET assay of quercetin on PEDV 3C-like protease, quercetin showed dose-dependent inhibition of PEDV 3C-like protease. In another FRET assay concerning the effect of tannic acid on PEDV 3C-like protease, tannic acid similarly showed dose-dependent inhibition of PEDV 3C-like protease. Therefore, the FRET assay is widely used to investigate the interaction between compounds and PEDV 3C-like protease. This invention also employs the FRET assay to investigate the inhibitory effect of compounds on PEDV 3C-like protease, finding that the three designed novel isoflavone compounds showed dose-dependent inhibition of PEDV 3C-like protease activity. Furthermore, at a concentration of 100 μM, compound 2 showed a particularly significant inhibitory effect on PEDV 3C-like protease.

[0279] Therefore, based on the experimental results of Example 3, compound 2 can be considered as a candidate compound for PEDV inhibitors. It has the potential to conduct in vivo experiments to further verify its antiviral ability and has the potential value to become an effective anti-PEDV drug. This provides a certain reference for the development and innovation of isoflavone compounds.

Claims

1. An isoflavone compound with the following structural formula: R1 is one of hydrogen, methyl, or halogen; R2 is hydrogen or nitro; R3 is one of hydrogen, methoxy, benzyloxy, cyano, hydroxyl, halogen, amino, bromomethyl, trifluoromethyl, or -NHSO2CH3; R4 is hydrogen or halogen; and R5 is hydrogen or fluorine.

2. The isoflavone compound according to claim 1, characterized in that, The compound is selected from one of the following compounds:

3. A method for preparing the isoflavone compound according to claim 1 or 2, comprising the following steps: (1) Add resorcinol, substituted phenylacetic acid and boron trifluoride diethyl ether to the reaction vessel in sequence, heat in an oil bath to 80°C, stir magnetically, and after the reaction is complete, obtain mixture I; (2) Add PCl5 and DMF to another reaction vessel, heat to 55°C, stir and react for 20 min to obtain mixture II; (3) Add mixture II to mixture I in batches, keep the reaction temperature not exceeding 40°C, and react with stirring. After the reaction is complete, pour into 0.1N hydrochloric acid, heat to 70°C, stir for 10 min, and let stand overnight at room temperature; filter the product, and recrystallize the filter cake with methanol to obtain the isoflavone compounds. The substituted phenylacetic acid is 4. The use of the isoflavone compounds of claim 1 or 2 in the preparation of drugs or feed additives for the prevention and / or treatment of porcine epidemic diarrhea.

5. The use of the isoflavone compounds according to claim 1 or 2 in the preparation of drugs or feed additives for treating porcine epidemic diarrhea virus.

6. The application according to claim 5, characterized in that, The application is in the preparation of drugs or feed additives that reduce the transcription levels of the N and M genes of porcine epidemic diarrhea virus.

7. The application according to claim 5, characterized in that, The application is in the preparation of drugs or feed additives that reduce the expression of N protein in porcine epidemic diarrhea virus.

8. The use of the isoflavone compound of claim 1 or 2 in the preparation of a medicament or feed additive for the prevention and / or treatment of PEDV-induced inflammation.

9. The use of the isoflavone compound according to claim 1 or 2 in the preparation of a porcine epidemic diarrhea virus 3C-like protease inhibitor.

10. A drug for treating porcine epidemic diarrhea virus infection, characterized in that, It is prepared by the isoflavone compound of claim 1 or 2 alone, or by the mixture of the isoflavone compound with a pharmaceutically acceptable carrier.

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