Application of an isoflavone compound in the preparation of anti-COVID-19 drugs

Abstract

This invention discloses the application of isoflavone compounds in the preparation of antiviral drugs against COVID-19, relating to the field of pharmaceutical formulation technology. The isoflavone compounds are multi-target, dual-function antiviral components, meaning they can target the coronavirus replication target (SARS-CoV-2M)... pro These isoflavones (and / or RdRp) exert direct antiviral effects and can also target p17 inflammatory factors to exert anti-inflammatory effects, thus possessing dual antiviral and anti-inflammatory functions. Given the complex pathophysiology of COVID-19, its treatment should include antiviral infection and regulation of the body's immune-inflammatory system imbalance. Therefore, the aforementioned isoflavone compounds with dual functions are promising candidates for anti-COVID-19 drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, specifically relating to the application of an isoflavone compound in the preparation of anti-COVID-19 drugs. Background Technology

[0002] The 2019 novel coronavirus (COVID-19, hereinafter referred to as the novel coronavirus) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a highly infectious enveloped positive RNA virus that can cause respiratory illness, fever, and pneumonia. Currently, anti-COVID-19 drugs are under development and have achieved significant success, including Remdesivir and Molnupiravir (RNA-dependent RNA polymerase RdRp inhibitors), Paxlovid and Xocova (Ernstavivir) (SARS-CoV-2 major protease-M...). pro Inhibitors and tocilizumab-interleukin (IL)-6 receptor inhibitors).

[0003] In thousands of years of clinical practice and disease control, traditional Chinese medicine has accumulated a large number of effective Chinese herbal prescriptions for the treatment of epidemic diseases through the summary of clinical experience. Based on ancient classic prescriptions, the "Three Prescriptions and Three Medicines" (Jinhua Qinggan Granules, Lianhua Qingwen Granules, Xuebijing Injection, Qingfei Paidu Decoction, Xuanfei Baidu Decoction, and Huashi Baidu Decoction) were derived.

[0004] Patent (CN112168899A) discloses a vine tea extract that can inhibit the 3CL proteolytic enzyme of the novel coronavirus or the 3CL proteolytic enzyme of the SARS coronavirus. The vine tea extract is a water extract, an alcohol extract, or a flavonoid extract of vine tea. The vine tea extract can be used to treat novel coronavirus or SARS coronavirus infection.

[0005] However, because the active ingredients and mechanisms of action of these prescriptions are unclear, they struggle to gain recognition from the world and mainstream medicine. Therefore, natural active substances with well-defined structures and components have become increasingly popular both domestically and internationally.

[0006] Patent (CN114409626A) discloses the preparation and antiviral application of baicalin derivatives. The baicalin derivatives are prepared from baicalin (5,6,7-trihydroxyflavone) via an electrophilic substitution reaction. The reaction solvent is anhydrous acetone after drying and dehydration. The reaction is carried out under ice bath (0°C) conditions for 1 hour, followed by quenching with ice water, and then filtering to obtain a pale yellow or off-white crude product. The half-maximal inhibitory concentrations (IC50) of the baicalin derivatives against influenza virus neuraminidase NA and the main protease Mpro of the novel coronavirus are both less than 100 μM. Furthermore, the synthesis method of the derivatives is simple and easy to perform. During the reaction, the two hydroxyl groups at C6-OH and C7-OH of baicalin are replaced by various sulfonyl chloride compounds.

[0007] Patent (CN 112402402 A) discloses the application of xanthohumol in the preparation of inhibitors against the novel coronavirus. Targeting the Mpro protease, which causes lung inflammation in the novel coronavirus, xanthohumol, a highly active antiviral compound with strong binding affinity to Mpro protease, was obtained through structure-based virtual screening. Xanthohumol inhibits Mpro protease activity by binding to the target Mpro protease, thereby inhibiting viral replication and transcription, achieving an antiviral effect against novel coronavirus infection. Xanthohumol can be used in the preparation of inhibitors against the novel coronavirus Mpro protease, thus enabling its application in the preparation of drugs for the prevention and / or treatment of novel coronavirus inflammation. The binding energy of xanthohumol to Mpro protease in this invention is -63.427 KJ / mol, and the IC50 of Mpro protease... 50 The value was 4.97 ± 1.79 μM, and the inhibition constant Ki value was 2.14 μM.

[0008] Patent (CN115192566A) discloses the application of flavonoid monomer compounds in the preparation of drugs for the prevention or treatment of novel coronavirus. The provided flavonoid monomer compounds, due to their high structural similarity to nucleotide substrates, can effectively bind to the NTPs entry channels of RNA polymerase, thereby blocking the entry of normal nucleotide substrates into RNA polymerase. In other words, the flavonoid monomer compounds occupy the nucleotide substrate binding sites, preventing the nucleotide substrate from binding to the RNA polymerase. Consequently, normal NTPs can no longer enter the enzyme's active site, ultimately exhibiting an inhibitory effect on the biological function of RNA polymerase. This patent is suitable for the prevention or treatment of novel coronavirus using flavonoid monomer compounds.

[0009] However, the therapeutic effects of current natural active drugs against COVID-19 remain very limited. Due to the high mutation and recombination rates of SARS-CoV-2, there is an urgent need for more effective antiviral drugs. In this context, combination therapy targeting multiple targets remains an indispensable direction for the research and development of anti-COVID-19 drugs. Therefore, this invention focuses on the main pharmacodynamic mechanisms (anti-inflammatory and antiviral), using integrative pharmacology to identify active ingredients with antiviral and anti-inflammatory effects. This allows for the efficient discovery and screening of active compounds against COVID-19, leading to the development of natural product drugs with clearly defined components and mechanisms that can act on multiple targets. Summary of the Invention

[0010] This invention addresses the problems existing in the prior art by providing an application of isoflavone compounds in the preparation of antiviral drugs against COVID-19. The isoflavone compounds are multi-target, dual-function antiviral components, meaning they can target the coronavirus replication target (SARS-CoV-2M)... pro These isoflavones exert direct antiviral effects and can also target p17 inflammatory factors to exert anti-inflammatory effects, thus possessing dual antiviral and anti-inflammatory functions. Given the complex pathophysiology of COVID-19, its treatment should include antiviral infection and regulation of the body's immune-inflammatory system imbalance. These isoflavones, with their dual functions, are promising candidate drugs for treating COVID-19.

[0011] To achieve the above objectives, in a first aspect, the present invention provides the application of isoflavone compounds in the preparation of anti-COVID-19 drugs, wherein the isoflavone compounds are selected from one or more of compounds of formula (I) or their stereoisomers, magnolol, emodin, glycyrrhizin chalcone, quercetin, and glycyrrhizin chalcone B.

[0012] The structure of the compound of formula (Ⅰ) is as follows:

[0013]

[0014] R1 and R2 are each independently selected from H, hydroxyalkyl or isopentenyl, wherein the hydroxyalkyl group is selected from hydroxymethyl or hydroxyethyl; R3 is selected from H or hydroxy; R4, R5, R6 and R7 are each independently selected from H, hydroxy or alkoxy-OR8, wherein R8 is selected from C1-4 alkyl, preferably methyl.

[0015] The structural formulas of magnolol, emodin, glycyrrhizin chalcone, quercetin, and glycyrrhizin chalcone B are as follows:

[0016]

[0017] In some preferred embodiments, the compound of formula (I) is selected from:

[0018]

[0019]

[0020] In some preferred embodiments, the isoflavone compound is selected from the following compounds or combinations thereof:

[0021]

[0022] In some preferred embodiments, the isoflavone compounds have dual antiviral and anti-inflammatory functions.

[0023] In some preferred embodiments, the isoflavone compounds target the coronavirus replication target SARS-CoV-2M. pro And / or RdRp exert direct antiviral effects and exert anti-inflammatory effects by targeting p17 inflammatory factors.

[0024] In some preferred embodiments, the functional groups hydroxyl-OH and carbonyl-CO- of the isoflavone compounds form hydrogen bonds with ASN-781, SER-784, TYR-129, THR-14 or LYS-47.

[0025] In some preferred embodiments, the functional groups hydroxyl-OH, carbonyl-CO-, and ether-O- of the isoflavone compounds form hydrogen bonds with LYS-5, ARG-4, TRP-207, or PHE-3.

[0026] In some preferred embodiments, the dosage form of the anti-COVID-19 drug is selected from granules, oral liquids, tablets, or capsules.

[0027] In some preferred embodiments, the isoflavone compounds are formulated into anti-COVID-19 drugs by adding appropriate pharmaceutically acceptable excipients.

[0028] In some preferred embodiments, the dosage of the anti-COVID-19 drug is an effective concentration of 1-300 μM.

[0029] Secondly, the present invention provides an anti-COVID-19 drug comprising isoflavone compounds, wherein the isoflavone compounds are selected from one or more of compounds of formula (I) or their stereoisomers, magnolol, emodin, glycyrrhizin chalcone, quercetin, and glycyrrhizin chalcone B.

[0030] The structure of the compound of formula (Ⅰ) is as follows:

[0031]

[0032] R1 and R2 are each independently selected from H, hydroxyalkyl or isopentenyl, wherein the hydroxyalkyl group is selected from hydroxymethyl or hydroxyethyl; R3 is selected from H or hydroxy; R4, R5, R6 and R7 are each independently selected from H, hydroxy or alkoxy-OR8, wherein R8 is selected from C1-4 alkyl.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The isoflavone compounds provided by this invention are multi-target, dual-function antiviral components, capable of targeting the coronavirus replication target (SARS-CoV-2M). pro It can exert direct antiviral effects with RdRp, and can also target p17 inflammatory factors to exert anti-inflammatory effects, thus having dual functions of antiviral and anti-inflammatory.

[0035] 2. The isoflavone compounds provided by this invention have excellent antiviral and anti-inflammatory effects and are promising candidate drugs for anti-COVID-19; among them, magnolol, glycyrrhizin isoflavone, glycyrrhizin A, emodin, licorice chalcone, quercetin and glycyrrhizin B all show superior performance in antiviral and anti-inflammatory activities. Attached Figure Description

[0036] Figure 1 Molecular docking virtual calculation results: (A) Molecular docking results; (B) Compound structure and potential pharmacologically active functional groups; Elliptical coils represent functional groups that form hydrogen bonds with protein amino acid residues; Line thickness indicates the number of hydrogen bonds formed between the compound and target protein residues; Functional groups a, -OH (hydroxyl), b, -CO- (carbonyl), c, -O- (ether bond); Compound and SARS-CoV-2M pro The polar interactions between / RdRp proteins are represented by dashed lines. Data are mean ± SEM values ​​from three independent experiments.

[0037] Figure 2 (A) Initial screening results of the ectopic nucleocapsid expression cell line (Caco-2-N) infected with SARS-CoV-2 virus-like particles (trVLPs) at 100 μM; (B) EC50 / CC50 of compounds with inhibition rates >90%, Caco-2 cells were infected with SARS-CoV-2 (MOI = 0.1) in the presence of different concentrations of isoflavone compounds. Cell supernatants were collected 24 hours after infection, and the viral copy number in the cell supernatant was determined by qRT-PCR. Data represent three independent experiments, mean ± SEM, and statistical significance was determined by ANOVA. ### P<0.001; (C) In vitro enzyme activity inhibition rate of candidate active ingredient against SARS-CoV-2Mpro; (D) In ​​vitro enzyme activity inhibition rate of candidate active ingredient against RdRp.

[0038] Figure 3 Screening of anti-inflammatory active components in TNP-1PMA cells infected with Bunyavirus (SFTSV): (A) THP-1 macrophages were incubated with SFTSV (MOI=5) for 1 hour and treated with 10 μM isoflavones; cells and supernatants were collected 48 hours after infection; the level of P17 in the supernatant and the expression level of Pro-IL-1β or NP in cell lysates were detected by Western blotting; (BC) Glycyrrhiza chalcone B, Northwest glycyrrhiza isoflavones and prickly glycyrrhiza chalcone inhibited SFTSV-induced P17 secretion in a dose-dependent manner; (B) THP-1 macrophages were incubated with SFTSV (MOI=5) for 1 hour and treated with 1.1, 3.3 and 10 μM concentrations; cells and supernatants were collected 48 hours after infection. The levels of P17 in the supernatant and the expression levels of Pro-IL-1β or NP in cell lysates were detected by Western blotting; (C) the inhibition rate was statistically assessed by the gray values ​​of the P17 band; data represent two independent experiments, mean ± SEM, and statistical significance was determined by ANOVA. # P<0.05, ## P<0.01; (DE) Anti-inflammatory effects of glycyrrhizin B, glycyrrhizin isoflavone, and prickly glycyrrhizin chalcone on SARS-CoV-2 infection-induced P17 release; Calu-3 cells were infected with SARS-CoV-2 (MOI=0.1) in the presence of glycyrrhizin B, glycyrrhizin isoflavone, and prickly glycyrrhizin at concentrations of 1.1, 3.3, 10, and 30 μM, with GSK-872 (5 μM) as a positive control; cells and supernatant were collected 48 hours after infection; (D) Western blotting was used to detect the level of P17 in the supernatant and the expression levels of Pro-IL-1β or NP in cell lysates; (E) The inhibition rate was assessed by gray value analysis of the P17 band; data represent three independent experiments, mean ± SEM, statistical significance was determined by ANOVA. ### P<0.001, #### P<0.0001. Detailed Implementation

[0039] It is worth noting that all raw materials used in this invention are common commercially available products, and their sources are not specifically limited. The English and Chinese equivalents of the isoflavone compounds used in this invention are as follows:

[0040]

[0041] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1: Virtual Calculation of Molecular Docking

[0043] The binding affinity between isoflavones and target proteins was detected using molecular docking virtual computation. In the D3Docking module of the D3Targets-2019-nCoV platform, isoflavones, along with the Mpro-positive drug Ensitrelvir and the RdRp-positive drug Remdesivir, were docked with the Mpro / RdRp targets, respectively. Specific target information is as follows:

[0044] 3CLpro / Mpro-Dimer PDB ID:6y2g;

[0045] 3CLpro / Mpro-Monomer PDB ID:5r82;

[0046] RdRp-+RNA&+Mg PDB ID:7bv2;

[0047] RdRp PDB ID:6m71;

[0048] The docking fraction is expressed in Kcal / mol.

[0049] Table 1 shows the binding scores of isoflavone compounds to their targets. It is evident that glycyrrhizin isoflavones, glycyrrhizin A, quercetin, prickly glycyrrhizin chalcone, magnolol, emodin, and glycyrrhizin chalcone B exhibit good binding affinity to the Mpro target protein, while the same compounds bind well to the RdRp target protein. Furthermore, glycyrrhizin isoflavones, glycyrrhizin A, quercetin, prickly glycyrrhizin chalcone, emodin, and glycyrrhizin chalcone B show good binding affinity to both Mpro and RdRp target proteins. pro Arg4 and Lys5 residues form hydrogen bonds; Northwest glycyrrhizin isoflavones form hydrogen bonds with Asn781, Ser784, and Thr141 residues of RdRp; and glycyrrhizin isoflavone A forms hydrogen bonds with Tyr129 and Lys47 residues. See details... Figure 1 And Table 2.

[0050] Table 1. Isoflavone compound docking scores with target sites

[0051]

[0052] Table 2. Potential pharmacodynamic functional groups of isoflavones and their target binding sites.

[0053]

[0054] Example 2: Antiviral activity of isoflavone compounds

[0055] Test method: A safe and convenient method that can be carried out in a BSL-2 laboratory was selected—the screening system of SARS-CoV-2 virus-like particles (trVLPs) infected nucleocapsid ectopic expression cell line (Caco-2-N) (see reference: Yu Y, Ju X, & Ding Q (2021) A Nucleocapsid-based Transcomplementation Cell Culture System of SARS-CoV-2 to Recapitulate the Complete Viral Life Cycle. Bio-protocol 11(21):e4257) to test the antiviral activity of isoflavone compounds.

[0056] The results show ( Figure 2 A) Magnolol, glycyrrhizin isoflavones, glycyrrhizin chalcone B, glycyrrhizin isoflavone A, emodin, salicyrrhizin chalcone, isoglycyrrhizin, rhein, quercetin, and magnolol all exhibit significant antiviral activity, with an inhibition rate >90% at a concentration of 100 μM. Based on preliminary screening results, the half-maximal effective concentration (EC50) of the first 10 chemical components was [not specified in the original text]. 50 ) and median lethal dose (50% cytotoxic concentration, CC) 50 The selectivity index (SI) was measured. 50 With EC 50 (CC 50 / EC 50 The ratio of SI to 5.0 is a method for measuring the potential therapeutic window of a drug. Six chemical components with an SI value greater than 5.0 were ultimately selected, including magnolol, glycyrrhizin isoflavones, glycyrrhizin isoflavone A, emodin, chalcone arachidone, and quercetin, indicating that these components have a large therapeutic window and good drug sensitivity.

[0057] SARS-CoV-2M proRdRp plays a crucial role in viral replication and is a promising therapeutic target in antiviral drug development. Therefore, the above isoflavone compounds were determined using fluorescence resonance energy transfer (FRET) protease assay and in vitro polymerase activity assay, and the half-maximal inhibitory concentration (IC50) was calculated based on dose-response curves. 50 ) value. For example Figure 2 As shown in C, quercetin and glycyrrhizin chalcone are effective against SARS-CoV-2M. pro It has a moderate inhibition rate (micromole concentration), IC50 50 The concentrations were 35.14 and 22.47 μM, respectively. Furthermore, glycyrrhizin and glycyrrhizin A significantly inhibited RdRp, with IC50 values ​​of 35.14 and 22.47 μM, respectively. 50 The values ​​were 28.90 and 47.31 μM, respectively. Figure 2 D).

[0058] Example 3: Anti-inflammatory activity of isoflavone compounds

[0059] Anti-inflammatory treatment is an effective approach for severe COVID-19 patients with excessive inflammatory immune responses (cytokine storm). Severe fever with thrombocytopenia syndrome (SFTSV), similar to SARS-CoV-2, promotes the production and secretion of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, etc.), leading to an inflammatory cytokine storm in critically ill patients. SFTSV-infected THP-1 macrophages are a successfully constructed inflammatory cell model, and the production and secretion of IL-1β can be used to evaluate virus-induced inflammation. Therefore, a preliminary screening of the anti-inflammatory activity of flavonoids at a concentration of 10 μM on SFTSV-infected THP-1 macrophages was conducted (see Appendix). Figure 3 ).

[0060] Through the secretion of mature IL-1β (P17), glycyrrhizin B, glycyrrhizin isoflavones, and licorice chalcone showed significant anti-inflammatory activity (inhibition rate >90%), while glycyrrhizin isoflavone A showed good anti-inflammatory activity (inhibition rate approximately 50%). Figure 3 A and 3B). THP-1 macrophages were infected with SFTSV (MOI=5), and the inhibitory effects of the above three chemical components on P17 secretion at concentrations of 1.1, 3.3, and 10 μM were examined. The results showed that the anti-inflammatory activity of the above chemical components was dose-dependent. Figure 3 (C and 3D). To further confirm the inhibitory effect of the above components on SARS-CoV-2-induced inflammation, screening was conducted in a SARS-CoV-2-infected Calu-3 cell system, with GSK-872 serving as a positive control (which significantly inhibited the secretion of IL-1βP17 induced by SARS-CoV-2 infection). The results showed that the above three components inhibited the release of IL-1βP17 in a dose-dependent manner. Figure 3 (D and 3E).

[0061] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of an isoflavone compound in the preparation of an anti-COVID-19 drug, characterized in that, The isoflavone compound is Northwest licorice isoflavone; the structural formula of the Northwest licorice isoflavone is shown in formula (Ⅰ). Equation (Ⅰ).

2. The application as described in claim 1, characterized in that, The isoflavone compounds have both antiviral and anti-inflammatory functions.

3. The application as described in claim 1, characterized in that, The isoflavone compounds target the coronavirus replication target SARS-CoV-2 M. pro And / or RdRp exert direct antiviral effects and exert anti-inflammatory effects by targeting p17 inflammatory factors.

4. The application as described in claim 1, characterized in that, The functional groups hydroxyl-OH and carbonyl-CO- of the isoflavone compounds form hydrogen bonds with ASN-781, SER-784, TYR-129, THR-14 or LYS-47.

5. The application as described in claim 1, characterized in that, The dosage form of the anti-COVID-19 drug is selected from granules, oral liquids, tablets, or capsules.

6. The application as described in claim 1, characterized in that, The isoflavone compounds, with the addition of appropriate pharmaceutically acceptable excipients, are used to formulate an anti-COVID-19 drug.

7. The application as described in claim 1, characterized in that, The effective concentration of the anti-COVID-19 drug is 1-300 μM.

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