Application of isoflavone compound in preparation of anti-new coronavirus medicine

Abstract

The invention discloses application of an isoflavone compound in preparation of a new coronavirus resisting medicine, and relates to the technical field of medicine preparations. The isoflavone compound is an antiviral component with multiple target points and double functions, not only can achieve a direct antiviral effect by targeting a coronavirus replication target (SARS-CoV-2 Mpro and / or RdRp), but also can achieve an anti-inflammatory effect by targeting a p17 inflammatory factor, and has double functions of antiviral and anti-inflammatory. In view of complex pathophysiology of the COVID-19, the treatment method of the COVID-19 comprises the steps of resisting virus infection and regulating immune-inflammation system imbalance of an organism, so that the isoflavone compound with dual functions is a promising anti-COVID-19 candidate drug.

Description

[0001] This application is a divisional application of the original application with the application number 202410224575.5 and the original filing date of February 29, 2024, and the entire contents of the original application are incorporated herein by reference.

[0002] This application claims priority to the Chinese patent application with the application number 202310261018.6 and the application title “Application of isoflavone compound in preparation of anti-new coronavirus drug” filed on March 14, 2023 with the Chinese Patent Office, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the technical field of pharmaceutical preparations, and specifically relates to the application of an isoflavone compound in the preparation of an anti-new coronavirus drug. BACKGROUND

[0004] The novel coronavirus (COVID-19, hereinafter referred to as the new coronavirus) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is a highly infectious enveloped positive-RNA virus that can cause respiratory disease, fever, and pneumonia. Currently, anti-COVID-19 drugs are being developed and have achieved significant success, including Remdesivir and Molnupiravir (RNA-dependent RNA polymerase RdRp inhibitors), Paxlovid and Xocova (Ensitrelvir) (SARS-CoV-2 main protease-M pro inhibitors and Tocilizumab-Interleukin (IL)-6 receptor inhibitors).

[0005] In the thousands of years of Chinese medicine clinical practice and disease resistance, a large number of effective traditional Chinese medicine prescriptions for treating epidemic diseases have been accumulated through the summary of Chinese medicine clinical experience. Based on the ancient classic prescriptions, “three prescriptions and three medicines” (Jinhuang Qianggan granules, Lianhua Qingwen granules, Xuebijing injection, Qingfei Paidu decoction, Xuanfei BaiDu decoction, and Huashi BaiDu decoction) have been derived.

[0006] Patent (CN112168899A) discloses a kudzu vine tea extract that can inhibit the 3CL protease of the novel coronavirus or the 3CL protease of the SARS coronavirus. The kudzu vine tea extract is a kudzu vine tea water extract, a kudzu vine tea alcohol extract, or a kudzu vine tea flavonoid extract. The kudzu vine tea extract can be used to treat novel coronavirus or SARS coronavirus infection.

[0007] However, due to the unclear effective substances and unclear mechanism of these prescriptions, it is difficult to obtain the recognition of the world and mainstream medicine for traditional Chinese medicine. Therefore, natural active substances with clear structure and composition are favored by domestic and foreign countries.

[0008] Patent (CN114409626A) discloses the preparation and antiviral application of baicalein derivatives. Baicalein derivatives are prepared from baicalein (5,6,7-trihydroxyflavone) by electrophilic substitution reaction. The reaction solvent is anhydrous acetone after drying and dehydration. The reaction process is as follows: after reacting for 1 hour under ice bath (0℃), the reaction solution is quenched by adding ice water, then filtered to obtain light yellow or white crude product. The half maximal inhibitory concentration of baicalein derivatives on influenza virus neuraminidase NA and novel coronavirus main protease Mpro is less than 100 μM. The synthesis method of the derivatives is simple and easy to operate. The two hydroxyl groups of baicalein C6-OH and C7-OH are substituted by various sulfonyl chloride compounds during the reaction process.

[0009] Patent (CN112402402A) proposes the application of xanthohumol in preparing novel coronavirus inhibitors. The Mpro protease causing lung inflammation in novel coronavirus is taken as the target, and the active compound xanthohumol with strong binding ability to Mpro protease is obtained through structure-based virtual screening. Xanthohumol inhibits the activity of Mpro protease by binding to the target Mpro protease, thereby inhibiting the replication and transcription of the virus, achieving the effect of resisting novel coronavirus infection. Xanthohumol can be applied in preparing novel coronavirus Mpro protease inhibitors, thereby being applied in preparing drugs for preventing and / or treating novel coronavirus inflammation. The binding energy of xanthohumol to Mpro protease is-63.427 KJ / mol, the IC 50 of xanthohumol to Mpro protease is 4.97±1.79 μM, and the inhibition constant Ki value is 2.14 μM.

[0010] Patent (CN115192566A) discloses the application of flavone monomer compounds in preparing drugs for preventing or treating novel coronavirus. The provided flavone monomer compounds can better bind to the NTPs entering channel of RNA polymerase due to their high structural similarity with nucleotide substrates, thereby blocking the entry of normal nucleotide substrates in RNA polymerase, i.e. the flavone monomer compounds occupy the site of nucleotide substrates, making the nucleotide substrate lose the site of RNA polymerase, and then the normal NTPs substrate cannot enter the active site of the enzyme, finally showing the inhibition effect on the biological function of RNA polymerase, which is suitable for preventing or treating novel coronavirus by using flavone monomer compounds.

[0011] However, the current natural active drugs have limited therapeutic effect on the new coronavirus, and due to the characteristics of high mutation rate and recombination rate of SARS-CoV-2, more effective antiviral drugs are urgently needed. In this case, combination therapy acting on multiple targets is still an indispensable direction for the research and development of anti-COVID-19 drugs. Therefore, the present application focuses on the main pharmacodynamic links (anti-inflammatory, antiviral), based on integrative pharmacology to carry out identification research on active ingredients with antiviral and anti-inflammatory effects, efficiently discover and screen active compounds against COVID-19, and develop natural product drugs with clear components and mechanisms that can act on multiple targets. SUMMARY

[0012] The present application provides the application of an isoflavone compound in the preparation of an anti-new coronavirus drug. The isoflavone compound is a multiple target bifunctional antiviral ingredient, that is, it can not only exert a direct antiviral effect by targeting coronavirus replication targets (SARS-CoV-2 M pro and RdRp), but also target p17 inflammatory factors to exert an anti-inflammatory effect, and has dual functions of antiviral and anti-inflammatory. In view of the complex pathophysiology of COVID-19, its treatment method should include anti-viral infection and regulation of the imbalance of the body's “immune-inflammation” system, and the isoflavone compound with dual functions is a promising candidate drug for anti-COVID-19.

[0013] To achieve the above-mentioned purpose, in a first aspect, the present application provides the application of an isoflavone compound in the preparation of an anti-new coronavirus drug, wherein the isoflavone compound is selected from one or more of a compound of formula (I) or its stereoisomer, magnolol, emodin, picraldehyde and glychappuol B, The structure of the compound of formula (I) is as follows: Formula (I); wherein R1 and R2 are each independently selected from H, hydroxyalkyl or isopentenyl, the hydroxyalkyl is selected from hydroxymethyl or hydroxyethyl; R3 is selected from H or hydroxyl; R4, R5, R6 and R7 are each independently selected from H, hydroxyl or alkoxy-OR8, R8 is selected from C1-4 alkyl, preferably methyl; and the compound of formula (I) is not the following compound: .

[0014] The structures of the magnolol, emodin, picraldehyde and glychappuol B are as follows:

[0015] In some preferred embodiments, the compound of formula (I) is selected from: .

[0016] In some preferred embodiments, the isoflavonoid compound is selected from the following compounds or a combination thereof:

[0017] In some preferred embodiments, the isoflavonoid compound has a dual function of anti-virus and anti-inflammation.

[0018] In some preferred embodiments, the isoflavonoid compound exerts a direct anti-virus effect by targeting the coronavirus replication target SARS-CoV-2 M pro and / or RdRp, and an anti-inflammatory effect by targeting the p17 inflammatory factor.

[0019] In some preferred embodiments, the functional groups hydroxyl -OH and carbonyl -CO- of the isoflavonoid compound form hydrogen bonds with ASN-781, SER-784, TYR-129, THR-14 or LYS-47.

[0020] In some preferred embodiments, the functional groups hydroxyl -OH, carbonyl -CO- and ether bond -O- of the isoflavonoid compound form hydrogen bonds with LYS-5, ARG-4, TRP-207 or PHE-3.

[0021] In some preferred embodiments, the dosage form of the anti-novel coronavirus drug is selected from granules, oral liquid, tablets or capsules.

[0022] In some preferred embodiments, the isoflavonoid compound is formulated into an anti-novel coronavirus drug with an appropriate amount of a pharmaceutically acceptable excipient.

[0023] In some preferred embodiments, the dosage of the anti-novel coronavirus drug is an effective concentration of 1-300 μM.

[0024] In a second aspect, the present application provides an anti-novel coronavirus drug comprising an isoflavonoid compound, the isoflavonoid compound being selected from one or more of the following compounds of formula (I) or a stereoisomer thereof, magnolol, emodin, picraline and glychol B, The structure of the compound of formula (I) is as follows: Formula (I); wherein R1and R2are each independently selected from H, hydroxyalkyl or isopentenyl, the hydroxyalkyl being selected from hydroxymethyl or hydroxyethyl; R3is selected from H or hydroxyl; R4, R5, R6and R7are each independently selected from H, hydroxyl or alkoxy-OR8, R8being selected from C1-4alkyl; and the compound of formula (I) is not the following compound: .

[0025] Compared with the prior art, the present application has the following beneficial effects: The isoflavone compound provided by the present application is a multi-target bifunctional antiviral component, which can not only play a direct antiviral role by targeting coronavirus replication targets (SARS-CoV-2 M pro and RdRp), but also can target p17 inflammatory factors to play an anti-inflammatory role, and has the dual functions of antiviral and anti-inflammatory. The isoflavone compound provided by the present application has excellent antiviral and anti-inflammatory effects, and is a promising anti-COVID-19 candidate drug; among them, magnolol, glycyrrhizin isoflavone A, emodin, stinging glycyrrhizin chalcone and glycyrrhizin chalcone B have better performances in antiviral activity and anti-inflammatory activity. BRIEF DESCRIPTION OF DRAWINGS Figure 1 Molecular docking virtual calculation results (A) molecular docking results; (B) compound structure and potential pharmacophore functional groups; the elliptical coil is the functional group forming a hydrogen bond with the protein amino acid residue; the line thickness represents the amount of hydrogen bond formed between the compound and the target protein residue; functional groups a, -OH (hydroxyl), b, -CO- (carbonyl), c, -O- (ether bond); the polar interaction between the compound and the SARS-CoV-2 M pro / RdRp protein is represented by a dashed line, and the data is mean ± SEM, three independent experiments.

[0026] Figure 2 (A) The initial screening results under 100 μM based on SARS-CoV-2 virus-like particles (trVLPs) infected nucleocapsid ectopic expression cell line (Caco-2-N); (B) EC50 / CC50 of compounds with inhibition rate > 90%; Caco-2 cells were infected with SARS-CoV-2 (MOI = 0.1) in the presence of different concentrations of isoflavone compounds, and the cell supernatant was collected after 24 hours after infection, and the viral copy number in the cell supernatant was determined by qRT-PCR, the data represents three independent experiments, mean ± SEM, and the statistical significance was analyzed by ANOVA ### P < 0.001; (C) Inhibition rate of candidate active ingredients on SARS-CoV-2 Mpro in vitro enzyme activity; (D) Inhibition rate of candidate active ingredients on RdRp in vitro enzyme activity.

[0027] Figure 3Anti-inflammatory activity of ingredients screening based on Bunyavirus (SFTSV) infection of THP-1 PMA cells (A) THP-1 macrophages were incubated with SFTSV (MOI = 5) for 1 h and treated with isoflavones at a concentration of 10 μM; cells and supernatants were collected 48 h post-infection; the level of P17 in the supernatants and the expression level of Pro-IL-1β or NP in the cell lysates were detected by Western blotting; (B-C) Dose-dependent inhibition of P17 secretion induced by SFTSV infection by licoricidin, licurze and spinacet; (B) THP-1 macrophages were incubated with SFTSV (MOI = 5) for 1 h and treated with licoricidin, licurze and spinacet at a concentration of 1.1, 3.3, 10 μM; cells and supernatants were collected 48 h post-infection. The level of P17 in the supernatants and the expression level of Pro-IL-1β or NP in the cell lysates were detected by Western blotting; (C) Inhibition rate was evaluated by statistical analysis of the gray value of P17 bands; data represent two independent experiments, mean ± SEM, statistical significance was determined by ANOVA # P < 0.05, ## P < 0.01; (D-E) Anti-inflammatory effect of licoricidin, licurze and spinacet on P17 release induced by SARS-CoV-2 infection; Calu-3 cells were infected with SARS-CoV-2 (MOI = 0.1) in the presence of licoricidin, licurze and spinacet at a concentration of 1.1, 3.3, 10, 30 μM, GSK-872 (5 μM) as a positive control; cells and supernatants were collected 48 h post-infection; (D) The level of P17 in the supernatants and the expression level of Pro-IL-1β or NP in the cell lysates were detected by Western blotting; (E) Inhibition rate was evaluated by statistical analysis of the gray value of P17 bands; data represent three independent experiments, mean ± SEM, statistical significance was determined by ANOVA, ### P < 0.001, #### P < 0.0001. DETAILED DESCRIPTION

[0028] It is worth noting that the raw materials used in the present application are all ordinary commercially available products, and their sources are not specifically limited. The raw materials of isoflavones used in the present application correspond to the following English and Chinese: .

[0029] The technical solutions of the present application will be clearly and completely described below in combination with specific embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0030] Example 1: Virtual Calculation of Molecular Docking 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: 3CLpro / Mpro-DimerPDB ID: 6y2g; 3CLpro / Mpro-MonomerPDB ID: 5r82; RdRp- +RNA&+MgPDB ID:7bv2; RdRpPDB ID:6m71; The docking fraction is expressed in Kcal / mol. 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.

[0031] Table 1. Isoflavone compound docking scores with target sites

[0032] Table 2. Potential pharmacodynamic functional groups of isoflavones and their target binding sites.

[0033] Example 2: Antiviral activity of isoflavones 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.

[0034] The results show that ( 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 arachidonicum, and quercetin, indicating that these components have a large therapeutic window and good drug sensitivity.

[0035] SARS-CoV-2 M 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).

[0036] Example 3: Anti-inflammatory activity of isoflavones 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) is similar to SARS-CoV-2 virus, promoting 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 virus-induced inflammation can be evaluated by detecting IL-1β production and secretion. 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 ).

[0037] Through the secretion of mature IL-1β (P17), glycyrrhizin B, northwestern glycyrrhizin isoflavones, and prickly glycyrrhizin 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 3C 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-1β P17 induced by SARS-CoV-2 infection). The results showed that the above three components inhibited the release of IL-1β P17 in a dose-dependent manner (C and 3D). Figure 3 (D and 3E).

[0038] 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 use of an isoflavone compound in the preparation of an anti-SARS-CoV-2 drug, characterized in that, The isoflavones are selected from one or more of the following: a compound of formula (I) or a stereoisomer thereof, magnolol, emodin, picraline and glyyurin B, The compound of formula (I) has the following structure: Formula (I); wherein R1 and R2 are each independently selected from H, hydroxyalkyl or isopentenyl, the hydroxyalkyl is selected from hydroxymethyl or hydroxyethyl; R3 is selected from H or hydroxyl; R4, R5, R6 and R7 are each independently selected from H, hydroxyl or alkoxy-OR8, R8 is selected from C1-4 alkyl; and the compound of formula (I) is not the following compound: 。 2. Use according to claim 1, wherein R8 is methyl.

3. The use according to claim 1, wherein The compound of formula (I) is selected from: 。 4. The use according to claim 3, wherein the compound is ###0002### The isoflavones are selected from one or more of the following: a compound of formula (I) or a stereoisomer thereof, magnolol, emodin, picraline and glyyurin B, 。 5. The use according to claim 1, wherein The isoflavones have dual functions of anti-virus and anti-inflammation.

6. The use according to claim 1, wherein The isoflavones exert a direct antiviral effect by targeting the coronavirus replication target SARS-CoV-2 M pro and / or RdRp, an anti-inflammatory effect by targeting the p17 inflammatory factor.

7. The use according to claim 1, wherein The functional groups of the isoflavones, hydroxyl-OH and carbonyl-CO-, form hydrogen bonds with ASN-781, SER-784, TYR-129, THR-14 or LYS-47.

8. The use according to claim 1, wherein The functional groups of the isoflavones, hydroxyl-OH, carbonyl-CO- and ether bond-O-, form hydrogen bonds with LYS-5, ARG-4, TRP-207 or PHE-3.

9. The use according to claim 1, wherein The dosage form of the anti-novel coronavirus drug is selected from granules, oral liquid, tablets or capsules.

10. The use according to claim 1, wherein The isoflavones are mixed with an appropriate amount of pharmaceutically acceptable excipients to form an anti-novel coronavirus drug.

11. The use according to claim 1, wherein The effective concentration of the anti-novel coronavirus drug is 1-300 μM.

12. An anti-SARS-CoV drug, characterized in that, The isoflavones are selected from one or more of the following: a compound of formula (I) or a stereoisomer thereof, magnolol, emodin, picraline and glyyurin B, The compound of formula (I) has the following structure: Formula (I); wherein R1 and R2 are each independently selected from H, hydroxyalkyl or isopentenyl, the hydroxyalkyl is selected from hydroxymethyl or hydroxyethyl; R3 is selected from H or hydroxyl; R4, R5, R6 and R7 are each independently selected from H, hydroxyl or alkoxy-OR8, R8 is selected from C1-4 alkyl; and the compound of formula (I) is not the following compound: 。

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