Application of 4,3',5'-trihydroxy resveratrol compounds in the preparation of coronavirus 3CL protease inhibitors

Abstract

The present invention provides the use of 4,3',5'-trihydroxy resveratrol compounds in the preparation of coronavirus 3CL protease inhibitors. The present invention discovers that 4,3',5'-trihydroxy resveratrol and its analogs have the activity of inhibiting coronavirus 3CL protease, and can play an anti-coronavirus role by inhibiting the activity of coronavirus 3CL protease. Among them, piceatannol, oxyresveratrol, physcion, 2,6,3',5'-tetrahydroxy stilbene, cis-tartriloside, 4-hydroxy resveratrol, and 4,3',5'-trihydroxy resveratrol have a half-maximal inhibitory concentration (IC50) against SARS-CoV and SARS-CoV-2 3CL proteases lower than 10 μM, showing good anti-coronavirus 3CL protease effect, effectively inhibiting the activity of coronaviruses, and can potentially be used for the prevention and / or treatment of coronavirus infections, and further for the preparation of drugs for the prevention and / or treatment of coronavirus infections.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the application of 4,3',5'-trihydroxyveratrol and its analogs in the preparation of coronavirus 3CL protease inhibitors. Background Art

[0002] As of August 22, 2022, the cumulative number of confirmed COVID-19 cases globally has exceeded 590 million, with more than 6.44 million cumulative deaths. COVID-19 is caused by the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Among the numerous targets of the novel coronavirus (SARS-CoV-2), 3CL protease (3CLpro, 3C-like protease, also known as the main protease Mpro) has attracted much attention from researchers due to its extremely crucial role in the replication and transcription of progeny viruses. 3CL protease has high structural similarity and conservation in multiple coronaviruses and novel coronavirus variants, and is one of the most attractive targets for the development of coronavirus-targeted drugs. 4,3',5'-Trihydroxyveratrol and its analogs are antioxidants that can reduce blood viscosity, inhibit platelet aggregation and vasodilation, keep blood flowing smoothly, prevent the occurrence and development of cancer, and have the effects of preventing and treating atherosclerosis, coronary heart disease, ischemic heart disease, and hyperlipidemia. They also have tumor inhibitory and estrogen-like effects and can be used to treat diseases such as Chemicalbook breast cancer. They can delay aging and prevent cancer. In recent years, the activities of 4,3',5'-trihydroxyveratrol and its analogs in antibacterial and anti-tumor aspects have attracted the attention of many pharmaceutical workers. However, there is currently no report on the inhibition of 3CL pro activity by 4,3',5'-trihydroxyveratrol and its analogs and coronaviruses. Summary of the Invention

[0003] In view of the problems existing in the prior art, the present invention provides the application of 4,3',5'-trihydroxyveratrol and its analogs in the preparation of coronavirus 3CL protease inhibitors and in the preparation of anti-SARS-CoV-2 drugs, and is expected to solve the current situation where there is no anti-coronavirus drug.

[0004] The present invention is achieved through the following technical solutions:

[0005] The present invention provides the application of 4,3',5'-trihydroxyveratrol compounds in the preparation of coronavirus 3CL protease inhibitors.

[0006] The present invention provides the application of 4,3',5'-trihydroxyveratrol compounds in the preparation of drugs for preventing and / or treating diseases caused by coronaviruses.

[0007] Preferably, the structural formula of the 4,3',5'-trihydroxy resveratrol compound is as shown in Formula I:

[0008]

[0009] Formula I

[0010] Wherein, R1, R2, R3, R4, R5, R6, R7, and R8 can independently be selected from H, OH, or OCH3.

[0011] Preferably, the 4,3',5'-trihydroxy resveratrol compound is one of the following structural formulas:

[0012]

[0013] Preferably, the 4,3',5'-trihydroxy resveratrol compound is piceatannol, oxyresveratrol, rhapontigenin, 2,6,3',5'-tetrahydroxystilbene, cis-tzivonianin, 4-hydroxy resveratrol, or 4,3',5'-trihydroxy resveratrol.

[0014] Preferably, the 4,3',5'-trihydroxy resveratrol compound is 4,3',5'-trihydroxy resveratrol or its analogs, or a pharmaceutically acceptable acid, base, salt, ester, solvate, stereoisomer, tautomer, or prodrug of 4,3',5'-trihydroxy resveratrol and its analogs.

[0015] Preferably, the coronavirus is: SARS-CoV, MERS-CoV, or SARS-CoV-2.

[0016] Preferably, the disease caused by the coronavirus is an infectious disease or its complication caused by SARS-CoV, MERS-CoV, or SARS-CoV-2.

[0017] Preferably, the coronavirus 3CL protease is SARS-CoV 3CL pro or SARS-CoV-2 3CL pro .

[0018] Another aspect of the present invention provides a drug for treating and / or preventing a disease caused by a coronavirus, which comprises a 4,3',5'-trihydroxy resveratrol compound and a pharmaceutically acceptable excipient, preferably comprising one or several of 4,3',5'-trihydroxy resveratrol and its analogs themselves or pharmaceutically acceptable acids, bases, salts, esters, solvates, stereoisomers, tautomers, prodrugs of 4,3',5'-trihydroxy resveratrol and its analogs.

[0019] Preferably, the dosage form of the drug includes one or more of capsules, tablets, granules, gels, sustained-release agents, oral liquids, dripping pills, emulsions, injections, and nano-formulations.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention for the first time discovers that 4,3',5'-trihydroxyveratrol and its analogs have the activity of inhibiting coronavirus 3CL protease, and can play an anti-coronavirus role by inhibiting the activity of coronavirus 3CL protease. Among them, piceatannol, oxyresveratrol, rhapontigenin, 2,6,3',5'-tetrahydroxystilbene, cis-trijuglaconoside, 4-hydroxyresveratrol, and 4,3',5'-trihydroxyveratrol all have a half inhibitory concentration against SARS-CoV and SARS-CoV-2 3CL protease lower than 10 μM, and have a good effect on anti-coronavirus 3CL protease, thus effectively inhibiting the activity of the coronavirus. Therefore, it can potentially be used for preventing and / or treating coronavirus infection, and further for preparing drugs for preventing and / or treating coronavirus infection. Description of the Drawings

[0022] Figure 1 It shows the inhibitory effect of piceatannol on SARS-CoV 3CL protease in the present invention;

[0023] Figure 2 It shows the inhibitory effect of oxyresveratrol on SARS-CoV 3CL protease in the present invention;

[0024] Figure 3 It shows the inhibitory effect of rhapontigenin on SARS-CoV 3CL protease in the present invention;

[0025] Figure 4 It shows the inhibitory effect of 2,6,3',5'-tetrahydroxystilbene on SARS-CoV 3CL protease in the present invention;

[0026] Figure 5 It shows the inhibitory effect of cis-trijuglaconoside on SARS-CoV 3CL protease in the present invention;

[0027] Figure 6 It shows the inhibitory effect of 4-hydroxyresveratrol on SARS-CoV 3CL protease in the present invention;

[0028] Figure 7 It shows the inhibitory effect of 4,3',5'-trihydroxyveratrol on SARS-CoV 3CL protease in the present invention;

[0029] Figure 8It is the inhibitory effect of piceatannol on SARS-CoV-2 3CL protease in the present invention;

[0030] Figure 9 It is the inhibitory effect of oxyresveratrol on SARS-CoV-2 3CL protease in the present invention;

[0031] Figure 10 It is the inhibitory effect of rhaponticin on SARS-CoV-2 3CL protease in the present invention;

[0032] Figure 11 It is the inhibitory effect of 2,6,3',5'-tetrahydroxystilbene on SARS-CoV-2 3CL protease in the present invention;

[0033] Figure 12 It is the inhibitory effect of cis-tartary stilbene glycoside on SARS-CoV-2 3CL protease in the present invention;

[0034] Figure 13 It is the inhibitory effect of 4-hydroxypiceatannol on SARS-CoV-2 3CL protease in the present invention;

[0035] Figure 14 It is the inhibitory effect of 4,3',5'-trihydroxystilbene on SARS-CoV-2 3CL protease in the present invention. Detailed implementation manners

[0036] To further understand the present invention, the present invention will be described below in conjunction with embodiments. These descriptions are only to further explain the features and advantages of the present invention and are not intended to limit the claims of the present invention.

[0037] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0038] Term explanation:

[0039] The so-called "pharmaceutically acceptable": within the scope of correct medical judgment, a compound, material, composition, and / or dosage form that is suitable for contact with human and animal tissues without causing additional toxicity, irritation, allergic reactions, or other problems and is commensurate with a reasonable benefit / risk ratio.

[0040] The term "pharmaceutically acceptable salt": A salt that retains the biological effectiveness and properties of the compounds of the present invention and is not biologically or otherwise undesirable. In some cases, the compounds in the present invention are capable of forming salts in the presence of phenol and / or carboxyl groups. Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases include, for example: sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, and magnesium salts. Salts derived from organic bases include, for example: primary amines, secondary amines, and tertiary amines.

[0041] The term "prodrug": An inactive form of a compound that must be metabolized in vivo, such as by biological fluids or enzymes and / or by the metabolism within an individual after administration, to the active form of the compound to produce the desired pharmacological effect. A prodrug can be metabolized before absorption, during absorption, after absorption, or at a specific site. The prodrug form of a compound can be utilized to enhance bioavailability; improve individual acceptability, such as masking or reducing unpleasant characteristics: such as bitterness, odor, or gastrointestinal irritation; alter solubility; provide extended or slow release or delivery; facilitate formulation; and / or provide site-specific delivery of the compound. The compounds mentioned in the present invention include the prodrug forms of the compounds.

[0042] It is worth noting that the raw materials used in the present invention are all ordinary commercially available products, and no specific limitation is made on their sources.

[0043] The following raw material sources are for illustrative purposes:

[0044] Assay Reagent (Assay Buffer: SARS-CoV M pro / 3CL pro =90:1), Substrate (100 μM Dabcyl-KTSAVLQSGFRKME-Edans) were all purchased from Beyotime Biotechnology Co., Ltd. in Shanghai

[0045] Example 14,3',5'-Trihydroxyveratrol and its analogues in vitro enzyme activity inhibition assay against SARS-CoV 3CL protease

[0046] A 3CLpro inhibitor screening kit using the principle of fluorescence resonance energy transfer (PRET) can simply, rapidly and sensitively complete the test of the inhibitory activity of the test drug. According to the number of samples, appropriate amounts of Assay Reagent and positive control drug are prepared according to the instructions. Then, according to the groups of blank control, 100% enzyme activity control, positive inhibitor control, and test drug, the detection reagents and sample solvents are added successively in a 96-well plate. Using a multichannel pipette, 4 μL of Substrate is quickly added to each well at low temperature, mixed evenly, and incubated at 37 °C in the dark for 5 minutes. Then, fluorescence measurement is performed using a multifunctional microplate reader (λ excitation = 340 nm, λ emission = 490 nm). The relative fluorescence units (RFU) in each well and the blank control well are calculated and recorded as RFU blank control, RFU100% enzyme activity control, RFU positive control, and RFU test drug respectively. From this, the percentage inhibition rate (%) and IC50 value of the target compound are calculated to evaluate the inhibitory ability of the inhibitor on 3CLpro activity.

[0047] × 100%

[0048] The experimental results are shown in Table 1. The 22 compounds in Example 2 all have inhibitory activity against SARS-CoV 3CL protease. Among them, piceatannol, oxyresveratrol, rhaponticin, 2,6,3',5'-tetrahydroxystilbene, cis-tartary glycoside, 4-hydroxyresveratrol, and 4,3',5'-trihydroxystilbene have strong inhibitory effects on SARS-CoV 3CL protease, and their IC 50 values are all below 10 μM. Among them, the IC 50 value of 4,3',5'-trihydroxystilbene reaches 1.69 μM ( Figures 1-7 respectively showing the inhibitory effects of piceatannol, oxyresveratrol, rhaponticin, 2,6,3',5'-tetrahydroxystilbene, cis-tartary glycoside, 4-hydroxyresveratrol, and 4,3',5'-trihydroxystilbene on SARS-CoV 3CL protease).

[0049] Table 1 Inhibitory activity of 22 compounds against SARS-CoV3CL protease

[0050]

[0051] Example 2 Detection of in vitro enzyme activity inhibition of 4,3',5'-trihydroxystilbene and its analogs against SARS-CoV-2 3CL protease

[0052] A 3CLpro inhibitor screening kit using the principle of fluorescence resonance energy transfer (PRET) can simply, rapidly, and sensitively complete the test of the inhibitory activity of the test drug. According to the number of samples, appropriate amounts of Assay Reagent and positive control drug are configured according to the instructions. Then, according to the groups of blank control, 100% enzyme activity control, positive inhibitor control, and test drug, the detection reagents and sample solvents are added successively in a 96-well plate. Using a multichannel pipette, 4 μL of Substrate is quickly added to each well at low temperature, mixed well, and incubated at 37 °C in the dark for 5 minutes. Then, fluorescence measurement is performed using a multifunctional microplate reader (λ excitation = 340 nm, λ emission = 490 nm). The relative fluorescence units (RFU) in each well and the blank control well are calculated and recorded as RFU blank control, RFU100% enzyme activity control, RFU positive control, and RFU test drug respectively. From this, the percentage inhibition rate (%) and IC50 value of the target compound are calculated to evaluate the inhibitory ability of the inhibitor on 3CLpro activity. × 100%

[0053] The experimental results are shown in Table 2. The 22 compounds in Example 2 all have inhibitory activity against SARS-CoV-2 3CL protease. Among them, piceatannol, oxyresveratrol, rhaponticin, 2,6,3',5'-tetrahydroxystilbene, cis-tartary glycoside, 4-hydroxyresveratrol, and 4,3',5'-trihydroxystilbene have strong inhibitory effects on SARS-CoV-2 3CL protease, and their IC 50 values are all below 10 μM. Among them, the IC 50 value of 4,3',5'-trihydroxystilbene reaches 1.65 μM ( Figures 8-14 which respectively show the inhibitory effects of piceatannol, oxyresveratrol, rhaponticin, 2,6,3',5'-tetrahydroxystilbene, cis-tartary glycoside, 4-hydroxyresveratrol, and 4,3',5'-trihydroxystilbene on SARS-CoV-2 3CL protease).

[0054] Table 2 Inhibitory activity of 22 compounds against SARS-CoV-2 3CL protease

[0055]

[0056] In summary, piceatannol, oxyresveratrol, rhapontigenin, 2,6,3',5'-tetrahydroxystilbene, cis-tartaric acid stilbene glycoside, 4-hydroxyveratrol, and 4,3',5'-trihydroxystilbene all showed excellent inhibitory activity against the 3CL proteases of both SARS-CoV and SARS-CoV-2 coronaviruses, and can be developed and studied as anti-coronavirus drugs.

Claims

1. Use of 4,3',5'-trihydroxyveratryl alcohol compounds in the preparation of a drug for preventing and / or treating diseases caused by coronaviruses, characterized in that, The 4,3',5'-trihydroxy resveratrol compound is physcion, and the coronavirus is SARS-CoV.

2. The application according to claim 1, wherein The 4,3',5'-trihydroxy resveratrol compound is a pharmaceutically acceptable salt of physcion.

3. The application according to claim 1, characterized in that, The disease caused by the coronavirus is an infectious disease caused by SARS-CoV.

Images