Application of 2-[(7-hydroxy-8-methyl-2-oxopigmene-4-yl) methylsulphonase]-4, 6-dimethylpyridine-3-carbonitrile in resisting Ebola virus

Abstract

The invention relates to application of 2-[(7-hydroxy-8-methyl-2-oxopigmene-4-yl) methylsulphonase]-4, 6-dimethyl pyridine-3-carbonitrile in resisting Ebola virus, and belongs to the technical field of medicine. The invention provides 2-[(7-hydroxy-8-methyl-2-oxopigmene-4-yl) methylsulphonase]-4, 6-dimethylpyridine-3-carbonitrile which can effectively inhibit the replication of Ebola viruses in a non-toxic range in order to solve the technical problem of lack of an effective Ebola virus resisting drug in the prior art, and also provides a preparation method of the 2-[(7-hydroxy-8-methyl-2-oxopigmene-4-yl) methylsulphonase]-4, 6-dimethylpyridine-3-carbonitrile. The compound has a good anti-infection effect on SUDV, BDBV and TAFV viruses, and can be applied to preparation of safe and effective broad-spectrum anti-Ebola virus drugs.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and specifically relates to the application of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfonyl]-4,6-dimethylpyridine-3-carbonitrile in anti-Ebola virus. Background Art

[0002] Ebola virus disease (EVD) is a severe and usually fatal disease caused by Ebola virus (EBOV); EBOV infection can cause acute infections in humans and non-human primates, characterized by a high fatality rate and weak clinical treatment capabilities. It mainly prevails in Central Africa and West Africa, with a mortality rate as high as 50%-90%. Although EBOV infection has geographical limitations, with the expansion of human activities and the increasing frequency of cross-border exchanges, the probability of natural or artificial EBOV infection has been greatly increased. Coupled with the lack of treatment measures and vaccination options, EBOV has become a deadly biological threat pathogen. Therefore, to ensure human health and safety and improve the prevention and control capabilities against this virus infection, it is of great significance to reserve drugs for the prevention and treatment of EBOV.

[0003] EBOV is a member of the Filoviridae family, with a genome of single-stranded negative-sense RNA, encoding 7 structural proteins and 2 non-structural proteins. The gene order is: 3'-end - NP - VP35 - VP40 - GP - VP30 - VP24 - L - 5'-end, encoding 7 structural genes respectively. Among them, NP is the nucleocapsid protein of the virus, VP30 and VP35 are viral structural proteins, GP is a type I transmembrane protein, VP24 is a small membrane protein, VP40 is the matrix protein associated with the virion inner membrane, and L is an RNA polymerase; among them, VP30, NP, VP35, and L proteins form a transcription initiation complex, which is responsible for the RNA synthesis of EBOV. In addition, during the transcription of the GP gene, RNA editing occurs to generate frameshift translation, thereby producing two other non-structural proteins (sGP and ssGP).

[0004] Currently, there is no relevant patent report on 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfonyl]-4,6-dimethylpyridine-3-carbonitrile, nor its application in the prevention or treatment of EBOV. Summary of the Invention

[0005] In order to solve the technical problem of the lack of effective anti-EBOV drugs in the prior art, the present invention provides the application of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfonyl]-4,6-dimethylpyridine-3-carbonitrile in anti-Ebola virus.

[0006] One of the purposes of the present invention is to provide the use of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile in the preparation of anti-Ebola virus drugs.

[0007] In a preferred embodiment of the present invention, the structural formula of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile is:

[0008]

[0009] In a preferred embodiment of the present invention, the anti-Ebola virus drug refers to a drug that inhibits the replication of the Ebola virus.

[0010] In a preferred embodiment of the present invention, the inhibition of Ebola virus replication means that 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile binds to the Ebola virus nucleoprotein NP and inhibits the replication of the Ebola virus.

[0011] In a preferred embodiment of the present invention, the molecular formula of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile is C 19 H 16 N2O3S.

[0012] In a preferred embodiment of the present invention, the molecular weight of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile is 352.4 g / mol.

[0013] Another purpose of the present invention is to provide an anti-Ebola virus drug, which comprises 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile and a pharmaceutically acceptable excipient.

[0014] In a preferred embodiment of the present invention, the pharmaceutically acceptable excipient is selected from one or more of a dispersant, a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a preservative, a suspending agent, an emulsifier or a flavoring agent.

[0015] Advantages of the present invention: The present invention provides the use of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile in the preparation of anti-Ebola virus (EBOV) drugs. The molecular formula of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile is C 19 H 16 N2O3S, and the molecular weight is 352.4 g / mol.

[0016] 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile provided by the present invention has good anti-infection effects against Sudan virus (SUDV), Bundibugyo virus (BDBV), and Taï Forest virus ( Forest virus, TAFV), and the IC 50 values are 0.23 μM, 0.44 μM, and 3.35 μM, respectively. In the case of transfecting the viral minigenome, 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile also has a good inhibitory effect on the replication of the EBOV minigenome, and has no cytotoxicity at the concentration that can produce 100% inhibition of live virus infection, indicating that 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile can also be used in the preparation of safe and effective broad-spectrum anti-EBOV drugs.

[0017] The present invention conducts cytotoxicity and virus infection experiments in Huh-7 and Vero-E6 cell lines. The results show that 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile can effectively inhibit the replication and infection of EBOV within the non-toxic range; 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile can effectively inhibit the infection of EBOV on Huh-7 and Vero-E6 cells. Among them, the IC 50 values are 0.08 μM (Huh-7 cells) and 0.35 μM (Vero-E6 cells), and the SI values are 1250.00 and >285.71, respectively.

[0018] The 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbonitrile provided by the invention can be used to prepare anti-EBOV drugs, thereby improving the utilization rate of 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbonitrile and expanding the application scope of the compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a graph showing the effect of compounds inhibiting the replication of the EBOV mini-genome; the ordinate Percent of inhibition is the inhibition rate, and the abscissa Concentration is the concentration;

[0020] Figure 2 This is a graph showing the inhibitory effect of the compound on EBOV infection in Huh-7 cells;

[0021] Figure 3 This is a graph showing the inhibitory effect of the compound on EBOV infection in Vero-E6 cells;

[0022] Figure 4 This is a diagram showing the effect of the compound in inhibiting SUDV infection;

[0023] Figure 5 This is a diagram showing the effect of the compound in inhibiting BDBV infection;

[0024] Figure 6 This is a diagram showing the effect of the compound in inhibiting TAFV infection;

[0025] Figure 7 This is a molecular docking detection diagram of the compound and the Ebola virus NP protein; among them, the Ebola virus NP protein C skeleton is displayed in green, N atoms are displayed in blue, O atoms are displayed in bright red, H atoms are displayed in white, 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbonitrile is displayed as a bean paste-colored stick, and the hydrogen bond length is displayed as a purple dotted line. DETAILED DESCRIPTION

[0026] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0027] To make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with specific embodiments and the accompanying drawings of the specification. Unless otherwise specified, the test methods used in the following embodiments are all conventional methods. The materials, reagents, methods and instruments used, unless otherwise specified, are all conventional materials, reagents, methods and instruments in the art, and those skilled in the art can obtain them through commercial channels.

[0028] The following English abbreviations involved in the following embodiments:

[0029] Ebola virus: Ebola virus, EBOV;

[0030] Sudan virus: Sudan Ebola Virus, SUDV;

[0031] Bundibugyo virus: Bundibugyo Ebola Virus, BDBV;

[0032] Tai Forest virus: Tai Forest Ebola Virus, TAFV.

[0033] The EBOV, SUDV, BDBV and TAFV viruses used in the following embodiments are all provided by the National Virus Recource Center.

[0034] Example 1: Application of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile in the preparation of anti-EBOV drugs

[0035] (1) Drug inhibition of EBOV minigenome replication experiment

[0036] Transfect the EBOV minigenome system into a 96-well plate. At 6 h after transfection, add 20, 2, 0.4 μM of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile (diluted with DMEM containing 2% FBS and 1% double antibody) respectively. At the same time, set a group adding the same amount of DMSO as a control. After continuing to incubate in an incubator at 37 °C for 48 h, aspirate the supernatant, collect the cells and lyse them, and read the luciferase activity to obtain the replication of the EBOV minigenome after treatment with different concentrations of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile.

[0037] The results are as Figure 1As shown, 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile provided by the present invention can effectively inhibit the replication of EBOV minigenome in cells, and the inhibitory activity has a dose-dependent relationship with the content of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile.

[0038] (2) Drug cytotoxicity experiment and inhibition of EBOV infection experiment

[0039] The cytotoxicity of the drug was measured using a CCK-8 kit. WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt) in the kit can be reduced by some dehydrogenases in mitochondria to highly water-soluble orange-yellow formazan under the action of the electron coupling carrier 1-Methoxy PMS. The color intensity of the generated formazan is directly proportional to cell viability and inversely proportional to cytotoxicity.

[0040] After incubating 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile at different concentration gradients with Huh-7 and Vero-E6 for corresponding times, CCK-8 was added, and the absorbance value was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. This measured value can indirectly reflect the number of viable cells.

[0041] Using the Huh-7 and Vero-E6 cell lines as virus infection models, Huh-7 and Vero-E6 cells were infected at an MOI of 0.1. At 1 h post-infection, the culture medium was aspirated. After washing the cells twice with PBS buffer, different concentrations of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile (diluted with DMEM containing 2% FBS and 1% penicillin-streptomycin) were added respectively. At the same time, a control group without the drug was set up. After culturing in an incubator at 37 °C for 72 h, the cell supernatant after culture was collected, and the viral RNA in the supernatant was extracted using a kit. The viral content in the cells and cell supernatant treated with different drugs was detected by RT-qPCR, and the inhibition rate of the drug was calculated.

[0042] The results are as Figure 2 shown, 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile provided by the present invention can effectively inhibit the infection of EBOV in Huh-7 cells, and the IC 50 is 0.08 μM, and the CC 50> 100.00 μM, with an SI value of 1250.00. The set drug concentrations are 10, 2.5, 0.63, 0.16, 0.04, and 0.001 μM respectively.

[0043] The results are as Figure 3 shown. 2-[(7-Hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile provided by the present invention can effectively inhibit the infection of EBOV in Vero-E6 cells. The IC 50 is 0.35 μM, and the CC 50 > 100.00 μM, with an SI value > 285.71. The set drug concentrations are 10, 2.5, 0.63, 0.16, 0.04, and 0.001 μM respectively.

[0044] (3) Determination of drug broad-spectrum

[0045] Using the Huh-7 cell line as a virus infection model, Huh-7 cells (MOI = 0.1) were infected with SUDV, BDBV, and TAFV respectively. At 1 h post-infection, the culture medium was aspirated, and the cells were washed twice with PBS buffer. Then, different concentrations of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile (diluted with DMEM containing 2% FBS and 1% double antibody) were added respectively. At the same time, a control group without adding the drug was set up. After continuing to culture in an incubator at 37 °C for 72 h, the cell supernatant was collected; the viral RNA in the supernatant was extracted using a kit, and the viral content in the supernatant after different drug treatments was detected by RT-qPCR, and the inhibition rate of the drug was calculated.

[0046] The results are as Figures 4 - 6 shown. 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile provided by the present invention can effectively inhibit the infection of SUDV, BDBV, and TAFV. The IC 50 are 0.23 μM, 0.44 μM, and 3.35 μM respectively, and the CC 50 > 100.00 μM. The set drug concentrations are 10, 2.5, 0.63, 0.16, 0.04, 0.001, and 0.0003 μM respectively.

[0047] (4) Molecular docking detection of the drug and EBOV

[0048] In this example, the binding mode of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile and the EBOV nucleoprotein NP was detected by molecular docking through MedChemExpress. The results are asFigure 7 as shown

[0049] It can be seen that 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile can form two hydrogen bond interactions with EBOZM and NP protein; the amino group of the compound can form one hydrogen bond interaction with GLN238 of the target protein; the carbonyl group of the compound can form one hydrogen bond interaction with LYS248 of the target protein; the hydrogen bond interactions are shown as purple dashed lines, and the longer the bond length, the weaker the hydrogen bond interaction.

[0050] In summary, 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile provided by the present invention can bind to the EBOV nucleoprotein NP, thereby achieving the purpose of inhibiting EBOV replication.

[0051] The content not detailedly described in the specification of the present invention is the well-known technology to those skilled in the art. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Use of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile in the preparation of anti-Ebola virus drugs.

2. The application according to claim 1, wherein The structural formula of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile is:

3. The application according to claim 1, characterized in that The anti-Ebola virus drug refers to a drug that inhibits the replication of the Ebola virus.

4. The application according to claim 2, wherein The inhibition of Ebola virus replication means that 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile binds to the Ebola virus nucleoprotein NP and inhibits the replication of the Ebola virus.

5. The application according to claim 1, wherein The molecular formula of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile is C 19 H 16 N2O3S.

6. The application according to claim 1, characterized in that, The molecular weight of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile is 352.4 g / mol.

7. A drug against Ebola virus, characterized in that, The drug contains 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile and pharmaceutically acceptable excipients.

8. The anti-Ebola virus drug according to claim 7, characterized in that, The pharmaceutically acceptable excipients are selected from one or more of dispersants, diluents, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers or flavoring agents.

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