2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfanyl]-4,6-dimethylpyridine-3-carbonitrile for use in the treatment of ebola virus

Abstract

The application of 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate in the treatment of Ebola virus belongs to the field of pharmaceutical technology. This invention addresses the technical problem of the lack of effective anti-Ebola virus drugs in the prior art by providing 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate, which can effectively inhibit Ebola virus replication within a non-toxic range. The compound exhibits good anti-infective effects against SUDV, BDBV, and TAFV viruses and can be used to prepare safe and effective broad-spectrum anti-Ebola virus drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate in the treatment of Ebola virus. Background Technology

[0002] Ebola virus disease (EVD) is a severe and often fatal disease caused by Ebola virus (EBOV). EBOV infection can cause acute infection in humans and non-human primates, characterized by a high mortality rate and limited clinical treatment capabilities. It is mainly prevalent in Central and West Africa, with a mortality rate as high as 50%-90%. Although EBOV infection is geographically limited, the expansion of human activity and increasingly frequent cross-border exchanges have greatly increased the probability of natural or human-induced EBOV infection. Coupled with the lack of treatment options and vaccination choices, EBOV has become a deadly biological threat. Therefore, to protect human health and safety and improve the ability to control this viral infection, stockpiling antiviral drugs for EBOV is of great significance.

[0003] EBOV is a member of the Filoviridae family. Its genome is a single-stranded negative-sense RNA that encodes seven structural proteins and two non-structural proteins. The gene sequence is: 3' end - NP - VP35 - VP40 - GP - VP30 - VP24 - L - 5' end, encoding the seven structural proteins. NP is the viral nucleocapsid protein, VP30 and VP35 are viral structural proteins, GP is a type I transmembrane protein, VP24 is a small membrane protein, VP40 is a matrix protein associated with the virion's inner membrane, and L is an RNA polymerase. VP30, NP, VP35, and L proteins constitute the transcription initiation complex, which is responsible for EBOV RNA synthesis. Furthermore, during transcription, the GP gene undergoes RNA editing to produce frameshift translation, resulting in two additional non-structural proteins (sGP and ssGP).

[0004] Currently, there are no patent reports related to 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate, nor are there any reports on its application in the prevention or treatment of EBOV. Summary of the Invention

[0005] To address the technical problem of the lack of effective anti-EBOV drugs in the prior art, this invention provides the application of 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate in the treatment of Ebola virus.

[0006] One of the objectives of this invention is to provide the use of 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate 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-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate 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 refers to the binding of 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate to the Ebola virus nucleoprotein NP, thereby inhibiting the replication of the Ebola virus.

[0011] In a preferred embodiment of the present invention, the molecular formula of the 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate is C2 19 H 16 N2O3S.

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

[0013] A second objective of this invention is to provide an anti-Ebola virus drug comprising 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate and pharmaceutically acceptable excipients.

[0014] In a preferred embodiment of the invention, the pharmaceutically acceptable excipient is selected from one or more of dispersants, diluents, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers, or flavoring agents.

[0015] Beneficial effects of the present invention: The present invention provides the application of 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate in the preparation of anti-Ebola virus (EBOV) drugs, wherein the molecular formula of 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate is C 19 H 16 N2O3S has a molecular weight of 352.4 g / mol.

[0016] The present invention provides 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate for Sudan virus (SUDV), Bundibugyo virus (BDBV), and Tai Forest virus (THB). Both Forest virus (TAFV) and IC50 have good anti-infective effects. 50 The concentrations were 0.23 μM, 0.44 μM, and 3.35 μM, respectively. In the case of transfection with the viral mini-genome, 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate also exhibited good inhibitory effects on the replication of the EBOV mini-genome, and showed no cytotoxicity at the concentration that produced 100% inhibition of live virus infection. This indicates that 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate can also be used to prepare safe and effective broad-spectrum anti-EBOV drugs.

[0017] This invention conducted cytotoxicity and viral infection experiments in Huh-7 and Vero-E6 cell lines. The results showed that 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate effectively inhibited EBOV replication and infection within a non-toxic range; 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate effectively inhibited EBOV infection on Huh-7 and Vero-E6 cells, wherein the IC50 value was [not specified in the original text]. 50 The concentrations were 0.08 μM (Huh-7 cells) and 0.35 μM (Vero-E6 cells), with SI values ​​of 1250.00 and >285.71, respectively.

[0018] The 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate provided by this invention can be used to prepare anti-EBOV drugs, improving the utilization rate of 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate and expanding the application range of this compound. Attached Figure Description

[0019] Figure 1 This is a graph showing the effect of the compound on inhibiting EBOV mini-genome replication; the vertical axis represents the percentage of inhibition, and the horizontal axis represents the concentration.

[0020] Figure 2 The graph shows the inhibitory effect of the compound on EBOV infection in Huh-7 cells;

[0021] Figure 3 The graph shows the inhibitory effect of the compound on EBOV infection in Vero-E6 cells;

[0022] Figure 4 The diagram shows the effect of the compound in inhibiting SUDV infection;

[0023] Figure 5 The diagram shows the effect of the compound in inhibiting BDBV infection.

[0024] Figure 6 The diagram shows 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; in the diagram, the C backbone of the Ebola virus NP protein is shown in green, the N atom is shown in blue, the O atom is shown in bright red, the H atom is shown in white, 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate is shown in a light brown stick, and the hydrogen bond length is shown as a purple-red dashed line. Detailed Implementation

[0026] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, which can be obtained commercially by those skilled in the art.

[0028] The following examples use the following abbreviations:

[0029] Ebola virus: EBOV;

[0030] Sudan Ebola Virus (SUDV)

[0031] Bundibugyo Ebola Virus (BDBV)

[0032] Tai Forest Ebola Virus (TAFV)

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

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

[0035] (1) Drug inhibition of EBOV mini-genome replication experiment

[0036] The EBOV mini-genome system was transfected into 96-well plates. Six hours after transfection, 20, 2, and 0.4 μM of 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbonitrile (diluted with DMEM containing 2% FBS and 1% penicillin) were added, respectively. A control group with the same amount of DMSO was also included. After incubation at 37°C for 48 hours, the supernatant was discarded, cells were collected and lysed, and luciferase activity was read to obtain the replication status of the EBOV mini-genome after treatment with different concentrations of 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbonitrile.

[0037] The results are as follows Figure 1As shown, the 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbonitrile provided by the present invention can effectively inhibit the replication of the EBOV mini-genome in cells, and this inhibitory activity is dose-dependent on the content of 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbonitrile.

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

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

[0040] Different concentration gradients of 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate were co-incubated with Huh-7 and Vero-E6 for the corresponding time. CCK-8 was then added, and the absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. This measurement value can indirectly reflect the number of live cells.

[0041] Using Huh-7 and Vero-E6 cell lines as viral infection models, Huh-7 and Vero-E6 cells were infected with an MOI of 0.1. One hour after infection, the culture medium was discarded. After washing the cells twice with PBS buffer, different concentrations of 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbonitrile (diluted with DMEM containing 2% FBS and 1% penicillin antibody) were added. A control group without the drug was also set up. After incubation at 37°C for 72 hours, the cell supernatant was collected, and viral RNA was extracted from the supernatant using a kit. The viral content in cells and cell supernatant treated with different drugs was detected by RT-qPCR, and the drug inhibition rate was calculated.

[0042] The results are as follows Figure 2 As shown, the 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate provided by this invention can effectively inhibit EBOV infection in Huh-7 cells, IC50. 50 0.08μM, CC 50>100.00μM, SI value 1250.00, drug set concentrations were 10, 2.5, 0.63, 0.16, 0.04 and 0.001μM respectively.

[0043] The results are as follows Figure 3 As shown, the 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate provided by this invention can effectively inhibit EBOV infection in Vero-E6 cells, IC50. 50 0.35μM, CC 50 >100.00μM, SI value >285.71, drug set concentrations were 10, 2.5, 0.63, 0.16, 0.04 and 0.001μM respectively.

[0044] (3) Assay for broad-spectrum drug use

[0045] Using the Huh-7 cell line as a viral infection model, Huh-7 cells were infected with SUDV, BDBV, and TAFV (MOI = 0.1). One hour after infection, the culture medium was discarded, and the cells were washed twice with PBS buffer. Different concentrations of 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate (diluted with DMEM containing 2% FBS and 1% penicillin antibody) were added. A control group without drug treatment was also set up. After culturing at 37°C for 72 hours, the cell supernatant was collected. Viral RNA was extracted from the supernatant using a kit, and the viral content in the supernatant after different drug treatments was detected by RT-qPCR. The drug inhibition rate was calculated.

[0046] The results are as follows Figure 4-6 As shown, the 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate provided by this invention can effectively inhibit SUDV, BDBV, and TAFV infections, IC50. 50 The concentrations were 0.23 μM, 0.44 μM, and 3.35 μM, respectively, and the CC concentrations were... 50 >100.00 μM, with drug concentration settings of 10, 2.5, 0.63, 0.16, 0.04, 0.001 and 0.0003 μM.

[0047] (4) Detection of molecular docking between drug and EBOV

[0048] In this embodiment, molecular docking detection of the binding mode between 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate and EBOV nucleoprotein NP was performed using MCE (MedChemExpress). The results are as follows: Figure 7 As shown.

[0049] As can be seen, 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate can form two hydrogen bonds with EBOZM and NP proteins; the amino group of the compound can form one hydrogen bond with GLN238 of the target protein; the carbonyl group of the compound can form one hydrogen bond with LYS248 of the target protein; the hydrogen bond interaction is shown as a purple-red dashed line, and the longer the bond length, the weaker the hydrogen bond interaction.

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

[0051] The contents not described in detail in this specification are well-known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. Use of 2-[(7-hydroxy-8-methyl-2-oxochromen-4-yl)methylsulfinyl]-4,6-dimethylpyridine-3-carbonitrile for the preparation of an antiebolavirus medicament, characterized in that, The structural formula of the 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate is: 。 2. Use according to claim 1, characterized in that, The anti-Ebola virus drugs mentioned refer to drugs that inhibit the replication of the Ebola virus.

3. Use according to claim 2, characterized in that, The inhibition of Ebola virus replication refers to the binding of 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate to the Ebola virus nucleoprotein NP, thereby inhibiting the replication of the Ebola virus.

4. Use according to claim 1, characterized in that, The molecular formula of the 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate is C 19 H 16 N2O3S.

5. Use according to claim 1, characterized in that, The molecular weight of the 2-[(7-hydroxy-8-methyl-2-oxochrome-4-yl)methylthiosulfase]-4,6-dimethylpyridine-3-carbamate is 352.4 g / mol.

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