Use of 4,4'-(benzo[c][1,2,5]thiadiazole-4,7-diyl)diphenylamine compounds in the preparation of anti-ebola virus drugs

Abstract

4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)dianiline compound in the preparation of anti-Ebola virus drugs belongs to the technical field of medicines.The present application provides 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)dianiline compound to solve the technical problem of lack of effective anti-Ebola virus drugs in the prior art, and the molecular formula of the compound is C 18 H 14 N4S.The 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)dianiline compound provided by the present application can effectively inhibit the replication and infection of Ebola virus in the non-toxic range, and has good anti-infection effect on Sudan virus SUDV, Bundibugyo virus BDBV and Taï Forest virus TAFV, and the compound can be applied to prepare safe and effective broad-spectrum anti-Ebola virus drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of a 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound in preparation of an anti-Ebola virus drug. BACKGROUND

[0002] Ebola virus (EBOV) is a kind of RNA virus with a capsule, single negative strand and no segment, and can cause very serious, usually fatal hemorrhagic fever with fever disease after infecting human beings, thus named Ebola hemorrhagic fever (EHF), also known as Ebola virus disease (EVD); symptoms include fever, headache, abdominal pain, severe weakness and fatigue; diarrhea, bloody stool, internal and external bleeding, multiple organ failure, and even death. In order to protect human health and safety, and improve the prevention and control ability of the virus infection, it is of great significance to reserve the prevention and control drugs for EBOV.

[0003] EBOV is a single negative strand RNA virus, and the genome encodes seven structural proteins and one non-structural protein. The gene order is: 3' end-NP-VP35-VP40-GP-VP30-VP24-L-5' end, the VP30, NP, VP35 and L proteins of Ebola virus form a large complex, i.e. a transcription initiation complex, which is responsible for the RNA synthesis of EBOV.

[0004] At present, there is no related patent report about 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine, and there is also no application thereof in preventing or treating Ebola virus disease. SUMMARY

[0005] The application provides application of a 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound in preparation of an anti-Ebola virus drug, so as to solve the technical problem that there is no effective anti-Ebola virus drug in the prior art.

[0006] One of the purposes of the application is to provide application of a 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound in preparation of an anti-Ebola virus drug.

[0007] In a preferred embodiment of the application, the anti-Ebola virus drug refers to inhibition of Ebola virus replication.

[0008] In a preferred embodiment of the present invention, the inhibition of Ebola virus replication refers to the binding of the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound to the Ebola virus NP protein, thereby inhibiting the replication of the Ebola virus.

[0009] In a preferred embodiment of the present invention, the CAS number of the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound is 1203707-77-0.

[0010] In a preferred embodiment of the present invention, the structural formula of the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound is as follows:

[0011]

[0012] In a preferred embodiment of the present invention, the molecular formula of the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound is C 18 H 14 N4S.

[0013] In a preferred embodiment of the present invention, the molecular weight of the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound is 318.4.

[0014] A second objective of this invention is to provide an anti-Ebola virus drug comprising a 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound and pharmaceutically acceptable excipients.

[0015] Beneficial effects of the present invention: The present invention provides the application of 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound in the preparation of anti-Ebola virus drugs. The CAS number of the compound is 1203707-77-0, and the molecular formula is C 18 H 14 N4S has a molecular weight of 318.4.

[0016] The 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound provided by this invention is effective against Sudan virus (SUDV), Bundibugyo virus (BDBV), and Tai Forest virus (…). It has good anti-infective effects against viruses (TAFV), IC 500.14 μM, 2.36 μM and 2.97 μM, respectively. In the case of transfection of the virus minigenome, the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)dianiline compound also has a good inhibitory effect on the replication of the EBOV minigenome, and has no cytotoxicity at the concentration capable of producing 100% inhibition on the live virus infection, indicating that the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)dianiline compound can also be applied to prepare a safe and effective broad-spectrum anti-Ebola virus drug.

[0017] The present application carries out cytotoxicity and virus infection experiments in Huh-7, Hela and Vero-E6 cell lines, and the results show that the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)dianiline compound can effectively inhibit the replication and infection of the Ebola virus in a non-toxic range; the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)dianiline compound can effectively inhibit the infection of EBOV in the supernatant of Huh-7, Hela and Vero-E6 cells, wherein the IC 50 3.54 μM, 0.13 μM and 2.08 μM, respectively, and the SI values are 50.34, 750.19 and >48.05, respectively.

[0018] The 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)dianiline compound provided by the present application can be applied to prepare an anti-Ebola virus drug, improves the utilization rate of the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)dianiline compound, and expands the application range of the compound. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is an effect diagram of the compound inhibiting the replication of the EBOV minigenome; the vertical coordinate Percentage of inhibition is the inhibition rate, and the horizontal coordinate Concentration is the concentration;

[0020] Figure 2 is an effect diagram of the compound inhibiting the infection of EBOV in the supernatant of Huh-7 cells;

[0021] Figure 3 is an effect diagram of the compound inhibiting the infection of EBOV in the supernatant of Hela cells;

[0022] Figure 4 is an effect diagram of the compound inhibiting the infection of EBOV in the supernatant of Vero-E6 cells;

[0023] Figure 5 is an effect diagram of the compound inhibiting the infection of SUDV;

[0024] Figure 6 Graph of the effect of a compound on inhibition of BDBV infection;

[0025] Figure 7 Graph of the effect of a compound on inhibition of TAFV infection;

[0026] Figure 8 Graph of the molecular docking of a compound with Ebola virus NP protein; wherein the Ebola virus NP protein C backbone is shown in light purple, N atoms are shown in blue, O atoms are shown in large red, H atoms are shown in white, the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound is shown in grass green stick, and the hydrogen bond length is shown in yellow dotted line. DETAILED DESCRIPTION

[0027] Those skilled in the art can modify the process parameters according to the content herein. It is particularly pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the related personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content and scope of the present application, to realize and apply the present application technology.

[0028] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with specific embodiments and the drawings of the specification. The test methods used in the following examples are conventional methods, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art, and can be obtained by commercial channels by those skilled in the art.

[0029] English abbreviations involved in the following examples:

[0030] Ebola virus: Ebola virus, EBOV;

[0031] Sudan Ebola virus: Sudan Ebola Virus, SUDV;

[0032] Bundibugyo Ebola virus: Bundibugyo Ebola Virus, BDBV;

[0033] Tai Forest Ebola virus: Tai Forest Ebola Virus, TAFV.

[0034] The EBOV, SUDV, BDBV and TAFV viruses used in the following examples were provided by the National Virus Recource Center.

[0035] The compound described in the following examples is a 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound.

[0036] Example 1: Application of 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound in preparation of anti-Ebola virus drugs

[0037] (1) Drug inhibits EBOV minigenome replication experiment

[0038] The EBOV minigenome system was transfected into a 96-well plate, and 100, 20, 4, 0.8, 0.16 and 0.032 μM of 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound (diluted with DMEM containing 2% FBS, 1% double antibody) were added at 6 h after transfection, and the same amount of DMSO group was set as a control, and incubated at 37°C for 48 h, then the supernatant was discarded, the cells were collected and lysed, and the luciferase activity was read to obtain the replication of EBOV minigenome after treatment with different concentrations of 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound.

[0039] The results are shown in Table 1. Figure 1 The 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound provided by the present application can effectively inhibit the replication of EBOV minigenome in cells, and the IC 50 is 0.01 μM.

[0040] (2) Drug cytotoxicity experiment and EBOV infection inhibition experiment

[0041] The CCK-8 kit was used to determine the cytotoxicity of the drug. WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazole monosodium salt) in the kit can be reduced to a highly water-soluble orange yellow formazan by some dehydrogenases in the mitochondria under the action of an electronic coupling carrier 1-methoxy PMS, and the color depth of the generated formazan is proportional to the cell activity and inversely proportional to the cytotoxicity.

[0042] Different concentration gradients of 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compounds were co-incubated with HeLa, Huh-7, and Vero-E6 for the corresponding time. CCK-8 was then added, and the absorbance was measured at 450 nm using a microplate reader. This measurement value can indirectly reflect the number of live cells.

[0043] HeLa, Huh-7, and Vero-E6 cell lines were used as viral infection models. HeLa cells were infected with an MOI of 1, and 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 concentration gradients of 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-dimethyl)diphenylamine compounds (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 treated with different drugs and in the cell supernatant was detected by RT-qPCR, and the drug inhibition rate was calculated.

[0044] The results are as follows Figure 2 As shown, the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound provided by this invention can effectively inhibit EBOV infection in Huh-7 cell supernatant (extracellular), IC50. 50 3.54 μM, CC 50 The effective concentration was 576.1 μM, with an SI value of 50.34. The drug concentration settings were 100, 33, 11, 3.7, 1.2, 0.41, and 0.14 μM.

[0045] The results are as follows Figure 3 As shown, the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound provided by this invention can effectively inhibit EBOV infection in HeLa cell supernatant (extracellular), IC50. 50 The concentration was 0.13 μM, the SI value was 750.19, and the drug set concentrations were 100, 20, 4, 0.8, 0.16 and 0.032 μM.

[0046] The results are as follows Figure 4 As shown, the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound provided by this invention can effectively inhibit EBOV infection in Vero-E6 cell supernatant (extracellular), IC50. 50 2.08μM, CC 50>100μM, SI value >48.05, drug set concentrations were 100, 20, 4, 0.8, 0.16, 0.032 and 0.0064μM.

[0047] (3) Assay for broad-spectrum drug use

[0048] 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 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-dimethyl)diphenylamine (diluted with DMEM containing 2% FBS and 1% penicillin antibody) were added. A control group without drug treatment was also set up. After incubation 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.

[0049] The results are as follows Figures 5-7 As shown, the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound provided by this invention can effectively inhibit SUDV, BDBV and TAFV infections, IC50 50 The concentrations were 0.14 μM, 2.36 μM, and 2.97 μM, respectively, and the CC concentrations were... 50 The drug concentration was 576.1 μM, and the set concentrations were 100, 20, 4, 0.8, 0.16, 0.032 and 0.0064 μM.

[0050] (4) Detection of molecular docking between drugs and Ebola virus

[0051] In this embodiment, molecular docking detection of the binding mode between 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine and Ebola virus nucleoprotein NP was performed using MCE (MedChemExpress). The results are as follows: Figure 8 As shown.

[0052] It is evident that the amino group of the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound can form three hydrogen bonds with the target protein's ARG240 / GLN238 / ASN335; in addition, the nitrogen atom on the thiadiazole ring of the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound can form one hydrogen bond with the target protein's GLY243.

[0053] In conclusion, the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound provided by the application can be combined with the Ebola virus NP protein, and the docking score is-5.841, thereby achieving the purpose of inhibiting the replication of the Ebola virus.

[0054] The content not described in detail in the specification of the present application is the technology known to those skilled in the art. Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and anyone skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. Use of a 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound in the preparation of an anti-Ebola virus drug.

2. Use according to claim 1, characterized in that, The anti-Ebola virus drug refers to inhibition of Ebola virus replication.

3. Use according to claim 2, characterized in that, The inhibition of Ebola virus replication refers to that the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound binds to Ebola virus nucleoprotein NP to inhibit the replication of Ebola virus.

4. Use according to claim 1, characterized in that, The CAS number of the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound is 1203707-77-0.

5. The use according to claim 1, characterized in that, The structural formula of the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound is 。 6. Use according to claim 1, characterized in that, The molecular formula of the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound is C 18 H 14 N4S.

7. The use according to claim 1, characterized in that, The molecular weight of the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound is 318.4.

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