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

Abstract

The invention discloses application of a 2, 4 '-(benzo [C] [1, 2, 5] thiadiazole-4, 7-diyl) diphenylamine compound in preparation of an anti-Ebola virus drug, and belongs to the technical field of medicines. The invention provides a 4, 4 '-(benzo [C] [1, 2, 5] thiadiazole-4, 7-diyl) diphenylamine compound, and the molecular formula of the compound is C18H14N4S in order to solve the technical problem that no effective Ebola virus resisting medicine exists in the prior art. The 4, 4 '-(benzo [C] [1, 2, 5] thiadiazole-4, 7-diyl) diphenylamine compound provided by the invention can be used for effectively inhibiting replication and infection of Ebola virus in a non-toxic range, and has a good anti-infection effect on sudan virus SUDV, BDBV and TAFV; the compound can be applied to preparation of safe and effective broad-spectrum anti-Ebola virus drugs.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to the application of 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compounds in the preparation of anti-Ebola virus drugs. Background Art

[0002] Ebola virus (EBOV) is an enveloped, single-stranded, non-segmented RNA virus that can cause very severe, usually fatal hemorrhagic and febrile illnesses after infecting humans, hence the name Ebola hemorrhagic fever (EHF), also known as Ebola virus disease (EVD); symptoms include: fever, headache, abdominal pain, severe weakness and fatigue; diarrhea, bloody stools, internal and external bleeding, multiple organ failure, and even death. In order to protect human health and safety and improve the ability to prevent and control the virus infection, it is of great significance to reserve drugs for prevention and treatment of EBOV.

[0003] EBOV is a single-stranded negative-strand RNA virus, and its genome encodes seven structural proteins and one non-structural protein. The gene sequence 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, the transcription initiation complex, which is responsible for the RNA synthesis of EBOV.

[0004] At present, there are no patent reports on 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine, and there is no application thereof in the prevention or treatment of Ebola virus disease. Summary of the invention

[0005] The present invention solves the technical problem of lack of effective anti-Ebola virus drugs in the prior art and provides the use of 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound in the preparation of anti-Ebola virus drugs.

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

[0007] In a preferred embodiment of the present invention, the anti-Ebola virus drug is a drug that inhibits the replication of Ebola virus.

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

[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 N 4 S.

[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] The second object of the present invention is to provide an anti-Ebola virus drug, which comprises a 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound and a pharmaceutically acceptable excipient.

[0015] Beneficial effects of the present invention: The present invention provides the use 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, the molecular formula is C 18 H 14 N 4 S, molecular weight is 318.4.

[0016] The 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound provided by the present invention is effective against Sudan virus (SUDV), Bundibugyo virus (BDBV) and Tai Forest virus ( virus, TAFV) have good anti-infection effects, IC 50The concentrations of 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compounds were 0.14μM, 2.36μM and 2.97μM respectively. In the case of transfecting the virus mini-genome, the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound also had a good inhibitory effect on the replication of the EBOV mini-genome, and had no cytotoxicity at a concentration that could produce 100% inhibition of live virus infection, indicating that the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound can also be used to prepare safe and effective broad-spectrum anti-Ebola virus drugs.

[0017] The present invention conducts 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)diphenylamine compound can effectively inhibit the replication and infection of Ebola virus within a non-toxic range; the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound can effectively inhibit the infection of EBOV in the supernatant of Huh-7, Hela and Vero-E6 cells, wherein IC 50 They were 3.54 μM, 0.13 μM and 2.08 μM respectively, and the SI values ​​were 50.34, 750.19 and >48.05 respectively.

[0018] The 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound provided by the present invention can be used to prepare drugs against Ebola virus, thereby improving the utilization rate of the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound 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 effect of the compound on inhibiting EBOV infection in the supernatant of Huh-7 cells;

[0021] Figure 3 This is a graph showing the effect of the compound on inhibiting EBOV infection in the supernatant of Hela cells;

[0022] Figure 4 This is a graph showing the effect of the compound on inhibiting EBOV infection in the supernatant of Vero-E6 cells;

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

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

[0025] Figure 7 This is a diagram showing the effect of the compound in inhibiting TAFV infection;

[0026] Figure 8 This is a molecular docking detection diagram of the compound and the Ebola virus NP protein; the Ebola virus NP protein C skeleton is displayed in light purple, the N atom is displayed in blue, the O atom is displayed in bright red, the H atom is displayed in white, the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound is displayed as a grass green stick, and the hydrogen bond length is displayed as a yellow dotted line. DETAILED DESCRIPTION

[0027] 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.

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

[0029] The English abbreviations involved in the following embodiments are:

[0030] Ebola virus: Ebola virus, EBOV;

[0031] Sudan 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 all 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 the preparation of anti-Ebola virus drugs

[0037] (1) Experiment on drug inhibition of EBOV mini-genome replication

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

[0039] The results are as follows Figure 1 As shown, the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound provided by the present invention can effectively inhibit the replication of the EBOV mini-genome in cells, IC 50 0.01μM.

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

[0041] The CCK-8 kit was used to measure the cytotoxicity of the drug. The 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 a highly water-soluble orange-yellow formazan by some dehydrogenases in the mitochondria under the action of the electron coupling carrier 1-Methoxy PMS. The color depth of the generated formazan is proportional to the cell activity and inversely proportional to the cytotoxicity.

[0042] After incubating Hela, Huh-7 and Vero-E6 with different concentration gradients of 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compounds for corresponding periods of time, CCK-8 was added and the absorbance was measured at a wavelength of 450nm using an enzyme reader. The measured value can indirectly reflect the number of living cells.

[0043] Hela, Huh-7 and Vero-E6 cell lines were used as virus infection models. Hela cells were infected with MOI=1, and Huh-7 and Vero-E6 cells were infected with MOI=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-diyl)diphenylamine compounds (diluted with DMEM containing 2% FBS and 1% double antibody) were added respectively. At the same time, a control group without drug addition was set up. After continued culture in a 37°C incubator for 72 hours, the cell supernatant after culture was collected, and the viral RNA in the supernatant was extracted using a kit. The viral content in cells and cell supernatants treated with different drugs 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 the present invention can effectively inhibit the infection of EBOV in the supernatant of Huh-7 cells (extracellular), IC 50 3.54μM, CC 50 It was 576.1 μM, the SI value was 50.34, and the drug set concentrations were 100, 33, 11, 3.7, 1.2, 0.41 and 0.14 μM, respectively.

[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 the present invention can effectively inhibit the infection of EBOV in the supernatant of Hela cells (extracellular), IC 50 The concentration of the drug was 0.13 μM, the SI value was 750.19, and the set concentrations of the drug were 100, 20, 4, 0.8, 0.16, and 0.032 μM, respectively.

[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 the present invention can effectively inhibit the infection of EBOV in the supernatant of Vero-E6 cells (extracellular), IC 50 2.08μM, CC 50>100 μM, SI value >48.05, and the drug set concentrations were 100, 20, 4, 0.8, 0.16, 0.032, and 0.0064 μM, respectively.

[0047] (3) Determination of broad-spectrum drug activity

[0048] The Huh-7 cell line was used as a virus infection model. Huh-7 was infected with SUDV, BDBV and TAFV (MOI = 0.1), respectively. One hour after infection, the culture medium was aspirated and the cells were washed twice with PBS buffer. Different concentrations of 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine (diluted with DMEM containing 2% FBS and 1% double antibody) were added, and a control group without drug addition was set up. After continued culture in a 37°C incubator for 72 hours, 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 drug inhibition rate was calculated.

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

[0050] (4) Molecular docking detection of drugs and Ebola virus

[0051] In this example, MCE (MedChemExpress) conducted molecular docking detection on the binding mode of 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound and Ebola virus nucleoprotein NP. The results are as follows Figure 8 shown.

[0052] It can be seen 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 ARG240 / GLN238 / ASN335 of the target protein; 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 GLY243 of the target protein.

[0053] In summary, the 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound provided by the present invention can bind to the Ebola virus NP protein with a docking score of -5.841, thereby achieving the purpose of inhibiting the replication of the Ebola virus.

[0054] The contents not described in detail in the specification of the present invention are well-known technologies for those skilled in the art. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the definition of the claims.

Claims

1. Application of 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compounds in the preparation of anti-Ebola virus drugs.

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

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

4. The 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. The 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.

8. An anti-Ebola virus drug, characterized in that: The drug comprises a 4,4'-(benzo[C][1,2,5]thiadiazole-4,7-diyl)diphenylamine compound and a pharmaceutically acceptable auxiliary material.

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