Use of isoxazole compounds in the preparation of anti-zika virus drugs

Abstract

The application provides application of an isoxazole compound or a pharmaceutical salt thereof in preparation of a drug for resisting or preventing ZIKV, wherein a structural formula of the isoxazole compound is shown as formula 1. The application shows for the first time that the isoxazole compound shown as formula 1 has extremely strong antiviral activity on ZIKV, can significantly inhibit replication of ZIKV on host cells A549, improve cell survival rate, can inhibit the process of virus entering the host cells A549, and has extremely strong antiviral effect at the cell level, has obvious anti-ZIKV effect, and the isoxazole compound shown as formula 1 can more effectively inhibit the virus when used before or during invasion of ZIKV.

Description

Technical Field

[0001] This invention patent relates to the field of pharmaceutical technology, and in particular to the application of an isoxazole compound in the preparation of anti-Zika virus drugs. Background Technology

[0002] Zika virus (ZIKV) is a member of the Flavivirus genus within the Flaviviridae family. It is the most common pathogen causing microcephaly and Guillain-Barré syndrome in newborns. Since its first report in 1947, ZIKV-related infectious diseases have experienced numerous outbreaks and epidemics worldwide. Currently, prevention and treatment of viral diseases primarily rely on vaccines and medications. However, there is no vaccine available to prevent ZIKV infection, and treatment options for ZIKV-related infectious diseases are quite limited. The main treatment methods are symptomatic treatment, supportive care, and broad-spectrum antiviral therapy; however, these methods exhibit significant individual variability and are difficult to implement universally.

[0003] Therefore, it is imperative to develop specific and effective anti-ZIKV drugs, and it is very necessary to develop an effective anti-Zika virus drug. Summary of the Invention

[0004] The present invention aims to at least partially solve one of the technical problems existing in the prior art. Therefore, in a first aspect, the present invention provides the use of isoxazole compounds or pharmaceutically acceptable salts thereof in the preparation of anti-Zika virus drugs, wherein the structural formula of the isoxazole compound is shown in Formula 1.

[0005]

[0006] During ZIKV invasion of host cells, it hijacks cell signaling pathways and degrades specific host proteins to promote its own replication. Therefore, viral-supporting cell signaling pathways may be promising novel antiviral targets. The RAF / MEK / ERK signaling pathway belongs to the classic mitogen-activated protein kinase (MAPK) cascade pathway and plays an important role in cell growth and development. The RAF family includes A-RAF, B-RAF, and C-RAF, which are serine / threonine protein kinases that can form RAF homodimers or heterodimers, activating downstream MEK1 / 2 proteins through phosphorylation; MEK kinases activate downstream ERK1 / 2 through MEK phosphorylation.

[0007] HSP90 is a crucial host factor in the viral life cycle, playing a vital role in the invasion of host cells by various viruses. For example, human cytomegalovirus infection regulates HSP90, which activates AKT, modulates downstream signaling molecules involved in viral replication, and promotes the expression of essential viral genes. Chikungunya virus infection promotes viral replication by activating the RAF / ERK signaling pathway through HSP90. Simultaneously, HSP90 also activates the AKT / NF-κB and MAPK / ERK pathways in virus-infected cells. These studies collectively demonstrate that HSP90 expression is positively correlated with AKT / RAF phosphorylation, indicating that HSP90 is a key host factor in viral replication signaling pathways. This suggests that HSP90 is a feasible ideal therapeutic target, providing a theoretical basis for using HSP90 inhibitors to treat viral infections.

[0008] Small molecule isoxazole compounds (the compounds shown in Formula 1) are potent HSP90 inhibitors and are among the most effective HSP90 ligands. They can anchor to the ATP site at the end of HSP90 nh2 and inhibit the activation of AKT and RAF by HSP90, thereby inhibiting the role of HSP90 in viral replication and cell growth.

[0009] In one or more embodiments of the present invention, the concentration of the isoxazole compound is 0.16-20 μM.

[0010] In one or more embodiments of the present invention, the concentration of the isoxazole compound is 2.23-20 μM.

[0011] In one or more embodiments of the present invention, the concentration of the isoxazole compound is 20 μM.

[0012] When the concentration of isoxazole compound (the compound shown in Formula 1) is 2.23 μM, an inhibition rate of 50% against ZIKV can be achieved.

[0013] When the concentration of the isoxazole compound shown in Formula 1 is 20 μM, the inhibition of ZIKV virus reaches approximately 70%.

[0014] In a second aspect, the present invention provides the use of isoxazole compounds in the preparation of drugs for the prevention of Zika virus infection, wherein the structural formula of the isoxazole compound is shown in Formula 1.

[0015]

[0016] In one or more embodiments of the present invention, the drug for preventing Zika virus infection is a drug for preventing severe Zika virus infection.

[0017] In one or more embodiments of the present invention, the concentration of the isoxazole compound is 0.16-20 μM.

[0018] Preferably, the application of the first aspect and / or the second aspect includes the combination of isoxazole compounds (the compounds shown in Formula 1) and ribavirin.

[0019] In a third aspect, the present invention provides the use of a pharmaceutical composition in the preparation of an anti-Zika virus drug, said pharmaceutical composition comprising a compound shown in Formula 1.

[0020]

[0021] In one or more embodiments of the present invention, the pharmaceutical composition is prepared into a pharmaceutical formulation by adding pharmaceutically acceptable excipients and a carrier, wherein the excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, and colorants.

[0022] In one or more embodiments of the present invention, the pharmaceutical preparation is selected from granules, tablets, pills, capsules, and injections.

[0023] The inventors evaluated the inhibitory activity of isoxazole compounds (the compounds shown in Formula 1) against ZIKV using standard viral activity assays. Extensive biological experiments revealed that the isoxazole compounds (the compounds shown in Formula 1) exhibited good inhibitory activity against ZIKV entry into cells. This demonstrates that isoxazole compounds (the compounds shown in Formula 1) have an inhibitory effect on ZIKV.

[0024] The beneficial effects of this invention are as follows:

[0025] 1. This invention provides the application of isoxazole compounds (the compounds shown in Formula 1) in the preparation of anti-Zika virus drugs. This invention is the first to discover that isoxazole compounds (the compounds shown in Formula 1) can be used to treat infectious diseases caused by ZIKV. Experiments show that isoxazole compounds (the compounds shown in Formula 1) have extremely strong antiviral activity against ZIKV, can significantly inhibit the replication of ZIKV in host cells A549, improve cell survival rate, and inhibit the process of virus entering host cells A549. They exhibit extremely strong antiviral effects at the cellular level and have obvious anti-ZIKV effects.

[0026] 2. This invention provides the application of isoxazole compounds (the compounds shown in Formula 1) in the preparation of drugs for preventing Zika virus infection. This invention is the first to discover that isoxazole compounds (the compounds shown in Formula 1) administered before or during Zika virus invasion can more effectively inhibit the virus. Therefore, it suggests that isoxazole compounds (the compounds shown in Formula 1) can be used in the preparation of drugs for preventing Zika virus infection. Attached Figure Description

[0027] Figure 1 This is a graph showing the A549 cell cytotoxicity test results of the isoxazole compounds shown in Formula 1 of Example 1 of the present invention against ZIKV.

[0028] Figure 2 This is a graph showing the safety and efficacy results of isoxazole compounds as antiviral drugs in Example 2 of the present invention (Symbol 1).

[0029] Figure 3 Immunofluorescence diagrams of blank (MOCK), ZIKV virus infection (ZIKV), inhibition of ZIKV virus by isoxazole compounds of Formula 1 at a concentration of 0.1 μM (ZIKV + 0.1 μM 1), inhibition of ZIKV virus by isoxazole compounds of Formula 1 at a concentration of 1 μM (ZIKV + 1 μM 1), and inhibition of ZIKV virus by isoxazole compounds of Formula 1 at a concentration of 10 μM (ZIKV + 10 μM 1);

[0030] Figure 4 The graph shows the inhibitory effect of different concentrations of isoxazol compounds (as shown in Formula 1) on the expression level of ZIKV viral proteins.

[0031] Figure 5 The graph shows the inhibitory effect of different concentrations of isoxazole compounds (as shown in Formula 1) on ZIKV mRNA levels.

[0032] Figure 6 Graph showing the inhibitory effect of different concentrations of ribavirin on ZIKV viral protein expression levels;

[0033] Figure 7 This is a schematic diagram of the time axis for treatment with isoxazole compounds as shown in Equation 1.

[0034] Figure 8 The effect of isoxazol compounds, as shown in Formula 1, on the expression level of ZIKV viral proteins at different temperatures and stages in ZIKV-infected cells.

[0035] Figure 9 The inhibitory effects of isoxazole compounds, as shown in Formula 1, on ZIKV-infected cells at different temperatures and stages are illustrated. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments and accompanying drawings. However, the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the following embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the methods used are conventional methods known in the art, and the consumables and reagents used are commercially available. Unless otherwise stated, the technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be applied to the present invention.

[0037] In the following embodiments, this invention combines viral RNA level analysis, titer determination, and Alamar Blue cell viability assay to study the anti-ZIKV activity of the isoxazole compounds shown in Formula 1. Data analysis in these embodiments was performed using GraphPadPrism 9.0 software. It is understood that further optimization using the isoxazole compounds shown in Formula 1 as lead compounds for the preparation of drugs to treat ZIKV infectious diseases also falls within the scope of this invention. The isoxazole compounds shown in Formula 1 were purchased from MedChemExpress (MCE).

[0038] The structures of the isoxazole compounds shown in Formula 1 are as follows.

[0039]

[0040] Example 1: Cytotoxicity detection of isoxazole compounds shown in Formula 1

[0041] The cytotoxicity of the isoxazole compounds shown in Formula 1 was assessed in A549 cells. A549 cells were cultured at a concentration of 1 × 10⁻⁶ cells / year. 4 Cells were evenly seeded per well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 12-16 hours. The cell culture medium was then discarded, and culture medium containing different concentrations of isoxazole compounds (as shown in Formula 1) at 2% FBS was added for further culture. Each group had six replicates, with an equal volume of 2% FBS culture medium used as a control. After 48 hours of incubation, the cells were stained with Alamar Blue and incubated at 37°C for 2 hours. Fluorescence values ​​were measured using a multi-mode microplate reader (fluorescence detection: excitation at 560 nm, emission wavelength at 590 nm) to analyze cell viability.

[0042] Results Analysis: The test results are as follows: Figure 1As shown, GraphPad Prism 9.0 software calculates the median cyctoxic concentration (CC50) of the drug for cells. The CC50 of the isoxazole compound shown in Equation 1 is 44.87 μmol / L. In subsequent implementation examples, the maximum concentration of the isoxazole compound shown in Equation 1 used was 20 μM, which is within the safe and non-toxic range.

[0043] Example 2: Safety and efficacy analysis of isoxazole compounds shown in Formula 1 as antiviral drugs

[0044] A549 cells were uniformly seeded in 6-well cell culture plates and cultured at 37°C in a 5% CO2 incubator for 12–16 h. After infection with ZIKV at MOI=2 for 1 h, cells were treated with cell maintenance media containing different concentrations of isoxazole compounds as shown in Formula 1. The negative control was treated with the same volume of 2% FBS culture medium. After 24 h of treatment, the effect of inhibiting viral proliferation was detected by RT-qPCR.

[0045] The inhibition rates of isoxazole compounds (as shown in Equation 1) against ZIKV at different concentrations were calculated based on the viral copy number obtained by the absolute quantification method. Nonlinear regression analysis was performed on the inhibition rates using GraphPad Prism 9.0 to calculate the half-maximal inhibitory concentration (IC50). The IC50 of the isoxazole compounds shown in Equation 1 was 2.23 μmol / L. The selectivity index (SI) is a reference index used to determine the safety range of drug efficacy. Its value in in vitro experiments is equal to the ratio of CC50 / EC50. A selectivity index greater than 1 is considered effective, and a higher index value indicates greater safety. Figure 2 This is a graph showing the safety and efficacy results of isoxazole compounds as antiviral drugs in Example 2 of the present invention, as shown in Formula 1.

[0046] Results analysis: Based on the previous cytotoxicity test results (CC50 = 44.87 μmol / L), the selectivity index of the isoxazole compound shown in Formula 1 can be calculated to be 20.12. This result further illustrates that the isoxazole compound shown in Formula 1 has great safety and effectiveness in inhibiting ZIKV.

[0047] Example 3: Detection of antiviral activity of different concentrations of isoxazole compounds (Formula 1) against ZIKV

[0048] A549 cells were seeded in 6-well cell culture plates and cultured at 37°C in a 5% CO2 incubator for 12–16 h. After infection with ZIKV at MOI=2 for 1 h, cells were treated with cell maintenance media containing isoxazole compounds of Formula 1 at concentrations of 0.16 μM, 0.8 μM, 4 μM, and 20 μM. A negative control was added to the same volume of 2% FBS culture medium. After 24 h of treatment, viral infection was detected using immunofluorescence. Subsequently, the same experiments were performed with different concentrations of isoxazole compounds of Formula 1 (0.16 μM, 0.8 μM, 4 μM, and 20 μM). Viral protein expression levels were detected by Western blotting, and the inhibitory effect of the isoxazole compounds of Formula 1 on viral proliferation was detected by RT-qPCR. Ribavirin was used as a positive control to detect viral protein expression levels.

[0049] Results Analysis: The test results are as follows: Figure 3 As shown, Figure 3 This is a schematic diagram of immunofluorescence assays for blank (MOCK), ZIKV virus infection (ZIKV), inhibition of ZIKV virus at a concentration of 0.1 μM (ZIKV + 0.1 μM), inhibition of ZIKV virus by isoxazole compounds shown in Formula 1 at a concentration of 1 μM (ZIKV + 1 μM), and inhibition of ZIKV virus by isoxazole compounds shown in Formula 1 at a concentration of 10 μM (ZIKV + 10 μM). Figure 4 The inhibitory effect of different concentrations of isoxazole compounds (as shown in Formula 1) on the expression level of ZIKV viral proteins; Figure 5 The inhibitory effects of different concentrations of isoxazole compounds (as shown in Formula 1) on ZIKV mRNA levels are illustrated. Figure 6 This study illustrates the inhibitory effect of different concentrations of the positive control (Ribavirin) on ZIKV viral protein expression levels. Under different concentration conditions, the isoxazole compound shown in Formula 1 inhibited ZIKV proliferation, and the inhibitory effect became more pronounced with increasing concentration of the isoxazole compound shown in Formula 1. When the concentration of the isoxazole compound shown in Formula 1 was 20 μM, the inhibition of ZIKV virus reached approximately 70%.

[0050] Example 4: Detection of the antiviral activity of the isoxazole compounds shown in Formula 1 against different stages of ZIKV-treated cells.

[0051] A549 cells were seeded in 6-well cell culture plates and cultured at 37°C in a 5% CO2 incubator for 12–16 h. The cells were then infected with ZIKV at an MOI of 2. At different temperatures and stages of ZIKV treatment, A549 cells were treated with cell maintenance medium containing 1 μM of the isoxazole compound shown in Formula 1. A negative control was prepared with the same volume of 2% FBS. After 24 h of treatment, the inhibitory effect of the isoxazole compound shown in Formula 1 on viral proliferation was detected by RT-qPCR, and the expression levels of viral proteins were detected by immunoblotting.

[0052] Results Analysis: The antiviral activity of isoxazole compounds (as shown in Equation 1) against ZIKV-treated cells at different temperatures and stages is illustrated in the figure. Figure 7 This is a schematic diagram of the time axis of treatment with isoxazole compounds as shown in Equation 1. Figure 8 The effect of isoxazole compounds, as shown in Formula 1, on the expression levels of ZIKV viral proteins at different stages of ZIKV infection. Figure 9 The study investigated the inhibitory effects of the isoxazole compounds shown in Formula 1 on the adsorption and cell invasion stages of ZIKV at 4°C, and on the inhibitory effects of the isoxazole compounds shown in Formula 1 on ZIKV after cell invasion and throughout the entire cell invasion stage at 37°C. The results showed that the isoxazole compounds shown in Formula 1 had a significant inhibitory effect on ZIKV after cell invasion.

[0053] In summary, the isoxazole compounds shown in Formula 1 can significantly inhibit the activity of ZIKV invasion, can significantly suppress the expression level of viral proteins after A549 infection with ZIKV, and inhibit the replication of viral RNA, thereby enhancing cell survival rate. They have the potential to be further developed into a clinically effective drug against ZIKV infection.

[0054] The present invention demonstrates, through the above embodiments, the role of isoxazole compounds of Formula 1 in the fight against ZIKV. Isoxazole compounds of Formula 1 can inhibit the expression level of viral proteins and RNA replication level of ZIKV in host cells A549, and enhance cell survival rate; they have the potential to be developed into drugs for the effective treatment of ZIKV infection.

[0055] This invention further provides the application of the isoxazole compound of Formula 1 in the preparation of anti-ZIKV infection drugs. The application refers to the addition of pharmaceutically acceptable excipients and carriers to the isoxazole compound of Formula 1 for the preparation of anti-ZIKV formulations. The excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, or colorants, with different excipients selected according to the requirements of the drug dosage form. The formulation is a granule, tablet, pill, capsule, injection, or dispersant.

[0056] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention, all of which should be included within the protection scope of the present invention.

Claims

1. Use of an isoxazole compound or a pharmaceutically acceptable salt thereof in the manufacture of an anti-Zika virus drug, characterized in that, The structural formula of the isoxazole compound is shown in Formula 1. 1。 2.The isoxazole compound for use in the preparation of an anti-Zika virus drug according to claim 1, characterized in that, The concentration of the isoxazole compounds is 0.16-20 μM.

3. The isoxazole compound according to claim 2, for use in the preparation of an anti-Zika virus medicament, characterized in that, The concentration of the isoxazole compound is 20 μM.

4. Use of a pharmaceutical composition in the manufacture of a medicament for the treatment of Zika virus, characterized in that, The active ingredient of the pharmaceutical composition is a compound represented by Formula 1. 1。 5. Use according to claim 4, characterized in that, The pharmaceutical composition is prepared into a pharmaceutical formulation by adding pharmaceutically acceptable excipients and carriers, wherein the excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, and colorants.

6. Use according to claim 5, characterized in that, The pharmaceutical preparation is selected from one of the following: granules, tablets, pills, capsules, and injections.

Images