Application of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate in the preparation of products for inhibiting influenza virus

Abstract

The present invention belongs to the field of biological preparation technology and specifically relates to the use of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate in the preparation of products for inhibiting influenza viruses. The present invention is the first to discover that (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate has a significant inhibitory effect on influenza A virus at the cellular level. The inhibition efficiency reaches 95% without affecting cell activity, which makes it have great potential for clinical application.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological preparations, and particularly relates to the use of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate in the preparation of a product for inhibiting influenza virus. Background Art

[0002] Autophagy is involved in nearly all cellular processes and is closely linked to the development and progression of numerous human diseases. Research has shown that autophagy is closely linked to viral infection. Autophagy is an intracellular self-digestive process that maintains cellular homeostasis by degrading intracellular substances. In viral infection, autophagy plays a dual role: on the one hand, it serves as a host cell defense mechanism, limiting viral replication by clearing infected cellular components; on the other hand, viruses also exploit autophagy to promote their own replication and survival. Autophagy as a host defense mechanism: During viral infection, host cells activate autophagy to clear infected cellular components, thereby limiting viral replication. For example, during infection, Streptococcus pyogenes is sequestered in autophagosomes and ultimately degraded in lysosomes. Furthermore, interferon can upregulate autophagy, further enhancing the host cell's antiviral capacity. Viruses utilize autophagy to promote their own replication: Certain viruses also exploit autophagy to promote their own replication. For example, influenza virus nucleoprotein NP can also induce mitophagy and promote viral replication, which is a strategy by which viruses hijack the host metabolic system to maintain their own replication.

[0003] In summary, autophagy is closely linked to viral infection, serving as both a host defense mechanism and exploited by viruses to promote their own survival and replication. This interplay reflects the complex interactions between cells and viruses and is of great significance for understanding the mechanisms of viral infection and developing effective antiviral strategies.

[0004] Influenza viruses are commonly referred to as influenza viruses. Based on differences in the nucleoprotein (NP) and matrix protein (M), influenza viruses can be divided into four types: A (A), B (B), C (C), and D (D). Influenza A and B viruses can cause outbreaks and epidemics, while influenza C causes mild infections. Only influenza A virus (IAV) can cause global pandemics. IAVs cause an estimated 290,000 to 650,000 deaths worldwide each year and are listed by the WHO as one of the top ten threats to global health.

[0005] IAV has a segmented negative-sense single-stranded RNA genome and exhibits a wide host range. This allows IAV to circulate continuously in nature and produce genetically diverse variants through evolution. These mutations can lead to seasonal epidemics, unpredictable pandemics, and outbreaks of zoonotic diseases. The extremely high gene recombination efficiency of IAV has seriously affected the effectiveness of influenza vaccines, and anti-influenza drugs have become the key to clinical treatment. At present, drugs for the treatment of influenza mainly use some neuraminidase inhibitors and ion channel M2 protein inhibitors. However, with the mutation of the virus, existing anti-influenza drugs face a severe drug resistance situation. Widespread antiviral resistance limits the clinical application of these inhibitors in the prevention and treatment of influenza infections. Therefore, in order to meet the urgent needs of clinical influenza patients for treatment, the development of new anti-influenza virus drugs is imminent.

[0006] The molecular formula of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate is C 13 H 10 N₄O₄, with a molecular weight of 286.247, is composed of the functional groups amino(pyridin-2-yl)methylidene and amino 2-nitrobenzoate. Currently, there are no reports of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate inhibiting influenza virus activity. Summary of the Invention

[0007] In order to solve the problem that there has been no report in the prior art on the inhibitory effect of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on influenza virus, the present invention provides a small molecule compound (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate and its use in the preparation of a product for inhibiting influenza virus.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention provides the use of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate in the preparation of a product for inhibiting influenza virus. The influenza virus is influenza A virus, including the H1N1 (A / PR / 8 / 34) strain.

[0010] Among them, the products include reagents used for laboratory research on influenza viruses or other products that inhibit influenza viruses.

[0011] The present invention also provides the use of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate in the preparation of a drug for treating or preventing influenza A virus infection, and the use of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate in the preparation of an anti-influenza A virus drug.

[0012] The present invention also provides the use of the (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate in the preparation of a drug for promoting cell autophagy.

[0013] Among them, the molecular formula of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate is C 13 H 10 N4O4, molecular weight is 286.247, and the structural formula is as follows:

[0014] .

[0015] Among them, the English name of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate is [amino(pyridin-2-yl)methylidene]amino 2-nitrobenzoate.

[0016] The present invention has discovered that (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate has a significant inhibitory effect on influenza A virus at the cellular level, achieving an inhibition efficiency of 95% without affecting cell viability. This indicates that the compound has significant potential for clinical application. Therefore, the present invention provides an effective small molecule compound for the clinical treatment of influenza A virus.

[0017] Preferably, the drug contains the (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate as an active ingredient and is supplemented with a pharmaceutically acceptable carrier.

[0018] Preferably, the carrier comprises any one or more of glycerol, lecithin and cholesterol.

[0019] Preferably, the effective concentration of the (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate in the drug is 10 μM to 40 μM.

[0020] Preferably, the drug includes oral dosage forms, injection dosage forms and inhalation dosage forms.

[0021] Among them, the oral dosage form is to form (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate with excipients into a solid or liquid preparation and then take it orally, which is convenient to carry and take.

[0022] Injectable dosage forms include normal saline injection and sterile injection of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate powder, which are rapidly absorbed and effective.

[0023] The inhalation dosage form comprises (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate and its solution, along with a suitable propellant, encapsulated in a pressure-resistant, sealed container with a custom valve system. During use, the propellant's pressure is used to spray the contents in a mist-like form. The drug has a faster onset of action than oral medications and relatively fewer side effects. The present invention can prepare (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate into the following specific pharmaceutical dosage forms, but is not limited thereto. Any pharmaceutical dosage form that can be used to prevent and treat rheumatoid arthritis, prepared from (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate, is available in the pharmaceutical preparation field.

[0024] Preferably, the drug is in the form of a solution, tablet or granule.

[0025] According to the actual treatment purpose and treatment plan, (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate can be prepared into different types of drugs to achieve the effects of preventing and treating influenza A virus infection.

[0026] Preferably, the drug is a solution. Solutions offer high dispersibility, rapid absorption, rapid action, and good bioavailability. The active ingredients are evenly dispersed, allowing for accurate dosing, making them particularly suitable for children and the elderly. The dosage is easily adjustable and controllable, and can reduce the local irritation of certain readily soluble drugs and increase the stability and safety of certain drugs. Therefore, the drug provided herein is in the form of a solution.

[0027] The solvent in the solution is DMSO; the effective concentration of the (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate in the drug is 10 μM to 40 μM.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention provides the use of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate in products for inhibiting influenza viruses. Research results in the present invention demonstrate that (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate has a significant inhibitory effect on influenza A virus at the cellular level, achieving an inhibition efficiency of 95% without affecting cell viability. This suggests that the compound has significant potential for clinical application. This novel discovery is enabling the development of a drug for treating or preventing influenza A virus infection.

[0030] (Z)-[Amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate is a small molecule compound with minimal toxicity to A549 cells. At a concentration of 40 μM, the cell survival rate is over 80%, indicating that it is safe and has low cytotoxicity.

[0031] When (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate acts on A549 cells at a concentration of 40μM, the inhibition rate of viral nucleic acid copy number of influenza A virus H1N1 (PR8) replication reaches more than 99%, indicating that it has highly effective anti-influenza properties.

[0032] 2. The present invention also discovered through research that (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate has the effect of promoting cell autophagy and can be used to prepare drugs that promote cell autophagy, and is expected to play an important role in the treatment of tumors, viral infections, obesity, diabetes and other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the cytotoxicity of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on A549 cells in the present invention.

[0034] Figure 2 Schematic diagram of the Western Blot detection effect of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate against influenza A virus at the cellular level in the present invention; wherein, Figure 2 A in the figure is a Western Blot showing the effect of different concentrations of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on the proliferation of influenza A virus at the cellular level. The weaker the band, the lower the virus content. GADPH is the cell internal reference control. The lanes from left to right represent the working concentrations of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate: 0 μM, 10 μM, 20 μM, and 40 μM, respectively. Figure 2B in the figure is the ratio of the grayscale value of the influenza A (H1N1) virus nucleoprotein (NP) band to the grayscale value of the cellular reference (GADPH) band. The smaller the ratio, the lower the virus content. The horizontal axis values ​​from left to right represent the working concentrations of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate as 0μM, 20μM, 30μM, and 40μM, respectively.

[0035] Figure 3 The figure is a schematic diagram of the Western Blots detection results of the (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate in the present invention at the cellular level to inhibit influenza A virus at different time points; wherein. Figure 3 Figure A is a Western Blot result showing the effect of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate at a concentration of 40 μM on the proliferation of influenza A virus at different time points. The weaker the band, the lower the virus content. GADPH is the cell internal control. The lanes from left to right represent 12 hours, 24 hours, and 36 hours after PR8 influenza virus infection, respectively. Figure 3 B in the figure is the ratio of the gray value of the influenza A (H1N1) virus nucleoprotein (NP) band to the gray value of the cell internal reference (GADPH) band. The smaller the ratio, the lower the virus content.

[0036] Figure 4 Schematic diagram of Western Blots detection results of different concentrations of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate at the cellular level promoting the degradation of autophagy marker protein p62, wherein: Figure 4 A in the figure is the Western Blot results, showing the effect of different concentrations of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on the autophagy marker protein p62 after 24 hours of treatment. The weaker the band, the stronger the promotion of autophagy. GADPH is the cell internal control. The lanes from left to right represent the concentrations of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate: 0, 10, 20, and 40 μM, respectively. Figure 4 Figure B is the ratio of the grayscale values ​​of the autophagy marker protein (p62) and the cellular internal reference (GADPH). The smaller the ratio, the stronger the degree of autophagy.

[0037] Figure 5 Schematic diagram of the promotion of GFP-LC3 spot formation by (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate at a concentration of 40 μM in the present invention, wherein: Figure 5 A in the figure is a related picture taken under a laser confocal microscope; Figure 5B in the figure is the statistical result of the number of GF-LC3 spots formed in each cell. The greater the number, the stronger the degree of promoting autophagy. DETAILED DESCRIPTION

[0038] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0039] At present, antiviral drug evaluation models are mainly divided into in vitro models and in vivo models.

[0040] Among them, in vitro models mainly use various cell lines to evaluate drugs. Their advantages are that they can provide a large number of cells with the same genetic characteristics as research objects, are easy to operate, can eliminate the influence of other external factors, and can detect drug toxicity, effective concentration, etc., providing more basis for later mechanism research.

[0041] The advantage of in vivo models is that they allow for a realistic and systematic evaluation of the effects of candidate drugs in vivo.

[0042] The antiviral drug evaluation model used in the present invention is an in vitro model, specifically the human non-small cell lung cancer cell line A549 is used to evaluate the in vitro anti-influenza A virus effect of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate.

[0043] The experimental materials used in the following examples are as follows:

[0044] (1) Cell lines, experimental animals, and viruses required for the experiment

[0045] Cell line: The human non-small cell lung cancer cell line A549 is maintained by the Henan Provincial Key Laboratory of Immunology and Targeted Drugs, Xinxiang Medical College. The human non-small cell lung cancer cell line A549 is also known as A549 cells.

[0046] Jurkat cells were obtained from ATCC.

[0047] Strain: The influenza A H1N1 virus (A / PR / 8 / 34 strain) model virus was preserved by the Immunology Research Center of Xinxiang Medical College.

[0048] (2) Drugs required for the experiment.

[0049] (Z)-[Amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate was purchased from Shanghai Taoshu Biotechnology Co., Ltd.

[0050] In the actual cell test in the example, (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate was dissolved in DMSO to prepare a (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate solution, which was then diluted to a working concentration with DMEM / F12 culture medium to obtain (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate drug solutions of different concentrations for use in the test.

[0051] Among them, DMEM / F12 medium is a serum-free basic medium purchased from GIBCO.

[0052] (3) Reagents required for the experiment:

[0053] DMEM / F12 culture medium and fetal bovine serum (FBS) were purchased from GIBCO.

[0054] Cell activity detection kit: TransDetect Cell Counting Kit (CCK) was purchased from Beijing Quanshijin Biotechnology.

[0055] Example 1: Toxicity test of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on cells

[0056] A549 cells were used in the present invention and seeded into 96-well plates. When the cell density reached 80%, the cells were incubated for 48 hours with (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate solution at final concentrations of 0 μM, 10 μM, 20 μM, 40 μM, 80 μM, and 160 μM. After incubation, the cell viability was determined using a TransDetect cell counting kit according to the kit instructions, and the OD was measured. 450nm The specific implementation process is as follows:

[0057] 1. Cell culture

[0058] After thawing, A549 cells were passaged twice and then expanded in DMEM / F12 medium containing 10% fetal bovine serum and double antibodies.

[0059] Among them, the dual antibiotics are penicillin 100U / mL and streptomycin 100ug / mL.

[0060] 2. Cytotoxicity test of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate

[0061] Take well-growing A549 cells for digestion and passage, and adjust the cell density to 2×10 6 / mL, and then inoculated into a 96-well plate, with 100 μL in each well; 100 μL of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate drug solution prepared in culture medium was added to each well and mixed.

[0062] The above-mentioned (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate drug solution was set to 6 concentration gradients, with 3 replicates for each gradient concentration, and the final concentrations were 0 μM, 10 μM, 20 μM, 40 μM, 80 μM, and 160 μM, respectively. At the same time, a cell control was set up and placed in a 37°C, 5% CO2 incubator for culture. After 48 hours of culture, the cell activity was determined using the TransDetect cell counting box. 10 μL of CCK-8 reagent was added to the 96-well plate cells after treatment and culture, and the cells were incubated in the dark at 37°C for 2 hours. The OD was detected by a microplate reader. 450nm Read the count and calculate the cell viability.

[0063] The cell growth medium is DMEM / F12 medium + 10% fetal bovine serum + double antibody.

[0064] The culture medium was DMEM / F12 medium + 10% serum + double antibody.

[0065] Cell viability (%) = OD of drug-treated group 450nm / OD of untreated control group 450nm *100%

[0066] Test results:

[0067] The results are as follows Figure 1 As shown in Table 1, the cell viability assay can reflect the cytotoxic effect of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on A549 cells.

[0068] Table 1 Effects of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on cell viability

[0069]

[0070] from Figure 1 As can be seen from Table 1, after A549 cells were treated with (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate at a concentration of 40 μM for 48 h, the cell viability was maintained at 83.7% compared with the control group, indicating that (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate had low cytotoxicity at this concentration.

[0071] Example 2: Inhibitory effect of different concentrations of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on influenza A virus at the cellular level

[0072] The present invention uses Western Blots and viral nucleic acid copy number in culture supernatant to detect the effect of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on the proliferation of influenza A virus (PR8) at the cellular level. The specific steps are as follows:

[0073] 1. Cells were treated with different concentrations of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate and infected with influenza A virus (PR8)

[0074] 1) Digest and passage A549 cells that are in good growth condition, and adjust the cell density to 1×10 5 / mL, 1mL per well was inoculated into a 12-well plate to grow into a monolayer.

[0075] 2) Add different concentrations of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate solution to the cells, with the final concentrations being 0 μM, 10 μM, 20 μM, and 40 μM. The 0 μM working concentration serves as the DMSO control.

[0076] 3) After treatment with various concentrations of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate solution for 24 h, the cells were infected with PR8 influenza virus and incubated at an MOI of 0.05 for 1 h. The virus solution was discarded and the cells were cultured in the aforementioned DMEM / F12 cell growth medium containing various concentrations of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate.

[0077] 4) 24 hours after infection, collect supernatant and cell lysate. Western blots are used to detect influenza virus NP protein in the lysate. Fluorescence quantitative PCR is used to detect viral nucleic acid copies in the supernatant to reflect the drug's inhibition of the virus.

[0078] 2. Western Blots were used to detect the proliferation of influenza A virus PR8 on cells under the action of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate.

[0079] Western Blots were performed on cell lysates infected with influenza virus to detect the effect of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on the proliferation of influenza A virus PR8. The specific steps are as follows:

[0080] The virus-infected cell lysate collected above was added with 5x protein loading, boiled in boiling water for 10 minutes, and centrifuged briefly for use.

[0081] SDS-PAGE electrophoresis: prepare a separating gel with a volume fraction of 10% polyamide gel, spot the sample and run electrophoresis at a constant voltage of 80 V for 30 min for the stacking gel and 120 V for 1.5 h for the separating gel.

[0082] Transfer: When transferring, pre-cool the transfer solution in advance, place the PAGE gel after electrophoresis into the transfer tank, and transfer at a constant voltage of 100V for 1 hour.

[0083] Blocking: After transfer, remove the NC membrane and block it with blocking solution for 1 hour.

[0084] After the primary and secondary antibody incubation is completed, color development is performed using Thermo's ECL color development kit.

[0085] 3. Determination of viral nucleic acid copy number in influenza A virus proliferation fluid by fluorescent quantitative PCR

[0086] The viral load in the culture supernatant after influenza virus infection was determined by measuring the influenza virus NP gene copy number. Specifically, 100 μL of the culture supernatant from virus-infected A549 cells was aspirated and viral nucleic acid was extracted using Magen's Viral Nucleic Acid Extraction Kit (R4171-02). A standard curve for the influenza virus NP plasmid was generated using quantitative PCR, and the influenza virus NP nucleic acid level in the supernatant was calculated based on the Ct value of the quantitative PCR results.

[0087] The steps of fluorescence quantitative PCR detection are reverse transcription first, followed by fluorescence quantitative amplification, as detailed below:

[0088] Extracted RNA was reverse transcribed in 40 µL of the following reaction system: 1.0 µL AMV reverse transcriptase, 8.0 µL 5× AMV buffer, 0.5 µL RNase inhibitor, 2.5 µL 10 pmol Oligo d(T)18, 1 µg RNA template, 2.5 µL 10 mM dNTPs. Add ddH2O to 40 µL, mix gently, and reverse transcribe in a PCR instrument: 42°C for 60 min, 72°C for 15 min, and store at 4°C. The reaction product was used directly or stored at -20°C until needed.

[0089] The fluorescence quantitative PCR system was as follows: 5 μL 2× SYBR Green Master Mix (Rox), 1 μL cDNA, 0.25 μL of 10 mM upstream and downstream primers, and 3.5 μL of DEPC-treated water to a total volume of 10 μL. The reaction conditions were as follows: hot start at 95°C for 15 s; followed by denaturation at 95°C for 5 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, for a total of 40 cycles; melting curve amplification. Each sample was replicated three times. The transcript level of each gene was averaged using the internal reference GAPDH and was determined by 2 −△△Ct Calculated by formula.

[0090] Test results:

[0091] The results are as follows Figure 2 Western Blot results show that (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate significantly inhibited the replication of PR8 influenza virus. After virus infection, the gray value of viral NP protein in the cell fluid treated with 40μM concentration of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate was significantly lower than that of the control group without drug addition, and Figure 2 It can be seen that the inhibitory effect of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on PR8 influenza virus is dose-dependent, and the inhibitory effect on the virus increases with the increase in drug concentration. In addition, the results of viral nucleic acid detection in the culture supernatant of A549 cells treated with different concentrations of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate after infection with influenza virus are shown in Table 2. The results in Table 2 also show that (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate at concentrations of 10μM to 40μM has a significant inhibitory effect on the proliferation of PR8 influenza virus.

[0092] Table 2 Effects of different concentrations of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on the proliferation of PR8 influenza virus

[0093]

[0094] Example 3: Inhibitory effect of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on influenza A virus at different time points

[0095] The present invention uses Western Blots and viral nucleic acid copy number in culture supernatant to detect the effect of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on the proliferation of influenza A virus (PR8) at the cellular level. The specific steps are as follows:

[0096] 1. Cells were treated with (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate and infected with influenza A virus (PR8)

[0097] 1) A549 cells in good growth condition were digested and passaged, and the cell density was adjusted to 1×10 5 / mL, 1mL per well was inoculated into a 12-well plate to grow into a monolayer.

[0098] 2) Add 40 μM (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate solution to set up a drug-free control for the DMSO group.

[0099] 3) After 24 h of treatment with the (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate drug solution, the cells were infected with PR8 influenza virus at an MOI of 0.05 for 1 h. The virus solution was discarded, and the cells were cultured in the aforementioned DMEM / F12 cell growth medium containing 40 μM (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate.

[0100] 4) Supernatant and cell lysate were collected 12 hours, 24 hours, and 36 hours after virus infection. The influenza virus NP protein in the lysate was detected by Western Blots, and the virus titer in the supernatant was detected by fluorescence quantitative PCR.

[0101] The steps of Western Blots detection of influenza virus NP protein in the lysate were the same as those in Example 2.

[0102] The steps of detecting the virus titer in the supernatant by fluorescent quantitative PCR are the same as those in Example 3.

[0103] 2. Western Blots detection of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate proliferation of influenza A virus PR8 on cells at different time points

[0104] Western Blots were performed on cell lysates infected with influenza virus to detect the effect of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on the proliferation of influenza A virus PR8. The specific steps are as follows:

[0105] 1) Add 5x protein loading to the collected virus-infected cell lysate, boil in boiling water for 10 minutes, and centrifuge briefly for later use.

[0106] 2) SDS-PAGE electrophoresis: Prepare 10% polyamide gel for separation gel electrophoresis. Run the sample at a constant voltage of 80 V for 30 min on the stacking gel and 120 V for 1.5 h on the separating gel.

[0107] 3) Transfer: When transferring, pre-cool the transfer solution in advance, place the PAGE gel after electrophoresis into the transfer tank, and transfer at a constant voltage of 100V for 1 hour.

[0108] 4) Blocking: After transfer, remove the NC membrane and block it with blocking solution for 1 hour.

[0109] 5) After the primary and secondary antibody incubation is completed, color development is performed using Thermo's ECL color development kit.

[0110] 3. Fluorescence quantitative PCR was used to determine the nucleic acid copy number of influenza A virus after treatment with (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate for different time periods.

[0111] The supernatant of the A549 cells infected with influenza virus was used to quantitatively detect the viral copy number in the supernatant using fluorescence. The specific steps are as follows:

[0112] 1) 100 μL of supernatant was aspirated and viral RNA was extracted using a viral nucleic acid extraction kit produced by Magen. The steps for detecting the viral nucleic acid copy number by fluorescent quantitative PCR were the same as those in Example 3.

[0113] 2) Convert the test results into copy numbers according to the instructions and calculate the viral inhibition rate.

[0114] Test results:

[0115] The results are as follows Figure 3 Western blot results show that (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate significantly inhibited PR8 influenza virus replication. Following viral infection, the grayscale values ​​of viral NP protein in cell fluids treated with 40 μM (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate were significantly lower than those in the untreated DMSO control at 12, 24, and 36 hours. Furthermore, viral nucleic acid detection results in the supernatant are shown in Table 3.

[0116] Table 3 Effects of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on PR8 influenza virus proliferation at different time points

[0117]

[0118] The results in Table 3 also show that (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate has a significant inhibitory effect on the proliferation of PR8 influenza virus.

[0119] Example 4: (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate promotes cellular autophagy

[0120] 1. Western Blots to detect the effect of the small molecule compound (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on cell autophagy:

[0121] In the present invention, Jurkat cells were seeded in 12-well plates, and then different concentrations of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate drug solutions were added. After incubation for 24 hours, the cell lysate was collected and the autophagy marker p62 (SQSTM1) was detected by Western Blots. The specific steps are as follows:

[0122] 1) Add 5x protein loading to the collected virus-infected cell lysate, boil in boiling water for 10 minutes, and centrifuge briefly for later use.

[0123] 2) SDS-PAGE electrophoresis: Prepare 10% polyamide gel for separation gel electrophoresis. Run the sample at a constant voltage of 80 V for 30 min on the stacking gel and 120 V for 1.5 h on the separating gel.

[0124] 3) Transfer: When transferring, pre-cool the transfer solution in advance, place the PAGE gel after electrophoresis into the transfer tank, and transfer at a constant voltage of 100V for 1 hour.

[0125] 4) Blocking: After transfer, remove the NC membrane and block it with blocking solution for 1 hour.

[0126] 5) After the primary and secondary antibody incubation is completed, color development is performed using Thermo's ECL color development kit.

[0127] Test results:

[0128] The results are as follows Figure 4 Western blot results show that treatment with (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate significantly promoted the degradation of the autophagy marker p62 protein. As the concentration of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate increased, the amount of p62 protein gradually decreased, suggesting that (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate promotes cellular autophagy.

[0129] 2. Laser confocal microscopy observation of the small molecule compound (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on the formation of GFP-LC3 spots:

[0130] In the present invention, A549 cells were seeded in a confocal microplate and transfected with a GFP-LC3 plasmid after cell attachment. 24 hours later, a 40 μM solution of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate or an equal volume of DMSO was added. After 24 hours, the effect of the small molecule compound (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on GFP-LC3 spot formation was observed using laser confocal microscopy. The specific steps are as follows:

[0131] 1) Plate A549 cells onto a confocal microplate dish and culture overnight until the cells adhere. Overnight culture refers to a culture time of ≥12 hours.

[0132] 2) Each well was transfected with 1 μg of GFP-LC3 plasmid. 24 hours after transfection, 40 μM (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate drug solution or an equal volume of DMSO control was added.

[0133] 3) After 24 hours, the culture supernatant was removed and the cells were fixed with paraformaldehyde for 10 minutes, followed by permeabilization with 0.1% Triton X-100 for 15 minutes.

[0134] 4) Wash three times with PBS, add DAPI staining solution, incubate for 10 minutes, then wash one side with PBS, and then observe GFP-LC3 spots using a confocal microscope.

[0135] Test results:

[0136] The results are as follows Figure 5 As shown in the results, treatment with the (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate compound significantly increased GFP-LC3 puncta formation.

[0137] The above experimental results indicate that (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate has a significant inhibitory effect on influenza virus replication and has great potential in preventing or treating influenza virus infection. Furthermore, we also found that the small molecule (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate significantly promotes cellular autophagy. Given the close correlation between autophagy and influenza virus replication, we speculate that the effect of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate on influenza virus replication is closely related to cellular autophagy. Furthermore, changes in cellular autophagic activity are associated with tumors, aging, neurodegenerative diseases, cardiovascular diseases, and autoimmune diseases. The small molecule (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate has similar research value in the treatment of these diseases.

[0138] The present invention provides the use of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate in the preparation of drugs for inhibiting influenza A virus, and provides a new method for treating or preventing influenza A virus infection.

[0139] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0140] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. Use of (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate in the preparation of a product for inhibiting influenza virus, characterized in that: The structural formula of the (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate is shown below: ； The influenza virus is influenza A virus.

2. The use according to claim 1, characterized in that The influenza viruses include A / PR / 8 / 34 (H1N1).

3. Use of the (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate according to claim 1 in the preparation of a medicament for treating or preventing influenza A virus infection.

4. The use according to claim 1 or 3, characterized in that The product or the medicine uses the (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate as an active ingredient and is supplemented with a pharmaceutically acceptable carrier.

5. The use according to claim 4, characterized in that The carrier includes any one or more of glycerol, lecithin and cholesterol.

6. The use according to claim 4, characterized in that The effective concentration of the (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate in the drug is 10 μM to 40 μM.

7. The use according to claim 4, characterized in that The medicine includes oral dosage form, injection dosage form and inhalation dosage form.

8. The use according to claim 7, characterized in that The medicine is in the form of solution, tablet or granule.

9. The use according to claim 8, characterized in that The drug is a solution, and the solvent in the solution is DMSO; The effective concentration of the (Z)-[amino(pyridin-2-yl)methylene]amino-2-nitrobenzoate in the drug is 10 μM to 40 μM.

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