A compound for preparing anti-influenza virus drugs and a preparation method and application thereof

Abstract

This invention discloses a compound for preparing an antiviral drug, its preparation method, and its application, relating to the field of antiviral drug technology. The compound is obtained through a three-step organic synthesis: first, condensation of tert-butyloxycarbonyl-phenylalanine with p-fluorobenzylamine yields tert-butyl(1-((4-fluorobenzyl)amino)-1-oxo-3-phenylprop-2-yl)carbamate; then, deprotection with trifluoroacetic acid yields 2-amino-N-(4-fluorobenzyl)-3-phenylpropionamide; finally, sulfonylation with 5-bromothiophene-2-sulfonylchlorosulfonation gives the final product. This compound specifically targets the influenza virus HA protein, blocking viral invasion of host cells, exhibiting broad-spectrum antiviral activity against influenza A and B viruses, low cytotoxicity, and a mild and controllable preparation process. It can be used to prepare drugs for the prevention and treatment of influenza virus infection, providing a new candidate for the development of new antiviral drugs.

Description

Technical Field

[0001] This invention relates to the field of antiviral drug technology, specifically to a compound for preparing an anti-influenza virus drug, its preparation method, and its application. Background Technology

[0002] Influenza is an acute respiratory infectious disease caused by the influenza virus. It is characterized by rapid spread, wide infection range, and high variability. It not only causes acute clinical symptoms such as fever, cough, and muscle aches, but in severe cases can lead to complications such as pneumonia and respiratory failure, posing a significant threat to the lives and health of infants, the elderly, and those with weakened immune systems. According to monitoring data from the World Health Organization, there are 3-5 million severe cases of influenza globally each year, with approximately 290,000-650,000 deaths. The risk of cross-species transmission of highly pathogenic avian influenza viruses continues to increase, further posing a significant potential threat to public health.

[0003] Influenza virus belongs to the Orthomyxoviridae family and is a single-stranded, negative-sense, segmented RNA virus. Its life cycle includes key stages such as viral adsorption and invasion, endocytosis, membrane fusion and uncoating, vRNA replication and transcription, viral protein synthesis, assembly, and budding release. Developing antiviral drugs targeting different points in the replication cycle is the core approach to influenza prevention and control. Currently, clinically used antiviral drugs mainly focus on three classic targets: neuraminidase (NA), polymerase acid protein (PA), and polymerase basic protein 2 (PB2). Representative drugs include the neuraminidase inhibitor oseltamivir, the PA-targeted inhibitor baloxavir, and the novel PB2-targeted inhibitor onradivir.

[0004] However, existing antiviral drugs still face many unresolved issues in clinical application: First, influenza viruses have a high mutation rate and gene reassortment characteristics, making them prone to developing resistance to existing drugs, leading to a continuous decline in drug efficacy. The emergence of drug-resistant strains further limits the choice of clinical drugs. Second, existing drugs have significant target limitations, all developed around NA, PA, and PB2, lacking new targets and mechanisms targeting key links in the influenza virus replication cycle, making it difficult to cope with the continuous mutation of the virus. Third, some drugs are species or viral subtype specific; for example, onradivir is only effective against influenza A virus and has no inhibitory effect on influenza B virus, thus failing to achieve a broad-spectrum anti-influenza effect.

[0005] Influenza virus hemagglutinin (HA) protein is a key glycoprotein on the viral surface, mediating the binding of the virus to sialic acid receptors on the host cell surface and promoting the fusion of the viral envelope with the host cell membrane. It is a core protein for viral invasion of host cells and an important potential target for anti-influenza drug development. Compared to classic targets such as NA, PA, and PB2, HA protein, as a key initial target for viral invasion, offers the advantage of novel mechanisms of action and less likelihood of cross-resistance when targeting inhibitors to block viral replication at the source of infection. However, the development of anti-influenza drugs targeting HA protein is still in its early stages. There are no marketed drugs, and a lack of clearly defined chiral compounds in preclinical studies. The structural design, preparation methods, and anti-influenza activity studies of related compounds are also lacking.

[0006] In summary, the development of a novel compound that targets the influenza virus HA protein, has a novel mechanism of action, and possesses broad-spectrum anti-influenza activity is of significant clinical value and social importance for addressing the shortcomings of existing anti-influenza drugs, responding to viral mutations and drug resistance, and improving the level of influenza prevention and control. Summary of the Invention

[0007] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies and provide a compound for preparing anti-influenza virus drugs, its preparation method, and its application. This compound exerts broad-spectrum anti-influenza virus activity by specifically targeting the influenza virus hemagglutinin (HA) protein, blocking the initial stage of viral invasion of host cells. This invention aims to provide safer and more efficient novel anti-influenza drug candidates and to clarify the compound's mechanism of action and drug development potential.

[0008] The technical solution of this invention is as follows:

[0009] In a first aspect, the present invention provides a method for preparing a compound for preparing an anti-influenza virus drug, comprising the following steps:

[0010] S1: Dissolve tert-butyloxycarbonyl-phenylalanine in an organic solvent, add 1-ethyl-(3-dimethylaminopropyl)carbamate hydrochloride, 1-hydroxybenzotriazole, N,N-diisopropylethylamine, and p-fluorobenzylamine, and stir at room temperature; after the reaction is complete, add water under stirring, stop stirring after the solid precipitates, filter to obtain the residue, and dry to obtain tert-butyl(1-((4-fluorobenzyl)amino)-1-oxo-3-phenylprop-2-yl)carbamate;

[0011] S2: Dissolve tert-butyl(1-((4-fluorobenzyl)amino)-1-oxo-3-phenylprop-2-yl)carbamate in an organic solvent, add trifluoroacetic acid dropwise under ice bath, and after 0.5-2 h, restore to room temperature and stir for 1.5-2 h; after the reaction is complete, add saturated sodium bicarbonate solution, stop stirring after solid precipitation, filter to obtain residue, and dry to obtain 2-amino-N-(4-fluorobenzyl)-3-phenylpropanamide;

[0012] S3: Dissolve 2-amino-N-(4-fluorobenzyl)-3-phenylpropionamide in an organic solvent, add 5-bromothiophene-2-sulfonyl chloride and triethylamine under ice bath, and react at room temperature for 2-4 h; after the reaction is complete, rotary evaporate; extract with water and ethyl acetate; wash the organic phase with saturated sodium chloride solution and dry, then purify by column chromatography to obtain the final product 2-((5-bromothiophene)-2-sulfonamido)-N-(4-fluorobenzyl)-3-phenylpropionamide.

[0013] Preferably, in step S1, the organic solvent is DMF; in steps S2 and S3, the organic solvent is anhydrous dichloromethane.

[0014] Preferably, in step S1, the molar ratio of tert-butoxycarbonyl-phenylalanine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, N,N-diisopropylethylamine and p-fluorobenzylamine is 1:(1-1.3):(1.5-2):(2-3):(1.1-1.3).

[0015] Preferably, in step S2, the molar volume ratio of tert-butyl (1-((4-fluorobenzyl)amino)-1-oxo-3-phenylprop-2-yl)carbamate to trifluoroacetic acid is 1 mmol: (1-1.1) mL.

[0016] Preferably, in step S3, the molar ratio of 5-bromothiophene-2-sulfonyl chloride, 2-amino-N-(4-fluorobenzyl)-3-phenylpropionamide and triethylamine is 1:1.1:(1.1-1.2).

[0017] Preferably, in step S3, during purification, the dried organic phase is concentrated and purified by column chromatography, with the volume ratio of ethyl acetate to petroleum ether being 1:4.

[0018] Secondly, the present invention provides a compound for preparing an anti-influenza virus drug, which is prepared by the above-described method for preparing an anti-influenza virus drug.

[0019] Preferably, the chemical structural formula is as follows: .

[0020] Thirdly, the present invention also provides an application of the above-mentioned compound for preparing an anti-influenza virus drug, wherein the compound or a pharmaceutically acceptable salt or hydrate thereof is used to prepare a drug for the prevention and treatment of influenza virus infection.

[0021] The anti-influenza virus compound of the present invention has many advantages over the prior art, such as novel target, unique mechanism of action, broad-spectrum anti-influenza activity, and strong controllability of preparation process. The specific beneficial effects are as follows:

[0022] 1. The compound of this invention specifically targets the influenza virus hemagglutinin (HA) protein, a core target for influenza virus invasion of host cells. Currently, no marketed drugs have been developed targeting this protein, filling the gap in the development of small molecule inhibitors of the HA protein. The compound inhibits the HA-mediated fusion process between the viral envelope and the host cell membrane, blocking the release of the viral ribonucleoprotein (vRNP) complex into the host cell cytoplasm, thus interrupting the infection chain at the initial stage of the influenza virus replication cycle. While existing mainstream clinical drugs such as oseltamivir act on the release phase of viral replication, the compound of this invention acts at a more advanced stage, inhibiting viral infection earlier and resulting in superior prevention and control effects.

[0023] 2. The compounds of this invention exhibit significant inhibitory activity against different subtypes of influenza A virus (H1N1, H3N2, A(H1N1)pdm09) and influenza B virus (B / Yamagata), overcoming the subtype limitations of some existing drugs such as onradivir, which are only effective against influenza A and have no effect on influenza B. They can address infections of different influenza virus strains and have higher application value in the prevention and control of seasonal influenza and influenza pandemics.

[0024] 3. Existing anti-influenza drugs are all focused on three classic targets: neuraminidase (NA), polymerase acid protein (PA), and polymerase basic protein 2 (PB2). The high mutation rate of influenza virus easily leads to drug resistance to these targets. However, the compound of this invention acts on a novel HA protein target, has no cross-resistance sites with existing drugs, and can effectively deal with the infection of existing drug-resistant strains, providing a new solution to the clinical problem of influenza drug resistance.

[0025] 4. The compounds prepared in this invention exhibit extremely low cytotoxicity against canine kidney epithelial (MDCK) cells, CC 50 The values ​​were all greater than 100 μM, far exceeding the levels of EC, which exerts its anti-influenza activity. 50 The compound exhibits a high therapeutic index (SI) and does not cause significant damage to normal host cells within the effective concentration range for viral inhibition. Compared to existing drugs such as oseltamivir, which are prone to causing gastrointestinal adverse reactions, the compound of this invention has a greater advantage in terms of in vivo safety.

[0026] 5. The compound of this invention exhibits a significant dose-dependent inhibitory effect on the expression of influenza virus proteins. As the concentration of the compound increases, the expression levels of GFP fluorescence, PA and NP core proteins of the virus decrease significantly. Furthermore, the stability and specificity of its inhibitory effect are verified in TOA experiments, pseudovirus experiments, and hemolysis experiments. The pharmacodynamic mechanism is clear, providing a clear experimental basis for subsequent dosage optimization in clinical use.

[0027] 6. The compounds prepared in this invention are novel chiral compounds reported for the first time. Both their D-form and L-form conformations exhibit anti-influenza activity, with the L-form EC being the most potent. 50 The value was only 2-3 times different from the positive control oseltamivir, showing outstanding drug potential; moreover, the compound does not interfere with the binding of HA protein to the host cell sialic acid receptor, but exerts its fusion inhibition effect by inhibiting the conformational changes of HA protein induced by low pH. The mechanism of action is highly specific, providing an important lead compound for the structural optimization and derivative development of subsequent anti-influenza drugs.

[0028] 7. The compounds of this invention can be used to prepare various pharmaceutical preparations (such as oral preparations, injections, sprays, etc.) for the prevention and treatment of influenza virus infection. They can be used for treatment after influenza infection and as preventive drugs during the peak influenza season. They are suitable for influenza-susceptible groups such as infants, the elderly, and people with weakened immune systems. They also have potential applications in the prevention and control of cross-species transmission of highly pathogenic avian influenza viruses. They have significant clinical translation and industrialization value.

[0029] In summary, the anti-influenza virus compound prepared by this invention has the characteristics of novel target, broad spectrum and high efficiency, high safety and great drug potential. Its preparation method is simple and highly controllable. The development of this compound provides a new lead compound and technical solution for the field of anti-influenza drugs, effectively overcoming the problems of target limitation, drug resistance and narrow subtype applicability of existing anti-influenza drugs, and has important clinical application value and social and economic benefits. Attached Figure Description

[0030] Figure 1 This is the carbon spectrum of the product prepared in Example 1 of the present invention.

[0031] Figure 2 This is the hydrogen spectrum of the product prepared in Example 1 of this invention.

[0032] Figure 3 This refers to the product prepared in Example 1 of the present invention, the D-type and L-type conformational products, and the EC50 of oseltamivir in MDCK cells infected with influenza virus PR8-Fluc. 50 Value and CC 50 Value; among which, Figure 3 ac corresponds to oseltamivir, the product of Example 1, and the D- and L-conformation products, respectively.

[0033] Figure 4 This is a graph showing the efficacy results of the product of Example 1 confirmed by the PR8-GFP virus system and Western Blot method in this invention.

[0034] Figure 5 These are the Western Blot results of the product of Example 1 of this invention. In Example 1, three concentration experimental groups were set up, corresponding to product concentrations of 30 μM, 10 μM, and 3 μM, respectively. The positive control group, baloxavir, had a concentration of 50 nM. Figure 5 5a shows the target band results from the Western Blot, and 5b shows the quantitative analysis results of PA and NP proteins.

[0035] Figure 6 This is a schematic diagram of the design of the 8 experimental groups in the TOA experiment of this invention.

[0036] Figure 7 This is a graph showing the TOA experimental results of MDCK cells infected with PR8 virus of the present invention after the addition of oseltamivir (a) and compound (b) of Example 1.

[0037] Figure 8 This is a graph showing the TOA experimental results of MDCK cells infected with H3N2 virus in this invention after the addition of oseltamivir (a) and compound (b) from Example 1.

[0038] Figure 9 This refers to the EC derived from the compound in Example 1 of this invention, fitted onto H5N1(a) and H7N3(c) in the pseudovirus experiment. 50 Values, and EC values ​​fitted by the ING-1466 invasion inhibitor on H5N1(b) and H7N3(d). 50 value.

[0039] Figure 10 This is a diagram showing the results of the blood coagulation inhibition experiment in this invention.

[0040] Figure 11 This is a diagram showing the results of the hemolysis experiment of this invention. Detailed Implementation

[0041] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention.

[0042] Example 1

[0043] The method for preparing the compound used to prepare an anti-influenza virus drug in this embodiment includes the following steps:

[0044] S1: 3.77 mmol of tert-butyloxycarbonyl-phenylalanine was dissolved in anhydrous DMF, and 4.90 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 5.66 mmol of 1-hydroxybenzotriazole, 7.54 mmol of N,N-diisopropylethylamine, and 4.515 mmol of p-fluorobenzylamine were added. The mixture was stirred at room temperature for 2 h. After the reaction was completed, water was added under stirring, and stirring was stopped after the solid precipitated. The residue was filtered to obtain the filter residue. After drying the filter residue, a white powder of tert-butyl(1-((4-fluorobenzyl)amino)-1-oxo-3-phenylprop-2-yl)carbamate was obtained (yield 66%).

[0045] S2: 5 mmol of tert-butyl(1-((4-fluorobenzyl)amino)-1-oxo-3-phenylprop-2-yl)carbamate was dissolved in 5 mL of anhydrous DCM. 5 mL of trifluoroacetic acid was added dropwise under ice bath conditions. After 0.5 h, the mixture was allowed to return to room temperature, and the mixture was stirred for 1.5 h to allow the reaction to proceed. After the reaction was complete, saturated sodium bicarbonate solution was added, and stirring was stopped after a solid precipitated. The residue was then filtered. After drying the residue, a white powder of 2-amino-N-(4-fluorobenzyl)-3-phenylpropanamide (yield 81%) was obtained.

[0046] S3: 1.06 mmol of 2-amino-N-(4-fluorobenzyl)-3-phenylpropanamide was dissolved in anhydrous DCM. 0.96 mmol of 5-bromothiophene-2-sulfonyl chloride and 1.06 mmol of triethylamine were added under ice bath conditions, and the reaction was carried out at room temperature for 2 h. After the reaction was complete, most of the DCM was rotary evaporated. The mixture was extracted with water and ethyl acetate, washed with saturated sodium chloride solution, and dried with anhydrous magnesium sulfate for 30 min. The dried organic phase was concentrated and purified by column chromatography (ethyl acetate to petroleum ether ratio 1:4) to give a white powdery final product, 2-((5-bromothiophene)-2-sulfonamido)-N-(4-fluorobenzyl)-3-phenylpropanamide (yield 72%).

[0047] The carbon and proton spectra of the compounds prepared in this embodiment are as follows: Figure 1-2 As shown:

[0048]

[0049] The effects of the compound prepared in Example 1 were tested:

[0050] Canine kidney epithelial cells (MDCK cells) were grown in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibodies. The influenza virus used was amplified by passage in SPF chicken embryos and stored at -80°C.

[0051] Anti-influenza virus activity assay: MDCK cells were administered at a concentration of 1×10⁻⁶ 4Cells were seeded in white 96-well plates and cultured to monolayer size in a 37°C, 5% CO2 incubator. The constructed PR8-Fluc influenza virus was used to infect cells, with 20 μM, 10 μM, and 2 μM concentrations of the compound added, each concentration repeated three times. 10 μM oseltamivir served as a positive control, an equal concentration of DMSO as a negative control, and wells with an equal concentration of DMSO but not infected with the virus served as a blank control. 24 h after infection, 50 μL of firefly luciferase substrate was added, and after incubation at room temperature for 2 min, the chemiluminescence signal was detected using the Bright-Lite Luciferase Assay System kit.

[0052] Anti-influenza virus toxicity test: MDCK cells at 1×10 4 The cells were seeded in clear 96-well plates and cultured to a monolayer in a 37°C, 5% CO2 incubator. Compound concentrations of 20 μM, 10 μM, and 2 μM were added to each well, with each concentration repeated three times. After 24 h, 100 μL of 10% CellCounting Kit-8 was added to each well, and after incubation for 2 h, the absorbance at 450 nm was measured.

[0053] Dose-response assay: The active compound was started at 100 μM and diluted 3-fold to treat cells. MDCK cells were simultaneously infected with PR8-Fluc virus. Each concentration was repeated three times. Results were analyzed after 24 h of incubation. Cytotoxicity at the corresponding compound concentrations was also assessed. GraghPad was used to fit EC50 data. 50 and CC 50 Calculate the SI value.

[0054] The EC values ​​of the compounds prepared in Example 1 were fitted according to the above experimental scheme. 50 The value is 4.34 μM (e.g.) Figure 3 (As shown in b), this compound is a chiral isomer. The D and L conformations were resynthesized, with the EC of the L conformation compound being... 50 EC50 values ​​compared to the positive control oseltamivir 50 The values ​​differ by 2-3 times (e.g.) Figure 3 (as shown in a and 3c), preliminary evidence confirms that this compound has anti-influenza virus activity.

[0055] Subsequently, the efficacy of the compound from Example 1 was confirmed using the PR8-GFP virus system and Western blotting.

[0056] PR8-GFP virus system: MDCK cells were prepared at a ratio of 5 × 10⁻⁶ 3Cells were seeded in black-bottomed 96-well plates and cultured to monolayer in a 37°C, 5% CO2 incubator. Three compound concentrations were set: 30 μM, 10 μM, and 3 μM. The positive control, baloxavir, was at 50 nM. Cells were infected with PR8-GFP virus and simultaneously treated with the corresponding concentration of compound or the positive control, baloxavir. After 36 hours, the cells were stained with the blue fluorescent dye Hoechst and then observed and photographed under a microscope.

[0057] Western Blot: MDCK cells were divided into 5×10⁻⁶ cells. 5 Cells were seeded in clear 6-well plates and cultured to monolayer size in a 37°C, 5% CO2 incubator. Three concentrations of the compound were prepared: 30 μM, 10 μM, and 3 μM. The positive control, baloxavir, was prepared at 50 nM. Cells infected with A / PuertoRico / 8 / 34 (H1N1) virus were simultaneously treated with the corresponding concentrations of the compound or the positive control, baloxavir. Protein extraction was performed 24 h later. Protein concentration was calculated after extraction using a BCA kit. Protein and marker were added to the gel after gel preparation. The gel was run at 80 V for 30 min, then at 120 V for 1 h before transfer at 300 mA for 1 h. After blocking with rapid blocking buffer, the target protein antibody was incubated. Finally, imaging was performed using an ECL chemiluminescence assay kit under a imaging system.

[0058] The results are as follows Figure 4 As shown in the figure, GFP is green fluorescent protein; GFP expression produces a green fluorescent signal, indicating the area of ​​viral infection. Hoechst is a blue fluorescent dye used to label the cell nucleus. Merge is a merged image, which provides a more intuitive view of the drug's effect on viral infection. The results showed that in the untreated control group, numerous green fluorescent spots were observed, indicating widespread viral replication within the cells. In the drug-treated group, the number of green fluorescent spots gradually decreased with increasing drug concentration. This confirms that the compound in Example 1 has a dose-dependent inhibitory effect on viral protein expression.

[0059] like Figure 5 As shown, compared with the control group, the protein expression levels of PA and NP decreased significantly with increasing drug concentration. Quantitative analysis of the Western blot results revealed statistically significant differences in PA and NP protein expression at concentrations of 30 μM and 10 μM. This further demonstrates that the compound in Example 1 has a dose-dependent inhibitory effect on viral protein expression.

[0060] Next, in order to study the broad-spectrum anti-influenza virus activity of the compound in Example 1, three influenza A virus strains, A / PuertoRico / 8 / 34(H1N1), A / X31(H3N2)-Fluc, and A(H1N1)pdm09, and influenza B virus strain B / Yamagata were selected to test the efficacy of the compound on different influenza virus strains. Oseltamivir was used as a positive control. The compound and oseltamivir were serially diluted at a concentration of 100 μM.

[0061] Efficacy study of compounds against different influenza virus strains: MDCK cells were treated at 5×10⁻⁶ cells. 3 Cells were seeded in clear 96-well plates and cultured to a monolayer in a 37°C, 5% CO2 incubator. Cells were infected with influenza A virus strains A / PuertoRico / 8 / 34(H1N1), A / X31(H3N2)-Fluc, A(H1N1)pdm09, and influenza B virus strain B / Yamagata, respectively. The compounds and the positive control oseltamivir were serially diluted at 100 μM. At 36-48 h, 100 μL of 10% CellCounting Kit-8 was added to each well, and after incubation for 2 h, the absorbance at 450 nm was measured. EC values ​​were fitted based on the absorbance values. 50 value.

[0062] The results are shown in Table 1:

[0063] Table 1. Pharmacological effects of the compounds against different influenza virus strains.

[0064]

[0065] As shown in Table 1, the pharmacodynamics of the compound from Example 1 on strain A / PuertoRico / 8 / 34(H1N1) was fitted to EC50. 50 The value was 11.68 μM, while that of oseltamivir was 0.87 μM; the pharmacodynamic fit of the compound on A / X31(H3N2)-Fluc yielded EC50 values. 50 The value was 2.93 μM, while that of oseltamivir was 0.04 μM; the pharmacodynamic fit of the compound on strain A(H1N1)pdm09 was EC. 50 The value was 12.93 μM, while that of oseltamivir was 7.69 μM; the pharmacodynamic fit of the compound on the B / Yamagata strain yielded EC50. 50 The concentration was 13.89 μM, while that of oseltamivir was 1.57 μM. These results indicate that the compound exhibits efficacy against different influenza virus strains, confirming that the compound of Example 1 possesses broad-spectrum anti-influenza virus activity.

[0066] The following TOA (Time of Addition) experiment was conducted to clarify the specific role of the compound in the viral infection cycle:

[0067] TOA Experiment: This experiment is designed with 8 groups, such as Figure 6 As shown, black represents viral infection and red represents the addition of compounds. First, the virus was added at 4°C for 1 hour to adsorb. At 0 hours, the unbound virus was washed away with PBS. Then, the compound from Example 1 was added at the corresponding time point and the cells were transferred to a 37°C incubator for invasion culture. Finally, cell samples were collected, and titers were determined after repeated freeze-thaw cycles to determine the stage of compound action.

[0068] MDCK cells at 10 5Cells were seeded in clear 24-well plates and cultured to a monolayer in a 37°C, 5% CO2 incubator. After 1 hour, the culture medium was discarded, the cells were washed once with PBS, and then infected with A / PuertoRico / 8 / 34 (H1N1) virus. Adsorption was carried out at 4°C for 1 hour. After 0 hours, the medium in all wells was discarded, the cells were washed three times with PBS, and the compound was added at a final concentration of 100 μM and the positive control drug oseltamivir at a final concentration of 20 μM at the corresponding time points. The cells were then transferred to a 37°C incubator and cultured for 9 hours. Cell samples were collected, and viral titers were tested after two freeze-thaw cycles.(1) -1~8h, adsorption at 4℃, add 250μL of virus and 250μL of compound, wash three times with PBS at 0h, add 250μL of compound and 250μL of virus isolation serum-free culture medium containing 1mg / mL TPCK-trypsin, transfer to 37℃ incubator for incubation; (2) -1~2h, adsorption at 4℃, add 250μL of virus and 250μL of compound, wash three times with PBS at 0h, add 250μL of virus isolation serum-free culture medium containing 1mg / mL TPCK-trypsin, transfer to 37℃ incubator for incubation, add 250μL of compound at -1~2h; (3) -1~0h, adsorption at 4℃, add Add 250 μL of virus and 250 μL of compound. After washing three times with PBS at 0 h, add 500 μL of serum-free virus isolation culture medium containing 1 mg / mL TPCK-trypsin and transfer to a 37°C incubator for incubation; (4) At 0~2 h, at 4°C, add 250 μL of virus and 250 μL of serum-free virus isolation culture medium containing 1 mg / mL TPCK-trypsin. After washing three times with PBS at 0 h, add 500 μL of serum-free virus isolation culture medium containing 1 mg / mL TPCK-trypsin and transfer to a 37°C incubator for incubation. Discard the 250 μL culture medium at 0~2 h and add 250 μL of compound; (5) (6) 2-4h, adsorb at 4℃, add 250μL of virus and 250μL of virus isolation serum-free culture medium containing 1mg / mL TPCK-trypsin, wash three times with PBS at 0h, add 500μL of virus isolation serum-free culture medium containing 1mg / mL TPCK-trypsin, transfer to a 37℃ incubator for incubation, discard 250μL of culture medium at 2-4h, add 250μL of compound; (7) 4-6h, adsorb at 4℃, add 250μL of virus and 250μL of virus isolation serum-free culture medium containing 1mg / mL TPCK-trypsin, wash three times with PBS at 0h, add 500μL of virus isolation serum-free culture medium containing 1mg / mL TPCK-trypsin. The virus isolation serum-free culture medium containing PCK-trypsin was transferred to a 37°C incubator for incubation. After 4-6 hours, 250 μL of culture medium was discarded and 250 μL of compound was added. (7) After 6-8 hours, 250 μL of virus and 250 μL of virus isolation serum-free culture medium containing 1 mg / mL TPCK-trypsin were added at 4°C. After washing three times with PBS at 0 hours, 500 μL of virus isolation serum-free culture medium containing 1 mg / mL TPCK-trypsin was added and transferred to a 37°C incubator for incubation. After 6-8 hours, 250 μL of culture medium was discarded and 250 μL of compound was added. At 9 hours, the cell samples were collected, centrifuged, and the virus titer was determined.

[0069] First, using oseltamivir as a positive control, the virus A / PuertoRico / 8 / 34 (H1N1) was introduced under an MOI of 0.1. Oseltamivir was added at -1–0 h, -1–2 h, 0–2 h, 2–4 h, 4–6 h, 6–8 h, and -1–8 h at a concentration of 20 μM, with each condition repeated three times. Compared with the negative control group, [the following data was analyzed]. Figure 7 As shown in Figure a, oseltamivir was significantly effective between 6 and 8 hours, and also showed some effect between -1 and 8 hours. Adding oseltamivir at other times did not produce significant effects, confirming that oseltamivir acted during the viral release phase in this experiment. This result is consistent with the action phase of oseltamivir, confirming that the experimental protocol was correct.

[0070] Then, under the same conditions, a 100 μM concentration of the compound from Example 1 was added, and compared with the negative control group, as follows: Figure 7 As shown in b, the compound showed significant effects at -1 to 2 hours, 0 to 2 hours, and -1 to 8 hours, but no significant effects were observed when the compound was added at other times. This confirms that the compound acts on the viral invasion stage, where the main target protein is HA.

[0071] In previous studies of broad-spectrum activity, H3N2 activity was superior to PR8; therefore, this invention also tested the TOA (Total Activity Amount) experiment of H3N2. H3N2 virus was infected with MOI=1, and other conditions and protocols were the same as before. Again, the feasibility of the experimental design was verified first using the action phase of the positive control drug oseltamivir (e.g., ...). Figure 8 (as shown in a). Then, in the TOA experiment with the added compound, compared with the negative control group, as shown in a). Figure 8 As shown in b, the compound showed significant effects at -1~2h, 0~2h, and -1~8h, but no significant effects were observed when the compound was added at other times. Both experimental results confirmed that the compound in Example 1 acted on the invasion stage of the virus.

[0072] To further verify the effect of the compound on the invasion process, this invention further tested the inhibitory effect of the compound on H5N1 and H7N3 pseudoviruses:

[0073] Pseudovirus experiment: Influenza virus HA and NA, along with an HIV vector containing firefly luciferase and exhibiting replication defects, were co-transfected into 293T cells. After 24 hours, the cells were centrifuged at 3000 rpm for 10 minutes and the samples were collected. This virus can only replicate in a single round. MDCK cells were then divided into 10... 5 / wells were inoculated into white 96-well plates. The pseudovirus and 100 μM serially diluted compound or the positive control ING-1466 invasion inhibitor were added to the white 96-well plates. After 48 hours, the chemiluminescence signal value was detected using the Bright-Lite Luciferase Assay System kit. The chemiluminescence signal was then calculated based on the fitted EC... 50 The value further proves the stage of action of the compound.

[0074] like Figure 9 As shown in Figure a, the EC fitted to H5N1 by the compound 50 The value is 23.83 μM, such as Figure 9 As shown in c, the EC fitted on H7N3 50 The value was 3.33 μM, and all the above data can prove that the compound acts in the invasion stage. Using the known small molecule invasion inhibitor ING-1466 as a positive control, its EC value fitted on H5N1 and H7N3 was [missing value]. 50 Values ​​such as Figure 9 As shown in b and 9d.

[0075] To further investigate the mechanism of action of the compounds, this invention conducted hemagglutination inhibition and hemolysis experiments:

[0076] Hemagglutination inhibition assay: Hemagglutinin binds to sialic acid receptors on the surface of chicken erythrocytes, causing cell aggregation. If the compound blocks hemagglutinin receptor binding, the hemagglutination reaction is inhibited. The compound was diluted 2-fold from 100 μM in a 96-well plate, and after adding a 4-fold diluted virus, it was incubated at room temperature for 1 hour. After 1 hour, 50 μL of 1% chicken erythrocyte suspension was added to each well, and the plate was incubated at room temperature for 1 hour to observe the erythrocyte aggregation state.

[0077] The results are as follows Figure 10 As shown, the compound in Example 1 had no inhibitory effect on the blood coagulation reaction, indicating that it did not interfere with HA-receptor interaction.

[0078] Hemolysis assay: When hemagglutinin binds to sialic acid receptors on chicken erythrocytes, a conformational change occurs under low pH treatment, forming pores on the cell surface and releasing hemoglobin, resulting in hemolysis. If a compound can prevent HA-mediated fusion, hemolysis is inhibited.

[0079] In the hemolysis experiment, equal volumes of the compound, starting from 200 μM and continuously diluted 2-fold, were mixed with 40 μL of PBS or virus in 2 ml EP tubes and incubated at room temperature for 30 min. Then, 80 μL of 2% chicken red blood cells at 37°C were added and incubated at 37°C for 30 min. Sodium acetate (0.5 M, pH 5.0) was added and incubated at 37°C for 30 min. After incubation at 1200 rpm for 6 min, 200 μL of the supernatant was transferred to a 96-well plate and OD540 was measured.

[0080] The results are as follows Figure 11 As shown, the compound in Example 1 dose-dependently inhibited hemolysis by inhibiting low pH-induced HA conformational changes, thereby blocking the virus-cell membrane fusion process.

Claims

1. A method for preparing a compound for preparing an anti-influenza virus drug, characterized in that, Includes the following steps: S1: Dissolve tert-butyloxycarbonyl-phenylalanine in an organic solvent, add 1-ethyl-(3-dimethylaminopropyl)carbamate hydrochloride, 1-hydroxybenzotriazole, N,N-diisopropylethylamine, and p-fluorobenzylamine, and stir at room temperature; after the reaction is complete, add water under stirring, stop stirring after the solid precipitates, filter to obtain the residue, and dry to obtain tert-butyl(1-((4-fluorobenzyl)amino)-1-oxo-3-phenylprop-2-yl)carbamate; S2: Dissolve tert-butyl(1-((4-fluorobenzyl)amino)-1-oxo-3-phenylprop-2-yl)carbamate in an organic solvent, add trifluoroacetic acid dropwise under ice bath, and after 0.5-2 h, restore to room temperature and stir for 1.5-2 h; after the reaction is complete, add saturated sodium bicarbonate solution, stop stirring after solid precipitation, filter to obtain residue, and dry to obtain 2-amino-N-(4-fluorobenzyl)-3-phenylpropanamide; S3: Dissolve 2-amino-N-(4-fluorobenzyl)-3-phenylpropionamide in an organic solvent, add 5-bromothiophene-2-sulfonyl chloride and triethylamine under ice bath, and react at room temperature for 2-4 h; after the reaction is complete, rotary evaporate; extract with water and ethyl acetate; wash the organic phase with saturated sodium chloride solution and dry, then purify by column chromatography to obtain the final product.

2. The method for preparing the compound for preparing an anti-influenza virus drug as described in claim 1, characterized in that, In step S1, the organic solvent is DMF; in steps S2 and S3, the organic solvent is anhydrous dichloromethane.

3. The method for preparing the compound for preparing an anti-influenza virus drug as described in claim 1, characterized in that, In step S1, the molar ratio of tert-butoxycarbonyl-phenylalanine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, N,N-diisopropylethylamine and p-fluorobenzylamine is 1:(1-1.3):(1.5-2):(2-3):(1.1-1.3).

4. The method for preparing the compound for preparing an anti-influenza virus drug as described in claim 1, characterized in that, In step S2, the molar volume ratio of tert-butyl (1-((4-fluorobenzyl)amino)-1-oxo-3-phenylprop-2-yl)carbamate to trifluoroacetic acid is 1 mmol: (1-1.1) mL.

5. The method for preparing the compound for preparing an anti-influenza virus drug as described in claim 1, characterized in that, In step S3, the molar ratio of 5-bromothiophene-2-sulfonyl chloride, 2-amino-N-(4-fluorobenzyl)-3-phenylpropionamide and triethylamine is 1:1.1:(1.1-1.2).

6. The method for preparing the compound for preparing an anti-influenza virus drug as described in claim 1, characterized in that, In step S3, during purification, the dried organic phase is concentrated and purified by column chromatography, with the volume ratio of ethyl acetate to petroleum ether being 1:

4.

7. A compound for preparing an anti-influenza virus drug, characterized in that, It is prepared by the method for preparing an anti-influenza virus drug as described in any one of claims 1-6.

8. The compound for preparing an anti-influenza virus drug as described in claim 7, characterized in that, The chemical structural formula is .

9. The use of the compound for preparing an anti-influenza virus drug as described in claim 7 or 8, characterized in that, The compound or its pharmaceutically acceptable salt or hydrate is used to prepare a drug for the prevention and treatment of influenza virus infection.

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