Use of a compound in the preparation of a medicament for the treatment of influenza virus

Abstract

This invention discloses the application of a compound in the preparation of anti-influenza virus drugs, namely, the application of the compound of formula I, its stereoisomer, its solvate or its pharmaceutically acceptable salt in the preparation of anti-influenza virus drugs: the compound of formula I of this invention has the effect of inhibiting the activity of influenza virus polymerase and can inhibit the proliferation of influenza virus, thereby achieving the effect of anti-influenza.

Description

Technical Field

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

[0002] Influenza viruses are a class of viruses that use humans, pigs, or birds as their natural hosts. They are highly contagious and have a high pathogenicity. Influenza viruses can cause severe respiratory infections, and in severe cases, can lead to acute lung injury or even death. The World Health Organization estimates that seasonal influenza epidemics infect approximately 10-20% of the global population each year, with 3-5 million people developing severe illness and 300,000-500,000 deaths, posing a significant threat to human life and health.

[0003] Influenza virus polymerase is a heterotrimeric complex composed of three subunits: PA, PB1, and PB2. It is an RNA-dependent RNA polymerase responsible for the transcription and replication of the influenza virus genome. The PB1 subunit possesses the classic RdRp domain, forming the catalytic center of the virus. The PA subunit exhibits endonuclease activity, and the PB2 subunit has a cap-binding domain that specifically binds to host mRNA precursors with methylated caps. The PA subunit cleaves the host mRNA to form a 10-15 nt capped RNA segment, which serves as a primer to initiate the transcription of the influenza virus genome. Influenza virus polymerase is highly conserved across different influenza virus subtypes and is often considered an ideal target for anti-influenza drugs. Therefore, anti-influenza drugs targeting influenza virus polymerase can be developed to inhibit influenza virus proliferation by suppressing the transcriptional and replication functions of the polymerase, thereby achieving an anti-influenza effect.

[0004] Currently, the main antiviral drugs on the market include transmembrane protein (M2) ion channel inhibitors, neuraminidase (NA) inhibitors, and polymerase inhibitors. The antigens on the surface of the influenza virus are prone to antigenic shift and antigenic drift, leading to viral mutations and drug resistance, rendering most antiviral drugs gradually ineffective. Currently, novel polymerase inhibitors can effectively inhibit influenza viruses resistant to adamantane-based (M2 ion channel inhibitors) and NAIs (NA enzyme inhibitors). However, problems such as viral resistance and teratogenic reactions have emerged during clinical trials, indicating that the development of novel influenza virus polymerase inhibitors still faces significant challenges. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide the use of a compound in the preparation of an anti-influenza virus drug.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A first aspect of the invention provides the use of compounds of formula I, their stereoisomers, their solvates, or pharmaceutically acceptable salts thereof in the preparation of antiviral drugs for influenza:

[0008]

[0009] The chemical name of the compound of formula I is 2-[4-hydroxy-5-(2-oxoindol-3-yl)-2-sulfanylidene-1,3-thiazol-3-yl]pentanoic acid, i.e., 2-[4-hydroxy-5-(2-oxoindol-3-yl)-2-thioalkyl-1,3-thiazol-3-yl]pentanoic acid; the compound code is STL035778.

[0010] A second aspect of the invention provides the use of a compound of formula I, its stereoisomers, its solvates, or pharmaceutically acceptable salts thereof in the preparation of a drug for inhibiting influenza virus polymerase.

[0011] A third aspect of the invention provides the use of a compound of formula I, its stereoisomers, its solvates, or pharmaceutically acceptable salts thereof in the preparation of a drug for inhibiting influenza A virus.

[0012] In some embodiments of the present invention, the influenza virus includes at least one of influenza A virus, influenza B virus, influenza C virus, or influenza D virus.

[0013] In some embodiments of the present invention, the influenza virus includes at least one of the following influenza A virus subtypes: H1N1, H2N2, H3N2, H5N1, H7N9, H7N7, H1N2, H9N2, H7N2, H7N3, H5N2, H10N7, H10N3, or H5N8.

[0014] In some preferred embodiments of the present invention, the pharmaceutically acceptable salt includes, but is not limited to, at least one of inorganic acid salts, organic acid salts, alkyl sulfonates, or aryl sulfonates; preferably, the inorganic acid salt includes, but is not limited to, at least one of hydrochloride, hydrobromide, nitrate, sulfate, or phosphate; preferably, the organic acid salt includes, but is not limited to, at least one of formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, or citrate; preferably, the alkyl sulfonate includes, but is not limited to, at least one of methyl sulfonate or ethyl sulfonate; and the aryl sulfonate includes, but is not limited to, at least one of benzene sulfonate or p-toluene sulfonate.

[0015] The term "stereoisomer" includes enantiomers and diastereomers, as well as cis-trans isomers and tautomers.

[0016] The term "solvent" refers to a complex of variable stoichiometry formed by a solute and a solvent. Such solvents used for the purposes of this invention may not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water. Solvates of solvent molecules, such as water, are commonly referred to as "hydrates." Hydrates include compositions containing a stoichiometry of water, as well as compositions containing a variable amount of water.

[0017] In some embodiments of the present invention, the therapeutically effective amount of the compound of formula I, its stereoisomers, its solvates or pharmaceutically acceptable salts thereof is 0.01 mg to 100 mg / kg body weight / day, such as 30 mg to 90 mg / kg body weight / day.

[0018] A fourth aspect of the invention provides a pharmaceutical composition comprising a compound of formula I, a stereoisomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, and optionally, a pharmaceutically acceptable excipient.

[0019] In some embodiments of the present invention, the content of compound of formula I in the pharmaceutical composition is 1 mg to 100 mg / unit dosage form, such as 10 mg to 100 mg / unit dosage form, 30 mg / unit dosage form, 40 mg / unit dosage form, 50 mg / unit dosage form, 60 mg / unit dosage form, 70 mg / unit dosage form, 80 mg / unit dosage form, and 90 mg / unit dosage form.

[0020] Pharmaceutically acceptable excipients are substances that are non-toxic, compatible with the active ingredient, and otherwise biologically suitable for use in organisms. The selection of specific excipients will depend on the route of administration or the type and state of disease in the treatment of a particular patient. Examples of pharmaceutically acceptable excipients include, but are not limited to, solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, binders, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, ion exchangers, release agents, coating agents, flavoring agents, and antioxidants, which are common in the pharmaceutical field. Flavoring agents, preservatives, and sweeteners may also be added to the pharmaceutical composition where necessary.

[0021] Pharmaceutically acceptable excipients include: ion exchangers; alumina; aluminum stearate; lecithin; serum proteins (e.g., human serum albumin); buffers (e.g., Twin 80, phosphates, glycine, sorbic acid, or potassium sorbate); partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts); colloidal silica; magnesium trisilicate; polyvinylpyrrolidone; polyacrylates; waxes; polyethylene-polyoxypropylene block copolymers; methylcellulose; hydroxypropyl methylcellulose; lanolin; sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; astragalus powder; malt; gelatin; slip Stone; excipients, such as cocoa butter and suppository wax; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; diols, such as propylene glycol or polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotropic physiological saline; Ringer's solution; ethanol; and phosphate buffer solutions; and, at the discretion of the formulator, other non-toxic and compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavoring agents, aromatizers, preservatives, and antioxidants may also be present in the composition.

[0022] The pharmaceutical compositions of the present invention may further comprise one or more second therapeutic agents, which may be combined with the compounds of Formula I described in this application for the treatment and / or prevention of related diseases caused by influenza viruses. The second therapeutic agents include substances known to have therapeutic activity against diseases caused by influenza viruses.

[0023] In some embodiments of the present invention, the second therapeutic active agent includes at least one of baloxavir, oseltamivir phosphate, favipiravir (CAS No. 259793-96-9), and an anti-influenza vaccine.

[0024] The pharmaceutical composition can be formulated using methods known in the art for the intended route of delivery, including intravenous, intramuscular, intraperitoneal, subcutaneous, intraocular, intrathecal, intra-articular, intrasynovial, intracisional, intrahepatic, intralesional, intracranial, infusion, and / or inhalation.

[0025] The pharmaceutical composition may be administered to the subject via any suitable route, including oral, transdermal, subcutaneous, intranasal, inhalation, intramuscular, and intravascular administration. It should be understood that the preferred route of administration and pharmaceutical formulation will vary depending on the subject's condition and age, the nature of the condition to be treated, the desired therapeutic effect, and the specific mitochondrial-targeting antioxidant used.

[0026] In some embodiments of the present invention, the dosage form of the pharmaceutical composition includes injections, powders, capsules, tablets, ointments, suppositories, aerosols, pills, drops, sustained-release tablets, suspensions, granules, lozenges, powders, pills, granules, solutions, creams, patches, tablets, or any combination thereof.

[0027] The pharmaceutical compositions of the present invention are preferably administered orally or by injection.

[0028] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier (e.g., sodium citrate or dicalcium phosphate) and / or the following: a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silica; b) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) humectants, such as glycerin; d) disintegrants, such as agar-agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate; e) solution blockers, such as paraffin; f) absorption accelerators, such as quaternary ammonium compounds; g) wetting agents, such as cetyl alcohol and glycerol monostearate; h) adsorbents, such as kaolin and bentonite; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also include a buffer.

[0029] Injectable formulations (e.g., sterile injectable aqueous or oily suspensions) can be formulated using suitable dispersants, wetting agents, and / or suspending agents according to known techniques. Sterile injectable formulations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic, non-enteric-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Among acceptable mediators and solvents, water, Ringer's solution, USP, and isotonic sodium chloride solution can be used. Furthermore, sterile non-volatile oils are routinely used as solvents or suspension media. For this purpose, any mild non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids (e.g., oleic acid) are used in the preparation of injectable formulations.

[0030] The beneficial effects of this invention are:

[0031] The compound of Formula I of this invention inhibits the activity of influenza virus polymerase and can inhibit the proliferation of influenza virus, thus achieving an anti-influenza effect. Therefore, this small molecule compound can be used as the active ingredient and combined with a pharmaceutically acceptable carrier to prepare an anti-influenza drug for the treatment of influenza. Attached Figure Description

[0032] Figure 1This invention demonstrates the inhibitory activity of compound I of formula I against influenza virus polymerase in Example 1 of the present invention.

[0033] Figure 2 This invention demonstrates the inhibitory activity of compound I of formula 2 against influenza virus.

[0034] Figure 3 This invention demonstrates the inhibitory activity of compound I of formula 3 against influenza virus protein expression. Detailed Implementation

[0035] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0036] Example 1

[0037] This embodiment evaluates the inhibitory effect of compound I (STL035778) on influenza virus polymerase activity. The specific process is as follows:

[0038] Minigenome reporter assay: The inhibitory activity of the compound against influenza virus polymerase was verified using a standard dual-luciferase reporter gene assay. The specific procedure is as follows:

[0039] 1) Cell line: HEK293T cell line, cultured in DMEM medium (Gibco) with 10% fetal bovine serum (Gibco), 1% penicillin (Gibco) and 1% streptomycin (Gibco) added to the medium.

[0040] 2) Instrument: Microplate reader (Bio-Tek, USA).

[0041] 3) Experimental method: After cell counting, 293T cells in good growth condition were subjected to a 3×10⁻⁶ splitting process. 5 Cells were seeded at a density of 100 μL / mL in 96-well plates, with 100 μL of cell suspension added to each well. The plates were incubated in a 5% CO2 incubator at 37°C. Once the cell density exceeded 80%, the cells were transfected with influenza virus polymerase expression plasmid and reporter gene expression plasmid.

[0042] Dilute 50 ng of WSN plasmids (pcDNA3.1-PA, pcDNA3.1-PB1, pcDNA3.1-PB2, pcDNA3.1-NP), pYH-Luci plasmid, and internal control pRL-TK plasmid to 15 μL of Opti-MEM, mix well, and incubate for 5 min. Dilute PEI transfection reagent by adding 0.9 μL of PEI transfection reagent to 15 μL of Opti-MEM at a plasmid:1 μg / mL PEI ratio of 1:3, mix well, and incubate for 5 min. Slowly add the plasmid to the PEI, gently pipette to mix, and let stand for 20 min. Aspirate 40 μL of culture medium from the 96-well plate and add 30 μL of plasmid-PEI mixture to each well.

[0043] Three hours after transfection, 10 μL of different concentrations of the compound were added to each well to achieve final concentrations of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, and 0.78125 μM. Three replicates were set up for each concentration, along with a positive control, a negative control, and a blank control. The mixture was incubated at 37°C with 5% CO2 for 24 hours. Fluorescence intensity was detected using a microplate reader according to the Dual Luciferase Reporter Assay Kit instructions, and the data were processed using GraphPad Prism 9.5 software.

[0044] The results are as follows Figure 1 As shown, compound I exhibits inhibitory activity against influenza virus polymerase, EC 50 The value is 31.17 μM.

[0045] Example 2

[0046] This embodiment evaluates the anti-influenza virus activity of compound I (STL035778), and the specific process is as follows:

[0047] Cytopathic Effect Reduction Assay: This assay uses standard cell viability assays to verify the inhibitory effect of compounds on cytopathic effects induced by influenza virus infection. The specific procedure is as follows:

[0048] 1) Cell line: MDCK(NBL-2)(ATCC-34TM) cell line, cultured in MEM medium (Gibco) with 10% fetal bovine serum (Gibco), 1% penicillin (Gibco) and 1% streptomycin (Gibco) added to the medium.

[0049] 2) Virus strain: Influenza A virus A / Puerto Rico / 8 / 1934(PR8)(H1N1), stored in a laboratory freezer at -80℃. When used, it was amplified and propagated using 9-day-old SPF chicken embryos, and the viral titer after amplification was determined using MDCK cells.

[0050] 3) Instrument: Microplate reader (Bio-Tek, USA).

[0051] 4) Experimental method: After cell counting, MDCK cells in good growth condition were subjected to a 2×10⁻⁶ splitting process. 5 Influenza A virus was seeded at a density of 100 μL / mL in 96-well plates, with each well containing 100 μL of cell suspension. After incubation for 12 hours in a 5% CO2, 37°C incubator, the culture medium was discarded, and the cells were washed twice with PBS. Influenza A virus was then diluted with MEM medium containing only 1 μg / mL TPCK-trypsin (Gibco) to adjust the virus to a multiplicity of infection (MOI) of 0.1 for 1 hour of cell infection. The final concentrations of the compound were then adjusted to 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, and 0.78125 μM. After discarding the viral infection solution, 100 μL of the compound was added to each well, with five replicates per compound. Positive and negative control groups were also set up, each with five replicates. After culturing in a 37℃, 5% CO2 incubator for 48 hours, 10 μL of CCK8 was added to each well, and the mixture was placed back into the incubator for 1–4 hours. Once the solution in the wells turned darker, the OD value of each well was measured using a microplate reader, and the inhibition rate of the compound was calculated using the Bliss method.

[0052] The results are as follows Figure 2 As shown, compound I has an effect on the EC50 of influenza A virus. 50 It is 29.18 μM.

[0053] Example 3

[0054] This embodiment evaluates the inhibitory effect of compound I (STL035778) on influenza virus protein expression levels. The specific process is as follows:

[0055] Western Blotting Assay: This assay uses standard immunoblotting techniques to verify the inhibition of influenza virus protein expression by a compound. The specific procedure is as follows:

[0056] 1) Cell line: MDCK(NBL-2)(ATCC-34TM) cell line, cultured in MEM medium (Gibco) with 10% fetal bovine serum (Gibco), 1% penicillin (Gibco) and 1% streptomycin (Gibco) added to the medium.

[0057] 2) Virus strain: Influenza A virus A / Puerto Rico / 8 / 1934(PR8)(H1N1), stored in a laboratory freezer at -80℃. When used, it was amplified and propagated using 9-day-old SPF chicken embryos, and the viral titer after amplification was determined using MDCK cells.

[0058] 3) Instrument: Chemiluminescence analyzer (iBright 1500).

[0059] 4) Experimental method: After cell counting, MDCK cells in good growth condition were subjected to a 2×10⁻⁶ splitting process. 5 Influenza A virus was seeded at a density of 100 μL / mL in 6-well plates, with each well containing 100 μL of cell suspension. After incubation at 37°C with 5% CO2 for 12 hours, the culture medium was discarded, and the cells were washed twice with PBS. Influenza A virus was then diluted with MEM medium containing only 1 μg / mL TPCK-trypsin (Gibco) to adjust the virus to a multiplicity of infection (MOI) of 0.1 for 1 hour of cell infection. 1 mL of 100 nM baloxavir and compounds (50 μM and 25 μM) were added to each well, and the cells were incubated at 37°C for 24 hours.

[0060] Discard the culture medium in the 6-well plate, wash three times with pre-cooled PBS, then add 200 μL of cell lysis buffer (add PMSF to a final concentration of 1 mM), and incubate at 4°C with shaking for 30 min. Transfer the cell lysis buffer to a 1.5 mL EP tube, sonicate, and centrifuge at 14,000 rpm for 15 min at 4°C. Transfer the supernatant to a new EP tube, add 5x SDS loading buffer, mix well, and boil at 100°C for 5 min. After cooling, store at -20°C for later use.

[0061] Prepare a 10% protein gel. Add 10 μL of protein sample and 8 μL of marker to each well. Perform electrophoresis at 80V for 30 min. When the protein sample and marker in the upper gel are observed to enter the lower separating gel, reduce the voltage to 120V and continue electrophoresis until the bromophenol blue band reaches the bottom of the separating gel and is about to escape. Turn off the power and stop electrophoresis. Equilibrate the filter paper and NC membrane by immersing them in pre-cooled transfer buffer. Perform transfer at a constant current of 300mA for 90 min. Transfer the NC membrane to TBST buffer containing 5% skim milk powder and block at room temperature for 1 hour. Add primary antibody (1:2000 dilution) and incubate at 4℃ for 18–24 h. Remove the primary antibody and wash the NC membrane three times with TBST for 5 min each time. Add fluorescent secondary antibody and incubate at room temperature for 1 h. Remove the secondary antibody and wash the NC membrane three times with TBST for 5 min each time. Expose the protein bands and analyze the results.

[0062] The results are as follows Figure 3As shown, compound I (STL035778) can inhibit the expression of influenza virus proteins. At 50 μM, this compound can significantly and effectively inhibit the expression of NP and PA proteins of influenza A virus.

[0063] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of drugs against influenza A virus: 。 2. The application according to claim 1, characterized in that: The compound of Formula I or a pharmaceutically acceptable salt thereof inhibits influenza A virus polymerase.

3. The application according to claim 1 or 2, characterized in that: The influenza A virus includes at least one of the following influenza A virus subtypes: H1N1, H2N2, H3N2, H5N1, H7N9, H7N7, H1N2, H9N2, H7N2, H7N3, H5N2, H10N7, H10N3, or H5N8.

4. The application according to claim 3, characterized in that: The therapeutically effective amount of the compound of Formula I or a pharmaceutically acceptable salt thereof is 0.01 mg to 100 mg / kg body weight / day.

5. The application according to claim 1, characterized in that: The drug is a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and optionally, a pharmaceutically acceptable excipient.

6. The application according to claim 1, characterized in that: The content of compound I in the pharmaceutical composition is 10mg~100mg / unit dosage form.

7. The application according to claim 1, characterized in that: The pharmaceutical composition further includes one or more second therapeutic agents, the second therapeutic agents including at least one of baloxavir, oseltamivir phosphate, favipiravir, and an anti-influenza vaccine.

8. The application according to claim 5, characterized in that: The dosage forms of the pharmaceutical composition include injections, powders, capsules, tablets, suppositories, aerosols, pills, drops, suspensions, granules, lozenges, solutions, tablets, or any combination thereof.

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