WO2022270478A1 - 抗ウイルス剤 - Google Patents

抗ウイルス剤 Download PDF

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Publication number
WO2022270478A1
WO2022270478A1 PCT/JP2022/024596 JP2022024596W WO2022270478A1 WO 2022270478 A1 WO2022270478 A1 WO 2022270478A1 JP 2022024596 W JP2022024596 W JP 2022024596W WO 2022270478 A1 WO2022270478 A1 WO 2022270478A1
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compound
cells
antiviral agent
group
nmr
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French (fr)
Japanese (ja)
Inventor
健次 門出
勇太 村井
靖之 五十嵐
靖剛 臼杵
洋文 澤
靖子 大場
道仁 佐々木
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Hokkaido University NUC
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Hokkaido University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
    • A01N31/08—Oxygen or sulfur directly attached to an aromatic ring system
    • A01N31/16—Oxygen or sulfur directly attached to an aromatic ring system with two or more oxygen or sulfur atoms directly attached to the same aromatic ring system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
    • A01N35/04—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing aldehyde or keto groups, or thio analogues thereof, directly attached to an aromatic ring system, e.g. acetophenone; Derivatives thereof, e.g. acetals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/222—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin with compounds having aromatic groups, e.g. dipivefrine, ibopamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals

Definitions

  • the present invention relates to antiviral agents that are effective as therapeutic agents against coronaviruses such as SARS-CoV-2.
  • coronaviruses such as SARS-CoV-2.
  • a coronavirus is an enveloped virus whose gene is a positive single-stranded RNA.
  • Coronaviruses that infect humans include, in addition to viruses that cause common colds, those that cause acute respiratory syndrome.
  • SARS-CoV which is the cause of the severe acute respiratory syndrome (SARS) epidemic in 2003
  • SARS-CoV-2 which is the cause of the new coronavirus infection COVID-19.
  • SARS-CoV-19 there are still few therapeutic drugs for COVID-19, and the development of more effective therapeutic drugs is required.
  • TMPRSS2 pathway The entry of SARS-CoV-2 into cells is divided into a pathway (TMPRSS2 pathway) by the activity of the spike protein (S) after adsorption by the host TMPRSS2 and a pathway in which it is taken up into cells by endosomes and propagated by cathepsins in lysosomes ( Cathepsin pathway) are mainly mentioned (Non-Patent Document 1).
  • Compounds having inhibitory activity against the TMPRSS2 pathway and the cathepsin pathway are expected as candidate compounds for therapeutic agents for SARS-CoV-2, and searches for such compounds are actively carried out worldwide.
  • remdesivir has inhibitory activity on these pathways and has been approved as a therapeutic agent for COVID-19 (Non-Patent Document 2).
  • Remdesivir is a monophosphoramidate prodrug of GS-441524, an antiviral agent consisting of a nucleic acid compound developed as a treatment for Ebola hemorrhagic fever and Marburg virus infection.
  • the purpose of the present invention is to provide an antiviral agent that is effective as a therapeutic drug for viral infectious diseases including COVID-19.
  • Edible natural products are expected to have the advantage of ensuring safety for humans and sufficiently lowering the hurdles for application to pharmaceuticals. Therefore, the present inventors screened a library of compounds derived from edible natural products, and identified compounds having inhibitory activity against the TMPRSS2 pathway and the cathepsin pathway as candidate compounds for antiviral agents against SARS-CoV-2. The present invention was completed by selecting as
  • the present invention provides the following antiviral agents and the like. [1] the following general formula (1) or (2)
  • R 1 and R 2 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an acyl group having 1 to 6 carbon atoms, a benzyl group, or a benzoyl group; , each independently is 1 or 2; r is an integer from 2 to 12.
  • An antiviral agent comprising a compound represented by or a derivative thereof, a salt of these compounds, or a solvate thereof.
  • the general formula (1) is represented by the following general formulas (1-1) to (1-4)
  • R 1 , R 2 and r are the same as defined above.
  • the antiviral agent of [1] above which is any one of [3]
  • [4] The antiviral agent of any one of [1] to [3], which has the ability to suppress cell invasion of coronavirus.
  • a pharmaceutical composition comprising the antiviral agent according to any one of [1] to [4] as an active ingredient.
  • the pharmaceutical composition according to [5] above which is used for treating infections caused by coronaviruses.
  • the pharmaceutical composition of [6] above which is used for treatment of COVID-19.
  • the antiviral agent according to the present invention contains, as an active ingredient, a compound with high antiviral activity against viruses such as SARS-CoV-2 that enter host cells via the TMPRSS2 pathway or the cathepsin pathway. Therefore, a pharmaceutical composition containing the antiviral agent as an active ingredient is suitable as an in vivo or ex vivo antiviral agent, and is used for the treatment or prevention of infectious diseases caused by coronaviruses such as COVID-19. It is very useful as an active ingredient of a pharmaceutical composition.
  • FIG. 10 shows the measurement results of the total area ( ⁇ m 2 ) of cells having an area of 500 ⁇ m 2 or more per cell in the cell fusion inhibitory effect test of Example 4.
  • FIG. 10 is a diagram showing the measurement results of the number of cells having an area of 500 ⁇ m 2 or more per cell in the cell fusion inhibitory effect test of Example 4.
  • FIG. 10 is a diagram showing the measurement results of the number of cells having an area of 500 ⁇ m 2 or more per cell in the cell fusion inhibitory effect test of Example 4.
  • X1 to X2 (X1 and X2 are real numbers that satisfy X1 ⁇ X2)" means "X1 or more and X2 or less”.
  • the antiviral agent according to the present invention is a compound represented by the following general formula (1) (hereinafter sometimes referred to as “compound (1)”) or a compound represented by the following general formula (2) (hereinafter referred to as Sometimes referred to as “compound (2)”).
  • r is an integer of 2-12.
  • r is preferably an integer of 4-12, more preferably an integer of 6-10.
  • R 1 and R 2 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an acyl group having 1 to 6 carbon atoms, a benzyl group, or a benzoyl group.
  • a plurality of R 1 in one molecule may be the same group or different groups.
  • a plurality of R 2 in one molecule may be the same group or different groups.
  • R 1 and R 2 are alkyl groups having 1 to 6 carbon atoms
  • the alkyl groups may be linear or branched. More specifically, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, n-pentyl group, sec-pentyl group, isopentyl group,
  • An alkyl group having 1 to 6 carbon atoms such as an n-hexyl group, a sec-hexyl group and an isohexyl group is preferable, an alkyl group having 1 to 4 carbon atoms is more preferable, an alkyl group having 1 to 3 carbon atoms is even more preferable, and methyl groups, ethyl groups are even more preferred.
  • the acyl group may be an alkyl group having 1 to 5 carbon atoms, and may be linear. It may be branched. More specifically, the alkyl group moiety is methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, n-pentyl group, sec-pentyl Alkyl groups having 1 to 5 carbon atoms such as groups and isopentyl groups are preferable, alkyl groups having 1 to 4 carbon atoms are more preferable, alkyl groups having 1 to 3 carbon atoms are more preferable, and methyl and ethyl groups are even more preferable. .
  • compound (1) includes compounds represented by general formulas (1-1) to (1-4).
  • Compound (2) includes compounds represented by general formulas (2-1) to (2-4).
  • R 1 , R 2 and r are the same as in general formula (1).
  • a compound represented by the general formula (1-1) (hereinafter sometimes referred to as “compound (1-1)") is preferable, and among the compounds (1-1), r is Compounds 6-10 are particularly preferred.
  • a compound represented by the general formula (2-1) hereinafter sometimes referred to as “compound (2-1)”
  • compound (2-1) is preferable, and among the compounds (2-1), r is Compounds 6-10 are particularly preferred.
  • Compound (1-1) includes compounds represented by general formulas (1-1-1) to (1-1-25).
  • Compound (2-1) includes compounds represented by general formulas (2-1-1) to (2-1-25).
  • r is the same as in general formula (1), and 4 to 12 is preferred, and 6-10 is more preferred.
  • R 3 is a methyl group, an ethyl group, an n-propyl group, or an isopropyl group, preferably a methyl group.
  • a plurality of R 3 in one molecule may be the same group or different groups.
  • Ac is an acetyl group
  • Bz is a benzoyl group
  • Bn is a benzyl group.
  • compound (1) or compound (2) compounds represented by the following formulas (C-1) to (C-11) are particularly preferable.
  • Compound (C-1) is Malabaricone C, a component of the spices nutmeg and mace, and is guaranteed to be safe for humans to ingest.
  • Compounds (C-2) to (C-9) can be synthesized by appropriately modifying the hydroxy group of compound (C-1) or by reducing the ketone group to a hydroxy group.
  • Compounds (C-10) to (C-11) can be synthesized, for example, according to the synthetic routes shown in Examples described later.
  • compound (1) and compound (2) may form a salt, and the acid or base that forms the salt includes mineral acids such as hydrochloric acid and sulfuric acid; Acids, organic acids such as citric acid; alkali metals such as sodium and potassium; alkaline earth metals such as calcium and magnesium; Compound (1) and compound (2) may also be in the form of solvates such as hydrates.
  • Derivatives of compound (1) and compound (2) may be used as antiviral agents according to the present invention.
  • Derivatives of compound (1) or compound (2) are preferably derivatives from which compound (1) or compound (2) is produced by enzymatic treatment or the like in vivo.
  • the derivative of compound (1) or compound (2) that can be used as an antiviral agent is obtained, for example, by prodrug conversion, which is carried out for pharmaceuticals containing a low-molecular-weight compound as an active ingredient. Derivatives are more preferred.
  • a monophosphoramidite derivative in which the hydroxy group is substituted with a monophosphoramidite group and a monophosphorothioate derivative in which the hydroxy group is substituted with a monophosphorothioate group etc.
  • the derivative of compound (1) and the derivative of compound (2) may form a salt or may be in the form of a solvate such as a hydrate.
  • the salt those listed above can be used.
  • Compound (1), derivative of compound (1), compound (2), derivative of compound (2), salts of these compounds, or solvates thereof has the ability to suppress entry into host cells via the TMPRSS2 pathway or the cathepsin pathway. Due to this ability to suppress cell entry, the compounds of the present invention have antiviral activity against viruses, particularly viruses such as SARS-CoV-2 that enter host cells via the TMPRSS2 pathway or the cathepsin pathway. , is useful as an active ingredient of a pharmaceutical composition used for treatment or prevention of viral infections.
  • the compound according to the present invention is particularly useful as an active ingredient of pharmaceutical compositions used for treating or preventing coronavirus infectious diseases including COVID-19. Since the compound according to the present invention is a low-molecular-weight compound, there is no problem such as immunogenicity. In addition, since the route of administration is not so limited, it is particularly useful as an active ingredient of medicines for mammals including humans.
  • one or more compounds according to the present invention are contained in a pharmaceutical composition, they can be mixed with a pharmaceutically acceptable carrier, if necessary, and various dosage forms can be adopted depending on the purpose of prevention or treatment. is. Examples of such forms include oral agents, injections, sprays, suppositories, ointments, patches, etc., but oral agents are preferred. Each of these dosage forms can be produced by a conventional formulation method known to those skilled in the art.
  • Pharmaceutically acceptable carriers include excipients, binders, disintegrants, lubricants, and coloring agents in solid preparations; solvents, solubilizers, suspending agents, tonicity agents, buffers in liquid preparations and analgesics, etc. are used.
  • Formulation additives such as preservatives, antioxidants, colorants, sweeteners, stabilizers, etc. may also be used as necessary.
  • excipients are added to the compounds of the present invention, and if necessary, binders, disintegrants, lubricants, coloring agents, flavoring agents, etc. are added, and the Tablets, coated tablets, granules, powders, capsules, etc. can be produced by
  • flavoring agents When preparing oral liquid preparations, flavoring agents, buffering agents, stabilizing agents, flavoring agents, etc. are added to the compounds of the present invention, and oral liquid preparations, syrups, elixirs, etc. are produced by conventional methods. can be done.
  • the compound according to the present invention When preparing an injection, the compound according to the present invention is added with a pH adjusting agent, a buffering agent, a stabilizer, a tonicity agent, a local anesthetic, etc., and injected subcutaneously, intramuscularly or intravenously by a conventional method. agent can be manufactured.
  • a pH adjuster, a buffering agent, a stabilizer, a tonicity agent, a local anesthetic, etc. are added to the compound according to the present invention to prepare a liquid preparation, which is filled in a nebulizer to prepare a nebulizer. can do.
  • Suppositories can be prepared by conventional methods after adding pharmaceutical carriers known in the art such as polyethylene glycol, lanolin, cocoa butter, fatty acid triglycerides, etc. to the compound of the present invention.
  • pharmaceutical carriers known in the art such as polyethylene glycol, lanolin, cocoa butter, fatty acid triglycerides, etc.
  • bases, stabilizers, wetting agents, preservatives and the like that are commonly used for the compounds of the present invention are blended, if necessary, and mixed and formulated by conventional methods.
  • the above-mentioned ointments, creams, gels, pastes, etc. may be applied to a conventional support by a conventional method.
  • the content of the compound according to the present invention in each of the above formulations varies depending on the patient's condition, dosage form, etc., but is generally about 0.001 to 1000 mg for oral formulations and about 0.001 to 500 mg for injections. , about 0.01 to 1000 mg for suppositories.
  • the daily dose of these formulations varies depending on the patient's symptoms, body weight, age, sex, etc., and cannot be determined indiscriminately. 0.01 to 1000 mg is preferable, and it is preferable to administer this amount once a day or in 2 to 3 divided doses.
  • the animal to which the antiviral agent and pharmaceutical composition containing the compound of the present invention as an active ingredient is administered is not particularly limited, and may be a human or a non-human animal.
  • Non-human animals include mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters and guinea pigs, and birds such as chickens, quails and ducks.
  • the compound according to the present invention is preferably added not only to pharmaceutical compositions but also to foods.
  • a preventive effect against viral infectious diseases such as COVID-19 can be expected by ingesting food containing the compound of the present invention.
  • foods include health foods, functional foods, supplements, and other foods that are ingested with the expectation of contributing to the maintenance and promotion of health.
  • Foods containing the compound of the present invention include, for example, forms in which the compound of the present invention is inoculated in the form of tablets, liquids, capsules, and the like.
  • the compound according to the present invention can also be in the form of powder, granules, or liquid (solution, suspension) and added to other foods for ingestion, such as so-called seasonings.
  • the compound according to the present invention may be contained in foods for animals other than humans.
  • Animal food includes, for example, livestock feed and pet food (pet food).
  • pet food pet food
  • it can be produced by a conventional method, except that the compound according to the present invention is blended together with other raw materials.
  • the compound according to the present invention can also be used as an active ingredient of a disinfectant.
  • a disinfectant containing the compound of the present invention it is possible to disinfect or disinfect viruses present in the sprayed area.
  • marabacholine C compound (C-1)
  • sprays containing Marabacholine C as an active ingredient are safe even in places and environments where sprayed liquids can be absorbed into the body, such as dining tables, food materials, food manufacturing plants, nursery schools, schools, and hospitals. can be used for
  • SARS-CoV-2 was the WK-521 strain (Wuhan strain) (hCoV-19/Japan/TY/WK-521/2020) provided by the National Institute of Infectious Diseases, ⁇ strain (UK strain) (hCoV-19/Japan/QK002/2020), and ⁇ strain (Brazilian strain) (hCoV-19/Japan/TY7-501/2021).
  • TMPRSS2-expressing gene was introduced into MA104 cells, a cultured cell line derived from African green monkey kidney cells, to produce MA104T cells that constitutively express TMPRSS2. These MA104T cells were infected with SARS-CoV-2 (WK-521) and cultured while periodically exchanging the medium. After viral infection, many cells died and detached due to cytopathic effects. Cells surviving after long-term culture were collected and used as SARS-CoV-2 persistently infected cells.
  • 293TA cells An ACE2 expression gene was introduced into a cultured cell line HEK-293T cells derived from human embryonic kidney cells to prepare 293TA cells in which ACE2 was constitutively expressed.
  • 293TA-G cells 293TA-G cells were prepared by introducing a GFP-expressing gene into HEK-293T cells (293TA cells) in which ACE2 was expressed.
  • 293TA-M cells 293TA-M cells were prepared by introducing an M-Cherry expression gene into 293TA cells.
  • Vero E6 T cells A TMPRSS2-expressing gene was introduced into Vero E6 cells, a cultured cell line derived from African green monkey kidney cells, to prepare Vero E6 T cells in which TMPRSS2 was constantly expressed.
  • All of these cultured cells were cultured in a medium (2 % FBS-containing MEM). In addition, when used in subsequent experiments, the cells were adjusted in advance to an appropriate cell concentration using a culture medium.
  • MTT solution 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (Merck) was dissolved in PBS to 5 ⁇ g/mL, and then diluted to 0.45 ⁇ m or 0.45 ⁇ m. Filtered through a 22 ⁇ m filter.
  • ⁇ Cell lysate (virus inactivation solution) Prepared by adding 50 mL Triton X, 4 mL hydrochloric acid (12 mol/L) in 500 mL isopropanol.
  • test compound was previously diluted with DMSO or a culture medium (MEM containing 2% FBS) to an appropriate concentration, and a 2-fold serial dilution series was prepared in a 96-well plate (50 ⁇ L/well).
  • MTT assay Cells (2.0 to 2.5 ⁇ 10 5 cells/mL) adjusted to an appropriate cell number were dispensed at 100 ⁇ L/well into a 96-well plate containing the test compound. Next, 50 ⁇ L/well of a SARS-CoV-2 solution that had been previously diluted to an appropriate concentration with a culture medium (MEM containing 2% FBS) was dispensed into each well of the 96-well plate. After mixing with a plate mixer, the plate was cultured in a CO 2 incubator for 2-3 days.
  • MEM containing 2% FBS culture medium
  • the 96-well plate cultured for 2 to 3 days was observed with the naked eye or under a microscope to confirm the morphology of cells, the presence or absence of crystals, and the like. Then, 30 ⁇ L of the MTT solution was dispensed into each well and cultured in a CO 2 incubator for 4-6 hours. After culturing, 140 ⁇ L of the supernatant was removed from each well while taking care not to absorb the cells. After 140 ⁇ L of cell lysate (virus inactivation solution) was dispensed into each well, the plate was wrapped with plastic wrap so as not to dry and left overnight at room temperature.
  • EC 50 is calculated based on the following formula from two points A-High (High OD, High conc.) and B-Low (Low OD, Low conc.) sandwiching the 50% OD value on the absorbance and drug concentration curve. did.
  • OD means absorbance
  • conc means concentration of drug (test compound).
  • test compound was previously diluted with dimethyl sulfoxide (DMSO) or a culture medium (MEM containing 2% FBS) to an appropriate concentration, and a 2- to 5-fold serial dilution series was prepared in a 96-well plate (50 ⁇ L/well).
  • DMSO dimethyl sulfoxide
  • MEM containing 2% FBS culture medium
  • SARS-CoV-2 persistently infected cells adjusted to an appropriate cell number were dispensed into 96-well plates containing test compounds at 100 ⁇ L/well, and allowed to react at room temperature for 1 hour. After that, 293TA-G cells and 293TA-M cells were adjusted to an appropriate cell number, added to each well, mixed with a plate mixer, and cultured in a CO 2 incubator for 24 hours.
  • Example 1 A derivative of malavaricon C was synthesized. Malavaricon C purchased from Carbosynth was used. In addition, a compound having a methylene chain length different from that of Marabacholine C was also synthesized.
  • Example 2 The antiviral activity against SARS-CoV-2 of malavaricon C and its structural analogues and their derivatives was examined by MTT assay.
  • WK-521 Wild strain
  • ⁇ strain British strain
  • ⁇ strain Brain strain
  • malavaricon C manufactured by Carbosynth
  • compound (C-1) compounds (C-2) to (C-11) synthesized in Example 1
  • RD remdesivir
  • MK-4482 molnupiravir
  • test compound A 2-fold serial dilution series of the test compound was prepared at 50 ⁇ L/well in a 96-well plate, and Vero E6T cells adjusted to 2.0 ⁇ 10 5 cells/mL were dispensed at 100 ⁇ L/well. Then, 50 ⁇ L/well of SARS-CoV-2 (WK-521) solution, which had been diluted to an appropriate concentration in advance, was dispensed into each well, mixed with a plate mixer, and placed in a CO 2 incubator for 2 to 2 hours. Cultured for 3 days.
  • Example 3 It was investigated by MTT assay whether malavaricon C and its structural analogues and their derivatives also have antiviral activity against various mutants of SARS-CoV-2.
  • SARS-CoV-2 ⁇ strain (British strain) or ⁇ strain (Brazilian strain) was used, and Vero E6T cells or 293TA cells were used as cells to be infected in the same manner as in Example 2. Then, the cells were infected with the virus and the MTT assay was performed. The results are shown in Tables 2 and 3. In the table, "ND" indicates no data.
  • the compounds according to the present invention also showed antiviral activity against SARS-CoV-2 other than the WK-521 strain (Wuhan strain).
  • Example 4 The ability of malavaricon C and its derivatives to suppress SARS-CoV-2 entry into host cells was examined by a cell fusion inhibitory effect test.
  • SARS-CoV-2 the WK-521 strain (Wuhan strain) provided by the National Institute of Infectious Diseases was used.
  • Compound (C-1), compound (C-3), and compound (C-5) were used as test compounds.
  • the compound (C-1) (malavaricon C) was purchased from Carbosynth, and the others were synthesized in Example 1.
  • neutralizing antibody Ab1 and neutralizing antibody Ab2 were used. These antibodies are known to inhibit adhesion of SARS-CoV-2 to host cells and suppress entry of SARS-CoV-2 into host cells.
  • Compound (C-1), compound (C-3), and compound (C-5) were added to 6 wells each in a 2-fold serial dilution series from 25 ⁇ M.
  • Neutralizing antibody Ab1 and neutralizing antibody Ab2 were added to 6 wells each in a 5-fold serial dilution series from 25 ⁇ g/mL.
  • 3 wells (CC) to which neither the virus nor the test compound was added, and 3 wells (VC) to which only the virus was added without the test compound were set.
  • FIG. 1 shows the measurement results of the total area ( ⁇ m 2 ) of cells having an area of 500 ⁇ m 2 or more per cell
  • FIG. 2 shows the measurement results of the number of cells.
  • compound (C-1), compound (C-3), compound (C-5), neutralizing antibody Ab1, and neutralizing antibody Ab2 all decreased the number of fused cells in a concentration-dependent manner. , the total area of fused cells was also smaller.
  • compound (C-1), compound (C-3), and compound (C-5) are similar to neutralizing antibody Ab1 and neutralizing antibody Ab2, cell fusion by SARS-CoV-2, That is, it was confirmed that entry of SARS-CoV-2 into host cells could be suppressed.

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