Novel compounds and anti-coronavirus agents

Abstract

To provide an anti-coronavirus agent having sufficient antiviral action.SOLUTION: The present invention discloses an anti-coronavirus agent that contains a compound illustrated below as an active ingredient and has an antiviral action on coronavirus. The target coronavirus is SARS-CoV-2.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to a novel compound and an anti-coronavirus agent, in particular an anti-coronavirus agent having an antiviral effect against SARS-CoV-2. [Background technology]

[0002] SARS-CoV-2 is a novel coronavirus that was detected in a pneumonia patient in Wuhan, Hubei Province, China in December 2019 (Non-Patent Document 1), and has since spread throughout the world, with the World Health Organization declaring it a pandemic in March 2020. As of October 2020, the number of infected people is 37.4 million, and the death toll exceeds 1.08 million. Novel coronavirus disease (COVID-19) is a disease that humanity must contain in its spread.

[0003] SARS-CoV-2 is a single-stranded positive-stranded RNA virus with petal-shaped spike protein (S) protrusions on the envelope (lipid bilayer) on the surface of the virus particle. SARS-CoV-2 is further composed of envelope protein (E), matrix protein (M), and nucleoprotein (N).

[0004] Researchers around the world are developing treatments and vaccines, and it has been suggested that some commercially available drugs (remdesivir, ciclesonide, favipiravir, etc.) are effective in treating COVID-19. General antiviral drugs work by inhibiting viral polymerase or nuclease, and remdesivir, which was approved in Japan in May 2020 as a treatment for COVID-19, inhibits viral replication by inhibiting polymerase. On the other hand, no nuclease inhibitors have been approved for COVID-19 at present, but it has recently been reported that the active metabolite of ciclesonide, Cic2, binds to the endonuclease (Nsp15) of SARS-CoV-2 and inhibits the replication of genomic RNA (Non-Patent Documents 2, 3). Nsp15 is known to act as an enzyme that protects the virus itself from the host's immune system, and to have structural similarities in multiple coronaviruses (95.7% homology with Nsp15 of SARS-CoV, which was prevalent in 2003) (Non-Patent Document 4). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] N Engl J Med. 2020;382:727-733 [Non-Patent Document 2] Matsuyama, S., Kawase, M., Nao, N., Shirato, K., Ujike, M., Kamitani, W., Shimojima, M., and Fukushi, S., bioRxiv, https: / / doi.org / 10.1101 / 2020.03.11.987016. [Non-Patent Document 3] Matsuyama S, Kawase M, Nao N, Shirato K, Ujike M, Kamitani W, Shimojima M, Fukushi S. J. Allergy Clin. Immunol., in press. https: / / doi.org / 10.1016 / j.jaci.2020.05.029 [Non-Patent Document 4] Y. Kim, R. Jedrzejczak, NI Maltseva, M. Wilamowski, M. Endres, A. Godzik, K. Michalska, A. Joachimiak, Protein Sci, 2020, 29, 1596-1605. Summary of the Invention [Problem to be solved by the invention]

[0006] However, the antiviral effects of both ciclesonide (Cicle) and its active metabolite (Cic2) are insufficient.

[0007] The present invention has been made in consideration of such problems, and an object of the present invention is to provide an anti-coronavirus agent having sufficient antiviral activity. [Means for solving the problem]

[0008] The novel compound according to the present invention has the following structural formula:

[0009] [ka]

[0010] The anti-coronavirus agent of the present invention contains the following compound as an active ingredient and is characterized by having an antiviral effect against coronaviruses:

[0011] [ka]

[0012] The anti-coronavirus agent of the present invention contains the following compound as an active ingredient and is characterized by having an antiviral effect against coronaviruses:

[0013] [ka]

[0014] The anti-coronavirus agent of the present invention contains the following compound as an active ingredient and is characterized by having an antiviral effect against coronaviruses:

[0015] [ka]

[0016] The anti-coronavirus agent of the present invention contains the following compound as an active ingredient and is characterized by having an antiviral effect against coronaviruses:

[0017] [ka] Effect of the Invention

[0018] According to the present invention, an anti-coronavirus agent having sufficient antiviral activity can be obtained. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is a diagram examining the binding analysis of the ciclesonide derivative Cic2 by docking simulation. [Diagram 2] FIG. 1 shows the results of a cytotoxicity test for each compound. [Diagram 3] FIG. 1 shows the viral proliferation inhibitory effect of each compound. [Figure 4] FIG. 1 shows the viral proliferation inhibitory effect of administration of compounds Cic4 and Cic6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Hereinafter, an embodiment of the present invention will be described in detail with reference to the attached drawings. However, the embodiment is intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the embodiment described below. Other embodiments in which a person skilled in the art appropriately replaces the configuration of the embodiment described below are also included in the scope of the present invention.

[0021] (1) New compound The novel compound according to the present invention has the following structural formula:

[0022] [ka]

[0023] The above ciclesonide analog Cic4 can be synthesized by the following route.

[0024] Cic2, a solution of triethylamine in dichloromethane is added to a solution of methanesulfonyl chloride in dichloromethane. After stirring at room temperature, the reaction mixture is diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate and saturated saline. After drying over anhydrous sodium sulfate, the mixture is filtered and the solvent is removed under reduced pressure. The resulting residue is purified to obtain Cic3.

[0025] Next, sodium azide was added to the acetonitrile solution of Cic3 and stirred. The reaction solution was diluted with ethyl acetate, washed with water and saturated saline, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting residue was purified to obtain Cic4.

[0026] [ka]

[0027] (2) Anti-coronavirus agents SARS-CoV has a specific gene sequence known as nonstructural protein 15 (nsp15), which has been shown to have 95.7% similarity to the gene sequence of SARS-CoV-2. Examination of the sequence and structure of nsp15 has revealed that nsp15 is an endonuclease enzyme essential for disease development and has structural similarities among multiple coronaviruses. This endonuclease has a high probability of performing a common function in protecting coronaviruses from the host immune system. It is believed that nsp15 inhibitors will be effective not only against SARS-CoV and SARS-CoV-2, but also against any new coronaviruses.

[0028] We investigated the binding analysis of the ciclesonide derivative Cic2 with viral proteins involved in the genome replication of the novel coronavirus SARS-CoV-2 by using advanced bioinformatics technology and docking simulations using high-performance computers. From the ligand binding analysis, we designed ciclesonide analogs Cic3 to Cic11 in which the primary hydroxyl groups corresponding to Ser294 and Pro344, which are thought to be important for protein binding, were replaced with functional groups (Figure 1). Although ciclesonide did not act on the novel coronavirus genome replicase (RNA-dependent RNA polymerase), it has been found to bind to the active center of nsp15 and inhibit the accurate replication of the viral genome.

[0029] The anti-coronavirus agent of the present invention contains the following compound as an active ingredient and has an antiviral effect against coronaviruses.

[0030] [ka]

[0031] The anti-coronavirus agent of the present invention contains the following compound as an active ingredient and has an antiviral effect against coronaviruses.

[0032] [ka]

[0033] The anti-coronavirus agent of the present invention contains the following compound as an active ingredient and has an antiviral effect against coronaviruses.

[0034] [ka]

[0035] The anti-coronavirus agent of the present invention contains the following compound as an active ingredient and has an antiviral effect against coronaviruses.

[0036] [ka]

[0037] The coronavirus targeted by the anti-coronavirus agent of the present invention is not particularly limited, but may be, for example, a variant of a wild-type coronavirus.

[0038] The coronavirus targeted by the anti-coronavirus agent of the present invention is not particularly limited, but is, for example, a wild-type coronavirus.

[0039] Wild-type coronaviruses are made up of coronaviruses that belong to the Coronavirinae subfamily. Coronaviruses are classified in the order Nidovirales, which is divided into the Coronaviridae family and the subfamily Coronavirinae and Torovirinae. The Coronavirinae subfamily is further divided into three genera: Alphacoronavirus, Betacoronavirus, and Gammacoronavirus.

[0040] Examples of coronaviruses belonging to the Coronavirinae subfamily include, but are not limited to, SARS-CoV-2.

[0041] Examples of coronaviruses belonging to the Coronavirinae subfamily include, but are not limited to, SARS coronavirus, MERS coronavirus, human coronavirus 229E, human coronavirus OC43, human coronavirus HKU1, human coronavirus NL63, feline infectious peritonitis virus, canine coronavirus, avian infectious bronchitis virus, bovine coronavirus, and porcine coronaviruses including transmissible gastroenteritis virus, porcine deltacoronavirus, porcine epidemic diarrhea virus, porcine respiratory coronavirus, and porcine hemagglutinating encephalomyelitis coronavirus.

[0042] The anti-coronavirus agent of the present invention has anti-coronavirus activity, and the anti-coronavirus activity includes at least one of the following: killing the coronavirus; or, even if the coronavirus remains, acting on its surface protein to cause it to lose its infectivity (ability to grow).

[0043] It is also possible to define an anti-coronavirus composition as one containing the anti-coronavirus agent of the present invention and at least one component selected from the group consisting of alcohol, surfactant, antibacterial agent, moisturizer, and cosmetic oils and fats.

[0044] The form is not particularly limited, but typical examples include the following: - Alcohol preparations containing anti-coronavirus agents and alcohol A cleaning composition containing an anti-coronavirus agent and a surfactant Disinfecting composition containing an anti-coronavirus agent and an antibacterial agent -Lotion, milky lotion or cream containing an anti-coronavirus agent and a moisturizer and / or cosmetic oils and fats The cleaning composition is a composition capable of removing dirt from food, tableware, cooking utensils, the hands and fingers of workers, clothing, etc., and disinfecting coronaviruses, and is provided, for example, as a liquid or solid detergent. The alcohol preparation and disinfecting composition are compositions used to inactivate coronaviruses and bacteria attached to food, tableware, cooking utensils, the hands and fingers of workers, or utensils that have handled the dirt of coronavirus patients, and are provided, for example, as a spray similar to conventional ethanol preparations. The lotion, cream, and emulsion are compositions (basic cosmetics) that can be applied to the hands and fingers of workers that are easily roughened by water work, etc., to provide skin care and disinfect coronaviruses.

[0045] In the above-mentioned anti-coronavirus composition, the alcohol, surfactant, bactericide, moisturizer, and cosmetic oils and fats may be used in combination of two or more. For example, it is preferable to further blend a surfactant such as a fatty acid ester in an alcohol preparation to further enhance antibacterial properties. The above-mentioned cleaning composition can take the form of a hand soap containing a bactericide and alcohol in addition to a surfactant, and the above-mentioned cream can take the form of a hand skin protecting component and an antibacterial agent and alcohol for keeping the hand skin clean.

[0046] The above-mentioned anti-coronavirus compositions can be appropriately blended with various components for imparting desired performance and improving the quality of each composition, such as thickeners (xanthan gum, locust bean gum, sodium polyacrylate, etc.), antioxidants, fragrances, colorants, etc., and in the case of cosmetics such as lotions, skin roughness prevention agents, anti-inflammatory agents, etc.

[0047] The content of the anti-coronavirus agent in the above-mentioned anti-coronavirus composition can be appropriately adjusted depending on the aspects of the composition, such as the component configuration and method of use, within the range in which anti-coronavirus activity is expressed.

[0048] The concentration of the active ingredient of the anti-coronavirus agent of the present invention can be appropriately adjusted within the range in which anti-norovirus activity is expressed, and is, for example, 1 μM to 80 μM.

[0049] In the case of an anti-coronavirus agent containing the compound Cic4 as an active ingredient, the concentration is, for example, 5 μM to 80 μM, preferably 7 μM to 40 μM, and more preferably 6 μM to 20 μM.

[0050] In the case of an anti-coronavirus agent containing the compound Cic6 as an active ingredient, the concentration is, for example, 1 μM to 80 μM, preferably 5 μM to 40 μM, and more preferably 6 μM to 20 μM.

[0051] [ka]

[0052] The anti-coronavirus agent of the present invention can also be defined as an anti-coronavirus agent for medicinal use for preventing coronavirus infection and / or treating diseases caused by said coronavirus.

[0053] The administration site of the anti-coronavirus agent for pharmaceutical use can include oral administration, oral administration, intratracheal administration, subcutaneous administration, intramuscular administration, and intravascular (intravenous) administration. When the anti-coronavirus agent of the present invention is used as a pharmaceutical, it is formulated into various forms by known methods, such as injections, capsules, tablets, syrups, granules, patches, drips, ointments, etc. The anti-coronavirus agent of the present invention can be administered alone or together with a pharmacologically acceptable carrier.

[0054] These preparations contain excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, low-substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, calcium carboxymethylcellulose, and internally crosslinked sodium carboxymethylcellulose; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate, etc.), binders (e.g., the above-mentioned excipients; gelatin; polyvinylpyrrolidone; macrogol, etc.), disintegrants (e.g., the above-mentioned excipients; chemically modified starches and cellulose derivatives such as croscarmellose sodium, sodium carboxymethyl starch, and crosslinked polyvinylpyrrolidone, etc.), lubricants (e.g., talc; stearin, etc.), and the like. Acids; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; veegum; waxes such as beeswax and geranium; boric acid; glycol; fumaric acid; carboxylic acids such as adipic acid; sodium carboxylates such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; starch derivatives in the above excipients, etc.; stabilizers (e.g., methylparaben, propane, etc.); They are produced by well-known methods using additives such as paraoxybenzoic acid esters such as propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenol; phenols such as cresol; thimerosal; acetic anhydride; sorbic acid, etc.), flavorings (e.g., commonly used sweeteners, acidulants, fragrances, etc.), suspending agents (e.g., polysorbate 80, sodium carboxymethylcellulose, etc.), diluents, and formulation solvents (e.g., water, ethanol, glycerin, etc.).Furthermore, when the anti-coronavirus agent of the present invention is used as an injection, examples of the storage container include ampoules, vials, prefilled syringes, cartridges for pen-type syringes, and drip infusion bags.

[0055] The dosage of the anti-coronavirus agent of the present invention as a medicine is not particularly limited as long as the desired therapeutic or preventive effect is obtained, and can be appropriately determined depending on symptoms, sex, age, etc. The dosage is preferably 1 ng / kg to 10 mg / kg, more preferably 10 ng / kg to 1 mg / kg, even more preferably 5 to 500 μg / kg, still more preferably 10 to 100 μg / kg, and most preferably 10 to 30 μg / kg. EXAMPLES

[0056] (1) Molecular design of ciclesonide analogues A docking study of the ciclesonide metabolite Cic2 was performed using MOE (Molecular Operating Environment; CCG) with Nsp15 (PDB: 6VWW), whose X-ray crystal structure has been revealed.

[0057] [ka]

[0058] First, the crystal water was removed, hydrogen atoms were added to the protein, and the amino acid side chain structure was corrected, and the ligand-binding region was detected using Site Finder. Next, the ligand-binding mode was analyzed by docking simulation (force field: AMBER10:EHT) between Nsp15 and Cic2 (Figure 1). Based on the ligand-binding mode, we designed ciclesonide analogs Cic3-Cic11 in which the primary hydroxyl groups corresponding to Ser294 and Pro344, which are thought to be important for protein binding, were replaced with the respective functional groups.

[0059] [ka]

[0060] [ka]

[0061] [ka]

[0062] [ka]

[0063] [ka]

[0064] [ka]

[0065] [ka]

[0066] [ka]

[0067] [ka]

[0068] (2) Synthesis of ciclesonide analogues Next, ciclesonide analogues Cic3 to Cic11 were synthesized using Cic2 as a starting material according to the scheme shown below.

[0069] [ka]

[0070] (Synthesis of Cic3) To a dichloromethane solution (2 mL) of Cic2 (250 mg, 0.5 mmol) and triethylamine (154 μL, 1.1 mmol), a dichloromethane solution (0.5 mL) of methanesulfonyl chloride (43 μL, 0.55 mmol) was added at 0°C. After stirring at room temperature for 30 minutes, the reaction solution was diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate and saturated saline. After drying over anhydrous sodium sulfate, the solution was filtered and the solvent was removed under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (hexane / ethyl acetate = 4:1 to 1:1) to give Cic3 as a colorless foamy solid (270 mg, 99%). 1H NMR (600 MHz, CDCl3): δ 7.25 (d, J = 10.2 Hz, 1H), 6.29 (dd, J = 10.2, 1.2 Hz, 1H), 6.03 (s, 1H), 5.00 (d, J = 1.8 Hz, 2H), 4.86 (d, J = 4.8 Hz, 1H), 4.51 (t, J = 3.0 Hz, 1H), 4.31 (d, J = 4.8 Hz, 1H), 3.25 (s, 3H), 2.57 (ddd, J = 13.2, 13.2, 4.8 Hz, 1H), 2.35 (dd, J = 13.2, 3.0Hz, 1H), 2.20-2.14 (m, 1H), 2.08-2.04 (m, 1H), 1.79-1.70 (m, 3H), 1.66-1.56 (m, 9H), 1.45 (s, 3H), 1.26-1.04 (m, 7H), 0.96 (s, 3H). 13C NMR (151 MHz, CDCl3): δ 202.8, 186.5, 169.5, 155.8, 128.0, 122.6, 107.7, 97.2, 81.9, 71.6, 69.9, 55.0, 49.7, 46.1, 43.9, 40.9, 40.6, 39.5, 33.9, 33.3, 31.8, 30.3, 27.1, 26.2, 25.5, 21.1, 17.1.

[0071] (Synthesis of Cic4) Sodium azide (156 mg, 2.4 mmol) was added to a solution of Cic3 (265 mg, 0.48 mmol) in acetonitrile (2.4 mL) and the mixture was stirred at 60°C for 48 hours. The reaction mixture was returned to room temperature, diluted with ethyl acetate, washed with water and saturated saline, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (hexane / ethyl acetate = 4:1 to 1:1) to give Cic4 as a white foamy solid (115 mg, 48%). 1H NMR (600 MHz, CDCl3): δ 7.25 (d, J = 10.8 Hz, 1H), 6.29 (dd, J = 10.8, 1.8 Hz, 1H), 6.04 (s, 1H), 4.89 (d, J = 4.8 Hz, 1H), 4.52 (t, J = 3.0 Hz, 1H), 4.29 (d, J = 4.8 Hz, 1H), 4.17 (d, J = 18.6 Hz, 1H), 3.92 (d, J = 18.6 Hz, 1H), 2.57 (ddd, J = 12.6, 12.6, 4.8 Hz, 1H), 2.35 (dd, J = 13.2, 3.0 Hz, 1H), 2.18-2.12 (m, 1H), 2.11-2.06 (m, 1H), 1.79-1.54 (m, 12H), 1.46 (s, 3H), 1.26-1.04 (m, 7H), 0.94 (s, 3H). 13C NMR (151 MHz, CDCl3): δ 204.6, 186.5, 169.6, 155.8, 128.0, 122.6, 107.5, 97.3, 81.7, 70.0, 55.9, 55.1, 49.8, 45.8, 43.9, 41.2, 40.6, 33.9, 33.2, 31.8, 30.3, 27.1, 26.2, 25.5, 21.1, 17.5. HRMS (ESI) m / z calculated for C28H38N3O5+ 496.2806 [M + H]+ found 496.2802.

[0072] (Synthesis of Cic5) To a solution (267 μL) of Cic4 (12 mg, 0.02 mmol) in tetrahydrofuran, 1M hydrochloric acid (133 μL) was added, followed by triphenylphosphine (9 mg, 0.03 mmol), and the mixture was stirred at room temperature for 12 hours. The reaction mixture was evaporated to dryness under reduced pressure, and the residue was azeotroped twice with acetonitrile. The resulting residue was dissolved in dichloromethane (460 μL), and isobutyric anhydride (8 μL, 0.046 mol) and triethylamine (13 μL, 0.092 mmol) were added at 0°C. After stirring at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by flash silica gel column chromatography (hexane / ethyl acetate = 4 : 1 to 1 : 2) to give Cic5 as a white foamy solid (9.5 mg, 77%). 1H NMR (600 MHz, CDCl3): δ 7.27 (d, J = 9.6 Hz, 1H), 6.28 (dd, J = 9.6, 1.8 Hz, 1H), 6.17 (br.t, J = 4.2 Hz, 1H), 6.03 (br.s, 1H), 4.87 (d, J = 6.0 Hz, 1H), 4.50 (d, J = 3.0 Hz, 1H), 4.30 (dd, J = 14.4, 4.2 Hz, 1H), 4.27 (t, J = 4.2 Hz, 1H), 4.12 (dd, J = 14.4, 4.2 Hz, 1H), 2.56 (ddd, J = 13.2, 13.2, 4.8 Hz, 1H), 2.46 (quint., J = 7.2 Hz, 1H), 2.34 (dd, J = 13.8, 3.0 Hz, 1H), 2.15 (ddd, J = 10.8, 10.8, 2.4 Hz, 1H), 2.09 (dd, J = 13.8, 2.4 Hz, 1H), 1.93 (dd, J = 13.8, 3.0 Hz, 1H), 1.85 (dd, J = 14.4, 2.4 Hz, 2H), 1.80 - 1.70 (m, 5H), 1.67 - 1.53 (m, 4H), 1.45 (s, 3H), 1.26 - 1.01 (m, 7H), 1.20 (d, J = 7.2 Hz, 3H), 1.18 (d, J = 6.6 Hz, 3H), 0.91 (s, 3H). 13C NMR (151 MHz, CDCl3): δ 206.2, 186.5, 177.4, 169.6, 155.9, 128.0, 122.6, 107.4, 97.4, 81.6, 69.9, 55.1, 49.8, 47.7, 45.9, 44.0, 40.8, 40.6, 35.4, 34.0, 33.3, 31.9, 30.4, 27.1, 26.2, 25.5, 21.1, 19.5, 19.5, 17.1.

[0073] (Synthesis of Cic6) Lithium chloride (9.7 mg, 0.23 mmol) was added to a solution of Cic3 (25.0 mg, 0.046 mmol) in N,N-dimethylformamide (1 mL) and the mixture was stirred at 60°C for 3 hours. The reaction mixture was returned to room temperature, diluted with ethyl acetate, washed with water and saturated saline, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (hexane / ethyl acetate = 3:1) to give Cic6 as a white solid (19.2 mg, 86%). 1H NMR (600 MHz, CDCl3): δ 7.26 (d, J = 4.2 Hz, 1H), 6.28 (dd, J = 9.6, 1.8 Hz, 1H), 6.02 (br.s, 1H), 4.89 (d, J = 4.8 Hz, 1H), 4.51 (br.s, 1H), 4.40 (d, J = 16.2 Hz, 1H), 4.28 (d, J = 4.2 Hz, 1H), 4.22 (d, J = 17.4 Hz, 1H), 2.56 (td, J = 8.4, 6.0 Hz, 1H), 2.34 (dd, J =13.8, 3.0Hz, 1H), 2.16-2.07 (m, 3H), 1.77-1.59 (m, 10H), 1.45 (s, 3H), 1.21-1.08 (m, 8H), 0.92 (s, 3H). 13C NMR (151 MHz, CDCl3): δ 201.4, 186.5, 169.8, 155.9, 128.0, 122.5, 107.5, 97.8, 81.9, 69.9, 55.1, 49.7, 47.6, 45.6, 44.0, 41.1, 40.6, 33.9, 33.2, 31.8, 30.3, (27.1, 27.1), 26.2, 25.5 (1C overlapped), 21.1, 17.6.

[0074] (Synthesis of Cic7) Lithium bromide (23.4 mg, 0.27 mmol) was added to a solution of Cic3 (37.0 mg, 0.067 mmol) in N,N-dimethylformamide (1 mL) and stirred at 60°C for 1 hour. The reaction mixture was returned to room temperature, diluted with ethyl acetate, washed with water and saturated saline, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (hexane / ethyl acetate = 3:1) to give Cic7 as a white solid (30 mg, 85%). 1H NMR (600 MHz, CDCl3): δ 7.24 (d, J = 10.2 Hz, 1H), 6.30 (d, J = 1.8 Hz, 1H), 6.03 (br.s, 1H), 4.90 (d, J = 4.8 Hz, 1H), 4.52 (br.s, 1H), 4.32 (d, J = 4.2 Hz, 1H), 4.22 (d, J = 14.4 Hz, 1H), 4.06 (d, J = 14.4 Hz, 1H), 2.56 (td, J = 9.6, 8.4 Hz, 1H), 2.35 (dd, J =10.8, 3.0 Hz, 1H), 2.14-2.04 (m, 3H), 1.77-1.58 (m, 10H), 1.45 (s, 3H), 1.34 (br.s, 1H), 1.20-1.09 (m, 7H), 0.93 (s, 3H). 13C NMR (151 MHz, CDCl3): δ 201.4, 186.5, 169.6, 155.8, 128.0, 122.6, 107.5, 98.0, 81.9, 70.0, 55.1, 49.6, 45.7, 43.9, 41.3, 40.6, 33.9, 33.3, 33.2, 31.8, 30.3, 27.2, 27.1, 26.2, 25.5 (1C overlapped), 21.1, 17.7.

[0075] (Synthesis of Cic8) Sodium iodide (15 mg, 0.10 mmol) was added to a solution of Cic3 (27.4 mg, 0.05 mmol) in N,N-dimethylformamide (0.3 mL) and stirred at 60°C for 1 hour. The reaction mixture was cooled to room temperature, diluted with ethyl acetate, washed with water and saturated saline, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (hexane / ethyl acetate = 4:1 to 1:1) to give Cic8 as a white foamy solid (14 mg, 48%). 1H NMR (600 MHz, CDCl3): δ 7.26 (d, J = 10.2 Hz, 1H), 6.29 (dd, J = 10.2, 1.8 Hz, 1H), 6.04 (s, 1H), 4.89 (d, J = 5.4 Hz, 1H), 4.53 (br. s, 1H), 4.41 (d, J = 4.8 Hz, 1H), 4.09 (d, J = 11.4 Hz, 1H), 3.92 (d, J = 11.4 Hz, 1H), 2.57 (ddd, J = 13.8, 13.8, 5.4 Hz, 1H), 2.35 (dd, J = 13.2, 3.0Hz, 1H), 2.18-2.05 (m, 3H), 1.77-1.56 (m, 10H), 1.46 (s, 3H), 1.23-1.05 (m, 7H), 0.93 (s, 3H). 13C NMR (151 MHz, CDCl3): δ 203.2, 186.5, 169.7, 155.9, 128.0, 122.6, 107.3, 98.1, 81.7, 70.0, 55.1, 49.4, 45.9, 45.6, 43.9, 41.3, 40.7, 33.9, 33.3, 31.8, 30.4, 27.2, 26.2, 25.5, 21.1, 17.9.

[0076] (Synthesis of Cic9) Cic7 (27.4 mg, 0.05 mmol) was dissolved in dimethylamine solution (2 M tetrahydrofuran) (400 μL) and stirred for 12 hours at 60° C. The reaction solution was evaporated under reduced pressure, and the resulting residue was purified by flash silica gel column chromatography (dichloromethane / methanol = 100 : 0 to 90 : 10) to give Cic9 as a pale yellow oil (4.5 mg, 18%). 1H NMR (600 MHz, CDCl3): δ 7.23 (d, J = 9.6 Hz, 1H), 6.28 (dd, J = 9.6, 1.8 Hz, 1H), 6.01 (dd, J = 6.0, 1.8 Hz, 1H), 4.86 (d, J = 6.3 Hz, 1H), 4.49 (d, J = 3.0 Hz, 1H), 4.23 (d, J = 4.2 Hz, 1H), 3.67 (t, J = 6.3 Hz, 1H), 3.51 (d, J = 19.2 Hz, 1H), 3.17 (d, J = 19.2 Hz, 1H), 2.54 (ddd, J = 13.8, 13.8, 5.4 Hz, 1H), 2.30 (s, 6H), 2.15-2.04 (m, 2H), 1.75-1.51 (m, 12H), 1.44 (s, 3H), 1.27-1.02 (m, 7H), 0.90 (s, 3H). 13C NMR (151 MHz, CDCl3): δ 207.2, 186.6, 169.7, 155.8, 128.1, 122.7, 107.2, 97.6, 81.5, 70.3, 63.0, 55.3, 50.0, 45.9, 45.6, 44.1, 41.6, 40.8, 34.1, 33.4, 32.0, 30.5, 27.3, 26.4, 25.7(1C overlapped), 21.3, 17.7.

[0077] (Synthesis of Cic10) To a solution (580 μL) of Cic4 (24.8 mg, 0.05 mmol), 1M hydrochloric acid (290 μL) was added, followed by triphenylphosphine (20 mg, 0.075 mmol), and the mixture was stirred at room temperature for 12 hours. The reaction mixture was evaporated to dryness under reduced pressure, and the residue was suspended in water (2 mL) and washed with dichloromethane (1 mL x 2). The aqueous layer was concentrated in vacuum to give a white solid (22 mg, 85%). 1H NMR (600 MHz, MeOH-d4): δ 7.44 (d, J = 10.2 Hz, 1H), 6.25 (dd, J = 10.2, 1.8 Hz, 1H), 6.01 (s, 1H), 4.44 (dd, J = 6.6, 3.0 Hz, 1H), 4.38 (d, J = 4.8 Hz, 1H), 4.17 (d, J = 18.6 Hz, 1H), 3.85 (d, J = 18.6 Hz, 1H), 2.64 (ddd, J = 13.2, 13.2, 1.5 Hz, 1H), 2.37 (dd, J = 13.2, 3.0 Hz, 1H), 2.26-2.19 (m, 1H), 2.15-2.11 (m, 1H), 1.98 (dd, J = 13.2, 3.6 Hz, 1H), 1.80-1.72 (m, 6H), 1.68-1.56 (m, 4H), 1.48 (s, 3H), 1.27-0.99 (m, 8H), 0.97 (s, 3H). 13C NMR (151 MHz, MeOH-d4): δ 204.5, 188.8, 174.1, 159.5, 128.0, 122.7, 108.6, 98.6, 83.2, 70.3, 57.2, 51.4, 47.5, 47.5, 45.9, 42.1, 41.4, 35.5, 34.3, 33.0, 31.7, 28.3, 27.4, 26.7, 21.5, 17.6.

[0078] (Synthesis of Cic11-1 and Cic11-2) Cic3 (89.9 mg, 0.164 mmol) was dissolved in N,N-dimethylformamide (1 mL) and stirred at room temperature for 24 hours. The mixture was diluted with ethyl acetate, washed with water and saturated saline, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The residue was purified by flash silica gel column chromatography (hexane / ethyl acetate = 3:1 to 1:1) to give Cic11-1 and Cic11-2 as white solids (Cic11-1, 19.9 mg, 25%) (Cic11-2, 8.4 mg, 11%). Cic11-1 1H NMR (600 MHz, CDCl3): δ 7.29 (d, J = 10.2 Hz, 1H), 6.30 (dd, J = 10.2, 1.8 Hz, 1H), 6.04 (s, 1H), 4.68 (d, J = 4.2 Hz, 1H), 4.51 (br.s, 1H), 4.17 (d, J = 5.4 Hz, 1H), 3.21 (d, J = 4.8 Hz, 1H), 3.16 (d, J = 4.8 Hz, 1H), 2.57 (td, J = 8.4, 6.0 Hz, 1H), 2.34 (dd, J = 12.9, 2.4 Hz, 1H), 2.18-2.15 (m, 2H), 2.04-2.00 (m, 1H), 1.75-1.61 (m, 9H), 1.52-1.47 (s, 5H), 1.27-1.01 (m, 10H). 13C NMR (151 MHz, CDCl3): δ 186.6, 169.6, 156.0, 128.0, 122.6, 108.4, 91.0, 79.3, 70.0, 55.1, 49.2, 49.0, 46.9, 46.5, 44.0, 41.4, 40.9, 33.9, 33.2, 31.8, 31.5, 30.1, 27.3, 27.1, 26.2, 25.6 (1C overlapped), 21.1, 16.6. Cic11-2 1H NMR (600 MHz, CDCl3): δ 7.27 (d, J = 10.2 Hz, 1H), 6.29 (dd, J = 9.9, 1.8 Hz, 1H), 6.03 (s, 1H), 4.70 (d, J = 3.6 Hz, 1H), 4.48 (d, J = 3.0 Hz, 1H), 4.25 (d, J = 6.0 Hz, 1H), 3.27 (d, J = 4.8 Hz, 1H), 3.01 (d, J = 4.8 Hz, 1H), 2.56 (td, J = 13.2, 8.4 Hz, 1H), 2.34 (dd, J = 16.2, 3.0Hz, 1H), 2.15-2.00 (m, 3H), 1.81-1.41 (m, 14H), 1.27-1.05 (m, 10H). 13C NMR (151 MHz, CDCl3): δ 186.5, 169.5, 155.8, 128.0, 122.6, 107.9, 91.4, 79.8, 70.0, 55.1, 50.2, 50.0, 48.9, 46.7, 44.0, 41.0, 40.8, 33.9, 33.4, 31.8, 30.1, 27.2, 27.0, 26.2, 25.6 (1C overlapped), 21.1, 17.3.

[0079] (3) Toxicity testing Vero E6 / TMPRSS2 (JCRB 1819) cells were cultured for 18 hours in medium (DMEM supplemented with 2% FCS, penicillin (100 units / mL), and streptomycin (100 μg / mL)) containing test compounds at different final concentrations (2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM). Cytotoxicity in the presence of each test compound was evaluated using the Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories). For quantification, absorbance at 480 nm was measured using a Nivo multimode microplate reader (PerkinElmer). N=3 was used for each test compound and each concentration. DMSO was used as a control.

[0080] The cytotoxicity of the test compounds was evaluated by a cytotoxicity test using LDH as an index when they were coexisted with Vero E6 / TMPRSS2 cells at different concentrations for 18 hours. As a result, out of a total of 10 compounds including ciclesonide (Cicle), seven compounds, which are metabolites of ciclesonide, Cic2, Cic3, Cic7, Cic8, Cic9, Cic10, and Cic11-2, showed cytotoxicity at a concentration of 40 μM, while four compounds (Cicle, Cic4, Cic6, and Cic11-1) showed less than 10% LDH release even at a concentration of 40 μM, indicating that Cic4, Cic6, and Cic11-1 had the same cytotoxicity as Cicle (Figure 2).

[0081] (4) Viral infection experiments The SARS-CoV-2 JPN / TY / WK-521 strain was cultured in Vero E6 / TMPRSS2 cells described above at 37°C under 5% CO2 in Dulbecco's modified Eagle's medium (DMEM, low glucose; Fujifilm Wako Pure Chemical Industries) containing 5% heat-inactivated fetal bovine serum (FBS; SAFC Biosciences, Lenexa, KS, USA), penicillin (100 units / mL), and streptomycin (100 μg / mL).

[0082] For virus infection experiments, Vero E6 / TMPRSS2 cells were plated in 96-well flat-bottom tissue culture plates (TPPs) at 1.5 x 10 cells per well. 4After inoculation and culturing for 20 hours, the supernatant was removed by aspiration, and 100 μL of medium containing the test compound (DMEM with 2% FBS, penicillin (100 units / mL), and streptomycin (100 μg / mL)) was quickly added to each well (three wells were used for each test compound and each concentration). Then, 100 μL of SARS-CoV-2 suspension was added to each well, with the concentration adjusted to 0.1 using DMEM with 2% FBS, penicillin (100 units / mL), and streptomycin (100 μg / mL) (final concentrations of test substance: 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM). After culturing for 18 hours at 37℃ under 5% CO2, the entire culture supernatant was collected, centrifuged, and used for the following virus titer test. At the same time, RNA was extracted from the cells after removing the supernatant. DMSO was used as a control.

[0083] RNA was extracted from 100 μL of cell culture supernatant using Maxwell RSC Viral Total Nucleic Acid Purification Kit (Promega). TM Direct RNA Prep Kit for RT-PCR (Real Time) (Takara Bio) was used.

[0084] Real-time PCR was performed using 2.5 μL of RNA extract as a template, TaqMan® Fast Virus 1-Step Master Mix (Thermo Fisher Scientific) as the master mix, and ABI7500 (Thermo Fisher Scientific) as the thermal cycler. The primers and probes, reaction solution composition, and reaction conditions were in accordance with the N2 set of the "National Institute of Infectious Diseases Pathogen Detection Manual 2019-nCoV Ver. 2.6". For viral RNA quantification, a calibration curve was created in advance using 2019-nCoV_N_Positive Control (IDT), and the number of viral RNA copies in each RNA sample was determined by absolute quantification using this. The above-mentioned 2019-nCoV_N_Positive Control was used as the positive control during the reaction, and RNA extracted from the DMSO-administered group was used as the negative control.

[0085] VeroE6 / TMPRSS2 cells were seeded in a 12-well tissue culture plate (TPP) using DMEM supplemented with 2% FBS, penicillin (100 units / mL), and streptomycin (100 μg / mL) and cultured for 18 hours at 37°C under 5% CO2. The infected cell supernatant samples were serially diluted 10-fold with DMEM supplemented with 2% FBS, penicillin (100 units / mL), and streptomycin (100 μg / mL), and 50 μL of each dilution was added per well and incubated at 37°C under 5% CO2 for 1 hour. The supernatant was then aspirated and DMEM containing 1% methylcellulose, 2% FBS, penicillin (100 units / mL), and streptomycin (100 μg / mL) was added and cultured at 37°C under 5% CO2 for 72 to 96 hours. After incubation, the wells were fixed with 10% neutral buffered formalin, 0.5 ml of 0.05% methylene blue solution was added, and stained for 1 hour. The number of plaques per well was counted, and the number of plaques per 1 ml of sample solution was calculated. N=3 was used for each test substance and concentration, and a sigmoid curve was created using the XLfit program (IDBS) based on the obtained values, and the concentrations required to reduce the initial virus titer to 1 / 100 and 1 / 1000 were calculated.

[0086] The amount of viral RNA in the supernatant was evaluated by real-time PCR when SARS-CoV-2 JPN / TY / WK-521 strain was infected for 18 hours in a medium containing a test compound at a concentration of 10 μM. As a result, the amount of viral RNA in the control group (no treatment; no compound) was 4.8 x 10 10 The Cicle group had a 5.2x10 8 Among the compounds that showed cytotoxicity equivalent to that of Cicle, the viral RNA level in the Cic4 group was 2.1x10 5 copy / well, 4.6x10 in the Cic6-treated group 5 The number of copies / well and the viral load were significantly reduced compared to the Cicle-treated group (Figure 3). On the other hand, the viral RNA load in the Cic11-1-treated group was 1.3x10 9 The number of copies / well in the Cicle-treated group was 100, and no significant reduction was observed compared to the Cic-treated group (Figure 3). In addition, Cic2, Cic3, and Cic7 were listed as compounds that showed a significant reduction in the amount of viral RNA compared to the Cicle-treated group, but since their cytotoxicity in Figure 3 was significantly higher than that of the Cicle-treated group, they were excluded from further evaluation.

[0087] To further evaluate the viral growth inhibitory effect of Cic4 and Cic6 administration, cells were infected with SARS-CoV-2 JPN / TY / WK-521 strain in medium containing different concentrations of the test compounds for 18 hours, and the viral titer in the cell supernatant was evaluated by plaque assay. As shown in Figure 4, Cicle, Cic2, Cic4, and Cic6 all showed a concentration-dependent viral growth inhibitory effect, and the viral titer (approximately 4.6x10) observed in the control group (DMSO) was 1.2x10 5 The concentrations required to reduce the viral proliferation rate (PFU / mL) to 1 / 100 or 1 / 1000 were 5.86 μM and >80.00 μM for Cicle, 24.55 μM and 60.95 μM for Cic2, whereas the concentrations were 6.51 μM and 7.36 μM for Cic4, and 5.95 μM and 6.50 μM for Cic6, indicating that Cic4 and Cic6 have a higher viral proliferation inhibitory effect than Cicle and Cic2. [Industrial Applicability]

[0088] It can be used to prevent and / or treat the new coronavirus.

Claims

1. A new compound having the following structural formula: 【Chemistry 1】

2. An anti-coronavirus agent comprising the following compound as an active ingredient and having an antiviral effect against coronaviruses: 【Chemistry 2】

3. An anti-coronavirus agent comprising the following compound as an active ingredient and having an antiviral effect against coronaviruses: 【Chemistry 3】

4. An anti-coronavirus agent comprising the following compound as an active ingredient and having an antiviral effect against coronaviruses: 【Chemistry 4】

5. An anti-coronavirus agent comprising the following compound as an active ingredient and having an antiviral effect against coronaviruses: 【Chemistry 5】

6. The anti-coronavirus agent according to any one of claims 2 to 5, wherein the coronavirus is a variant of a wild-type coronavirus.

7. The anti-coronavirus agent according to any one of claims 2 to 5, wherein the coronavirus is a wild-type coronavirus.

8. The anti-coronavirus agent according to claim 7, characterized in that the wild-type coronavirus is a coronavirus belonging to the Coronavirinae subfamily.

9. The anti-coronavirus agent according to claim 8, characterized in that the coronavirus belonging to the coronavirus subfamily is SARS-CoV-2.

10. The anti-coronavirus agent according to claim 8, characterized in that the coronavirus belonging to the coronavirus subfamily is selected from the group consisting of SARS coronavirus, MERS coronavirus, human coronavirus 229E, human coronavirus OC43, human coronavirus HKU1, human coronavirus NL63, feline infectious peritonitis virus, canine coronavirus, avian infectious bronchitis virus, bovine coronavirus, and porcine coronaviruses including transmissible gastroenteritis virus, porcine deltacoronavirus, porcine epidemic diarrhea virus, porcine respiratory coronavirus, and porcine hemagglutinating encephalomyelitis coronavirus.

11. The anti-coronavirus agent described in any one of claims 2 to 10, characterized in that the concentration of the active ingredient of the anti-coronavirus agent is 1 μM to 80 μM.

12. The anti-coronavirus agent according to claim 2, characterized in that the concentration of the active ingredient of the anti-coronavirus agent is 7 μM to 40 μM.

13. The anti-coronavirus agent described in claim 2, characterized in that the concentration of the active ingredient of the anti-coronavirus agent is 6 μM to 20 μM.

14. The anti-coronavirus agent described in claim 3, characterized in that the concentration of the active ingredient of the anti-coronavirus agent is 5 μM to 40 μM.

15. The anti-coronavirus agent described in claim 3, characterized in that the concentration of the active ingredient of the anti-coronavirus agent is 6 μM to 20 μM.

16. The anti-coronavirus agent according to any one of claims 2 to 15, characterized in that the anti-coronavirus agent is an anti-coronavirus agent for medicinal use for preventing infection with the coronavirus and / or treating a disease caused by the coronavirus.

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