JP2022084098A - Novel compound and anti-coronavirus agent - Google Patents

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

The present invention relates to novel compounds and anti-coronavirus agents, and more particularly to anti-coronavirus agents having antiviral activity against SARS-CoV-2.

SARS-CoV-2 is a new type of coronavirus detected in a patient with pneumonia that occurred in Wuhan City, Hubei Province, China in December 2019 (Non-Patent Document 1), and then spread worldwide in March 2020. Has been declared a pandemic by the World Health Organization. As of October 2020, the number of infected people has exceeded 37.4 million and the number of deaths has exceeded 1.08 million. Coronavirus infection (COVID-19) is a disease that should be controlled for humankind.

SARS-CoV-2 is a single-strand plus-strand RNA virus, and petal-shaped spike protein (S) protrusions are present on the envelope (lipid bilayer membrane) on the surface of virus particles. SARS-CoV-2 is further composed of envelope protein (E), matrix protein (M), and nucleoprotein (N).

Researchers around the world are developing therapeutic agents and vaccines, suggesting that several over-the-counter drugs (remdesivir, ciclesonide Cicle, favipiravir, etc.) are effective in treating COVID-19. General antiviral drugs are effective by inhibiting the polymerase or nuclease of the virus, and remdesivir, which was approved in Japan as a COVID-19 therapeutic drug in May 2020, suppresses viral replication by inhibiting the polymerase. do. On the other hand, at present, no nuclease inhibitor for COVID-19 has been approved, but recently, the ciclesonide active metabolite Cic2 binds to the SARS-CoV-2 endonuclease (Nsp15) and suppresses genomic RNA replication. It was reported (Non-Patent Documents 2 and 3). It is known that Nsp15 acts as an enzyme that protects the virus itself from the host's immune system and has structural similarities in multiple coronaviruses (95.7% similarity to SARS-CoV Nsp15, which was prevalent in 2003). (Non-Patent Document 4).

N Engl J Med. 2020; 382: 727-733 S. Matsuyama, M. Kawase, N. Nao, K. Shirato, M. Ujike, W. Kamitani, M. Shimojima, S. Fukushi, bioRxiv, https://doi.org/10.1101/2020.03.11.987016. S. Matsuyama, M. Kawase, N. Nao, K. Shirato, M. Ujike, W. Kamitani, M. Shimojima, S. Fukushi, J. Allergy Clin. Immunol., In press. Https://doi.org /10.1016/j.jaci.2020.05.029 Y. Kim, R. Jedrzejczak, N. I. Maltseva, M. Wilamowski, M. Endres, A. Godzik, K. Michalska, A. Joachimiak, Protein Sci, 2020, 29, 1596-1605.

However, neither the ciclesonide Cicle nor the ciclesonide active metabolite Cic2 has sufficient antiviral activity.

The present invention has been made in view of such problems, and an object of the present invention is to provide an anticoronavirus agent having a sufficient antiviral activity.

The novel compound according to the present invention has the following structural formula.

The anti-coronavirus agent according to the present invention is characterized by having the following compound as an active ingredient and having an antiviral action against coronavirus.

The anti-coronavirus agent according to the present invention is characterized by having the following compound as an active ingredient and having an antiviral action against coronavirus.

The anti-coronavirus agent according to the present invention is characterized by having the following compound as an active ingredient and having an antiviral action against coronavirus.

The anti-coronavirus agent according to the present invention is characterized by having the following compound as an active ingredient and having an antiviral action against coronavirus.

According to the present invention, an anticoronavirus agent having a sufficient antiviral activity can be obtained.

It is a figure which examines the binding analysis of the ciclesonide derivative Cic2 by the docking simulation. It is a figure which shows the result of the cytotoxicity test of each compound. It is a figure which shows the virus growth inhibitory effect of each compound. It is a figure which shows the virus growth inhibitory effect by administration of compounds Cic4 and Cic6.

Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings, but the embodiments are for facilitating the understanding of the principles of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.

(1) New compound The new compound according to the present invention has the following structural formula.

The ciclesonide analog Cic4 can be synthesized by the following route.

Add the dichloromethane solution of methanesulfonyl chloride to the dichloromethane solution of Cic2 and triethylamine. After stirring at room temperature, the reaction solution is diluted with dichloromethane and washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine. After drying over anhydrous sodium sulfate, the mixture was filtered and the solvent was distilled off under reduced pressure. The obtained residue is purified to obtain Cic3.

Next, sodium azide was added to the acetonitrile solution of Cic3, and the mixture was stirred. The reaction mixture is diluted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent is evaporated under reduced pressure. The obtained residue is purified to obtain Cic4.

(2) Anti-coronavirus agent
SARS-CoV has a specific gene sequence known as nonstructural protein 15 (nsp15), which has been found to be 95.7% similar to the SARS-CoV-2 gene sequence. Examination of the sequence and structure of nsp15 has revealed that nsp15 is an endonuclease enzyme essential for the onset of disease and has structural similarities in multiple coronaviruses. This endonuclease has a high probability of performing a common function of protecting the coronavirus itself from the host's immune system. The nsp15 inhibitor appears to be effective against all new coronaviruses, not just SARS-CoV and SARS-CoV-2.

We investigated the binding analysis of the ciclesonide derivative Cic2 using advanced bioinformatics technology and docking simulation using a high-performance computer to investigate the interaction between the viral protein involved in the genome replication of the new coronavirus SARS-CoV-2 and ciclesonide. Ciclesonide analogs Cic3 to Cic11 were designed by substituting the primary hydroxyl groups corresponding to Ser294 and Pro344, which are considered to be important for protein binding from ligand binding analysis, with each functional group (Fig. 1). Although ciclesonide did not act on the new coronavirus genome replicating enzyme (RNA-dependent RNA polymerase), it was found to bind to the active center of nsp15 and inhibit accurate replication of the viral genome.

The anti-coronavirus agent according to the present invention contains the following compound as an active ingredient and has an antiviral action against coronavirus.

The anti-coronavirus agent according to the present invention contains the following compound as an active ingredient and has an antiviral action against coronavirus.

The anti-coronavirus agent according to the present invention contains the following compound as an active ingredient and has an antiviral action against coronavirus.

The anti-coronavirus agent according to the present invention contains the following compound as an active ingredient and has an antiviral action against coronavirus.

The coronavirus targeted by the anti-coronavirus agent according to the present invention is not particularly limited, but is, for example, a variant of wild-type coronavirus.

The coronavirus targeted by the anti-coronavirus agent according to the present invention is not particularly limited, but is, for example, a wild-type coronavirus.

The wild-type coronavirus consists of coronaviruses belonging to the subfamily Coronavirinae. Coronaviruses are classified into the order Nidovirals, among which are the Coronaviridae, and the Coronaviridae and Trovirus subfamily. The subfamily Coronavirinae is further classified into three genera: alpha coronavirus, betacoronavirus, and gamma coronavirus.

The coronavirus belonging to the subfamily Coronavirinae is not particularly limited, but is, for example, SARS-CoV-2.

The coronavirus belonging to the subfamily coronavirus is not particularly limited, but for example, SARS coronavirus, MERS coronavirus, human coronavirus 229E, human coronavirus OC43, human coronavirus HKU1, human coronavirus NL63, and cat transmission. Sexual peritonitis virus, canine coronavirus, chicken infectious bronchitis virus, bovine coronavirus, and infectious gastroenteritis virus, porcine delta coronavirus, porcine epidemic diarrhea virus, porcine respiratory coronavirus, porcine blood cell aggregate brain Selected from the group consisting of porcine coronaviruses, including myelitis coronavirus.

The anti-coronavirus agent according to the present invention has anti-coronavirus activity, and the anti-coronavirus activity is when the coronavirus is killed or acts on the surface protein of the coronavirus even if it remains. It shall include at least one of cases where the infectivity (proliferation ability) is lost.

A composition for anti-coronavirus by containing the anti-coronavirus agent according to the present invention and at least one component selected from the group consisting of alcohols, surfactants, antibacterial agents, moisturizers and oils and fats for cosmetics. It is also possible to specify that.

The embodiment is not particularly limited, but typical examples thereof include the following.
-Alcohol preparation containing anti-coronavirus agent and alcohol-Cleaning composition containing anti-coronavirus agent and surfactant-Disinfecting composition containing anti-coronavirus agent and antibacterial agent-Anti-coronavirus Lotions, emulsions or creams containing agents and moisturizers and / or cosmetic oils and fats Cleaning compositions clean foods, tableware, cookware, workers' fingers and clothing, and disinfect coronavirus. It is a composition of an aspect capable of being provided, for example, as a liquid or solid detergent. Alcohol preparations and disinfectant compositions are used to inactivate coronaviruses and bacteria adhering to foods, tableware, kitchenware, workers' fingers, or instruments that handle coronavirus patient filth. The composition is provided as a spraying agent similar to that of a conventional ethanol preparation, for example. Lotions, creams and milky lotions are compositions (basic cosmetics) that can be applied to the hands and fingers of workers who are prone to get rough due to water work or the like for skin care and disinfecting coronavirus.

In the above-mentioned anti-coronavirus composition, alcohol, a surfactant, a bactericidal agent, a moisturizer, and cosmetic oils and fats may be used in combination of two or more. For example, for alcohol preparations, it is also preferable to further add a surfactant such as a fatty acid ester in order to further enhance the antibacterial property. In addition, the cleaning composition can take the form of a hand soap containing a bactericide or alcohol in addition to a surfactant, and the cream keeps the hands clean together with the ingredients that protect the hands. It is possible to take an embodiment in which an antibacterial agent or alcohol is blended.

The above-mentioned anti-coronavirus compositions are provided with various components for imparting desired performance and enhancing the quality of each composition, such as thickeners (xanthan gum, locust bean gum, sodium polyacrylate, etc.), antioxidants, etc. , Fragrances, pigments and the like, and in the case of cosmetics such as lotions, rough skin preventive agents, anti-inflammatory agents and the like can be appropriately blended.

The content of the anti-coronavirus agent in the above-mentioned anti-coronavirus composition can be appropriately adjusted according to the composition of the composition, the method of use, and the like within the range in which the anti-coronavirus activity is exhibited.

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 exhibited, and is, for example, 1 μM to 80 μM.

The anti-coronavirus agent containing the compound Cic4 as an active ingredient is, for example, 5 μM to 80 μM, preferably 7 μM to 40 μM, and more preferably 6 μM to 20 μM.

The anti-coronavirus agent containing the compound Cic6 as an active ingredient is, for example, 1 μM to 80 μM, preferably 5 μM to 40 μM, and more preferably 6 μM to 20 μM.

The anti-coronavirus agent of the present invention can also be defined as a medicinal anti-coronavirus agent for preventing coronavirus infection and / or treating a disease caused by the coronavirus.

Examples of the administration site of the pharmaceutical anti-coronavirus agent include oral administration, oral administration, intraairway administration, subcutaneous administration, intramuscular administration, intravascular (intravenous) administration and the like. When the anti-coronavirus agent of the present invention is used as a pharmaceutical, it is formulated into various embodiments by a known method, and for example, injections, capsules, tablets, syrups, granules, patches, infusions, ointments and the like. Can be mentioned. The anti-coronavirus agent of the present invention may be administered alone or in combination with a pharmacologically acceptable single substance.

These formulations are excipients (eg, sugar derivatives such as lactose, sucrose, glucose, mannit, sorbit; starch derivatives such as corn starch, horse bell starch, α-starch, dextrin, carboxymethyl starch; crystalline cellulose. , Low Substitution Hydroxylpropyl Cellulose, Hydroxypropylmethyl Cellulose, Carboxymethyl Cellulose, Carboxymethyl Cellulose Calcium, Internally Crosslinked Cellular Derivatives such as Sodium Carboxymethyl Cellulose; Silate derivatives such as; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate; etc.), binders (eg, said excipients; gelatin; polyvinyl). Pyrrolidone; macrogol, etc.), disintegrants (eg, the excipients described above; chemically modified, such as croscarmellose sodium, carboxymethyl starch sodium, crosslinked polyvinylpyrrolidone, starch, cellulose derivatives, etc.), lubricants (eg, For example, talc; stearic acid; calcium stearate, metal stearate such as magnesium stearate; colloidal silica; bee gum; beeswax, waxes such as gay wax; boric acid; glycol; fumaric acid; carboxylic acid such as adipic acid. Acids: Sodium Carboxymethyl Salts Like Sodium Savorate; Sulfates Like Sodium Sulfate; Leucine; Lauryl Sulfates Like Sodium Lauryl Sulfate, Magnesium Lauryl Sulfate; Silicas such as Anhydrous Silicic Acid, Silicic Acid Hydrate ; Stabilizers (eg, paraoxybenzoic acid esters such as methylparapen, propylparapen; alcohols such as chlorobutanol, benzyl alcohol, phenylethyl alcohol; benzalconium chloride; Phenol; phenols such as cresol; thimerosal; anhydrous acetic acid; sorbic acid, etc.), flavoring agents (eg, commonly used sweeteners, acids, fragrances, etc.), suspending agents (eg, polysorbate 80, etc.) It is produced by a well-known method using additives such as sodium carboxymethyl cellulose, a diluent, and a solvent for preparation (for example, water, ethanol, glycerin, etc.). When the anti-coronavirus agent of the present invention is used as an injection, examples of the storage container include ampoules, vials, prefilled syringes, pen-type syringe cartridges, and drip bags.

The pharmaceutical dose of the anti-coronavirus agent of the present invention is not particularly limited as long as it is a dose at which a desired therapeutic effect or preventive effect can be obtained, and can be appropriately determined depending on symptoms, gender, age and the like. The dose is preferably 1 ng / kg to 10 mg / kg, more preferably 10 ng / kg to 1 mg / kg, still more preferably 5 to 500 μg / kg, and even more preferably 10 to. It is 100 μg / kg, most preferably 10 to 30 μg / kg.

(1) Molecular design of ciclesonide analogs
A docking study of the ciclesonide metabolite Cic2 was performed using MOE (Molecular Operating Environment; CCG) for Nsp15 (PDB: 6VWW) whose X-ray crystal structure has been clarified.

First, water of crystallization was eliminated, 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, ligand binding mode analysis was performed by docking simulation of Nsp15 and Cic2 (force field; AMBER10: EHT) (Fig. 1). A ciclesonide analog Cic3-Cic11 was designed in which the primary hydroxyl groups corresponding to Ser294 and Pro344, which are considered to be important for protein binding based on the binding mode of the ligand, were substituted with each functional group.

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

(Synthesis of Cic3)
Dichloromethane sulfonyl chloride (43 μL, 0.55 mmol) in dichloromethane (0.5 mL) was added to a solution of Cic2 (250 mg, 0.5 mmol) and triethylamine (154 μL, 1.1 mmol) in dichloromethane (2 mL) at 0 ° C. .. After stirring at room temperature for 30 minutes, the reaction solution was diluted with dichloromethane and washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine. After drying over anhydrous sodium sulfate, the mixture was filtered and the solvent was distilled off under reduced pressure. The obtained residue was purified by flash silica gel column chromatography (hexane / ethyl acetate = 4: 1 to 1: 1) to give Cic3 as a colorless foam 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, 3.0 Hz, 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.

(Cic4 composition)
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 brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The obtained residue was purified by flash silica gel column chromatography (hexane / ethyl acetate = 4: 1 to 1: 1) to give Cic4 as a white foam 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.

(Cic5 composition)
After adding 1M hydrochloric acid (133 μL) to a solution of Cic4 (12 mg, 0.02 mmol) in tetrahydrofuran (267 μL), triphenylphosphine (9 mg, 0.03 mmol) was added, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was dried under reduced pressure, and the residue was azeotroped twice with acetonitrile. The resulting residue was dissolved in dichloromethane (460 μL) and isobutyric acid anhydride (8 μL, 0.046 mol) was added at 0 ° C., followed by triethylamine (13 μL, 0.092 mmol). After stirring at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure, the obtained residue was purified by flash silica gel column chromatography (hexane / ethyl acetate = 4: 1 to 1: 2), and Cic5 was a white foam solid. Obtained as (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.

(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 brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The obtained 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, 3.0 Hz, 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.

(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 the mixture was stirred at 60 ° C. for 1 hour. The reaction mixture was returned to room temperature, diluted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The obtained 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.

(Cic8 composition)
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 the mixture was stirred at 60 ° C. for 1 hour. The reaction mixture was returned to room temperature, diluted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The obtained residue was purified by flash silica gel column chromatography (hexane / ethyl acetate = 4: 1 to 1: 1) to give Cic8 as a white foam 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, 3.0 Hz, 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.

(Synthesis of Cic9)
Cic7 (27.4 mg, 0.05 mmol) was dissolved in a dimethylamine solution (2Mtetrahydrofuran) (400 μL) and stirred at 60 ° C. for 12 hours. After distilling off the reaction solution under reduced pressure, the obtained residue was purified by flash silica gel column chromatography (dichloromethane / methanol = 100: 0 to 90: 10) to obtain Cic9 as a pale yellow oily substance (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.

(Synthesis of Cic10)
After adding 1M hydrochloric acid (290 μL) to a solution of Cic4 (24.8 mg, 0.05 mmol) in tetrahydrofuran (580 μL), triphenylphosphine (20 mg, 0.075 mmol) was added, and the mixture was stirred at room temperature for 12 hours. After the reaction was dried under reduced pressure, the residue was suspended in water (2 mL) and washed with dichloromethane (1 mL x 2). The aqueous layer was concentrated under 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.

(Synthesis of Cic11-1,11-2)
Cic3 (89.9 mg, 0.164 mmol) was dissolved in N, N-dimethylformamide (1 mL) and stirred at room temperature for 24 hours. It was diluted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The obtained residue was purified by flash silica gel column chromatography (hexane / ethyl acetate = 3: 1 to 1: 1) to obtain Cic11-1 and Cic11-2 as white solids (Cic11-1, 19.9 mg, Cic11-1, 19.9 mg,). 25%) (Cic 11-2, 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, 3.0 Hz, 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.

(3) Toxicity test
Vero E6 / TMPRSS2 (JCRB 1819) cells in medium (2% FCS, penicillin (100 units / mL), streptomycin (100 μg / mL) containing test compounds at different final concentrations (2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM) ) Was cultured for 18 hours using DMEM). Cytotoxicity LDH Assay Kit-WST (Dojin Kagaku Kenkyusho) was used to evaluate the cytotoxicity in the presence of each test compound. For quantification, the absorbance at 480 nm was measured using a Nivo multimode microplate reader (PerkinElmer). N = 3 was set for each test compound and each concentration. DMSO was used as the control group.

The cytotoxicity of Vero E6 / TMPRSS2 cells co-existing with different concentrations of test compounds for 18 hours was evaluated by a cytotoxicity test using LDH as an index. As a result, of the total of 10 compounds containing cyclesonide (Cicle), the cyclesonide metabolites Cic2, Cic3, Cic7, Cic8, Cic9, Cic10 and Cic11-2 showed cytotoxicity at a concentration of 40 μM, while The four compounds (Cicle, Cic4, Cic6, Cic11-1) released less than 10% LDH even at a concentration of 40 μM, indicating that Cic4, Cic6 and Cic11-1 remain cytotoxic to the same level as Cicle (Fig. 2). ).

(4) Virus infection experiment
The above-mentioned Vero E6 / TMPRSS2 cells were used for culturing the SARS-CoV-2 JPN / TY / WK-521 strain. The cell line contains Dulbecco's modified Eagle's medium (DMEM, low) containing 5% heat-inactivated fetal bovine serum (FBS; SAFC Biosciences, Lenexa, KS, USA), penicillin (100 units / mL), and streptomycin (100 μg / mL). Glucose; Fuji Film Wako Pure Chemical Industries, Ltd.) was used for culturing at 37 ° C. under 5% CO ₂ .

In the virus infection experiment, Vero E6 / TMPRSS2 cells were inoculated into a 96-well flat-bottomed tissue culture plate (TPP) so as to be 1.5 x 10 ⁴ cells per well, and after culturing for 20 hours, the supernatant was removed by suction. Immediately, 100 μL of medium containing the test compound (2% FBS, penicillin (100 units / mL), streptomycin (100 μg / mL) -added DMEM) was added per well (3 wells were used for each test compound and each concentration). .. Then, using 2% FBS, penicillin (100units / mL), and streptomycin (100 μg / mL) -added DMEM, 100 μL of SARS-CoV-2 suspension adjusted to a concentration of 0.1 for MOI (Multiplicity of Infection) was prepared. It was added to each well (final concentrations of test substance were 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM). After culturing at 37 ° C. under 5% CO ₂ for 18 hours, the entire culture supernatant was recovered, centrifuged, and used in the following virus titer test. At the same time, RNA was extracted from the cells after removing the supernatant. DMSO was used as the control group.

RNA was extracted from 100 μL of cell culture supernatant using the Maxwell RSC Viral Total Nucleic Acid Purification Kit (Promega). CellAmp ^TM Direct RNA Prep Kit for RT-PCR (Real Time) (Takara Bio) was used for RNA extraction from cells after supernatant removal.

Real-time PCR was performed using 2.5 μL of RNA extract as a template, using TaqMan (registered trademark) Fast Virus 1-Step Master Mix (Thermo Fisher Scientific) for the master mix, and ABI7500 (Thermo Fisher Scientific) for the thermal cycler. .. The primer / probe, reaction solution composition and reaction conditions were in accordance with the N2 set of "National Institute of Infectious Diseases Pathogen Detection Manual 2019-nCoV Ver.2.6". For viral RNA quantification, a calibration curve was prepared 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 calibration curve. The above-mentioned 2019-nCoV_N_Positive Control was used as a positive control during the reaction, and RNA extracted from the DMSO-administered group was used as a negative control.

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) at 37 ° C. under 5% CO ₂ . Incubated for 18 hours. Infected cell supernatant sample is diluted 10-fold with 2% FBS, penicillin (100 units / mL), streptomycin (100 μg / mL) and DMEM, and then 50 μL of each diluted solution is added per well at 37 ° C., 5%. It was kept warm for 1 hour under CO ₂ . Then, the supernatant is removed by suction, DMEM containing 1% methylcellulose, 2% FBS, penicillin (100 units / mL) and streptomycin (100 μg / mL) is added, and the mixture is added at 37 ° C. and 5% CO ₂ for 72 to 96 hours. It was cultured. After culturing, the cells were fixed with a 10% neutral buffered formalin solution, 0.5 ml of a 0.05% methylene blue solution was added, and after staining for 1 hour, the number of plaques per well was counted to determine the number of plaques per 1 mL of the sample solution. For each test substance / concentration, N = 3 was tested, and based on the obtained values, a sigmoid curve was created using the XLfit program (IDBS), and the initial virus titer was reduced to 1/100 and 1/1000. The concentration required for the virus was calculated.

The amount of viral RNA in the supernatant after infection with SARS-CoV-2 JPN / TY / WK-521 strain for 18 hours in a medium containing a test compound having a concentration of 10 μM was evaluated by real-time PCR. As a result, the amount of viral RNA in the control group (No treatment; in the absence of the compound) was 4.8x10 ¹⁰ copy / well, while that in the Cicle-administered group was 5.2x10 ⁸ copy / well, showing a significant decrease. .. Among the compounds showing cytotoxicity equivalent to that of Cicle, the amount of viral RNA in the Cic4 administration group was 2.1x10 ⁵ copy / well, and the amount of virus in the Cic6 administration group was 4.6x10 ⁵ copy / well. (Fig. 3). On the other hand, the amount of viral RNA in the Cic11-1 administration group was 1.3x10 ⁹ copy / well, which was not significantly reduced as compared with the Cic administration group (Fig. 3). In addition, Cic2, Cic3, and Cic7 were mentioned as compounds that showed a significant reduction in the amount of viral RNA between the Cicle-administered group, but the cytotoxicity shown in FIG. 3 was significantly higher than that of the Cicle-administered group. Was obtained, so it was excluded from the subsequent evaluation targets.

In order to further evaluate the viral growth inhibitory effect of Cic4 and Cic6 administration, the SARS-CoV-2 JPN / TY / WK-521 strain was infected with the SARS-CoV-2 JPN / TY / WK-521 strain in a medium containing different concentrations of the test compound for 18 hours, and then the virus in the cell supernatant. Titer was assessed by plaque assay. As shown in FIG. 4, Cicle, Cic2, Cic4, and Cic6 all showed a virus growth inhibitory effect in a concentration-dependent manner, and the virus titer (about 4.6x10 ⁵ PFU / mL) observed in the control group (DMSO) was 1 /. The concentrations required to reduce to 100 or 1/1000 were 5.86 μM,> 80.00 μM for Cicle, 24.55 μM, 60.95 μM for Cic2, 6.51 μM, 7.36 μM for Cic4, 5.95 for Cic6. It was shown that Cic4 and Cic6 have a higher virus growth inhibitory effect than Cicle and Cic2, such as μM and 6.50 μM.

It can be used for the prevention and / or treatment of the new coronavirus.

Claims (16)

A novel compound having the following structural formula.

An anti-coronavirus agent comprising the following compound as an active ingredient and having an antiviral action against coronavirus.

An anti-coronavirus agent comprising the following compound as an active ingredient and having an antiviral action against coronavirus.

An anti-coronavirus agent comprising the following compound as an active ingredient and having an antiviral action against coronavirus.

An anti-coronavirus agent comprising the following compound as an active ingredient and having an antiviral action against coronavirus.

The anti-coronavirus agent according to any one of claims 2 to 5, wherein the coronavirus is a variant of wild-type coronavirus. The anti-coronavirus agent according to any one of claims 2 to 5, wherein the coronavirus is a wild-type coronavirus. The anti-coronavirus agent according to claim 7, wherein the wild-type coronavirus comprises a coronavirus belonging to the subfamily Coronavirinae. The anti-coronavirus agent according to claim 8, wherein the coronavirus belonging to the coronavirinae subfamily is SARS-CoV-2. The coronaviruses belonging to the subfamily coronavirus are SARS coronavirus, MERS coronavirus, human coronavirus 229E, human coronavirus OC43, human coronavirus HKU1, human coronavirus NL63, feline infectious peritonitis virus, canine coronavirus, and chicken. Infectious bronchitis virus, bovine coronavirus, and porcine coronavirus including infectious gastroenteritis virus, porcine delta coronavirus, porcine epidemic diarrheavirus, porcine respiratory coronavirus, porcine blood cell-aggregating encephalomyelitis coronavirus The anti-coronavirus agent according to claim 8, wherein the anti-coronavirus agent is selected from the group consisting of. The anticoronavirus agent according to any one of claims 2 to 10, which is 1 μM to 80 μM. The anti-coronavirus agent according to claim 2, which is 7 μM to 40 μM. The anti-coronavirus agent according to claim 2, which is 6 μM to 20 μM. The anti-coronavirus agent according to claim 3, wherein the size is 5 μM to 40 μM. The anti-coronavirus agent according to claim 3, which is 6 μM to 20 μM. Any 1 of claims 2 to 15, wherein the anti-coronavirus agent is a medicinal anti-coronavirus agent for preventing infection with the coronavirus and / or treating a disease caused by the coronavirus. The anti-coronavirus agent described in the section.

Images