Pharmaceutical composition containing uracil derivative

Abstract

To provide a compound that exhibits coronavirus growth-inhibiting activity.SOLUTION: There are provided a compound represented by formula (II), or a pharmaceutically acceptable salt thereof. (In the formula, X is a single bond or -CH2-; R2 is a substituted or unsubstituted aromatic carbocyclic group; R3c is a substituted or unsubstituted aromatic carbocyclic group, substituted or unsubstituted non-aromatic carbocyclic group, substituted or unsubstituted aromatic heterocyclic group, substituted or unsubstituted non-aromatic heterocyclic group, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted amino; R1 is a substituted or unsubstituted aromatic heterocyclic group or substituted or unsubstituted non-aromatic heterocyclic group; m is 0 or 1; each R5a is independently a hydrogen atom or a substituted or unsubstituted alkyl; and each R5b is independently a hydrogen atom or a substituted or unsubstituted alkyl.)SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition containing a compound exhibiting coronavirus 3CL protease inhibitory activity.

Background Art

[0002] Coronaviruses belonging to the subfamily Orthocoronavirinae of the family Coronaviridae, order Nidovirales, have a genome size of about 30 kilobases and are the largest single-stranded + strand RNA viruses among known RNA viruses. Coronaviruses are classified into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. As coronaviruses that infect humans, a total of seven types are known, including two types (HCoV-229E, HCoV-NL63) of the genus Alphacoronavirus and five types (HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, SARS-CoV-2) of the genus Betacoronavirus. Among these, four types (HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC43) are pathogens of the common cold, while the remaining three types are severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), and novel coronavirus (SARS-CoV-2), which cause severe pneumonia.

[0003] The novel coronavirus disease (COVID-19) that emerged in December 2019 rapidly spread across the international community and was declared a pandemic by the WHO on March 11, 2020. Droplet infection, contact infection, and aerosol infection have been reported as the main routes of infection of SARS-CoV-2, and it has been confirmed that SARS-CoV-2 can continue to float in the air with aerosols for about 3 hours and maintain its infectivity (Non-Patent Document 1). The incubation period is about 2 to 14 days, and typical cold-like symptoms such as fever (87.9%), dry cough (67.7%), fatigue (38.1%), and sputum (33.4%) are common (Non-Patent Document 2). In severe cases, respiratory failure due to acute respiratory distress syndrome, acute lung injury, interstitial pneumonia, etc. occurs. In addition, multiple organ failure such as renal failure and liver failure has also been reported.

[0004] In Japan, from the drug repositioning of existing drugs, remdesivir, an antiviral drug, dexamethasone, an anti-inflammatory drug, and baricitinib, a rheumatism drug, have been approved as therapeutic drugs for COVID-19, and tocilizumab, an anti-IL-6 receptor antibody, was additionally approved in January 2022. In addition, in July 2021, ronapreve (casirivimab / imdevimab), an antibody cocktail therapy, was approved under special circumstances, sotrovimab was approved under special circumstances in September 2021, and molnupiravir was approved under special circumstances in December 2021. Sufficient evidence has not been obtained regarding the efficacy and safety of these drugs. Therefore, the development of therapeutic drugs for COVID-19 is an urgent task.

[0005] When the coronavirus infects a cell, it synthesizes two polyproteins. These two polyproteins contain a replication complex that makes the viral genome and two proteases. The protease cleaves the polyprotein synthesized from the virus and plays an essential role in enabling each protein to function. Of the two proteases, 3CL protease (main protease) is responsible for most of the cleavage of the polyprotein (Non-Patent Document 3). As a COVID-19 therapeutic targeting 3CL protease, in June 2021, the completion of the Phase 1b trial of Lufotrelvir (PF-07304814), a prodrug of PF-00835231 by Pfizer, was posted on ClinicalTrials.gov (NCT04535167). Also in March 2021, Pfizer announced the start of a Phase 1 trial of PF-07321332, a therapeutic for COVID-19. The structural formulas of PF-00835231, Lufotrelvir, and PF-07321332 are as shown below, and they have different chemical structures from the compounds used in the present invention (Non-Patent Documents 4, 8, and 9, and Patent Documents 1 and 2). PF-00835231:

Chem.

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[0006] Also, as a COVID-19 therapeutic targeting 3CL protease, in August 2021, the start of the Phase 1 trial of PBI-0451 by Pardes Biosciences was posted on ClinicalTrials.gov (NCT05011812). The structural formula of PBI-0451 is as shown below, and it has a different chemical structure from the compounds used in the present invention (Non-Patent Document 12).

Chem.

[0007] Furthermore, Ensitrelvir Fumarate, the active ingredient of Zocova (registered trademark), was approved under emergency authorization in Japan on November 22, 2022, and under regular authorization on March 5, 2024, as a COVID-19 therapeutic targeting 3CL protease (Non-Patent Documents 17 and 18). The active ingredient of Zocova is Ensitrelvir Fumarate, and its structural formula is as shown below, with a chemical structure different from the compounds used in the present invention (Patent Documents 10 to 13).

Chemical formula

[0008] On the other hand, sufficient evidence has not been obtained regarding resistance mutations to COVID-19 therapeutics targeting 3CL protease.

[0009] Compounds having 3CL protease inhibitory activity are disclosed in Non-Patent Documents 4 to 7 and 13 to 16, but neither the compounds used in the present invention nor any suggestions thereof are described in any of these documents. Also, compounds having 3CL protease inhibitory activity are disclosed in Patent Documents 14 and 15, but neither pharmaceutical compositions containing the compounds according to the present invention nor any suggestions thereof are described in any of these documents. Ρ2X3 and / or Ρ2X 2 / 3 Compounds having P2X3 and / or P2X receptor inhibitory effects are disclosed in Patent Documents 3 to 9, but neither the 3CL protease inhibitory activity nor the antiviral effect is described or suggested in any of these documents. Compounds having HIV-1 reverse transcriptase inhibitory effects are described in Non-Patent Document 11, but neither the 3CL protease inhibitory activity nor the anti-coronavirus effect is described or suggested.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

[0011] [Non-Patent Document 1] The NEW ENGLAND JOURNAL of MEDICINE (2020), Vol. 382, pp. 1564 - 1567 [Non-Patent Document 2] "Report of the WHO-China Joint Mission on Coronavirus Disease 2019 (COVID-19)", [online], February 28, 2020, WHO, [searched on March 16, 2023], Internet <URL:https: / / www.who.int / docs / default-source / coronaviruse / who-china-joint-mission-on-covid-19-final-report.pdf> [Non-Patent Document 3] Science (2003), Vol. 300, pp. 1763 - 1767 [Non-Patent Document 4] "A comparative analysis of SARS-CoV-2 antivirals characterizes 3CLpro inhibitor PF-00835231 as a potential new treatment for COVID-19", Journal of Virology, 2021 Mar 10;95(7), e01819 - 20 [Non-Patent Document 5] Cell Research (2020), Vol. 30, pp. 678 - 692 [Non-Patent Document 6] Science (2020), Vol. 368, pp. 409 - 412 [Non-Patent Document 7] ACS Central Science (2021), Vol. 7, No. 3, pp. 467 - 475 [Non-Patent Document 8] 261st Am Chem Soc (ACS) Natl Meet · 2021 - 04 - 05 / 2021 - 04 - 16 · Virtual, N / A · Abst 243 [Non-Patent Document 9] Science (2021), Vol. 374, pp. 1586 - 1593 [Non-Patent Document 10] "Pfizer’s Novel COVID-19 Oral Antiviral Treatment Candidate Reduced Risk Of Hospitalization Or Death By 89% In Interim Analysis Of Phase 2 / 3 EPIC-HR Study", [online], November 5, 2021, Pfizer Press Release, [searched on March 16, 2023], Internet <URL:https: / / www.pfizer.com / news / press-release / press-release-detail / pfizers-novel-covid-19-oral-antiviral-treatment-candidate>

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

[0012] An object of the present invention is to provide a pharmaceutical composition containing a compound having a coronavirus 3CL protease inhibitory activity. Preferably, the present invention provides a medicament containing a compound having an antiviral action, particularly an inhibitory action on the growth of coronavirus. [Means for Solving the Problems]

[0013] The present invention relates to the following. (1) A pharmaceutical composition containing a compound represented by formula (I-1): [Chemical formula] or a pharmaceutically acceptable salt thereof. (2) The pharmaceutical composition according to item (1) above, which is a 3CL protease inhibitor. (3) The pharmaceutical composition according to item (1) or (2) above, which is used for inhibiting the viral growth of SARS-CoV-2. (4) The pharmaceutical composition according to any one of items (1) to (3) above, which is a therapeutic agent and / or a prophylactic agent for coronavirus disease 2019 (COVID-19). (5) The pharmaceutical composition according to any one of items (1) to (4) above, which is used for suppressing the exacerbation of an infection caused by SARS-CoV-2. (6) The pharmaceutical composition according to any one of items (1) to (4) above, which is used such that administration is started within 72 hours after the onset of symptoms of an infection caused by SARS-CoV-2 or from the positive determination of SARS-CoV-2. The pharmaceutical composition according to any one of the above items (1) to (4), which is used to start administration within 24 hours after the onset of symptoms of the infection caused by SARS-CoV-2 or after the determination of being positive for SARS-CoV-2. The pharmaceutical composition according to any one of the above items (1) to (4), which is used to start administration within 120 hours after the onset of symptoms of the infection caused by SARS-CoV-2 or after the determination of being positive for SARS-CoV-2. The pharmaceutical composition according to any one of the above items (1) to (4), which is used to start administration within 48 hours after the onset of symptoms of the infection caused by SARS-CoV-2 or after the determination of being positive for SARS-CoV-2. The pharmaceutical composition according to any one of the above items (1) to (4), which is used to suppress the viral transmission of SARS-CoV-2. The pharmaceutical composition according to any one of the above items (1) to (4), wherein the symptoms of the infection caused by SARS-CoV-2 are mild or moderate symptoms of type I.

[0014] A method for inhibiting the viral growth of SARS-CoV-2, which comprises administering to an individual in need of treatment and / or prevention of coronavirus disease 2019 (COVID-19) a compound represented by formula (I-1):

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[0015] (21) Use of a compound represented by formula (I-1):

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[0016] Furthermore, the present invention relates to the following. (101) A medicament for the prevention of coronavirus disease (COVID-19), which is used for suppressing the onset after exposure to SARS-CoV-2, and has the following formula (I-1):

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[0017] Furthermore, the present invention relates to the following. (201) A medicament for preventing coronavirus disease 2019 (COVID-19), which is used for pre-exposure prophylaxis of SARS-CoV-2, and has the following formula (I-1):

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[0018] Also, as one embodiment, the present invention includes the following. (301) Formula (II):

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Advantages of the Invention

[0019] The compound according to the present invention has inhibitory activity against coronavirus 3CL protease, and the pharmaceutical composition containing the compound according to the present invention is useful as a therapeutic agent and / or a prophylactic agent for coronavirus infections.

Brief Description of the Drawings

[0020]

Figure 1

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Figure 7

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Mode for Carrying Out the Invention

[0021] The meanings of the terms used in this specification are explained below. Unless otherwise specified, each term is used with the same meaning whether used alone or in combination with other terms. The term "consisting of" means having only the constituent elements. The terms "comprising" and "including" mean not being limited to the constituent elements and not excluding elements not described. Hereinafter, the present invention will be described while showing embodiments. Throughout this specification, it should be understood that singular expressions also include the concepts of their plural forms unless otherwise specified. Therefore, singular articles (for example, "a", "an", "the", etc. in English) should be understood to also include the concepts of their plural forms unless otherwise specified. Also, it should be understood that the terms used in this specification are used in the meanings commonly used in the above field unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technological terms used in this specification have the same meanings as generally understood by those skilled in the art to which the present invention pertains. In case of contradiction, this specification (including definitions) shall prevail.

[0022] In the compound represented by formula (II) in this specification, R 1 , R 2 , R 3c , R 5a and R 5b The definitions of the terms used in can incorporate WO 2023 / 195529 by reference, and its disclosure is incorporated herein by reference. Similarly, regarding the definitions of the substituents used in each term, WO 2023 / 195529 can be incorporated by reference, and its disclosure is incorporated herein by reference.

[0023] The compounds represented by formula (I-1) or formula (II) are not limited to specific isomers, and include all possible isomers (e.g., keto-enol isomers, imine-enamine isomers, diastereoisomers, optical isomers, rotational isomers, etc.), racemates or mixtures thereof.

[0024] One or more hydrogens, carbons and / or other atoms of the compound represented by formula (I-1) or formula (II) may each be substituted with an isotope of hydrogen, carbon and / or other atoms. Examples of such isotopes include, respectively 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, 123 I and 36 Cl, etc., including hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine. The compounds represented by formula (I-1) or formula (II) also include compounds substituted with such isotopes (e.g., deuterium-converted products, etc.). The compounds substituted with such isotopes are also useful as pharmaceuticals. The compounds represented by formula (I-1) or formula (II) include all radiolabeled substances substituted with radioisotopes contained in such isotopes. Also included in the present invention is a "radioisotope labeling method" for producing the "radiolabeled substance", and the "radiolabeled substance" is useful as a tool for metabolic pharmacokinetics research, research in binding assays and / or diagnosis.

[0025] Examples of the deuterium-converted product of the compound represented by formula (I-1) include the following structures.

Chemical formula

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[0026] The radiolabeled form of the compound represented by formula (I-1) or formula (II) can be prepared by methods well known in the art. For example, a tritium-labeled compound represented by formula (I-1) or formula (II) can be prepared by introducing tritium into a specific compound represented by formula (I-1) or formula (II) by a catalytic dehalogenation reaction using tritium. This method involves reacting a compound represented by formula (I-1) or formula (II) with a tritium gas in the presence or absence of a base, in the presence of a suitable catalyst such as Pd / C, with the compound represented by formula (I-1) or formula (II) being appropriately halogen-substituted. Other suitable methods for preparing tritium-labeled compounds can be found by referring to "Isotopes in the Physical and Biomedical Sciences, Vol. 1, Labeled Compounds (Part A), Chapter 6 (1987)". 14 The C-labeled compound can be 14 prepared by using a starting material having C carbon.

[0027] The compounds represented by formula (I-1) or formula (II) of the present invention may form prodrugs, and the present invention also includes such various prodrugs. A prodrug is a derivative of a compound of the present invention having a group that can be decomposed chemically or metabolically, and is a compound that becomes a pharmaceutically active compound of the present invention in vivo by solvolysis or under physiological conditions. Prodrugs include compounds that are enzymatically oxidized, reduced, hydrolyzed, etc. under physiological conditions in vivo to be converted into the compound represented by formula (I-1) or formula (II), compounds that are hydrolyzed by gastric acid, etc. to be converted into the compound represented by formula (I-1) or formula (II), and the like. Methods for selecting and producing appropriate prodrug derivatives are described, for example, in "Design of Prodrugs, Elsevier, Amsterdam, 1985". A prodrug may itself have activity.

[0028] As used herein, the "compound represented by formula (I-1) or formula (II)" may form a salt, a co-crystal, or a solvate thereof. As used herein, the "compound represented by formula (I-1) or formula (II), its pharmaceutically acceptable salt, or a solvate thereof" also includes such various salts, co-crystals, and solvates thereof.

[0029] As used herein, the "salt" means, for example, that the counter molecule is regularly arranged within the same crystal lattice as the "compound represented by formula (I-1) or formula (II)", and may contain any number of counter molecules. It refers to those via an ionic bond by proton transfer between the compound and the counter molecule in the crystal lattice.

[0030] As used herein, "cocrystal" means that counter molecules (co-former molecules) are regularly arranged within the same crystal lattice, and may contain any number of counter molecules (co-former molecules). Further, a cocrystal refers to a case where the intermolecular interaction between a compound and counter molecules (co-former molecules) is mediated by non-covalent and non-ionic chemical interactions such as hydrogen bonds and van der Waals forces.

[0031] Generally, it is considered that proton transfer occurs between a compound and counter molecules in a salt, but it is also known that in some cases, the proton transfer may not be complete. Since this state is not a true salt, it may be called a cocrystal. It is also known that proton transfer may change continuously depending on temperature. Therefore, the "pharmaceutically acceptable salt of the compound represented by formula (I-1) or formula (II)" as used herein includes a cocrystal and refers to a pharmaceutically acceptable salt or cocrystal of the compound represented by formula (I-1) or formula (II).

[0032] One aspect herein is a pharmaceutically acceptable salt or cocrystal of the compound represented by formula (I-1) or formula (II) with hydrofluoric acid, hydrochloric acid, hydrobromic acid, orthophosphoric acid, hydroiodic acid, nitric acid, phosphoric acid, boric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, trifluoromethylbenzenesulfonic acid, chlorobenzenesulfonic acid, methoxybenzenesulfonic acid, acetic acid, propionic acid, lactic acid, citric acid, fumaric acid, malonic acid, malic acid, succinic acid, salicylic acid, maleic acid, glycerophosphoric acid, tartaric acid, benzoic acid, glutamic acid, aspartic acid, 2-naphthalenesulfonic acid, hexanoic acid, acetylsalicylic acid, etc.

[0033] Studies on salt formation and cocrystal formation provide means to alter the physicochemical and resulting biological characteristics of a drug without changing its chemical structure. Salt formation and cocrystal formation can have a dramatic impact on the properties of a drug. In the selection of an appropriate salt or cocrystal, hygroscopicity, stability, solubility and processing characteristics are also important aspects. The solubility of a salt or cocrystal can affect its suitability for use as a drug. If the aqueous solubility is low, the dissolution rate in in vivo administration is rate-limiting in the absorption process and can result in low bioavailability. Also, due to low water solubility, administration by injection can be difficult, which can limit the choice of an appropriate administration route.

[0034] The "compound represented by formula (I-1) or formula (II)" can form a solvate with water (i.e., hydrate) or a solvate with a common organic solvent. The "pharmaceutically acceptable salt of the compound represented by formula (I-1) or formula (II)" can form a solvate with water (i.e., hydrate) or a solvate with a common organic solvent.

[0035] As used herein, "solvate" refers to, for example, a compound represented by formula (I-1) or formula (II) that is regularly arranged with any number of solvent molecules. Examples of the solvent molecule include ethyl acetate, water, ethanol, acetone, 1,1 - diethoxypropane, 1,1 - dimethoxymethane, 2,2 - dimethoxypropane, isooctane, isopropyl ether, methyl isopropyl ketone, methyl tetrahydrofuran, petroleum ether, trichloroacetic acid, trifluoroacetic acid, acetic acid, anisole, 1 - butanol, 2 - butanol, n - butyl acetate, t - butyl methyl ether, cumene, dimethyl sulfoxide, diethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3 - methyl - 1 - butanol, methyl ethyl ketone, methyl isobutyl ketone, 2 - methyl - 1 - propanol, pentane, 1 - pentanol, 1 - propanol, 2 - propanol, propyl acetate, tetrahydrofuran, acetonitrile, chlorobenzene, chloroform, cyclohexane, 1,2 - dichloroethene, dichloromethane, 1,2 - dimethoxyethane, N,N - dimethylacetamide, N,N - dimethylformamide, 1,4 - dioxane, 2 - ethoxyethanol, ethylene glycol, formamide, hexane, methanol, 2 - methoxyethanol, methyl butyl ketone, methylcyclohexane, N - methylpyrrolidone, nitromethane, pyridine, sulfolane, tetralin, toluene, 1,1,2 - trichloroethene, xylene, and t - butanol. Preferably, ethyl acetate, water, ethanol, acetone, 1,1 - diethoxypropane, 1,1 - dimethoxymethane, 2,2 - dimethoxypropane, isooctane, isopropyl ether, methyl isopropyl ketone, methyl tetrahydrofuran, petroleum ether, trichloroacetic acid, trifluoroacetic acid, acetic acid, anisole, 1 - butanol, 2 - butanol, n - butyl acetate, t - butyl methyl ether, cumene, dimethyl sulfoxide, diethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3 - methyl - 1 - butanol, methyl ethyl ketone, methyl isobutyl ketone, 2 - methyl - 1 - propanol, pentane, 1 - pentanol, 1 - propanol, 2 - propanol, propyl acetate, and tetrahydrofuran are included. More preferably, ethyl acetate, water, ethanol, acetone, 1,1 - diethoxypropane, 1,1 - dimethoxymethane, 2,2 - dimethoxypropane, isooctane, isopropyl ether, methyl isopropyl ketone, methyl tetrahydrofuran, petroleum ether, trichloroacetic acid, trifluoroacetic acid, etc. may be mentioned. In addition, when the "compound represented by formula (I - 1) or formula (II)" is left in the air, it may absorb moisture and adsorbed water may adhere, or a hydrate may be formed.

[0036] Note that the "crystal" used in this specification means a solid in which atoms, ions, molecules, etc. that make it up are arranged regularly in three dimensions, and is distinguished from an amorphous solid that does not have such a regular internal structure. The crystals used in this specification may be single crystals, twins, polycrystals, etc. Furthermore, there may be "crystal polymorphs" in the "crystal" that have the same composition but different arrangements in the crystal, and these are included in the "crystal form". The "compound represented by formula (I - 1) or formula (II), its pharmaceutically acceptable salt, or their solvates" includes their crystal polymorphs. The crystal of the compound according to the present invention may be a deuterium - converted form. The crystals used in this specification may be labeled with isotopes (e.g., 2 H, 3 H, 14 C, 35 S, 125 I, etc.). The crystal form and / or crystallinity can be confirmed by spectroscopic methods such as X - ray diffraction, Raman spectroscopy, infrared absorption spectroscopy, solid - state NMR, etc. Also, the physical properties of the crystal can be confirmed by many techniques such as differential scanning calorimetry, moisture adsorption - desorption measurement, dissolution characteristics, etc.

[0037] One aspect in this specification is an anhydrous crystal of the compound represented by formula (I - 1). The "anhydride" used in this specification is synonymous with "non - solvate", "non - solvated", "anhydrate" and "non - hydrated". The anhydrous crystals of the compound represented by formula (I-1) have a theoretical content of crystal water of 0% by weight. However, in the analysis of the water content and / or solvent content, it may take a value higher than the theoretical content of crystal water due to the influence of the adhering water and / or adhering solvent attached to the crystal surface.

[0038] One aspect herein is the anhydrous crystals of the compound represented by formula (I-1), which have characteristic peaks at diffraction angles (2θ): 6.5° ± 0.2°, 15.6° ± 0.2°, 17.4° ± 0.2°, 19.9° ± 0.2° and 20.3° ± 0.2° in the powder X-ray diffraction pattern (CuKα ray, λ = 1.5418 Å).

[0039] One aspect herein is when a single crystal diffraction experiment is carried out at 298 K (25 °C) with CuKα ray (λ = 1.5418 Å). The following crystallographic data: Space group: Pbca a = 14.67 Å ± 0.05 Å b = 11.83 Å ± 0.05 Å c = 27.10 Å ± 0.05 Å α = 90° β = 90° γ = 90° The anhydrous crystals of the compound represented by formula (I-1), which are characterized by the above.

[0040] One aspect herein is the anhydrous crystals of the compound represented by formula (I-1), which have a melting point of 261.3 °C ± 2 °C in differential scanning calorimetry (DSC). One aspect herein is the anhydrous crystals of the compound represented by formula (I-1), which have a melting point of 265.6 °C ± 2 °C in simultaneous differential thermal - thermogravimetric measurement (TG / DTA).

[0041] One aspect herein is in the Raman spectrum, at 415.2 cm -1 ± 2 cm -1 、502.7 cm -1 ± 2 cm -1 、1431.4 cm -1 ± 2 cm -1, 1714.8 cm -1 ±2 cm -1 and 3065.4 cm -1 ±2 cm -1 It is an anhydrous crystal of the compound represented by formula (I-1), having peaks characteristic of

[0042] (Powder X-ray diffraction (XRPD)) Powder X-ray diffraction (XRPD) is one of the most sensitive analytical methods for measuring the crystal form and crystallinity of solids. When X-rays irradiate a crystal, they are reflected by the crystal lattice planes, interfere with each other, and show diffraction lines corresponding to the periodicity of the structure. On the other hand, for amorphous solids, since they usually do not have an ordered repeating period in their structure, the diffraction phenomenon does not occur, and they show a broad XRPD pattern without features (also called a halo pattern).

[0043] The crystal form of the compound represented by formula (I-1) or formula (II) can be identified by a powder X-ray diffraction pattern and characteristic diffraction peaks. The crystal form of the compound represented by formula (I-1) or formula (II) can be distinguished from other crystal forms by the presence of characteristic diffraction peaks. The characteristic diffraction peaks used in this specification are peaks selected from the observed diffraction pattern. The characteristic diffraction peaks are preferably selected from about 10 peaks, more preferably about 5 peaks, and even more preferably about 3 peaks in the diffraction pattern. In distinguishing multiple crystals, peaks confirmed in that crystal and not confirmed in other crystals are preferred characteristic peaks for identifying that crystal rather than the peak intensity. Even one or two such characteristic peaks can characterize the crystal. By comparing the measured charts, if these characteristic peaks match, it can be said that the powder X-ray diffraction patterns substantially match.

[0044] Generally, since an error can occur in the diffraction angle (2θ) in powder X-ray diffraction within a range of ±0.2°, the value of the diffraction angle in powder X-ray diffraction should be understood to include numerical values within a range of about ±0.2°. Therefore, not only crystals with exactly matching diffraction angles of peaks in powder X-ray diffraction but also crystals with matching diffraction angles of peaks with an error of about ±0.2° are included in the present invention.

[0045] The peak intensities shown in the following tables and figures generally can vary due to many factors, such as the effect of the preferred orientation of crystals with respect to the X-ray beam, the influence of coarse particles, the purity of the substance being analyzed, or the crystallinity of the sample. Also, the peak position can shift based on the variation in the sample height. Furthermore, different shifts are obtained according to the Bragg's law (nλ = 2dsinθ) when measuring using different wavelengths, and another XRPD pattern obtained by using such different wavelengths is also included in the scope of the present invention.

[0046] (Single crystal structure analysis) One method for identifying a crystal is to obtain crystallographic parameters in the crystal, and further, atomic coordinates (values indicating the spatial positional relationship of each atom) and a three-dimensional structure model. Refer to "Guide to X-ray Structure Analysis" written by Toshio Sakurai, published by Shoeka Publishing Co., Ltd. (1983), "X-Ray Structure Determination: A Practical Guide" written by Stout & Jensen, Macmillan Co., New York (1968), etc. Single crystal structure analysis is useful when identifying the structures of crystals of complexes, salts, optical isomers, tautomers, geometric isomers as in the present invention.

[0047] (Raman spectroscopy) The Raman spectrum shows the characteristics of the vibrations of molecules or composite systems. Its origin lies in the inelastic collisions between molecules and photons, which are the particles of light that include the light beam. The collision between a molecule and a photon results in an energy exchange, and as a result, the energy changes, causing the wavelength of the photon to change. That is, since the Raman spectrum is a very narrow spectral line emitted when photons are incident on the target molecule, a laser or the like is used as the light source. The wavelength of each Raman line is represented by the wavenumber shift from the incident light, which is the difference between the reciprocal of the wavelength of the Raman line and that of the incident light. The Raman spectrum measures the vibrational state of a molecule, which is determined by its molecular structure. Generally, the Raman spectrum peak (cm -1 ) may have an error within the range of ±2 cm -1 . Therefore, the value of the above Raman spectrum peak needs to be understood to include numerical values within the range of about ±2 cm -1 . Thus, not only crystals with exactly matching Raman spectrum peaks in the Raman spectrum, but also crystals with Raman spectrum peaks matching with an error of about ±2 cm -1 are included in the present invention.

[0048] (Differential Scanning Calorimetry (DSC)) DSC is one of the main measurement methods in thermal analysis and is a method for measuring the thermal properties of substances as aggregates of atoms and molecules. By DSC, the change in the amount of heat related to the temperature or time of the pharmaceutical active ingredient is measured, and a differential scanning calorimetry curve is obtained by plotting the obtained data against the temperature or time. From the differential scanning calorimetry curve, information on the onset temperature when the pharmaceutical active ingredient melts, the maximum value of the endothermic peak curve accompanying melting, and the enthalpy can be obtained. Regarding DSC, it is known that the observed temperature can depend on the rate of temperature change, the sample preparation technique used, and the specific apparatus. Therefore, the "melting point" in DSC refers to the onset temperature that is less affected by the sample preparation technique. The error range at the onset temperature obtained from the differential scanning calorimetry curve is approximately ±2°C. In the identification of crystal identity, not only the melting point but also the overall pattern is important, and it can vary somewhat depending on the measurement conditions and the measuring instrument.

[0049] (Thermogravimetry-Differential Thermal Analysis (TG / DTA)) TG / DTA is one of the main measurement methods in thermal analysis and is a method for measuring the weight and thermal properties of substances as aggregates of atoms and molecules. TG / DTA is a method for measuring the changes in weight and heat quantity of a pharmaceutical active ingredient with respect to temperature or time, and by plotting the obtained data against temperature or time, TG (thermogravimetry) and DTA (differential thermal analysis) curves can be obtained. From the TG / DTA curve, information on the weight and heat quantity changes related to the decomposition, dehydration, oxidation, reduction, sublimation, and evaporation of the pharmaceutical active ingredient can be obtained. Regarding TG / DTA, it is known that the observed temperature and weight change can depend on the rate of temperature change, the sample preparation technique used, and the specific apparatus. Therefore, the "melting point" in TG / DTA refers to the onset temperature that is less affected by the sample preparation technique. In the identification of crystal identity, not only the melting point but also the overall pattern is important, and it can vary somewhat depending on the measurement conditions and the measuring instrument.

[0050] Since the compound according to the present invention has coronavirus 3CL protease inhibitory activity, it is useful as a therapeutic agent and / or prophylactic agent for diseases involving coronavirus 3CL protease. In the present invention, the term "therapeutic agent and / or prophylactic agent" includes symptom relievers. In the present invention, the term "prevention" includes suppressing the onset after exposure to SARS-CoV-2. In the present invention, when referring to "prevention", it includes suppressing the onset of symptoms caused by SARS-CoV-2 by administering the pharmaceutical composition of the present invention to an individual who has been exposed to SARS-CoV-2 and requires suppression of onset. In the present invention, when referring to "prevention", it includes pre-exposure prophylaxis against SARS-CoV-2. In the present invention, when referring to "prevention", it includes suppressing the onset of symptoms caused by SARS-CoV-2 by administering the pharmaceutical composition of the present invention to an individual who has been pre-exposed to SARS-CoV-2 and requires suppression of onset. When referring to "prevention" of the present invention, it includes suppressing the onset and exacerbation after exposure to SARS-CoV-2.

[0051] Diseases involving coronavirus 3CL protease include viral infectious diseases, preferably coronavirus infectious diseases. In one aspect, coronaviruses include human-infecting coronaviruses. Human-infecting coronaviruses include HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and / or SARS-CoV-2. In one aspect, coronaviruses include alphacoronaviruses and / or betacoronaviruses, more preferably betacoronaviruses, and even more preferably sarbecoviruses. In one aspect, alphacoronaviruses include HCoV-229E and HCoV-NL63. Particularly preferably, HCoV-229E is included. In one aspect, betacoronaviruses include HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and / or SARS-CoV-2. Preferably HCoV-OC43 or SARS-CoV-2, and particularly preferably SARS-CoV-2. In one aspect, examples of beta coronaviruses include beta coronavirus lineage A, beta coronavirus lineage B, and beta coronavirus lineage C. More preferably, beta coronavirus lineage A and beta coronavirus lineage B are included, and particularly preferably, beta coronavirus lineage B is included. Examples of beta coronavirus lineage A include, for example, HCoV-HKU1 and HCoV-OC43, and preferably, HCoV-OC43. Examples of beta coronavirus lineage B include, for example, SARS-CoV and SARS-CoV-2, and preferably, SARS-CoV-2. Examples of beta coronavirus lineage C preferably include MERS-CoV. In one aspect, examples of coronaviruses include HCoV-229E, HCoV-OC43, and / or SARS-CoV-2, and particularly preferably, SARS-CoV-2. It is generally known that viruses mutate during repeated growth and infection. The above-mentioned coronaviruses include not only known mutants in the art but also mutants that will appear in the future, as long as the compounds according to the present invention can exhibit coronavirus 3CL protease inhibitory activity against the strain. Examples of known mutants of SARS-CoV-2 include, for example, the mutants used in the examples of this specification. Examples of coronavirus infections include those caused by HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV, MERS-CoV, and / or SARS-CoV-2. Preferably, infections caused by HCoV-229E, HCoV-OC43, and / or SARS-CoV-2 are included, and particularly preferably, infections caused by SARS-CoV-2 are included. Among coronavirus infections, particularly preferably, novel coronavirus infection (COVID-19) is included.

[0052] The severity classification of novel coronavirus-infected patients is exemplified as follows. (Reference: Guidelines for the Diagnosis and Treatment of Coronavirus Disease 2019 (COVID-19), 10.0th Edition (Ministry of Health, Labour and Welfare)) (Mild) The oxygen saturation is 96% or higher. Clinically, there are no respiratory symptoms, or there is only cough without dyspnea, and in either case, no pneumonia findings are observed. (Moderate I) The oxygen saturation is less than 96% and more than 93%. There is dyspnea and pneumonia findings. (Moderate II) The oxygen saturation is less than 93%. There is respiratory failure and oxygen administration is required. (Severe) The patient is admitted to the ICU or requires a ventilator. The above classification is the definition of severity classification in Japan, and for example, it is possible to adopt the severity classification in China or the NIH in the United States. In addition, asymptomatic SARS-CoV-2-infected patients mean asymptomatic pathogen carriers. For example, those who do not show 14 or 12 symptoms of COVID-19 are included. (14 symptoms of COVID-19: malaise (fatigue), muscle pain or body ache, headache, chills / sweating, feverishness or fever, taste disorder, smell disorder, runny nose or nasal congestion, sore throat, cough, shortness of breath (dyspnea), nausea, vomiting, diarrhea)

[0053] In this specification, the 12 symptoms of COVID-19 include (lassitude (sense of fatigue), muscle pain or body pain, headache, chills / sweating, feverishness or fever, runny nose or nasal congestion, sore throat, cough, shortness of breath (dyspnea), nausea, vomiting, diarrhea).

[0054] In this specification, suppressing disease progression includes suppressing the progression of asymptomatic SARS-CoV-2 infected individuals to a severity level classified as mild, moderate I, moderate II, or severe. In this specification, suppressing disease progression includes suppressing the progression of asymptomatic or mild SARS-CoV-2 infected individuals to a severity level classified as moderate I, moderate II, or severe. In this specification, suppressing disease progression includes suppressing the progression of asymptomatic, mild, or moderate I SARS-CoV-2 infected individuals to a severity level classified as moderate II or severe. In this specification, suppressing disease progression includes suppressing the progression of asymptomatic, mild, moderate I, or moderate II SARS-CoV-2 infected individuals to a severity level classified as severe. In this specification, suppressing disease progression includes reducing the risk of hospitalization and death of SARS-CoV-2 infected individuals through the virus growth inhibitory effect of this agent. In this specification, suppressing disease progression includes reducing inflammation in the lungs of SARS-CoV-2 infected individuals through the virus growth inhibitory effect of this agent. In this specification, suppressing disease progression includes suppressing pneumonia caused by SARS-CoV-2 viral infection through the virus growth inhibitory effect of this agent. In this specification, suppressing disease progression includes suppressing the excessive immune response of the host caused by SARS-CoV-2 viral infection through the virus growth inhibitory effect of this agent.

[0055] As one embodiment, the pharmaceutical composition of the present invention can be administered to SARS-CoV-2 infected patients who have at least one risk factor for severe disease. For information on underlying medical conditions related to severe disease, reference can be made to the summary by the US CDC (https: / / www.cdc.gov / coronavirus / 2019-ncov / hcp / clinical-care / underlyingconditions.html).

[0056] As one embodiment, the pharmaceutical composition of the present invention can be administered to SARS-CoV-2 infected patients who have at least one of the following risk factors for severe disease. Risk factors for severe disease: malignant tumors, metabolic diseases, cardiovascular diseases, respiratory diseases, liver diseases, kidney diseases, mental and neurological diseases, lack of exercise, pregnancy, smoking, children, genetic diseases, immunodeficiency

[0057] As one embodiment, the pharmaceutical composition of the present invention is administered to infected patients with at least one risk factor for severe disease and is used to suppress the progression of COVID-19 symptoms.

[0058] As one embodiment, the pharmaceutical composition of the present invention is used for patients with pneumonia caused by SARS-CoV-2.

[0059] As used herein, "prevention" includes suppressing the onset after exposure to SARS-CoV-2. For example, the pharmaceutical composition of the present invention can be administered to cohabiting family members or household contacts of patients with COVID-19. For example, after contact with a COVID-19 patient, the pharmaceutical composition of the present invention can be promptly administered (e.g., within 72 hours) to the contacts. For example, the onset of asymptomatic SARS-CoV-2 infected patients can be suppressed by administering the pharmaceutical composition of the present invention. Also, "prevention" includes pre-exposure prophylaxis against SARS-CoV-2. For example, in cases where there is a risk of SARS-CoV-2 infection, such as during the COVID-19 pandemic, the pharmaceutical composition of the present invention can be administered to medical staff, the elderly, and those with risk factors for severe disease.

[0060] (Method for producing the compound represented by formula (I-1)) The compound represented by formula (I-1) can be produced, for example, by the general synthetic methods shown below. Extraction, purification, etc. may be performed by the processes used in ordinary organic chemistry experiments. Synthesis can be carried out with reference to methods known in the art. Extraction, purification, etc. may be performed by the processes used in ordinary organic chemistry experiments. The compound represented by formula (I-1) can be produced with reference to methods known in the art. For example, it can be produced with reference to WO2012 / 020742, WO2013 / 118855, WO2023 / 195529, WO2023 / 195530, etc. (Method for producing the compound represented by formula (II)) The compound represented by formula (II) can be produced, for example, with reference to WO2023 / 195529.

[0061] The compounds according to the present invention (e.g., the compound represented by formula (I-1), the compound represented by formula (I-2), and the compound represented by formula (II)) have coronavirus 3CL protease inhibitory activity, and thus are useful as therapeutic agents and / or prophylactic agents for viral infections. Furthermore, the compounds according to the present invention have utility as pharmaceuticals, and preferably have any one or a plurality of the following excellent characteristics. a) They have weak inhibitory effects on CYP enzymes (e.g., CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, etc.). b) They exhibit good pharmacokinetics such as high bioavailability and moderate clearance. c) They have high metabolic stability. d) They do not show irreversible inhibitory effects on CYP enzymes (e.g., CYP3A4) within the concentration range of the measurement conditions described herein. e) It has no mutagenicity. f) It has a low cardiovascular risk. g) It shows high solubility. h) It has a high protein unbound fraction (fu value). i) It has high selectivity for coronavirus 3CL protease. j) It has high coronavirus growth inhibitory activity. For example, it has high coronavirus growth inhibitory activity in the presence of human serum (HS) or human serum albumin (HSA). k) It also has high growth inhibitory activity against 3CL protease inhibitor-resistant viruses. As the coronavirus growth inhibitor, for example, in the CPE suppression effect confirmation test (SARS-CoV-2) described later, for example, EC 50 is 10 μM or less, preferably 1 μM or less, more preferably 100 nM or less.

[0062] The pharmaceutical composition of the present invention can be administered by either oral or parenteral methods. Examples of parenteral administration methods include transdermal, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, transmucosal, inhalation, intranasal, ophthalmic, otic, intravaginal administration, etc.

[0063] In the case of oral administration, it may be prepared into any of the commonly used dosage forms such as solid preparations for internal use (for example, tablets, powders, granules, capsules, pills, films, etc.), liquid preparations for internal use (for example, suspensions, emulsions, elixirs, syrups, lemonades, spirits, aromatic waters, extracts, decoctions, tinctures, etc.) according to conventional methods and then administered. Tablets may be sugar-coated tablets, film-coated tablets, enteric-coated tablets, sustained-release tablets, troches, sublingual tablets, buccal tablets, chewable tablets or orally disintegrating tablets. Powders and granules may be dry syrups. Capsules may be soft capsules, microcapsules or sustained-release capsules.

[0064] In the case of parenteral administration, it can be preferably administered in any of the commonly used dosage forms such as injections, drip infusions, external preparations (e.g., eye drops, nasal drops, ear drops, aerosol agents, inhalants, lotions, injections, coating agents, gargles, enemas, ointments, plasters, jelly agents, creams, patches, cataplasms, external powders, suppositories, etc.). The injection may be an emulsion such as O / W, W / O, O / W / O, W / O / W type, etc.

[0065] An effective amount of the compound according to the present invention can be mixed with various pharmaceutical additives such as excipients, binders, disintegrants, lubricants, etc. suitable for the dosage form as needed to form a pharmaceutical composition. Further, the pharmaceutical composition can be made into a pharmaceutical composition for pediatric, elderly, critically ill patients or surgical use by appropriately changing the effective amount of the compound according to the present invention, the dosage form and / or various pharmaceutical additives. For example, a pharmaceutical composition for pediatric use can be administered to patients such as neonates (less than 4 weeks after birth), infants (4 weeks to less than 1 year after birth), toddlers (over 1 year old and less than 7 years old), children (7 years old and over and less than 15 years old) or patients aged 15 to 18 years. For example, a pharmaceutical composition for the elderly can be administered to patients aged 65 years or older.

[0066] The dosage of the pharmaceutical composition of the present invention is preferably set in consideration of the patient's age, weight, type and degree of disease, administration route, etc. However, in the case of oral administration, as the compound according to the present invention, it is usually 0.01 to 100 mg / kg / day, preferably in the range of 0.05 to 50 mg / kg / day. In the case of parenteral administration, it varies greatly depending on the administration route, but as the compound according to the present invention, it is usually 0.005 to 200 mg / kg / day, preferably in the range of 0.01 to 100 mg / kg / day. It can be administered in divided doses once to several times a day.

[0067] The compounds according to the present invention may be used in combination with, for example, therapeutic agents for other coronavirus diseases (COVID-19) (such therapeutic agents include approved drugs and drugs under development or to be developed in the future) (hereinafter referred to as combination drugs) for the purpose of enhancing the action of the compounds or reducing the dosage of the compounds. At this time, the administration timing of the compounds according to the present invention and the combination drugs is not limited, and they may be administered simultaneously to the administration subject or at intervals. Further, the compounds according to the present invention and the combination drugs may be administered as two or more types of preparations containing their respective active ingredients, or may be administered as a single preparation containing their active ingredients.

[0068] The dosage of the combination drug can be appropriately selected based on the clinically used dosage. Further, the mixing ratio of the compound according to the present invention and the combination drug can be appropriately selected depending on the administration subject, administration route, target disease, symptoms, combination, etc. For example, when the administration subject is a human, 0.01 to 100 parts by weight of the combination drug may be used with respect to 1 part by weight of the compound according to the present invention.

Examples

[0069] The present invention will be described in more detail below with reference to Examples, Reference Examples, and Test Examples, but the present invention is not limited thereto.

[0070] In addition, the abbreviations used in this specification have the following meanings. FBS: Fetal bovine serum mM: mmol / L nM: nmol / L μM: μmol / L

[0071] (Method for identifying compounds) The NMR analysis obtained in each example was performed at 400 MHz and measured using DMSO-d6 and CDCl3. Further, when showing NMR data, there are cases where not all the measured peaks are described. The "RT" in the specification represents the retention time in LC / MS: liquid chromatography / mass spectrometry, and was measured under the following conditions. (Measurement condition A) Column: ACQUITY UPLC (registered trademark) BEH C18 (1.7μm i.d. 2.1x50mm) (Waters) Flow rate: 0.8 mL / min UV detection wavelength: 254 nm Mobile phase: [A] is an aqueous solution containing 0.1% formic acid, [B] is an acetonitrile solution containing 0.1% formic acid Gradient: A linear gradient of 5% - 100% solvent [B] was carried out for 3.5 minutes, and then 100% solvent [B] was maintained for 0.5 minutes.

[0072] Powder X-ray diffraction experiment (XRPD) According to the powder X-ray diffraction measurement method described in the general test method of the Japanese Pharmacopoeia, powder X-ray diffraction measurement of the solid form (crystal) obtained in the example was carried out. The measurement conditions are shown below. Measurement condition 1: Powder X-ray diffractometer: SmartLab manufactured by Rigaku Measurement method: Reflection method Wavelength used: CuKα ray (λ = 1.5418 Å) Tube current: 200 mA Tube voltage: 45 kV Sample plate: Aluminum Incident angle of X-ray: 2.5° Sampling width: 0.02° Detector: HyPix-3000 (two-dimensional detection mode)

[0073] Measurement and analysis method of single crystal structure analysis Single crystal structure analysis of the crystal obtained in the example was carried out. The measurement conditions and analysis methods are shown below. (Equipment) XtaLAB P200 MM007 manufactured by Rigaku (Measurement conditions) Measurement temperature: 25 °C Temperature controller: Sample spraying low-temperature device manufactured by Rigaku Wavelength used: CuKα line (λ = 1.5418 Å) Software: CrysAlisPro 1.171.39.46e (Rigaku Oxford Diffraction, 2018) (Data processing) Software: CrysAlisPro 1.171.39.46e (Rigaku Oxford Diffraction, 2018) The data was subjected to Lorentz and polarization corrections and absorption correction. (Crystal structure analysis) The phase determination was carried out using the direct method program ShelXT (Sheldrick, G.M., 2015), and the refinement was performed using ShelXL (Sheldrick, G.M., 2015) with the full-matrix least-squares method. The temperature factors of non-hydrogen atoms were all refined anisotropically. Hydrogen atoms were introduced by calculation using the default parameters of ShelXL unless otherwise specified and were treated as riding atoms. Also, the hydrogen atoms were refined with isotropic parameters. For the drawing of the following structural diagrams, PLATON (Spek, 1991) / ORTEP (Johnson, 1976) was used (30% PROBABILITY level).

[0074] Measurement of Raman spectrum The measurement conditions for the measurement of the Raman spectrum of the crystal obtained in the example and the baseline correction are shown below. Measurement condition 1 Measurement method: Microscopic laser Raman spectroscopy Laser wavelength: 671 nm Number of accumulations: 1 time Exposure time: 1 second

[0075] Differential scanning calorimetry (DSC) The DSC measurement of the crystal obtained in the example was carried out. The sample was weighed into an aluminum pan, simply sealed, and measured. The measurement conditions are shown below. Note that the measurement by differential scanning calorimetry (DSC) may have an error within the range of ±2 °C. Apparatus: Discovery DSC / TA Instrument Measurement temperature range: -10°C - 270°C Heating rate: 10°C / min Atmosphere: N2 50 mL / min

[0076] Simultaneous differential thermal - thermogravimetric measurement (TG / DTA) Simultaneous differential thermal - thermogravimetric measurement (TG / DTA) of the solid form (crystal) obtained in the example was performed. The sample obtained in the example was weighed and filled into an aluminum pan, and the measurement was carried out in an open system. The measurement conditions are as follows. Apparatus: Hitachi High - Technologies TG / DTA STA7200RV Measurement temperature range: room temperature - 350°C Heating rate: 10°C / min

[0077] HPLC measurement Equipment: Agilent 1290 Infinity VL Column: Waters Acquity UPLC BEH C18, 1.7 μm, 2.1×100 mm Column temperature: constant temperature near 40°C UV detection wavelength: 300 nm Mobile phase: [A] is an aqueous solution containing 0.1% formic acid, [B] is an acetonitrile solution containing 0.1% formic acid Gradient: Maintain 20% solvent [B] for 1 minute, perform a linear gradient from 20% - 80% solvent [B] in 32 minutes, and then maintain 20% solvent [B] for 7 minutes. Flow rate: 0.4 mL / min Injection volume: 3 μL

Example

[0078] Synthesis of compound (I - 1)

Chem.

[0079] Step 2 Synthesis of compound 4 Acetic acid (40 mL) and concentrated hydrochloric acid (41 mL) were added to compound 3 (14.8 g, 48.8 mmol), and the mixture was stirred at 110 °C for 5 hours. After the reaction solution was cooled to room temperature, water (80 ml) was added. The precipitate was collected by filtration and washed with water. After air drying, compound 4 (11.6 g, 42.2 mmol) was obtained. 1 1H-NMR (DMSO-d6) δ: 7.31 (ddd, J = 8.8, 4.9, 2.1 Hz, 1H), 7.45 (t, J = 8.8 Hz, 1H), 7.53 (dd, J = 7.3, 2.1 Hz, 1H), 11.57 (s, 1H), 12.24 (brs, 1H) LC / MS (ESI): m / z = 275, RT = 1.81 min, LC / MS measurement condition A

[0080] Step 3 Synthesis of compound 5 Compound 4 (1.00 g, 3.64 mmol), 5-chloropyridine-3-boronic acid (1.14 g, 7.27 mmol), copper(II) acetate (0.99 g, 5.45 mmol), acetonitrile (10 mL), triethylamine (5.04 mL, 36.4 mmol) and pyridine (7.34 mL, 91.0 mmol) were mixed, and the solution was stirred at room temperature overnight. Saturated aqueous sodium hydrogen carbonate solution (5 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with water, dried over sodium sulfate and filtered. The filtrate was concentrated, and the obtained residue was purified by silica gel column chromatography (chloroform:methanol = 100:0 to 90:10), and the solvent was distilled off under reduced pressure. The obtained residue was dried under reduced pressure to obtain Compound 5 (1.15 g, 2.97 mmol, yield 82%). 1 1H-NMR (DMSO-d6) δ: 7.35 - 7.37 (1H, m), 7.49 (1H, t, J = 9.0 Hz), 7.54 - 7.56 (1H, m), 8.07 (1H, t, J = 2.1 Hz), 8.54 (1H, d, J = 2.0 Hz), 8.70 (1H, d, J = 2.3 Hz). LC / MS (ESI): m / z = 386, RT = 1.98 min, LC / MS measurement condition A

[0081] Step 4 Synthesis of Compound 6 To a solution of Compound 5 (520 mg, 1.345 mmol), N,N-diisopropylethylamine (0.705 mL, 4.04 mmol) and DMF (5.2 mL) was added 2-bromoacetonitrile (269 μL, 4.04 mmol), and the mixture was stirred at room temperature overnight. Under ice-cooling, 2 mol / L hydrochloric acid (2 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with water, dried over sodium sulfate and filtered. The filtrate was concentrated, and the obtained residue was purified by silica gel column chromatography (chloroform:methanol = 100:0 to 99:1), and the solvent was distilled off under reduced pressure. The obtained residue was dried under reduced pressure to obtain Compound 6 (291 mg, 0.684 mmol, yield 51%). 1H-NMR (CDCl3) δ: 5.13 (2H, s), 7.23 - 7.24 (2H, m), 7.42 (1H, d, J = 7.3 Hz), 7.67 (1H, t, J = 2.1 Hz), 8.45 (1H, d, J = 2.3 Hz), 8.67 (1H, d, J = 2.3 Hz). LC / MS (ESI): m / z = 425, RT = 2.17 min, LC / MS measurement condition A

[0082] Step 5 Synthesis of compound (I-1) Compound 6 (25.0 mg, 0.059 mmol), 6,6-difluoro-2-azaspiro[3.3]heptane trifluoroacetate (17.4 mg, 0.070 mmol), N,N-diisopropylethylamine (20.5 μL, 0.117 mmol), and DMF (0.5 mL) were mixed, and the solution was stirred at 60 °C for 2 hours. Water (2 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with water, dried over sodium sulfate, and filtered. The filtrate was concentrated, and ethyl acetate (0.05 mL), hexane (0.125 mL), and diisopropyl ether (0.125 mL) were added. The resulting precipitate was collected by filtration and washed with diisopropyl ether. The obtained solid was dried under reduced pressure to obtain anhydrous crystals of compound (I-1) (22.0 mg, 0.042 mmol, yield 72%). 1 H-NMR (CDCl3) δ: 2.75 (4H, t, J = 12.0 Hz), 4.02 (4H, s), 4.74 (2H, s), 7.16 - 7.18 (2H, m), 7.32 - 7.35 (1H, m), 7.65 (1H, t, J = 2.1 Hz), 8.43 (1H, d, J = 2.3 Hz), 8.61 (1H, d, J = 2.3 Hz). LC / MS (ESI): m / z = 522, RT = 2.27 min, LC / MS measurement condition A

[0083] (Grinding of anhydrous crystals of the compound represented by formula (I-1)) The anhydrous crystals of the compound represented by formula (I-1) obtained in Example 1 were sieved through a 1000 μM mesh and then ground under the following conditions. Apparatus: A-O Jet Mill (Seishin Enterprise Co., Ltd.) Supply method: Feeder Supply rate: 20 g / hour Crushing pressure: 0.30 MPa Supply pressure: 0.40 MPa

[0084] (Powder X-ray diffraction experiment of the anhydrous crystal of the compound represented by formula (I-1)) For the anhydrous crystal of the compound represented by formula (I-1) after crushing, a powder X-ray diffraction experiment was carried out under the measurement conditions described above. The powder X-ray diffraction pattern is shown in Figure 7, and the peak list of the powder X-ray diffraction pattern is shown in Figure 8. Hereinafter, in the table of the peak list of the powder X-ray diffraction pattern, Position represents 2θ (°) and Intensity represents the intensity. In the powder X-ray diffraction pattern, peaks were observed at diffraction angles (2θ): 6.5° ± 0.2°, 10.1° ± 0.2°, 13.0° ± 0.2°, 14.1° ± 0.2°, 15.3° ± 0.2°, 15.6° ± 0.2°, 16.2° ± 0.2°, 17.4° ± 0.2°, 18.9° ± 0.2°, 19.9° ± 0.2°, 20.3° ± 0.2°, 21.7° ± 0.2°, 23.0° ± 0.2°, 23.8° ± 0.2°, 25.8° ± 0.2°, 28.8° ± 0.2° and 30.6° ± 0.2°. The anhydrous crystal of the compound represented by formula (I-1) showed characteristic peaks at diffraction angles (2θ): 6.5° ± 0.2°, 15.6° ± 0.2°, 17.4° ± 0.2°, 19.9° ± 0.2° and 20.3° ± 0.2° in the powder X-ray diffraction pattern.

[0085] (Single crystal structure analysis of the anhydrous crystal of the compound represented by formula (I-1)) <Method for preparing single crystal> To 1 mg of the crystal of the compound represented by formula (I-1), 400 μL of methanol was added and heated to 50 °C to dissolve it. The solution was dispensed into a 1.5 mL HPLC vial, the lid of the HPLC vial was put on, a syringe needle was pierced into the lid, and it was left standing at room temperature. Single crystals were prepared by the solvent evaporation method. <Single crystal structure analysis> The single crystal diffraction experiment and analysis were carried out by the method described above. Since Cl1 and Cl7C, and H5CA and H6CA are in a disorder relationship, they were analyzed with an occupancy of Cl1:Cl7C = 0.75:0.25 and H5CA:H6CA = 0.25:0.75.

[0086] The results of the single crystal structure analysis are shown below. R1(I>2.00s(I)) was 0.0555, and it was confirmed that there were no deficiencies or misplacements of electron density from the final difference Fourier.

[0087] The crystallographic data are shown in Table 1.

Table 1

[0088] The fractional coordinates x, y, z (Å×10 4 ) and the equivalent isotropic temperature factor U(eq) (Equivalent Isotropic Displacement Parameters, Å 2 ×10 3 ) of non-hydrogen atoms are shown in Table 2. Here, U(eq) is defined as one-third of the trace of the rectified U ij tensor. Note that the numbers of non-hydrogen atoms in Table 2 correspond to the numbers described in Figure 9 respectively.

Table 2

[0089] Next, the atomic coordinates x, y, z (Å×10 4 ) and the isotropic temperature factor U(eq) (Isotropic Displacement Parameters, Å 2 ×10 3 ) of hydrogen atoms are shown in Table 3.

Table 3

[0090] The structure in the asymmetric unit of the crystal structure is shown in Fig. 9. Note that the label numbers of the non-hydrogen atoms described in Fig. 9 correspond to the numbers of the non-hydrogen atoms in Table 2.

[0091] Since only one molecule of the compound represented by the formula (I-1) was present in the asymmetric unit of the crystal structure, it was identified as an anhydrous crystal of the compound represented by the formula (I-1).

[0092] From the crystal structure, the powder X-ray diffraction pattern (λ = 1.5418 Å) calculated using Mercury (The Cambridge Crystallographic Data Centre, Ver. 4.0.0) was confirmed to be generally consistent with the above powder X-ray diffraction pattern (Fig. 7).

[0093] (Differential Scanning Calorimetry of the Anhydrous Crystal of the Compound Represented by the Formula (I-1)) Approximately 2 mg of the anhydrous crystal of the compound represented by the formula (I-1) after pulverization was weighed into an aluminum pan and measured by the method described above. The results are shown in Fig. 10. An endothermic peak with an onset temperature of about 261.3 °C was shown.

[0094] (Simultaneous Differential Thermal-Thermogravimetric Measurement of the Anhydrous Crystal of the Compound Represented by the Formula (I-1)) The anhydrous crystal of the compound represented by the formula (I-1) after pulverization was measured by the method described above. The results are shown in Fig. 11. An endothermic peak with an onset temperature of about 265.6 °C was shown. Also, no weight loss was confirmed.

[0095] (Raman Spectrum Measurement of the Anhydrous Crystal of the Compound Represented by the Formula (I-1)) The anhydrous crystal of the compound represented by the formula (I-1) after pulverization was measured for its Raman spectrum under the above-described measurement condition 1. The results are shown in Fig. 12. Also, the main Raman peaks are shown below. [Table 4] The anhydrous crystal of the compound represented by formula (I-1) shows characteristic peaks in the Raman spectrum at 415.2 cm -1 ±2 cm -1 , 502.7 cm -1 ±2 cm -1 , 1431.4 cm -1 ±2 cm -1 , 1714.8 cm -1 ±2 cm -1 , and 3065.4 cm -1 ±2 cm -1 .

[0096] (HPLC measurement of the anhydrous crystal of the compound represented by formula (I-1)) The anhydrous crystal of the compound represented by formula (I-1) after pulverization was measured by the method described above. The results are shown in Figure 13 (about 96.5 pa%).

Example

[0097] Synthesis of compound (I-2)

Chemical formula

[0098] Step 1: Synthesis of compound 8 Compound 7 (2.6 g, 14.2 mmol), DMF (13 mL) and methyl alcohol-O 18 (1.18 mL, 29.1 mmol) were mixed, and sodium hydride (1.42 g, 35.4 mmol) was slowly added under ice cooling. The reaction solution was warmed to room temperature and stirred for 2 hours. The reaction solution was cooled in an ice bath, quenched by adding water (26 mL) and ethyl acetate (26 mL), and extracted with ethyl acetate. The organic layer was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (hexane:ethyl acetate = 10:0 to 9:1), and the solvent was distilled off under reduced pressure to obtain compound 8 (1.57 g, 8.79 mmol, yield 62%). LC / MS (ESI): m / z = 179, RT = 1.57 min, LC / MS measurement condition A 11H-NMR (CDCl3) δ: 3.97 (s, 3H), 4.00 (s, 3H), 6.41 (s, 1H)

[0099] Step 2: Synthesis of Compound 10 Compound 9 (0.5 g, 3.43 mmol), heavy water (17.1 mL, 945 mmol), and 5 mol / L DCl (0.69 mL, 3.43 mmol) were mixed and heated at 180 °C for 30 minutes using microwave. The same operation was repeated 10 times. All the reaction solutions were mixed, 50 mL of ethyl acetate and 51.5 mL of 1 mol / L sodium hydroxide solution were added, and the mixture was extracted with ethyl acetate and then washed with water and saturated brine. The organic layer was concentrated under reduced pressure to obtain Compound 10 (5.02 g, 34 mmol, yield 99%). 1 1H-NMR (CDCl3) δ: 3.59 (brs, 2H), 6.87 - 6.95 (m, 1H)

[0100] Step 3: Synthesis of Compound 11 Compound 10 (5 g, 33.9 mmol) and water (11.5 mL) were mixed, and hydrogen bromide (11.5 mL, 102 mmol) was added under ice-cooling. Then, NaNO2 (2.384 g, 34.6 mmol) was dissolved in water (7.5 mL) and added slowly. The reaction solution was heated to 30 °C, CuBr (6.32 g, 44.0 mmol) was dissolved in hydrogen bromide (13.42 mL, 119 mmol) and added, and the addition was carried out at an internal temperature of 30 - 40 °C. After stirring for 30 minutes, 200 mL of hexane and 67.8 mL of 5 mol / L aqueous sodium hydroxide solution (339 mmol) were added to the reaction solution. The reaction solution was filtered through celite, and the filtrate was extracted with hexane. The organic layer was washed with water and saturated brine, dried over magnesium sulfate, and filtered. The organic layer was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (hexane:ethyl acetate = 10:0 - 9:1), and the solvent was distilled off under reduced pressure to obtain Compound 11 (4.35 g, 15.1 mmol, 73.5 wt%). Since Compound 11 has a low boiling point, it was used in the next reaction without completely distilling off the solvent. 1 1H-NMR (CDCl3) δ: 7.00 - 7.07 (m, 2H)

[0101] Synthesis of Compound 12 in Step 4 A solution of Compound 8 (0.75 g, 4.2 mmol) in tetrahydrofuran (70 mL) was slowly added dropwise to a mixed solution of 2.76 mol / L hexane solution of n-butyllithium (1.8 mL, 5.04 mmol) and tetrahydrofuran (2.6 mL) at -78 °C over 5 minutes. The reaction solution was stirred at -78 °C for 1 hour. To the reaction solution, a 2-methyltetrahydrofuran solution of 1.9 mol / L zinc chloride (2.6 mL, 82.4 mmol) was added dropwise. The reaction solution was stirred at room temperature for 2 hours. To the reaction solution, Compound 11 (1.2 g, 4.2 mmol) and tetrakis(triphenylphosphine)palladium (243 mg, 0.21 mmol) were added, and the mixture was stirred at 90 °C for 1.5 hours. The reaction solution was cooled to room temperature, water (7.5 mL) was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated brine, dried over magnesium sulfate, and filtered. The organic layer was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (hexane:ethyl acetate = 10:0 to 9:1), and the solvent was distilled off under reduced pressure to obtain Compound 12 (374 mg, 1.2 mmol, yield 29%). 1 1H-NMR (CDCl3) δ: 3.95 (s, 3H), 4.05 (s, 3H), 7.20 (d, J = 9.0 Hz, 2H) LC / MS (ESI): m / z = 309, RT = 2.51 min, LC / MS measurement condition A

[0102] Synthesis of Compound 13 in Step 5 Compound 12 (374 mg, 1.2 mmol), acetonitrile (7.5 mL), NaI (181 mg, 1.2 mmol), and TMSCl (464 μL) were mixed, and the reaction solution was heated at 40 °C. NaI (181 mg, 1.2 mmol) and TMSCl (464 μL) were added approximately every 30 minutes until the raw materials almost disappeared. After completion of the reaction, water (15 mL) was added to the reaction solution, and a solid precipitated. After partially concentrating the reaction solution, the solid was collected by filtration. The solid was washed with cold acetonitrile to obtain Compound 13 (246 mg, 0.88 mmol). 1H-NMR (DMSO-d6) δ: 7.43 (d, J = 9.5 Hz, 2H), 7.53 (dd, J = 7.3, 2.1 Hz, 1H), 11.53 (s, 1H), 12.23 (br s, 1H) LC / MS (ESI): m / z = 281, RT = 1.44 min, LC / MS measurement condition A

[0103] Step 6 Synthesis of Compound 14 Compound 13 (250 mg, 0.89 mmol), 5-chloropyridine-3-boronic acid (280 mg, 1.78 mmol), copper(II) acetate (242 mg, 1.33 mmol), acetonitrile (7.5 mL), triethylamine (1.23 mL, 8.89 mmol) and pyridine (1.8 mL, 22.2 mmol) were mixed, and the solution was stirred at room temperature overnight. 2 mol / L hydrochloric acid (44.5 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with water, dried over magnesium sulfate and filtered. The filtrate was concentrated, and the obtained residue was carried on to the next step without purification.

[0104] Step 7 Synthesis of Compound 15 To a solution of Compound 14 (330 mg, 0.84 mmol), DIPEA (440 μL, 2.52 mmol) and DMF (3.3 mL) was added 2-bromoacetonitrile (168 μL, 2.52 mmol). The resulting solution was stirred at room temperature overnight. Water (14 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated brine, dried over magnesium sulfate and filtered. The filtrate was concentrated, and the obtained residue was purified by silica gel column chromatography (hexane:ethyl acetate = 10:0 to 2:3), and the solvent was distilled off under reduced pressure to obtain Compound 15 (307 mg, 0.71 mmol, yield 85%). 1 H-NMR (CDCl3) δ: 5.13 (s, 2H), 7.21 - 7.25 (m, 1H), 7.67 (1H, t, J = 2.3 Hz), 8.45 (d, J = 2.1 Hz, 1H), 8.66 (d, J = 2.1 Hz, 1H). LC / MS (ESI): m / z = 431, RT = 2.13 min, LC / MS measurement condition A

[0105] Synthesis of Engineering 8 Compound (I-2) Compound 15 (48.8 mg, 0.113 mmol), 6,6-difluoro-2-azaspiro[3.3]heptane trifluoro hydrochloride (33.5 mg, 0.136 mmol), DMF (0.5 mL) and N,N-diisopropylethylamine (59.2 μL, 0.339 mmol) were mixed and stirred at 60 °C for 2 hours. Water (2 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with water, dried over magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (hexane:ethyl acetate = 10:0 to 3:7). The solvent was distilled off under reduced pressure, and diisopropyl ether was added. The obtained precipitate was collected by filtration and washed with diisopropyl ether. The obtained solid was dried under reduced pressure to obtain Compound (I-2) (34.3 mg, 0.065 mmol, yield 57%). 1 1H-NMR (DMSO-d6) δ: 2.83 (t, J = 12.5 Hz, 4H), 4.07 (s, 4H), 4.86 (s, 2H), 7.44 (d, J = 9.4 Hz, 1H), 8.01 (t, J = 2.2 Hz, 1H), 8.50 (d, J = 2.0 Hz, 1H), 8.73 (d, J = 2.0 Hz, 1H). LC / MS (ESI): m / z = 528, RT = 2.19 min, LC / MS measurement condition A

[0106] (Reference Example 1) Synthesis of Compound (II-1)

Chemical Structure

[0107] (Reference Example 2) In the same manner as in Reference Example 1 and International Publication No. WO2023 / 195529, the following compounds can be synthesized.

Chemical Structure

Chemical Structure

[0108] The following describes the biological test examples of the compounds according to the present invention. The compound according to the present invention has an inhibitory effect on coronavirus 3CL protease and inhibits coronavirus 3CL protease. Specifically, in the evaluation method described below, the IC 50 is preferably 50 μM or less, more preferably 1 μM or less, and even more preferably 100 nM or less. The EC 50 is preferably 10 μM or less, more preferably 1 μM or less, and even more preferably 100 nM or less.

[0109] Test Example 1: Confirmation test of the inhibitory effect of Cytopathic effect (CPE) using human TMPRSS2 and ACE2-expressing HEK293T cells (HEK293T / ACE2-TMPRSS2 cells) <Operation procedure> · Dilution and dispensing of the test sample Dilute the test sample to an appropriate concentration with DMSO in advance. After preparing a 2- to 5-fold serial dilution series, dispense it into a 384-well plate. · Dilution and dispensing of cells and SARS-CoV-2 Mix HEK293T / ACE2-TMPRSS2 cells (GCP-SL222, 5×10 3 cells / well) and SARS-CoV-2 (200-600 TCID 50 / well) with the medium (MEM, 2% FBS, penicillin-streptomycin), dispense the mixture into the wells containing the test sample, and then culture it in a CO2 incubator for 3 days. · Dispensing of CellTiter-Glo® 2.0 and measurement of the luminescence signal After returning the plate cultured for 3 days to room temperature, dispense CellTiter-Glo® 2.0 into each well and mix it with a plate mixer. After standing for a certain period of time, measure the luminescence signal (Lum) with a plate reader. <Calculation of each measurement item value> · Calculation of the 50% inhibitory concentration of SARS-CoV-2-infected cell death (EC 50 ) When x is the logarithm of the compound concentration and y is %Efficacy, the inhibition curve is approximated by the following Logistic regression equation, and the value of x when y = 50(%) is substituted is EC 50 which is calculated as follows. y = min + (max - min) / {1 + (X50 / x) ^Hill} %Efficacy = {(Sample - virus control) / (cell control - virus control)} * 100% cell control: the average of Lum of cell control wells virus control: the average of Lum of virus control wells min: lower limit value of the y-axis, max: upper limit value of the y-axis, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max

[0110] The compound according to the present invention was essentially tested as described above. EC 50 values are shown below. (Results) Compound I-1: 1.66 nM Compound I-2: 2.36 nM

[0111] Test Example 2-1: Inhibition Activity Test against SARS-CoV-2 3CL Protease (Materials) · Commercially available Recombinant SARS-CoV-2 3CL Protease · Commercially available substrate peptide Dabcyl-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu(Edans)-NH2 (SEQ ID NO: 1) · Internal Standard peptide Dabcyl-Lys-Thr-Ser-Ala-Val-Leu( 13 C6,15 N)-Gln (SEQ ID NO: 2) Dabcyl-Lys-Thr-Ser-Ala-Val-Leu( 13 C6, 15 N)-Gln can be synthesized with reference to the literature (Atherton, E.; Sheppard, R. C., "In Solid Phase Peptide Synthesis, A Practical Approach", IRL Press at Oxford University Press, 1989 and Bioorg. Med. Chem., Vol. 5, No. 9, 1997, pp. 1883-1891, etc.). An example is shown below. Using Rink amide resin, by Fmoc solid-phase synthesis, H-Lys-Thr-Ser-Ala-Val-Leu( 13 C6, 15 N)-Glu(resin)-OαOtBu (the Lys side chain is protected by Boc, the Thr side chain is protected by a tert-butyl group, the Ser side chain is protected by a tert-butyl group, the C-terminal OH of Glu is protected by a tert-butyl group, and the carboxylic acid of the Glu side chain is condensed with the resin) is synthesized. The modification of the N-terminal Dabcyl group is carried out by condensing 4-dimethylaminoazobenzene-4'-carboxylic acid (Dabcyl-OH) on the resin using EDC / HOBT. Final deprotection and cleavage from the resin are performed by treatment with TFA / EDT = 95:5. Then, it is purified by reverse-phase HPLC. ·RapidFire Cartridge C4 typeA <Operation Procedure> ·Preparation of Assay Buffer In this test, an assay buffer consisting of 20 mM Tris-HCl, 1 mM EDTA, 10 mM DTT, and 0.01% BSA is used. ·Dilution and Dispensing of Test Samples The test samples are previously diluted to an appropriate concentration with DMSO, a 2- to 5-fold serial dilution series is prepared, and then dispensed into a 384-well plate. ·Addition of Enzyme and Substrate, Enzyme Reaction To the prepared compound plate, add 8 μM of substrate and 6 nM or 0.6 nM of enzyme solution, and incubate at room temperature for 3 to 5 hours. Then, add the reaction stop solution (0.067 μM Internal Standard, 0.1% formic acid, 10 or 25% acetonitrile) to stop the enzyme reaction. · Measurement of reaction products The completed reaction plate is measured using a RapidFire System 360 and a mass spectrometer (Agilent, 6550 iFunnel Q-TOF), or a Rapid Fire System 365 and a mass spectrometer (Agilent, 6495C Triple Quadrupole). As the mobile phase during measurement, use Solution A (75% isopropanol, 15% acetonitrile, 5 mM ammonium formate) and Solution B (0.01% trifluoroacetic acid, 0.09% formic acid). The reaction products detected by the mass spectrometer are calculated using a RapidFire Integrator or a program capable of equivalent analysis to obtain the Product area value. At the same time, the simultaneously detected Internal Standard is also calculated to obtain the Internal Standard area value. <Calculation of each measurement item value> · Calculation of P / IS Calculate the P / IS by calculating the area value obtained in the previous item according to the following formula. P / IS = Product area value / Internal Standard area value · 50% SARS-CoV-2 3CL protease inhibitory concentration (IC 50 ) calculation When x is the logarithmic value of the compound concentration and y is %Inhibition, approximate the inhibition curve with the following Logistic regression equation, and calculate the value of x when y = 50 (%) as IC 50 and calculate it as such. y = min + (max - min) / {1 + (X50 / x) ^ Hill} %Inhibition = {1-(Sample - Control(-)) / Control(+)-Control(-))} * 100 Control(-): the average of P / IS of enzyme inhibited condition wells Control(+): the average of P / IS of DMSO control wells min: lower limit value of the y-axis, max: upper limit value of the y-axis, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max

[0112] The compound of the present invention was tested essentially as described above. IC 50 values are shown below. (Results) Compound I-1: 0.00036 μM Compound I-2: 0.00033 μM Compound II-1: 0.00068 μM

[0113] Test Example 2-2: Inhibition Activity Test against SARS-CoV-2 3CL Protease <Materials> · Commercially available Recombinant SARS-CoV-2 3CL Protease · SARS-CoV-2 3CL Protease P132H · Commercially available substrate peptide Dabcyl-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu(Edans)-NH2 (SEQ ID NO: 1) · Internal Standard peptide Dabcyl-Lys-Thr-Ser-Ala-Val-Leu( 13 C6, 15 N)-Gln (SEQ ID NO: 2) Dabcyl-Lys-Thr-Ser-Ala-Val-Leu( 13 C6, 15N)-Gln can be synthesized with reference to the literature (Atherton, E.; Sheppard, R. C., “In Solid Phase Peptide Synthesis, A Practical Approach”, IRL Press at Oxford University Press, 1989 and Bioorg. Med. Chem., Vol. 5, No. 9, 1997, pp. 1883-1891, etc.). An example is shown below. Using Rink amide resin, by Fmoc solid-phase synthesis, H-Lys-Thr-Ser-Ala-Val-Leu( 13 C6, 15 N)-Glu(resin)-OαOtBu (the Lys side chain is protected by Boc, the Thr side chain is protected by a tert-butyl group, the Ser side chain is protected by a tert-butyl group, the C-terminal OH of Glu is protected by a tert-butyl group, and the carboxylic acid of the Glu side chain is condensed with the resin) is synthesized. The modification of the N-terminal Dabcyl group is carried out by condensing 4-dimethylaminoazobenzene-4'-carboxylic acid (Dabcyl-OH) on the resin using EDC / HOBT. Final deprotection and cleavage from the resin are performed by treatment with TFA / EDT = 95:5. Then, it is purified by reverse-phase HPLC. ·RapidFire Cartridge C4 typeA <Operation Procedure> ·Preparation of assay buffer In this test, an assay buffer consisting of 20 mM Tris-HCl, 1 mM EDTA, 10 mM DTT, and 0.01% BSA is used. ·Dilution and dispensing of test samples The test samples are diluted to an appropriate concentration with DMSO in advance. After preparing a three-fold serial dilution series, they are dispensed into a 384-well plate. ·Addition of enzyme and substrate, enzyme reaction To the prepared compound plate, a substrate with a final concentration of 4 μM and an enzyme of 0.3 nM are added, and incubation is carried out at room temperature for 4 hours. Then, a reaction stop solution (7.2 nM Internal Standard, 0.1% formic acid, 10% acetonitrile) is added to stop the enzyme reaction. ·Measurement of reaction products The plates after the reaction is completed are measured using a Rapid Fire System 365 and a mass spectrometer (Agilent, 6495C Triple Quadrupole). As the mobile phase during measurement, Solution A (75% isopropanol, 15% acetonitrile, 5 mM ammonium formate) and Solution B (0.01% trifluoroacetic acid, 0.09% formic acid) are used. The reaction products detected by the mass spectrometer are calculated using RapidFire Integrator to obtain the Product area value. At the same time, the Internal Standard detected simultaneously is also calculated to obtain the Internal Standard area value. <Calculation of each measurement item value> ·Calculation of P / IS The area value obtained in the previous item is calculated by the following formula to calculate P / IS. P / IS = Product area value / Internal Standard area value ·50% SARS-CoV-2 3CL protease inhibitory concentration (IC 50 ) calculation When x is the logarithmic value of the compound concentration and y is %Inhibition, the inhibition curve is approximated by the following Logistic regression equation, and the value of x when y = 50 (%) is substituted is calculated as IC 50 y = min + (max - min) / {1 + (X50 / x) ^ Hill} %Inhibition = {1 - (Sample - Control(-)) / Control(+) - Control(-))} * 100 Control(-): the average of P / IS ratio in the wells without SARS-CoV-2 3CL protease and test substance ​Control(+): the average of P / IS ratio in the wells with SARS-CoV-2 3CL protease and without test substance min: lower limit value of the y-axis, max: upper limit value of the y-axis, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max

[0114] The compound according to the present invention was essentially tested as described above. The results are shown below.

Table 5

[0115] Test Example 3: Confirmation test of Cytopathic effect (CPE) inhibitory effect using human TMPRSS2-expressing Vero E6 cells (Vero E6 / TMPRSS2 cells) <Operation procedure> · Dilution and dispensing of the test sample The test sample is previously diluted to an appropriate concentration with DMSO, a three-fold serial dilution series is prepared, and then dispensed into a 96-well plate. · Dilution and dispensing of cells and SARS-CoV-2 VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4cells / well) and SARS-CoV-2 hCoV-19 / Japan / TY / WK-521 / 2020 (Ancestral), hCoV-19 / Japan / QHN001 / 2020 (Alpha), hCoV-19 / Japan / TY8-612 / 2021 (Beta), hCoV-19 / Japan / TY7-501 / 2021 (Gamma), hCoV-19 / Japan / TY11-927 / 2021 (Delta), hCoV-19 / Japan / TY38-871 / 2021 (Omicron BA.1.1), hCoV-19 / Japan / TY40-385 / 2022 (Omicron BA.2), hCoV-19 / Japan / TY41-721 / 2022 (Omicron BA.2.12.1), hCoV-19 / Japan / TY41-716 / 2022 (Omicron BA.2.75), hCoV-19 / Japan / TY41-703 / 2022 (Omicron BA.4.1), hCoV-19 / Japan / TY41-763 / 2022 (Omicron BA.4.6), hCoV-19 / Japan / TY41-702 / 2022 (Omicron BE.1 / BA.5-like), hCoV-19 / Japan / TY41-704 / 2022 (Omicron BA.5.2.1), hCoV-19 / Japan / TY41-820 / 2022 (Omicron BF.7), hCoV-19 / Japan / TY41-828 / 2022 (Omicron BF.7.4.1), hCoV-19 / Japan / TY41-796 / 2022 (Omicron BQ.1.1), hCoV-19 / Japan / TY41-832 / 2022 (Omicron CH.1.1.11), hCoV-19 / Japan / TY41-795 / 2022 (Omicron XBB.1), hCoV-19 / Japan / 23-018 / 2022 (Omicron XBB.1.5), hCoV-19 / Japan / TY-41951 / 2023 (Omicron XBB.1.9.1), hCoV-19 / Japan / TY41-984 / 2023 (Omicron XBB.1.16)、hCoV-19 / Japan / TY41-831 / 2022 (Omicron XBF), hCoV-19 / Japan / TY41-686 / 2022 (Omicron XE) (30 - 3000 TCID. 50 / well) was mixed with medium (MEM, 2% FBS, penicillin-streptomycin) and dispensed into wells containing test samples, and then cultured in a CO2 incubator for 3 days or 4 days. · Dispensing of CellTiter-Glo® 2.0 and measurement of luminescence signal After returning the plates cultured for 3 days to room temperature, CellTiter-Glo® 2.0 was dispensed into each well and mixed with a plate mixer. After standing for a certain period of time, the luminescence signal (Lum) was measured with a plate reader.

[0116] <Calculation of each measurement item value> · 50% SARS-CoV-2 infection cell death inhibition concentration (EC 50 ) calculation When x is the logarithm of the compound concentration and y is % Efficacy, the inhibition curve was approximated by the following Logistic regression equation, and the value of x when y = 50 (%) was substituted was calculated as EC 50 and calculated as. y = min + (max - min) / {1 + (X50 / x) ^ Hill} % Efficacy = {(Sample - virus control) / (cell control - virus control)} * 100% cell control: the average of Lum of cell control wells virus control: the average of Lum of virus control wells min: lower limit value of the y-axis, max: upper limit value of the y-axis, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max

[0117] The compound according to the present invention was essentially tested as described above. The results are shown below.

Table 6

[0118] Test Example 5: Antiviral effect against HCoV-OC43 <Operation procedure> ·Dilution and dispensing of test samples The test sample was previously diluted to an appropriate concentration with DMSO, a three-fold serial dilution series was prepared, and then dispensed into a 96-well plate and diluted with maintenance medium (MEM, 2% FBS, penicillin-streptomycin). ·Dilution and dispensing of cells and HCoV-OC43 MRC-5 cells (2×10 4 cells / well) suspended in passage medium (DMEM, 10% FBS, penicillin-streptomycin) were seeded in a 96-well plate one day before infection. The next day, HCoV-OC43 (300 TCID 50 / well) suspended in maintenance medium (MEM, 2% FBS, penicillin-streptomycin) was infected for 1 hour. Then, the virus solution was removed, maintenance medium containing the test reagent was added, and the cells were cultured in a CO2 incubator for 72 hours. In addition, in order to examine the cytotoxicity of the test reagent, the same operation was carried out in the absence of the virus. ·Dispensing of CellTiter-Glo (registered trademark) 2.0 and measurement of luminescence signal After the plate cultured for 72 hours was returned to room temperature, CellTiter-Glo (registered trademark) 2.0 was dispensed into each well and mixed with a plate mixer. After standing for a certain time, the luminescence signal (Lum) was measured with a plate reader.

[0119] <Calculation of each measurement item value> ·Calculation of 50% HCoV-OC43-infected cell death inhibition concentration (EC 50 ) When x is the logarithmic value of the compound concentration and y is % Efficacy, the inhibition curve is approximated by the following Logistic regression equation, and the value of x when y = 50 (%) is substituted is EC 50It is calculated as follows. y = min + (max - min) / {1 + (X50 / x) ^Hill} %Efficacy = {(Sample - virus control) / (cell control - virus control)} * 100% cell control: the average of Lum of cell control wells virus control: the average of Lum of virus control wells min: lower limit value of the y-axis, max: upper limit value of the y-axis, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max ·Calculation of 50% cytotoxic concentration When x is the logarithm of the compound concentration and y is %Cytotoxicity, the inhibition curve is approximated by the following Logistic regression equation, and the value of x when y = 50(%) is substituted is CC 50 It is calculated as follows. y = min + (max - min) / {1 + (X50 / x) ^Hill} %Cytotoxicity = {(Sample - medium control) / (cell control - medium control)} * 100% cell control: the average of Lum of cell control wells medium control: the average of Lum of medium control wells min: lower limit value of the y-axis, max: upper limit value of the y-axis, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max

[0120] The compound according to the present invention was essentially tested as described above. The results are shown below. Compound I-1: EC 50 92.0 ± 21.0 nM, CC 50 > 100 μM

[0121] Test Example 6: Antiviral effect against HCoV-229E <Procedure> · Dilution and dispensing of test samples The test sample is previously diluted to an appropriate concentration with DMSO, a three-fold serial dilution series is prepared, and then dispensed into a 96-well plate and diluted with maintenance medium (MEM, 2% FBS, penicillin-streptomycin). · Dilution and dispensing of cells and HCoV-229E MRC-5 cells (2 × 10 4 cells / well) suspended in subculture medium (DMEM, 10% FBS, penicillin-streptomycin) are seeded into a 96-well plate one day before infection. The next day, HCoV-229E (1000 TCID 50 / well) suspended in maintenance medium (MEM, 2% FBS, penicillin-streptomycin) is infected for 1 hour. Then, the virus solution is removed, maintenance medium containing the test reagent is added, and cultured in a CO2 incubator for 72 hours. · Dispensing of CellTiter-Glo® 2.0 and measurement of luminescence signal After the plate cultured for 72 hours is returned to room temperature, CellTiter-Glo® 2.0 is dispensed into each well and mixed with a plate mixer. After standing for a certain time, the luminescence signal (Lum) is measured with a plate reader.

[0122] <Calculation of each measurement item value> · Calculation of 50% HCoV-229E infected cell death inhibition concentration (EC 50 ) When x is the logarithmic value of the compound concentration and y is % Efficacy, the inhibition curve is approximated by the following Logistic regression equation, and the value of x when y = 50 (%) is substituted is calculated as EC 50 . y = min + (max - min) / {1 + (X50 / x) ^Hill} %Efficacy = {(Sample - virus control) / (cell control - virus control)} * 100% cell control: the average of Lum of cell control wells virus control: the average of Lum of virus control wells min: Lower limit value of the y-axis, max: Upper limit value of the y-axis, X50: x-coordinate of the inflection point, Hill: Slope of the curve at the midpoint between min and max

[0123] The compound according to the present invention was essentially tested as described above. The results are shown below. Compound I-1: EC 50 3570±510nM

[0124] Test Example 7: Effects of human serum, mouse serum, and hamster serum on anti-SARS-CoV-2 activity <Operation procedure> · Dilution and dispensing of the test sample Dilute the test sample to an appropriate concentration with DMSO in advance. After preparing a three-fold serial dilution series, dispense it into a 96-well plate. · Addition of serum-supplemented medium Prepare the medium (MEM, 2% FBS, penicillin-streptomycin) to a final concentration of 0, 12.5, 25, 50% human serum or mouse serum or hamster serum, dispense it into the wells containing the test sample, and incubate at room temperature for 1 hour. · Dilution and dispensing of cells and SARS-CoV-2 VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4Cells / well) were seeded on a 96-well plate the day before infection, and the next day, SARS-CoV-2 hCoV-19 / Japan / TY7-501 / 2021 (1000 TCID 50 / well) was used to infect for 1 hour. Then, the virus solution was removed, and the test reagent and medium supplemented with human serum, mouse serum, or hamster serum were added, and cultured in a CO2 incubator for 1 day. ·Quantification of virus amount The supernatant of the plate cultured for 1 day was removed, and a cell lysate prepared by mixing Trizol LS and maintenance medium at a ratio of 3:1 was added, and RNA was extracted using the Direct-zol-96 RNA Kit (ZYMO RESEARCH). The extracted RNA solution was quantified by real-time PCR (Applied BioSystems QuantStudio5). The following probes and primers were used.

Table 7

[0125] <Calculation of each measurement item value> ·90% SARS-CoV-2 virus replication inhibition concentration (EC 90 ) calculation Let x be the logarithm of the compound concentration and y be the virus copy number (Log 10 copies / mL). In the concentration-dependent curve, the value corresponding to a 1 log 10 reduction from the virus control is calculated by the two-point method from the copy numbers of the two concentrations before and after that. That is, X = the lowest concentration at the mean reduction of viral RNA copy number from that of virus control is less than z x = the highest concentration at which the mean reduction of viral RNA copy number from that of the virus control is z or more. Y = the mean of the logarithmic reduction of viral RNA copy number from that of the virus control at X y = the mean of the logarithmic reduction of viral RNA copy number from that of the virus control at x EC 90 values were calculated by the following formula. EC 90 = 10[log(x) + (log(X) - log(x)) × (y - z) / (y - Y)] z = log 10 (10 / 100) ·Protein-Adjusted EC 90 (PA-EC 90 ) calculation EC at each serum concentration 90 After calculating the EC at each serum concentration, the PA-EC value under 100% serum conditions was calculated by linear regression. 90 value was calculated.

[0126] The compound according to the present invention was tested essentially as described above. The results are shown below. Compound I-1: Human serum PA-EC 90 7.90 nM Mouse serum PA-EC 90 19.8 nM Hamster serum PA-EC 90 8.17 nM

[0127] Test Example 8: Confirmation Test of Viral Growth Inhibitory Effect Using human airway epithelial cells Cells <Operation Procedure> ·Dilution and Dispensing of Test Samples The test samples were previously diluted to an appropriate concentration with DMSO, a three-fold serial dilution series was prepared, then diluted 200-fold with MucilAirTM culture medium, and dispensed into 24-well plates. ·SARS-CoV-2 Infection MucilAir seeded in Transwell TM (Nasal cavity, approximately 5.0×10 5 cells / well) was infected with SARS-CoV-2 hCoV-19 / Japan / TY41-702 / 2022 (Omicron BE.1 / BA.5-like) (5000 TCID 50 / well) and cultured in a CO2 incubator for 2 hours. Then, it was washed with MucilAir TM culture medium, the Transwell was placed on the well containing the test sample, and cultured in a CO2 incubator. Two days after infection, MucilAir TM culture medium was added to the Transwell, and the supernatant was collected. ·Measurement of Viral Titer in Supernatant The collected supernatant was serially diluted 10-fold with medium (MEM, 2% FBS, penicillin-streptomycin), then mixed with VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4 cells / well) and seeded in a 96-well plate. After culturing in a CO2 incubator for 4 days, the Cytopathic effect (CPE) was observed, and the viral titer contained in the supernatant was calculated.

[0128] <Calculation of Each Measured Item Value> ·Calculation of 90% SARS-CoV-2 Virus Production Inhibitory Concentration (EC 90 ) Let x be the logarithm of the compound concentration and y be the viral titer (Log 10 TCID 50 / mL). In the concentration-dependent curve, 1 log from the virus control10 The value corresponding to reduction is calculated by the two-point method from the virus titers at two concentrations before and after that.

Number

[0129] The compound according to the present invention was essentially tested as described above. The results are shown below.

Table 8

[0130] Test Example 9: Test for inhibiting the growth of virus titer in the lung homogenate of SARS-CoV-2-infected mice by delayed administration of Compound I-1 <Materials and Methods> ·Compound Compound I-1 according to the present invention was used as a test sample using polyethylene glycol 400 (PEG400) containing N,N-dimethylacetamide (DMA) and 0.5 w / v% poly(1-vinylpyrrolidone-co-vinyl acetate) (PVPVA) (DMA:PEG400 containing 0.5 w / v% PVPVA = 1:9). The administration volume was 5 mL / kg. ·Virus The SARS-CoV-2 hCoV-19 / Japan / TY7-501 / 2021 strain isolated at the National Institute of Infectious Diseases was used. ·Mouse lung infection, dosing, lung recovery Five-week-old female BALB / c mice (CLEA Japan, Inc.) free of specific pathogens were used in this study. At the time of virus inoculation, the mice were anesthetized by intramuscular injection of 100 μL of anesthetic solution containing 0.03 mg / mL of medetomidine hydrochloride, 0.4 mg / mL of midazolam, and 0.5 mg / mL of butorphanol tartrate in saline. The mice were given, under anesthesia, 50 μL of hCoV-19 / Japan / TY7-501 / 2021 (1.00×10 4 TCID 50They were inoculated nasally. Starting from 1 day after virus infection, mice (n = 5 / group) were orally administered Compound I-1 at doses of 0.1, 0.3, 1, 3, and 10 mg / kg twice a day. Control mice were orally administered DMA / 0.5 w / v% PVPVA in PEG400 twice a day. The compound administration was carried out for 2 days starting from the start of administration. Three days after infection, mouse lungs were collected, 2 mL of PBS was added, homogenized, and the supernatant after centrifugation was collected. · Measurement of virus titer in lung homogenate After preparing a 10-fold dilution series of the lung homogenate in medium (MEM, 2% FBS, penicillin-streptomycin), it was mixed with VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4 cells / well) and seeded in a 96-well plate. After culturing in a CO2 incubator for 4 days, the cytopathic effect (CPE) was observed, and the virus titer contained in the lung homogenate was calculated.

[0131] The compound according to the present invention was tested essentially as described above. The results are shown below. The virus titer in the lung homogenate 3 days after infection was 6.45-log 10 TCID 50 / mL in the DMA / 0.5 w / v% PVPVA in PEG400 administration group, and 6.27, 5.39, 3.97, 2.51, 2.47-log 10 TCID 50 / mL in the Compound I-1 0.1, 0.3, 1, 3, 10 mg / kg administration groups, respectively. Since the virus titer in the lung homogenate was lower in the Compound I-1 administration groups than in the DMA / 0.5 w / v% PVPVA in PEG400 administration group, it was suggested that there was an effect of reducing the virus titer in the lung homogenate even when there was a period from infection to administration (Figure 1).

[0132] Test Example 10: Inhibitory test of virus titer growth and inhibitory test of lung weight increase in lung and turbinate homogenates of SARS-CoV-2-infected hamsters by delayed administration of Compound I-1 <Materials and Methods> · Compound The compound I-1 according to the present invention was used as a test sample with N,N-dimethylacetamide (DMA) and polyethylene glycol 400 (PEG400) containing 0.5 w / v% poly(1-vinylpyrrolidone-co-vinyl acetate) (PVPVA) (DMA: PEG400 containing 0.5 w / v% PVPVA = 1:9). The administration volume was 2.5 mL / kg. · Virus The SARS-CoV-2 hCoV-19 / Japan / TY41-702 / 2022 strain (Omicron BE.1 / BA.5-like) isolated at the National Institute of Infectious Diseases was used. · Hamster lung infection, dosing, lung, turbinate collection Six-week-old male Syrian hamsters (Japan SLC, Inc.) free of specific pathogens were used in this study. At the time of virus inoculation, the hamsters were anesthetized by subcutaneous administration of an anesthetic solution containing 0.07 mg / mL of medetomidine hydrochloride, 6.98 mg / mL of alphaxalone, and 1.16 mg / mL of butorphanol tartrate at 3 mL / kg, and 100 μL of hCoV-19 / Japan / TY41-702 / 2022 (1.00×10 4 TCID 50 ) was inoculated intranasally. Infected hamsters were orally administered compound I-1 at doses of 0.1, 1, and 10 mg / kg twice a day starting 1 day after virus inoculation. Control infected and non-infected hamsters were orally administered DMA / 0.5 w / v% PVPVA in PEG400 twice a day. The compound administration was for 5 days from the start of administration. The lungs and turbinates of infected hamsters were collected 3 and 4 days after infection, 5 mL or 1 mL of PBS was added respectively, homogenized, and the supernatant after centrifugation was collected. Also, the lungs of infected and non-infected hamsters were collected 7 days after infection, and the lung weights were measured. · Measurement of virus titer in lung and turbinate homogenate After preparing a 10-fold dilution series of the lung and turbinate homogenate in medium (MEM, 2% FBS, penicillin-streptomycin), VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4Cells were seeded at (cells / well). After culturing for 4 days in a CO2 incubator, the cytopathic effect (CPE) was observed, and the viral titer contained in the lung or turbinate homogenate was calculated.

[0133] The compound according to the present invention was essentially tested as described above. The results are shown below. The viral titer in the lung homogenate was 5.4-log 10 TCID 50 / mL in the DMA / 0.5 w / v% PVPVA in PEG400 administration group 3 days after infection, and 5.5, 3.5, 2.5-log 10 TCID 50 / mL in the groups administered with compound I-1 at 0.1, 1, 10 mg / kg, respectively. 4 days after infection, it was 5.6-log 10 TCID 50 / mL in the DMA / 0.5 w / v% PVPVA in PEG400 administration group, and 4.6, 2.4, 1.9-log 10 TCID 50 / mL in the groups administered with compound I-1 at 0.1, 1, 10 mg / kg, respectively. The viral titer in the turbinate homogenate was 5.0-log 10 TCID 50 / mL in the DMA / 0.5 w / v% PVPVA in PEG400 administration group 3 days after infection, and 4.8, 3.2, 2.7-log 10 TCID 50 / mL in the groups administered with compound I-1 at 0.1, 1, 10 mg / kg, respectively. 4 days after infection, it was 3.8-log 10 TCID 50 / mL in the DMA / 0.5 w / v% PVPVA in PEG400 administration group, and 4.0, 2.2, 1.8-log 10 TCID 50 / mL in the groups administered with compound I-1 at 0.1, 1, 10 mg / kg, respectively. In the group administered with Compound I-1, the viral titers in the lung and turbinate homogenates showed dose-dependently lower values compared to those in the group administered with DMA / 0.5 w / v% PVPVA in PEG400. This suggests that even when there is a period between infection and administration, it has the effect of reducing the viral titers in the lung and turbinate homogenates (Figs. 2-1, 2-2). Also, the lung weight / body weight 7 days after infection was 5.26 mg / g in non-infected hamsters, 8.96 mg / g in the group of infected hamsters administered with DMA / 0.5 w / v% PVPVA in PEG400, and 8.24, 5.75, and 5.57 mg / g in the groups administered with Compound I-1 at 0.1, 1, and 10 mg / kg, respectively. Since the lung weight / body weight in the group administered with Compound I-1 showed dose-dependently lower values compared to those in the group administered with DMA / 0.5 w / v% PVPVA in PEG400, it was suggested that administration of Compound I-1 has the effect of suppressing the increase in lung weight caused by SARS-CoV-2 infection (Fig. 3).

[0134] Test Example 11: Test for suppressing weight loss in SARS-CoV-2-infected hamsters by administration of Compound I-1 <Materials and Methods> · Compound Compound I-1 according to the present invention was used as a test sample using N,N-dimethylacetamide (DMA) and polyethylene glycol 400 (PEG400) containing 0.5 w / v% poly(1-vinylpyrrolidone-co-vinyl acetate) (PVPVA) (DMA: PEG400 containing 0.5 w / v% PVPVA = 1:9). The administration volume was 2.5 mL / kg. · Virus The SARS-CoV-2 hCoV-19 / Japan / TY41-702 / 2022 strain (Omicron BE.1 / BA.5-like) isolated at the National Institute of Infectious Diseases was used. · Hamster lung infection, drug administration, body weight measurement Six-week-old male Syrian hamsters (Japan SLC, Inc.) without specific pathogens were used in this study. At the time of virus inoculation, hamsters were anesthetized by subcutaneous administration of an anesthetic solution containing 0.07 mg / mL medetomidine hydrochloride, 6.98 mg / mL alphaxalone, and 1.16 mg / mL butorphanol tartrate at 3 mL / kg, and 100 μL of hCoV-19 / Japan / TY41-702 / 2022 (1.00×10 4 TCID 50 ) was inoculated intranasally. Infected hamsters were orally administered Compound I-1 at doses of 0.1, 1, and 10 mg / kg twice a day starting from 1 day after virus inoculation. Control infected and non-infected hamsters were orally administered DMA / 0.5 w / v% PVPVA in PEG400 twice a day. Compound administration was carried out for 5 days from the start of administration. Body weight was monitored once a day.

[0135] The compound according to the present invention was tested essentially as described above. The results are shown below. Weight gain was observed in non-infected hamsters, but in infected hamsters, body weight decreased from 3 days after infection and reached the lowest value 6 days after infection. At this time, in the groups administered Compound I-1 at 1 and 10 mg / kg, all animals survived until 10 days after infection, and the decrease in body weight was suppressed (Figure 4). From the above results, it was suggested that administration of Compound I-1 has an effect of suppressing body weight loss.

[0136] Test Example 12: Test for suppressing body weight loss and virus growth in the lungs and nasal turbinate homogenates of SARS-CoV-2-infected hamsters by prophylactic administration of Compound I-1 <Materials and Methods> · Compound Compound I-1 was used as a test sample using a 0.5% methylcellulose (0.5% MC) solution. The administration volume was 10 mL / kg. · Virus The SARS-CoV-2 hCoV-19 / Japan / TY41-702 / 2022 strain (Omicron BE.1 / BA.5-like) isolated at the National Institute of Infectious Diseases was used. · Hamster lung infection, drug administration, body weight measurement Six-week-old male Syrian hamsters (Japan SLC, Inc.) free of specific pathogens were used in this study. At the time of virus inoculation, the hamsters were anesthetized by subcutaneous administration of an anesthetic solution containing 0.07 mg / mL medetomidine hydrochloride, 6.98 mg / mL alphaxalone, and 1.16 mg / mL butorphanol tartrate at 3 mL / kg, and 100 μL of hCoV-19 / Japan / TY41-702 / 2022 (1.00×10 4 TCID 50 ) was inoculated intranasally. In the body weight loss suppression test, Compound I-1 was administered subcutaneously once at 3 and 10 mg / kg 1 day or 3 days before virus infection. In the virus growth inhibition test in lung and turbinate homogenates, Compound I-1 was administered subcutaneously once at 10 and 30 mg / kg 1 day before virus infection. The control hamsters were administered 0.5% MC subcutaneously once 1 day before virus infection. The body weight was monitored once a day. One and two days after infection, the lungs and turbinates of the infected hamsters were collected, 5 mL or 1 mL of PBS was added respectively, homogenized, and the supernatant after centrifugation was collected. · Measurement of virus titer in lung and turbinate homogenates After preparing a 10-fold dilution series of lung and turbinate homogenates in medium (MEM, 2% FBS, penicillin-streptomycin), they were seeded onto VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4 cells / well) that had been pre-cultured in a 96-well plate. After culturing in a CO2 incubator for 4 days, the cytopathic effect (CPE) was observed, and the virus titer contained in the lung or turbinate homogenate was calculated.

[0137] The compound according to the present invention was tested essentially as described above. The results are shown below. In infected hamsters, the body weight decreased from 3 days after infection, and reached the lowest value 6 days after infection. At this time, in the groups administered Compound I-1 at 3 and 10 mg / kg 1 day before infection and the group administered 10 mg / kg 3 days before infection, all animals survived until 7 days after infection, and the body weight loss was suppressed (Figure 5). The viral titer in the lung homogenate was 5.59-log 10 TCID 50 / mL in the 0.5% MC administration group 1 day after infection, and 3.34 and 1.84-log 10 TCID 50 / mL in the compound I-1 10 and 30 mg / kg administration groups, respectively. 2 days after infection, it was 6.18-log 10 TCID 50 / mL in the 0.5% MC administration group, and 5.29 and 3.55-log 10 TCID 50 / mL in the compound I-1 10 and 30 mg / kg administration groups, respectively (Figure 6-1). The viral titer in the turbinate homogenate was 5.71-log 10 TCID 50 / mL in the 0.5% MC administration group 1 day after infection, and 4.80 and 3.36-log 10 TCID 50 / mL in the compound I-1 10 and 30 mg / kg administration groups, respectively. 2 days after infection, it was 5.80-log 10 TCID 50 / mL in the 0.5% MC administration group, and 5.38 and 5.01-log 10 TCID 50 / mL in the compound I-1 10 and 30 mg / kg administration groups, respectively (Figure 6-2). In addition, when the plasma concentration immediately before infection was measured, it was 6.1, 12.5, 5.9, and 3.0 ng / mL in the 3 and 10 mg / kg administration groups 1 day before infection and in the 3 and 10 mg / kg administration groups 3 days before infection, respectively. From the above results, prophylactic administration of compound I-1 showed an effect of suppressing weight loss when the plasma concentration at the time of viral infection was approximately 6 ng / mL or more, and an effect of suppressing viral growth in the lung and turbinate homogenates when it was approximately 12 ng / mL or more.

[0138] The above test results indicate that the prophylactic subcutaneous administration of Compound I-1 suppressed the progression of the disease state and viral growth caused by viral infection, supporting the usefulness of Compound I-1 as a prophylactic agent for infectious diseases caused by SARS-CoV-2.

[0139] Test Example 13: Viral titer clearance test in nasal lavage fluid of SARS-CoV-2-infected hamsters by delayed administration of Compound I-1 <Materials and Methods> · Compound Compound I-1 according to the present invention was used as a test sample using N,N-dimethylacetamide (DMA) and polyethylene glycol 400 (PEG400) containing 0.5 w / v% poly(1-vinylpyrrolidone-co-vinyl acetate) (PVPVA) (DMA: PEG400 containing 0.5 w / v% PVPVA = 1:9). The administration volume was 2.5 mL / kg. · Virus The SARS-CoV-2 hCoV-19 / Japan / TY41-702 / 2022 strain (Omicron BE.1 / BA.5-like) isolated at the National Institute of Infectious Diseases was used. · Hamster nasal infection, drug administration, and collection of nasal lavage fluid Six-week-old male Syrian hamsters (Japan SLC, Inc.) free of specific pathogens were used in this study. At the time of virus inoculation, the hamsters were anesthetized by subcutaneous administration of an anesthetic solution containing 0.07 mg / mL of medetomidine hydrochloride, 6.98 mg / mL of alphaxalone, and 1.16 mg / mL of butorphanol tartrate at 3 mL / kg, and 100 μL of hCoV-19 / Japan / TY41-702 / 2022 (1.00×10 4 TCID 50They were inoculated intranasally. Infected hamsters were orally administered Compound I-1 at doses of 1 and 10 mg / kg twice a day starting 2 or 3 days after virus inoculation. Control infected hamsters were orally administered DMA / 0.5 w / v% PVPVA in PEG400 twice a day. The compound administration was carried out for 5 days starting from the start of administration. 2-6 days after infection, the infected hamsters were anesthetized with isoflurane and nasal lavage fluid was collected with 400 μL of DPBS, and the supernatant after centrifugation was collected. The experiment was performed with n = 4 in each group. · Measurement of virus titer in nasal lavage fluid The supernatant of the nasal lavage fluid was serially diluted 10-fold with medium (MEM, 2% FBS, penicillin-streptomycin), and then seeded onto VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4 cells / well) that had been pre-cultured in a 96-well plate. After culturing in a CO2 incubator for 4 days, the cytopathic effect (CPE) was observed, and the virus titer contained in the nasal lavage fluid was calculated.

[0140] The compound of the present invention was tested essentially as described above. The results are shown below. The virus titer in the nasal lavage fluid in the group administered the compound starting 2 days after virus inoculation was 4.80, 4.90, 4.88-log 10 TCID 50 / mL at the start of administration (2 days after infection, day 1 of administration) in the DMA / 0.5 w / v% PVPVA in PEG400 administration group and the Compound I-1 1, 10 mg / kg administration groups, respectively, 3.75, 3.63, 3.68-log 10 TCID 50 / mL 3 days after infection (day 2 of administration), and 2.96, <1.80, <1.80-log 10 TCID 50 / mL 5 days after infection (day 4 of administration), respectively (Figure 14). In the group administered the compound starting 3 days after virus inoculation, at the start of administration (3 days after infection, day 1 of administration) in the DMA / 0.5 w / v% PVPVA in PEG400 administration group and the Compound I-1 1, 10 mg / kg administration groups, they were 3.40, 2.93, 3.13-log 10TCID 50 / mL, on the 4th day after infection (1 day after the start of administration), they were 2.97, 1.84, and 2.13-log respectively 10 TCID 50 / mL, on the 6th day after infection (4 days after the start of administration), they were 2.43, <1.80, <1.80-log respectively 10 TCID 50 / mL (Figure 15). In the groups administered with 1 and 10 mg / kg of Compound I-1, the viral titer in the nasal lavage fluid showed lower values compared to the group administered with DMA / 0.5 w / v% PVPVA in PEG400, regardless of whether the start of administration was 2 days or 3 days after infection. This indicates that even if there is a period between infection and administration, there is an effect of reducing the virus in vivo.

[0141] Test Example 14: Inhibition Test of Virus Transmission from SARS-CoV-2 Infected Animals to Non-infected Animals by Delayed Administration of Compound I-1 <Materials and Methods> · Compound Compound I-1 according to the present invention was used as a test sample using polyethylene glycol 400 (PEG400) containing N,N-dimethylacetamide (DMA) and 0.5 w / v% poly(1-vinylpyrrolidone-co-vinyl acetate) (PVPVA) (DMA: PEG400 containing 0.5 w / v% PVPVA = 1:9). The administration volume was 2.5 mL / kg. · Virus SARS-CoV-2 hCoV-19 / Japan / TY11-927 / 2021 isolated at the National Institute of Infectious Diseases was used. · Hamster Nasal Infection, Medication, Cohabitation, Nasal Lavage Fluid and Lung Recovery Six-week-old male Syrian hamsters (Japan SLC, Inc.) free of specific pathogens were used in this study. At the time of virus inoculation, the hamsters were anesthetized by subcutaneous administration of 3 mL / kg of anesthetic solution containing 0.07 mg / mL of medetomidine hydrochloride, 6.98 mg / mL of alphaxalone, and 1.16 mg / mL of butorphanol tartrate, and 100 μL of hCoV-19 / Japan / TY11-927 / 2021 (1.00×10 3 TCID50 ) was inoculated intranasally. Starting 8 hours after virus inoculation in the infected hamsters (Index), Compound I-1 was orally administered twice a day at doses of 0.1, 1, and 10 mg / kg. The control infected hamsters (Index) were orally administered DMA / 0.5 w / v% PVPVA in PEG400 twice a day. The compound administration was performed a total of 3 times starting from the start of administration. From 1 day to 2 days after infection, for 12 hours at night, one infected hamster (Index) and one non-infected hamster (Contact) that was not inoculated with the virus were placed in separate stainless steel cages in the same cage at a distance of 2 cm apart so that they could not come into direct contact and cohabited. After the cohabitation ended, they were individually housed. Four days after the start of cohabitation (3 days after the end of cohabitation), nasal lavage fluid and lungs were collected from the non-infected hamsters (Contact). The nasal lavage fluid was collected with 400 μL of DPBS under isoflurane anesthesia, and the supernatant after centrifugation was collected. For the lungs, 5 mL of DPBS was added, homogenized, and the supernatant after centrifugation was collected. The experiment was performed with n = 6 in each group. · Measurement of virus titer in nasal lavage fluid and lung homogenate supernatant After preparing a 10-fold dilution series of nasal lavage fluid or lung homogenate supernatant in medium (MEM, 2% FBS, penicillin-streptomycin), it was inoculated into VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4 cells / well) that had been pre-cultured in a 96-well plate. After culturing for 4 days in a CO2 incubator, the cytopathic effect (CPE) was observed, and the virus titer contained in the nasal lavage fluid or lung homogenate supernatant was calculated.

[0142] The compound of the present invention was tested essentially as described above. The results are shown below. The virus titer in the nasal lavage fluid or lung homogenate supernatant of the non-infected hamsters (Contact) 4 days after the start of cohabitation (3 days after the end of cohabitation) with the infected hamsters (Index) was below the detection limit (1.8-log 10 TCID 50Individuals with a titer of 1.0×10⁶ TCID₅₀ / mL or higher were 6 / 6 in the DMA / 0.5 w / v% PVPVA administration group, 6 / 6 in the compound I-1 0.1 mg / kg administration group, 0 / 6 in the compound I-1 1 mg / kg administration group, and 0 / 6 in the compound I-1 10 mg / kg administration group, showing a dose-dependent virus transmission inhibitory effect (Figure 16). From these results, it was shown that administering compound I-1 to infected hamsters (Index) after infection has the effect of suppressing virus transmission to non-infected hamsters (Contact).

[0143] Test Example 15: Prevention Test of Virus Transmission from SARS-CoV-2 Infected Animals by Prophylactic Administration of Compound I-1 to Non-infected Animals <Materials and Methods> ·Compound Compound I-1 was used as the test sample with a 0.5% methylcellulose (0.5% MC) solution. The administration volume was 5 mL / kg. ·Virus SARS-CoV-2 hCoV-19 / Japan / TY11-927 / 2021 (Delta) isolated at the National Institute of Infectious Diseases was used. ·Hamster Nasal Infection, Medication, Cohabitation, Nasal Wash and Lung Recovery Six-week-old male Syrian hamsters (Japan SLC, Inc.) free of specific pathogens were used in this study. At the time of virus inoculation, the hamsters were anesthetized by subcutaneous administration of an anesthetic solution containing 0.07 mg / mL medetomidine hydrochloride, 6.98 mg / mL alphaxalone, and 1.16 mg / mL butorphanol tartrate at 3 mL / kg, and 100 μL of hCoV-19 / Japan / TY11-927 / 2021 (1.00×10 3 TCID 50It was inoculated intranasally. Compound I-1 was administered subcutaneously once to non-infected hamsters (Contact) 12 hours before cohabitation at doses of 3, 10, 30, and 90 mg / kg. 0.5% MC was administered subcutaneously once to control non-infected hamsters (Contact). From 1 day to 2 days after infection of the infected hamsters (Index), one infected hamster (Index) and one non-infected hamster (Contact) administered with the compound were placed in separate stainless steel cages 2 cm apart in the same cage so that they could not come into direct contact and cohabited. After the cohabitation ended, they were individually housed. The nasal wash fluid and lungs were collected from non-infected hamsters (contact) 4 days after the start of cohabitation (3 days after the end of cohabitation). The nasal wash fluid was collected with 400 μL of DPBS under isoflurane anesthesia, and the supernatant after centrifugation was collected. The lungs were added with 3 mL of DPBS, homogenized, the supernatant after centrifugation was collected, and diluted 2-fold with DPBS. Each group was performed with n = 6. · Measurement of virus titer in nasal wash fluid and lung homogenate supernatant After preparing a 10-fold dilution series of nasal wash fluid or lung homogenate supernatant in medium (MEM, 2% FBS, penicillin-streptomycin), it was inoculated into VeroE6 / TMPRSS2 cells (JCRB1819, 1.50×10 4 cells / well) that had been previously cultured in a 96-well plate. After culturing for 4 days in a CO2 incubator, the cytopathic effect (CPE) was observed, and the virus titer contained in the nasal wash fluid or lung homogenate supernatant was calculated.

[0144] The compound of the present invention was tested essentially as described above. The results are shown below. The virus titer in the nasal wash fluid or lung homogenate supernatant of non-infected hamsters (Contact) administered with the compound 4 days after the start of cohabitation (3 days after the end of cohabitation) with the infected hamsters (Index) was at the detection limit (1.8-log 10 TCID 50Individuals with a titer of 103 TCID50 / mL or higher were 6 / 6 in the 0.5% MC administration group, 5 / 6 in the compound I-1 3 mg / kg administration group, 6 / 6 in the compound I-1 10 mg / kg administration group, 3 / 6 in the compound I-1 30 mg / kg administration group, and 0 / 6 in the compound I-1 90 mg / kg administration group, showing a dose-dependent infection prevention effect (Figure 17). From these results, it was suggested that prophylactic administration of compound I-1 has the effect of preventing virus transmission from infected hamsters (Index).

[0145] Test Example 16: Lethality and body weight loss suppression test of SARS-CoV-2-infected aged hamsters by delayed administration of compound I-1 <Materials and Methods> · Compound Compound I-1 according to the present invention was used as a test sample using polyethylene glycol 400 (PEG400) containing N,N-dimethylacetamide (DMA) and 0.5 w / v% poly(1-vinylpyrrolidone-co-vinyl acetate) (PVPVA) (DMA: 0.5 w / v% PVPVA-containing PEG400 = 1:9). The administration volume was 1.25 mL / kg. · Virus SARS-CoV-2 hCoV-19 / Japan / TY11-927 / 2021 isolated at the National Institute of Infectious Diseases was used. · Hamster nasal infection, dosing, cohabitation, nasal lavage fluid, and lung recovery Specific pathogen-free 11-month-old (aged) male Syrian hamsters (Japan SLC, Inc.) were used in this study. At the time of virus inoculation, the hamsters were anesthetized by subcutaneous administration of an anesthetic solution containing 0.07 mg / mL of medetomidine hydrochloride, 6.98 mg / mL of alphaxalone, and 1.16 mg / mL of butorphanol tartrate at 3 mL / kg, and 100 μL of hCoV-19 / Japan / TY11-927 / 2021 (1.00×10 4 TCID 50It was inoculated intranasally. Starting from 1 day after virus inoculation, Compound I-1 was orally administered twice a day at doses of 0.1, 1, and 10 mg / kg. The control infected hamsters were orally administered DMA / 0.5 w / v% PVPVA in PEG400 twice a day. The compound administration was carried out for 5 days starting from the start of administration. The body weight was monitored once a day, and in the evaluation of survival rate, if it was less than 80% based on the body weight immediately before infection, it was regarded as death. It was carried out with n = 6 in each group.

[0146] The compound of the present invention was tested essentially as described above. The results are shown below. In the group administered with DMA / 0.5 w / v% PVPVA in PEG400 of the infected hamsters, 2 animals died 8 days after infection, and the survival rate was 66.7%. At this time, in the groups administered with 1 and 10 mg / kg of Compound I-1, all animals survived until 10 days after the observed infection, and the body weight loss was suppressed (Figs. 18 and 19). From the above results, it was suggested that even in aged hamsters, administration of Compound I-1 had an effect of suppressing lethality and body weight loss.

[0147] In rodents, it is also known that with aging, similar to humans, the expression level of the ACE2 receptor, which is known as a receptor for SARS-CoV-2, increases (References: Scientific Reports (2020)10:22401 and Molecular Therapy Methods & Clinical Development, Vol. 18, P1-6, 2020). Also, because the normal immune response that defends the body from the infection source and eliminates them decreases, it is assumed that it is likely to deteriorate due to virus infection. As shown above, in the SARS-CoV-2 infection model using aged hamsters, administration of Compound I-1 was found to have an effect of suppressing body weight loss and lethality. The non-clinical trial using aged hamsters is positioned as one of the non-clinical evaluation systems that mimics the process of high-risk patients with underlying diseases leading to severe illness. Therefore, as a treatment option for patients at high risk of severe illness, the usefulness of Compound I-1 is supported. In addition, the above test results suggest that the exacerbation caused by viral infection was suppressed in the compound I-1 administration group, supporting not only the antiviral effect of compound I-1 against SARS-CoV-2 but also its usefulness as a medicine for suppressing the exacerbation of infectious diseases caused by SARS-CoV-2.

[0148] Test Example 17: In vitro combination effect confirmation test <Operation procedure> · Test sample Ensitrelvir fumarate, nirmatrelvir, remdesivir, EIDD-1931, sotrovimab, and tixagevimab / cilgavimab were used as test samples to be combined with compound I-1. · Dilution and dispensing of test samples Each test sample was diluted to an appropriate concentration with DMSO or DPBS and medium (MEM, 2% FBS, penicillin-streptomycin), and a serial dilution series was prepared in a 96-well plate. · Dilution and dispensing of cells and SARS-CoV-2 A549 / ACE2-TMPRSS2 cells (Invivogen, a549-hace2tpsa, 1.5×10 4 cells / well) and SARS-CoV-2 hCoV-19 / Japan / TY11-927 / 2021 (100 TCID 50 / well) were mixed with medium (MEM, 2% FBS, penicillin-streptomycin) and dispensed into the wells containing the test samples, followed by culturing in a CO2 incubator for 2 days. · Dispensing of CellTiter-Glo® 2.0 and measurement of luminescence signal After returning the plates cultured for 3 days to room temperature, CellTiter-Glo® 2.0 was dispensed into each well and mixed with a plate mixer. After standing for a certain period of time, the luminescence signal (Lum) was measured with a plate reader.

[0149] <Analysis of combination effect> The Synergy volume and Antagonism volume were calculated by MacSynergyII. These numerical values can be calculated with reference to previously reported papers (Antiviral Research, 1990, Volume 14, p. 181-206), etc. · Judgment of combined effect The combined effect was judged according to the following criteria for the Synergy volume and Antagonism volume at a 99% confidence level of MacSynergyII. synergy volume ≦ 25: additive 25 < synergy volume ≦ 50: minor synergy 50 < synergy volume ≦ 100: moderate synergy 100 < synergy volume: strong synergy -25 ≦ antagonism volume: additive -50 ≦ antagonism volume < -25: minor antagonism -100 ≦ antagonism volume < -50: moderate antagonism antagonism volume < -100: strong antagonism

[0150] Essentially, the tests were conducted as described above. The results are shown in the following table.

[0151]

Table 9

[0152] From the above results, the combination of Compound I-1 with RNA-dependent RNA polymerase inhibitors (EIDD-1931 and remdesivir), 3CL protease inhibitors (ensitrelvir, nirmatrelvir), and anti-SARS-CoV-2 monoclonal antibodies (sotrovimab, tixagevimab / cilgavimab) showed additive to synergistic SARS-CoV-2 growth inhibitory effects without showing antagonistic effects.

[0153] The following formulation examples are merely illustrative and are not intended to limit the scope of the invention in any way. The compounds according to the present invention can be administered as pharmaceutical compositions by any conventional route, particularly enterally, for example, orally, for example, in the form of tablets or capsules, or parenterally, for example, in the form of injection solutions or suspensions, topically, for example, in the form of lotions, gels, ointments or creams, or in nasal form or suppository form. Pharmaceutical compositions containing the compounds of the present invention in free form or in the form of pharmaceutically acceptable salts, together with at least one pharmaceutically acceptable carrier or diluent, can be produced by conventional methods, by mixing, granulating or coating methods. For example, oral compositions can be made into tablets, granules, capsules containing excipients, disintegrants, binders, lubricants, etc. and active ingredients, etc. Also, injection compositions can be made into solutions or suspensions, may be sterilized, and may contain preservatives, stabilizers, buffering agents, etc.

Industrial Applicability

[0154] The compounds according to the present invention have inhibitory activity against coronavirus 3CL protease, and pharmaceutical compositions containing the compounds according to the present invention are useful as therapeutic agents and / or prophylactic agents for coronavirus infections.

Claims

1. Formula (II): 【Chemical 1】 wherein X is a single bond or -CH ２ -; R ２ is a substituted or unsubstituted aromatic carbocyclic group; R ３ｃ is a substituted or unsubstituted aromatic carbocyclic group, a substituted or unsubstituted non-aromatic carbocyclic group, a substituted or unsubstituted aromatic heterocyclic group, a substituted or unsubstituted non-aromatic heterocyclic group, a halogen, a substituted or unsubstituted alkyl or a substituted or unsubstituted amino; R １ is a substituted or unsubstituted aromatic heterocyclic group or a substituted or unsubstituted non-aromatic heterocyclic group; m is 0 or 1; R ５ａ each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group; R ５ｂ each independently represents a hydrogen atom or a substituted or unsubstituted alkyl), provided that the following compounds: [Chemical 2] excluding), or a pharmaceutically acceptable salt thereof.

2. X is a single bond; R ２ is phenyl substituted by halogen; R ３ｃ is a non-aromatic heterocyclic group substituted with 1 to 3 substituents selected from substituent group a or an unsubstituted non-aromatic heterocyclic group (substituent group a: halogen, hydroxy, C1-C3 alkyl, C1-3 alkyloxy, halo C1-3 alkyl, halo C1-3 alkyloxy, unsubstituted 5-6 membered aromatic heterocyclic group and 5-6 membered aromatic heterocyclic group substituted with halogen) or an alkyl substituted with 1 to 3 substituents selected from substituent group b or an unsubstituted alkyl (substituent group b: halogen, hydroxy, C1-3 alkyloxy and halo C1-3 alkyloxy); R １ is a pyridyl substituted with a halogen; The compound according to claim 1, wherein m is 0, or a pharmaceutically acceptable salt thereof.

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