Pharmaceutical composition containing uracil derivative

Abstract

The present invention provides a pharmaceutical composition containing a compound that exhibits coronavirus growth–inhibiting activity. Provided is a pharmaceutical composition containing a compound represented by formula (I-1) or a pharmaceutically acceptable salt thereof.

Description

Pharmaceutical compositions containing uracil derivatives

[0001] The present invention relates to pharmaceutical compositions containing compounds that exhibit coronavirus 3CL protease inhibitory activity.

[0002] Coronaviruses, which belong to the Orthocoronavirus subfamily of the Coronaviridae family of the Nidovirales order, have a genome size of approximately 30 kilobases and are among the largest single-stranded positive-strand RNA viruses known. Coronaviruses are classified into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. Seven types of coronaviruses that infect humans are known: two types in the Alphacoronavirus genus (HCoV-229E and HCoV-NL63) and five types in the Betacoronavirus genus (HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2). Of these, four (HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43) are pathogens that cause the common cold, while the remaining three 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 internationally, and on March 11, 2020, the WHO declared it a pandemic. The main routes of SARS-CoV-2 transmission have been reported to be droplet, contact, and aerosol transmission, and it has been confirmed that SARS-CoV-2 remains airborne with aerosols for approximately three hours, maintaining its infectiousness (Non-Patent Document 1). The incubation period is approximately two to 14 days, and typical symptoms include cold-like fever (87.9%), dry cough (67.7%), fatigue (38.1%), and phlegm (33.4%) (Non-Patent Document 2). In severe cases, respiratory failure due to acute respiratory distress syndrome, acute lung injury, and interstitial pneumonia occurs. Multiple organ failure, including renal and liver failure, has also been reported.

[0004] In Japan, through drug repositioning of existing drugs, the antiviral drug remdesivir, the anti-inflammatory drug dexamethasone, and the rheumatism drug baricitinib have been approved as treatments for COVID-19, with the anti-IL-6 receptor antibody tocilizumab receiving additional approval in January 2022. Furthermore, the antibody cocktail therapy Lonaprive (casirivimab / imdevimab) received special approval in July 2021, sotrovimab in September 2021, and molnupiravir in December 2021. There is insufficient evidence regarding the efficacy and safety of these drugs. Therefore, there is an urgent need to develop treatments for COVID-19.

[0005] When coronaviruses infect cells, they synthesize two polyproteins. These polyproteins contain the replication complex that creates the viral genome and two proteases. Proteases cleave the polyproteins synthesized by the virus, playing an essential role in enabling each protein to function. Of the two proteases, the 3CL protease (main protease) is responsible for most of the polyprotein cleavage (Non-Patent Document 3). In June 2021, the completion of Phase 1b trials of Pfizer's Lufotrelvir (PF-07304814), a prodrug of PF-00835231, a COVID-19 treatment targeting 3CL protease, was published in ClinicalTrials.gov (NCT04535167). Additionally, in March 2021, Pfizer announced the initiation of Phase 1 trials for PF-07321332, a therapeutic drug for COVID-19. The structural formulae of PF-00835231, Lufotrelvir, and PF-07321332 are shown below, and 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: Lufotrelvir (PF-07304814): PF-07321332: In December 2021, PAXLOVID™ was granted emergency use authorization in the United States, and on February 10, 2022, PAXLOVID® Pak was granted special approval in Japan.

[0006] Furthermore, in August 2021, the start of Phase 1 trials of PBI-0451 by Pardes Biosciences, a COVID-19 treatment targeting 3CL protease, was published in ClinicalTrials.gov (NCT05011812). The structural formula of PBI-0451 is shown below, and its chemical structure is different from that of the compound used in the present invention (Non-Patent Document 12).

[0007] Furthermore, Zocova (registered trademark) was approved in Japan on an emergency basis on November 22, 2022, as a COVID-19 treatment targeting 3CL protease, and was approved on March 5, 2024 (Non-Patent Documents 17 and 18). The active ingredient of Zocova is ensitrevir fumarate, and its structural formula is shown below, and its chemical structure is different from that of the compound used in the present invention (Patent Documents 10 to 13).

[0008] On the other hand, there is insufficient evidence regarding resistance mutations to COVID-19 drugs targeting 3CL proteases.

[0009] Compounds having 3CL protease inhibitory activity are disclosed in Non-Patent Documents 4 to 7 and 13 to 16, but none of these documents describe or suggest the compounds to be used in the present invention. Furthermore, compounds having 3CL protease inhibitory activity are disclosed in Patent Documents 14 and 15, but none of these documents describe or suggest pharmaceutical compositions containing the compounds according to the present invention. ３ and / or P2X ２／３Compounds having receptor inhibitory activity are disclosed in Patent Documents 3 to 9, but none of these documents describe or suggest 3CL protease inhibitory activity or antiviral effects. Non-Patent Document 11 describes a compound having HIV-1 reverse transcriptase inhibitory activity, but none of these documents describe or suggest 3CL protease inhibitory activity or anti-coronavirus effects.

[0010] International Publication No. 2021 / 205298 International Publication No. 2021 / 250648 International Publication No. 2012 / 020742 International Publication No. 2013 / 118855 Chinese Patent Application Publication No. 113620888 Chinese Patent Application Publication No. 113666914 Chinese Patent Application Publication No. 113735838 Chinese Patent Application Publication No. 113773300 Chinese Patent Application Publication No. 113801097 International Publication No. 2022 / 138987 International Publication No. 2022 / 138988 International Publication No. 2023 / 054292 International Publication No. 2023 / 054732 International Publication No. 2023 / 195529 International Publication No. 2023 / 195530

[0011] The New England Journal of Medicine (2020), Vol. 382, ​​pp. 1564-1567, "Report of the WHO-China Joint Mission on Coronavirus Disease 2019 (COVID-19)", [online], February 28, 2020, WHO, [Retrieved March 16, 2023], Internet <URL:https: / / www.who.int / docs / default-source / coronaviruse / who-china-joint-mission-on-covid-19-final-report.pdf> Science (2003), Vol. 300, pp. 1763-1767 "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 Cell Research (2020), Vol. 30, pp. 678-692 Science (2020), Vol. 368, pp. 409-412 ACS Central Science (2021), Vol. 7, No. 3, pp. 467-475 261st Am Chem Soc (ACS) Natl Meet ・ 2021-04-05 / 2021-04-16 ・ Virtual, N / A ・ Abst 243Science (2021), Vol. 374, pp. 1586-1593, "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, [Retrieved March 16, 2023], Internet <URL: https: / / www.pfizer.com / news / press-release / press-release-detail / pfizers-novel-covid-19-oral-antiviral-treatment-candidate> Synthetic Communications (2006), Vol. 36, No. 19, pp. 2913-2920 "Discovery and Development of PBI-0451", [online], March 24, 2022, 35th International Conference on Antiviral Research (ICAR), [Retrieved March 16, 2023], Internet <URL: https: / / ir.pardesbio.com / static-files / fc7c4f8c-e0bd-4b97-8c9c-eff09bafd4db> Molecules (2020), Vol. 25, p. 3193 Molecules (2020), Vol. 25, p. 3920 European Journal of Medicinal Chemistry (2020), Vol. 206, pp. 112711; Journal of the American Chemical Society (2022), Vol. 144, pp. 2905-2920; Shionogi & Co., Ltd. Press Release (November 22, 2022); Shionogi & Co., Ltd. Press Release (March 5, 2024).

[0012] An object of the present invention is to provide a pharmaceutical composition containing a compound having coronavirus 3CL protease inhibitory activity. Preferably, the present invention provides a medicament containing a compound having antiviral activity, particularly an activity of inhibiting coronavirus proliferation.

[0013] The present invention relates to the following: (1) Formula (I-1): or a pharmaceutically acceptable salt thereof. (2) The pharmaceutical composition according to the above item (1), which is a 3CL protease inhibitor. (3) The pharmaceutical composition according to the above item (1) or (2), which is used for inhibiting the proliferation of SARS-CoV-2 virus. (4) The pharmaceutical composition according to any of the above items (1) to (3), which is a therapeutic agent and / or preventive agent for novel coronavirus disease 2019 (COVID-19). (5) The pharmaceutical composition according to any of the above items (1) to (4), which is used for suppressing the aggravation of infection caused by SARS-CoV-2. (6) The pharmaceutical composition according to any of the above items (1) to (4), which is used so as to start administration within 72 hours after the onset of symptoms of infection caused by SARS-CoV-2 or after a positive diagnosis of SARS-CoV-2. (7) The pharmaceutical composition according to any one of items (1) to (4) above, which is used so that administration is initiated within 24 hours after the onset of symptoms of SARS-CoV-2 infection or a positive diagnosis of SARS-CoV-2. (8) The pharmaceutical composition according to any one of items (1) to (4) above, which is used so that administration is initiated within 120 hours after the onset of symptoms of SARS-CoV-2 infection or a positive diagnosis of SARS-CoV-2. (9) The pharmaceutical composition according to any one of items (1) to (4) above, which is used so that administration is initiated within 48 hours after the onset of symptoms of SARS-CoV-2 infection or a positive diagnosis of SARS-CoV-2. (10) The pharmaceutical composition according to any one of items (1) to (4) above, which is used to suppress viral transmission of SARS-CoV-2. (11) The pharmaceutical composition according to any one of the above items (1) to (4), wherein the symptoms of infection with SARS-CoV-2 are mild or moderate I symptoms.

[0014] (12) A method for inhibiting SARS-CoV-2 viral proliferation, comprising administering to an individual in need of treatment and / or prevention of novel coronavirus infection (COVID-19) a compound represented by formula (I-1): (13) A method for treating and / or preventing novel coronavirus disease (COVID-19), comprising administering to an individual in need of treatment and / or prevention of novel coronavirus disease (COVID-19), a compound represented by formula (I-1): (14) A method for suppressing the aggravation of infection caused by SARS-CoV-2, comprising administering to an individual in need of treatment and / or prevention of novel coronavirus infection (COVID-19), a compound represented by formula (I-1): or a pharmaceutically acceptable salt thereof. (15) A method for treating novel coronavirus disease (COVID-19), comprising administering to an individual in need of treatment for novel coronavirus disease (COVID-19) a compound represented by formula (I-1): (16) A method for treating novel coronavirus disease (COVID-19), comprising administering a compound represented by formula (I-1): or a pharmaceutically acceptable salt thereof to an individual in need of treatment for novel coronavirus disease (COVID-19), within 72 hours after the onset of symptoms of infection with SARS-CoV-2 or after a positive diagnosis of SARS-CoV-2. or a pharmaceutically acceptable salt thereof within 24 hours after the onset of symptoms of infection with SARS-CoV-2 or after a positive diagnosis of SARS-CoV-2. (17) A method for treating novel coronavirus infection (COVID-19), comprising administering a compound represented by formula (I-1): or a pharmaceutically acceptable salt thereof within 120 hours after the onset of symptoms of infection with SARS-CoV-2 or after a positive diagnosis of SARS-CoV-2. (18) A method for treating novel coronavirus infection (COVID-19), comprising administering a compound represented by formula (I-1): (19) A method for treating novel coronavirus disease (COVID-19), comprising administering a compound represented by formula (I-1) or a pharmaceutically acceptable salt thereof to an individual in need of treatment for novel coronavirus disease (COVID-19) within 48 hours after the onset of symptoms of infection with SARS-CoV-2 or after a positive diagnosis of SARS-CoV-2. (20) A method for treating novel coronavirus disease (COVID-19), wherein the symptoms of an individual in need of treatment for novel coronavirus disease (COVID-19) are mild or moderate I symptoms, and the individual is administered a compound represented by formula (I-1): or a pharmaceutically acceptable salt thereof.

[0015] (21) A compound of formula (I-1): for inhibiting SARS-CoV-2 viral growth: (22) Use of a compound represented by formula (I-1) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment and / or prevention of novel coronavirus infection (COVID-19): (23) Use of a compound represented by formula (I-1) or a pharmaceutically acceptable salt thereof for suppressing the aggravation of infection caused by SARS-CoV-2. (23') Use of a compound represented by formula (I-1) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for suppressing the aggravation of novel coronavirus infection (COVID-19): (24) Use of a compound represented by formula (I-1): or a pharmaceutically acceptable salt thereof for administration within 72 hours after the onset of symptoms of infection with SARS-CoV-2 or after a positive diagnosis of SARS-CoV-2. (24') Use of a compound represented by formula (I-1) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating novel coronavirus disease (COVID-19): or a pharmaceutically acceptable salt thereof, wherein the medicament is administered within 72 hours after the onset of symptoms of SARS-CoV-2 infection or after a positive diagnosis of SARS-CoV-2. (25) A compound of formula (I-1): (25') Use of a compound represented by formula (I-1) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating novel coronavirus disease (COVID-19): or a pharmaceutically acceptable salt thereof, wherein the medicament is administered within 24 hours after the onset of symptoms of SARS-CoV-2 infection or after a positive diagnosis of SARS-CoV-2. (26) A compound of formula (I-1): (26') Use of a compound represented by formula (I-1) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating novel coronavirus disease (COVID-19): or a pharmaceutically acceptable salt thereof, wherein the medicament is administered within 120 hours after the onset of symptoms of infection with SARS-CoV-2 or after a positive diagnosis of SARS-CoV-2. (27) A compound of formula (I-1): (27') Use of a compound represented by formula (I-1) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating novel coronavirus disease (COVID-19): (28) Use of a compound represented by formula (I-1): or a pharmaceutically acceptable salt thereof, wherein the medicament is administered within 48 hours after the onset of symptoms of infection with SARS-CoV-2 or after a positive diagnosis of SARS-CoV-2. (28') Use of a compound represented by formula (I-1) or a pharmaceutically acceptable salt thereof for producing a medicament for suppressing the spread of novel coronavirus infection (COVID-19): (29) Use of a compound represented by formula (I-1) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating novel coronavirus disease (COVID-19): or a pharmaceutically acceptable salt thereof, wherein the symptoms of an individual in need of treatment for novel coronavirus disease (COVID-19) are mild or moderate I symptoms. (29') Use of a compound represented by formula (I-1): or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of novel coronavirus disease (COVID-19): or a pharmaceutically acceptable salt thereof, wherein the symptoms of an individual in need of treatment for novel coronavirus disease (COVID-19) are mild or moderate I symptoms.

[0016] The present invention also relates to the following: (101) A preventive drug for novel coronavirus infection (COVID-19), which is used to suppress the onset of SARS-CoV-2 after exposure, comprising a compound represented by the following formula (I-1): (102) A method for preventing novel coronavirus infection (COVID-19), comprising administering to an individual in need of suppression of onset after exposure to SARS-CoV-2 a compound represented by formula (I-1): (103) A method for preventing novel coronavirus disease (COVID-19), comprising administering a compound represented by formula (I-1): or a pharmaceutically acceptable salt thereof for suppressing the onset of SARS-CoV-2 after exposure. (104) Use of a compound represented by formula (I-1) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for suppressing the onset of SARS-CoV-2 after exposure. or a pharmaceutically acceptable salt thereof.

[0017] Furthermore, the present invention relates to the following: (201) A preventive drug for novel coronavirus infection (COVID-19), which is used for pre-exposure prophylaxis against SARS-CoV-2, comprising a compound represented by the following formula (I-1): (202) A method for preventing novel coronavirus infection (COVID-19), comprising administering to an individual in need of pre-exposure prophylaxis against SARS-CoV-2 a compound represented by formula (I-1): or a pharmaceutically acceptable salt thereof. (203) A method for preventing novel coronavirus disease (COVID-19), comprising administering a compound represented by formula (I-1): or a pharmaceutically acceptable salt thereof for pre-exposure prophylaxis against SARS-CoV-2. (204) Use of a compound represented by formula (I-1) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for pre-exposure prophylaxis against SARS-CoV-2. or a pharmaceutically acceptable salt thereof.

[0018] In one embodiment, the present invention also includes the following: (301) Formula (II): (Wherein, X is a single bond or —CH ２ - and 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, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted amino; R１ is a substituted or unsubstituted aromatic heterocyclic group or a substituted or unsubstituted non-aromatic heterocyclic group; m is 0 or 1; R ５ａ are each independently a hydrogen atom or a substituted or unsubstituted alkyl; R ５ｂ are each independently a hydrogen atom or a substituted or unsubstituted alkyl) (provided that the following compounds: (302) X is a single bond; R ２ is phenyl substituted with 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, haloC1-3 alkyl, haloC1-3 alkyloxy, unsubstituted 5- to 6-membered aromatic heterocyclic group and 5- to 6-membered aromatic heterocyclic group substituted with halogen), or an alkyl or unsubstituted alkyl substituted with 1 to 3 substituents selected from substituent group b (substituent group b: halogen, hydroxy, C1-3 alkyloxy and haloC1-3 alkyloxy); R １ is pyridyl substituted with halogen; and m is 0, or a pharmaceutically acceptable salt thereof.

[0019] 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 and / or preventive agents for coronavirus infections.

[0020] hCoV-19 / Japan / TY7-501 / 2021-infected mice (1.00 × 10 ４ TCID ５０ The graph shows the virus titer in the lung homogenate 3 days after infection when the vehicle and the compound represented by formula (I-1) were orally administered twice daily for 2 days starting from 1 day after infection for hCoV-19 / Japan / TY41-702 / 2022-infected hamsters (1.00 x 10 ４T.C.I.D. ５０ The graph shows the virus titer in the lung homogenate 3-4 days after infection when a vehicle and the compound represented by formula (I-1) were orally administered twice daily for 2-3 days starting 1 day after infection for hamsters (inoculated intranasally with hCoV-19 / Japan / TY41-702 / 2022). The vertical axis shows the virus titer in the lung homogenate, and the horizontal axis shows each administration group and the number of days after infection. ４ T.C.I.D. ５０ The figure shows the virus titer in the nasal turbinate homogenate 3-4 days after infection when a vehicle and the compound represented by formula (I-1) were orally administered twice daily for 2-3 days starting 1 day after infection for hamsters (inoculated intranasally). The vertical axis shows the virus titer in the nasal turbinate homogenate, and the horizontal axis shows each administration group and the number of days after infection. hamsters (inoculated intranasally with hCoV-19 / Japan / TY41-702 / 2022) infected with hCoV-19 / Japan / TY41-702 / 2022 (1.00 x 10 ４ T.C.I.D. ５０ The graph shows lung weight / body weight ratio 7 days after infection when a vehicle and a compound represented by formula (I-1) were orally administered twice daily for 5 days starting from 1 day after infection for hamsters (inoculated intranasally with hCoV-19 / Japan / TY41-702 / 2022). The vertical axis shows lung weight / body weight, and the horizontal axis shows each administration group. ４ T.C.I.D. ５０ The graph shows the weight change up to 10 days after infection when a vehicle and the compound represented by formula (I-1) were orally administered twice daily for 5 days starting from 1 day after infection to hamsters (inoculated intranasally with hCoV-19 / Japan / TY41-702 / 2022). The vertical axis shows the weight change (%) when the weight on the day of infection is taken as 100%, and the horizontal axis shows the number of days after infection. ４ T.C.I.D. ５０ The graph shows the weight change up to 7 days after infection when a vehicle and the compound represented by formula (I-1) were administered subcutaneously once 3 days or 1 day before infection to hamsters infected with hCoV-19 / Japan / TY41-702 / 2022 (1.00 x 10 ４ T.C.I.D. ５０The graph shows the virus titer in the lung homogenate 1-2 days after infection when a vehicle and the compound represented by formula (I-1) were administered subcutaneously once 1 day before infection in hamsters infected with hCoV-19 / Japan / TY41-702 / 2022 (1.00 x 10 ４ T.C.I.D. ５０

[0047] Figure 1 shows the virus titer in the nasal turbinate homogenate 1-2 days after infection when a single subcutaneous administration of a vehicle or the compound represented by formula (I-1) was administered 1 day before infection to hamsters (nasally inoculated with the compound represented by formula (I-1)). The vertical axis shows the virus titer in the nasal turbinate homogenate, and the horizontal axis shows each administration group and the number of days since infection.

[0048] Figure 1 shows the powder X-ray diffraction pattern of the anhydrous crystal of the compound represented by formula (I-1). The horizontal axis shows 2θ (°), and the vertical axis shows intensity.

[0049] Figure 2 shows a peak list for the powder X-ray diffraction pattern in Figure 7. In the table, position shows 2θ (°), and intensity shows intensity.

[0050] Figure 1 shows the crystal structure (structure in the asymmetric unit) of the anhydrous crystal of the compound represented by formula (I-1).

[0051] Figure 1 shows the results of differential scanning calorimetry (DSC) of the anhydrous crystal of the compound represented by formula (I-1). The horizontal axis shows temperature (°C), and the vertical axis shows heat (W / g). The figure shows the results of simultaneous differential thermal analysis and thermogravimetry (TG / DTA) of the anhydrous crystal of the compound represented by formula (I-1). The vertical axis shows the amount of heat (μV) or the weight change (%), and the horizontal axis shows the temperature (°C). Cel in the figure means degrees Celsius (°C). The figure shows the Raman spectrum of the anhydrous crystal of the compound represented by formula (I-1). The horizontal axis shows the Raman shift (cm －１ ), and the vertical axis represents peak intensity. The upper part shows the HPLC measurement results of the anhydrous crystals of the compound represented by formula (I-1). The lower part shows the HPLC peak table. ４ T.C.I.D. ５０The graph shows the virus titer in nasal lavage fluid (NALF) 3-7 days after infection when a vehicle and the compound represented by formula (I-1) were orally administered twice daily for 1-5 days starting 2 days after infection for hamsters (inoculated intranasally with hCoV-19 / Japan / TY41-702 / 2022). The vertical axis shows the virus titer in NALF, and the horizontal axis shows the number of days after infection. ４ T.C.I.D. ５０ The graph shows the virus titer in nasal lavage fluid (NALF) 4-8 days after infection when a vehicle and a compound represented by formula (I-1) were orally administered twice daily for 1-5 days starting 3 days after infection for hamsters (inoculated intranasally with hCoV-19 / Japan / TY11-927 / 2021). The vertical axis shows the virus titer in NALF, and the horizontal axis shows the number of days after infection. ３ T.C.I.D. ５０ The graph shows the virus titers in the lung homogenate and nasal lavage fluid (NALF) of the uninfected hamsters (Contact) four days after the start of cohabitation when a compound was administered to a hamster (Contact) via nasal inoculation with hCoV-19 / Japan / TY11-927 / 2021 (Index) and a non-infected hamster (Contact) was housed together for 12 hours overnight from day 1 to day 2 after infection. The infected hamsters (Index) were orally administered a vehicle and a compound represented by formula (I-1) twice daily starting 8 hours after infection. The vertical axis shows the virus titers in the lung homogenate and NALF, and the horizontal axis shows each administration group. hCoV-19 / Japan / TY11-927 / 2021-infected hamsters (1.00 x 10 ３ T.C.I.D. ５０ The graph shows the virus titers in the lung homogenate and nasal lavage fluid (NALF) of the uninfected hamster (Contact) four days after the start of cohabitation when the uninfected hamster (Contact) was cohabited with a non-infected hamster (Contact) administered with the compound for 12 hours overnight from day 1 to day 2 after infection. The uninfected hamster (Contact) was subcutaneously administered a vehicle and a compound represented by formula (I-1) 12 hours before the start of cohabitation. The vertical axis shows the virus titers in the lung homogenate and NALF, and the horizontal axis shows each administration group. Aged hamsters infected with hCoV-19 / Japan / TY11-927 / 2021 (1.00 x 10 ４ T.C.I.D. ５０The graph shows the survival rate up to 10 days after infection when a vehicle and a compound represented by formula (I-1) were orally administered twice daily for 5 days starting one day after infection for 11-month-old hamsters (inoculated intranasally with hCoV-19 / Japan / TY11-927 / 2021). The vertical axis shows the survival rate (%), and the horizontal axis shows the number of days after infection. ４ T.C.I.D. ５０

[0063] Figure 1 shows the weight change up to 10 days after infection when a vehicle or the compound of formula (I-1) was orally administered twice daily for 5 days starting one day after infection to 11-month-old hamsters (inoculated intranasally). The vertical axis shows the weight change (%) when the weight on the day of infection is taken as 100%, and the horizontal axis shows the number of days after infection.

[0021] The meaning of each term used in this specification is explained below. Unless otherwise specified, each term has the same meaning whether used alone or in combination with other terms. The term "consisting of" means having only the constituent elements. The terms "comprise" and "include" mean that the term is not limited to the constituent elements and does not exclude unrecited elements. The present invention will be described below with reference to exemplary embodiments. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, terms used in this specification should be understood to have the meaning commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0022] In the compounds of formula (II) herein, R １ , R ２ , R ３ｃ , R ５ａ and R ５ｂThe definitions of each term used in the above may refer to WO 2023 / 195529, the disclosure of which is incorporated herein by reference. Similarly, the definitions of substituents used in each term may refer to WO 2023 / 195529, the disclosure of which 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, rotamers, etc.), racemates, or mixtures thereof.

[0024] One or more hydrogen, carbon and / or other atoms of the compounds represented by formula (I-1) or formula (II) may be replaced with isotopes of hydrogen, carbon and / or other atoms, respectively. Examples of such isotopes include 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 The isotopes of the compounds represented by formula (I-1) or formula (II) include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as Cl. The compounds represented by formula (I-1) or formula (II) also include compounds substituted with such isotopes (e.g., deuterium-substituted compounds, etc.). The isotope-substituted compounds are also useful as pharmaceuticals. The compounds represented by formula (I-1) or formula (II) include all radiolabeled compounds substituted with radioactive isotopes contained in the isotopes. Also included in the present invention is a "radiolabeling method" for producing the "radiolabeled compound," and the "radiolabeled compound" is useful as a research and / or diagnostic tool in metabolism pharmacokinetic studies and binding assays.

[0025] Deuterium-substituted derivatives of the compound represented by formula (I-1) include the following structures.

[0026] Radiolabeled compounds of formula (I-1) or (II) can be prepared by methods well known in the art. For example, tritium-labeled compounds of formula (I-1) or (II) can be prepared by introducing tritium into a specific compound of formula (I-1) or (II) via catalytic dehalogenation using tritium. This method involves reacting a precursor of a compound of formula (I-1) or (II) that has been appropriately halogen-substituted with tritium gas in the presence of a suitable catalyst, such as Pd / C, with or without a base. Other suitable methods for preparing tritium-labeled compounds can be found in "Isotopes in the Physical and Biomedical Sciences, Vol. 1, Labeled Compounds (Part A), Chapter 6 (1987)." １４ C-labeled compounds are １４ It can be prepared by using a raw material having C carbon.

[0027] The compounds of formula (I-1) or (II) of the present invention may form prodrugs, and the present invention also encompasses such various prodrugs. Prodrugs are derivatives of the compounds of the present invention having chemically or metabolically decomposable groups, and are compounds that become pharmaceutically active compounds of the present invention in vivo by solvolysis or under physiological conditions. Prodrugs include compounds that are converted to compounds of formula (I-1) or (II) by enzymatic oxidation, reduction, hydrolysis, etc. under physiological conditions in vivo, and compounds that are converted to compounds of formula (I-1) or (II) by hydrolysis with gastric acid, etc. Methods for selecting and preparing appropriate prodrug derivatives are described, for example, in "Design of Prodrugs, Elsevier, Amsterdam, 1985." Prodrugs may themselves be active.

[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), a pharmaceutically acceptable salt thereof, or a solvate thereof" also encompasses such various salts, co-crystals, and solvates thereof.

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

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

[0031] Generally, salts are considered to be those in which proton transfer occurs between a compound and a counter molecule; however, it is known that in some cases, proton transfer may not be complete. This state is sometimes called a cocrystal, as it is not a true salt. It is also known that proton transfer may change continuously depending on temperature. Therefore, as used herein, "a pharmaceutically acceptable salt of a compound represented by formula (I-1) or formula (II)" encompasses cocrystals and refers to a pharmaceutically acceptable salt or cocrystal of a compound represented by formula (I-1) or formula (II).

[0032] One aspect of the present specification is a pharmaceutically acceptable salt or co-crystal of a 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, sulfuric 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, or the like.

[0033] The study of salt and co-crystal formation provides a means to modify the physicochemical and resulting biological characteristics of a drug without altering its chemical structure. Salt and co-crystal formation can dramatically affect the properties of a drug. Hygroscopicity, stability, solubility, and processing characteristics are also important considerations in selecting an appropriate salt or co-crystal. The solubility of a salt or co-crystal can affect its suitability for use as a drug. If aqueous solubility is low, the dissolution rate upon in vivo administration may be rate-limited by the absorption process, resulting in low bioavailability. In addition, low water solubility can make administration by injection difficult, limiting the selection of an appropriate administration route.

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

[0035] The term "solvate" as used herein refers to, for example, a compound represented by formula (I-1) or formula (II) in which any number of solvent molecules are regularly arranged. Examples of solvent molecules 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, pentane, pentane-1-one, pentane-2-one ... Examples of suitable solvents include ethanol, 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.Preferred examples 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, and tetrahydrofuran. More preferred examples 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, and trifluoroacetic acid. Furthermore, 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 to it or a hydrate may be formed.

[0036] As used herein, the term "crystal" refers to a solid in which constituent atoms, ions, molecules, etc. are arranged in a three-dimensional order, and is distinguished from amorphous solids which do not have such an orderly internal structure. The term "crystal" as used herein may be a single crystal, twin crystal, polycrystal, etc. Furthermore, "crystals" may have "crystalline polymorphs" which have the same composition but different arrangements within the crystal, and these are all referred to as "crystalline forms." "Compounds represented by formula (I-1) or formula (II), pharmaceutically acceptable salts thereof, or solvates thereof" encompass these crystalline polymorphs. The crystals of the compounds according to the present invention may be deuterium-converted forms. As used herein, "crystals" refer to isotopes (e.g., ２ H. ３ H. １４ C. ３５ S. １２５The 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. The physical properties of the crystals can also be confirmed by a number of techniques such as differential scanning calorimetry, moisture adsorption / desorption measurements, and dissolution characteristics.

[0037] One embodiment herein is an anhydrous crystal of the compound represented by formula (I-1). As used herein, "anhydrous" is synonymous with "solvate-free," "nonsolvate," "anhydrate," and "nonhydrate." The theoretical content of water of crystallization in an anhydrous crystal of the compound represented by formula (I-1) is 0% by weight. However, in analyzing the amount of water and / or the amount of solvent, the value may be higher than the theoretical content of water of crystallization due to the influence of the water and / or solvent attached to the crystal surface.

[0038] One embodiment of the present specification is an anhydrous crystal of the compound represented by formula (I-1), which has 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 a powder X-ray diffraction pattern (CuKα radiation, λ=1.5418 Å).

[0039] One embodiment of the present specification is an anhydrous crystal of the compound represented by formula (I-1), characterized by the following crystallographic data when a single crystal diffraction experiment was carried out using CuKα radiation (λ=1.5418 Å) at 298 K (25° C.): space group: Pbca a=14.67 ű0.05 Å b=11.83 ű0.05 Å c=27.10 ű0.05 Å α=90° β=90° γ=90°

[0040] One embodiment of the present specification is an anhydrous crystalline form of the compound represented by formula (I-1) having a melting point of 261.3°C ± 2°C as measured by differential scanning calorimetry (DSC). Another embodiment of the present specification is an anhydrous crystalline form of the compound represented by formula (I-1) having a melting point of 265.6°C ± 2°C as measured by simultaneous differential thermal analysis-thermogravimetry (TG / DTA).

[0041] One aspect of the present specification is a Raman spectrum of 415.2 cm－１ ±2cm －１ , 502.7 cm －１ ±2cm －１ , 1431.4 cm －１ ±2cm －１ , 1714.8 cm －１ ±2cm －１ and 3065.4 cm －１ ±2cm －１ The anhydrous crystal of the compound represented by formula (I-1) has a characteristic peak at

[0042] X-ray Powder Diffraction (XRPD) X-ray powder diffraction (XRPD) is one of the most sensitive analytical methods for measuring the crystalline form and crystallinity of solids. When X-rays are irradiated onto a crystal, they reflect off the crystal lattice planes and interfere with each other, producing ordered diffraction lines corresponding to the periodicity of the structure. On the other hand, amorphous solids typically do not exhibit diffraction patterns because they do not have an ordered repeating period in their structure, resulting in a featureless, broad XRPD pattern (also known as a halo pattern).

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

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

[0045] It is known that the intensities of the peaks displayed in the following tables and figures may generally vary depending on many factors, such as the effect of preferred orientation of the crystal relative to the X-ray beam, the influence of large particles, the purity of the material being analyzed, or the crystallinity of the sample. Peak positions may also shift based on variations in sample height. Furthermore, measurements using different wavelengths will result in different shifts according to the Bragg equation (nλ=2d sin θ), and other XRPD patterns obtained using such different wavelengths are also within the scope of the present invention.

[0046] (Single crystal structure analysis) This is one of the methods for identifying a crystal, and it is possible to obtain the crystallographic parameters of the crystal, as well as atomic coordinates (values ​​indicating the spatial relationship of each atom) and a three-dimensional structural model. See, for example, "X-Ray Structure Analysis Handbook" by Toshio Sakurai, published by Shokabo (1983), and "X-Ray Structure Determination: A Practical Guide" by Stout & Jensen, Macmillan Co., New York (1968). Single crystal structure analysis is useful for identifying the crystal structures of complexes, salts, optical isomers, tautomers, and geometric isomers such as those of the present invention.

[0047] (Raman spectroscopy) Raman spectra show the vibrational characteristics of molecules or complex systems. Its origin is the inelastic collision between molecules and photons, which are light particles that comprise light rays. The collision between a molecule and a photon results in an exchange of energy, which changes the energy and thus the wavelength of the photon. In other words, Raman spectra are spectral lines with extremely narrow wavelengths that are emitted when a photon is incident on a molecule of interest, so a laser or similar light source is used. The wavelength of each Raman line is expressed by the wavenumber shift from the incident light, which is the difference between the Raman line and the reciprocal of the wavelength of the incident light. Raman spectra measure the vibrational state of a molecule, which is determined by its molecular structure. Generally, Raman spectrum peaks (cm －１ ) is ±2cm －１ Since there may be an error within the range of ±2 cm －１ Therefore, it should be understood that the Raman spectrum includes not only crystals in which the Raman spectrum peaks completely coincide, but also crystals in which the Raman spectrum peaks are within ±2 cm. －１ Crystals that match within a certain degree of error are also included in the present invention.

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

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

[0050] The compounds of the present invention have coronavirus 3CL protease inhibitory activity and are therefore useful as therapeutic and / or prophylactic agents for diseases associated with coronavirus 3CL protease. In the present invention, the term "therapeutic and / or prophylactic agents" encompasses agents for improving symptoms. In the present invention, the term "prevention" encompasses suppressing the onset of symptoms caused by SARS-CoV-2 after exposure. In the present invention, the term "prevention" encompasses suppressing the onset of symptoms caused by SARS-CoV-2 after exposure to SARS-CoV-2 by administering the pharmaceutical composition of the present invention to an individual in need of suppression of onset. In the present invention, the term "prevention" encompasses pre-exposure prophylaxis of SARS-CoV-2. In the present invention, the term "prevention" encompasses suppressing the onset of symptoms caused by SARS-CoV-2 before exposure to SARS-CoV-2 by administering the pharmaceutical composition of the present invention to an individual in need of suppression of onset. The term "prevention" in the present invention includes suppressing the onset and aggravation of SARS-CoV-2 after exposure.

[0051] Diseases associated with coronavirus 3CL protease include viral infections, preferably coronavirus infections. In one embodiment, the coronavirus includes a coronavirus that infects humans. Examples of coronaviruses that infect humans include HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and / or SARS-CoV-2. In one embodiment, the coronavirus includes an alphacoronavirus and / or a betacoronavirus, more preferably a betacoronavirus, and even more preferably a sarbecovirus. In one embodiment, the alphacoronavirus includes HCoV-229E and HCoV-NL63. Particularly preferred is HCoV-229E. In one embodiment, the betacoronavirus includes HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and / or SARS-CoV-2. Preferably, HCoV-OC43 or SARS-CoV-2 is used, and particularly preferably SARS-CoV-2. In one embodiment, the betacoronavirus includes betacoronavirus lineage A (β-coronavirus lineage A), betacoronavirus lineage B (β-coronavirus lineage B), and betacoronavirus lineage C (β-coronavirus lineage C). More preferably, betacoronavirus lineage A and betacoronavirus lineage B are used, with betacoronavirus lineage B being particularly preferred. Examples of betacoronavirus lineage A include HCoV-HKU1 and HCoV-OC43, preferably HCoV-OC43. Examples of betacoronavirus lineage B include SARS-CoV and SARS-CoV-2, preferably SARS-CoV-2.A preferred example of the betacoronavirus C lineage (β-coronavirus lineage C) is MERS-CoV. In one embodiment, the coronavirus includes HCoV-229E, HCoV-OC43, and / or SARS-CoV-2, with SARS-CoV-2 being particularly preferred. It is generally known that viruses mutate during repeated proliferation and infection. The coronaviruses described above include not only mutant strains known in the art, but also mutant strains that may emerge in the future, as long as the compounds according to the present invention are capable of exhibiting coronavirus 3CL protease inhibitory activity. Known mutant strains of SARS-CoV-2 include, for example, the mutant strains used in the Examples herein. Examples of coronavirus infections include infections 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. Particularly preferably, examples of coronavirus infections include novel coronavirus disease (COVID-19).

[0052] The severity classification of novel coronavirus infection patients is exemplified below. (Reference: Novel Coronavirus Infection COVID-19 Medical Care Guide, Version 10.0 (Ministry of Health, Labour and Welfare)) (Mild): Oxygen saturation is 96% or higher. Clinical condition is no respiratory symptoms, or only coughing without dyspnea, and in either case, no signs of pneumonia are observed. (Moderate I): Oxygen saturation is less than 96% but more than 93%. Dyspnea and signs of pneumonia are present. (Moderate II): Oxygen saturation is less than 93%. Respiratory failure occurs and oxygen administration is required. (Severe): The patient is admitted to an ICU or requires a ventilator. The above classification is the definition of severity classification in Japan; for example, severity classifications from China and the NIH in the United States can also be used. Furthermore, an asymptomatic SARS-CoV-2 infected patient refers to an asymptomatic pathogen carrier. For example, this includes those who do not exhibit 14 or 12 of the COVID-19 symptoms. (14 symptoms of COVID-19: fatigue, muscle or body aches, headache, chills / sweats, fever or fever, loss of taste or smell, runny or stuffy nose, sore throat, cough, shortness of breath (difficulty breathing), nausea, vomiting, diarrhea)

[0053] As used herein, the 12 symptoms of COVID-19 include fatigue (fatigue), muscle or body aches, headache, chills / sweats, fever or fever, runny or stuffy nose, sore throat, cough, shortness of breath (difficulty breathing), nausea, vomiting, and diarrhea.

[0054] As used herein, "suppressing the progression of severity" includes preventing an asymptomatic SARS-CoV-2 infected person from being upgraded to a severity classified as mild, moderate I, moderate II, or severe. As used herein, "suppressing the progression of severity" includes preventing an asymptomatic SARS-CoV-2 infected person or a mild SARS-CoV-2 infected person from being upgraded to a severity classified as moderate I, moderate II, or severe. As used herein, "suppressing the progression of severity" includes preventing an asymptomatic SARS-CoV-2 infected person, a mild SARS-CoV-2 infected person, or a moderate I SARS-CoV-2 infected person from being upgraded to a severity classified as moderate II or severe. As used herein, "suppressing the progression of severity" includes upgrading the severity of an asymptomatic SARS-CoV-2-infected patient, a mild SARS-CoV-2-infected patient, a moderate I SARS-CoV-2-infected patient, or a moderate II SARS-CoV-2-infected patient to a severe level. As used herein, "suppressing the progression of severity" includes reducing the risk of hospitalization or death in a SARS-CoV-2-infected patient through the viral replication inhibitory effect of the drug. As used herein, "suppressing the progression of severity" includes reducing inflammation in the lungs of a SARS-CoV-2-infected patient through the viral replication inhibitory effect of the drug. As used herein, "suppressing the progression of severity" includes suppressing pneumonia caused by SARS-CoV-2 viral infection through the viral replication inhibitory effect of the drug. As used herein, suppression of aggravation includes suppressing the excessive immune response of the host caused by SARS-CoV-2 viral infection through the viral proliferation inhibitory effect of the drug.

[0055] In one embodiment, the pharmaceutical composition of the present invention is administered to SARS-CoV-2 infected individuals who have at least one risk factor for severe illness. For information on underlying conditions associated with severe illness, please refer to the U.S. CDC summary (https: / / www.cdc.gov / coronavirus / 2019-ncov / hcp / clinical-care / underlyingconditions.html).

[0056] In one embodiment, the pharmaceutical composition of the present invention is administered to SARS-CoV-2 infected individuals who have at least one of the following risk factors for aggravation: malignant tumor, metabolic disease, cardiovascular disease, respiratory disease, liver disease, kidney disease, neuropsychiatric disease, lack of exercise, pregnancy, smoking, childhood, genetic disease, and immunodeficiency.

[0057] In one embodiment, the pharmaceutical composition of the present invention is administered to an infected individual who has at least one risk factor for aggravation, and is used to suppress the aggravation of COVID-19 symptoms.

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

[0059] As used herein, "prevention" includes the suppression of onset of SARS-CoV-2 after exposure. For example, the pharmaceutical composition of the present invention can be administered to family members or cohabitants of a patient with COVID-19. For example, the pharmaceutical composition of the present invention can be administered to a contact person promptly (e.g., within 72 hours) after contact with a patient with COVID-19. For example, the pharmaceutical composition of the present invention can be administered to an asymptomatic person infected with SARS-CoV-2 to suppress onset of the disease. Furthermore, "prevention" also includes pre-exposure prophylaxis of SARS-CoV-2. For example, in cases where there is a risk of SARS-CoV-2 infection, such as during the COVID-19 epidemic, the pharmaceutical composition of the present invention can be administered to healthcare workers, the elderly, and individuals with risk factors for severe illness.

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

[0061] The compounds according to the present invention (e.g., compounds represented by formula (I-1), compounds represented by formula (I-2), and compounds represented by formula (II)) have coronavirus 3CL protease inhibitory activity and are therefore useful as therapeutic and / or preventive agents for viral infections. Furthermore, the compounds according to the present invention are useful as pharmaceuticals and preferably have one or more of the following excellent characteristics: a) weak inhibitory effect on CYP enzymes (e.g., CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, etc.); b) good pharmacokinetics such as high bioavailability and moderate clearance; c) high metabolic stability; d) no irreversible inhibitory effect on CYP enzymes (e.g., CYP3A4) within the concentration range of the measurement conditions described herein; e) no mutagenicity; f) low cardiovascular risk; g) high solubility; h) high protein unbound rate (fu value). i) High coronavirus 3CL protease selectivity. j) High coronavirus growth inhibitory activity. For example, high coronavirus growth inhibitory activity is observed under the addition of human serum (HS) or human serum albumin (HSA). k) High growth inhibitory activity is also observed against 3CL protease inhibitor-resistant viruses. As a coronavirus growth inhibitor, for example, in the CPE inhibitory effect confirmation test (SARS-CoV-2) described below, ５０ is 10 μM or less, preferably 1 μM or less, and more preferably 100 nM or less.

[0062] The pharmaceutical composition of the present invention can be administered orally or parenterally, including transdermal, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, transmucosal, inhalation, nasal, ophthalmic, otic, and vaginal administration.

[0063] For oral administration, the composition may be prepared and administered in any of the commonly used dosage forms, such as solid preparations for internal use (e.g., tablets, powders, granules, capsules, pills, films, etc.) and liquid preparations for internal use (e.g., suspensions, emulsions, elixirs, syrups, lemonades, spirits, perfumes, extracts, decoctions, tinctures, etc.), according to conventional methods. 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; and capsules may be soft capsules, microcapsules, or sustained-release capsules.

[0064] In the case of parenteral administration, the compound can be suitably administered in any of the commonly used dosage forms, such as injections, infusions, and topical preparations (e.g., eye drops, nasal drops, ear drops, aerosols, inhalants, lotions, infusions, liniments, mouthwashes, enemas, ointments, plasters, jellies, creams, patches, poultices, powders for topical use, suppositories, etc.). Injections may be emulsions such as O / W, W / O, O / W / O, and W / O / W types.

[0065] Pharmaceutical compositions can be prepared by mixing an effective amount of the compound of the present invention with various pharmaceutical additives, such as excipients, binders, disintegrants, and lubricants, appropriate for the dosage form, as needed. Furthermore, by appropriately modifying the effective amount of the compound of the present invention, the dosage form, and / or the various pharmaceutical additives, the pharmaceutical composition can also be prepared as a pharmaceutical composition for pediatrics, the elderly, critically ill patients, or surgical patients. For example, pediatric pharmaceutical compositions can be administered to newborns (less than 4 weeks old), infants (4 weeks old to less than 1 year old), toddlers (1 year old to less than 7 years old), children (7 years old to less than 15 years old), or patients aged 15 to 18 years. For example, pharmaceutical compositions 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 desirably determined taking into consideration the patient's age, body weight, type and severity of the disease, route of administration, etc., but when administered orally, it is usually 0.01 to 100 mg / kg / day, preferably 0.05 to 50 mg / kg / day, of the compound of the present invention. When administered parenterally, it varies greatly depending on the route of administration, but it is usually 0.005 to 200 mg / kg / day, preferably 0.01 to 100 mg / kg / day, of the compound of the present invention. This dosage can be administered once or in divided doses per day.

[0067] The compound of the present invention may be used in combination with, for example, another therapeutic agent for novel coronavirus disease (COVID-19) (such therapeutic agents include approved agents and agents under development or to be developed in the future) (hereinafter referred to as a concomitant drug) for the purpose of enhancing the effect of the compound or reducing the dosage of the compound. In this case, the administration timing of the compound of the present invention and the concomitant drug is not limited, and they may be administered to the subject simultaneously or at staggered times. Furthermore, the compound of the present invention and the concomitant drug may be administered as two or more types of preparations containing the respective active ingredients, or as a single preparation containing those active ingredients.

[0068] The dose of the concomitant drug can be appropriately selected based on the dose used clinically. The compounding ratio of the compound of the present invention to the concomitant 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 concomitant drug may be used per 1 part by weight of the compound of the present invention.

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

[0070] 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) NMR analysis obtained in each example was carried out at 400 MHz, and DMSO-d ６ , CDCl ３ Measurement was performed using a NMR spectrometer. When NMR data is presented, not all measured peaks may be listed. In the specification, RT 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 (1.7 μm i.d. 2.1 x 50 mm) (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 performed over 3.5 minutes, followed by maintaining 100% solvent [B] for 0.5 minutes.

[0072] Powder X-ray Diffraction Experiment (XRPD) Powder X-ray diffraction measurements were carried out on the solid forms (crystals) obtained in the examples according to the powder X-ray diffraction measurement method described in the general test methods of the Japanese Pharmacopoeia. The measurement conditions are as follows. Measurement Condition 1: Powder X-ray diffractometer: SmartLab manufactured by Rigaku Corporation Measurement method: Reflection method Wavelength used: CuKα ray (λ=1.5418 Å) Tube current: 200 mA Tube voltage: 45 kV Sample plate: Aluminum X-ray incident angle: 2.5° Sampling width: 0.02° Detector: HyPix-3000 (two-dimensional detection mode)

[0073] Measurement and Analysis Method for Single Crystal Structure Analysis Single crystal structure analysis was performed on the crystals obtained in the examples. The measurement conditions and analysis method are shown below. (Apparatus) Rigaku XtaLAB P200 MM007 (Measurement conditions) Measurement temperature: 25°C Temperature controller: Rigaku sample spray cryogenic device Wavelength used: CuKα ray (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 Lorentz and polarization corrected, and absorption corrected. (Crystal structure analysis) Phase determination was performed using the direct method program ShelXT (Sheldrick, G.M., 2015), and refinement was performed using full-matrix least-squares using ShelXL (Sheldrick, G.M., 2015). All non-hydrogen atom temperature factors were refined anisotropically. Hydrogen atoms were introduced by calculation using the default parameters of ShelXL unless otherwise specified and treated as riding atoms. Hydrogen atoms were also refined using isotropic parameters. PLATON (Spek, 1991) / ORTEP (Johnson, 1976) was used to draw the following structure diagram (30% PROBABILITY level).

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

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

[0076] Simultaneous Differential Thermal Analysis - Thermogravimetry (TG / DTA) Simultaneous differential thermal analysis - thermogravimetry (TG / DTA) was carried out on the solid form (crystal) obtained in the examples. The samples obtained in the examples were weighed, placed in aluminum pans, and measured in an open system. The measurement conditions were 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 around 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: 20% solvent [B] was maintained for 1 minute, followed by a linear gradient of 20% - 80% solvent [B] over 32 minutes, followed by maintaining 20% ​​solvent [B] for 7 minutes. Flow rate: 0.4 mL / min Injection volume: 3 μL

[0078] Synthesis of Compound (I-1) Step 1: Synthesis of Compound 3 To a mixed solution of 2.64 mol / L n-butyllithium in hexane (31 mL, 82.4 mmol) and tetrahydrofuran (20 mL), a solution of compound 1 (12 g, 68.7 mmol) in tetrahydrofuran (70 mL) was added dropwise over 15 minutes at −78°C. The mixture was stirred at −78°C for 1 hour. A 1.9 mol / L solution of zinc chloride in 2-methyltetrahydrofuran (43 mL, 82.4 mmol) was added dropwise over 5 minutes. The mixture was stirred at room temperature for 2 hours. Compound 2 (9.1 mL, 75.6 mmol) and tetrakis(triphenylphosphine)palladium (4.0 g, 3.44 mmol) were added, and the mixture was stirred at 80°C for 1.5 hours. The reaction mixture was cooled to room temperature, and water (80 mL) and 2 mol / L hydrochloric acid (40 mL) were added, followed by extraction with ethyl acetate. The organic layer was concentrated under reduced pressure, and isopropanol (40 mL) was added to the resulting residue. The precipitate was collected by filtration, washed with isopropanol, and air-dried to give compound 3 (14.8 g, 49 mmol). １ H-NMR (CDCl ３) δ: 3.95 (s, 3H), 4.05 (s, 3H), 7.18-7.23 (m, 2H), 7.35 (d, J = 6.8Hz, 1H) LC / MS (ESI): m / z = 303, RT = 2.70min, LC / MS measurement conditions A

[0079] Step 2: Synthesis of Compound 4 Compound 3 (14.8 g, 48.8 mmol) was added with acetic acid (40 mL) and concentrated hydrochloric acid (41 mL), and the mixture was stirred at 110°C for 5 hours. The reaction mixture was cooled to room temperature, and then water (80 mL) was added. The precipitate was collected by filtration and washed with water. The mixture was air-dried to obtain Compound 4 (11.6 g, 42.2 mmol). １ H-NMR (DMSO-d ６ ) δ: 7.31 (ddd, J=8.8, 4.9, 2.1Hz, 1H), 7.45 (t, J=8.8Hz, 1H), 7.53 (dd, J=7.3, 2.1Hz, 1H), 11.57 (s, 1H), 12.24 (brs, 1H) LC / MS (ESI): m / z = 275, RT = 1.81 min, LC / MS measurement conditions 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 bicarbonate 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 resulting residue was purified by silica gel column chromatography (chloroform:methanol = 100:0 to 90:10), and the solvent was evaporated under reduced pressure. The resulting residue was dried under reduced pressure to give compound 5 (1.15 g, 2.97 mmol, 82% yield). １ H-NMR (DMSO-d ６) δ: 7.35-7.37 (1H, m), 7.49 (1H, t, J = 9.0Hz), 7.54-7.56 (1H, m), 8 .07 (1H, t, J=2.1Hz), 8.54 (1H, d, J=2.0Hz), 8.70 (1H, d, J=2.3Hz). LC / MS (ESI): m / z = 386, RT = 1.98 min, LC / MS measurement conditions A

[0081] Step 4: Synthesis of Compound 6 To a mixed solution of compound 5 (520 mg, 1.345 mmol), N,N-diisopropylethylamine (0.705 mL, 4.04 mmol), and DMF (5.2 mL), 2-bromoacetonitrile (269 μL, 4.04 mmol) was added and stirred at room temperature overnight. 2 mol / L hydrochloric acid (2 mL) was added to the reaction mixture under ice cooling, 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 resulting residue was purified by silica gel column chromatography (chloroform:methanol = 100:0 to 99:1), and the solvent was evaporated under reduced pressure. The resulting residue was dried under reduced pressure to give compound 6 (291 mg, 0.684 mmol, yield 51%). １ H-NMR (CDCl ３ ) δ: 5.13 (2H, s), 7.23-7.24 (2H, m), 7.42 (1H, d, J = 7.3Hz), 7.67 (1H, t, J = 2.1Hz), 8.45 (1H, d, J = 2.3Hz), 8.67 (1H, d, J = 2.3Hz). LC / MS (ESI): m / z = 425, RT = 2.17 min, LC / MS measurement conditions 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 resulting solid was dried under reduced pressure to give anhydrous crystals of compound (I-1) (22.0 mg, 0.042 mmol, yield 72%). １ H-NMR (CDCl ３ ) δ: 2.75 (4H, t, J = 12.0Hz), 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=2.1Hz), 8.43 (1H, d, J=2.3Hz), 8.61 (1H, d, J=2.3Hz). LC / MS (ESI): m / z = 522, RT = 2.27 min, LC / MS measurement conditions A

[0083] (Crushing 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 crushed under the following conditions: Apparatus: A-O jet mill (Seishin Enterprise Co., Ltd.) Feeding method: feeder Feeding rate: 20 g / hour Crushing pressure: 0.30 MPa Feeding pressure: 0.40 MPa

[0084] (Powder X-ray Diffraction Experiment of Anhydrous Crystals of the Compound Represented by Formula (I-1)) A powder X-ray diffraction experiment was carried out on the pulverized anhydrous crystals of the compound represented by formula (I-1) under the measurement conditions described above. The powder X-ray diffraction pattern is shown in FIG. 7, and a peak list of the powder X-ray diffraction pattern is shown in FIG. 8. In the table below listing the peaks of the powder X-ray diffraction pattern, Position indicates 2θ (°), and Intensity indicates 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 crystals 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 Anhydrous Crystals of the Compound Represented by Formula (I-1)) <Single Crystal Preparation Method> 400 μL of methanol was added to 1 mg of crystals of the compound represented by formula (I-1), and the mixture was heated to 50°C to dissolve. The solution was dispensed into 1.5 mL HPLC vials, the HPLC vials were capped, a syringe needle was pierced into the cap, and the mixture was allowed to stand at room temperature. Single crystals were prepared by the solvent evaporation method. <Single Crystal Structure Analysis> Single crystal diffraction experiments and analysis were carried out by the method described above. Note that, since Cl1 and Cl7C, and H5CA and H6CA are in a disordered relationship, analysis was carried out at occupancy ratios 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. １ (I>2.00s(I)) was 0.0555, and the final difference Fourier confirmed that there were no missing or misplaced electron densities.

[0087] The crystallographic data are shown in Table 1. Here, V means the unit cell volume, and Z means the number of molecules in the unit cell.

[0088] Atomic fraction coordinates x, y, z (Å × 10 ４ ) and the equivalent isotropic temperature factor U(eq) (Equivalent Isotropic Displacement Parameters, Å ２ x10 ３ ) are shown in Table 2. Here, U(eq) is the orthogonalized U ｉｊ The numbers of the non-hydrogen atoms in Table 2 correspond to the numbers in Figure 9.

[0089] Next, the atomic coordinates x, y, z of the hydrogen atom (Å×10 ４ ) and isotropic temperature factor U(eq) (Isotropic Displacement Parameters, Å ２ x10 ３ ) are shown in Table 3.

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

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

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

[0093] (Differential scanning calorimetry of the anhydrous crystals of the compound represented by formula (I-1)) Approximately 2 mg of the anhydrous crystals of the compound represented by formula (I-1) after pulverization was weighed into an aluminum pan and measured by the method described above. The results are shown in Figure 10. An endothermic peak was observed with an onset temperature of approximately 261.3°C.

[0094] (Simultaneous Differential Thermal and Thermogravimetric Measurement of Anhydrous Crystals of the Compound of Formula (I-1)) The anhydrous crystals of the compound of formula (I-1) after pulverization were measured by the method described above. The results are shown in Figure 11. An endothermic peak with an onset temperature of approximately 265.6°C was observed. No weight loss was observed.

[0095] (Raman Spectroscopic Measurement of Anhydrous Crystals of the Compound Represented by Formula (I-1)) The anhydrous crystals of the compound represented by formula (I-1) after pulverization were subjected to Raman spectroscopy under the measurement condition 1 described above. The results are shown in Figure 12. The main Raman peaks are shown below. The anhydrous crystal of the compound represented by formula (I-1) exhibits a Raman spectrum of 415.2 cm －１ ±2cm －１ , 502.7 cm －１ ±2cm －１ , 1431.4 cm －１ ±2cm －１ , 1714.8 cm －１ ±2cm －１ , and 3065.4 cm －１ ±2cm －１ showed a characteristic peak.

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

[0097] Synthesis of Compound (I-2)

[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 the addition of water (26 mL) and ethyl acetate (26 mL), and extracted with ethyl acetate. The organic layer was concentrated under reduced pressure, and the resulting 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 １ H-NMR (CDCl ３ ) 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 DC1 (0.69 mL, 3.43 mol) were mixed and heated at 180 °C for 30 minutes using a microwave. The same procedure was repeated 10 times, and all the reaction solutions were mixed. 50 mL of ethyl acetate and 1 mol / L sodium hydroxide solution (51.5 mL) were added, 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 H-NMR (CDCl ３ ) δ: 3.59 (brs, 2H), 6.87 to 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. ２(2.384 g, 34.6 mmol) was dissolved in water (7.5 mL) and slowly added. The reaction solution was heated to 30°C, and CuBr (6.32 g, 44.0 mmol) was dissolved in hydrogen bromide (13.42 mL, 119 mmol) and added, with the internal temperature at 30-40°C. After stirring for 30 minutes, 200 mL of hexane and 5 mol / L aqueous sodium hydroxide solution (67.8 mL, 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 resulting residue was purified by silica gel column chromatography (hexane:ethyl acetate = 10:0 to 9:1), and the solvent was evaporated 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 H-NMR (CDCl ３ ) δ: 7.00-7.07 (m, 2H)

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

[0102] Step 5: Synthesis of Compound 13 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 were almost completely consumed. After the reaction was completed, water (15 mL) was added to the reaction solution, and a solid precipitated. The reaction solution was partially concentrated, and the solid was collected by filtration. The solid was washed with chilled acetonitrile to obtain compound 13 (246 mg, 0.88 mmol). １ H-NMR (DMSO-d ６ ) δ: 7.43 (d, J=9.5Hz, 2H), 7.53 (dd, J=7.3, 2.1Hz, 1H), 11.53 (s, 1H), 12.23 (br s, 1H) LC / MS (ESI): m / z = 281, RT = 1.44 min, LC / MS measurement conditions 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 resulting residue was used in the next step without purification.

[0104] Step 7 Synthesis of Compound 15 To a mixed 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, followed by extraction 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 resulting residue was purified by silica gel column chromatography (hexane:ethyl acetate=10:0 to 2:3). The solvent was evaporated under reduced pressure to give compound 15 (307 mg, 0.71 mmol, yield 85%). １ H-NMR (CDCl ３ ) δ: 5.13 (s, 2H), 7.21-7.25 (m, 1H), 7.67 (1H, t, J = 2.3Hz), 8.45 (d, J = 2.1Hz, 1H), 8.66 (d, J = 2.1Hz, 1H). LC / MS (ESI): m / z = 431, RT = 2.13 min, LC / MS measurement conditions A

[0105] Step 8: Synthesis of Compound (I-2) Compound 15 (48.8 mg, 0.113 mmol), 6,6-difluoro-2-azaspiro[3.3]heptane trifluorohydrochloride (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 resulting residue was purified by silica gel column chromatography (hexane:ethyl acetate = 10:0 to 3:7). The solvent was evaporated under reduced pressure, and diisopropyl ether was added. The resulting precipitate was collected by filtration and washed with diisopropyl ether. The resulting solid was dried under reduced pressure to give compound (I-2) (34.3 mg, 0.065 mmol, yield 57%). １ H-NMR (DMSO-d ６) δ: 2.83 (t, J = 12.5Hz, 4H), 4.07 (s, 4H), 4.86 (s, 2H), 7.44 (d, J = 9.4Hz, 1H), 8.01 (t, J=2.2Hz, 1H), 8.50 (d, J=2.0Hz, 1H), 8.73 (d, J=2.0Hz, 1H). LC / MS (ESI): m / z = 528, RT = 2.19 min, LC / MS measurement conditions A

[0106] Reference Example 1: Synthesis of Compound (II-1) Step 1: Synthesis of Compound (II-1) To a mixed solution of compound 16 (150 mg, 0.376 mmol; see International Publication No. WO 2023 / 195529 for the synthesis method), N,N-diisopropylethylamine (0.197 mL, 1.13 mmol), and DMF (1.2 mL), propargyl bromide (90 μL, 1.13 mmol) was added and stirred at 80°C for 4 hours. 6,6-Difluoro-2-azaspiro[3.3]heptane trifluorohydrochloride (186 mg, 0.753 mmol) and N,N-diisopropylethylamine (0.197 mL, 1.13 mmol) were added, and the solution was stirred at 80°C for 3 hours. A 0.1 mol / L aqueous hydrochloric acid solution (10 mL) was added to the reaction solution, followed by extraction with ethyl acetate. The organic layer was washed with water, dried over sodium sulfate, and filtered. The filtrate was concentrated, the resulting residue was purified by silica gel column chromatography (hexane:ethyl acetate=9:1 to 3:7), and the solvent was evaporated under reduced pressure to obtain compound (II-1) (57 mg, 0.109 mmol, yield 29%). １ H-NMR (CDCl ３ ) δ: 2.73-2.79 (4H, m), 3.45 (1H, s), 4.06 (4H, s), 4.57 (2H, s), 7.18-7.21 (2H, m), 7. 36-7.43 (2H, m), 7.97 (1H, t, J = 2.0Hz), 8.44 (1H, d, J = 2.0Hz), 8.66 (1H, d, J = 2.4Hz). LC / MS (ESI): m / z = 521, RT = 2.37 min, LC / MS measurement conditions A

[0107] Reference Example 2 The following compound can be synthesized in the same manner as in Reference Example 1 and WO 2023 / 195529.

[0108] The following describes examples of biological tests of the compounds according to the present invention. The compounds according to the present invention have an inhibitory effect on coronavirus 3 CL protease and inhibit coronavirus 3 CL protease. Specifically, in the evaluation method described below, IC ５０ is preferably 50 μM or less, more preferably 1 μM or less, and even more preferably 100 nM or less. 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: Cytopathic effect (CPE) inhibitory effect confirmation test using human TMPRSS2 and ACE2-expressing HEK293T cells (HEK293T / ACE2-TMPRSS2 cells) <Procedure> - Dilution and dispensing of test sample: The test sample is diluted to an appropriate concentration with DMSO in advance, and a 2- to 5-fold serial dilution series is prepared, and then dispensed into a 384-well plate. - Dilution and dispensing of cells and SARS-CoV-2: HEK293T / ACE2-TMPRSS2 cells (GCP-SL222, 5 x 10 ３ cells / well) and SARS-CoV-2 (200-600 TCID ５０ / well) was mixed with a medium (MEM, 2% FBS, penicillin-streptomycin), and the mixture was dispensed into the wells containing the test samples. ２ Culture in an incubator for 3 days. Dispensing CellTiter-Glo® 2.0 and measuring luminescence signals After 3 days of culture, return the plate to room temperature, dispense CellTiter-Glo® 2.0 into each well, and mix using a plate mixer. After a certain period of time, measure the luminescence signal (Lum) using a plate reader. <Calculation of values ​​for each measurement item> 50% SARS-CoV-2-infected cell death inhibitory concentration (EC ５０ ) Calculation 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 the EC ５０Calculated 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 of y-axis, max: upper limit of y-axis, X50: x-coordinate of inflection point, Hill: slope of the curve at the midpoint between min and max

[0110] Compounds of the invention were tested essentially as described above. ５０ The values ​​are shown below. (Results) Compound I-1: 1.66 nM Compound I-2: 2.36 nM

[0111] Test Example 2-1: Inhibitory 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)-NH ２ (SEQ ID NO: 1) Internal Standard Peptide Dabcyl-Lys-Thr-Ser-Ala-Val-Leu ( １３ C ６ , １５ N)-Gln (SEQ ID NO: 2) Dabcyl-Lys-Thr-Ser-Ala-Val-Leu ( １３ C ６ , １５N)-Gln can be synthesized with reference to the literature (Atherton, E.; Sheppard, RC, "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. H-Lys-Thr-Ser-Ala-Val-Leu ( １３ C ６ , １５N)-Glu(resin)-OαOtBu (Lys side chain is protected with Boc, Thr side chain is protected with a tert-butyl group, Ser side chain is protected with a tert-butyl group, the C-terminal OH of Glu is protected with a tert-butyl group, and the carboxylic acid of the Glu side chain is condensed to the resin) is synthesized. The N-terminal Dabcyl group is modified 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. The product is then purified by reverse-phase HPLC. RapidFire Cartridge C4 Type A <Procedure> Assay buffer preparation: This test uses an assay buffer consisting of 20 mM Tris-HCl, 1 mM EDTA, 10 mM DTT, and 0.01% BSA. Test sample dilution and dispensing: Test samples are diluted to an appropriate concentration with DMSO, and a 2- to 5-fold serial dilution series is prepared and dispensed into a 384-well plate. Enzyme and substrate addition: Enzyme reaction: 8 μM substrate and 6 nM or 0.6 nM enzyme solution are added to the prepared compound plate and incubated at room temperature for 3 to 5 hours. The enzyme reaction is then stopped by adding a reaction stop solution (0.067 μM internal standard, 0.1% formic acid, 10 or 25% acetonitrile). Measurement of reaction products The plate after the reaction is completed is measured using a RapidFire System 360 and a mass spectrometer (Agilent, 6550 iFunnel Q-TOF) or a RapidFire System 365 and a mass spectrometer (Agilent, 6495C Triple Quadruple). Solution A (75% isopropanol, 15% acetonitrile, 5 mM ammonium formate) and solution B (0.01% trifluoroacetic acid, 0.09% formic acid) are used as the mobile phase during measurement. The reaction products detected by the mass spectrometer are calculated using RapidFire Integrator or an equivalent analysis program to obtain the product area value. At the same time, the internal standard detected is also calculated and used as 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 using 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 logarithm 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 used to calculate the IC ５０ Calculated as follows: 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 of y-axis, max: upper limit of y-axis, X50: x-coordinate of inflection point, Hill: slope of the curve at the midpoint between min and max

[0112] Compounds of the invention were tested essentially as described above. ５０ The 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: Inhibitory 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)-NH ２ (SEQ ID NO: 1) Internal Standard Peptide Dabcyl-Lys-Thr-Ser-Ala-Val-Leu ( １３ C ６, １５ N)-Gln (SEQ ID NO: 2) Dabcyl-Lys-Thr-Ser-Ala-Val-Leu ( １３ C ６ , １５ N)-Gln can be synthesized with reference to 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. H-Lys-Thr-Ser-Ala-Val-Leu ( １３ C ６ , １５N)-Glu(resin)-OαOtBu (Lys side chain is protected with Boc, Thr side chain is protected with a tert-butyl group, Ser side chain is protected with a tert-butyl group, the C-terminal OH of Glu is protected with a tert-butyl group, and the carboxylic acid of the Glu side chain is condensed to the resin) is synthesized. The N-terminal Dabcyl group is modified 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. The product is then purified by reverse-phase HPLC. RapidFire Cartridge C4 Type A <Procedure> Assay buffer preparation: This test uses an assay buffer consisting of 20 mM Tris-HCl, 1 mM EDTA, 10 mM DTT, and 0.01% BSA. Dilution and dispensing of test samples: Test samples are diluted to an appropriate concentration with DMSO in advance, and a 3-fold serial dilution series is prepared and dispensed into a 384-well plate. Addition of enzyme and substrate: Enzyme reaction: Substrate and enzyme at a final concentration of 4 μM and 0.3 nM are added to the prepared compound plate and incubated at room temperature for 4 hours. The enzyme reaction is then stopped by adding a reaction stop solution (7.2 nM internal standard, 0.1% formic acid, 10% acetonitrile). Measurement of reaction products The plate after the reaction is completed is measured using a Rapid Fire System 365 and a mass spectrometer (Agilent, 6495C Triple Quadruple). Solution A (75% isopropanol, 15% acetonitrile, 5 mM ammonium formate) and solution B (0.01% trifluoroacetic acid, 0.09% formic acid) are used as the mobile phase during measurement. The reaction products detected by the mass spectrometer are calculated using a Rapid Fire Integrator to obtain the product area value. The internal standard detected at the same time 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 using 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 logarithm 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 used to calculate the IC ５０ It is calculated as follows. 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 of y-axis, max: upper limit of y-axis, X50: x-coordinate of inflection point, Hill: slope of curve at midpoint between min and max

[0114] Compounds of the present invention were tested essentially as described above, with the results shown below.

[0115] Test Example 3: Cytopathic effect (CPE) inhibitory effect confirmation test using human TMPRSS2-expressing Vero E6 cells (Vero E6 / TMPRSS2 cells) <Procedure> - Dilution and dispensing of test sample: The test sample is diluted to an appropriate concentration with DMSO in advance, and a 3-fold serial dilution series is prepared and then dispensed into a 96-well plate. - Dilution and dispensing of cells and SARS-CoV-2: Vero E6 / TMPRSS2 cells (JCRB1819, 1.5 x 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), ]CoV-19 / JLO / TY-385 / 2022 / OOcr- BA.2 / / OOO / OYO-72 / / OOOO BA.2.12.1), ]CoV-19 / JLL / TY--716 / 2022 / OOcr5BA.2.75 / / CoV-9 / JOO / OOO-703 / 2022 BA.4.1)]CoV-19 / Jap / TY1-763 / 2022OOmcrs BA.4.6), ]CoV-19 / JLL / TIO-702 / 2022 / OOcrsBE. BA.5.2.1), ]CoV-19 / JAL / TY-820 / 2022(Omcr-B.7 / )]CoV-19 / JOO / OOO-820 / 2022 BF.7.4.1),]CoV-19 / Jap / TY1-796 / 2022OOmcrs BQ.1.1), ]CoV-19 / JLL / TY-832 / 2022(Omcrs CH.O.1.11)]CoV-19 / JOO / OEO-795 / 2022 ^BB.1), ]CoV-19 / JOO / 23-018 / 2022OOO]r-EBBO.5 / / OOO / TY-1195 / OOOOOOOOOO The ^BB.1.16)]CoV-19 / Jap / TY1-831 / 2022(Omcrrs^BF)hCoV-19 / Japan / TY41-686 / 2022 (Omicron XE) (30-3000TCID, ５０ / well) was mixed with a medium (MEM, 2% FBS, penicillin-streptomycin), and the mixture was dispensed into the wells containing the test samples. ２ Culture in an incubator for 3 or 4 days. Dispensing CellTiter-Glo® 2.0 and measuring luminescence signals After allowing the 3-day culture plate to return to room temperature, dispense CellTiter-Glo® 2.0 into each well and mix using a plate mixer. After leaving the plate for a certain period of time, measure the luminescence signal (Lum) using a plate reader.

[0116] <Calculation of each measurement item value> 50% SARS-CoV-2 infected cell death inhibitory concentration (EC ５０ ) Calculation 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 the EC ５０ 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 of y-axis, max: upper limit of y-axis, X50: x-coordinate of inflection point, Hill: slope of the curve at the midpoint between min and max

[0117] Compounds of the present invention were tested essentially as described above, with the results shown below.

[0118] Test Example 5: Antiviral effect against HCoV-OC43 <Procedure> - Dilution and dispensing of test sample Test sample was diluted to an appropriate concentration with DMSO in advance, and a 3-fold serial dilution series was prepared. The sample was 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 x 10 ４ cells / well) were seeded onto a 96-well plate the day before infection, and the next day, HCoV-OC43 (300 TCID ５０ The virus solution was then removed, and a maintenance medium containing the test reagent was added. ２ The plates are cultured in an incubator for 72 hours. To assess the cytotoxicity of the test reagent, the same procedure is performed in the absence of virus. Dispensing CellTiter-Glo® 2.0 and measuring the luminescence signal: After 72 hours of culture, the plate is returned to room temperature, and CellTiter-Glo® 2.0 is dispensed into each well and mixed using a plate mixer. After a certain period of time, the luminescence signal (Lum) is measured using a plate reader.

[0119] <Calculation of each measurement item value> 50% HCoV-OC43 infected cell death inhibitory concentration (EC ５０ ) Calculation 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 the EC ５０Calculation is made 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 of y-axis, max: upper limit of y-axis, X50: x coordinate of inflection point, Hill: slope of the curve at the midpoint between min and max Calculation of 50% cytotoxicity 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 calculated as CC ５０ 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 of y-axis, max: upper limit of y-axis, X50: x-coordinate of inflection point, Hill: slope of the curve at the midpoint between min and max

[0120] Compounds of the present invention were tested essentially as described above, and the results are shown below. Compound I-1: EC ５０ 92.0±21.0nM, CC ５０ >100 μM

[0121] Test Example 6: Antiviral effect against HCoV-229E <Procedure> - Dilution and dispensing of test sample Test sample was diluted to an appropriate concentration with DMSO in advance, and a 3-fold serial dilution series was prepared. The sample was 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 x 10 ４ cells / well) were seeded onto a 96-well plate the day before infection, and the next day, HCoV-229E (1000 TCID ５０ The cells (1 well / well) were infected for 1 hour. After that, the virus solution was removed, and a maintenance medium containing the test reagent was added. ２ Culture in an incubator for 72 hours. Dispensing of CellTiter-Glo® 2.0 and measurement of luminescence signal After 72 hours of culture, return the plate to room temperature, dispense CellTiter-Glo® 2.0 into each well, and mix using a plate mixer. After leaving for a certain period of time, measure the luminescence signal (Lum) using a plate reader.

[0122] <Calculation of each measurement item value> 50% HCoV-229E infected cell death inhibitory concentration (EC ５０ ) Calculation 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 the EC ５０Calculated 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 of y-axis, max: upper limit of y-axis, X50: x-coordinate of inflection point, Hill: slope of the curve at the midpoint between min and max

[0123] Compounds of the present invention were tested essentially as described above, and the results are shown below. Compound I-1: EC ５０ 3570±510nM

[0124] Test Example 7: Effect of human serum, mouse serum, and hamster serum on anti-SARS-CoV-2 activity <Procedure> - Dilution and dispensing of test sample: Test sample is diluted to an appropriate concentration with DMSO in advance, and a 3-fold serial dilution series is prepared and dispensed into a 96-well plate. - Addition of serum-supplemented medium: Prepare a final concentration of 0, 12.5, 25, or 50% human serum, mouse serum, or hamster serum in medium (MEM, 2% FBS, penicillin-streptomycin), dispense into 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 x 10 ４ cells / well) were seeded onto a 96-well plate the day before infection, and the next day, SARS-CoV-2 hCoV-19 / Japan / TY7-501 / 2021 (1000 TCID ５０ After that, the virus solution was removed, and a test reagent and a medium containing human serum, mouse serum, or hamster serum were added. ２Culture in an incubator for one day. Quantification of viral load: After one day of culture, the supernatant of the plate is removed, and a cell lysis solution made by mixing Trizol LS and maintenance medium at a ratio of 3:1 is added. RNA is extracted using the Direct-zol-96 RNA Kit (ZYMO RESEARCH). The extracted RNA solution is quantified using real-time PCR (Applied BioSystems QuantStudio5). The following probes and primers are used.

[0125] <Calculation of each measurement item value> 90% SARS-CoV-2 virus replication inhibitory concentration (EC ９０ ) calculation, where x is the logarithm of the compound concentration and y is the number of copies of the virus (Log １０ In the concentration-dependent curve, the virus control was measured as 1 log １０ The value corresponding to reduction is calculated using the two-point method from the copy numbers at two concentrations before and after it: X = the lowest concentration at which the mean reduction of viral RNA copy number from that of the virus control is less than zx = the highest concentration at which the mean reduction of viral RNA copy number from that of the virus control is z or moreY = the mean of logarithmic reduction of viral RNA copy number from that of the virus control at Xy = the mean of logarithmic reduction of viral RNA copy number from that of the virus control at xEC 90 values ​​were calculated by the following formula. 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 of EC at each serum concentration ９０ After calculating the PA-EC under 100% serum conditions by linear regression, ９０ values ​​were calculated.

[0126] Compounds of the present invention were tested essentially as described above, with the results 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: Test to confirm the effect of inhibiting viral proliferation using human airway epithelial cells <Procedure> - Dilution and dispensing of test sample The test sample was diluted to an appropriate concentration with DMSO in advance, and a 3-fold serial dilution series was prepared. Then, the test sample was diluted 200-fold with MucilAir™ culture medium and dispensed into a 24-well plate. - SARS-CoV-2 infection MucilAir™ culture medium seeded in a Transwell was used. ＴＭ (Nasal cavity, approximately 5.0 x 10 ５ SARS-CoV-2 hCoV-19 / Japan / TY41-702 / 2022 (Omicron BE.1 / BA.5-like) (5000TCID ５０ / well) and CO ２ The mixture was cultured in an incubator for 2 hours. ＴＭ After washing with culture medium, the transwell was placed on the well containing the test sample and then incubated with CO ２ Two days after infection, the cells were cultured in an incubator. ＴＭThe culture medium was added, and the supernatant was collected. Measurement of virus titer in the supernatant: The collected supernatant was diluted 10-fold in a medium (MEM, 2% FBS, penicillin-streptomycin) and then cultured in VeroE6 / TMPRSS2 cells (JCRB1819, 1.5 × 10 ４ The cells were mixed with 1000 cells / well and seeded onto a 96-well plate. ２ After culturing in an incubator for 4 days, cytopathic effect (CPE) was observed, and the virus titer contained in the supernatant was calculated.

[0128] <Calculation of each measurement item value> 90% SARS-CoV-2 virus production inhibitory concentration (EC ９０ ) calculation, where x is the logarithm of the compound concentration, and y is the virus titer (Log １０ TCID ５０ / mL) from the virus control in the concentration-dependent curve １０ The value corresponding to the reduction is calculated by the two-point method from the virus titers at two concentrations before and after the reduction.

[0129] Compounds of the present invention were tested essentially as described above, with the results shown below.

[0130] Test Example 9: Test for inhibiting viral titer growth in 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 N,N-dimethylacetamide (DMA) and 0.5 w / v% poly(1-vinylpyrrolidone-co-vinyl acetate) (PVPVA)-containing polyethylene glycol 400 (PEG400) (DMA: 0.5 w / v% PVPVA-containing PEG400 = 1:9). The administration volume was 5 mL / kg. Virus SARS-CoV-2 hCoV-19 / Japan / TY7-501 / 2021 strain isolated by the National Institute of Infectious Diseases was used. Mouse lung infection, dosing, and lung recovery. Specific pathogen-free, 5-week-old female BALB / c mice (CLEA Japan, Inc.) were used in this study. For virus inoculation, mice were anesthetized by intramuscular administration of 100 μL of an anesthetic solution containing 0.03 mg / mL medetomidine hydrochloride, 0.4 mg / mL midazolam, and 0.5 mg / mL butorphanol tartrate in saline. Under anesthesia, mice were inoculated with 50 μL of hCoV-19 / Japan / TY7-501 / 2021 (1.00 × 10 ４ TCID ５０ ) was inoculated intranasally. Starting one day after virus infection, compound I-1 was orally administered twice daily to mice (n=5 / group) at doses of 0.1, 0.3, 1, 3, and 10 mg / kg. Control mice were orally administered DMA / 0.5 w / v% PVPVA in PEG400 twice daily. Compound administration was for two days from the start of administration. Three days after infection, mouse lungs were collected, 2 mL of PBS was added, homogenized, and the supernatant was collected after centrifugation. Measurement of virus titer in lung homogenate The lung homogenate was diluted 10-fold in culture medium (MEM, 2% FBS, penicillin-streptomycin) and then incubated with VeroE6 / TMPRSS2 cells (JCRB1819, 1.5 x 10 ４ The cells were mixed with 1000 cells / well and seeded onto a 96-well plate. ２ After culturing in an incubator for 4 days, cytopathic effect (CPE) was observed and the virus titer contained in the lung homogenate was calculated.

[0131] Compounds of the present invention were tested essentially as described above. The results are shown below. Three days after infection, the virus titer in the lung homogenate was 6.45-log in the DMA / 0.5% w / v PVPVA in PEG400 group. １０ TCID ５０ / mL, and in the groups administered 0.1, 0.3, 1, 3, and 10 mg / kg of compound I-1, the values ​​were 6.27, 5.39, 3.97, 2.51, and 2.47-log １０ TCID ５０ The virus titer in the lung homogenate in the compound I-1-administered group was lower than that in the DMA / 0.5 w / v% PVPVA in PEG400-administered group, suggesting that compound I-1 is effective in reducing the virus titer in the lung homogenate even if there is a long period between infection and administration (FIG. 1).

[0132] Test Example 10: Test for inhibiting viral titer growth in lung and nasal turbinate homogenates of SARS-CoV-2-infected hamsters and test for inhibiting lung weight gain 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 0.5 w / v% poly(1-vinylpyrrolidone-co-vinyl acetate) (PVPVA)-containing polyethylene glycol 400 (PEG400) (DMA: 0.5 w / v% PVPVA-containing PEG400 = 1:9). The administration volume was 2.5 mL / kg. Virus 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, medication, lung and nasal turbinate collection. Specific pathogen-free, 6-week-old male Syrian hamsters (Japan SLC, Inc.) were used in this study. For virus inoculation, the hamsters were anesthetized with an anesthetic solution containing 0.07 mg / mL medetomidine hydrochloride, 6.98 mg / mL alphaxalone, and 1.16 mg / mL butorphanol tartrate, administered subcutaneously at a dose of 3 mL / kg. 100 μL of hCoV-19 / Japan / TY41-702 / 2022 (1.00 × 10 ４ TCID ５０) was inoculated intranasally. Compound I-1 was orally administered twice daily at doses of 0.1, 1, or 10 mg / kg to the infected hamsters, starting one day after virus inoculation. DMA / 0.5 w / v% PVPVA in PEG400 was orally administered twice daily to the control infected and non-infected hamsters. Compound administration was for 5 days from the start of administration. 3 and 4 days after infection, the lungs and nasal turbinates of the infected hamsters were collected, and 5 mL or 1 mL of PBS was added, respectively, followed by homogenization. The supernatant was collected after centrifugation. 7 days after infection, the lungs of the infected and non-infected hamsters were collected, and the lung weights were measured. Measurement of virus titer in lung and nasal turbinate homogenates. The lung and nasal turbinate homogenates were diluted 10-fold in medium (MEM, 2% FBS, penicillin-streptomycin) and then cultured in a 96-well plate with VeroE6 / TMPRSS2 cells (JCRB1819, 1.5 x 10 ４ The cells were seeded onto a 1000 x 1000 x 1000 cells / well. ２ After culturing in an incubator for 4 days, cytopathic effect (CPE) was observed, and the virus titer contained in the lung or nasal turbinate homogenate was calculated.

[0133] Compounds of the present invention were tested essentially as described above. The results are shown below. The virus titer in the lung homogenate was 5.4-log in the DMA / 0.5% w / v PVPVA in PEG400 group 3 days after infection. １０ T.C.I.D. ５０ / mL, and in the groups administered with compound I-1 at 0.1, 1, and 10 mg / kg, the values ​​were 5.5, 3.5, and 2.5-log / mL, respectively. １０ T.C.I.D. ５０ Four days after infection, the DMA / 0.5 w / v% PVPVA in PEG400 administration group showed a 5.6-log １０ T.C.I.D. ５０ / mL, and the compound I-1 0.1, 1, and 10 mg / kg groups showed 4.6, 2.4, and 1.9-log / mL, respectively. １０ T.C.I.D. ５０ The virus titer in the nasal turbinate homogenate was 5.0-log / mL in the DMA / 0.5 w / v% PVPVA in PEG400 administration group 3 days after infection. １０ T.C.I.D.５０ / mL, and in the groups administered with compound I-1 at 0.1, 1, and 10 mg / kg, the values ​​were 4.8, 3.2, and 2.7-log / mL, respectively. １０ TCID ５０ Four days after infection, the DMA / 0.5 w / v% PVPVA in PEG400 administration group showed a 3.8-log １０ TCID ５０ / mL, and the compound I-1 0.1, 1, and 10 mg / kg groups showed 4.0, 2.2, and 1.8-log １０ TCID ５０ The viral titers in the lung and nasal turbinate homogenates of the compound I-1-treated group were dose-dependently lower than those of the DMA / 0.5 w / v% PVPVA in PEG400-treated group, suggesting that compound I-1 is effective in reducing the viral titers in the lung and nasal turbinate homogenates even after a long period of time between infection and treatment (Figures 2-1 and 2-2). Seven days after infection, the lung weight / body weight ratio was 5.26 mg / g in uninfected hamsters, 8.96 mg / g in the DMA / 0.5 w / v% PVPVA in PEG400-treated group, and 8.24, 5.75, and 5.57 mg / g in the 0.1, 1, and 10 mg / kg compound I-1-treated groups, respectively. The lung weight / body weight ratio in the compound I-1-treated group was dose-dependently lower than that in the DMA / 0.5 w / v% PVPVA in PEG400-treated group, suggesting that administration of compound I-1 has an inhibitory effect on the increase in lung weight induced by SARS-CoV-2 infection (Figure 3).

[0134] Test Example 11: Test for suppression of 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 0.5 w / v% poly(1-vinylpyrrolidone-co-vinyl acetate) (PVPVA)-containing polyethylene glycol 400 (PEG400) (DMA: 0.5 w / v% PVPVA-containing PEG400 = 1:9). The administration volume was 2.5 mL / kg. Virus 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, medication, and weight measurement. Specific pathogen-free, 6-week-old male Syrian hamsters (Japan SLC, Inc.) were used in this study. For 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 a rate of 3 mL / kg. 100 μL of hCoV-19 / Japan / TY41-702 / 2022 (1.00 × 10 ４ TCID ５０ ) was inoculated intranasally. Compound I-1 was orally administered twice daily at doses of 0.1, 1, or 10 mg / kg to the infected hamsters, starting one day after virus inoculation. DMA / 0.5 w / v% PVPVA in PEG400 was orally administered twice daily to the control infected and uninfected hamsters. Compound administration was continued for five days from the start of administration. Body weight was monitored once daily.

[0135] The compounds of the present invention were tested essentially as described above. The results are shown below. While uninfected hamsters showed weight gain, infected hamsters began to lose weight 3 days after infection, reaching their lowest point 6 days after infection. In the groups administered 1 or 10 mg / kg of compound I-1, all animals survived until 10 days after infection, and weight loss was suppressed (Figure 4). These results suggest that administration of compound I-1 has an inhibitory effect on weight loss.

[0136] Test Example 12: Test for the inhibition of weight loss in SARS-CoV-2-infected hamsters and the inhibition of viral proliferation in lung and nasal turbinate homogenates 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 SARS-CoV-2 hCoV-19 / Japan / TY41-702 / 2022 strain (Omicron BE.1 / BA.5-like) isolated by the National Institute of Infectious Diseases was used. Hamster Lung Infection, Medication, and Weight Measurement Specific pathogen-free 6-week-old male Syrian hamsters (Japan SLC, Inc.) were used in this study. For virus inoculation, hamsters were anesthetized by subcutaneous administration of 3 mL / kg of an anesthetic solution containing 0.07 mg / mL medetomidine hydrochloride, 6.98 mg / mL alphaxalone, and 1.16 mg / mL butorphanol tartrate. 100 μL of hCoV-19 / Japan / TY41-702 / 2022 (1.00 × 10 ４ TCID ５０ ) was inoculated intranasally. In the weight loss inhibition test, compound I-1 was administered subcutaneously once at 3 or 10 mg / kg one day or three days before virus infection. In the virus growth inhibition test in lung and nasal turbinate homogenates, compound I-1 was administered subcutaneously once at 10 or 30 mg / kg one day before virus infection. 0.5% MC was administered subcutaneously once one day before virus infection to the control hamsters. Body weight was monitored once daily. One and two days after infection, the lungs and nasal turbinates of the infected hamsters were collected, and 5 mL or 1 mL of PBS was added, respectively, followed by homogenization. The supernatant was collected after centrifugation. Measurement of virus titer in lung and nasal turbinate homogenates. The lung and nasal turbinate homogenates were diluted 10-fold in medium (MEM, 2% FBS, penicillin-streptomycin) and then cultured in a 96-well plate with VeroE6 / TMPRSS2 cells (JCRB1819, 1.5 x 10 ４ The cells were seeded onto a 1000 x 1000 x 1000 cells / well. ２ After culturing in an incubator for 4 days, cytopathic effect (CPE) was observed, and the virus titer contained in the lung or nasal turbinate homogenate was calculated.

[0137] The compounds of the present invention were tested essentially as described above. The results are shown below. Infected hamsters began to lose weight 3 days after infection, reaching a minimum weight 6 days after infection. In the groups administered compound I-1 at 3 or 10 mg / kg 1 day before infection and the group administered compound I-1 at 10 mg / kg 3 days before infection, all animals survived until 7 days after infection, and weight loss was suppressed (Figure 5). The virus titer in the lung homogenate was 5.59-log in the 0.5% MC group 1 day after infection. １０ T.C.I.D. ５０ / mL, and in the compound I-1 10 mg / kg and 30 mg / kg groups, the values ​​were 3.34 and 1.84-log １０ T.C.I.D. ５０ On day 2 after infection, the 0.5% MC administration group showed a 6.18-log １０ T.C.I.D. ５０ / mL, and in the compound I-1 10 mg / kg and 30 mg / kg groups, the values ​​were 5.29 and 3.55-log １０ T.C.I.D. ５０ The virus titer in the nasal turbinate homogenate was 5.71-log / mL in the 0.5% MC group on day 1 post-infection. １０ T.C.I.D. ５０ / mL, and in the compound I-1 10 mg / kg and 30 mg / kg groups, the values ​​were 4.80 and 3.36-log １０ T.C.I.D. ５０ On day 2 after infection, the 0.5% MC administration group showed a 5.80-log １０ T.C.I.D. ５０ / mL, and in the compound I-1 10 mg / kg and 30 mg / kg groups, the values ​​were 5.38 and 5.01-log １０ T.C.I.D. ５０ / mL (Figure 6-2). When plasma concentrations were measured immediately before infection, they were 6.1, 12.5, 5.9, and 3.0 ng / mL in the 3 and 10 mg / kg groups administered 1 day before infection and the 3 and 10 mg / kg groups administered 3 days before infection, respectively. These results demonstrate that prophylactic administration of compound I-1 exhibited a weight loss inhibitory effect when plasma concentrations at the time of viral infection were approximately 6 ng / mL or higher, and an inhibitory effect on viral proliferation in lung and nasal turbinate homogenates was observed when plasma concentrations were approximately 12 ng / mL or higher.

[0138] The above test results indicate that prophylactic subcutaneous administration of compound I-1 inhibited the progression of the disease and viral proliferation caused by viral infection, supporting the usefulness of compound I-1 as a prophylactic drug for infection with SARS-CoV-2.

[0139] Test Example 13: Test for viral titer clearance in nasal wash 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 0.5 w / v% poly(1-vinylpyrrolidone-co-vinyl acetate)-containing polyethylene glycol 400 (PEG400) (PVPVA) (DMA: 0.5 w / v% PVPVA-containing PEG400 = 1:9). The administration volume was 2.5 mL / kg. Virus 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. Nasal infection of hamsters, medication, and collection of nasal washes. Specific pathogen-free, 6-week-old male Syrian hamsters (Japan SLC, Inc.) were used in this study. For 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 a rate of 3 mL / kg. Then, 100 μL of hCoV-19 / Japan / TY41-702 / 2022 (1.00 × 10 ４ T.C.I.D. ５０) was inoculated intranasally. Compound I-1 was orally administered to the infected hamsters at doses of 1 or 10 mg / kg twice daily, starting 2 or 3 days after virus inoculation. DMA / 0.5 w / v% PVPVA in PEG400 was orally administered to the control infected hamsters twice daily. Compound administration was for 5 days from the start of administration. 2-6 days after infection, the infected hamsters were anesthetized with isoflurane, and nasal washes were collected with 400 μL of DPBS, and the supernatants were collected after centrifugation. Each group was used with n=4. Measurement of virus titer in nasal washes The supernatant of the nasal washes was diluted 10-fold in medium (MEM, 2% FBS, penicillin-streptomycin) and diluted with VeroE6 / TMPRSS2 cells (JCRB1819, 1.5 × 10 ４ The cells were seeded onto a 1000 x 1000 x 1000 cells / well. ２ After culturing in an incubator for 4 days, cytopathic effect (CPE) was observed and the virus titer contained in the nasal wash was calculated.

[0140] The compounds of the present invention were tested essentially as described above. The results are shown below. In the groups in which the compounds were administered starting 2 days after virus inoculation, the virus titers in the nasal washes were 4.80, 4.90, and 4.88-log at the start of administration (2 days after infection, 1 day after administration) in the DMA / 0.5 w / v% PVPVA in PEG400 group and the 1 and 10 mg / kg Compound I-1 groups, respectively. １０ T.C.I.D. ５０ / mL, and 3 days after infection (2 days after the start of administration), the values ​​were 3.75, 3.63, and 3.68-log １０ T.C.I.D. ５０ / mL, and 5 days after infection (4 days after the start of administration), the values ​​were 2.96, <1.80, and <1.80-log １０ T.C.I.D. ５０ In the groups administered with the compounds starting 3 days after virus inoculation, the DMA / 0.5 w / v% PVPVA in PEG400 group and the compound I-1 1 and 10 mg / kg groups showed 3.40, 2.93, and 3.13-log / mL, respectively, at the start of administration (3 days after infection, 1 day after the start of administration). １０ T.C.I.D. ５０ / mL, and 4 days after infection (1 day after the start of administration), the values ​​were 2.97, 1.84, and 2.13-log １０ T.C.I.D. ５０ / mL, and 6 days after infection (4 days after the start of administration), the values ​​were 2.43, <1.80, and <1.80-log １０ T.C.I.D. ５０ / mL (Figure 15). In the groups administered with 1 or 10 mg / kg of compound I-1, the virus titer in the nasal wash was lower than that in the group administered with DMA / 0.5 w / v% PVPVA in PEG400, regardless of whether administration was initiated 2 or 3 days after infection, demonstrating that compound I-1 has the effect of reducing the virus in vivo even if there is a long period between infection and administration.

[0141] Test Example 14: Test for inhibiting viral 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 prepared as a test sample using N,N-dimethylacetamide (DMA) and 0.5 w / v% poly(1-vinylpyrrolidone-co-vinyl acetate) (PVPVA)-containing polyethylene glycol 400 (PEG400) (DMA: 0.5 w / v% PVPVA-containing PEG400 = 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 Wash, and Lung Collection Specific pathogen-free 6-week-old male Syrian hamsters (Japan SLC, Inc.) were used in this study. For 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 a rate of 3 mL / kg. 100 μL of hCoV-19 / Japan / TY11-927 / 2021 (1.00 × 10 ３ T.C.I.D. ５０) was inoculated intranasally. Compound I-1 was orally administered twice daily at doses of 0.1, 1, or 10 mg / kg to the infected hamsters (Index), starting 8 hours after virus inoculation. DMA / 0.5 w / v% PVPVA in PEG400 was orally administered twice daily to the control infected hamsters (Index). Compound administration was performed a total of three times from the start of administration. For 12 hours overnight from day 1 to day 2 after infection, one infected hamster (Index) and one uninfected hamster (Contact) that had not been inoculated with the virus were housed together in the same cage, but in separate stainless steel cages separated by 2 cm to prevent direct contact. After the end of cohabitation, the hamsters were housed individually. Nasal washes and lungs were collected from the uninfected hamster (Contact) four days after the start of cohabitation (three days after removal from cohabitation). Nasal washes were collected with 400 μL of DPBS under isoflurane anesthesia, and the supernatant was collected after centrifugation. 5 mL of DPBS was added to the lungs, which were then homogenized and the supernatant was collected after centrifugation. n=6 samples were used for each group. Measurement of virus titers in nasal washes and lung homogenate supernatants. A 10-fold dilution series of nasal washes or lung homogenate supernatants was prepared in medium (MEM, 2% FBS, penicillin-streptomycin), and then the diluted 10-fold dilution series was added to VeroE6 / TMPRSS2 cells (JCRB1819, 1.5 × 10 cells) previously cultured in a 96-well plate. ４ The cells were inoculated into a 1000 x 1000 x 1000 cells / well. ２ After culturing in an incubator for 4 days, cytopathic effect (CPE) was observed, and the virus titer contained in the nasal wash or lung homogenate supernatant was calculated.

[0142] The compounds of the present invention were tested essentially as described above. The results are shown below. The virus titer in the nasal wash or lung homogenate supernatant of the uninfected hamster (Contact) was below the detection limit (1.8-log １０ TCID ５０The number of individuals with a viral load of ≥ 1000 mg / mL was 6 / 6 in the DMA / 0.5 w / v% PVPVA in PEG400 group, 6 / 6 in the compound I-1 0.1 mg / kg group, 0 / 6 in the compound I-1 1 mg / kg group, and 0 / 6 in the compound I-1 10 mg / kg group, demonstrating a dose-dependent inhibitory effect on virus transmission (Figure 16). These results demonstrate that administration of compound I-1 to infected hamsters (Index) after infection is effective in suppressing virus transmission to uninfected hamsters (Contact).

[0143] Test Example 15: Test for prevention of virus transmission from SARS-CoV-2-infected animals by prophylactic administration of compound I-1 to uninfected animals <Materials and Methods> Compound Compound I-1 was used as the test sample using 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 by the National Institute of Infectious Diseases was used. Nasal infection of hamsters, administration, cohabitation, nasal wash, and lung collection Specific pathogen-free 6-week-old male Syrian hamsters (Japan SLC, Inc.) were used in this study. For virus inoculation, hamsters were anesthetized by subcutaneous administration of 3 mL / kg of an anesthetic solution containing 0.07 mg / mL medetomidine hydrochloride, 6.98 mg / mL alphaxalone, and 1.16 mg / mL butorphanol tartrate. 100 μL of hCoV-19 / Japan / TY11-927 / 2021 (1.00 × 10 ３ TCID ５０) was inoculated intranasally. Compound I-1 was administered subcutaneously once to uninfected hamsters (Contact) at doses of 3, 10, 30, or 90 mg / kg 12 hours before cohabitation. Control uninfected hamsters (Contact) were administered subcutaneously once with 0.5% MC. One infected hamster (Index) and one uninfected hamster (Contact) administered with the compound were housed together in separate stainless steel cages, separated by 2 cm, for 12 hours overnight, one day after infection with the infected hamsters (Index) and two days after infection, respectively, in the same cage to prevent direct contact. After cohabitation, the hamsters were housed individually. Nasal washes and lungs were collected from uninfected hamsters (Contact) four days after cohabitation began (three days after cohabitation was discontinued). Nasal washes were collected in 400 μL of DPBS under isoflurane anesthesia, and the supernatant was collected after centrifugation. The lungs were homogenized with 3 mL of DPBS, and the supernatant was collected after centrifugation and diluted 2-fold with DPBS. Six samples were used for each group. Measurement of virus titers in nasal washes and lung homogenate supernatants. A 10-fold serial dilution of the nasal washes or lung homogenate supernatants was prepared in medium (MEM, 2% FBS, penicillin-streptomycin), and then diluted with VeroE6 / TMPRSS2 cells (JCRB1819, 1.50 x 10 ４ The cells were inoculated into a 1000 x 1000 x 1000 cells / well. ２ After culturing in an incubator for 4 days, cytopathic effect (CPE) was observed, and the virus titer contained in the nasal wash or lung homogenate supernatant was calculated.

[0144] The compounds of the present invention were tested essentially as described above. The results are shown below. The virus titers in the nasal washes or lung homogenate supernatants of uninfected hamsters (Contact) administered with the compounds four days after the start of cohabitation with infected hamsters (Index) (three days after the release of cohabitation) were below the detection limit (1.8-log １０ TCID ５０The number of animals with a viral load of ≥ 1000 mg / mL was 6 / 6 in the 0.5% MC group, 5 / 6 in the 3 mg / kg group, 6 / 6 in the 10 mg / kg group, 3 / 6 in the 30 mg / kg group, and 0 / 6 in the 90 mg / kg group, demonstrating a dose-dependent preventive effect (Figure 17). These results suggest that prophylactic administration of compound I-1 is effective in preventing viral transmission from infected hamsters (Index).

[0145] Test Example 16: Test for the prevention of lethality and weight loss in 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 prepared as a test sample using N,N-dimethylacetamide (DMA) and 0.5 w / v% poly(1-vinylpyrrolidone-co-vinyl acetate) (PVPVA)-containing polyethylene glycol 400 (PEG400) (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. Nasal infection of hamsters, medication, cohabitation, nasal wash, and lung collection. Specific pathogen-free, 11-month-old (aged) male Syrian hamsters (Japan SLC, Inc.) were used in this study. For 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 a rate of 3 mL / kg. 100 μL of hCoV-19 / Japan / TY11-927 / 2021 (1.00 × 10 ４ TCID ５０) was inoculated intranasally. Starting one day after virus inoculation, compound I-1 was orally administered twice daily at doses of 0.1, 1, or 10 mg / kg. DMA / 0.5 w / v% PVPVA in PEG400 was orally administered twice daily to control infected hamsters. Compound administration was continued for five days from the start of administration. Body weight was monitored once daily, and survival rates were assessed by considering animals dead when their body weight fell below 80% of the weight immediately before infection. Each group consisted of six animals.

[0146] The compounds of the present invention were tested essentially as described above. The results are shown below. In the infected hamster group treated with DMA / 0.5 w / v% PVPVA in PEG400, two animals died 8 days after infection, resulting in a survival rate of 66.7%. In contrast, in the groups treated with 1 and 10 mg / kg of compound I-1, all animals survived until 10 days after infection, and weight loss was suppressed (Figures 18 and 19). These results suggest that compound I-1 administration is effective in suppressing lethality and weight loss, even in aged hamsters.

[0147] Similar to humans, aging in rodents is known to increase the expression of the ACE2 receptor, a known receptor for SARS-CoV-2 (see Scientific Reports (2020)10:22401 and Molecular Therapy Methods & Clinical Development, Vol. 18, P1-6, 2020). Furthermore, the normal immune response that protects the body from and eliminates sources of infection is weakened, presumably making them more susceptible to severe viral infection. As shown above, in a SARS-CoV-2 infection model using aged hamsters, administration of compound I-1 suppressed weight loss and mortality. Preclinical studies using aged hamsters serve as a nonclinical evaluation system that mimics the progression of severe disease in high-risk patients with underlying diseases. Therefore, this study supports the usefulness of compound I-1 as a treatment option for patients at high risk of severe disease. Furthermore, the above test results suggest that the aggravation of viral infection was suppressed in the compound I-1-administered group, supporting not only the antiviral effect of compound I-1 against SARS-CoV-2 but also its usefulness as a pharmaceutical for suppressing the aggravation of SARS-CoV-2 infection.

[0148] Test Example 17: In vitro combination effect confirmation test <Procedure> Test sample Encitrervir fumarate, nilmatrervir, remdesivir, EIDD-1931, sotrovimab, and tixagevimab / silgavimab were used as test samples to be used in combination 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 x 10 ４ cells / well) and SARS-CoV-2 hCoV-19 / Japan / TY11-927 / 2021 (100TCID ５０ / well) was mixed with a medium (MEM, 2% FBS, penicillin-streptomycin), and the mixture was dispensed into the wells containing the test samples. ２ The plates were cultured in an incubator for two days. Dispensing of CellTiter-Glo® 2.0 and measurement of luminescence signals After the plates had been cultured for three days, they were returned to room temperature, and CellTiter-Glo® 2.0 was dispensed into each well and mixed using a plate mixer. After leaving the plates for a certain period of time, the luminescence signals (Lum) were measured using a plate reader.

[0149] <Analysis of Combination Effect> Synergy volume and antagonism volume were calculated using MacSynergy II. These values ​​can be calculated with reference to a previously published paper (Antiviral Research, 1990, Volume 14, pp. 181-206). - Evaluation of Combination Effect The combination effect was evaluated using the following criteria for synergy volume and antagonism volume at 99% confidence level using MacSynergy II. 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] Testing was carried out essentially as described above, and the results are shown in the table below.

[0151]

[0152] From the above results, the combination of compound I-1 with RNA-dependent RNA polymerase inhibitors (EIDD-1931 and remdesivir), 3CL protease inhibitors (ensitrervir, nilmatrervir), and anti-SARS-CoV-2 monoclonal antibodies (sotrolvimab, tixagevimab / silgavimab) showed additive to synergistic inhibitory effects on SARS-CoV-2 proliferation without showing antagonistic effects.

[0153] The following formulation examples are illustrative only and are not intended to limit the scope of the invention. The compounds of the present invention can be administered as pharmaceutical compositions by any conventional route, particularly enterally, e.g., orally, e.g., in the form of tablets or capsules, or parenterally, e.g., in the form of injection solutions or suspensions, topically, e.g., in the form of lotions, gels, ointments, or creams, or intranasally or in the form of suppositories. Pharmaceutical compositions containing the compounds of the present invention in free form or in the form of a pharmaceutically acceptable salt together with at least one pharmaceutically acceptable carrier or diluent can be prepared by conventional mixing, granulation, or coating methods. For example, oral compositions can be in the form of tablets, granules, or capsules containing excipients, disintegrants, binders, lubricants, etc., and active ingredients, etc. Injectable compositions can be in the form of solutions or suspensions, which may be sterilized and may contain preservatives, stabilizers, buffers, etc.

[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 and / or preventive agents for coronavirus infections.

Claims

1. Formula (I-1): or a pharma- ceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, which is a 3CL protease inhibitor.

3. The pharmaceutical composition according to claim 1 or 2, which is used to inhibit the viral proliferation of SARS-CoV-2.

4. The pharmaceutical composition according to any one of claims 1 to 3, which is a therapeutic and / or preventive agent for novel coronavirus disease (COVID-19).

5. The pharmaceutical composition according to any one of claims 1 to 4, which is used for suppressing the aggravation of infectious disease caused by SARS-CoV-2.

6. The pharmaceutical composition according to any one of claims 1 to 4, which is administered within 72 hours after the onset of symptoms of SARS-CoV-2 infection or after a positive diagnosis of SARS-CoV-2.

7. The pharmaceutical composition according to any one of claims 1 to 4, which is administered within 24 hours after the onset of symptoms of SARS-CoV-2 infection or after a positive diagnosis of SARS-CoV-2.

8. The pharmaceutical composition according to any one of claims 1 to 4, which is used to inhibit viral transmission of SARS-CoV-2.

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