Pharmaceutical compositions containing uracil derivatives

Abstract

The present invention provides a pharmaceutical composition containing a compound exhibiting coronavirus proliferation inhibitory activity. Formula (I-1): TIFF0007674711000062.tif4157 or a pharma- ceutically acceptable salt thereof.

Description

[Technical field]

[0001] The present invention relates to pharmaceutical compositions containing compounds exhibiting coronavirus 3CL protease inhibitory activity. [Background technology]

[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 the largest single-stranded + strand RNA viruses known. Coronaviruses are classified into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. There are seven known coronaviruses that infect humans: two types of Alphacoronavirus (HCoV-229E, HCoV-NL63) and five types of Betacoronavirus (HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, 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), which broke out in December 2019, spread rapidly throughout the international community, and on March 11, 2020, the WHO declared it a pandemic. Droplet infection, contact infection, and aerosol infection have been reported as the main routes of infection for SARS-CoV-2, and it has been confirmed that SARS-CoV-2 remains suspended in the air with aerosols for about three hours and maintains its infectiousness (Non-Patent Document 1). The incubation period is about 2 to 14 days, and cold-like symptoms such as fever (87.9%), dry cough (67.7%), fatigue (38.1%), and phlegm (33.4%) are typical (Non-Patent Document 2). In severe cases, respiratory failure due to acute respiratory distress syndrome, acute lung injury, interstitial pneumonia, etc. occurs. Multiple organ failure such as kidney failure 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, and the anti-IL-6 receptor antibody tocilizumab was additionally approved in January 2022. In addition, the antibody cocktail therapy Lonapriv (casirivimab / imdevimab) was specially approved in July 2021, sotrovimab was specially approved in September 2021, and molnupiravir was specially approved in December 2021. There is insufficient evidence regarding the efficacy and safety of these drugs. Therefore, the creation of treatments for COVID-19 is urgently needed.

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

[0006] In addition, as a COVID-19 treatment targeting 3CL protease, the start of Phase 1 trials of PBI-0451 by Pardes Biosciences was published on ClinicalTrials.gov in August 2021 (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). [ka]

[0007] In addition, 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 a regular basis on March 5, 2024 (Non-patent documents 17 and 18). The active ingredient of Zocoba 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). [ka]

[0008] On the other hand, there is insufficient evidence regarding resistance mutations to COVID-19 therapeutic 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 used in the present invention. In addition, compounds having 3CL protease inhibitory activity are disclosed in Patent Documents 14 and 15, but none of these documents describe or suggest a pharmaceutical composition containing the compound according to the present invention. P2X3 and / or P2X 2 / 3 Compounds having receptor inhibitory activity are disclosed in Patent Documents 3 to 9, but none of these documents describes or suggests 3CL protease inhibitory activity or antiviral effects. A compound having HIV-1 reverse transcriptase inhibitory activity is described in Non-Patent Document 11, but 3CL protease inhibitory activity and anti-coronavirus effect are neither described nor suggested. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2021 / 205298 [Patent Document 2] International Publication No. 2021 / 250648 [Patent Document 3] International Publication No. 2012 / 020742 [Patent Document 4] International Publication No. 2013 / 118855 [Patent Document 5] China Patent Publication No. 113620888 [Patent Document 6] China Patent Application Publication No. 113666914 [Patent Document 7] China Patent Application Publication No. 113735838 [Patent Document 8] China Patent Application Publication No. 113773300 [Patent Document 9] China Patent Application Publication No. 113801097 [Patent Document 10] International Publication No. 2022 / 138987 [Patent Document 11] International Publication No. 2022 / 138988 [Patent Document 12] International Publication No. 2023 / 054292 [Patent Document 13] International Publication No. 2023 / 054732 [Patent Document 14] International Publication No. 2023 / 195529 [Patent Document 15] International Publication No. 2023 / 195530 [Non-patent literature]

[0011] [Non-Patent Document 1] The NEW ENGLAND JOURNAL of MEDICINE (2020), Vol. 382, ​​pp. 1564-1567 [Non-Patent Document 2] "Report of the WHO-China Joint Mission on Coronavirus Disease 2019 (COVID-19)", [online], February 28, 2020, WHO, [March 16, 2023], インターネット<URL:https: / / www.who.int / docs / default-source / coronaviruse / who-china-joint-mission-on-covid-19-final-report.pdf> [Non-licensed document 3] Science (2003), vol. 300, pp. 1763-1767

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

[0012] The 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 pharmaceutical composition containing a compound having an antiviral effect, particularly an inhibitory effect on coronavirus proliferation. [Means for solving the problem]

[0013] The present invention relates to the following: (1) Formula (I-1): [ka] or a pharma- ceutically 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 item (1) or (2) above, which is used to inhibit the viral proliferation of SARS-CoV-2. (4) The pharmaceutical composition according to any one of items (1) to (3) above, which is a therapeutic and / or preventive agent for novel coronavirus disease (COVID-19). (5) The pharmaceutical composition according to any one of items (1) to (4) above, which is used for suppressing the aggravation of infectious diseases caused by SARS-CoV-2. (6) The pharmaceutical composition according to any one of items (1) to (4) above, 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 items (1) to (4) above, 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 items (1) to (4) above, which is administered within 120 hours after the onset of symptoms of SARS-CoV-2 infection or after a positive diagnosis of SARS-CoV-2. (9) The pharmaceutical composition according to any one of items (1) to (4) above, which is administered within 48 hours after the onset of symptoms of SARS-CoV-2 infection or after a positive diagnosis of SARS-CoV-2. (10) The pharmaceutical composition according to any one of items (1) to (4) above, which is used for suppressing viral transmission of SARS-CoV-2. (11) The pharmaceutical composition according to any one of items (1) to (4) above, 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 disease (COVID-19) a compound represented by the formula (I-1): [ka] or a pharma- ceutically acceptable salt thereof, Methods for inhibiting SARS-CoV-2 viral proliferation. (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 according to the formula (I-1): [ka] or a pharma- ceutically acceptable salt thereof, Methods for treating and / or preventing COVID-19. (14) A method for suppressing the aggravation of an infectious disease caused by SARS-CoV-2, comprising administering to an individual in need of treatment and / or prevention of a novel coronavirus disease (COVID-19) a compound represented by the formula (I-1): [ka] or a pharma- ceutically acceptable salt thereof, Methods to prevent the severity of SARS-CoV-2 infection. (15) A method for treating COVID-19, comprising administering to an individual in need of treatment for COVID-19 a compound of the formula (I-1): [ka] or a pharma- ceutical acceptable salt thereof within 72 hours after the onset of symptoms of SARS-CoV-2 infection or after a positive test for SARS-CoV-2. Treatment methods for novel coronavirus disease (COVID-19). (16) A method for treating COVID-19, comprising administering to an individual in need of treatment for COVID-19 a compound of the formula (I-1): [ka] or a pharma- ceutical acceptable salt thereof within 24 hours of onset of symptoms of SARS-CoV-2 infection or a positive test for SARS-CoV-2. Treatment methods for novel coronavirus disease (COVID-19). (17) A method for treating COVID-19, comprising administering to an individual in need of treatment for COVID-19 a compound of the formula (I-1): [ka] or a pharma- ceutical acceptable salt thereof within 120 hours after the onset of symptoms of SARS-CoV-2 infection or after a positive test for SARS-CoV-2. Treatment methods for novel coronavirus disease (COVID-19). (18) A method for treating COVID-19, comprising administering to an individual in need of treatment for COVID-19 a compound of the formula (I-1): [ka] or a pharma- ceutical acceptable salt thereof within 48 hours after the onset of symptoms of SARS-CoV-2 infection or after a positive test for SARS-CoV-2. Treatment methods for novel coronavirus disease (COVID-19). (19) A method for suppressing SARS-CoV-2 viral transmission, comprising administering to an individual in need of treatment for novel coronavirus disease (COVID-19) a compound of formula (I-1): [ka] or a pharma- ceutically acceptable salt thereof, How to suppress SARS-CoV-2 viral transmission. (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 symptoms I, comprising administering to said individual a compound of formula (I-1): [ka] or a pharma- ceutical acceptable salt thereof.

[0015] (21) For inhibiting viral proliferation of SARS-CoV-2, a compound of formula (I-1): [ka] or a pharma- ceutically acceptable salt thereof. (22) A compound according to the formula (I-1): for producing a medicament for treating and / or preventing novel coronavirus disease (COVID-19): [ka] or a pharma- ceutically acceptable salt thereof. (23) For suppressing the aggravation of infection caused by SARS-CoV-2, a compound according to the formula (I-1): [ka] or a pharma- ceutically acceptable salt thereof. (23') A compound according to the formula (I-1): [ka] or a pharma- ceutically acceptable salt thereof. (24) A compound of formula (I-1) for administration within 72 hours after the onset of symptoms of infection with SARS-CoV-2 or after a positive test for SARS-CoV-2: [ka] or a pharma- ceutically acceptable salt thereof. (24') A compound according to the formula (I-1): [ka] or a pharma- ceutical acceptable salt thereof, wherein the medicament is administered within 72 hours after the onset of symptoms of infection with SARS-CoV-2 or after a positive test for SARS-CoV-2. (25) A compound of formula (I-1) for administration within 24 hours after the onset of symptoms of infection with SARS-CoV-2 or after a positive test for SARS-CoV-2: [ka] or a pharma- ceutically acceptable salt thereof. (25') A compound according to the formula (I-1): [ka] or a pharma- ceutical acceptable salt thereof, wherein the medicament is administered within 24 hours after the onset of symptoms of infection with SARS-CoV-2 or after a positive test for SARS-CoV-2. (26) A compound of formula (I-1) for administration within 120 hours after the onset of symptoms of infection with SARS-CoV-2 or a positive test for SARS-CoV-2: [ka] or a pharma- ceutically acceptable salt thereof. (26') A compound according to the formula (I-1): [ka] or a pharma- ceutical 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 test for SARS-CoV-2. (27) A compound of formula (I-1) for administration within 48 hours after the onset of symptoms of infection with SARS-CoV-2 or after a positive test for SARS-CoV-2: [ka] or a pharma- ceutically acceptable salt thereof. (27') A compound according to the formula (I-1): [ka] or a pharma- ceutical 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 test for SARS-CoV-2. (28) A compound according to the formula (I-1) for inhibiting viral transmission of SARS-CoV-2: [ka] or a pharma- ceutically acceptable salt thereof. (28') A compound according to the formula (I-1): [ka] or a pharma- ceutically acceptable salt thereof. (29) A compound according to the formula (I-1): [ka] or a pharma- ceutical acceptable salt thereof, in which the symptoms of an individual in need of treatment for novel coronavirus disease (COVID-19) are mild or moderate I symptoms. (29') A compound according to the formula (I-1): [ka] or a pharma- ceutical acceptable salt thereof, in which 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 medicine for novel coronavirus disease (COVID-19), which is used for suppressing the onset of SARS-CoV-2 after exposure, comprising the following formula (I-1): [ka] or a pharma- ceutically acceptable salt thereof. (102) A method for preventing novel coronavirus disease (COVID-19), comprising administering to an individual in need of suppression of onset after exposure to SARS-CoV-2 a compound of the formula (I-1): [ka] or a pharma- ceutically acceptable salt thereof, How to prevent coronavirus disease (COVID-19). (103) For suppressing the onset of SARS-CoV-2 after exposure, a compound according to the formula (I-1): [ka] or a pharma- ceutically acceptable salt thereof. (104) A compound according to the formula (I-1): [ka] or a pharma- ceutically acceptable salt thereof.

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

[0018] In one embodiment, the present invention includes the following. (301) Formula (II): [ka] (In the formula, X is a single bond or -CH2-; 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; R 1 is a substituted or unsubstituted aromatic heterocyclic group or a substituted or unsubstituted non-aromatic heterocyclic group; m is 0 or 1; R 5a 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 compound: [ka] ) or a pharma- ceutically acceptable salt thereof. (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; The compound according to the above item (301), wherein m is 0, or a pharma- ceutically acceptable salt thereof. Effect of the Invention

[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. [Brief description of the drawings]

[0020] [Figure 1] The virus titer in the lung homogenate 3 days after infection is shown for mice infected with hCoV-19 / Japan / TY7-501 / 2021 (1.00×104 TCID50 / mouse inoculated intranasally) when the vehicle and the compound represented by formula (I-1) were orally administered twice a day for 2 days starting 1 day after infection. The vertical axis shows the virus titer in the lung homogenate, and the horizontal axis shows each administration group. [Figure 2-1] For hCoV-19 / Japan / TY41-702 / 2022 infected hamsters (1.00×104 TCID50 / hamster inoculated intranasally), the vehicle and the compound represented by formula (I-1) were orally administered twice a day for 2-3 days starting 1 day after infection, and the virus titer in the lung homogenate was measured 3-4 days after infection. 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. [Figure 2-2] For hCoV-19 / Japan / TY41-702 / 2022 infected hamsters (1.00×104 TCID50 / hamster inoculated intranasally), the vehicle and the compound represented by formula (I-1) were orally administered twice a day for 2-3 days starting 1 day after infection, and the virus titer in the nasal turbinate homogenate was measured 3-4 days after infection. 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. [Diagram 3]For hCoV-19 / Japan / TY41-702 / 2022-infected hamsters (1.00×104 TCID50 / hamster inoculated intranasally), the vehicle and the compound represented by formula (I-1) were orally administered twice a day for 5 days starting 1 day after infection, and the lung weight / body weight 7 days after infection is shown. The vertical axis shows the lung weight / body weight, and the horizontal axis shows each administration group. [Figure 4] For hamsters infected with hCoV-19 / Japan / TY41-702 / 2022 (1.00×104 TCID50 / hamster nasally inoculated), the vehicle and the compound represented by formula (I-1) were orally administered twice a day for 5 days starting 1 day after infection, and the weight change up to 10 days after infection is shown. 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. [Diagram 5] For hamsters infected with hCoV-19 / Japan / TY41-702 / 2022 (1.00×104 TCID50 / hamster nasally inoculated), the vehicle and the compound represented by formula (I-1) were administered subcutaneously once 3 days or 1 day before infection, and the weight change up to 7 days after infection is shown. 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. [Figure 6-1] For hCoV-19 / Japan / TY41-702 / 2022 infected hamsters (1.00×104 TCID50 / hamster nasally inoculated), the vehicle and the compound represented by formula (I-1) were administered subcutaneously once one day before infection, and the virus titer in the lung homogenate was measured 1-2 days after infection. 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. [Figure 6-2] For hCoV-19 / Japan / TY41-702 / 2022 infected hamsters (1.00×104 TCID50 / hamster nasally inoculated), the virus titer in the nasal turbinate homogenate 1-2 days after infection when the vehicle and the compound represented by formula (I-1) were administered subcutaneously once 1 day before infection is shown. 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. [Figure 7]1 shows the powder X-ray diffraction pattern of the anhydrous crystal of the compound represented by formula (I-1). The horizontal axis represents 2θ (°), and the vertical axis represents intensity. [Figure 8] The peak list of the powder X-ray diffraction pattern of Figure 7 is shown below. In the table, Position indicates 2θ(°) and Intensity indicates intensity. [Figure 9] The crystal structure (structure in the asymmetric unit) of the anhydrous crystal of the compound represented by formula (I-1) is shown. [Figure 10] 1 shows the results of differential scanning calorimetry (DSC) of the anhydrous crystals of the compound represented by formula (I-1).The horizontal axis represents temperature (°C), and the vertical axis represents heat (W / g). [Figure 11] The figure shows the results of simultaneous differential thermal analysis and thermogravimetry (TG / DTA) of the anhydrous crystals 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). In the figure, Cel means degrees Celsius (°C). [Figure 12] 1 shows the Raman spectrum of anhydrous crystals of the compound represented by formula (I-1), where the horizontal axis represents the Raman shift (cm-1) and the vertical axis represents the peak intensity. [Figure 13] The upper part shows the HPLC measurement results of the anhydrous crystals of the compound represented by formula (I-1), and the lower part shows the HPLC peak table. [Figure 14] For hCoV-19 / Japan / TY41-702 / 2022 infected hamsters (1.00×104 TCID50 / hamster inoculated intranasally), the vehicle and the compound represented by formula (I-1) were orally administered twice a day for 1-5 days starting 2 days after infection. The virus titer in the nasal lavage fluid (NALF) 3-7 days after infection is shown. The vertical axis shows the virus titer in the NALF, and the horizontal axis shows the number of days after infection. [Figure 15] For hCoV-19 / Japan / TY41-702 / 2022-infected hamsters (1.00×104TCID50 / hamster inoculated intranasally), the following shows the virus titer in nasal lavage fluid (NALF) 4-8 days after infection when the vehicle and the compound represented by formula (I-1) were orally administered twice a day for 1-5 days starting 3 days after infection. The vertical axis shows the virus titer in NALF, and the horizontal axis shows the number of days after infection. [Figure 16] Compounds were administered to hCoV-19 / Japan / TY11-927 / 2021-infected hamsters (1.00 x 103 TCID50 / hamster inoculated intranasally, Index) and uninfected hamsters (Contact) were housed together for 12 hours at night from day 1 to day 2 after infection. The virus titers in the lung homogenate and nasal lavage fluid (NALF) of uninfected hamsters (Contact) four days after the start of cohabitation are shown. Infected hamsters (Index) were orally administered the vehicle and the compound represented by formula (I-1) twice a day starting 8 hours after infection. The vertical axis indicates the virus titers in the lung homogenate and NALF, and the horizontal axis indicates each administration group. [Figure 17] The virus titers in the lung homogenate and nasal lavage fluid (NALF) of the non-infected hamster (Contact) 4 days after the start of cohabitation when a hCoV-19 / Japan / TY11-927 / 2021-infected hamster (1.00 × 103 TCID50 / hamster was inoculated intranasally, Index) and a non-infected hamster (Contact) administered a compound were cohabited for 12 hours at night from 1 day to 2 days after infection. The non-infected hamster (Contact) was subcutaneously administered the vehicle and the compound represented by formula (I-1) 12 hours before the start of cohabitation. The vertical axis indicates the virus titers in the lung homogenate and NALF, and the horizontal axis indicates each administration group. [Figure 18] The figure shows the survival rate up to 10 days after infection for aged hamsters infected with hCoV-19 / Japan / TY11-927 / 2021 (1.00 × 104 TCID50 / hamster inoculated intranasally, 11-month-old hamsters) when the vehicle and the compound represented by formula (I-1) were orally administered twice a day for 5 days starting 1 day after infection. The vertical axis shows the survival rate (%), and the horizontal axis shows the number of days after infection. [Figure 19] The weight change up to 10 days after infection is shown for aged hamsters infected with hCoV-19 / Japan / TY11-927 / 2021 (1.00×104 TCID50 / hamster intranasally inoculated, 11-month-old hamsters) when the vehicle and the compound represented by formula (I-1) were orally administered twice a day for 5 days starting 1 day after infection. 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The meaning of each term used in this specification will be explained below. Unless otherwise specified, each term has the same meaning whether it is used alone or in combination with other terms. The term "consisting of" means having only the constituent elements. The terms "comprise" and "include" are meant to be open ended and not excluding unrecited elements. Hereinafter, the present invention will be described with reference to the embodiments. Throughout this specification, it should be understood that the singular expression includes the plural concept unless otherwise specified. Therefore, it should be understood that the singular article (e.g., in the case of English, "a", "an", "the", etc.) includes the plural concept unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the above field unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In case of conflict, the present specification (including definitions) shall prevail.

[0022] In the compounds of formula (II) herein, R 1 , R 2 , R 3c , R 5a and R 5b The definition of each term used in the above can be referred to in International Publication No. 2023 / 195529, the disclosure of which is incorporated herein by reference. In addition, the definition of the substituent used in each term can be referred to in International Publication No. 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 a specific isomer, 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 As Cl, hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine are included. The compounds of formula (I-1) or formula (II) also include compounds substituted with such isotopes (e.g., deuterium exchangers, etc.). The compounds substituted with such isotopes are also useful as pharmaceuticals. The compounds of formula (I-1) or formula (II) include all radiolabeled products substituted with radioisotopes contained in the isotopes. The present invention also includes a "radiolabeling method" for producing the "radiolabeled product", and the "radiolabeled product" is useful as a research and / or diagnostic tool in metabolism pharmacokinetic studies, binding assays.

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

[0026] Radiolabeled compounds of formula (I-1) or formula (II) can be prepared by methods well known in the art. For example, tritium-labeled compounds of formula (I-1) or formula (II) can be prepared by introducing tritium into a particular compound of formula (I-1) or formula (II) by catalytic dehalogenation reaction with tritium. This method involves reacting a suitable halogen-substituted precursor of a compound of formula (I-1) or formula (II) with tritium gas in the presence of a suitable catalyst, such as Pd / C, in the presence or absence of 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 compound of the present invention represented by formula (I-1) or formula (II) may form a prodrug, and the present invention also includes such various prodrugs. A prodrug is a derivative of the compound of the present invention having a group that can be chemically or metabolically decomposed, and is a compound that becomes a pharmacologic active compound of the present invention in vivo by solvolysis or under physiological conditions. Prodrugs include compounds that are converted to a compound represented by formula (I-1) or formula (II) by enzymatic oxidation, reduction, hydrolysis, etc. under physiological conditions in vivo, and compounds that are converted to a compound represented by formula (I-1) or formula (II) by hydrolysis by gastric acid, etc. Methods for selecting and preparing suitable prodrug derivatives are described, for example, in "Design of Prodrugs, Elsevier, Amsterdam, 1985". Prodrugs may themselves have activity.

[0028] As used herein, the "compound represented by formula (I-1) or formula (II)" may form a salt, a cocrystal, or a solvate thereof. As used herein, the term "a compound represented by formula (I-1) or formula (II), a pharma- ceutically acceptable salt thereof, or a solvate thereof" also includes such various salts, cocrystals, and solvates thereof.

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

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

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

[0032] One embodiment in the present specification is a pharma- ceutically acceptable salt or cocrystal 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] Salt and co-crystal formation studies provide a means to modify the physicochemical and resulting biological characteristics of a drug without changing its chemical structure. Salt and co-crystal formation can dramatically affect the properties of a drug. Hygroscopicity, stability, solubility, and processing properties are also important aspects in the selection of an appropriate salt or co-crystal. The solubility of a salt or co-crystal can affect its suitability for use as a drug. If the aqueous solubility is low, the dissolution rate upon in vivo administration may be rate-limited by the absorption process, resulting in low bioavailability. Also, low water solubility may make administration by injection difficult, limiting the selection of an appropriate route of administration.

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

[0035] The term "solvate" as used herein refers to a compound of formula (I-1) or formula (II) regularly arranged with any number of solvent molecules. 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, 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, methylbutylketone, methylcyclohexane, N-methylpyrrolidone, nitromethane, pyridine, sulfolane, tetralin, toluene, 1,1,2-trichloroethene, xylene, and t-butanol. Preferred are 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 are 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 the compound or a hydrate may be formed.

[0036] In addition, the term "crystal" as used herein means a solid in which constituent atoms, ions, molecules, etc. are arranged in a three-dimensional order, and is distinguished from amorphous solids that do not have such an orderly internal structure. The term "crystal" as used herein may be a single crystal, a twin crystal, a polycrystal, etc. Furthermore, "crystals" can have "crystal polymorphs" that have the same composition but different arrangements within the crystal, and all of these are referred to as "crystal forms." The "compound represented by formula (I-1) or formula (II), a pharma- ceutically acceptable salt thereof, or a solvate thereof" includes their crystal polymorphs. The crystal of the compound of the present invention may be a deuterium conversion product. As used herein, a crystal is an isotope (e.g., 2 H, 3 H, 14 C. 35 S, 125 I, etc.) may be used. The crystal morphology and / or crystallinity can be confirmed by spectroscopic methods such as, for example, X-ray diffraction, Raman spectroscopy, infrared absorption spectroscopy, solid-state NMR, etc. Additionally, the physical properties of the crystals can be confirmed by a number of techniques such as differential scanning calorimetry, moisture adsorption / desorption measurements, dissolution characteristics, etc.

[0037] One embodiment of the present specification is an anhydrous crystal of the compound represented by formula (I-1). As used herein, "anhydrous" is synonymous with "nosolvate," "nonsolvate," "anhydrate," and "nonhydrate." The theoretical content of water of crystallization in the anhydrous crystal of the compound represented by formula (I-1) is 0% by weight. However, in the analysis of the water content and / or the solvent content, the value may be higher than the theoretical content of water of crystallization due to the influence of the water and / or the 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) having 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] In one embodiment of the present specification, when a single crystal diffraction experiment was carried out using CuKα radiation (λ=1.5418 Å) at 298 K (25° C.), Crystallographic data for: Space group: Pbca a=14.67ű0.05Å b = 11.83 Å ± 0.05 Å c=27.10ű0.05Å α=90° β=90° γ=90° The anhydrous crystal of the compound represented by formula (I-1) is characterized by:

[0040] One embodiment herein is an anhydrous crystal 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). One embodiment of the present specification is an anhydrous crystal 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] In one embodiment of the present invention, a Raman spectrum of 415.2 cm -1 ±2cm -1 , 502.7cm -1 ±2cm -1 , 1431.4cm -1 ±2cm -1, 1714.8cm -1 ±2cm -1 and 3065.4 cm -1 ±2cm -1 The anhydrous crystals of the compound represented by formula (I-1) have characteristic peaks.

[0042] (X-ray powder diffraction (XRPD)) X-ray powder diffraction (XRPD) is one of the most sensitive analytical techniques for measuring the crystalline morphology 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 that correspond to the periodicity of the structure. On the other hand, amorphous solids usually do not have an ordered repeating period in their structure, so no diffraction phenomenon occurs and they produce a featureless broad XRPD pattern (also called a halo pattern).

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

[0044] Generally, the diffraction angle (2θ) in powder X-ray diffraction can have an error within the range of ±0.2°, and therefore the value of the diffraction angle in powder X-ray diffraction should be understood to include a numerical value within the range of about ±0.2°. Therefore, not only crystals in which the diffraction angles of the peaks in powder X-ray diffraction are completely the same, but also crystals in which the diffraction angles of the peaks are the same 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 due to many factors, such as the effect of the preferred orientation of the crystals relative to the X-ray beam, the influence of coarse particles, the purity of the material being analyzed, or the crystallinity of the sample. The peak positions may also shift based on the variation of the sample height. Furthermore, measurements using different wavelengths will result in different shifts according to the Bragg equation (nλ=2dsinθ), and the use of different wavelengths to obtain different XRPD patterns is also within the scope of the present invention.

[0046] (Single crystal structure analysis) It 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 positional relationship of each atom) and a three-dimensional structure model. See, for example, "X-Ray Structure Analysis Handbook" by Toshio Sakurai, published by Shokabo Publishing (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 a molecule or complex system. It originates from inelastic collisions between molecules and photons, which are light particles that comprise a beam of light. Collisions between molecules and photons result in an exchange of energy, which changes the energy and therefore the wavelength of the photon. That is, Raman spectra are spectral lines with extremely narrow wavelengths that are emitted when photons are incident on a molecule of interest, so a laser or other light source is used. The wavelength of each Raman line is represented by a 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. In general, Raman spectrum peaks (cm -1 ) is ±2cm -1 Since there may be an error within the range of ±2 cm -1 It should be understood that the values ​​include values ​​within the range of about 1 cm. Therefore, not only crystals with perfectly matching Raman spectrum peaks in the Raman spectrum, but also crystals with Raman spectrum peaks within ±2 cm -1 Crystals that match to 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 a substance as an aggregate of atoms and molecules. DSC measures the change in heat quantity with respect to temperature or time of a pharmaceutical active ingredient, and the obtained data is plotted against temperature or time to obtain a differential scanning calorimetry curve. 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 accompanying melting, and enthalpy. It is known that for DSC, the observed temperatures may depend on the rate of temperature change as well as the sample preparation technique and the particular instrument used. Thus, the "melting point" in DSC refers to the onset temperature, which is less affected by the sample preparation technique. The error range in onset temperatures obtained from differential scanning calorimetry curves is approximately ±2°C. In determining the identity of a crystal, not only the melting point but also the overall pattern is important, which may vary somewhat depending on the measurement conditions and the measurement instrument.

[0049] (Differential thermal / thermogravimetric simultaneous measurement method (TG / DTA)) TG / DTA is one of the main methods of thermal analysis, and is used to measure the weight and thermal properties of a substance as an aggregate of atoms and molecules. TG / DTA is a method to measure the change in weight and heat quantity of a pharmaceutical active ingredient with respect to temperature or time, and the obtained data is plotted against temperature or time to obtain TG (thermogravimetry) and DTA (differential thermal) curves. From the TG / DTA curve, information on the change in weight and heat quantity related to the decomposition, dehydration, oxidation, reduction, sublimation, and evaporation of the pharmaceutical active ingredient can be obtained. It is known that the observed temperature and weight changes in TG / DTA may depend on the rate of temperature change as well as the sample preparation technique and the specific instrument used. Therefore, the "melting point" in TG / DTA refers to the onset temperature that is less affected by the sample preparation technique. In identifying the identity of a crystal, not only the melting point but also the overall pattern is important, and may vary somewhat depending on the measurement conditions and the measurement instrument.

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

[0051] Diseases in which coronavirus 3CL protease is involved include viral infections, preferably coronavirus infections. In one embodiment, the coronavirus includes a coronavirus that infects humans, including 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, and particularly preferably SARS-CoV-2. In one embodiment, the beta coronavirus includes beta coronavirus lineage A, beta coronavirus lineage B, and beta coronavirus lineage C. More preferably, the beta coronavirus lineage A and beta coronavirus lineage B are included, and particularly preferably, the beta coronavirus lineage B is included. Examples of the beta-coronavirus A lineage include HCoV-HKU1 and HCoV-OC43, preferably HCoV-OC43. Examples of the beta-coronavirus B lineage include SARS-CoV and SARS-CoV-2, preferably SARS-CoV-2. Examples of the beta-coronavirus C lineage include 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 above coronaviruses include not only mutant strains known in the art, but also mutant strains that will appear in the future, as long as the compound according to the present invention is a strain that can exhibit coronavirus 3CL protease inhibitory activity. Known mutant strains of SARS-CoV-2 include, for example, the mutant strains used in the examples of this specification. 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, and more preferably infections caused by SARS-CoV-2. A particularly preferred example of the coronavirus infection is novel coronavirus disease (COVID-19).

[0052] The severity classification of novel coronavirus infections is, for example, as follows. (Reference: Novel Coronavirus COVID-19 Treatment Guide, Version 10.0 (Ministry of Health, Labor and Welfare)) (Mild symptoms) Oxygen saturation is 96% or higher. Clinical condition is no respiratory symptoms, or only coughing and no dyspnea, and in either case no signs of pneumonia are observed. (Moderate I) Oxygen saturation is less than 96% but more than 93%. There is difficulty breathing and signs of pneumonia. (Moderate II) Oxygen saturation is less than 93%. Respiratory failure occurs and oxygen supplementation is required. (Severe) They are in the ICU or require mechanical ventilation. The above classification is the definition of the severity classification used in Japan, and it is possible to refer to the severity classifications used in China and the US NIH, for example. Additionally, an asymptomatic SARS-CoV-2 infected person refers to a pathogen carrier who is asymptomatic, such as a person who does not exhibit any of the 14 or 12 symptoms of COVID-19. (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 (malaise (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 progression to severe illness includes preventing an asymptomatic SARS-CoV-2 infected individual from progressing to a severity classified as mild, moderate I, moderate II, or severe. As used herein, suppressing progression to severe disease includes preventing an asymptomatic or mildly symptomatic SARS-CoV-2 infected individual from progressing to a severity classified as moderate I, moderate II, or severe. As used herein, suppressing progression to severe disease includes preventing an asymptomatic SARS-CoV-2 infected person, a SARS-CoV-2 infected person with mild symptoms, or a SARS-CoV-2 infected person with moderate I symptoms from being classified as moderate II symptoms or severe symptoms. As used herein, suppression of progression includes upgrading 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 of severity. In this specification, suppression of progression includes reducing the risk of hospitalization and death in SARS-CoV-2-infected patients through the drug's effect of inhibiting viral proliferation. In this specification, suppression of aggravation includes reducing inflammation in the lungs of patients infected with SARS-CoV-2 through the drug's effect of inhibiting viral proliferation. In this specification, suppression of aggravation includes suppressing pneumonia caused by SARS-CoV-2 viral infection through the viral proliferation inhibitory effect of the drug. In this specification, suppression of aggravation includes suppressing the host's excessive immune response 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 a SARS-CoV-2 infected individual who has at least one risk factor for severe symptoms. For information on underlying diseases related to severe symptoms, please refer to the US 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 a SARS-CoV-2-infected individual who has at least one of the following risk factors for severe illness: Risk factors for severe disease: malignant tumors, metabolic diseases, cardiovascular diseases, respiratory diseases, liver diseases, kidney diseases, neuropsychiatric diseases, lack of exercise, pregnancy, smoking, childhood, genetic diseases, immunodeficiency

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

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

[0059] As used herein, "prevention" includes suppressing the 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 novel coronavirus infection. For example, the pharmaceutical composition of the present invention can be administered to a person promptly (e.g., within 72 hours) after coming into contact with a patient infected with COVID-19. For example, the pharmaceutical composition of the present invention can be administered to an asymptomatic SARS-CoV-2 infected individual to suppress the onset of the disease. Additionally, "prevention" includes pre-exposure prophylaxis for SARS-CoV-2. For example, when there is a risk of infection with SARS-CoV-2, such as during a COVID-19 epidemic, the pharmaceutical composition of the present invention can be administered to medical workers, elderly people, and people with risk factors for developing severe disease.

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

[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 compound according to the present invention has pharmaceutical utility and preferably has one or more of the following excellent characteristics: a) It has a weak inhibitory effect on CYP enzymes (e.g., CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, etc.). b) It exhibits favorable pharmacokinetics, including high bioavailability and moderate clearance. c) High metabolic stability. d) It does not exhibit irreversible inhibitory effects on CYP enzymes (e.g., CYP3A4) within the concentration range of the measurement conditions described herein. e) It is not mutagenic. f) Lower cardiovascular risk. g) High solubility. h) The protein unbound rate (fu value) is high. i) It has high coronavirus 3CL protease selectivity. j) It has high coronavirus proliferation inhibitory activity. For example, it has high coronavirus proliferation inhibitory activity in the presence of human serum (HS) or human serum albumin (HSA). k) It has high proliferation inhibitory activity even against 3CL protease inhibitor-resistant viruses. As a coronavirus proliferation inhibitor, for example, in the CPE suppression effect confirmation test (SARS-CoV-2) described below, 50 In one embodiment, the concentration 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, ocular, otic, vaginal, etc.

[0063] In the case of 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.), liquid preparations for internal use (e.g., suspensions, emulsions, elixirs, syrups, lemonades, spirits, aromatic perfumes, extracts, decoctions, tinctures, etc.), etc., in accordance with the usual 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, drops, external preparations (e.g., eye drops, nasal drops, ear drops, aerosols, inhalants, lotions, injections, liniments, mouthwashes, enemas, ointments, plasters, jellies, creams, patches, poultices, external powders, suppositories, etc.) The injections may be emulsions such as O / W, W / O, O / W / O, and W / O / W types.

[0065] A pharmaceutical composition can be prepared by mixing an effective amount of the compound according to the present invention with various pharmaceutical additives suitable for the dosage form, such as excipients, binders, disintegrants, and lubricants, as necessary. Furthermore, the pharmaceutical composition can be prepared as a pharmaceutical composition for children, elderly people, critically ill patients, or surgical patients by appropriately changing the effective amount of the compound according to the present invention, the dosage form, and / or various pharmaceutical additives. For example, a pharmaceutical composition for children can be administered to newborns (less than 4 weeks after birth), infants (4 weeks to less than 1 year after birth), toddlers (1 year to less than 7 years), children (7 years to less than 15 years) or patients aged 15 to 18 years. For example, a pharmaceutical composition for the elderly can be administered to patients aged 65 years or older.

[0066] The dosage of the pharmaceutical composition of the present invention is preferably set in consideration of the age and body weight of the patient, the type and severity of the disease, the administration route, etc., but in the case of oral administration, the compound of the present invention is usually 0.01 to 100 mg / kg / day, preferably 0.05 to 50 mg / kg / day. In the case of parenteral administration, although it varies greatly depending on the administration route, the compound of the present invention is usually 0.005 to 200 mg / kg / day, preferably 0.01 to 100 mg / kg / day. This may be administered once or several times a day.

[0067] The compound according to the present invention may be used in combination with, for example, other therapeutic agents for novel coronavirus disease (COVID-19) (including approved drugs and drugs under development or to be developed in the future) (hereinafter referred to as concomitant drugs) for the purpose of enhancing the action of the compound or reducing the dosage of the compound. In this case, the administration time of the compound according to the present invention and the concomitant drug is not limited, and they may be administered to the subject at the same time or at different times. Furthermore, the compound according to 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 dosage of the concomitant drug can be appropriately selected based on the dosage used clinically. The compounding ratio of the compound according to 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 part by weight of the compound according to the present invention. EXAMPLES

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

[0070] Furthermore, 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 of identifying compounds) The NMR analysis obtained in each example was performed at 400 MHz using DMSO-d6 and CDCl3. In addition, when presenting NMR data, there are cases where not all measured peaks are listed. In the description, RT refers to retention time in LC / MS (liquid chromatography / mass spectrometry), and was measured under the following conditions. (Measurement condition A) Column: ACQUITY UPLC® BEH C18 (1.7 μm id2.1x50 mm) (Waters) Flow rate: 0.8mL / 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 from 5% to 100% solvent [B] in 3.5 min, followed by a hold at 100% solvent [B] for 0.5 min.

[0072] X-ray powder diffraction experiment (XRPD) According to the powder X-ray diffraction measurement method described in the general test method of the Japanese Pharmacopoeia, the solid form (crystal) obtained in the examples was subjected to powder X-ray diffraction measurement under the following measurement conditions. Measurement condition 1: Powder X-ray diffraction equipment: Rigaku SmartLab Measurement method: Reflection method Wavelength used: CuKα ray (λ=1.5418Å) Tube current: 200mA Tube voltage: 45kV Sample plate: Aluminum X-ray incidence angle: 2.5° Sampling width: 0.02° Detector: HyPix-3000 (2D detection mode)

[0073] Measurement and analysis method for single crystal structure analysis The crystals obtained in the examples were subjected to single crystal structure analysis. The measurement conditions and analysis method are shown below. (Device) Rigaku XtaLAB P200 MM007 (Measurement conditions) Measurement temperature: 25℃ Temperature controller: Rigaku sample spray low temperature device Wavelength used: CuKα ray (λ=1.5418Å) Software: CrysAlisPro 1.171.39.46e (Rigaku Oxford Diffraction, 2018) (Data Processing) Software: CrysAlisPro 1.171.39.46e (Rigaku Oxford Diffraction, 2018) The data were Lorentzian, polarization and absorption corrected. (Crystal structure analysis) Phase determination was performed using the direct method program ShelXT (Sheldrick, GM, 2015), and refinement was performed using full-matrix least-squares fitting with ShelXL (Sheldrick, GM, 2015). All non-hydrogen atom temperature factors were refined anisotropically. Hydrogen atoms were introduced by calculation using the default parameters of ShelXL unless otherwise noted, and were treated as riding atoms. Hydrogen atoms were also refined with isotropic parameters. The following structural diagram was drawn using PLATON (Spek, 1991) / ORTEP (Johnson, 1976) (30% PROBABILITY level).

[0074] Measurement of Raman spectra 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: 671nm Number of times accumulated: 1 Exposure time: 1 second

[0075] Differential Scanning Calorimetry (DSC) The crystals obtained in the examples were subjected to DSC measurement. The sample was weighed in an aluminum pan, sealed, and measured. The measurement conditions are shown below. Note that the measurement by differential scanning calorimetry (DSC) may have an error within the range of ±2°C. Instrument: Discovery DSC / TA Instrument Measurement temperature range: -10℃-270℃ Heating rate: 10℃ / min Atmosphere: N250mL / min

[0076] Simultaneous differential thermal and thermogravimetric measurement (TG / DTA) The solid form (crystal) obtained in the examples was subjected to simultaneous differential thermal analysis and thermogravimetry (TG / DTA). The samples obtained in the examples were weighed, placed in aluminum pans, and measured in an open system. The measurement conditions were as follows. Equipment: Hitachi High-Technologies TG / DTA STA7200RV Measurement temperature range: Room temperature - 350℃ Heating rate: 10℃ / min

[0077] HPLC measurements Equipment: Agilent 1290 Infinity VL Column: Waters Acquity UPLC BEH C18, 1.7 μm, 2.1 × 100 mm Column temperature: constant temperature around 40℃ 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] for 1 min, followed by a linear gradient of 20%-80% solvent [B] in 32 min, followed by a 20% solvent [B] hold for 7 min. Flow rate: 0.4mL / min Injection volume: 3μL EXAMPLES

[0078] Synthesis of compound (I-1) [ka] Step 1 Synthesis of compound 3 A solution of compound 1 (12 g, 68.7 mmol) in tetrahydrofuran (70 mL) was added dropwise to a mixed solution of 2.64 mol / L n-butyllithium in hexane (31 mL, 82.4 mmol) and tetrahydrofuran (20 mL) at -78 ° C over 15 minutes. The mixture was stirred at -78 ° C for 1 hour. A solution of 1.9 mol / L 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 solution was cooled to room temperature, water (80 mL) and 2 mol / L hydrochloric acid (40 mL) were added, and the mixture was extracted 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(CDCl3)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 Acetic acid (40 mL) and concentrated hydrochloric acid (41 mL) were added to compound 3 (14.8 g, 48.8 mmol), and the mixture was stirred at 110° C. for 5 hours. The reaction solution was cooled to room temperature, and water (80 ml) was added. The precipitate was collected by filtration and washed with water. Compound 4 (11.6 g, 42.2 mmol) was obtained by air drying. 1 H-NMR(DMSO-d6)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=1.81min, 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. A saturated aqueous solution of sodium bicarbonate (5 mL) was added to the reaction solution, and the solution was extracted with ethyl acetate. The organic layer was washed with water, dried over sodium sulfate, and filtered. The filtrate was concentrated, and the obtained residue was purified by silica gel column chromatography (chloroform:methanol = 100:0 to 90:10), and the solvent was distilled off under reduced pressure. The obtained residue was dried under reduced pressure to obtain compound 5 (1.15 g, 2.97 mmol, yield 82%). 1 H-NMR(DMSO-d6)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=1.98min, LC / MS measurement conditions A

[0081] Step 4 Synthesis of compound 6 2-Bromoacetonitrile (269μL, 4.04mmol) was added to a solution of compound 5 (520mg, 1.345mmol), N,N-diisopropylethylamine (0.705mL, 4.04mmol), and DMF (5.2mL), and the mixture was stirred at room temperature overnight. 2mol / L hydrochloric acid (2mL) was added to the reaction solution 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 obtained residue was purified by silica gel column chromatography (chloroform:methanol=100:0-99:1), and the solvent was distilled off under reduced pressure. The obtained residue was dried under reduced pressure to obtain compound 6 (291mg, 0.684mmol, yield 51%). 1H-NMR(CDCl3)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=2.17min, 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 it 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 obtained precipitate was collected by filtration and washed with diisopropyl ether. The obtained solid was dried under reduced pressure to obtain anhydrous crystals of compound (I-1) (22.0 mg, 0.042 mmol, yield 72%). 1 H-NMR(CDCl3)δ:2.75(4H,t,J=12.0Hz),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=2.27min, 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 pulverized under the following conditions. Equipment: AO Jet Mill (Seishin Enterprise Co., Ltd.) Supply method: Feeder Feeding rate: 20g / hour Grinding pressure: 0.30MPa Supply pressure: 0.40MPa

[0084] (X-ray powder diffraction experiment of anhydrous crystals of the compound represented by formula (I-1)) A powder X-ray diffraction experiment was carried out on the anhydrous crystals of the compound represented by formula (I-1) after pulverization under the measurement conditions described above. The powder X-ray diffraction pattern is shown in FIG. 7, and the peak list of the powder X-ray diffraction pattern is shown in FIG. 8. In the table of the peak list 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 in the powder X-ray diffraction pattern 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°.

[0085] (Single crystal structure analysis of the anhydrous crystal of the compound represented by formula (I-1)) <How to prepare single crystals> 400μL of methanol was added to 1mg of crystals of the compound represented by formula (I-1) and dissolved by heating to 50℃. The solution was dispensed into 1.5mL HPLC vials, the HPLC vials were capped, a syringe needle was inserted into the cap, and the solution 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 performed using the method described above. Note that since Cl1 and Cl7C, and H5CA and H6CA are in a disordered relationship, the analysis was performed with the 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: R1(I>2.00s(I)) was 0.0555, and the final difference Fourier confirmed that there was no missing or misplaced electron density.

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

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

[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 ×10 3 ) are shown in Table 3. [Table 3]

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

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

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

[0093] (Differential scanning calorimetry of 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 out in 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 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 11. An endothermic peak was observed with an onset temperature of about 265.6°C. No weight loss was observed.

[0095] (Raman Spectroscopic Measurement of Anhydrous Crystal 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 spectrum measurement under the above-described measurement condition 1. The results are shown in Figure 12. The main Raman peaks are shown below. [Table 4] The anhydrous crystal of the compound represented by formula (I-1) has a Raman spectrum of 415.2 cm -1 ±2cm -1 , 502.7cm -1 ±2cm -1 , 1431.4cm -1 ±2cm -1 , 1714.8cm -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, and the results are shown in Figure 13 (about 96.5 pa%). EXAMPLES

[0097] Synthesis of compound (I-2) [ka]

[0098] Step 1 Synthesis of compound 8 Compound 7 (2.6 g, 14.2 mmol), DMF (13 mL) and methyl alcohol-O 18 (1.18mL, 29.1mmol) was mixed, and sodium hydride (1.42g, 35.4mmol) was slowly added under ice cooling. The reaction solution was warmed to room temperature and stirred for 2 hours. The reaction solution was cooled in an ice bath, quenched by adding water (26mL) and ethyl acetate (26mL), 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 (1.57g, 8.79mmol, yield 62%). LC / MS(ESI): m / z=179, RT=1.57min, LC / MS measurement conditions A 1H-NMR(CDCl3)δ:3.97(s,3H), 4.00(s,3H), 6.41(s,1H)

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

[0100] Step 3 Synthesis of compound 11 Compound 10 (5 g, 33.9 mmol) and water (11.5 mL) were mixed, and hydrogen bromide (11.5 mL, 102 mmol) was added under ice cooling. Then, NaNO2 (2.384 g, 34.6 mmol) was dissolved in water (7.5 mL) and 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 at an internal temperature of 30 to 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 saline, 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 distilled off under reduced pressure to obtain compound 11 (4.35 g, 15.1 mmol, 73.5 wt%). Since compound 11 has a low boiling point, the solvent was not completely distilled off and the compound was used in the next reaction. 1 H-NMR(CDCl3)δ:7.00-7.07(m,2H)

[0101] Step 4 Synthesis of compound 12 A solution of compound 8 (0.75 g, 4.2 mmol) in tetrahydrofuran (70 mL) was slowly added dropwise to a mixed solution of 2.76 mol / L n-butyllithium in hexane (1.8 mL, 5.04 mmol) and tetrahydrofuran (2.6 mL) at -78°C over 5 minutes. The reaction solution was stirred at -78°C for 1 hour. A solution of 1.9 mol / L zinc chloride in 2-methyltetrahydrofuran (2.6 mL, 82.4 mmol) was added dropwise to the reaction solution. 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 saline, 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 distilled off under reduced pressure to obtain compound 12 (374 mg, 1.2 mmol, yield 29%). 1 H-NMR(CDCl3)δ:3.95(s,3H), 4.05(s,3H), 7.20(d, J=9.0Hz,2H) LC / MS(ESI): m / z=309, RT=2.51min, 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 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 filtered. The solid was washed with chilled acetonitrile to obtain compound 13 (246 mg, 0.88 mmol). 1H-NMR(DMSO-d6)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=1.44min, 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 for the next step without purification.

[0104] Step 7 Synthesis of compound 15 2-Bromoacetonitrile (168μL, 2.52mmol) was added to a solution of compound 14 (330mg, 0.84mmol), DIPEA (440μL, 2.52mmol), and DMF (3.3mL). The resulting solution was stirred at room temperature overnight. Water (14mL) was added to the reaction solution and extracted with ethyl acetate. The organic layer was washed with water and saturated saline, dried over magnesium sulfate, and filtered. The filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography (hexane:ethyl acetate=10:0-2:3), and the solvent was distilled off under reduced pressure to obtain compound 15 (307mg, 0.71mmol, yield 85%). 1 H-NMR(CDCl3)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=2.13min, 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 extracted with ethyl acetate. The organic layer was washed with water, dried over magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (hexane: ethyl acetate = 10: 0 to 3: 7), the solvent was distilled under reduced pressure, and diisopropyl ether was added. The obtained precipitate was collected by filtration and washed with diisopropyl ether. The obtained solid was dried under reduced pressure to obtain compound (I-2) (34.3 mg, 0.065 mmol, yield 57%). 1 H-NMR(DMSO-d6)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=2.19min, LC / MS measurement conditions A

[0106] (Reference example 1) Synthesis of compound (II-1) [ka] Step 1: Synthesis of compound (II-1) Compound 16 (150 mg, 0.376 mmol, see International Publication No. WO 2023 / 195529 for synthesis method), N,N-diisopropylethylamine (0.197 mL, 1.13 mmol), and DMF (1.2 mL) were mixed, to which 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), N,N-diisopropylethylamine (0.197 mL, 1.13 mmol) were added, and the solution was stirred at 80 ° C for 3 hours. 0.1 mol / L hydrochloric acid aqueous solution (10 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, the resulting residue was purified by silica gel column chromatography (hexane:ethyl acetate=9:1 to 3:7), and the solvent was distilled off under reduced pressure to obtain compound (II-1) (57 mg, 0.109 mmol, yield 29%). 1 H-NMR(CDCl3)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=2.37min, LC / MS measurement conditions A

[0107] (Reference example 2) The following compounds can be synthesized in the same manner as in Reference Example 1 and WO 2023 / 195529. [ka] [ka]

[0108] Biological test examples of the compounds according to the present invention are described below. The compounds according to the present invention have a coronavirus 3CL protease inhibitory effect and inhibit coronavirus 3CL 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) suppression test using human TMPRSS2 and ACE2 expressing HEK293T cells (HEK293T / ACE2-TMPRSS2 cells) <Operation Procedure> -Dilution and dispensing of test samples The test sample is diluted in advance with DMSO to an appropriate concentration, 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 × 10 3 cells / well) and SARS-CoV-2 (200-600TCID 50 / well) is mixed with culture medium (MEM, 2% FBS, penicillin-streptomycin), dispensed into wells containing test samples, and then cultured in a CO2 incubator for 3 days. Dispensing CellTiter-Glo® 2.0 and measuring luminescence signals After the plate was cultured for 3 days, it was returned to room temperature, and CellTiter-Glo® 2.0 was dispensed into each well and mixed with a plate mixer. After a certain period of time, the luminescence signal (Lum) was measured with a plate reader. <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 the %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 It is calculated as follows. y = min + (max - min) / {1 + (X50 / x) ^Hill} %Efficacy = {(Sample - virus control) / (cell control - virus control)} * 100% cell control: the average of Lum of cell control wells virus control: the average of Lum of virus control wells min: lower limit 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

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

[0111] Test Example 2-1: Inhibitory activity test against SARS-CoV-2 3CL protease <Material> Commercially available Recombinant SARS-CoV-2 3CL Protease Commercially available substrate peptides Dabcyl-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu(Edans)-NH2 (SEQ ID NO: 1) ·Internal Standard Peptides Dabcyl-Lys-Thr-Ser-Ala-Val-Leu( 13 C6,15 N)-Gln (SEQ ID NO: 2) Dabcyl-Lys-Thr-Ser-Ala-Val-Leu( 13 C6, 15 N)-Gln can be synthesized with reference to the literature (Atherton, E.; Sheppard, 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 C6, 15 N)-Glu(resin)-OαOtBu (Lys side chain is Boc protected, Thr side chain is protected with a tert-butyl group, Ser side chain is protected with a tert-butyl group, 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 typeA <Operation Procedure> Preparation of assay buffer In this test, an assay buffer consisting of 20 mM Tris-HCl, 1 mM EDTA, 10 mM DTT, 0.01% BSA is used. -Dilution and dispensing of test samples The test sample is diluted in advance with DMSO to an appropriate concentration, and a 2- to 5-fold serial dilution series is prepared and then dispensed into a 384-well plate. Addition of enzyme and substrate, enzyme reaction Add 8μM substrate and 6nM or 0.6nM enzyme solution to the prepared compound plate and incubate at room temperature for 3-5 hours. Then, add reaction stop solution (0.067μM internal standard, 0.1% formic acid, 10 or 25% acetonitrile) to stop the enzyme reaction. Measurement of reaction products The plate where the reaction was completed is measured using a RapidFire System 360 and a mass spectrometer (Agilent, 6550 iFunnel Q-TOF) or a Rapid Fire System 365 and a mass spectrometer (Agilent, 6495C Triple Quadrupole). 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 program capable of analysis, and the product area is calculated. The internal standard area is also calculated and the internal standard area is calculated. <Calculation of each measurement item value> ·P / IS calculation The area value obtained in the previous section is calculated using the following formula to calculate the 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 the % Inhibition, the inhibition curve is approximated by the following logistic regression equation, and the value of x when y = 50(%) is substituted is 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 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 curve at midpoint between min and max

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

[0113] Test Example 2-2: Inhibitory activity test against SARS-CoV-2 3CL protease <Material> Commercially available Recombinant SARS-CoV-2 3CL Protease ·SARS-CoV-2 3CL Protease P132H Commercially available substrate peptides Dabcyl-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu(Edans)-NH2 (SEQ ID NO: 1) ·Internal Standard Peptides Dabcyl-Lys-Thr-Ser-Ala-Val-Leu( 13 C6, 15 N)-Gln (SEQ ID NO: 2) Dabcyl-Lys-Thr-Ser-Ala-Val-Leu( 13 C6, 15N)-Gln can be synthesized with reference to the literature (Atherton, E.; Sheppard, 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 C6, 15 N)-Glu(resin)-OαOtBu (Lys side chain is Boc protected, Thr side chain is protected with a tert-butyl group, Ser side chain is protected with a tert-butyl group, 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 typeA <Operation Procedure> Preparation of assay buffer In this test, an assay buffer consisting of 20 mM Tris-HCl, 1 mM EDTA, 10 mM DTT, 0.01% BSA is used. -Dilution and dispensing of test samples The test sample is diluted in advance with DMSO to an appropriate concentration, and a 3-fold serial dilution series is prepared and then dispensed into a 384-well plate. Addition of enzyme and substrate, enzyme reaction Add the substrate (final concentration: 4 μM) and enzyme (0.3 nM) to the prepared compound plate and incubate at room temperature for 4 hours. Then, add a reaction stop solution (7.2 nM internal standard, 0.1% formic acid, 10% acetonitrile) to stop the enzyme reaction. 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 Quadrupole). 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 RapidFire Integrator to obtain the product area value. The internal standard area value is also calculated using the internal standard detected at the same time. <Calculation of each measurement item value> ·P / IS calculation The area value obtained in the previous section is calculated using the following formula to calculate the 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 the % Inhibition, the inhibition curve is approximated by the following logistic regression equation, and the value of x when y = 50(%) is substituted is 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 according to the invention were tested essentially as described above and the results are shown below. [Table 5]

[0115] Test Example 3: Cytopathic effect (CPE) suppression test using human TMPRSS2-expressing Vero E6 cells (Vero E6 / TMPRSS2 cells) <Operation Procedure> -Dilution and dispensing of test samples The test sample is diluted in advance with DMSO to an appropriate concentration, 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 VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4cells / well) and SARS-CoV-2 hCoV-19 / Japan / TY / WK-521 / 2020(Ancestral), hCoV-19 / Japan / QHN001 / 2020(Alpha), hCoV-19 / Japan / TY8-612 / 2021(Beta), hCoV-19 / Japan / TY7-501 / 2021(Gamma), hCoV-19 / Japan / TY11-927 / 2021(Delta), hCoV-19 / Japan / TY38-871 / 2021(Omicron BA.1.1), hCoV-19 / Japan / TY40-385 / 2022(Omicron BA.2), hCoV-19 / Japan / TY41-721 / 2022(Omicron BA.2.12.1)、hCoV-19 / Japan / TY41-716 / 2022(Omicron BA.2.75)、hCoV-19 / Japan / TY41-703 / 2022(Omicron BA.4.1)、hCoV-19 / Japan / TY41-763 / 2022(Omicron BA.4.6)、hCoV-19 / Japan / TY41-702 / 2022(Omicron BE.1 / BA.5-like)、hCoV-19 / Japan / TY41-704 / 2022(Omicron BA.5.2.1)、hCoV-19 / Japan / TY41-820 / 2022(Omicron BF.7)、hCoV-19 / Japan / TY41-828 / 2022(Omicron BF.7.4.1)、hCoV-19 / Japan / TY41-796 / 2022(Omicron BQ.1.1)、hCoV-19 / Japan / TY41-832 / 2022(Omicron CH.1.1.11)、hCoV-19 / Japan / TY41-795 / 2022(Omicron XBB.1)、hCoV-19 / Japan / 23-018 / 2022(Omicron XBB.1.5)、hCoV-19 / Japan / TY-41951 / 2023(Omicron XBB.1.9.1)、hCoV-19 / Japan / TY41-984 / 2023(Omicron XBB.1.1)16), hCoV-19 / Japan / TY41-831 / 2022(Omicron XBF), hCoV-19 / Japan / TY41-686 / 2022(Omicron XE)(30-3000TCID. 50 / well) is mixed with culture medium (MEM, 2% FBS, penicillin-streptomycin), dispensed into wells containing the test samples, and then cultured in a CO2 incubator for 3 or 4 days. Dispensing CellTiter-Glo® 2.0 and measuring luminescence signals After the plate was cultured for 3 days, it was returned to room temperature, and CellTiter-Glo® 2.0 was dispensed into each well and mixed with a plate mixer. After a certain period of time, the luminescence signal (Lum) was measured with a plate reader.

[0116] <Calculation of each measurement item value> ·50% SARS-CoV-2 infected cell death inhibitory concentration (EC 50 )calculation When x is the logarithm of the compound concentration and y is the %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 It is calculated as follows. y = min + (max - min) / {1 + (X50 / x) ^Hill} %Efficacy = {(Sample - virus control) / (cell control - virus control)} * 100% cell control: the average of Lum of cell control wells virus control: the average of Lum of virus control wells min: lower limit 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

[0117] Compounds according to the invention were tested essentially as described above and the results are shown below. [Table 6]

[0118] Test Case 5: Antiviral effect against HCoV-OC43 <Operation Procedure> -Dilution and dispensing of test samples The test sample is diluted in advance to an appropriate concentration with DMSO, and a 3-fold serial dilution series is prepared. The dilution is then dispensed into a 96-well plate and diluted with maintenance medium (MEM, 2% FBS, penicillin-streptomycin). Dilution and dispensing of cells and HCoV-OC43 MRC-5 cells (2 × 10 4 cells / well) were seeded onto a 96-well plate the day before infection, and the next day, HCoV-OC43 (300 TCID 50 The cells are infected with 10 ... Dispensing CellTiter-Glo® 2.0 and measuring luminescence signals After 72 hours of incubation, the plate is returned to room temperature, 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 the %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 50It is calculated as follows. y = min + (max - min) / {1 + (X50 / x) ^Hill} %Efficacy = {(Sample - virus control) / (cell control - virus control)} * 100% cell control: the average of Lum of cell control wells virus control: the average of Lum of virus control wells min: lower limit 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 Calculation of 50% cytotoxicity concentration When x is the logarithm of the compound concentration and y is the %Cytotoxicity, the inhibition curve is approximated by the following logistic regression equation, and the value of x when y = 50(%) is substituted is the CC 50 It is calculated as follows. y = min + (max - min) / {1 + (X50 / x) ^Hill} %Cytotoxicity = {(Sample - medium control) / (cell control - medium control)} * 100% cell control: the average of Lum of cell control wells medium control: the average of Lum of medium control wells min: lower limit 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

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

[0121] Test Example 6: Antiviral effect against HCoV-229E <Operation Procedure> -Dilution and dispensing of test samples The test sample is diluted in advance to an appropriate concentration with DMSO, and a 3-fold serial dilution series is prepared. The dilution is then dispensed into a 96-well plate and diluted with maintenance medium (MEM, 2% FBS, penicillin-streptomycin). Dilution and dispensing of cells and HCoV-229E MRC-5 cells (2 × 10 4 HCoV-229E (1000TCID 50 1 / well) for 1 hour. After that, the virus solution is removed, and a maintenance medium containing the test reagent is added, and the cells are cultured in a CO2 incubator for 72 hours. Dispensing CellTiter-Glo® 2.0 and measuring luminescence signals After 72 hours of incubation, the plate is returned to room temperature, 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.

[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 the %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 It is calculated as follows. y = min + (max - min) / {1 + (X50 / x) ^Hill} %Efficacy = {(Sample - virus control) / (cell control - virus control)} * 100% cell control: the average of Lum of cell control wells virus control: the average of Lum of virus control wells min: lower limit 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

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

[0124] Test Example 7: Effects of human serum, mouse serum, and hamster serum on anti-SARS-CoV-2 activity <Operation Procedure> -Dilution and dispensing of test samples The test sample is diluted in advance with DMSO to an appropriate concentration, and a 3-fold serial dilution series is prepared and then dispensed into a 96-well plate. Addition of serum-supplemented medium Human serum, mouse serum, or hamster serum is prepared in medium (MEM, 2% FBS, penicillin-streptomycin) to a final concentration of 0, 12.5, 25, or 50%, and dispensed into wells containing test samples and incubated at room temperature for 1 hour. Dilution and dispensing of cells and SARS-CoV-2 VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4cells / well) were seeded on a 96-well plate the day before infection, and the next day, SARS-CoV-2 hCoV-19 / Japan / TY7-501 / 2021 (1,000 TCID 50 The cells are infected with 10 ... Quantification of viral load After 1 day of incubation, the supernatant is removed, and a cell lysis solution made by mixing Trizol LS and maintenance medium in a 3:1 ratio 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. [Table 7]

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

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

[0127] Test Example 8: Viral proliferation inhibition test using human airway epithelial cells <Operation Procedure> -Dilution and dispensing of test samples The 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 diluted 200-fold with MucilAir™ culture medium and dispensed into a 24-well plate. ·SARS-CoV-2 infection MucilAir seeded in Transwell TM (Nasal cavity, approx. 5.0×10 5 cells / well) to SARS-CoV-2 hCoV-19 / Japan / TY41-702 / 2022(Omicron BE.1 / BA.5-like)(5000TCID 50 / well) and cultured in a CO2 incubator for 2 hours. TM After washing with culture medium, the transwell was placed on the well containing the test sample and cultured in a CO2 incubator. Two days after infection, the transwell was inoculated with MucilAir. TM Culture medium was added and the supernatant was collected. -Measurement of viral titer in supernatant The collected supernatant was diluted 10-fold in medium (MEM, 2% FBS, penicillin-streptomycin) and then incubated with VeroE6 / TMPRSS2 cells (JCRB1819, 1.5 × 10 4 The virus was mixed with 1000 μg / ml of 1000 cells / well and seeded onto a 96-well plate. After culturing for 4 days in a CO2 incubator, cytopathic effect (CPE) was observed and the virus titer in the supernatant was calculated.

[0128] <Calculation of each measurement item value> 90% SARS-CoV-2 virus production inhibitory concentration (EC 90 )calculation x is the logarithm of the compound concentration, and y is the virus titer (Log 10 TCID 50 The concentration-response curve was 1 log10 The value equivalent to the reduction is calculated using the two-point method from the virus titers of the two concentrations before and after it.

number

[0129] Compounds according to the invention were tested essentially as described above and the results are shown below. [Table 8]

[0130] Test Example 9: Test to inhibit viral titer proliferation in lung homogenates 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 by mixing 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% PPVVA-containing PEG400 = 1:9). The administration volume was 5 mL / kg. ·virus The SARS-CoV-2 hCoV-19 / Japan / TY7-501 / 2021 strain isolated at the National Institute of Infectious Diseases was used. Mouse lung infection, dosing, and lung harvest 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 anesthesia solution containing 0.03 mg / mL medetomidine hydrochloride, 0.4 mg / mL midazolam, and 0.5 mg / mL butorphanol tartrate in saline. 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 a day at doses of 0.1, 0.3, 1, 3, and 10 mg / kg to mice (n=5 / group). DMA / 0.5 w / v% PPVVA in PEG400 was orally administered twice a day to control mice. Compound administration was for 2 days from the start of administration. Three days after infection, mouse lungs were collected, 2 mL of PBS was added, homogenized, and the supernatant after centrifugation was collected. -Measurement of viral titer in lung homogenate The lung homogenate was diluted 10-fold in medium (MEM, 2% FBS, penicillin-streptomycin) and then transferred to VeroE6 / TMPRSS2 cells (JCRB1819, 1.5 × 10 4 The cells were mixed with 100% IgG4-heptane IgG4 (100% IgG4) and seeded onto a 96-well plate. After 4 days of culture in a CO2 incubator, cytopathic effect (CPE) was observed and the virus titer in the lung homogenate was calculated.

[0131] Compounds according to the invention were tested essentially as described above and the results are shown below. Three days after infection, the viral titer in the lung homogenate was 6.45-log in the DMA / 0.5w / v% PPVVA in PEG400 group. 10 TCID 50 / mL, and the compound I-1 0.1, 0.3, 1, 3, and 10 mg / kg groups 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-treated group was lower than that in the DMA / 0.5 w / v% PPVVA in PEG400-treated group, suggesting that the compound I-1-treated group has the effect of reducing the virus titer in the lung homogenate even if there is a long period between infection and administration (Figure 1).

[0132] Test Example 10: Test to inhibit viral titer proliferation in lung and nasal turbinate homogenates and lung weight increase in 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 by mixing 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% PPVVA-containing PEG400 = 1:9). The administration volume was 2.5 mL / kg. ·virus The SARS-CoV-2 hCoV-19 / Japan / TY41-702 / 2022 strain (Omicron BE.1 / BA.5-like) isolated at the National Institute of Infectious Diseases was used. Hamster pulmonary infection, medication, lungs, 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 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, and 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 a day at doses of 0.1, 1, and 10 mg / kg to the infected hamsters, starting from 1 day after the virus inoculation. DMA / 0.5 w / v% PPVVA in PEG400 was orally administered twice a day 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, 5 mL or 1 mL of PBS was added, homogenized, and the supernatant after centrifugation was collected. In addition, the lungs of the infected and non-infected hamsters were collected 7 days after infection, and the lung weights were measured. Measurement of virus titers 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 × 10 4After culturing for 4 days in a CO2 incubator, cytopathic effect (CPE) was observed and the virus titer in the lung or nasal turbinate homogenate was calculated.

[0133] Compounds according to the invention were tested essentially as described above and the results are shown below. The virus titer in the lung homogenate was 5.4-log in the DMA / 0.5w / v% PPVVA in PEG400-treated group 3 days after infection. 10 TCID 50 The values ​​are 5.5, 3.5, and 2.5-log / mL for the groups administered 0.1, 1, and 10 mg / kg of compound I-1, respectively. 10 TCID 50 / mL after 4 days of infection. The DMA / 0.5w / v%PVPVA in PEG400 group showed a 5.6-log 10 TCID 50 The values ​​were 4.6, 2.4, and 1.9-log / mL in the groups administered 0.1, 1, and 10 mg / kg of compound I-1, respectively. 10 TCID 50 / mL was shown. The virus titer in the nasal turbinate homogenate was 5.0-log in the DMA / 0.5w / v%PVPVA in PEG400-treated group 3 days after infection. 10 TCID 50 The values ​​were 4.8, 3.2, and 2.7-log / mL in the groups administered 0.1, 1, and 10 mg / kg of compound I-1, respectively. 10 TCID 50 / mL. Four days after infection, the DMA / 0.5 w / v% PPVVA in PEG400 group showed a 3.8-log 10 TCID 50 The values ​​were 4.0, 2.2, and 1.8-log / mL in the groups administered 0.1, 1, and 10 mg / kg of compound I-1, respectively. 10 TCID 50 / mL was shown. In the compound I-1 administration group, the virus titers in the lung and nasal turbinate homogenates were dose-dependently lower than those in the DMA / 0.5w / v% PPVVA in PEG400 administration group, suggesting that the compound I-1 has the effect of reducing the virus titers in the lung and nasal turbinate homogenates even if there is a long period between infection and administration (Figures 2-1 and 2-2). In addition, the lung weight / body weight 7 days after infection was 5.26 mg / g in uninfected hamsters, 8.96 mg / g in the infected hamsters treated with DMA / 0.5 w / v% PPVVA in PEG400, and 8.24, 5.75, and 5.57 mg / g in the groups treated with 0.1, 1, and 10 mg / kg of compound I-1, 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% PPVVA in PEG400-treated group, suggesting that administration of compound I-1 has an inhibitory effect on the increase in lung weight caused by SARS-CoV-2 infection (Figure 3).

[0134] Test Example 11: Inhibition 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 by mixing 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% PPVVA-containing PEG400 = 1:9). The administration volume was 2.5 mL / kg. ·virus The SARS-CoV-2 hCoV-19 / Japan / TY41-702 / 2022 strain (Omicron BE.1 / BA.5-like) isolated at the National Institute of Infectious Diseases was used. Hamster lung infection, medication, 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 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, and 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% PPVVA in PEG400 was orally administered twice daily to the control infected and non-infected hamsters. Compound administration was continued for 5 days from the start of administration. Body weight was monitored once daily.

[0135] Compounds according to the invention were tested essentially as described above and the results are shown below. Non-infected hamsters showed weight gain, but infected hamsters showed weight loss from 3 days after infection, reaching the lowest value 6 days after infection. In the groups administered 1 and 10 mg / kg of compound I-1, all hamsters survived until 10 days after infection, and weight loss was suppressed (Figure 4). These results suggest that administration of compound I-1 has an effect of suppressing weight loss.

[0136] Test Example 12: Study on the inhibition of weight loss and viral proliferation in lung and nasal turbinate homogenates in SARS-CoV-2-infected hamsters by prophylactic administration of compound I-1 Materials and Methods ·Compound Compound I-1 was dissolved in 0.5% methylcellulose (0.5% MC) to prepare a test sample, and the administration volume was 10 mL / kg. ·virus The SARS-CoV-2 hCoV-19 / Japan / TY41-702 / 2022 strain (Omicron BE.1 / BA.5-like) isolated at the National Institute of Infectious Diseases was used. Hamster lung infection, medication, 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 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, and 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 1 or 3 days before virus infection. In the virus proliferation inhibition test in lung and nasal turbinate homogenate, compound I-1 was administered subcutaneously once at 10 or 30 mg / kg 1 day before virus infection. 0.5% MC was administered subcutaneously once 1 day before virus infection to the control hamsters. Body weight was monitored once a day. 1 and 2 days after infection, the lungs and nasal turbinates of the infected hamsters were collected, 5 mL or 1 mL of PBS was added, homogenized, and the supernatant after centrifugation was collected. Measurement of virus titers 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 × 10 4 After culturing for 4 days in a CO2 incubator, cytopathic effect (CPE) was observed and the virus titer in the lung or nasal turbinate homogenate was calculated.

[0137] Compounds according to the invention were tested essentially as described above and the results are shown below. In the infected hamsters, weight loss began 3 days after infection, reaching its lowest value 6 days after infection. At this time, all hamsters in the groups administered 3 or 10 mg / kg of compound I-1 1 day before infection and the group administered 10 mg / kg 3 days before infection 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 at 1 day after infection. 10 TCID 50 / mL, and the compound I-1 10 and 30 mg / kg groups showed 3.34 and 1.84-log 10 TCID 50 / mL at 2 days after infection, the 0.5% MC group showed a 6.18-log 10 TCID 50 / mL, and the compound I-1 10 and 30 mg / kg groups showed 5.29 and 3.55-log 10 TCID 50 / mL (Figure 6-1). The virus titer in the nasal turbinate homogenate was 5.71-log in the 0.5% MC group at 1 day after infection. 10 TCID 50 / mL, and the compound I-1 10 and 30 mg / kg groups were 4.80 and 3.36-log 10 TCID 50 / mL at 2 days after infection, the 0.5% MC group showed a 5.80-log 10 TCID 50 / mL, and the compound I-1 10 and 30 mg / kg groups showed 5.38 and 5.01-log 10 TCID 50 / mL (Figure 6-2). In addition, when the plasma concentrations were measured immediately before infection, they were 6.1, 12.5, 5.9, and 3.0 ng / mL, respectively, in the 3 and 10 mg / kg groups administered 1 day before infection and in the 3 and 10 mg / kg groups administered 3 days before infection. These results indicate that prophylactic administration of compound I-1 inhibited weight loss when the plasma concentration during viral infection was approximately 6 ng / mL or higher, and inhibited viral proliferation in the lungs and nasal turbinate homogenates when the plasma concentration was approximately 12 ng / mL or higher.

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

[0139] Test Example 13: Viral titer clearance test in nasal wash 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 by mixing 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% PPVVA-containing PEG400 = 1:9). The administration volume was 2.5 mL / kg. ·virus The SARS-CoV-2 hCoV-19 / Japan / TY41-702 / 2022 strain (Omicron BE.1 / BA.5-like) isolated at the National Institute of Infectious Diseases was used. - Nasal infection in hamsters, administration of medication, collection of nasal washings 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 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, and 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 a day at doses of 1 or 10 mg / kg to the infected hamsters, starting 2 or 3 days after virus inoculation. DMA / 0.5 w / v% PPVVA in PEG400 was orally administered twice a day to the control infected hamsters. Compound administration was for 5 days from the start of administration. Two to six days after infection, the infected hamsters were anesthetized with isoflurane and nasal washings were collected with 400 μL of DPBS, and the supernatants were collected after centrifugation. Each group was performed with n=4. -Measurement of viral titer in nasal wash The supernatant of the nasal wash was 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 × 10 4 After culturing for 4 days in a CO2 incubator, cytopathic effect (CPE) was observed and the virus titer in the nasal wash was calculated.

[0140] Compounds of the invention were tested essentially as described above and the results are shown below. In the groups administered the compounds 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% PPVVA in PEG400 group and the 1 and 10 mg / kg compound I-1 groups, respectively. 10 TCID 50 / mL, and 3 days after infection (2 days after start of treatment), the IL-10 levels were 3.75, 3.63, and 3.68-log 10 TCID 50 / mL, and 5 days after infection (4 days after the start of treatment), the IL-10 levels were 2.96, <1.80, and <1.80-log 10 TCID 50 In the groups administered the compound 3 days after virus inoculation, the DMA / 0.5 w / v% PPVVA in PEG400 group and the compound I-1 1 and 10 mg / kg groups had 3.40, 2.93, and 3.13-log, respectively, at the start of administration (3 days after infection, 1 day after administration). 10TCID 50 / mL, and 4 days after infection (1 day after the start of treatment), the IL-10 levels were 2.97, 1.84, and 2.13-log 10 TCID 50 / mL, and 6 days after infection (4 days after the start of treatment), the mean serum IL-1 levels were 2.43, <1.80, and <1.80-log 10 TCID 50 / mL (Figure 15). In the groups administered 1 or 10 mg / kg of compound I-1, the virus titer in the nasal wash was lower than that in the group administered DMA / 0.5 w / v% PPVVA in PEG400, regardless of whether administration was initiated 2 or 3 days after infection, demonstrating that the compound I-1 has the effect of reducing the virus in the body even if there is a long period between infection and administration.

[0141] Test Example 14: Test to inhibit virus transmission from SARS-CoV-2 infected animals to non-infected animals by delayed administration of compound I-1 Materials and Methods ·Compound Compound I-1 according to the present invention was used as a test sample by mixing 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% PPVVA-containing PEG400 = 1:9). The administration volume was 2.5 mL / kg. ·virus The SARS-CoV-2 hCoV-19 / Japan / TY11-927 / 2021 isolated at the National Institute of Infectious Diseases was used. - Nasal infection of hamsters, administration of medication, cohabitation, collection of nasal washes and lung samples 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 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, and 100 μL of hCoV-19 / Japan / TY11-927 / 2021 (1.00 × 10 3 TCID50 ) was inoculated intranasally. Compound I-1 was orally administered twice a day at doses of 0.1, 1, and 10 mg / kg to the infected hamster (Index), starting 8 hours after virus inoculation. DMA / 0.5 w / v% PPVVA in PEG400 was orally administered twice a day to the control infected hamster (Index). Compound administration was performed a total of three times from the start of administration. One infected hamster (Index) and one non-infected hamster (Contact) that was not inoculated with the virus were housed together in the same cage for 12 hours at night from one day after infection to two days after infection, in separate stainless steel cages at a distance of 2 cm so that they could not come into direct contact. After the end of cohabitation, they were kept individually. Nasal washes and lungs were collected from the non-infected hamster (Contact) four days after the start of cohabitation (three days after cohabitation was released). Nasal washes were collected in 400 μL of DPBS under isoflurane anesthesia, and the supernatant after centrifugation was collected. The lungs were homogenized in 5 mL of DPBS and centrifuged to collect the supernatant. Measurement of viral titers in nasal washes and lung homogenate supernatants The nasal wash or lung homogenate supernatant was 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 × 10 4 After culturing for 4 days in a CO2 incubator, cytopathic effect (CPE) was observed, and the virus titer in the nasal wash or lung homogenate supernatant was calculated.

[0142] Compounds of the invention were tested essentially as described above and the results are shown below. The virus titer in the nasal wash or lung homogenate supernatant of the uninfected hamster (Contact) four days after cohabitation with the infected hamster (Index) (three days after removal from cohabitation) was at the detection limit (1.8-log 10 TCID 50The number of individuals with a viral load of 1000 or more (1000 / mL) was 6 / 6 in the DMA / 0.5w / v% PPVVA in PEG400 group, 6 / 6 in the compound I-1 0.1mg / kg group, 0 / 6 in the compound I-1 1mg / kg group, and 0 / 6 in the compound I-1 10mg / kg group, showing a dose-dependent effect of suppressing transmission (Figure 16). These results indicate that administration of compound I-1 to infected hamsters (Index) after infection has the effect of suppressing viral transmission to non-infected hamsters (Contact).

[0143] Test Example 15: Prevention of virus transmission from SARS-CoV-2-infected animals by prophylactic administration of compound I-1 to non-infected animals Materials and Methods ·Compound Compound I-1 was dissolved in 0.5% methylcellulose (0.5% MC) to prepare a test sample, and the administration volume was 5 mL / kg. ·virus The SARS-CoV-2 hCoV-19 / Japan / TY11-927 / 2021(Delta) isolated at the National Institute of Infectious Diseases was used. - Nasal infection of hamsters, administration of medication, cohabitation, collection of nasal washes and lung samples 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 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, and 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 non-infected hamsters (Contact) at doses of 3, 10, 30, and 90 mg / kg 12 hours before cohabitation. 0.5% MC was administered subcutaneously once to control non-infected hamsters (Contact). From 1 day after infection of the infected hamsters (Index) to 12 hours at night 2 days after infection, one infected hamster (Index) and one non-infected hamster (Contact) administered with the compound were housed together in the same cage, in separate stainless steel cages at a distance of 2 cm so that they could not come into direct contact. After the end of cohabitation, they were kept individually. Nasal washes and lungs were collected from non-infected hamsters (Contact) 4 days after the start of cohabitation (3 days after cohabitation was terminated). Nasal washes were collected in 400 μL of DPBS under isoflurane anesthesia, and the supernatant after centrifugation was collected. The lungs were homogenized with 3 mL of DPBS and centrifuged, and the supernatant was collected and diluted 2-fold with DPBS. Measurement of viral titers in nasal washes and lung homogenate supernatants The nasal wash or lung homogenate supernatant was diluted 10-fold in medium (MEM, 2% FBS, penicillin-streptomycin) and then cultured in a 96-well plate with VeroE6 / TMPRSS2 cells (JCRB1819, 1.50 × 10 4 After culturing for 4 days in a CO2 incubator, cytopathic effect (CPE) was observed, and the virus titer in the nasal wash or lung homogenate supernatant was calculated.

[0144] Compounds of the invention were tested essentially as described above and the results are shown below. The virus titer in the nasal wash or lung homogenate supernatant of the uninfected hamster (Contact) administered the compound 4 days after cohabitation with the infected hamster (Index) (3 days after cohabitation was discontinued) was below the detection limit (1.8-log 10 TCID 50The number of individuals with a virulence factor of 100 / mL or more was 6 / 6 in the 0.5% MC group, 5 / 6 in the compound I-1 3mg / kg group, 6 / 6 in the compound I-1 10mg / kg group, 3 / 6 in the compound I-1 30mg / kg group, and 0 / 6 in the compound I-1 90mg / kg group, showing a dose-dependent infection prevention 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 to prevent lethality and weight loss in aged hamsters infected with SARS-CoV-2 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 by mixing 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% PPVVA-containing PEG400 = 1:9). The administration volume was 1.25 mL / kg. ·virus The SARS-CoV-2 hCoV-19 / Japan / TY11-927 / 2021 isolated at the National Institute of Infectious Diseases was used. - Nasal infection of hamsters, administration of medication, cohabitation, collection of nasal washes and lung samples 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 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, and 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 a day at doses of 0.1, 1, and 10 mg / kg. DMA / 0.5 w / v% PPVVA in PEG400 was orally administered twice a day to the control infected hamsters. Compound administration was for 5 days from the start of administration. Body weight was monitored once a day, and for the evaluation of survival rate, death was considered when the body weight was less than 80% of the body weight immediately before infection. Each group was performed with n=6.

[0146] Compounds of the invention were tested essentially as described above and the results are shown below. In the infected hamsters administered DMA / 0.5w / v%PVPVA in PEG400, two died 8 days after infection, with a survival rate of 66.7%. In the groups administered 1 and 10mg / kg of compound I-1, all survived until 10 days after infection, and weight loss was suppressed (Figures 18 and 19). These results suggest that administration of compound I-1 has an inhibitory effect on lethality and weight loss even in aged hamsters.

[0147] It is known that aging in rodents, like humans, increases the expression of the ACE2 receptor, which is known as a receptor for SARS-CoV-2 (References: Scientific Reports (2020)10:22401 and Molecular Therapy Methods & Clinical Development, Vol. 18, P1-6, 2020). In addition, the normal immune response that protects the body from infection sources and eliminates them is weakened, making it more likely that viral infections will cause severe symptoms. As shown above, in a SARS-CoV-2 infection model using aged hamsters, administration of compound I-1 was found to have an inhibitory effect on weight loss and lethality. Non-clinical trials using aged hamsters are positioned as one of the non-clinical evaluation systems that mimic the process in which high-risk patients with underlying diseases become severe, and therefore support the usefulness of compound I-1 as a treatment option for patients at high risk of severe symptoms. In addition, the above test results suggest that the aggravation of viral infection was suppressed in the compound I-1-treated group, supporting not only the antiviral effect of compound I-1 against SARS-CoV-2, but also its usefulness as a medicine for suppressing the aggravation of SARS-CoV-2 infection.

[0148] Test Example 17: In vitro combination effect confirmation test <Operation Procedure> Test sample The test samples used in combination with compound I-1 were ensitrevir fumarate, nilmatrevir, remdesivir, EIDD-1931, sotrovimab, and tixagevimab / silgavimab. -Dilution and dispensing of test samples Each test sample was diluted to an appropriate concentration with DMSO or DPBS and medium (MEM, 2% FBS, penicillin-streptomycin), and a serial dilution series was prepared in a 96-well plate. Dilution and dispensing of cells and SARS-CoV-2 A549 / ACE2-TMPRSS2 cells (Invivogen, a549-hace2tpsa, 1.5×10 4 cells / well) and SARS-CoV-2 hCoV-19 / Japan / TY11-927 / 2021(100TCID 50 / well) was mixed with culture medium (MEM, 2% FBS, penicillin-streptomycin), dispensed into wells containing the test samples, and then cultured in a CO2 incubator for 2 days. Dispensing CellTiter-Glo® 2.0 and measuring luminescence signals After the plates were cultured for 3 days and returned to room temperature, 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 signal (Lum) was measured using a plate reader.

[0149] <Analysis of combined effects> Synergy volume and antagonism volume were calculated using MacSynergy II. These values ​​can be calculated by referring to previously published papers (Antiviral Research, 1990, Volume 14, p. 181-206). -Evaluation of combination effects The combined effect was evaluated based on the synergy volume and antagonism volume of MacSynergy II at 99% reliability according to the following criteria. 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] [Table 9]

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

[0153] The formulation examples shown below are merely illustrative and are not intended to limit the scope of the invention in any way. The compounds according to the present invention can be administered as pharmaceutical compositions by any conventional route, in particular 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 in the form of intranasal or suppositories. Pharmaceutical compositions containing the compounds of the present invention in free form or in the form of a pharma-ceutically acceptable salt together with at least one pharma-ceutically acceptable carrier or diluent can be prepared by conventional mixing, granulation or coating methods. For example, oral compositions can be tablets, granules or capsules containing excipients, disintegrants, binders, lubricants, etc. and active ingredients, etc. In addition, injectable compositions can be solutions or suspensions, which may be sterilized and may contain preservatives, stabilizers, buffers, etc. [Industrial Applicability]

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

Claims

1. Formula (I-1): 【Chemistry 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 for inhibiting the proliferation of SARS-CoV-2 virus.

4. The pharmaceutical composition according to claim 1 or 2, which is a therapeutic and / or preventive agent for novel coronavirus disease (COVID-19).

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

6. The pharmaceutical composition according to claim 1 or 2, 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 claim 1 or 2, 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 claim 1 or 2, which is used to suppress viral transmission of SARS-CoV-2.

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