Pharmaceutical composition containing triazine derivative

Abstract

The present invention relates to a pharmaceutical composition containing a triazine derivative. The present invention provides the use of a pharmaceutical composition containing a compound represented by the following formula or a pharmaceutically acceptable salt thereof in the manufacture of a drug for inhibiting the virus transmission of SARS-CoV-2. # imgabs0 #

Description

[0001] This application is a divisional application of an invention patent application with an application date of September 27, 2022, application number 202280007447.4 (PCT / JP2022 / 035803), and invention name “Pharmaceutical composition containing triazine derivatives”. Technical Field

[0002] The present invention relates to a pharmaceutical composition containing a compound that exhibits coronavirus 3CL protease inhibitory activity. Background Art

[0003] The coronavirus belonging to the subfamily Orthocoronavirus of the family Coronaviridae of the order Nidovirus has a genome size of about 30 kb and is the largest single-stranded + stranded RNA virus among the known RNA viruses. Coronaviruses can be classified into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. As coronaviruses that infect humans, there are 2 species of alphacoronavirus (HCoV-229E, HCoV-NL63) and 5 species of betacoronavirus (HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, SARS-CoV-2), a total of 7 species. Among them, 4 species (HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC43) are pathogens of colds, and the remaining 3 species are severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), and new coronavirus (SARS-CoV-2) that cause severe pneumonia.

[0004] The novel coronavirus infectious disease (COVID-19) was declared an epidemic by the WHO on March 11, 2020. As of September 21, 2022, the number of confirmed infections is more than 610 million, and the number of deaths is more than 6.5 million (Non-patent Document 1). As the main infection routes of SARS-CoV-2, droplet infection, contact infection and aerosol infection have been reported, and it has been confirmed that SARS-CoV-2 continues to float in the air with aerosols for about 3 hours and maintains infectivity (Non-patent Document 2). 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 sputum (33.4%) are typical (Non-patent Document 3). In severe cases, respiratory failure caused by acute respiratory distress syndrome, acute lung injury, interstitial pneumonia, etc. may occur. In addition, multiple organ failure such as renal failure and liver failure has also been reported.

[0005] In Japan, based on the repositioning of existing drugs, remdesivir as an antiviral drug, dexamethasone as an anti-inflammatory drug, and baricitinib as a rheumatism drug have been approved as treatment drugs for COVID-19, and tocilizumab as an anti-IL-6 receptor antibody was additionally approved in January 2022. In addition, in July 2021, Ronapreve (casirivimab / imdevimab) as an antibody cocktail therapy was specially approved, in September 2021, sotrovimab was specially approved, and in December 2021, molnupiravir was specially approved. There is not enough evidence on the effectiveness and safety of these drugs. Therefore, it is urgent to create treatment drugs for COVID-19, especially oral drugs.

[0006] If coronavirus infects cells, it synthesizes various proteins required for self-replication. Among them, there are 2 types of polyproteins, including the replication complex that makes the viral genome, and 2 types of proteases. Proteases cut the polyproteins synthesized by the virus and play an indispensable role in making various proteins function. Of the 2 types of proteases, 3CL protease (main protease) is responsible for most of the cutting of polyproteins (Non-patent Document 4).

[0007] As a COVID-19 therapeutic drug targeting 3CL protease, the completion of the Phase 1b clinical trial of Lufotrelvir (PF-07304814), a prodrug of PF-00835231, conducted by Pfizer in June 2021 was posted on ClinicalTrials.gov (NCT04535167). In addition, in March 2021, Pfizer announced the start of a Phase 1 clinical trial of PF-07321332, a therapeutic drug for novel coronavirus infectious diseases. The structural formulas of PF-00835231, Lufotrelvir, and PF-07321332 are shown below, and the chemical structures are different from the compounds involved in the present invention (Non-patent Documents 5, 12, and 13 and Patent Documents 5 and 6).

[0008] PF-00835231:

[0009] [Chemical formula 1]

[0010]

[0011] Lufotrelvir (PF-07304814):

[0012] [Chemical formula 2]

[0013]

[0014] PF-07321332:

[0015] [Chemical formula 3]

[0016]

[0017] In addition, ClinicalTrials.gov lists a Phase 2 / 3 clinical trial (NCT04960202) that began in July 2021 for the combined use of PF-07321332 and ritonavir in patients with high-risk COVID-19. In addition, in November 2021, on the homepage of Pfizer, it was reported that PAXLOVID(TM) (PF-07321332; ritonavir) reduced the risk of hospitalization or death by 89% in high-risk adult patients compared with placebo (Non-Patent Literature 14). In addition, in December 2021, PAXLOVID(TM) received emergency use authorization in the United States, and on February 10, 2022, the Paxlovid(registered trademark) combination package received special approval in Japan.

[0018] Although compounds having 3CL protease inhibitory activity are disclosed in Non-Patent Documents 5 to 8, none of the documents describes or suggests a pharmaceutical composition comprising the compound of the present invention.

[0019] Although Patent Documents 1 to 4 disclose a method having P2X3 and / or P2X 2 / 3 However, no document describes or suggests a pharmaceutical composition comprising a compound having 3CL protease inhibitory activity and antiviral effect.

[0020] Although non-patent documents 9 to 11 disclose triazine derivatives having antitumor effects, none of the documents describes coronavirus 3CL protease inhibitory activity and antiviral effects, and none of the documents describes or suggests a pharmaceutical composition containing the compound of the present invention.

[0021] Prior art literature

[0022] Patent Literature

[0023] Patent Document 1: International Publication No. 2012 / 020749

[0024] Patent Document 2: International Publication No. 2013 / 089212

[0025] Patent Document 3: International Publication No. 2010 / 092966

[0026] Patent Document 4: International Publication No. 2014 / 200078

[0027] Patent Document 5: International Publication No. 2021 / 205298

[0028] Patent Document 6: International Publication No. 2021 / 250648

[0029] Non-patent literature

[0030] Non-patent document 1: “COVID-19 Dashboard by the Center for Systems Science and Engineering at Johns Hopkins University”, [online], Johns Hopkins University, [retrieved on September 21, 2022], website<UR L:https: / / coronavirus.jhu.edu / map.html>

[0031] Non-patent document 2: The New England Journal of Medicine (2020), Vol. 382, ​​pp. 1564-1567

[0032] Non-patent document 3: “Report of the WHO-China Joint Mission on Coronavirus Disease 2019 (COVID-19)”, [online], February 28, 2020, WHO, [retrieved on September 21, 2022], URL <URL:https: / / www.wh o.int / docs / default-source / coronaviruse / who-china-joint-mission-on-covi d-19-final-report.pdf>

[0033] Non-patent document 4: Science (2003), Vol. 300, pp. 1763-1767

[0034] Non-patent literature 5: “A comparative analysis of SARS-CoV-2 anti-virals characterizes 3CLpro inhibitor PF-00835231 as a potential new treatment for COVID-19”, Journal of Virology, [online], February 23, 2021, [retrieved September 21, 2022], website <URL:https: / / jvi.as m.org / content / early / 2021 / 02 / 19 / JVI.01819-20><doi:10.1128 / JVI.01819-20>

[0035] Non-patent document 6: Cell Research (2020), Vol. 30, pp. 678-692

[0036] Non-patent document 7: Science (2020), Vol. 368, pp. 409-412

[0037] Non-patent document 8: ACS Central Science (2021), Vol. 7, No. 3, pp. 467-475

[0038] Non-patent document 9: Cancer Treatment Reviews (1984), Vol. 11, Supplement 1, pp. 99-110

[0039] Non-patent document 10: Contributions to Oncology (1984), Vol. 18, pp. 221-234

[0040] Non-patent document 11: Arzneimittel-Forschung (1984), Vol. 11, No. 6, pp. 663-668

[0041] Non-patent literature 12: 261st Am Chem Soc (ACS) Natl Meet·2021-04-05 / 2021-04-16·Virtual, N / A·Abst 243

[0042] Non-patent document 13: Science (2021), Vol. 374, pp. 1586-1593

[0043] Non-patent literature 14: “Pfizer's Novel COVID-19 Oral Antiviral Treatment Candidate Reduced Risk Of Hospitalization Or Death By 89% In Interim Analysis Of Phase 2 / 3 EPIC-HR Study”, [online], November 5, 2021, Pfizer Press Release, [retrieved on September 21, 2022], website <URL:https: / / www.pfizer.com / news / press-release / press-release-de tail / pfizers-novel-covid-19-oral-antiviral-treatment-candidate> Summary of the invention

[0044] Problems to be solved by the invention

[0045] 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 medicine containing a compound having an antiviral effect, especially a coronavirus proliferation inhibitory effect.

[0046] Means for solving problems

[0047] The present invention relates to the following contents. (1)

[0049] A pharmaceutical composition comprising a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0050] [Chemical formula 4]

[0051]

[0052] (where Y is N;

[0053] R 1 is a substituted or unsubstituted aromatic heterocyclic group;

[0054] R 2 is a substituted or unsubstituted 6-membered aromatic carbocyclic group;

[0055] R 3 is a substituted or unsubstituted aromatic heterocyclic group;

[0056] -X- is -NH-;

[0057] m is 0 or 1;

[0058] R 5a is a hydrogen atom;

[0059] R 5b is a hydrogen atom;

[0060] n is 1;

[0061] R 4a is a hydrogen atom;

[0062] R 4b for hydrogen atoms)

[0063] (2) The pharmaceutical composition according to item (1) above, wherein R 1 It is a substituted or unsubstituted 5- to 6-membered aromatic heterocyclic group.

[0064] (3) The pharmaceutical composition according to item (1) or (2) above, wherein R 2 is a 6-membered aromatic carbocyclic group substituted by 1, 2 or 3 substituents selected from the substituent group G;

[0065] The substituent group G is a group consisting of halogen, cyano and alkyl.

[0066] (4) The pharmaceutical composition according to any one of items (1) to (3) above, wherein R 3 It is a substituted or unsubstituted 9- to 10-membered aromatic heterocyclic group.

[0067] (5) A pharmaceutical composition comprising the compound according to item (1) above or a pharmaceutically acceptable salt thereof, wherein the compound represented by formula (I) is selected from the group consisting of compound I-003, compound I-005, compound I-017 and compound I-023.

[0068] (6) The pharmaceutical composition according to any one of items (1) to (5) above, which is a 3CL protease inhibitor.

[0069] (7) The pharmaceutical composition according to any one of items (1) to (6) above, which is used for inhibiting viral proliferation of SARS-CoV-2.

[0070] (8) The pharmaceutical composition according to any one of items (1) to (7) above, which is a therapeutic and / or preventive agent for the novel coronavirus infectious disease (COVID-19).

[0071] (9) The pharmaceutical composition according to any one of items (1) to (8) above, which is used for suppressing the severity of an infectious disease caused by SARS-CoV-2.

[0072] (10) The pharmaceutical composition according to any one of items (1) to (9) above, which is used in the following manner: administration is started after the symptoms of an infectious disease caused by SARS-CoV-2 appear, or within 72 hours from the time of determination of SARS-CoV-2 positivity.

[0073] (11) The pharmaceutical composition according to any one of items (1) to (9) above, which is used in the following manner: administration is started after the symptoms of an infectious disease caused by SARS-CoV-2 appear, or within 24 hours from the time of determination of SARS-CoV-2 positivity.

[0074] (12) The pharmaceutical composition according to any one of items (1) to (9) above, which is used in the following manner: administration is started after the symptoms of an infectious disease caused by SARS-CoV-2 appear, or within 120 hours from the time of determination of SARS-CoV-2 positivity.

[0075] (13) The pharmaceutical composition according to any one of items (1) to (12) above, wherein the symptoms of the infectious disease caused by SARS-CoV-2 include at least one of the 12 symptoms of COVID-19, and the symptom is of moderate or above severity.

[0076] (14) The pharmaceutical composition according to any one of items (1) to (9) above, which is used to inhibit the viral transmission of SARS-CoV-2.

[0077] (15) A method for inhibiting viral proliferation of SARS-CoV-2, comprising the step of administering a compound represented by formula (I) or a pharmaceutically acceptable salt thereof to an individual in need of treatment and / or prevention of the novel coronavirus infectious disease (COVID-19).

[0078] [Chemical formula 5]

[0079]

[0080] (where Y is N;

[0081] R 1 is a substituted or unsubstituted aromatic heterocyclic group;

[0082] R 2 is a substituted or unsubstituted 6-membered aromatic carbocyclic group;

[0083] R 3 is a substituted or unsubstituted aromatic heterocyclic group;

[0084] -X- is -NH-;

[0085] m is 0 or 1;

[0086] R 5a is a hydrogen atom;

[0087] R 5b is a hydrogen atom;

[0088] n is 1;

[0089] R 4a is a hydrogen atom;

[0090] R 4b for hydrogen atoms)

[0091] (16) The method for inhibiting viral proliferation of SARS-CoV-2 as described in item (15) above, wherein R 1 It is a substituted or unsubstituted 5- to 6-membered aromatic heterocyclic group.

[0092] (17) The method for inhibiting viral proliferation of SARS-CoV-2 according to item (15) or (16) above, wherein R 2 is a 6-membered aromatic carbocyclic group substituted by 1, 2 or 3 substituents selected from the substituent group G;

[0093] The substituent group G is a group consisting of halogen, cyano and unsubstituted alkyl.

[0094] (18) The method for inhibiting viral proliferation of SARS-CoV-2 according to any one of items (15) to (17) above, wherein R 3 It is a substituted or unsubstituted 9- to 10-membered aromatic heterocyclic group.

[0095] (19) The method for inhibiting viral proliferation of SARS-CoV-2 as described in item (15) above, wherein the compound represented by formula (I) is selected from the group consisting of compound I-003, compound I-005, compound I-017 and compound I-023.

[0096] (20) A method for treating and / or preventing the novel coronavirus infectious disease (COVID-19), comprising administering a compound represented by formula (I) or a pharmaceutically acceptable salt thereof to an individual in need of treatment and / or prevention of the novel coronavirus infectious disease (COVID-19).

[0097] [Chemical formula 6]

[0098]

[0099] (where Y is N;

[0100] R 1 is a substituted or unsubstituted aromatic heterocyclic group;

[0101] R 2 is a substituted or unsubstituted 6-membered aromatic carbocyclic group;

[0102] R 3 is a substituted or unsubstituted aromatic heterocyclic group;

[0103] -X- is -NH-;

[0104] m is 0 or 1;

[0105] R 5a is a hydrogen atom;

[0106] R 5b is a hydrogen atom;

[0107] n is 1;

[0108] R 4a is a hydrogen atom;

[0109] R 4b for hydrogen atoms)

[0110] (21) The method for treating and / or preventing the novel coronavirus infectious disease (COVID-19) as described in item (20) above, wherein R 1 It is a substituted or unsubstituted 5- to 6-membered aromatic heterocyclic group.

[0111] (22) The method for treating and / or preventing the novel coronavirus infectious disease (COVID-19) as described in item (20) or (21) above, wherein R 2 is a 6-membered aromatic carbocyclic group substituted by 1, 2 or 3 substituents selected from the substituent group G;

[0112] The substituent group G is a group consisting of halogen, cyano and unsubstituted alkyl.

[0113] (23) The method for treating and / or preventing the novel coronavirus infectious disease (COVID-19) according to any one of items (20) to (22) above, wherein R 3 It is a substituted or unsubstituted 9- to 10-membered aromatic heterocyclic group.

[0114] (24) A method for treating and / or preventing the novel coronavirus infectious disease (COVID-19) as described in item (20) above, wherein the compound represented by formula (I) is selected from the group consisting of compound I-003, compound I-005, compound I-017 and compound I-023.

[0115] (25) A method for inhibiting the severity of an infectious disease caused by SARS-CoV-2, the method comprising the step of administering a compound represented by formula (I) or a pharmaceutically acceptable salt thereof to an individual in need of treatment and / or prevention of the novel coronavirus infectious disease (COVID-19).

[0116] [Chemical formula 7]

[0117]

[0118] (where Y is N;

[0119] R 1 is a substituted or unsubstituted aromatic heterocyclic group;

[0120] R 2 is a substituted or unsubstituted 6-membered aromatic carbocyclic group;

[0121] R 3 is a substituted or unsubstituted aromatic heterocyclic group;

[0122] -X- is -NH-;

[0123] m is 0 or 1;

[0124] R 5a is a hydrogen atom;

[0125] R 5b is a hydrogen atom;

[0126] n is 1;

[0127] R 4a is a hydrogen atom;

[0128] R 4b for hydrogen atoms)

[0129] (26) The method for suppressing the severity of an infectious disease caused by SARS-CoV-2 as described in item (25) above, wherein R 1 It is a substituted or unsubstituted 5- to 6-membered aromatic heterocyclic group.

[0130] (27) The method for inhibiting the severity of an infectious disease caused by SARS-CoV-2 as described in item (25) or (26) above, wherein R 2 is a 6-membered aromatic carbocyclic group substituted by 1, 2 or 3 substituents selected from the substituent group G;

[0131] The substituent group G is a group consisting of halogen, cyano and unsubstituted alkyl.

[0132] (28) The method for inhibiting the severity of an infectious disease caused by SARS-CoV-2 according to any one of items (25) to (27) above, wherein R 3 It is a substituted or unsubstituted 9- to 10-membered aromatic heterocyclic group.

[0133] (29) The method for inhibiting the severity of an infectious disease caused by SARS-CoV-2 as described in item (25) above, wherein the compound represented by formula (I) is selected from the group consisting of compound I-003, compound I-005, compound I-017 and compound I-023.

[0134] (30) A method for treating the novel coronavirus infectious disease (COVID-19), which is a method for treating and / or preventing the novel coronavirus infectious disease (COVID-19), comprising the following steps: administering a compound represented by formula (I) or a pharmaceutically acceptable salt thereof to an individual who needs treatment and / or prevention of the novel coronavirus infectious disease (COVID-19) after the onset of symptoms of the infectious disease caused by SARS-CoV-2 or within 72 hours from the time of determining that the patient is positive for SARS-CoV-2.

[0135] [Chemical formula 8]

[0136]

[0137] (where Y is N;

[0138] R 1 is a substituted or unsubstituted aromatic heterocyclic group;

[0139] R 2 is a substituted or unsubstituted 6-membered aromatic carbocyclic group;

[0140] R 3 is a substituted or unsubstituted aromatic heterocyclic group;

[0141] -X- is -NH-;

[0142] m is 0 or 1;

[0143] R 5a is a hydrogen atom;

[0144] R 5b is a hydrogen atom;

[0145] n is 1;

[0146] R 4a is a hydrogen atom;

[0147] R 4b for hydrogen atoms)

[0148] (31) The method for treating the novel coronavirus infectious disease (COVID-19) as described in item (30) above, wherein R 1 It is a substituted or unsubstituted 5- to 6-membered aromatic heterocyclic group.

[0149] (32) The method for treating the novel coronavirus infectious disease (COVID-19) according to item (30) or (31), wherein R 2 is a 6-membered aromatic carbocyclic group substituted by 1, 2 or 3 substituents selected from the substituent group G;

[0150] The substituent group G is a group consisting of halogen, cyano and unsubstituted alkyl.

[0151] (33) The method for treating the novel coronavirus infectious disease (COVID-19) according to any one of items (30) to (32) above, wherein R 3 It is a substituted or unsubstituted 9- to 10-membered aromatic heterocyclic group.

[0152] (34) A method for treating the novel coronavirus infectious disease (COVID-19) as described in item (30) above, wherein the compound represented by formula (I) is selected from the group consisting of compound I-003, compound I-005, compound I-017 and compound I-023.

[0153] (35) A method for treating the novel coronavirus infectious disease (COVID-19), which is a method for treating and / or preventing the novel coronavirus infectious disease (COVID-19), comprising the following steps: administering a compound represented by formula (I) or a pharmaceutically acceptable salt thereof to an individual who needs treatment and / or prevention of the novel coronavirus infectious disease (COVID-19) after the onset of symptoms of the infectious disease caused by SARS-CoV-2 or within 24 hours from the time of determining that the patient is positive for SARS-CoV-2.

[0154] [Chemical formula 9]

[0155]

[0156] (where Y is N;

[0157] R 1 is a substituted or unsubstituted aromatic heterocyclic group;

[0158] R 2 is a substituted or unsubstituted 6-membered aromatic carbocyclic group;

[0159] R 3 is a substituted or unsubstituted aromatic heterocyclic group;

[0160] -X- is -NH-;

[0161] m is 0 or 1;

[0162] R 5a is a hydrogen atom;

[0163] R 5b is a hydrogen atom;

[0164] n is 1;

[0165] R 4a is a hydrogen atom;

[0166] R 4b for hydrogen atoms)

[0167] (36) The method for treating the novel coronavirus infectious disease (COVID-19) as described in item (35) above, wherein R 1 It is a substituted or unsubstituted 5- to 6-membered aromatic heterocyclic group.

[0168] (37) The method for treating the novel coronavirus infectious disease (COVID-19) according to item (35) or (36) above, wherein R 2 is a 6-membered aromatic carbocyclic group substituted by 1, 2 or 3 substituents selected from the substituent group G;

[0169] The substituent group G is a group consisting of halogen, cyano and unsubstituted alkyl.

[0170] (38) The method for treating the novel coronavirus infectious disease (COVID-19) according to any one of items (35) to (37) above, wherein R 3 It is a substituted or unsubstituted 9- to 10-membered aromatic heterocyclic group.

[0171] (39) A method for treating the novel coronavirus infectious disease (COVID-19) as described in item (35) above, wherein the compound represented by formula (I) is selected from the group consisting of compound I-003, compound I-005, compound I-017 and compound I-023.

[0172] (40) A method for inhibiting the viral transmission of SARS-CoV-2, comprising the step of administering a compound represented by formula (I) or a pharmaceutically acceptable salt thereof to an individual in need of treatment and / or prevention of the novel coronavirus infectious disease (COVID-19).

[0173] [Chemical formula 10]

[0174]

[0175] (where Y is N;

[0176] R 1 is a substituted or unsubstituted aromatic heterocyclic group;

[0177] R 2 is a substituted or unsubstituted 6-membered aromatic carbocyclic group;

[0178] R 3 is a substituted or unsubstituted aromatic heterocyclic group;

[0179] -X- is -NH-;

[0180] m is 0 or 1;

[0181] R 5a is a hydrogen atom;

[0182] R 5b is a hydrogen atom;

[0183] n is 1;

[0184] R 4a is a hydrogen atom;

[0185] R 4b for hydrogen atoms)

[0186] (41) The method for inhibiting the spread of SARS-CoV-2 as described in item (40) above, wherein R 1 It is a substituted or unsubstituted 5- to 6-membered aromatic heterocyclic group.

[0187] (42) The method for inhibiting the spread of SARS-CoV-2 according to item (40) or (41), wherein R 2 is a 6-membered aromatic carbocyclic group substituted by 1, 2 or 3 substituents selected from the substituent group G;

[0188] The substituent group G is a group consisting of halogen, cyano and unsubstituted alkyl.

[0189] (43) The method for inhibiting the spread of SARS-CoV-2 according to any one of items (40) to (42) above, wherein R 3 It is a substituted or unsubstituted 9- to 10-membered aromatic heterocyclic group.

[0190] (44) The method for inhibiting viral transmission of SARS-CoV-2 as described in the above item (40), wherein the compound represented by formula (I) is selected from the group consisting of compound I-003, compound I-005, compound I-017 and compound I-023.

[0191] (45) Use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof for inhibiting the viral proliferation of SARS-CoV-2.

[0192] [Chemical formula 11]

[0193]

[0194] (where Y is N;

[0195] R 1 is a substituted or unsubstituted aromatic heterocyclic group;

[0196] R 2 is a substituted or unsubstituted 6-membered aromatic carbocyclic group;

[0197] R 3 is a substituted or unsubstituted aromatic heterocyclic group;

[0198] -X- is -NH-;

[0199] m is 0 or 1;

[0200] R 5a is a hydrogen atom;

[0201] R 5b is a hydrogen atom;

[0202] n is 1;

[0203] R 4a is a hydrogen atom;

[0204] R 4b for hydrogen atoms)

[0205] (46) The use according to item (45) above, wherein R 1 It is a substituted or unsubstituted 5- to 6-membered aromatic heterocyclic group.

[0206] (47) The use according to item (45) or (46) above, wherein R 2 is a 6-membered aromatic carbocyclic group substituted by 1, 2 or 3 substituents selected from the substituent group G;

[0207] The substituent group G is a group consisting of halogen, cyano and unsubstituted alkyl.

[0208] (48) The use according to any one of items (45) to (47) above, wherein R 3 It is a substituted or unsubstituted 9- to 10-membered aromatic heterocyclic group.

[0209] (49) The use according to item (45) above, wherein the compound represented by formula (I) is selected from the group consisting of compound I-003, compound I-005, compound I-017 and compound I-023.

[0210] (50) Use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a drug for the treatment and / or prevention of the novel coronavirus infectious disease (COVID-19).

[0211] [Chemical formula 12]

[0212]

[0213] (where Y is N;

[0214] R 1 is a substituted or unsubstituted aromatic heterocyclic group;

[0215] R 2 is a substituted or unsubstituted 6-membered aromatic carbocyclic group;

[0216] R 3 is a substituted or unsubstituted aromatic heterocyclic group;

[0217] -X- is -NH-;

[0218] m is 0 or 1;

[0219] R 5a is a hydrogen atom;

[0220] R 5b is a hydrogen atom;

[0221] n is 1;

[0222] R 4a is a hydrogen atom;

[0223] R 4b for hydrogen atoms)

[0224] (51) The use according to item (50) above, wherein R 1 It is a substituted or unsubstituted 5- to 6-membered aromatic heterocyclic group.

[0225] (52) The use according to item (50) or (51) above, wherein R 2 is a 6-membered aromatic carbocyclic group substituted by 1, 2 or 3 substituents selected from the substituent group G;

[0226] The substituent group G is a group consisting of halogen, cyano and unsubstituted alkyl.

[0227] (53) The use according to any one of items (50) to (52) above, wherein R 3 It is a substituted or unsubstituted 9- to 10-membered aromatic heterocyclic group.

[0228] (54) The use according to item (50) above, wherein the compound represented by formula (I) is selected from the group consisting of compound I-003, compound I-005, compound I-017 and compound I-023.

[0229] (55) Use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof for inhibiting the aggravation of an infectious disease caused by SARS-CoV-2.

[0230] [Chemical formula 13]

[0231]

[0232] (where Y is N;

[0233] R 1 is a substituted or unsubstituted aromatic heterocyclic group;

[0234] R 2 is a substituted or unsubstituted 6-membered aromatic carbocyclic group;

[0235] R 3 is a substituted or unsubstituted aromatic heterocyclic group;

[0236] -X- is -NH-;

[0237] m is 0 or 1;

[0238] R 5a is a hydrogen atom;

[0239] R 5b is a hydrogen atom;

[0240] n is 1;

[0241] R 4a is a hydrogen atom;

[0242] R 4b for hydrogen atoms)

[0243] (56) The use according to item (55) above, wherein R 1 It is a substituted or unsubstituted 5- to 6-membered aromatic heterocyclic group.

[0244] (57) The use according to item (55) or (56) above, wherein R 2 is a 6-membered aromatic carbocyclic group substituted by 1, 2 or 3 substituents selected from the substituent group G;

[0245] The substituent group G is a group consisting of halogen, cyano and unsubstituted alkyl.

[0246] (58) The use according to any one of items (55) to (57) above, wherein R 3 It is a substituted or unsubstituted 9- to 10-membered aromatic heterocyclic group.

[0247] (59) The use according to item (55) above, wherein the compound represented by formula (I) is selected from the group consisting of compound I-003, compound I-005, compound I-017 and compound I-023.

[0248] (60) The compound represented by formula (I) or a pharmaceutically acceptable salt thereof is used for administration after the onset of symptoms of an infectious disease caused by SARS-CoV-2 or within 72 hours from the determination of SARS-CoV-2 positivity.

[0249] [Chemical formula 14]

[0250]

[0251] (where Y is N;

[0252] R 1 is a substituted or unsubstituted aromatic heterocyclic group;

[0253] R 2 is a substituted or unsubstituted 6-membered aromatic carbocyclic group;

[0254] R 3 is a substituted or unsubstituted aromatic heterocyclic group;

[0255] -X- is -NH-;

[0256] m is 0 or 1;

[0257] R 5a is a hydrogen atom;

[0258] R 5b is a hydrogen atom;

[0259] n is 1;

[0260] R 4a is a hydrogen atom;

[0261] R 4b for hydrogen atoms)

[0262] (61) The use according to item (60) above, wherein R 1 It is a substituted or unsubstituted 5- to 6-membered aromatic heterocyclic group.

[0263] (62) The use according to item (60) or (61) above, wherein R 2 is a 6-membered aromatic carbocyclic group substituted by 1, 2 or 3 substituents selected from the substituent group G;

[0264] The substituent group G is a group consisting of halogen, cyano and unsubstituted alkyl.

[0265] (63) The use according to any one of items (60) to (62) above, wherein R 3 It is a substituted or unsubstituted 9- to 10-membered aromatic heterocyclic group.

[0266] (64) The use according to item (60) above, wherein the compound represented by formula (I) is selected from the group consisting of compound I-003, compound I-005, compound I-017 and compound I-023.

[0267] (65) The compound represented by formula (I) or a pharmaceutically acceptable salt thereof is used for administration after the onset of symptoms of an infectious disease caused by SARS-CoV-2 or within 24 hours from the determination of SARS-CoV-2 positivity.

[0268] [Chemical formula 15]

[0269]

[0270] (where Y is N;

[0271] R 1 is a substituted or unsubstituted aromatic heterocyclic group;

[0272] R 2 is a substituted or unsubstituted 6-membered aromatic carbocyclic group;

[0273] R 3 is a substituted or unsubstituted aromatic heterocyclic group;

[0274] -X- is -NH-;

[0275] m is 0 or 1;

[0276] R 5a is a hydrogen atom;

[0277] R 5b is a hydrogen atom;

[0278] n is 1;

[0279] R 4a is a hydrogen atom;

[0280] R 4b for hydrogen atoms)

[0281] (66) The use according to item (65), wherein R 1 It is a substituted or unsubstituted 5- to 6-membered aromatic heterocyclic group.

[0282] (67) The use according to item (65) or (66), wherein R 2 is a 6-membered aromatic carbocyclic group substituted by 1, 2 or 3 substituents selected from the substituent group G;

[0283] The substituent group G is a group consisting of halogen, cyano and unsubstituted alkyl.

[0284] (68) The use according to any one of items (65) to (67) above, wherein R 3 It is a substituted or unsubstituted 9- to 10-membered aromatic heterocyclic group.

[0285] (69) The use according to item (65) above, wherein the compound represented by formula (I) is selected from the group consisting of compound I-003, compound I-005, compound I-017 and compound I-023.

[0286] (70) Use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof for inhibiting the viral transmission of SARS-CoV-2.

[0287] [Chemical formula 16]

[0288]

[0289] (where Y is N;

[0290] R 1 is a substituted or unsubstituted aromatic heterocyclic group;

[0291] R 2 is a substituted or unsubstituted 6-membered aromatic carbocyclic group;

[0292] R 3 is a substituted or unsubstituted aromatic heterocyclic group;

[0293] -X- is -NH-;

[0294] m is 0 or 1;

[0295] R 5a is a hydrogen atom;

[0296] R 5b is a hydrogen atom;

[0297] n is 1;

[0298] R4a is a hydrogen atom;

[0299] R 4b for hydrogen atoms)

[0300] (71) The use according to item (70) above, wherein R 1 It is a substituted or unsubstituted 5- to 6-membered aromatic heterocyclic group.

[0301] (72) The use according to item (70) or (71) above, wherein R 2 is a 6-membered aromatic carbocyclic group substituted by 1, 2 or 3 substituents selected from the substituent group G;

[0302] The substituent group G is a group consisting of halogen, cyano and unsubstituted alkyl.

[0303] (73) The use according to any one of items (70) to (72) above, wherein R 3 It is a substituted or unsubstituted 9- to 10-membered aromatic heterocyclic group.

[0304] (74) The use according to item (70) above, wherein the compound represented by formula (I) is selected from the group consisting of compound I-003, compound I-005, compound I-017 and compound I-023.

[0305] Effects of the Invention

[0306] The compounds of the present invention have inhibitory activity against coronavirus 3CL protease, and pharmaceutical compositions containing the compounds of the present invention are useful as therapeutic and / or preventive agents for coronavirus infectious diseases.

[0307] Furthermore, among the compounds according to the present invention, a pharmaceutical composition containing compound (I-003) or compound (I-005) is useful as a pharmaceutical substance.

[0308] In addition, a pharmaceutical composition containing a p-toluenesulfonate salt crystal of compound (I-003) or a fumaric acid co-crystal of compound (I-005) is very useful as a therapeutic agent for the novel coronavirus infectious disease (COVID-19). BRIEF DESCRIPTION OF THE DRAWINGS

[0309] [ Figure 1 ] shows the powder X-ray diffraction pattern of the p-toluenesulfonate type I crystal (Form I) of the compound represented by formula (IA). The horizontal axis represents 2θ (°) and the vertical axis represents intensity (Count).

[0310] [ Figure 2] shows the structure in the asymmetric unit of the p-toluenesulfonate type I crystal of the compound represented by formula (IA).

[0311] [ Figure 3 ] shows the powder X-ray diffraction pattern of the fumaric acid cocrystal Form I (Form I) of the compound represented by formula (IB). The horizontal axis represents 2θ (°) and the vertical axis represents intensity (Count).

[0312] [ Figure 4 ] shows the structure in the asymmetric unit of the fumaric acid cocrystal Form I (Form I) of the compound represented by formula (IB).

[0313] [ Figure 5A ] shows the results for hCoV-19 / Japan / TY7-501 / 2021 infected mice (at 1.00×10 4 TCID 50 / mice were inoculated intranasally), the virus titer in the lungs 1 day after infection when the vehicle was administered twice a day from immediately after infection or the fumaric acid cocrystal type I crystal of the compound shown in (IB) was administered once. The vertical axis represents the virus titer in the lungs, and the horizontal axis represents each administration group.

[0314] [ Figure 5B ] shows the results for hCoV-19 / Japan / TY7-501 / 2021 infected mice (at 1.00×10 4 TCID 50 / mouse was inoculated intranasally), and the virus titer in the lungs 1 day after infection was measured when the medium and the fumaric acid cocrystal type I crystal of the compound shown in (IB) were administered twice a day from immediately after infection. The vertical axis represents the virus titer in the lungs, and the horizontal axis represents each administration group.

[0315] [ Fig. 6A ] shows the results for hCoV-19 / Japan / TY7-501 / 2021 infected mice (at 1.00×10 4 TCID 50 / mouse was inoculated intranasally), and the medium and the fumaric acid cocrystal type I crystal of the compound shown in (IB) were administered twice a day from 1 day after infection for 2 days, and the viral titer in the lungs 1-3 days after infection. The vertical axis represents the viral titer in the lungs, and the horizontal axis represents the number of days from infection.

[0316] [ Figure 6B ] shows the results for hCoV-19 / Japan / TY7-501 / 2021 infected mice (at 1.00×10 4 TCID 50 / mouse was inoculated intranasally), and the medium and the fumaric acid cocrystal type I crystal of the compound shown in (IB) were administered twice a day for 2 days starting from 3 days after infection, and the viral titer in the lungs 1-5 days after infection. The vertical axis represents the viral titer in the lungs, and the horizontal axis represents the number of days from infection.

[0317] [ Figure 7 ] shows the results for hCoV-19 / Japan / TY7-501 / 2021 infected mice (at 1.00×10 4 TCID 50 / mouse was inoculated intranasally), the virus titer in the lungs 1-3 days after infection was measured when the medium and the fumaric acid cocrystal type I crystal of the compound shown in (IB) were administered three times a day from 1 day after infection for 2 days. The vertical axis represents the virus titer in the lungs, and the horizontal axis represents the number of days from infection.

[0318] [ Fig. 8A ] shows the results for hCoV-19 / Japan / TY7-501 / 2021 infected mice (at 1.00×10 5 TCID 50 / mouse was inoculated intranasally, and retired mice aged 35-45 weeks were administered with a medium and a fumaric acid cocrystal type I crystal of the compound shown in (IB) twice a day from 1 day after infection for 5 days, and the body weight change until 7 days after infection. The vertical axis represents the body weight change (%) when the body weight on the day of infection is set as 100%, and the horizontal axis represents the number of days from infection.

[0319] [ Figure 8B ] shows the results for hCoV-19 / Japan / TY7-501 / 2021 infected mice (at 1.00×10 5 TCID 50 / mouse was inoculated intranasally, and the survival rate was calculated from the nasal inoculation of 35-45 week-old retired mice) and the medium and the fumaric acid co-crystal type I crystal of the compound shown in (IB) were administered twice a day from 1 day after infection for 5 days until 7 days after infection. The vertical axis represents the survival rate (%), and the horizontal axis represents the number of days from infection.

[0320] [ Figure 8C ] shows the hCoV19 / Japan / TY / WK-521 / 2020 strain mouse domesticated strain MA-P10 infected mice (at 1.00×10 3 or 1.00 × 10 4 TCID 50 / mouse was inoculated intranasally, 15-week-old mice), and the medium and the fumaric acid cocrystal type I crystal of the compound shown in (IB) were administered twice a day from 1 day after infection for 5 days, and the body weight change until 7 days after infection. The vertical axis represents the body weight change (%) when the body weight on the day of infection is set as 100%, and the horizontal axis represents the number of days from infection.

[0321] [ Fig.8D ] shows the hCoV19 / Japan / TY / WK-521 / 2020 strain mouse domesticated strain MA-P10 infected mice (at 1.00×10 3 or 1.00 × 10 4 TCID 50 / mouse was inoculated intranasally, 15-week-old mice), the medium and the fumaric acid cocrystal type I crystal of the compound shown in (IB) were administered twice a day from 1 day after infection for 5 days, and the survival rate until 7 days after infection. The vertical axis represents the survival rate (%), and the horizontal axis represents the number of days from infection.

[0322] [ Fig. 8E ] shows the hCoV19 / Japan / TY / WK-521 / 2020 strain mouse domesticated strain MA-P10 infected mice (at 1.00×10 3 TCID 50 / mouse was inoculated intranasally, and retired mice aged 35-45 weeks were administered with a medium and a fumaric acid cocrystal type I crystal of the compound shown in (IB) twice a day from 1 day after infection for 5 days, and the body weight change until 7 days after infection. The vertical axis represents the body weight change (%) when the body weight on the day of infection is set as 100%, and the horizontal axis represents the number of days from infection.

[0323] [ Figure 8F ] shows the hCoV19 / Japan / TY / WK-521 / 2020 strain mouse domesticated strain MA-P10 infected mice (at 1.00×10 3 TCID 50 / mouse was inoculated intranasally, and the survival rate was calculated from the nasal inoculation of 35-45 week-old retired mice) and the medium and the fumaric acid co-crystal type I crystal of the compound shown in (IB) were administered twice a day from 1 day after infection for 5 days until 7 days after infection. The vertical axis represents the survival rate (%), and the horizontal axis represents the number of days from infection.

[0324] [ Fig. 9 ] shows that hCoV19 / Japan / TY11-927 / 2021 infected hamsters (at 5.00×10 3PFU / hamster was inoculated intranasally) when the hamsters were housed with the hamsters that were not inoculated with the virus. The hamsters were administered with the vehicle and the fumaric acid cocrystal type I crystal of the compound shown in (IB) twice a day from the time of infection. The vertical axis represents the viral titer in the lungs, and the horizontal axis represents each administration group.

[0325] [ Fig.10 ] shows that the compound was administered to hCoV19 / Japan / TY11-927 / 2021 infected hamsters (at 5.00×10 3 PFU / hamster for intranasal inoculation), and the viral titer in the lungs of infected (Infected) and non-infected (Contact) hamsters 6 days after infection when they were housed with non-infected hamsters that were not inoculated with the virus. The hamsters were administered with the vehicle and the fumaric acid cocrystal type I crystal of the compound shown in (IB) twice a day since just after infection. The vertical axis represents the viral titer in the lungs, and the horizontal axis represents each administration group.

[0326] [ Fig.11A ] shows that the compound was administered to hCoV19 / Japan / TY11-927 / 2021 infected hamsters (at 1.00×10 2 TCID 50 / hamsters were inoculated intranasally), and the lung virus titers of infected (Infected) and non-infected (Contact) hamsters 5 days after infection when they were housed with non-infected hamsters that were not inoculated with the virus from 2 days after infection. The hamsters were administered with the vehicle and the fumaric acid cocrystal type I crystal of the compound shown in (IB) twice a day from 1 day after infection. The vertical axis represents the lung virus titer, and the horizontal axis represents each administration group.

[0327] [ Fig. 11B ] shows that the compound was administered to hCoV19 / Japan / TY11-927 / 2021 infected hamsters (at 1.00×10 2 TCID 50 / hamsters were inoculated intranasally), and the virus titers in the lungs of infected (Infected) and non-infected (Contact) hamsters 5 days after infection when they were housed with non-infected hamsters that were not inoculated with the virus from 2 days after infection. The hamsters were administered with the vehicle and the fumaric acid cocrystal type I crystal of the compound shown in (IB) twice a day from 1 day after infection. The vertical axis represents the virus titer in the nasal concha, and the horizontal axis represents each administration group.

[0328] [ Fig. 12A ] shows the hCoV19 / Japan / TY / WK-521 / 2020 strain mouse domesticated strain MA-P10 infected mice (at 3.00×10 2 TCID50 / mouse was inoculated intranasally, and mice aged 37-57 weeks were subcutaneously administered with the medium and the compound shown in (IB) 24 hours before infection, and the body weight change until 14 days after infection. The vertical axis represents the body weight change (%) when the body weight on the day of infection is set as 100%, and the horizontal axis represents the number of days from infection.

[0329] [ Fig. 12B ] shows the hCoV19 / Japan / TY / WK-521 / 2020 strain mouse domesticated strain MA-P10 infected mice (at 3.00×10 2 TCID 50 / mouse was inoculated intranasally, and mice aged 37-57 weeks were subcutaneously administered with the medium and the compound shown in (IB) 24 hours before infection. The vertical axis represents the survival rate (%), and the horizontal axis represents the number of days from infection. DETAILED DESCRIPTION

[0330] The meaning of each term used in this specification is described below. Unless otherwise specified, each term has the same meaning when used alone or in combination with other terms.

[0331] The term "consisting of" means having only the constituent elements.

[0332] The term "comprising" does not limit the constituent elements and does not exclude undescribed elements.

[0333] Hereinafter, embodiments are shown to illustrate the present invention. Throughout the present specification, unless otherwise specified, expressions in the singular form should be understood as also including the concept of its plural form. Therefore, unless otherwise specified, articles in the singular form (for example, in English, "a", "an", "the", etc.) should be understood as also including the concept of its plural form.

[0334] In addition, in this specification, unless otherwise specified, the terms used should be understood to be used with the meaning commonly used in the art. Therefore, unless otherwise defined, all professional terms and scientific and technical terms used in this specification have the same meaning as those commonly understood by technicians in the field to which the present invention belongs. In the event of a conflict, this specification (including definitions) takes precedence.

[0335] The term "halogen" includes a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and a fluorine atom and a chlorine atom are particularly preferred.

[0336] The term "alkyl" refers to a linear or branched hydrocarbon group having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and still more preferably 1 to 4 carbon atoms. Examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl, and n-decyl.

[0337] Preferred embodiments of the "alkyl group" include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and n-pentyl. More preferred embodiments include methyl, ethyl, n-propyl, isopropyl and tert-butyl.

[0338] The term "alkenyl" refers to a linear or branched hydrocarbon group having 2 to 15 carbon atoms, preferably 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and further preferably 2 to 4 carbon atoms, and having one or more double bonds at any position. Examples thereof include vinyl, allyl, propenyl, isopropenyl, butenyl, isobutenyl, isoprenyl, butadienyl, pentenyl, isopentenyl, pentadienyl, hexenyl, isohexenyl, hexadienyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, and pentadecenyl.

[0339] Preferred embodiments of the "alkenyl group" include vinyl, allyl, propenyl, isopropenyl, and butenyl, and further preferred embodiments include vinyl and n-propenyl.

[0340] The term "alkynyl" refers to a linear or branched hydrocarbon group having 2 to 10 carbon atoms, preferably 2 to 8 carbon atoms, more preferably 2 to 6 carbon atoms, and more preferably 2 to 4 carbon atoms, and having one or more triple bonds at any position. It may also have a double bond at any position. For example, it includes ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, and the like.

[0341] Preferred embodiments of "alkynyl" include ethynyl, propynyl, butynyl and pentynyl. Further preferred embodiments include ethynyl and propynyl.

[0342] The "aromatic carbocyclic group" refers to a monocyclic or bicyclic or more cyclic aromatic hydrocarbon group, and examples thereof include phenyl, naphthyl, anthracenyl, and phenanthryl.

[0343] A preferred embodiment of the "aromatic carbocyclic group" includes a phenyl group.

[0344] The "6-membered aromatic carbocyclic group" refers to a monocyclic aromatic hydrocarbon group, and an example thereof is a phenyl group.

[0345] The term "non-aromatic carbocyclic group" refers to a monocyclic or bicyclic saturated hydrocarbon group or a cyclic non-aromatic unsaturated hydrocarbon group. A bicyclic or bicyclic "non-aromatic carbocyclic group" also includes a group obtained by condensing a ring in the above "aromatic carbocyclic group" to a monocyclic or bicyclic non-aromatic carbocyclic group.

[0346] Furthermore, the "non-aromatic carbocyclic group" also includes bridged groups or spiro ring-forming groups as described below.

[0347] [Chemical formula 17]

[0348]

[0349] The monocyclic non-aromatic carbocyclic group preferably has 3 to 16 carbon atoms, more preferably 3 to 12 carbon atoms, and still more preferably 4 to 8 carbon atoms. Examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclohexadienyl and the like.

[0350] The non-aromatic carbocyclic group having two or more rings preferably has 8 to 20 carbon atoms, and more preferably has 8 to 16 carbon atoms. Examples thereof include indanyl, indenyl, acenaphthyl, tetrahydronaphthyl, and fluorenyl.

[0351] The "aromatic heterocyclic group" refers to a monocyclic or bicyclic aromatic cyclic group having one or more heteroatoms selected arbitrarily from O, S and N, which are the same or different, in the ring.

[0352] The aromatic heterocyclic group having two or more rings also includes a group in which a ring in the above-mentioned "aromatic carbocyclic group" is fused to a monocyclic or bicyclic aromatic heterocyclic group, and the linking bond may be present on any ring.

[0353] The monocyclic aromatic heterocyclic group is preferably 5-8-membered, more preferably 5-membered or 6-membered. Examples of the 5-membered aromatic heterocyclic group include pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, isothiazolyl, thiazolyl, thiadiazolyl, etc. Examples of the 6-membered aromatic heterocyclic group include pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, etc.

[0354] The aromatic heterocyclic group having two rings is preferably 8-10-membered, more preferably 9-membered or 10-membered. For example, there can be mentioned indolyl, isoindolyl, indazolyl, indolizinyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, naphthyridinyl, quinoxalinyl, purinyl, pteridinyl, benzimidazolyl, benzisoxazolyl, benzoxazolyl, benzoxadiazolyl, benzisothiazolyl, benzothiazolyl, benzothiadiazolyl, benzofuranyl, isobenzofuranyl, benzothienyl, benzotriazolyl, imidazopyridinyl, triazolopyridinyl, imidazothiazolyl, pyrazinopyridazinyl, oxazolopyridinyl, thiazolopyridinyl and the like. Examples of the 9-membered aromatic heterocyclic group include indolyl, isoindolyl, indazolyl, indolizinyl, purinyl, benzimidazolyl, benzisoxazolyl, benzoxazolyl, benzoxadiazolyl, benzisothiazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzofuranyl, imidazopyridyl, triazolopyridyl, oxazolopyridyl, thiazolopyridyl, etc. Examples of the 10-membered aromatic heterocyclic group include quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, naphthyridinyl, quinoxalinyl, pteridinyl, pyrazinopyridazinyl, etc.

[0355] The aromatic heterocyclic group having three or more rings is preferably a 13- to 15-membered group and examples thereof include a carbazolyl group, an acridinyl group, a xanthenyl group, a phenothiazinyl group, a phenoxathiyl group, a phenoxazinyl group, and a dibenzofuranyl group.

[0356] The "5- to 6-membered aromatic heterocyclic group" refers to a 5- or 6-membered aromatic heterocyclic group among the above-mentioned "aromatic heterocyclic group".

[0357] The "9- to 10-membered aromatic heterocyclic group" refers to a 9- or 10-membered aromatic heterocyclic group among the above-mentioned "aromatic heterocyclic group".

[0358] The so-called "non-aromatic heterocyclic group" refers to a monocyclic or bicyclic non-aromatic cyclic group having one or more heteroatoms selected from O, S and N, which are the same or different. The bicyclic non-aromatic heterocyclic group also includes a group obtained by condensing each ring of the above-mentioned "aromatic carbocyclic group", "non-aromatic carbocyclic group", and / or "aromatic heterocyclic group" to a monocyclic or bicyclic non-aromatic heterocyclic group, and a group obtained by condensing a ring of the above-mentioned "aromatic heterocyclic group" to a monocyclic or bicyclic non-aromatic carbocyclic group, and the connecting bond may be present on any ring.

[0359] Furthermore, the "non-aromatic heterocyclic group" also includes a bridged group or a spiro ring-forming group as shown below.

[0360] [Chemical formula 18]

[0361]

[0362] The monocyclic non-aromatic heterocyclic group is preferably 3-8-membered, more preferably 5-membered or 6-membered.

[0363] As the 3-membered non-aromatic heterocyclic group, for example, thiirane, oxirane, and aziridine groups can be mentioned. As the 4-membered non-aromatic heterocyclic group, for example, oxetanyl and azetidinyl groups can be mentioned. As the 5-membered non-aromatic heterocyclic group, for example, oxathiolanyl, thiazolidinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, tetrahydrofuranyl, dihydrothiazolyl, tetrahydroisothiazolyl, dioxolyl, dioxolyl, tetrahydrothienyl, etc. can be mentioned. Examples of the 6-membered non-aromatic heterocyclic group include dioxane, thianyl, piperidinyl, piperazinyl, morpholinyl, morpholino, thiomorpholino, thiomorpholino, dihydropyridinyl, tetrahydropyridinyl, tetrahydropyranyl, dihydrooxazinyl, tetrahydropyridazinyl, hexahydropyrimidinyl, dioxazinyl, thiazinyl, and thiazinyl. Examples of the 7-membered non-aromatic heterocyclic group include hexahydroazepine, Tetrahydrodiazepine Oxanyl, oxepanyl.

[0364] The non-aromatic heterocyclic group having two or more rings is preferably 8-20-membered, more preferably 8-13-membered, and still more preferably 8-10-membered. Examples thereof include indolinyl, isoindolinyl, chromanyl, and isochromanyl.

[0365] In the present specification, “may be substituted by substituent group α” means “may be substituted by one or more groups selected from substituent group α”. The same applies to substituent groups β, γ, and γ′.

[0366] Substituent Group α: halogen, hydroxy, carboxyl, alkyloxy, haloalkyloxy, alkenyloxy, alkynyloxy, sulfanyl, and cyano.

[0367] Substituent group β: halogen, hydroxyl, carboxyl, cyano, alkyl which may be substituted by substituent group α, alkenyl which may be substituted by substituent group α, alkynyl which may be substituted by substituent group α, alkylcarbonyl which may be substituted by substituent group α, alkenylcarbonyl which may be substituted by substituent group α, alkynylcarbonyl which may be substituted by substituent group α, alkylsulfanyl which may be substituted by substituent group α, alkenylsulfanyl which may be substituted by substituent group α, alkynylsulfanyl which may be substituted by substituent group α, alkylsulfinyl which may be substituted by substituent group α, alkenylsulfinyl which may be substituted by substituent group α, alkynylsulfinyl which may be substituted by substituent group α, alkylsulfonyl which may be substituted by substituent group α, alkenylsulfonyl which may be substituted by substituent group α, alkynylsulfonyl which may be substituted by substituent group α,

[0368] an aromatic carbocyclic group which may be substituted with the substituent group γ, a non-aromatic carbocyclic group which may be substituted with the substituent group γ', an aromatic heterocyclic group which may be substituted with the substituent group γ, a non-aromatic heterocyclic group which may be substituted with the substituent group γ', an aromatic carbocyclic alkyl group which may be substituted with the substituent group γ, a non-aromatic carbocyclic alkyl group which may be substituted with the substituent group γ', an aromatic heterocyclic alkyl group which may be substituted with the substituent group γ, a non-aromatic heterocyclic alkyl group which may be substituted with the substituent group γ', an aromatic carbocyclic carbonyl group which may be substituted with the substituent group γ, a non-aromatic carbocyclic carbonyl group which may be substituted with the substituent group γ', an aromatic heterocyclic carbonyl group which may be substituted with the substituent group γ, a non-aromatic heterocyclic carbonyl group which may be substituted with the substituent group γ', an aromatic carbocyclic oxycarbonyl group which may be substituted with the substituent group γ, a non-aromatic carbocyclic oxycarbonyl group which may be substituted with the substituent group γ', a substituent group an aromatic heterocyclic oxycarbonyl group which may be substituted with the γ' substituent group, a non-aromatic heterocyclic oxycarbonyl group which may be substituted with the γ' substituent group, an aromatic carbocyclic sulfanyl group which may be substituted with the γ' substituent group, a non-aromatic carbocyclic sulfanyl group which may be substituted with the γ' substituent group, an aromatic heterocyclic sulfanyl group which may be substituted with the γ' substituent group, a non-aromatic heterocyclic sulfanyl group which may be substituted with the γ' substituent group, an aromatic carbocyclic sulfinyl group which may be substituted with the γ' substituent group, an aromatic heterocyclic sulfinyl group which may be substituted with the γ' substituent group, a non-aromatic heterocyclic sulfinyl group which may be substituted with the γ' substituent group, an aromatic carbocyclic sulfonyl group which may be substituted with the γ substituent group, a non-aromatic carbocyclic sulfonyl group which may be substituted with the γ' substituent group, an aromatic heterocyclic sulfonyl group which may be substituted with the γ substituent group, and a non-aromatic heterocyclic sulfonyl group which may be substituted with the γ' substituent group.

[0369] Substituent Group γ: Substituent Group α, alkyl, halogenated alkyl, hydroxyalkyl, alkenyl, alkynyl, alkylcarbonyl, halogenated alkylcarbonyl, alkenylcarbonyl, and alkynylcarbonyl.

[0370] Substituent group γ': substituent group γ and oxo group.

[0371] Examples of substituents on the "aromatic carbocyclic ring" and "aromatic heterocyclic ring" of the "substituted aromatic carbocyclic group" and "substituted aromatic heterocyclic group" include the following substituent group B. Atoms at any position on the ring may be bonded to one or more groups selected from the following substituent group B.

[0372] Substituent Group B: halogen, hydroxy, carboxyl, formyl, formyloxy, sulfanyl, sulfinyl, sulfo, thioformyl, thiocarboxyl, dithiocarboxyl, thiocarbamoyl, cyano, nitro, nitroso, azido, hydrazine, urea, amidino, guanidino, pentafluorosulfenyl, trialkylsilyl,

[0373] Alkyl which may be substituted by substituent group α, alkenyl which may be substituted by substituent group α, alkynyl which may be substituted by substituent group α, alkyloxy which may be substituted by substituent group α, alkenyloxy which may be substituted by substituent group α, alkynyloxy which may be substituted by substituent group α, alkylcarbonyloxy which may be substituted by substituent group α, alkenylcarbonyloxy which may be substituted by substituent group α, alkynylcarbonyloxy which may be substituted by substituent group α, alkylcarbonyl which may be substituted by substituent group α, alkenylcarbonyl which may be substituted by substituent group α, alkynylcarbonyl which may be substituted by substituent group α an alkyloxycarbonyl group which may be substituted by the α substituent group, an alkenyloxycarbonyl group which may be substituted by the α substituent group, an alkynyloxycarbonyl group which may be substituted by the α substituent group, an alkylsulfanyl group which may be substituted by the α substituent group, an alkenylsulfanyl group which may be substituted by the α substituent group, an alkynylsulfanyl group which may be substituted by the α substituent group, an alkylsulfinyl group which may be substituted by the α substituent group, an alkenylsulfinyl group which may be substituted by the α substituent group, an alkynylsulfinyl group which may be substituted by the α substituent group, an alkylsulfonyl group which may be substituted by the α substituent group, an alkenylsulfonyl group which may be substituted by the α substituent group, and an alkynylsulfonyl group which may be substituted by the α substituent group,

[0374] an amino group which may be substituted by a substituent group β, an imino group which may be substituted by a substituent group β, a carbamoyl group which may be substituted by a substituent group β, a sulfamoyl group which may be substituted by a substituent group β,

[0375] an aromatic carbocyclic group which may be substituted with a substituent group γ, a non-aromatic carbocyclic group which may be substituted with a substituent group γ', an aromatic heterocyclic group which may be substituted with a substituent group γ, a non-aromatic heterocyclic group which may be substituted with a substituent group γ', an aromatic carbocyclic oxy group which may be substituted with a substituent group γ, a non-aromatic carbocyclic oxy group which may be substituted with a substituent group γ', an aromatic heterocyclic oxy group which may be substituted with a substituent group γ, a non-aromatic heterocyclic oxy group which may be substituted with a substituent group γ', an aromatic carbocyclic carbonyloxy group which may be substituted with a substituent group γ, a non-aromatic carbocyclic carbonyloxy group which may be substituted with a substituent group γ', an aromatic heterocyclic carbonyloxy group which may be substituted with a substituent group γ, a non-aromatic heterocyclic carbonyloxy group which may be substituted with a substituent group γ', a substituent group an aromatic carbocyclic carbonyl group substituted with γ, a non-aromatic carbocyclic carbonyl group which may be substituted with γ' as a substituent group, an aromatic heterocyclic carbonyl group which may be substituted with γ as a substituent group, a non-aromatic heterocyclic carbonyl group which may be substituted with γ' as a substituent group, an aromatic carbocyclic oxycarbonyl group which may be substituted with γ as a substituent group, a non-aromatic carbocyclic oxycarbonyl group which may be substituted with γ' as a substituent group, an aromatic heterocyclic oxycarbonyl group which may be substituted with γ as a substituent group, a non-aromatic heterocyclic oxycarbonyl group which may be substituted with γ' as a substituent group, an aromatic carbocyclic alkyl group which may be substituted with γ as a substituent group, a non-aromatic carbocyclic alkyl group which may be substituted with γ' as a substituent group, an aromatic heterocyclic alkyl group which may be substituted with γ as a substituent group, a non-aromatic heterocyclic alkyl group which may be substituted with γ' as a substituent group, an aromatic carbocyclic alkyloxy group which may be substituted with γ as a substituent group, non-aromatic carbocyclic alkyloxy group which may be substituted with a substituent group γ', aromatic heterocyclic alkyloxy group which may be substituted with a substituent group γ, non-aromatic heterocyclic alkyloxy group which may be substituted with a substituent group γ', aromatic carbocyclic alkyloxycarbonyl group which may be substituted with a substituent group γ, non-aromatic carbocyclic alkyloxycarbonyl group which may be substituted with a substituent group γ', aromatic heterocyclic alkyloxycarbonyl group which may be substituted with a substituent group γ, non-aromatic heterocyclic alkyloxycarbonyl group which may be substituted with a substituent group γ', aromatic carbocyclic alkyloxyalkyl group which may be substituted with a substituent group γ, non-aromatic carbocyclic alkyloxyalkyl group which may be substituted with a substituent group γ', aromatic heterocyclic alkyloxyalkyl group which may be substituted with a substituent group γ, non-aromatic heterocyclic alkyloxyalkyl group which may be substituted with a substituent group γ' a non-aromatic heterocyclic sulfanyl group which may be substituted with the substituent group γ; an aromatic carbocyclic sulfanyl group which may be substituted with the substituent group γ'; an aromatic heterocyclic sulfanyl group which may be substituted with the substituent group γ; a non-aromatic heterocyclic sulfanyl group which may be substituted with the substituent group γ'; an aromatic carbocyclic sulfinyl group which may be substituted with the substituent group γ; a non-aromatic carbocyclic sulfinyl group which may be substituted with the substituent group γ'; an aromatic heterocyclic sulfinyl group which may be substituted with the substituent group γ; a non-aromatic heterocyclic sulfinyl group which may be substituted with the substituent group γ'; an aromatic carbocyclic sulfonyl group which may be substituted with the substituent group γ; a non-aromatic carbocyclic sulfonyl group which may be substituted with the substituent group γ'; an aromatic heterocyclic sulfonyl group which may be substituted with the substituent group γ; and a non-aromatic heterocyclic sulfonyl group which may be substituted with the substituent group γ'.

[0376] Examples of the substituents on the "non-aromatic carbocyclic ring" and "non-aromatic heterocyclic ring" of the "substituted non-aromatic carbocyclic group" and "substituted non-aromatic heterocyclic group" include the following substituent group C. Atoms at any position on the ring may be bonded to one or more groups selected from the following substituent group C.

[0377] Substituent Group C: Substituent Group B and oxo group.

[0378] When a "non-aromatic carbocyclic ring" or a "non-aromatic heterocyclic ring" is substituted with an "oxo group", it means a ring in which two hydrogen atoms on a carbon atom are substituted as follows.

[0379] [Chemical formula 19]

[0380]

[0381] As R 1 The substituents of the "substituted or unsubstituted aromatic heterocyclic group" or "substituted or unsubstituted 5- to 6-membered aromatic heterocyclic group" include, for example:

[0382] halogen;

[0383] Substituted or unsubstituted alkyl.

[0384] It may be substituted by one or more groups selected from these.

[0385] As R 1 The substituents of the "substituted or unsubstituted aromatic heterocyclic group" or "substituted or unsubstituted 5- to 6-membered aromatic heterocyclic group" include, for example:

[0386] halogen;

[0387] Substituted alkyl (hydroxyl as substituent); unsubstituted alkyl.

[0388] It may be substituted by one or more groups selected from these.

[0389] As R 2 The substituents of the "substituted or unsubstituted 6-membered aromatic carbocyclic group" include, for example:

[0390] Halogen; Cyano;

[0391] Substituted or unsubstituted alkyl.

[0392] It may be substituted by one or more groups selected from these.

[0393] As R 2 The substituents of the "substituted or unsubstituted 6-membered aromatic carbocyclic group" include, for example:

[0394] Halogen; Cyano;

[0395] Substituted alkyl (halogen as substituent); unsubstituted alkyl.

[0396] It may be substituted by one or more groups selected from these.

[0397] As R 3 The substituents of the "substituted or unsubstituted aromatic heterocyclic group" or the "substituted or unsubstituted 9- to 10-membered aromatic heterocyclic group" include, for example:

[0398] halogen;

[0399] a substituted or unsubstituted alkyl group;

[0400] A substituted or unsubstituted non-aromatic heterocyclic group.

[0401] It may be substituted by one or more groups selected from these.

[0402] As R 3 The substituents of the "substituted or unsubstituted aromatic heterocyclic group" or the "substituted or unsubstituted 9- to 10-membered aromatic heterocyclic group" include, for example:

[0403] halogen;

[0404] Substituted alkyl (halogen, hydroxyl, alkylcarbonylamino, non-aromatic heterocyclic group as substituent); unsubstituted alkyl;

[0405] Substituted non-aromatic heterocyclic group (alkylcarbonyl as substituent); unsubstituted non-aromatic heterocyclic group.

[0406] It may be substituted by one or more groups selected from these.

[0407] The following shows the compounds represented by formula (I): 1 , R 2 , R 3 As the compound represented by formula (I), all the combinations of the specific examples shown below can be exemplified. It should be noted that Y, -X-, R 5a , R 5b ,n,R 4a and R 4b As shown in item (1) above.

[0408] [Chemical formula 20]

[0409]

[0410] R 1Examples include substituted or unsubstituted aromatic heterocyclic groups (hereinafter referred to as A-1).

[0411] R 1 Examples include substituted or unsubstituted 5- to 6-membered aromatic heterocyclic groups (hereinafter referred to as A-2).

[0412] R 1 Examples thereof include an aromatic heterocyclic group substituted with a halogen, a substituted alkyl group (substituent: hydroxyl group) or an unsubstituted alkyl group or an unsubstituted aromatic heterocyclic group (hereinafter referred to as A-3).

[0413] R 1 Examples thereof include a 5- to 6-membered aromatic heterocyclic group substituted with a halogen, a substituted alkyl group (substituent: a hydroxyl group) or an unsubstituted alkyl group, and an unsubstituted 5- to 6-membered aromatic heterocyclic group (hereinafter referred to as A-4).

[0414] R 1 Examples thereof include an aromatic heterocyclic group substituted with an unsubstituted alkyl group or a halogen, and an unsubstituted aromatic heterocyclic group (hereinafter referred to as A-5).

[0415] R 1 Examples thereof include a 5- to 6-membered aromatic heterocyclic group substituted with an unsubstituted alkyl group or a halogen, and an unsubstituted 5- to 6-membered aromatic heterocyclic group (hereinafter referred to as A-6).

[0416] R 1 Examples include an aromatic heterocyclic group substituted with an unsubstituted alkyl group or a halogen (hereinafter referred to as A-7).

[0417] R 1 Examples include a 5- to 6-membered aromatic heterocyclic group substituted with an unsubstituted alkyl group or a halogen (hereinafter referred to as A-8).

[0418] R 1 Examples thereof include an aromatic heterocyclic group substituted with an unsubstituted alkyl group or an unsubstituted aromatic heterocyclic group (hereinafter referred to as A-9).

[0419] R 1 Examples thereof include a 5- to 6-membered aromatic heterocyclic group substituted with an unsubstituted alkyl group and an unsubstituted 5- to 6-membered aromatic heterocyclic group (hereinafter referred to as A-10).

[0420] R 1 An example is an aromatic heterocyclic group substituted with an unsubstituted alkyl group (hereinafter referred to as A-11).

[0421] R 1 Examples include a 5- to 6-membered aromatic heterocyclic group substituted with an unsubstituted alkyl group (hereinafter referred to as A-12).

[0422] R 2Examples include a substituted or unsubstituted 6-membered aromatic carbocyclic group (hereinafter referred to as B-1).

[0423] R 2 Examples include a 6-membered aromatic carbocyclic group substituted with a halogen, a cyano group, a substituted alkyl group (substituent: halogen) or an unsubstituted alkyl group (hereinafter referred to as B-2).

[0424] R 2 Examples include a 6-membered aromatic carbocyclic group substituted with a halogen, a cyano group, or an unsubstituted alkyl group (hereinafter referred to as B-3).

[0425] R 2 Examples include a 6-membered aromatic carbocyclic group substituted with 2 to 4 substituents selected from Substituent Group G (Substituent Group G: halogen, cyano and unsubstituted alkyl) (hereinafter referred to as B-4).

[0426] R 2 Examples include a 6-membered aromatic carbocyclic group substituted with 2 to 3 substituents selected from Substituent Group G (Substituent Group G: halogen, cyano and unsubstituted alkyl) (hereinafter referred to as B-5).

[0427] R 2 Examples include a 6-membered aromatic carbocyclic group substituted with 3 to 4 substituents selected from Substituent Group G (Substituent Group G: halogen, cyano and unsubstituted alkyl) (hereinafter referred to as B-6).

[0428] R 2 An example is a 6-membered aromatic carbocyclic group substituted with three halogens (hereinafter referred to as B-7).

[0429] R 3 Examples include substituted or unsubstituted aromatic heterocyclic groups (hereinafter referred to as C-1).

[0430] R 3 Examples include substituted or unsubstituted 9- to 10-membered aromatic heterocyclic groups (hereinafter referred to as C-2).

[0431] R 3 Examples include aromatic heterocyclic groups substituted with halogen or substituted or unsubstituted alkyl groups (hereinafter referred to as C-3).

[0432] R 3 Examples include a 9- to 10-membered aromatic heterocyclic group substituted with a halogen or a substituted or unsubstituted alkyl group (hereinafter referred to as C-4).

[0433] R 3 Examples include aromatic heterocyclic groups substituted with halogen or unsubstituted alkyl (hereinafter referred to as C-5).

[0434] R 3Examples include a 9- to 10-membered aromatic heterocyclic group substituted with a halogen or an unsubstituted alkyl group (hereinafter referred to as C-6).

[0435] R 3 Examples include indazolyl groups substituted with halogen or unsubstituted alkyl groups (hereinafter referred to as C-7).

[0436] R 3 Examples include indazolyl groups substituted with halogen and unsubstituted alkyl groups (hereinafter referred to as C-8).

[0437] m may be 0 or 1 (hereinafter referred to as D-1).

[0438] An example of m is 0 (hereinafter referred to as D-2).

[0439] Examples of m include 1 (hereinafter referred to as D-3).

[0440] Examples of the compound represented by formula (I) include the following.

[0441] (a-1)

[0442] R 1 is (A-12);

[0443] R 2 is (B-7);

[0444] R 3 is (C-8);

[0445] m is (D-2).

[0446] (a-2)

[0447] R 1 is (A-12);

[0448] R 2 is (B-7);

[0449] R 3 is (C-8);

[0450] m is (D-3).

[0451] (a-3)

[0452] R 1 is (A-12);

[0453] R 2 is (B-7);

[0454] R 3 is (C-8);

[0455] m is (D-1).

[0456] (a-4)

[0457] R 1 is (A-4);

[0458] R 2 is (B-4);

[0459] R 3 is (C-4);

[0460] m is (D-1).

[0461] The compound represented by formula (I) is preferably a compound represented by formula (IA) or a compound represented by formula (IB).

[0462] The chemical structural formulas represented by formula (I), formula (IA) and formula (IB) are also used uniformly in each formula.

[0463] The compound shown in formula (I) is not limited to a specific isomer, but includes all possible isomers (e.g., keto-enol isomers, imine-enamine isomers, diastereomers, optical isomers, rotational isomers, etc.), racemates or mixtures thereof. For example, the compound shown in formula (I) includes the following tautomers.

[0464] [Chemical formula 21]

[0465]

[0466] For example, the compound represented by formula (IA) or compound (I-003) includes the following tautomers and mixtures thereof.

[0467] [Chemical formula 22]

[0468]

[0469] For example, the compound represented by formula (IB) or compound (I-005) includes the following tautomers and mixtures thereof.

[0470] [Chemical formula 23]

[0471]

[0472] One or more hydrogen, carbon and / or other atoms of the compound represented by formula (I) may be substituted by isotopes of hydrogen, carbon and / or other atoms. Examples of such isotopes are: 2 H. 3 H. 11 C. 13 C. 14 C. 15 N. 18 O.17 O. 31 P. 32 P. 35 S. 18 F. 123 I and 36 Cl includes hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine. The compound shown in formula (I) also includes compounds substituted with such isotopes. The compounds substituted with isotopes are also useful as pharmaceuticals, including all radiolabeled forms of the compounds shown in formula (I). In addition, the "radiolabeling method" for producing the "radiolabeled form" is also included in the present invention, and the "radiolabeled form" is useful as a tool for research and / or diagnosis in metabolic drug dynamics studies, binding analysis.

[0473] In addition, the crystal of the present invention may also be a deuterated body. The crystal of the present invention may also be deuterated with isotopic elements (e.g., 3 H. 14 C. 35 S. 125 I, etc.) marking.

[0474] The radioactive labeled form of the compound represented by formula (I) can be prepared by a method known in the art. For example, the tritium-labeled compound represented by formula (I) can be prepared by introducing tritium into a specific compound represented by formula (I) through a catalytic dehalogenation reaction using tritium. The method comprises the following steps: reacting a precursor obtained by appropriately halogenating the compound represented by formula (I) 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 can be prepared by using 14 C carbon raw materials to prepare.

[0475] Examples of the pharmaceutically acceptable salt of the compound represented by formula (I) include salts of the compound represented by formula (I) with alkali metals (e.g., lithium, sodium, potassium, etc.), alkaline earth metals (e.g., calcium, barium, etc.), magnesium, transition metals (e.g., zinc, iron, etc.), ammonia, organic bases (e.g., trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, meglumine, ethylenediamine, pyridine, picoline, quinoline, etc.) and amino acids, or salts of the compound represented by formula (I) with inorganic acids (e.g., hydrochloric acid, sulfuric acid, nitric acid, carbonic acid, hydrobromic acid, phosphoric acid, hydroiodic acid, etc.) and organic acids (e.g., formic acid, acetic acid, propionic acid, trifluoroacetic acid, citric acid, lactic acid, tartaric acid, oxalic acid, maleic acid, fumaric acid, succinic acid, mandelic acid, glutaric acid, malic acid, benzoic acid, phthalic acid, ascorbic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, trifluoroacetic acid, etc.). These salts can be formed by commonly practiced methods.

[0476] As a pharmaceutically acceptable salt of the compound represented by formula (IA), for example, it is formed by the compound represented by formula (IA) and a counter molecule or counter ion, and may contain any number of counter molecules or counter ions. The pharmaceutically acceptable salt of the compound represented by formula (IA) refers to a salt that mediates an ionic bond by proton transfer between the compound and the counter molecule or counter atom.

[0477] The pharmaceutically acceptable salt of the compound represented by formula (IA) is preferably a p-toluenesulfonate of the compound represented by formula (IA).

[0478] In the preparation of the present invention, a complex of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof sometimes forms a solvate (e.g., a hydrate, etc.), a co-crystal and / or an inclusion compound, which are described as "complexes" in this specification.

[0479] The term "solvate" as used herein means that any number of solvent molecules (e.g., water molecules) can be coordinated with the compound represented by formula (I). The compound represented by formula (I) or a pharmaceutically acceptable salt thereof can absorb water by being placed in the atmosphere, and there is a case where water is adsorbed and attached, or a case where a hydrate is formed.

[0480] Examples of the solvent molecule include acetonitrile, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethylene, dichloromethane, 1,2-dimethoxyethane, N,N-dimethylacetamide, N,N-dimethylformamide, 1,4-dioxane, 2-ethoxyethanol, ethylene glycol, formamide, hexane, methanol, 2-methoxyethanol, methyl butyl ketone, methylcyclohexane, N-methylpyrrolidone, nitromethane, pyridine, sulfolane, tetralin, toluene, 1,1,2-trichloroethylene, xylene, acetic acid, anisole, 1 -Butanol, 2-butanol, n-butyl acetate, tert-butyl methyl ether, cumene, dimethyl sulfoxide, ethyl acetate, diethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, propyl acetate, tetrahydrofuran, water (i.e., hydrate), ethanol, acetone, 1,1-diethoxypropane, 1,1-dimethoxymethane, 2,2-dimethoxypropane, isooctane, isopropyl ether, methyl isopropyl ketone, methyltetrahydrofuran, petroleum ether, trichloroacetic acid and trifluoroacetic acid, preferably acetic acid, anisole, 1-butanol, 2-butanol, n-butyl acetate, tert-butyl methyl ether, cumene, dimethyl sulfoxide, ethyl acetate, diethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, propyl acetate, tetrahydrofuran, furan, water (i.e., hydrate), ethanol, acetone, 1,1-diethoxypropane, 1,1-dimethoxymethane, 2,2-dimethoxypropane, isooctane, isopropyl ether, methyl isopropyl ketone, methyltetrahydrofuran, petroleum ether, trichloroacetic acid and trifluoroacetic acid, and more preferably water (i.e., hydrate), ethanol, acetone, 1,1-diethoxypropane, 1,1-dimethoxymethane, 2,2-dimethoxypropane, isooctane, isopropyl ether, methyl isopropyl ketone, methyltetrahydrofuran, petroleum ether, trichloroacetic acid and trifluoroacetic acid can be mentioned.

[0481] The term "co-crystal" as used herein means that counter molecules are regularly arranged in the same crystal lattice and may contain any number of counter molecules. In addition, the term "co-crystal" means a substance in which the intermolecular interaction between a compound and a counter molecule is via non-covalent and non-ionic chemical interactions such as hydrogen bonds and van der Waals forces.

[0482] For example, a co-crystal of a compound represented by formula (IB) is composed of a compound represented by formula (IB) and a counter molecule, and may contain any number of counter molecules. Preferably, it is composed of a compound represented by formula (IB) and fumaric acid, and may contain any number of fumaric acids. More preferably, the co-crystal is a 1:1 molar ratio of the compound represented by formula (IB) and fumaric acid.

[0483] Co-crystals are distinguished from salts in that the compound remains essentially uncharged or neutral.

[0484] Co-crystals are distinguished from hydrates or solvates in that the countermolecule is not water or a solvent.

[0485] The term "crystal" as used herein refers to a solid whose constituent atoms, ions, molecules, etc. are regularly arranged in three dimensions, and is distinguished from an amorphous solid that does not have such a regular internal structure. The crystal of the present invention may be a single crystal, a twin crystal, a polycrystal, or the like.

[0486] In addition, a "crystal" may have "polymorphs" having the same composition but different arrangements in the crystal, and these are collectively referred to as "crystal forms".

[0487] The crystal form and crystallinity can be determined by a variety of techniques including, for example, powder X-ray diffraction, Raman spectroscopy, infrared absorption spectroscopy, water absorption and desorption, differential scanning calorimetry, and dissolution characteristics.

[0488] In addition, by recrystallizing the compound represented by formula (I), its pharmaceutically acceptable salt, or a complex thereof, "polymorphs" may be formed.

[0489] In the preparation of the present invention, various salts, complexes (hydrates, solvates, co-crystals, inclusion compounds), and polymorphs may be used, and a mixture of two or more thereof may also be used.

[0490] (X-ray powder diffraction (XRPD))

[0491] Powder X-ray diffraction (XRPD) is one of the most sensitive analytical methods for determining the crystal form and crystallinity of solids. When X-rays are irradiated to a crystal, they are reflected on the lattice planes and interfere with each other, showing ordered diffraction lines corresponding to the period of the structure. On the other hand, for amorphous solids, generally, there is no ordered repeating period in their structure, so no diffraction occurs, and a featureless broad XRPD pattern (also called a halo pattern) is shown.

[0492] The crystalline form of the compound represented by formula (IA) and formula (IB) can be identified by powder X-ray diffraction pattern and characteristic diffraction peaks. The crystalline form of the compound represented by formula (IA) and formula (IB) can be distinguished from other crystalline forms by the presence of characteristic diffraction peaks.

[0493] The characteristic diffraction peak used in this specification is a peak selected from the observed diffraction pattern. The characteristic diffraction peak is preferably selected from about 10, more preferably about 5, and further preferably about 3 peaks in the diffraction pattern.

[0494] In differentiating between multiple crystals, the peak that is confirmed in the crystal and not confirmed in other crystals becomes a preferred characteristic peak for determining the crystal compared to the intensity of the peak. If it is such a characteristic peak, one or two peaks can also characterize the crystal. Comparing the figures obtained by measuring, if these characteristic peaks are consistent, it can be said that the powder X-ray diffraction patterns are substantially consistent.

[0495] Generally, the diffraction angle (2θ) in powder X-ray diffraction may have an error within the range of ±0.2°, so it is necessary to understand that the value of the diffraction angle of powder X-ray diffraction also includes a numerical value within the range of about ±0.2°. Therefore, not only crystals whose peak diffraction angles in powder X-ray diffraction are completely consistent, but also crystals whose peak diffraction angles are consistent with an error of about ±0.2° are also included in the present invention.

[0496] It is known that the intensity of the peaks shown in the following tables and figures can usually be changed according to various factors, such as the effect of the preferred orientation of the crystal relative to the X-ray beam, the influence of coarse particles, the purity of the substance being analyzed, or the crystallinity of the sample. In addition, the peak position may also be shifted based on the change in sample height. In addition, when different wavelengths are used for measurement, different shifts can be obtained according to the Bragg formula (nλ=2dsinθ), but other XRPD patterns obtained by using other wavelengths are also included in the scope of the present invention.

[0497] (Single crystal structure analysis)

[0498] By one of the methods for determining the crystal, the crystallographic parameters, atomic coordinates (values ​​indicating the spatial positional relationship of each atom) and a three-dimensional structural model of the crystal can be obtained. Refer to "A Guide to X-Ray Structure Analysis" by Toshio Sakurai, published by Shokabo (1983), X-Ray Structure Determination: A Practical Guide by Stout & Jensen, Macmillan Co., New York (1968), etc. Single crystal structure analysis is useful in identifying the structure of crystals of complexes, salts, optical isomers, tautomers, and geometric isomers such as those of the present invention.

[0499] The compounds of the present invention have coronavirus 3CL protease inhibitory activity and are therefore useful as therapeutic and / or preventive agents for diseases in which coronavirus 3CL protease is involved. In the present invention, "therapeutic and / or preventive agents" also include symptom-improving agents. Diseases in which coronavirus 3CL protease is involved include viral infectious diseases, preferably coronavirus infectious diseases.

[0500] In one embodiment, coronaviruses that infect humans include coronaviruses that infect humans. Coronaviruses that infect humans include HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and / or SARS-CoV-2.

[0501] In one embodiment, examples of the coronavirus include alpha coronavirus and / or beta coronavirus, more preferably beta coronavirus, and even more preferably sarbecovirus.

[0502] In one embodiment, as alpha coronavirus, HCoV-229E and HCoV-NL63 can be mentioned. HCoV-229E is particularly preferred.

[0503] In one embodiment, beta coronaviruses include HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and / or SARS-CoV-2. HCoV-OC43 or SARS-CoV-2 is preferred, and SARS-CoV-2 is particularly preferred.

[0504] As one embodiment, as β-coronavirus, β-coronavirus lineage A, β-coronavirus lineage B, and β-coronavirus lineage C can be mentioned. β-coronavirus lineage A and β-coronavirus lineage B are more preferably mentioned, and β-coronavirus lineage B is particularly preferably mentioned.

[0505] As β-coronavirus lineage A, for example, HCoV-HKU1 and HCoV-OC43 can be mentioned, and HCoV-OC43 can be preferably mentioned. As β-coronavirus lineage B, for example, SARS-CoV and SARS-CoV-2 can be mentioned, and SARS-CoV-2 can be preferably mentioned. As β-coronavirus lineage C, MERS-CoV can be preferably mentioned.

[0506] In one embodiment, examples of coronavirus include HCoV-229E, HCoV-OC43, and / or SARS-CoV-2, and SARS-CoV-2 is particularly preferred.

[0507] It should be noted that it is generally known that viruses mutate during repeated proliferation and infection. Among the above coronaviruses, as long as the compounds of the present invention can exert coronavirus 3CL protease inhibitory activity, not only variants known in the art, but also variants that will appear in the future are included. As known variants of SARS-CoV-2, for example, the variants used in the examples of this specification can be cited.

[0508] As coronavirus infectious diseases, infectious diseases caused by HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV, MERS-CoV, and / or SARS-CoV-2 can be mentioned. Infectious diseases caused by HCoV-229E, HCoV-OC43, and / or SARS-CoV-2 are preferably mentioned, and infectious diseases caused by SARS-CoV-2 are particularly preferably mentioned.

[0509] As the coronavirus infectious disease, the novel coronavirus infectious disease (COVID-19) is particularly preferably mentioned.

[0510] The following is an example of the severity of the illness of people infected with the new coronavirus. (Reference: New Coronavirus Infectious Disease COVID-19 Diagnosis and Treatment Guidelines Version 5.2 (Ministry of Health, Labor and Welfare of Japan))

[0511] (mild)

[0512] Oxygen saturation was above 96%. Clinical status was: no respiratory symptoms, or only coughing, no dyspnea, and no pneumonia was found in any case.

[0513] (Medium I)

[0514] Oxygen saturation is less than 96% and greater than 93%. There is dyspnea and signs of pneumonia.

[0515] (Moderate II)

[0516] Oxygen saturation is less than 93%. There is respiratory failure and oxygen is required.

[0517] (severe)

[0518] Admission to the ICU or requiring artificial respiration.

[0519] The above classification is the definition of Japan's disease severity classification. For example, the disease severity classification of China and the United States NIH can be cited.

[0520] In addition, asymptomatic SARS-CoV-2 infected people refer to asymptomatic carriers of the pathogen. For example, people who have not confirmed any of the 14 symptoms of COVID-19 can be cited.

[0521] (14 symptoms of COVID-19: fatigue, muscle or body aches, headache, chills, feeling hot, abnormal taste, abnormal smell, runny or stuffy nose, sore throat, cough, shortness of breath, nausea, vomiting, diarrhea)

[0522] In this manual, the 12 symptoms of COVID-19 include fatigue, muscle or body pain, headache, chills, feeling hot, runny or stuffy nose, sore throat, cough, shortness of breath, nausea, vomiting, and diarrhea.

[0523] In this specification, suppression of severe symptoms means: suppressing the severity of the disease in asymptomatic SARS-CoV-2 infected persons from rising to the level of mild, moderate I, moderate II, or severe disease.

[0524] In this specification, the suppression of severe symptoms means: suppressing the severity of the disease of an asymptomatic SARS-CoV-2 infected person or a mild SARS-CoV-2 infected person from rising to the level of moderate disease I, moderate disease II, or severe disease.

[0525] In this specification, the term "suppression of severe symptoms" means: suppressing the severity of an asymptomatic SARS-CoV-2 infected person, a mild SARS-CoV-2 infected person, or a moderate I SARS-CoV-2 infected person from rising to a level of severity classified as moderate II or severe.

[0526] In this specification, the term "suppression of severe symptoms" means: suppressing the severity of an asymptomatic SARS-CoV-2 infected person, a mild SARS-CoV-2 infected person, a moderate I SARS-CoV-2 infected person, or a moderate II SARS-CoV-2 infected person from rising to a level of severity that is classified as severe.

[0527] In this specification, the suppression of severe symptoms means that the risk of hospitalization and death of SARS-CoV-2 infected patients is reduced due to the virus proliferation inhibitory effect of the agent of the present invention.

[0528] In this specification, the term "suppression of severe symptoms" means that inflammation in the lungs of patients infected with SARS-CoV-2 is reduced by the viral proliferation inhibitory effect of the agent of the present invention.

[0529] In this specification, the suppression of severe symptoms means that pneumonia caused by SARS-CoV-2 virus infection is suppressed by the virus proliferation inhibitory effect of the agent of the present invention.

[0530] In this specification, the suppression of severe symptoms means that the excessive immune response of the host caused by the viral infection of SARS-CoV-2 is suppressed by the viral proliferation inhibitory effect of the agent of the present invention.

[0531] As one embodiment, the pharmaceutical composition of the present invention is administered to a SARS-CoV-2 infected person who has at least one of the risk factors for severe symptoms listed below.

[0532] 50 years and above

[0533] Obesity (BMI 30 kg / m 2 above)

[0534] Cardiovascular disease (including cardiovascular system diseases including hypertension and congenital heart disease)

[0535] Chronic lung disease (including asthma, interstitial lung disease)

[0536] Type 1 or type 2 diabetes

[0537] Chronic renal impairment (including dialysis patients)

[0538] Chronic liver disease

[0539] Immunosuppressive conditions (e.g., cancer treatment, bone marrow or organ transplantation, immunodeficiency, poorly controlled HIV, AIDS, sickle cell anemia, thalassemia, long-term administration of immunosuppressants)

[0540] Chronic obstructive pulmonary disease (COPD)

[0541] Dyslipidemia

[0542] Smoking

[0543] Immunodeficiency after solid organ transplantation

[0544] ·Pregnancy

[0545] Patients with neurodevelopmental diseases and complex medical conditions (e.g. cerebral palsy, congenital diseases)

[0546] Patients with high medical dependency (e.g., tracheotomy, gastrostomy, positive pressure ventilation)

[0547] As one embodiment, the pharmaceutical composition of the present invention can be used to suppress the aggravation of COVID-19 symptoms by administering it to an infected person having at least one risk factor for severe symptoms.

[0548] As one embodiment, the pharmaceutical composition of the present invention can be used for patients suffering from pneumonia caused by SARS-CoV-2.

[0549] As one embodiment, the pharmaceutical composition of the present invention is administered to a SARS-CoV-2 infected person who has at least one of the risk factors for severe symptoms listed below.

[0550] Patients with ECMO (extracorporeal membrane oxygenation)

[0551] Patients on artificial respirators

[0552] Patients admitted to the ICU

[0553] Patients with oxygen saturation (SpO2) below 93% (room air) or requiring oxygen

[0554] As one embodiment, the pharmaceutical composition of the present invention is administered to a SARS-CoV-2 infected person who has at least one of the risk factors for severe symptoms listed below.

[0555] Oxygen saturation (SpO2) less than 94% (room air, sea level)

[0556] PaO2 / FiO2 less than 300 mmHg

[0557] Respiratory rate is more than 30 times per minute

[0558] Lung infiltration of more than 50%

[0559] (Method for producing the compound represented by formula (I))

[0560] The compound represented by formula (I) can be prepared, for example, by the usual synthesis method shown below. For extraction, purification, etc., the treatments performed in usual organic chemical experiments can be carried out. It can be synthesized with reference to methods known in the art. For extraction, purification, etc., the treatments performed in usual organic chemical experiments can be carried out.

[0561] The compound represented by formula (I) can be prepared by referring to methods known in the art. For example, it can be prepared by referring to WO2010092966, WO2012020749, WO2013089212, WO2014200078, WO2012020742 and WO2013118855.

[0562] (Method A)

[0563] [Chemical formula 24]

[0564]

[0565] (wherein, Alk is a C1-C3 alkyl group, Lg 1 is the leaving group, R 6is a hydrogen atom, and other symbols have the same meanings as above.)

[0566] (1st step)

[0567] Compound (A-1) or its hydrochloride or bromate is reacted with isocyanate (A-2) or 1-carbamoyl imidazole (A-2') in a solvent such as N,N-dimethylformamide, N,N-dimethylacetamide, N,N'-dimethylimidazolidinone, dimethyl sulfoxide, THF, in the presence of a base such as DBU, triethylamine, N,N-diisopropylethylamine, pyridine, etc. (preferably DBU) at -20°C to 50°C, preferably -10°C to ice-cooling. Subsequently, the reaction mixture is reacted with a carbonylating agent such as 1,1'-carbonyldiimidazole, phosgene, triphosgene, and a base such as DBU, triethylamine, N,N-diisopropylethylamine, pyridine, etc. (preferably DBU) at -20°C to 50°C, preferably -10°C to ice-cooling, to produce compound (A-3).

[0568] (Second step)

[0569] Compound (A-5) can be produced by reacting compound (A-3) with compound (A-4) in a solvent such as acetonitrile, acetone, DMF, DMSO, in the presence of a base such as potassium carbonate, sodium carbonate, N,N-diisopropylethylamine, at 50°C to reflux, preferably under reflux.

[0570] Examples of the leaving group include halogen and -OSO2 (C t F 2t+1 )(wherein t is an integer of 1 to 4) etc. As halogen, chlorine, iodine and bromine are preferred. As -OSO2(C t F 2t+1 ) group, preferably a -OTf group (trifluoromethanesulfonate).

[0571] (Step 3)

[0572] The compound represented by compound (I) can be produced by reacting compound (A-5) with compound (A-6) or compound (A-6') in a solvent such as NMP, DMF, DMA, DMSO, tert-butanol, 2-methyl-2-butanol, or the like, in the presence or absence of an acid such as acetic acid at 60°C to 150°C, preferably 80°C to 120°C.

[0573] By using the optically active isocyanate (A-2), a compound represented by the optically active compound (I) can be produced.

[0574] (Method B)

[0575] [Chemical formula 25]

[0576]

[0577] (wherein, Alk is a C1-C3 alkyl group, Pro is a C1-C4 alkyl group or a tert-butoxycarbonyl group, Lg 2 is the leaving group, R 6 is a hydrogen atom, and other symbols have the same meanings as above.)

[0578] (1st step)

[0579] Compound (B-2) can be produced from compound (B-1) in the same manner as in the second step of method A above.

[0580] (Second step)

[0581] Compound (B-3) can be produced by treating compound (B-2) with a strong acid such as TFA in the presence or absence of an organic solvent at -20°C to room temperature, preferably room temperature.

[0582] (Step 3)

[0583] Compound (B-4) can be produced from compound (B-3) in the same manner as in step 3 of method A above.

[0584] (Step 4)

[0585] Compound (IX) can be produced by a Goldberg amination reaction using compound (B-4) and compound (B-5).

[0586] Examples of the leaving group include the leaving groups described in the above-mentioned process A, step 1.

[0587] As the catalyst, for example, a commercially available copper catalyst such as copper iodide, copper cyanide, or copper bromide can be used.

[0588] As the ligand, 1,2-dimethylethylenediamine, trans-N,N′-dimethylcyclohexane-1,2-diamine, or the like can be used.

[0589] As the base, potassium carbonate, potassium phosphate or the like can be used.

[0590] As the solvent, NMP, dioxane, DMSO or the like can be used.

[0591] The reaction temperature may be from room temperature to a temperature at which the solvent is refluxed, and the reaction may be preferably carried out under heating under reflux.

[0592] (C Method)

[0593] [Chemical formula 26]

[0594]

[0595] (wherein, Alk is a C1-C3 alkyl group, Lg 3 is a leaving group, and other symbols have the same meanings as above. )

[0596] (1st step)

[0597] Compound (C-2) can be produced in the same manner as in the second step of the above-mentioned method A.

[0598] Examples of the leaving group include the leaving groups described in the above-mentioned process A, step 1.

[0599] (Second step)

[0600] The compound represented by compound (I) can be produced in the same manner as in step 3 of method A above.

[0601] The compounds of the present invention have coronavirus 3CL protease inhibitory activity and are therefore useful as therapeutic and / or preventive agents for viral infectious diseases.

[0602] Furthermore, the compounds according to the present invention have usefulness as medicines and preferably have any one or more of the following excellent characteristics.

[0603] a) The inhibitory effect on CYP enzymes (e.g., CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, etc.) is weak.

[0604] b) It exhibits good pharmacokinetics such as high bioavailability and moderate clearance.

[0605] c) High metabolic stability.

[0606] d) It does not show irreversible inhibitory action on CYP enzymes (e.g., CYP3A4) within the concentration range of the assay conditions described in the present specification.

[0607] e) Not mutagenic.

[0608] f) Low risk to the cardiovascular system.

[0609] g) Shows high solubility.

[0610] h) The protein unbound rate (fu value) is high.

[0611] i) High selectivity for coronavirus 3CL protease.

[0612] j) It has high coronavirus proliferation inhibition activity. For example, it has high coronavirus proliferation inhibition activity when human serum (HS) or human serum albumin (HSA) is added.

[0613] Examples of coronavirus proliferation inhibitors include those that have been tested in the CPE inhibition effect confirmation test (SARS-CoV-2) described below, such as EC 50 The concentration is 10 μM or less, preferably 1 μM or less, and more preferably 100 nM or less.

[0614] In addition, the salt, crystal, or complex (co-crystal) of the compound represented by formula (I) has usefulness as a medicine and preferably has any one or more of the following excellent characteristics.

[0615] A) It shows high bioavailability, moderate clearance, high AUC, high maximum blood concentration, etc., and good pharmacokinetics.

[0616] B) Shows high solubility, high chemical stability and low hygroscopicity.

[0617] The pharmaceutical composition of the present invention can be administered by any method, either orally or parenterally. Examples of parenteral administration methods include transdermal, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, transmucosal, inhalation, nasal, eye drops, ear drops, and intravaginal administration.

[0618] In the case of oral administration, it can be prepared into any commonly used dosage form 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, alcohols, aromatic waters, extracts, decoctions, tinctures, etc.) according to conventional methods. Tablets can be sugar-coated tablets, film-coated tablets, enteric-coated tablets, sustained-release tablets, lozenges, sublingual tablets, buccal tablets, chewable tablets or orally disintegrating tablets, powders and granules can be dry syrups, and capsules can be soft capsules, microcapsules or sustained-release capsules.

[0619] In the case of parenteral administration, it can be appropriately administered by any commonly used dosage form such as injection, drip, external preparation (e.g., eye drops, nose drops, ear drops, aerosol, inhalation, lotion, injection, coating, mouthwash, enema, ointment, plaster, jelly, cream, patch, cataplasm, external powder, suppository, etc.). Injection can be an emulsion such as O / W, W / O, O / W / O, W / O / W type.

[0620] According to the need, various pharmaceutical additives such as excipients, binders, disintegrants, lubricants suitable for the dosage form can be mixed in the effective amount of the compound of the present invention to prepare a pharmaceutical composition. In addition, the pharmaceutical composition can also be prepared into a pharmaceutical composition for children, the elderly, critically ill patients or surgery by appropriately changing the effective amount, dosage form and / or various pharmaceutical additives of the compound of the present invention. For example, a pharmaceutical composition for children can be administered to a newborn (less than 4 weeks after birth), an infant (4 weeks after birth and less than 1 year old), a toddler (over 1 year old and less than 7 years old), a child (over 7 years old and less than 15 years old) or a patient aged 15 to 18 years. For example, a pharmaceutical composition for the elderly can be administered to a patient over 65 years old.

[0621] The dosage of the pharmaceutical composition of the present invention (for example, a pharmaceutical composition comprising a p-toluenesulfonate type I crystal of the compound represented by formula (IA) or a pharmaceutical composition comprising a fumaric acid cocrystal type I of the compound represented by formula (IB)) is preferably set based on the patient's age, weight, type and degree of the disease, administration route, etc., and is generally within the range of 0.05 to 200 mg / kg / day, preferably 0.1 to 100 mg / kg / day in the case of oral administration. In the case of parenteral administration, although there are large differences depending on the administration route, it is generally within the range of 0.005 to 200 mg / kg / day, preferably 0.01 to 100 mg / kg / day. It can be administered once a day or divided into several times.

[0622] For the compound involved in the present invention, for the purpose of enhancing the effect of the compound or reducing the dosage of the compound, for example, it can be used in combination with other novel coronavirus infectious diseases (COVID-19) therapeutic drugs (including approved drugs as the therapeutic drugs, and drugs under development or in the future) (hereinafter referred to as combined drugs). At this time, the timing of administration of the compound involved in the present invention and the combined drug is not limited, and they can be administered to the object of administration at the same time, or they can be administered at intervals of time. In addition, the compound involved in the present invention and the combined drug can be administered in the form of more than two preparations containing each active ingredient, or can be administered in the form of a single preparation containing their active ingredients.

[0623] The dosage of the combined drug can be appropriately selected based on the dosage used clinically. In addition, the compound of the present invention and the combined drug ratio can be appropriately selected according to the administration object, administration route, target disease, symptoms, combination, etc. For example, when the administration object is human, 0.01 to 100 parts by weight of the combined drug can be used relative to 1 part by weight of the compound of the present invention.

[0624] Example

[0625] Hereinafter, the present invention will be described in more detail with reference to Examples, Reference Examples, and Test Examples, but the present invention is not limited thereto.

[0626] In addition, the abbreviations used in this specification have the following meanings.

[0627] Boc: tert-Butyloxycarbonyl

[0628] CDI: Carbonyldiimidazole

[0629] DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene

[0630] DIEA: N,N-diisopropylethylamine

[0631] DMA: N,N-dimethylacetamide

[0632] DMF: N,N-dimethylformamide

[0633] DMSO: dimethyl sulfoxide

[0634] DTT: dithiothreitol

[0635] EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0636] EDT: 1,2-ethanedithiol

[0637] EDTA: Ethylenediaminetetraacetic acid

[0638] FBS: Fetal bovine serum

[0639] HOBT: 1-Hydroxybenzotriazole

[0640] LHMDS: Lithium bis(trimethylsilyl)amide

[0641] MEM: Eagle's minimum essential medium

[0642] NMP: N-methylpyrrolidone

[0643] Pd(OAc)2: Palladium acetate

[0644] TFA: trifluoroacetic acid

[0645] TMSCl: trimethylchlorosilane

[0646] Xantphos: 4,5'-bis(diphenylphosphino)-9,9'-dimethylxanthene

[0647] mM:mmol / L

[0648] μM: μmol / L

[0649] nM: nmol / L

[0650] (Method for identification of compounds)

[0651] The NMR analysis obtained in each example was performed at 400 MHz using DMSO-d6, CDCl3, and MeOH-d4. In addition, when NMR data are shown, all the peaks measured may not be described.

[0652] In the specification, RT means retention time in LC / MS: liquid chromatography / mass spectrometry, and the measurement is performed under the following conditions.

[0653] (Measurement Condition 1)

[0654] Column: ACQUITY UPLC (registered trademark) BEH C18 (1.7 μm i.d. 2.1 x 50 mm) (Waters)

[0655] Flow rate: 0.8 mL / min

[0656] UV detection wavelength: 254nm

[0657] Mobile phase: [A] is an aqueous solution containing 0.1% formic acid, [B] is an acetonitrile solution containing 0.1% formic acid

[0658] Gradient: A linear gradient from 5% to 100% solvent [B] was performed over 3.5 minutes, and then 100% solvent [B] was maintained for 0.5 minutes.

[0659] (Measurement Condition 2)

[0660] Column: Shim-pack XR-ODS (2.2μm, id3.0x50mm) (Shimadzu)

[0661] Flow rate: 1.6mL / min

[0662] UV detection wavelength: 254nm

[0663] Mobile phase: [A] is an aqueous solution containing 0.1% formic acid, [B] is an acetonitrile solution containing 0.1% formic acid

[0664] Gradient: Linear gradient from 10% to 100% solvent [B] over 3 minutes, maintaining 100% solvent [B] for 0.5 minutes.

[0665] In addition, in this specification, the description of MS (m / z) means the value observed by mass spectrometry.

[0666] (Measurement of powder X-ray diffraction pattern)

[0667] The powder X-ray diffraction measurement of the crystals obtained in each example was performed according to the powder X-ray diffraction measurement method described in the General Test Methods of the Japanese Pharmacopoeia. The measurement conditions are as follows.

[0668] (Device)

[0669] SmartLab made by Rigaku Corporation

[0670] (Operation method)

[0671] Measurement method: Reflection method

[0672] Use wavelength: CuKα line

[0673] Tube current: 200mA

[0674] Tube voltage: 45kV

[0675] Sample plate: Aluminum

[0676] X-ray incident angle: 2.5°

[0677] Sampling width: 0.02°

[0678] Detector: HyPix-3000 (2D detection mode)

[0679] (Determination and analysis methods of single crystal structure analysis)

[0680] The measurement conditions and analysis method of the single crystal structure analysis are shown below.

[0681] (Device)

[0682] XtaLAB P200 MM007 manufactured by Rigaku Corporation

[0683] (Measurement conditions)

[0684] Measurement temperature: 25℃

[0685] Use wavelength: CuKα line

[0686] Software: CrysAlisPro 1.171.39.46e (Rigaku Oxford Diffraction, 2018)

[0687] (Data Processing)

[0688] Software: CrysAlisPro 1.171.39.46e (Rigaku Oxford Diffraction, 2018)

[0689] The data were subjected to Lorentz, polarization and absorption corrections.

[0690] (Crystal Structure Analysis)

[0691] Phase determination was performed using the direct method program ShelXT (Sheldrick, GM, 2015), and refinement was performed using ShelXL (Sheldrick, GM, 2015) using the full-matrix least squares method. The displacement parameters of non-hydrogen atoms were all refined anisotropically. Hydrogen atoms were introduced by calculation using the default parameters of ShelXL and treated as riding atoms. All hydrogen atoms were refined with isotropic parameters.

[0692] Figure 2 and Figure 4 PLATON (Spek, 1991) / ORTEP (Johnson, 1976) were used in the mapping.

[0693] [Example 1]

[0694] Synthesis of compound (I-003)

[0695] [Chemical formula 27]

[0696]

[0697] Step 1 Synthesis of Compound 14

[0698] 3,4,5-Trifluorobenzylamine (3.34 g, 20.7 mmol) was dissolved in dichloromethane (33.4 mL) and cooled in a water bath. Benzoyl isothiocyanate (2.93 mL, 21.8 mmol) was added to the reaction solution and stirred at room temperature for 30 minutes.

[0699] The solvent was distilled off, the residue was diluted with methanol, and 1 mol / L sodium hydroxide aqueous solution (7.45 mL, 7.45 mmol) was added. The reaction solution was stirred at room temperature for 30 minutes, and 2 mol / L hydrochloric acid aqueous solution was added. The aqueous layer was extracted with ethyl acetate, and the organic layer was washed with saturated sodium bicarbonate aqueous solution and saturated brine. The organic layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (8.3 g) of compound 14. The crude product was used in the next step as a yield of 100% without further purification.

[0700] LC / MS (ESI): m / z = 221, RT = 1.45 min, LC / MS measurement condition 2

[0701] Step 2 Synthesis of Compound 15

[0702] The crude product of compound 14 (8.3 g), DMF (85 mL), and iodomethane (4.84 mL, 77 mmol) were mixed, and the reaction solution was stirred at 50°C for 40 minutes. Water was added to the reaction solution, extracted with ethyl acetate, and washed with water. A 2 mol / L sodium hydroxide aqueous solution was added to the water layer, and the water layer was extracted with ethyl acetate. The organic layer was washed with water and saturated brine, and dried over sodium sulfate. The solvent was distilled off under reduced pressure to obtain a crude product of compound 15 (3.86 g, 16.5 mmol, yield 80%).

[0703] LC / MS (ESI): m / z = 235, RT = 0.84 min, LC / MS measurement condition 2

[0704] Step 3 Synthesis of Compound 16

[0705] Triphosgene (0.507 g, 1.71 mmol) and THF (6 mL) were mixed, and the reaction solution was cooled in an ice bath. 3-Amino-5-methylpyridine (0.462 g, 4.27 mmol) and triethylamine (1.48 mL, 10.7 mmol) were mixed in THF (6 mL), and the resulting solution was added dropwise to the reaction solution. The reaction solution was stirred at room temperature for 40 minutes, and then cooled in an ice bath. Compound 15 (1 g, 4.27 mmol) was added to the reaction solution and stirred at room temperature for 55 minutes. Water was added, the aqueous layer was extracted with ethyl acetate, and the organic layer was washed with water. The organic layer was dried with magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product of compound 16 (1.57 g, 4.26 mmol, yield: quantitative).

[0706] LC / MS (ESI): m / z = 369, RT = 1.52 min, LC / MS measurement conditions 1

[0707] Step 4 Synthesis of Compound 17

[0708] CDI (2.78 g, 17.2 mmol), compound 16 (1.58 g, 4.29 mmol) and DMF (12.6 mL) were mixed. Diisopropylethylamine (3.00 mL, 17.2 mmol) was added to the reaction solution, and microwaves were irradiated for 30 minutes while stirring at 110°C. The reaction solution was poured into ice, and the resulting precipitate was filtered out and washed with water. The obtained residue was dried under reduced pressure to obtain a crude product of compound 17 (649 mg, 1.51 mmol, yield 35%). LC / MS (ESI): m / z = 395, RT = 1.74 min, LC / MS measurement conditions 1

[0709] Step 5 Synthesis of Compound (I-003)

[0710] 6-Chloro-2-methyl-2H-indazole-5-amine (55.3 mg, 0.304 mmol), compound 17 (100 mg, 0.254 mmol) and THF (1 mL) were mixed. The reaction solution was cooled in an ice bath and LHMDS (0.761 mL, 0.761 mmol) was added. The reaction solution was stirred in an ice bath for 40 minutes and a saturated aqueous solution of ammonium chloride was added. The organic layer was extracted with ethyl acetate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform / methanol) to obtain compound (I-003) (80 mg, 0.145 mmol, yield 57.4%).

[0711] 1 H-NMR(Methanol-d4)δ:8.43(d,J=1.0Hz,1H),8.36(d,J=2.0Hz,1H),8.18(s,1H),7.74(s,1H),7.72(br s,1H),7.48-7.35(m,3H),5.32(s,2H),4.20(s,3H),2.42(s,3H).

[0712] LC / MS (ESI): m / z = 528, RT = 1.93 min, LC / MS measurement conditions 1

[0713] [Example 2]

[0714] Synthesis of compound (I-005)

[0715] [Chemical formula 28]

[0716]

[0717] Step 1 Synthesis of Compound 18

[0718] Compound 4 (926 mg, 4.04 mmol) (synthesis method, see WO2012020749, WO2013089212 and WO2014200078), acetonitrile (7.41 mL), potassium carbonate (726 mg, 5.25 mmol) and 2,4,5-trifluorobenzyl bromide (1000 mg, 4.44 mmol) were mixed. The reaction solution was stirred at 80 ° C for 40 minutes, left to cool, and then diluted with ethyl acetate. After filtering out the insoluble matter, the filtrate was concentrated to obtain a crude product of compound 18 (1.51 g, 4.04 mmol, yield: quantitative).

[0719] LC / MS (ESI): m / z = 374, RT = 2.54 min, LC / MS measurement conditions 1

[0720] Step 2 Synthesis of Compound 19

[0721] Compound 18 (1.51 g, 4.04 mmol) and TFA (3.02 mL) were mixed. The reaction solution was stirred at room temperature for 4 hours and allowed to stand overnight. TFA was distilled off under reduced pressure, and toluene was added to the residue for azeotropy. Isopropyl ether was added to the residue, and the mixture was suspended and filtered to obtain compound 19 (1.22 g, 3.84 mmol, yield 95%). LC / MS (ESI): m / z = 318, RT = 1.68 min, LC / MS measurement conditions 1

[0722] Step 3 Synthesis of Compound 20

[0723] Compound 19 (200 mg, 0.63 mmol), DMF (1.8 mL), potassium carbonate (261 mg, 1.89 mmol) and 3-(chloromethyl)-1-methyl-1H-1,2,4-triazole hydrochloride (159 mg, 0.946 mmol) were mixed. The reaction solution was stirred at 60°C for 2 hours, and a saturated aqueous solution of ammonium chloride was added. The aqueous layer was extracted with ethyl acetate, and the organic layer was washed with saturated brine. The organic layer was dried over magnesium sulfate, filtered, and concentrated. The residue was suspended in a mixed solvent of isopropyl ether, hexane, ethyl acetate, and chloroform, and filtered. The residue, DMF (1.8 mL), potassium carbonate (261 mg, 1.89 mmol) and 3-(chloromethyl)-1-methyl-1H-1,2,4-triazole hydrochloride (159 mg, 0.946 mmol) were mixed. The reaction solution was stirred at 60°C for 6 hours, and a saturated aqueous solution of ammonium chloride was added. The aqueous layer was extracted with ethyl acetate, and the organic layer was washed with saturated brine. The organic layer was dried over magnesium sulfate, filtered, and concentrated. The residue was suspended in a mixed solvent of isopropyl ether, hexane, ethyl acetate, and chloroform, and filtered to obtain Compound 20 (116 mg, 0.281 mmol, yield 45%).

[0724] LC / MS (ESI): m / z = 413, RT = 1.84 min, LC / MS measurement conditions: 1. Step 4 Synthesis of compound (I-005)

[0725] Compound 20 (115 mg, 0.279 mmol), THF (2.30 mL) and 6-chloro-2-methyl-2H-indazol-5-amine (60.8 mg, 0.335 mmol) were mixed. LHMDS (558 μL, 0.558 mmol) was added dropwise to the reaction solution at 0°C. The reaction solution was stirred at 0°C for 2.5 hours and at room temperature for 40 minutes, and a saturated aqueous solution of ammonium chloride was added. The organic layer was concentrated after extraction with chloroform. The residue was purified by silica gel column chromatography (chloroform / methanol) to obtain compound (I-005) (61.8 mg, 0.116 mmol, yield 42%).

[0726] 1 H-NMR (CDCl3) δ: 7.96 (s, 1H), 7.82 (d, J = 2.5Hz, 2H), 7.48 (br s,1H),7.45-7.37(m,1H),7.08(s,1H),6.97-6.88(m,1H),5.35(s,2H),5.17(s,2H),4.21(s,3H),3.89(s,3H).

[0727] LC / MS (ESI): m / z = 532, RT = 1.70 min, LC / MS measurement conditions 1

[0728] According to the above-mentioned common synthesis method and the method described in Examples 1 and 2, the compounds that can be used in the present invention are synthesized. Including the compounds manufactured in Examples 1 and 2, the structures and physical properties (LC / MS data) of each compound are shown in the following table. The compounds described in the table with amino structures sometimes also have imino structures, and the compounds shown with imino structures sometimes also have amino structures. That is, even if it is the same compound, according to the crystallization conditions, there are cases with imino structures and cases with amino structures, even if a salt or a complex is formed, according to the type of the countermolecule of the salt or the complex, there are cases with imino structures and cases with amino structures, even if it is the same countermolecule, according to the crystallization conditions, there are cases with imino structures and cases with amino structures. In addition, sometimes it is a mixture of a compound with an imino structure, a salt thereof, or a complex thereof and a compound with an amino structure, a salt thereof, or a complex thereof.

[0729] [Table 1]

[0730]

[0731] [Table 2]

[0732]

[0733] [Table 3]

[0734]

[0735] [Table 4]

[0736]

[0737] [Table 5]

[0738]

[0739] [Table 6]

[0740]

[0741] [Table 7]

[0742]

[0743] [Table 8]

[0744]

[0745] [Table 9]

[0746]

[0747] [Example 3]

[0748] To 1400 mg of compound (I-003), 7 mL of ethyl acetate and 557 μL (1.05 eq) of a 5 mol / L aqueous solution of p-toluenesulfonic acid were added. The mixture was stirred at 60°C for 15 minutes and then at 25°C for 2 hours. The solid was filtered and dried to obtain p-toluenesulfonate type I crystals of the compound represented by formula (IA) (1289.6 mg, 69%).

[0749] The results of single crystal structural analysis of type I crystal of the p-toluenesulfonate salt of the compound represented by formula (IA) are shown below.

[0750] R1 (I>2.00s(I)) was 0.0444, and the final difference Fourier analysis confirmed that there was neither loss nor misplacement of electron density.

[0751] The crystallographic data are shown in Table 10.

[0752] [Table 10]

[0753]

[0754] Here, volume refers to the unit cell volume, and Z refers to the number of molecules in the unit cell.

[0755] The structure of the p-toluenesulfonate salt type I crystal of the compound represented by formula (IA) in the asymmetric unit is shown in Figure 2 .

[0756] In the asymmetric unit, one molecule of the compound represented by formula (IA) and one molecule of p-toluenesulfonic acid were present. No hydrogen atom was bonded to the oxygen atoms represented by O3, O4, and O5 of p-toluenesulfonic acid, but a hydrogen atom was bonded to the nitrogen atom at N7 of the compound represented by formula (IA), thereby confirming the formation of a salt.

[0757] The bond length of N3-C9 is about The bond length of N4-C9 is about The bond length identified the compound represented by formula (IA) in the p-toluenesulfonate type I crystal as an imino structure:

[0758] [Chemical formula 29]

[0759]

[0760] In addition, the results of powder X-ray diffraction of type I crystals of the p-toluenesulfonate salt of the compound represented by formula (IA) are shown.

[0761] In the powder X-ray diffraction pattern, peaks were confirmed at diffraction angles (2θ): 9.1±0.2°, 11.5±0.2°, 14.6±0.2°, 15.2±0.2°, 18.8±0.2°, 20.2±0.2°, 23.6±0.2°, 24.2±0.2°, 24.9±0.2°, and 26.9±0.2°.

[0762] In the powder X-ray diffraction pattern, the peaks at diffraction angles (2θ): 9.1±0.2°, 15.2±0.2°, 18.8±0.2°, 23.6±0.2° and 24.9±0.2° are particular characteristics of the p-toluenesulfonate type I crystal of the compound represented by formula (IA).

[0763] [Example 4]

[0764] To 1170 mg of compound (I-005) were added 278 mg (1.1 eq) of fumaric acid and 5.85 mL of ethyl acetate, and the mixture was stirred at room temperature for 45 minutes. The solid was filtered and dried to obtain fumaric acid cocrystal type I crystals of the compound represented by formula (IB) (1369.4 mg, 94.6%).

[0765] The results of single crystal structural analysis of fumaric acid cocrystal Form I of the compound represented by formula (IB) are shown below.

[0766] R1 (I>2.00s(I)) was 0.0470, and the final difference Fourier analysis confirmed that there was neither loss nor misplacement of electron density.

[0767] The crystallographic data are shown in Table 11.

[0768] [Table 11]

[0769]

[0770] Here, volume refers to the unit cell volume, and Z refers to the number of molecules in the unit cell.

[0771] The structure of the asymmetric unit of the fumaric acid cocrystal type I of the compound represented by formula (IB) is shown in Figure 4 .

[0772] In the asymmetric unit, one molecule of the compound represented by formula (IB) and one molecule of fumaric acid were present. No ionic chemical interaction was confirmed, and a co-crystal with a molar ratio of 1:1 was confirmed.

[0773] The bond length of N10-C9 is about , the bond length of N16-C9 is about The bond length identified the compound represented by formula (IB) of fumaric acid cocrystal type I as an imino structure:

[0774] [Chemical formula 30]

[0775]

[0776] In addition, the results of powder X-ray diffraction of the fumaric acid cocrystal type I crystal of the compound represented by formula (IB) are shown.

[0777] In the powder X-ray diffraction pattern, peaks were confirmed at diffraction angles (2θ) of 7.8±0.2°, 9.5±0.2°, 10.1±0.2°, 10.9±0.2°, 13.8±0.2°, 14.7±0.2°, 18.6±0.2°, 22.6±0.2°, 23.5±0.2°, and 24.6±0.2°.

[0778] In the above powder X-ray diffraction pattern, the peaks of diffraction angles (2θ): 9.5±0.2°, 10.9±0.2°, 18.6±0.2°, 23.5±0.2° and 24.6±0.2° are special characteristics of the fumaric acid cocrystal type I crystal of the compound represented by formula (IB).

[0779] The biological test examples of the compounds of the present invention are described below. The compounds of the present invention can be tested essentially as in the following test examples.

[0780] The compound represented by formula (I) according to the present invention may be any compound as long as it has a coronavirus 3CL protease inhibitory effect and inhibits coronavirus 3CL protease.

[0781] Specifically, in the evaluation method described below, IC50 is preferably 50 μM or less, more preferably 1 μM or less, and even more preferably 100 nM or less.

[0782] Test Example 1: Cytopathic effect (CPE) inhibition test using human TMPRSS2-expressing Vero E6 cells (Vero E6 / TMPRSS2 cells)

[0783] <Operation steps>

[0784] Dilution and injection of test samples

[0785] The test sample was diluted to an appropriate concentration with DMSO in advance, and a 2- to 5-fold serial dilution series was prepared and dispensed into a 384-well plate.

[0786] Dilution and injection of cells and SARS-CoV-2

[0787] VeroE6 / TMPRSS2 cells (JCRB1819, 5×10 3 cells / well) and SARS-CoV-2 (100-300 TCID 50 / well) were mixed in a culture medium (MEM, 2% FBS, penicillin-streptomycin), dispensed into the wells containing the test samples, and cultured in a CO2 incubator for 3 days.

[0788] · CellTiter-Glo (registered trademark) 2.0 injection and luminescence signal measurement

[0789] After the plate was returned to room temperature after culturing for 3 days, CellTiter-Glo (registered trademark) 2.0 was dispensed into each well and mixed using a plate mixer. After standing for a certain period of time, the luminescence signal (Lum) was measured using a microplate reader.

[0790] <Calculation of each measurement item value>

[0791] 50% SARS-CoV-2 infected cell death inhibition concentration (EC 50 )calculate

[0792] When x is the logarithmic value of the compound concentration and y is the % efficacy, the inhibition curve is fitted by the following logistic regression equation, and the x value when y=50(%) is calculated as the EC 50 .

[0793] y=min+(max-min) / {1+(X50 / x)^Hill}

[0794] % efficacy = {(sample - virus control) / (cell control - virus control)}*100%

[0795] (%Efficacy={(Sample-virus control) / (cell control-virus control)}*100%)

[0796] Cell control: the average Lum of cell control wells

[0797] Virus control: the average Lum of virus control wells

[0798] min: y-axis lower limit, max: y-axis upper limit, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max

[0799] The compounds of the invention were tested essentially as described above. The results are shown in the table below.

[0800] It should be noted that EC 50 A value of less than 1 μM was recorded as "A", and a value of 1 μM or more and less than 10 μM was recorded as "B".

[0801] Compound I-003: 0.177 μM

[0802] Compound I-005: 0.328 μM

[0803] Compound I-006: 0.747 μM

[0804] Compound I-010: 0.306 μM

[0805] Compound I-012: 0.131 μM

[0806] Compound I-017: 0.0960 μM

[0807] Compound I-023: 1.03 μM

[0808] Compound I-035: 0.395 μM

[0809] [Table 12]

[0810]

[0811] Test Example 1-2: Test to confirm the cytopathic effect (CPE) inhibition effect using human TMPRSS2-expressing Vero E6 cells (Vero E6 / TMPRSS2 cells)

[0812] <Operation steps>

[0813] Dilution and injection of test samples

[0814] The test sample was diluted to an appropriate concentration with DMSO in advance, and a 3-fold serial dilution series was prepared and dispensed into a 96-well plate.

[0815] Dilution and injection of cells and SARS-CoV-2

[0816] VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4 cells / well) and SARS-CoV-2hCoV-19 / Japan / TY / WK-521 / 2020, hCoV-19 / Japan / QK002 / 2020, hCoV-19 / Japan / QHN001 / 2020, hCoV-19 / Japan / QHN002 / 2020, hCoV-19 / Japan / TY7-501 / 2021, hCoV-19 / Japan / TY7-503 / 2021, hCoV-19 / Japan / TY8-612 / 2021, hCoV-19 / Japan / TY11-927-P1 / 2021, hC oV-19 / Japan / TY33-456 / 2021, hCoV-19 / Japan / TY28-444 / 2021, hCoV-19 / Japan / TY26-717 / 2021 (30-3000TCID 50 / well) were mixed in a culture medium (MEM, 2% FBS, penicillin-streptomycin), dispensed into the wells containing the test samples, and cultured in a CO2 incubator for 3 or 4 days.

[0817] · CellTiter-Glo (registered trademark) 2.0 injection and luminescence signal measurement

[0818] After the plate was returned to room temperature after culturing for 3 days, CellTiter-Glo (registered trademark) 2.0 was dispensed into each well and mixed using a plate mixer. After standing for a certain period of time, the luminescence signal (Lum) was measured using a microplate reader.

[0819] <Calculation of each measurement item value>

[0820] 50% SARS-CoV-2 infected cell death inhibition concentration (EC 50 )calculate

[0821] When x is the logarithmic value of the compound concentration and y is the % efficacy, the inhibition curve is fitted by the following Logistic regression equation, and the x value when y=50(%) is calculated as the EC 50 .

[0822] y=min+(max-min) / {1+(X50 / x)^Hill}

[0823] % efficacy = {(sample - virus control) / (cell control - virus control)}*100%

[0824] Cell Control: Average Lum of cell control wells

[0825] Virus control: Average Lum of virus control wells

[0826] min: y-axis lower limit, max: y-axis upper limit, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max

[0827] The compounds of the invention were tested essentially as described above. The results are shown below.

[0828] [Table 13-1]

[0829]

[0830] Test Example 1-3: Test to confirm the cytopathic effect (CPE) inhibition effect using human TMPRSS2-expressing Vero E6 cells (Vero E6 / TMPRSS2 cells)

[0831] <Operation steps>

[0832] Dilution and injection of test samples

[0833] The test sample was diluted to an appropriate concentration with DMSO in advance, and a 3-fold serial dilution series was prepared and dispensed into a 96-well plate.

[0834] Dilution and injection of cells and SARS-CoV-2

[0835] VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4 cells / well) and SARS-CoV-2 hCoV-19 / Japan / TY38-873 / 2021, hCoV-19 / Japan / TY38-871 / 2021, hCoV-19 / Japan / TY40-385 / 2022, hCoV-19 / Japan / TY41-716 / 2022, hCoV-19 / Japan / TY41-703 / 2022, hCoV-19 / Japan / TY41-702 / 2022, hCoV-19 / Japan / TY41-686 / 2022 (300-3000 TCID 50 / well) were mixed in a culture medium (MEM, 2% FBS, penicillin-streptomycin), dispensed into the wells containing the test samples, and cultured in a CO2 incubator for 4 days.

[0836] · CellTiter-Glo (registered trademark) 2.0 injection and luminescence signal measurement

[0837] After the plate was returned to room temperature after culturing for 3 days, CellTiter-Glo (registered trademark) 2.0 was dispensed into each well and mixed using a plate mixer. After standing for a certain period of time, the luminescence signal (Lum) was measured using a microplate reader.

[0838] <Calculation of each measurement item value>

[0839] 50% SARS-CoV-2 infected cell death inhibition concentration (EC 50 )calculate

[0840] When x is the logarithmic value of the compound concentration and y is the % efficacy, the inhibition curve is fitted by the following Logistic regression equation, and the x value when y=50(%) is calculated as the EC 50 .

[0841] y=min+(max-min) / {1+(X50 / x)^Hill}

[0842] % efficacy = {(sample - virus control) / (cell control - virus control)}*100%

[0843] Cell Control: Average Lum of cell control wells

[0844] Virus control: Average Lum of virus control wells

[0845] min: y-axis lower limit, max: y-axis upper limit, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max

[0846] The compounds of the invention were tested essentially as described above. The results are shown below.

[0847] [Table 13-2]

[0848]

[0849] Test Example 2: Inhibitory activity test on SARS-CoV-2 3CL protease

[0850] <Materials>

[0851] Commercially available recombinant SARS-CoV-2 3CL protease

[0852] Commercially available substrate peptides

[0853] Dabcyl-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu(Edans)-NH2(SEQ ID NO: 1)

[0854] Internal Standard Peptide

[0855] Dabcyl-Lys-Thr-Ser-Ala-Val-Leu(13C6,15N)-Gln(SEQ ID NO: 2)

[0856] Dabcyl-Lys-Thr-Ser-Ala-Val-Leu(13C6,15N)-Gln can be synthesized by reference to literature (Atherton, E.; Sheppard, RC, "In Solid Phase Peptide Synthesis, A Practical Approach", published by 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.

[0857] H-Lys-Thr-Ser-Ala-Val-Leu(13C6,15N)-Glu(resin)-OαOtBu (Lys side chain is protected by Boc, Thr side chain is protected by tert-butyl, Ser side chain is protected by tert-butyl, C-terminal OH of Glu is protected by tert-butyl, and the carboxylic acid of Glu side chain is condensed with resin) was synthesized by Fmoc solid phase synthesis using Rink amide resin. The modification of N-terminal Dabcyl group was carried out by condensing 4-dimethylaminoazobenzene-4'-carboxylic acid (Dabcyl-OH) on the resin with EDC / HOBT. The final deprotection and excision from the resin were carried out by treatment with TFA / EDT=95:5. Then, purification was carried out by reverse phase HPLC.

[0858] ·RapidFire Cartridge C4 typeA

[0859] <Operation steps>

[0860] Preparation of Assay Buffer

[0861] In this test, the detection buffer composed of 20mM Tris-HCl, 100mM NaCl, 1mM EDTA, 10mM DTT, and 0.01% BSA was used. 50For compounds with a concentration of 10 nM or less, a detection buffer consisting of 20 mM Tris-HCl, 1 mM EDTA, 10 mM DTT, and 0.01% BSA was used.

[0862] Dilution and injection of test samples

[0863] The test sample was diluted to an appropriate concentration with DMSO in advance, and a 2- to 5-fold serial dilution series was prepared and dispensed into a 384-well plate.

[0864] Enzyme and substrate addition, enzyme reaction

[0865] 8 μM substrate and 6 or 0.6 nM enzyme solution were added to the prepared compound plate and incubated at room temperature for 3 to 5 hours. Then, a reaction stop solution (0.067 μM internal standard, 0.1% formic acid, 10 or 25% acetonitrile) was added to stop the enzyme reaction.

[0866] Determination of reaction products

[0867] After the reaction, the plate was measured using a RapidFire System 360 and a mass spectrometer (Agilent, 6550iFunnel Q-TOF) or a Rapid Fire System 365 and a mass spectrometer (Agilent, 6495C TripleQuadrupole). As the mobile phase for the measurement, solution A (75% isopropanol, 15% acetonitrile, 5 mM ammonium formate) and solution B (0.01% trifluoroacetic acid, 0.09% formic acid) were used.

[0868] The reaction product detected by the mass spectrometer is calculated using RapidFire Integrator or an equivalent analysis program as the product area value. In addition, the internal standard detected simultaneously is also calculated as the internal standard area value.

[0869] <Calculation of each measurement item value>

[0870] Calculation of P / IS

[0871] Calculate P / IS by using the following formula, by calculating the area value obtained in the previous item.

[0872] P / IS = product area value / internal standard area value

[0873] 50% SARS-CoV-2 3CL protease inhibition concentration (IC 50 )calculate

[0874] When x is the logarithmic value of the compound concentration and y is the % inhibition, the inhibition curve is fitted by the following logistic regression equation, and the x value when y=50(%) is calculated as IC 50 .

[0875] y=min+(max-min) / {1+(X50 / x)^Hill}

[0876] % Inhibition = {1-(sample-control (-)) / control (+)-control (-))}*100

[0877] (%Inhibition={1-(Sample-Control(-)) / Control(+)-Control(-))}*100)

[0878] Control (-): Average P / IS of wells in enzyme inhibition condition

[0879] (Control(-): the average of P / IS of enzyme inhibited condition wells)

[0880] Control (+): Average P / IS of DMSO control wells

[0881] (Control(+): the average of P / IS ofDMSO control wells)

[0882] min: y-axis lower limit, max: y-axis upper limit, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max

[0883] The compounds of the invention were tested essentially as described above. The results are shown in the table below.

[0884] It should be noted that IC 50 A value of less than 0.1 μM was recorded as “A”, a value of 0.1 μM or more and less than 1 μM was recorded as “B”, and a value of 1 μM or more and less than 10 μM was recorded as “C”.

[0885] Compound I-003: 0.014 μM

[0886] Compound I-005: 0.010 μM

[0887] Compound I-006: 0.0058 μM

[0888] Compound I-010: 0.0054 μM

[0889] Compound I-012: 0.0091 μM

[0890] Compound I-017: 0.0034 μM

[0891] Compound I-023: 0.0063 μM

[0892] Compound I-035: 0.0098 μM

[0893] [Table 14-1]

[0894]

[0895] Test Example 2-2: Inhibitory Activity Test on SARS-CoV-2 3CL Protease

[0896] <Materials>

[0897] Commercially available recombinant SARS-CoV-2 3CL protease

[0898] SARS-CoV-2 3CL protease variants (G15S, T21I, T24I, K88R, L89F, K90R, P108S, P132H, A193V, H246Y, A255V)

[0899] Commercially available substrate peptides

[0900] Dabcyl-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu(Edans)-NH2(SEQ ID NO: 1)

[0901] Internal standard peptide

[0902] Dabcyl-Lys-Thr-Ser-Ala-Val-Leu(13C6,15N)-Gln(SEQ ID NO: 2)

[0903] Dabcyl-Lys-Thr-Ser-Ala-Val-Leu(13C6,15N)-Gln can be synthesized by reference to literature (Atherton, E.; Sheppard, RC, "In Solid Phase Peptide Synthesis, A Practical Approach", published by 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.

[0904] H-Lys-Thr-Ser-Ala-Val-Leu(13C6,15N)-Glu(resin)-OαOtBu (Lys side chain is protected by Boc, Thr side chain is protected by tert-butyl, Ser side chain is protected by tert-butyl, C-terminal OH of Glu is protected by tert-butyl, and the carboxylic acid of Glu side chain is condensed with resin) was synthesized by Fmoc solid phase synthesis using Rink amide resin. The modification of N-terminal Dabcyl group was carried out by condensing 4-dimethylaminoazobenzene-4'-carboxylic acid (Dabcyl-OH) on the resin with EDC / HOBT. The final deprotection and excision from the resin were carried out by treatment with TFA / EDT=95:5. Then, purification was carried out by reverse phase HPLC.

[0905] ·RapidFire Cartridge C4 typeA

[0906] <Operation steps>

[0907] Preparation of Assay Buffer

[0908] In this test, a detection buffer composed of 20 mM Tris-HCl, 1 mM EDTA, 10 mM DTT, and 0.01% BSA was used.

[0909] Dilution and injection of test samples

[0910] The test sample was diluted to an appropriate concentration with DMSO in advance, and a 3-fold serial dilution series was prepared and dispensed into a 384-well plate.

[0911] Enzyme and substrate addition, enzyme reaction

[0912] Add the substrate at a final concentration of 2-4 μM and the enzyme solution at a final concentration of 2-6 nM to the prepared compound plate, incubate at room temperature for 2-3 hours, and then add the reaction stop solution (0.072 μM internal standard, 0.1% formic acid, 10% acetonitrile) to stop the enzyme reaction.

[0913] Determination of reaction products

[0914] After the reaction, the plate was measured using RapidFire System 360 and a mass spectrometer (Agilent, 6550iFunnel Q-TOF). As mobile phases for the measurement, solution A (75% isopropanol, 15% acetonitrile, 5 mM ammonium formate) and solution B (0.01% trifluoroacetic acid, 0.09% formic acid) were used.

[0915] The reaction product detected by the mass spectrometer was calculated using RapidFire Integrator as the product area value. In addition, the internal standard detected simultaneously was also calculated as the internal standard area value.

[0916] <Calculation of each measurement item value>

[0917] Calculation of P / IS

[0918] Calculate P / IS by using the following formula, by calculating the area value obtained in the previous item.

[0919] P / IS = product area value / internal standard area value

[0920] 50% SARS-CoV-2 3CL protease inhibition concentration (IC 50 )calculate

[0921] When x is the logarithmic value of the compound concentration and y is the % inhibition, the inhibition curve is fitted by the following Logistic regression equation, and the x value when y=50(%) is calculated as IC 50 .

[0922] y=min+(max-min) / {1+(X50 / x)^Hill}

[0923] % Inhibition = {1-(sample-control (-)) / control (+)-control (-))}*100

[0924] Control (-): Average P / IS ratio of wells without SARS-CoV-2 3CL protease and test substrate

[0925] (Control(-): the average of P / IS ratio in the wells without SARS-CoV-23CL protease and test substance)

[0926] Control (+): Average P / IS ratio of wells with SARS-CoV-2 3CL protease and no test substrate

[0927] (Control(+): the average of P / IS ratio in the wells with SARS-CoV-23CL protease and without test substance)

[0928] min: y-axis lower limit, max: y-axis upper limit, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max

[0929] The compounds of the invention were tested essentially as described above. The results are shown below.

[0930] [Table 14-2]

[0931]

[0932] Test Example 3: Inhibition test of viral titer proliferation in the lungs of SARS-CoV-2 infected mice by administering fumaric acid cocrystal type I crystals of the compound represented by formula (IB)

[0933] <Materials and Methods>

[0934] Compounds

[0935] Fumaric acid cocrystal type I crystal of the compound represented by formula (IB) was prepared using a 0.5% methylcellulose (0.5% MC) solution. The administration volume was set to 10 mL / kg. The administration amount represents the amount obtained by converting the free body.

[0936] ·Virus

[0937] The SARS-CoV-2hCoV19 / Japan / TY7-501 / 2021 strain isolated by the National Institute of Infectious Diseases was used.

[0938] Mouse lung infection, drug administration, and lung recovery

[0939] In this study, 5-week-old female BALB / c mice (CLEA Japan, Inc.) without specific pathogens were used. When inoculating the virus, the mice were anesthetized by intramuscular administration of 100 μL of anesthetic solution containing 0.03 mg / mL of medetomidine hydrochloride, 0.4 mg / mL of midazolam, and 0.5 mg / mL of butorphine tartrate in PBS. Under anesthesia, the mice were inoculated intranasally with 50 μL of hCoV19 / Japan / TY7-501 / 2021 (1.00×10 4 TCID 50). Taking the time immediately after virus infection as the starting point, the fumaric acid cocrystal type I crystal of the compound represented by formula (IB) was orally administered to mice (n=5-10 / group) at a dosage of 2 mg / kg, 4 mg / kg, 8 mg / kg, 16 mg / kg, 32 mg / kg, 64 mg / kg or twice a day at a dosage of 1 mg / kg, 2 mg / kg, 4 mg / kg, 8 mg / kg, 16 mg / kg, 32 mg / kg. For control mice, 0.5% MC was orally administered twice a day. One day after virus infection, the mouse lungs were recovered, 2 mL of PBS was added and homogenized, and the supernatant after centrifugation was recovered.

[0940] Determination of viral titer in lungs

[0941] The lung homogenate was diluted 10-fold in a culture medium (MEM, 2% FBS, penicillin-streptomycin) and incubated with VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4 The cells were mixed with 100 cells / well and inoculated into 96-well plates. After culturing in a CO2 incubator for 4 days, the cytopathic effect (CPE) was observed and the virus titer contained in the lung homogenate was calculated.

[0942] Statistical analysis

[0943] The intergroup differences in viral titers in lung homogenates were analyzed by Dunnett's test. Statistical analysis was performed using SAS version 9.4 for Windows (SAS Institute (Cary, NC)). Adjusted two-sided P values ​​less than 0.05 were considered statistically significant.

[0944] The compounds of the invention were tested essentially as described above. The results are shown below.

[0945] In the group administered with the fumaric acid cocrystal type I crystal of the compound represented by formula (IB), the viral titer in the lungs 1 day after infection was reduced in a dose-dependent manner in either single or twice-daily administration, and at all administration doses of 2 mg / kg or more, the viral titer was significantly lower than that in the group administered with 0.5% MC ( Figure 5A and Figure 5B In the 32 and 64 mg / kg administration groups for single administration and the 16 and 32 mg / kg administration groups for twice-daily administration, the viral titer in the lungs generally reached the lower limit of quantification (1.80-log 10 TCID 50 / mL).

[0946] Test Example 4: Inhibition test of viral titer proliferation in the lungs of SARS-CoV-2 infected mice by delayed administration of fumaric acid cocrystal type I crystal of the compound represented by formula (IB)

[0947] <Materials and Methods>

[0948] Compounds

[0949] The compound of the present invention (fumaric acid cocrystal type I crystal of the compound represented by formula (IB)) was prepared using a 0.5% methylcellulose (0.5% MC) solution. The administration volume was set to 10 mL / kg. The administration amount represents the amount obtained by converting the free body.

[0950] ·Virus

[0951] The SARS-CoV-2hCoV19 / Japan / TY7-501 / 2021 strain isolated by the National Institute of Infectious Diseases was used.

[0952] Mouse lung infection, drug administration, and lung recovery

[0953] In this study, 5-week-old female BALB / c mice (CLEA Japan, Inc.) without specific pathogens were used. When inoculating the virus, the mice were anesthetized by intramuscular administration of 100 μL of anesthetic solution containing 0.03 mg / mL of medetomidine hydrochloride, 0.4 mg / mL of midazolam, and 0.5 mg / mL of butorphine tartrate in PBS. Under anesthesia, the mice were inoculated intranasally with 50 μL of hCoV19 / Japan / TY7-501 / 2021 (1.00×10 4 TCID 50 ). Taking 1 day or 3 days after virus infection as the starting point, the fumaric acid cocrystal type I crystal of the compound represented by formula (IB) was orally administered to mice at a dosage of 30 mg / kg and 150 mg / kg twice a day (n=5 / group). For control mice, 0.5% MC was orally administered once a day. Compound administration was set to 2 days from the start of administration. Mouse lungs were recovered 2 days after the start of administration, 2 mL of PBS was added and homogenized, and the supernatant after centrifugation was recovered.

[0954] Determination of viral titer in lungs

[0955] The lung homogenate was diluted 10-fold in a culture medium (MEM, 2% FBS, penicillin-streptomycin) and incubated with VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4The cells were mixed with 100 cells / well and inoculated into 96-well plates. After culturing in a CO2 incubator for 4 days, the cytopathic effect (CPE) was observed and the virus titer contained in the lung homogenate was calculated.

[0956] The compounds of the invention were tested essentially as described above. The results are shown below.

[0957] In the group that was administered from day 1 after infection, the viral titer in the lungs 2 days after administration was 6.47-log in the 0.5% MC administration group. 10 TCID 50 / mL, and in the fumaric acid co-crystal type I crystal of the compound represented by formula (IB) 30 and 150 mg / kg administration groups, respectively, 4.47-log 10 TCID 50 / mL, 2.90-log 10 TCID 50 / mL( Fig. 6A ). In the group that was administered 3 days after infection, the viral titer in the lungs 2 days after the start of administration was 4.83-log in the 0.5% MC administration group. 10 TCID 50 / mL, and in the fumaric acid co-crystal type I crystal of the compound represented by formula (IB) 30 and 150 mg / kg administration groups, respectively, 3.63-log 10 TCID 50 / mL, 3.35-log 10 TCID 50 / mL( Figure 6B ).

[0958] In either case, whether the administration started 1 day or 3 days after infection, the viral titer in the lungs of the group administered with type I crystals of the fumaric acid cocrystal of the compound represented by formula (IB) was lower than that of the group administered with 0.5% MC, suggesting that the viral titer in the lungs can be reduced even if the period from infection to administration is left out.

[0959] Test Example 4-2: Inhibition test of viral titer proliferation in the lungs of SARS-CoV-2 infected mice by delayed administration of fumaric acid cocrystal type I crystal of the compound represented by formula (IB)

[0960] <Materials and Methods>

[0961] Compounds

[0962] Fumaric acid cocrystal type I crystal of the compound represented by formula (IB) was prepared using a 0.5% methylcellulose (0.5% MC) solution. The administration volume was set to 10 mL / kg. The administration amount represents the amount obtained by converting the free body.

[0963] ·Virus

[0964] The SARS-CoV-2hCoV19 / Japan / TY7-501 / 2021 strain isolated by the National Institute of Infectious Diseases was used.

[0965] Mouse lung infection, drug administration, and lung recovery

[0966] In this study, 5-week-old female BALB / c mice (CLEA Japan, Inc.) without specific pathogens were used. When inoculating the virus, the mice were anesthetized by intramuscular administration of 100 μL of anesthetic solution containing 0.03 mg / mL of medetomidine hydrochloride, 0.4 mg / mL of midazolam, and 0.5 mg / mL of butorphine tartrate in PBS. Under anesthesia, the mice were inoculated intranasally with 50 μL of hCoV19 / Japan / TY7-501 / 2021 (1.00×10 4 TCID 50 ). Taking one day after virus infection as the starting point, the fumaric acid cocrystal type I crystal of the compound represented by formula (IB) was orally administered to mice (n=5 / group) at a dosage of 8, 16, 32, and 64 mg / kg three times a day. For control mice, 0.5% MC was orally administered once a day. Compound administration was set to 2 days from the start of administration. Mouse lungs were recovered 2 days after the start of administration, 2 mL of PBS was added and homogenized, and the supernatant after centrifugation was recovered.

[0967] Determination of viral titer in lungs

[0968] The lung homogenate was diluted 10-fold in a culture medium (MEM, 2% FBS, penicillin-streptomycin) and incubated with VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4 The cells were mixed with 100 cells / well and inoculated into 96-well plates. After culturing in a CO2 incubator for 4 days, the cytopathic effect (CPE) was observed and the virus titer contained in the lung homogenate was calculated.

[0969] The compounds of the invention were tested essentially as described above. The results are shown below.

[0970] In the group that was administered from day 1 after infection, the viral titer in the lungs 2 days after the administration was 6.57-log in the 0.5% MC administration group. 10 TCID 50 / mL, and in the groups administered with 8, 16, 32, and 64 mg / kg of the fumaric acid cocrystal type I of the compound represented by formula (IB), the log values ​​were 5.55, 4.66, 2.85, and 2.40, respectively. 10TCID 50 / mL. In the group administered with the fumaric acid cocrystal type I crystal of the compound represented by formula (IB), the viral titer in the lungs was lower than that in the group administered with 0.5% MC, suggesting that even if the period from infection to administration was left blank, the viral titer in the lungs was reduced ( Figure 7 ).

[0971] Test Example 5: Lethality and weight loss inhibition test of SARS-CoV-2 infected mice by administering fumaric acid cocrystal type I crystal of the compound represented by formula (IB)

[0972] <Materials and Methods>

[0973] Compounds

[0974] The compound of the present invention (fumaric acid cocrystal type I crystal of the compound represented by formula (IB)) was prepared using a 0.5% methylcellulose (0.5% MC) solution. The administration volume was set to 10 mL / kg. The administration amount represents the amount obtained by converting the free body.

[0975] ·Virus

[0976] The SARS-CoV-2hCoV19 / Japan / TY7-501 / 2021 strain (TY7-501) isolated by the National Institute of Infectious Diseases and the SARS-Co V-2hCoV19 / Japan / TY / WK-521 / 2020 mouse-adapted strain MA-P10 isolated by Hokkaido University were used.

[0977] Mouse lung infection, drug administration, and body weight measurement

[0978] In this study, retired female BALB / c mice (CLEA Japan, Inc.) aged 15 or 35-45 weeks without specific pathogens were used. At the time of virus inoculation, mice were anesthetized by intramuscular administration of 100 μL of anesthetic solution containing 0.03 mg / mL of medetomidine hydrochloride, 0.4 mg / mL of midazolam, and 0.5 mg / mL of butorphine tartrate in PBS. Under anesthesia, mice were inoculated intranasally with 50 μL of TY7-501 (1.00×10 5 TCID 50 )、MA-P10(1.00×10 3 TCID 50 or 1.00 × 10 4 TCID 50). TY7-501 was used only in retired mice aged 35-45 weeks. Starting from one day after viral infection, the fumaric acid cocrystal type I crystal of the compound represented by formula (IB) was orally administered to mice at a dose of 50 mg / kg twice a day (n=4 / group). For control mice, 0.5% MC was orally administered twice a day. Body weight was monitored once a day, and in the evaluation of survival rate, it was considered to be dead when it became less than 80% based on the body weight immediately before infection.

[0979] The compounds of the invention were tested essentially as described above. The results are shown below.

[0980] Mice aged 35-45 weeks were infected with TY7-501 at a concentration of 1.00 × 10 5 TCID 50 In the case of , body weight decreased from 3 days after infection, and all test cases died 5 days after infection. At this time, in the group administered with 50 mg / kg of fumaric acid cocrystal type I crystal of the compound represented by formula (IB), all test cases survived until 7 days after infection, which was observed, and body weight loss was suppressed ( Fig. 8A and Figure 8B ). 15-week-old mice were infected with MA-P10 1.00×10 3 TCID 50 In the case of MA-P10 infection, weight loss was observed from 3 days after infection, but no mortality was observed. 4 TCID 50 In the case of , body weight decreased from 3 days after infection, death was observed in some individuals 4 days after infection, and all test cases died 6 days after infection. At this time, in the group administered with 50 mg / kg of fumaric acid cocrystal type I crystal of the compound represented by formula (IB), all test cases survived and no weight loss was observed until 7 days after infection when observation was performed ( Figure 8C and Fig.8D ).

[0981] Next, retired mice aged 35-45 weeks were infected with 1.00 × 10 MA-P10 3 TCID 50 In the case of , weight loss began 3 days after infection, and deaths were observed in some individuals 5 days after infection. All test cases died 6 days after infection, which suggests that retired mice aged 35-45 weeks are prone to severe symptoms even at a lower infection dose. At this time, in the group administered with 50 mg / kg of the fumaric acid cocrystal type I crystal of the compound represented by formula (IB), all test cases survived until 7 days after infection, which was observed, and weight loss was suppressed ( Fig. 8E and Figure 8F ).

[0982] The above results suggest that administration of the fumaric acid cocrystal type I crystal of the compound represented by formula (IB) has the effect of inhibiting mortality and weight loss in both 15-week-old mice and 35-45-week-old retired mice.

[0983] It is known that, in aged mice, the expression of ACE2 receptors known as receptors for SARS-CoV-2 increases, as in humans, when compared to young mice (references: Scientific Reports (2020) 10: 22401 and Molecular Therapy Methods & Clinical Development, Vol. 18, P1-6, 2020). In addition, the normal immune response that defends the body from damage by the infection source and eliminates them is reduced, and therefore, it is estimated that death will occur due to viral infection. However, as shown above, in the SARS-CoV-2 infection mouse model using aged mice, the survival rate of mice after 7 days of infection was 100% by administration of the fumaric acid cocrystal type I crystal of the compound represented by formula (IB). The non-clinical trial using aged mice is positioned as one of the non-clinical evaluation systems that mimics the process of high-risk patients with underlying diseases becoming seriously ill, and therefore, supports the usefulness of the fumaric acid cocrystal type I crystal of the compound represented by formula (IB) as a treatment option for patients with a high risk of becoming seriously ill.

[0984] In addition, the above test results suggest that the aggravation of symptoms caused by viral infection was suppressed in the group administered with the fumaric acid cocrystal type I crystal of the compound represented by formula (IB), which not only supports the antiviral effect of the fumaric acid cocrystal type I crystal of the compound represented by formula (IB) against SARS-CoV-2, but also supports its usefulness as a medicine for suppressing the aggravation of infectious diseases caused by SARS-CoV-2.

[0985] Test Example 6: Antiviral effect against SARS-CoV

[0986] <Materials>

[0987] Maintenance medium (MEM containing 2% FBS)

[0988] To 1000 mL of Minimum Essential Medium, 10 mL of 8.5% NaHCO 3 , 20 mL of FBS, and 10 mL of L-glutamine were added to prepare the medium.

[0989] MTT solution

[0990] 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide was dissolved in PBS to 5 μg / mL, and then filtered using a 0.45 μm or 0.22 μm filter.

[0991] Cell lysis solution (virus inactivation solution)

[0992] The solution was prepared by adding 50 mL of Triton X and 4 mL of hydrochloric acid (12 mol / L) to 500 mL of isopropanol.

[0993] Dimethyl sulfoxide (DMSO)

[0994] · Microplate reader (Thermo Fisher, Perkin Elmer, etc.)

[0995] <Operation steps>

[0996] Dilution and injection of test samples

[0997] The test sample was diluted in advance with DMSO to an appropriate concentration, and a 3-fold serial dilution series was prepared, and further diluted with a maintenance medium and dispensed into a 96-well plate at 50 μL / well.

[0998] ·Dilution and injection of cells and viruses

[0999] The virus (SARS-CoV Hanoi strain (1000TCID 50 Each well was diluted with a maintenance medium and dispensed at 50 μL / well into a 96-well plate containing the test sample.

[1000] VeroE6 / TMPRSS2 cells (1.5×10 4 100 μL / well of each solution (100 cells / well) was dispensed into a 96-well plate containing the test sample and virus, mixed using a plate mixer, and cultured in a CO2 incubator for 3 days.

[1001] ·Dispensing of MTT solution and cell lysis solution

[1002] Observe the 96-well plate under a microscope after 3 days of culture with the naked eye to confirm the morphology of the cells and the presence of crystals. Pour 30 μL of MTT solution into each well and culture in a CO2 incubator for 4 to 6 hours. Remove 150 μL of supernatant from each plate without sucking cells. Pour 150 μL of cell lysate (virus inactivation solution) into each well. Wrap the plate with plastic wrap so that it does not dry out and leave it at room temperature overnight.

[1003] Absorbance measurement (96-well plate)

[1004] The next day, the plate was mixed using a plate mixer. For the 96-well plate, absorbance at two wavelengths of 570 nm and 630 nm was measured using an ELISA reader.

[1005] <Calculation of each measurement item value>

[1006] 50% viral infection cell death inhibition concentration (EC 50 )calculate

[1007] When x is the logarithmic value of the compound concentration and y is the % efficacy, the inhibition curve is fitted by the following Logistic regression equation, and the x value when y=50(%) is calculated as the EC 50 .

[1008] y=min+(max-min) / {1+(X50 / x)^Hill}

[1009] % efficacy = {(sample - virus control) / (cell control - virus control)}*100%

[1010] Sample: the average of ODs of Sample wells

[1011] Cell control: the average OD of cell control wells

[1012] Virus control: the average OD of virus control wells

[1013] OD: OD(570nm)-OD(630nm)

[1014] min: y-axis lower limit, max: y-axis upper limit, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max

[1015] The compounds of the invention were tested essentially as described above. 50 As shown below.

[1016] Fumaric acid cocrystal type I of the compound represented by formula (IB): SARS-CoV 0.209 μM

[1017] Test Example 7: Antiviral effect against HCoV-OC43

[1018] <Operation steps>

[1019] Dilution and injection of test samples

[1020] 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 dispensed into a 96-well plate and diluted with a maintenance medium (MEM, 2% FBS, penicillin-streptomycin).

[1021] Dilution and injection of cells and HCoV-OC43

[1022] One day before infection, MRC-5 cells (2×10 4 Cells / well) were inoculated into 96-well plates and infected with HCoV-OC43 (100 TCID 50 / well). Then, after washing once with maintenance medium, maintenance medium with the test agent added was added and cultured in a CO2 incubator for 42 hours. In addition, in order to investigate the cytotoxicity of the test agent, the same operation was performed in the absence of virus.

[1023] Quantification of viral load

[1024] The supernatant of the plate after 42 hours of culture was collected and RNA was extracted using Quick-RNA Viral Kit (ZYMO RESEARCH, #R1041). The extracted RNA solution was quantified using real-time PCR (Applied BioSystems QuantStudio3). Primers were referenced in the literature (Journal of Clinical Microbiology., 2005, Jun 43(11), 5452-5456).

[1025] <Calculation of each measurement item value>

[1026] 90% HCoV-OC43 virus replication inhibition concentration (EC 90 )calculate

[1027] In the concentration-dependence curve where x is the logarithmic value of the compound concentration and y is the number of virus copies / mL, the value equivalent to 1 log reduction relative to the virus control is calculated by the two-point method based on the number of copies of the two concentrations before and after it. That is, X = log10 (high conc.)

[1028] x=log10(low conc.)

[1029] Y=log10(low copies / mL)-log10(control copies / mL)

[1030] y=log10(high copies / mL)-log10(control copies / mL)

[1031] EC was calculated by 90 value.

[1032] EC 90 =10[x+(-1-y)×(Xx) / (Yy)]

[1033] The compounds of the invention were tested essentially as described above. The results are shown below.

[1034] Fumaric acid cocrystal type I of the compound represented by formula (IB): EC 90 0.074μM

[1035] Test Example 8: Antiviral effect against HCoV-229E

[1036] <Operation steps>

[1037] Dilution and injection of test samples

[1038] 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 dispensed into a 96-well plate and diluted with a maintenance medium (MEM, 2% FBS, penicillin-streptomycin).

[1039] Dilution and injection of cells and HCoV-229E

[1040] One day before infection, MRC-5 cells (2×10 4 Cells / well) were inoculated into 96-well plates and infected with HCoV-229E (1000 TCID) suspended in maintenance medium (MEM, 2% FBS, penicillin-streptomycin) for 1 hour the next day. 50 / well). Then, the virus solution was removed, and the maintenance medium to which the test agent had been added was added, and the cells were cultured in a CO2 incubator for 72 hours. In addition, in order to investigate the cytotoxicity of the test agent, the same operation was performed in the absence of the virus.

[1041] · CellTiter-Glo (registered trademark) 2.0 injection and luminescence signal measurement

[1042] After the plate was cultured for 72 hours, it was returned to room temperature, and CellTiter-Glo (registered trademark) 2.0 was dispensed into each well and mixed using a plate mixer. After standing for a certain period of time, the luminescence signal (Lum) was measured using a microplate reader.

[1043] <Calculation of each measurement item value>

[1044] 50% HCoV-229E infected cell death inhibition concentration (EC 50 )calculate

[1045] When x is the logarithmic value of the compound concentration and y is the % efficacy, the inhibition curve is fitted by the following Logistic regression equation, and the x value when y=50(%) is calculated as the EC 50 .

[1046] y=min+(max-min) / {1+(X50 / x)^Hill}

[1047] % efficacy = {(sample - virus control) / (cell control - virus control)}*100%

[1048] Cell Control: Average Lum of cell control wells

[1049] Virus control: Average Lum of virus control wells

[1050] min: y-axis lower limit, max: y-axis upper limit, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max

[1051] Calculation of 50% cytotoxic concentration

[1052] When x is the logarithmic value of the compound concentration and y is the % cytotoxicity, the inhibition curve is fitted by the following logistic regression equation, and the value of x when y=50(%) is calculated as CC 50 .

[1053] y=min+(max-min) / {1+(X50 / x)^Hill}

[1054] % cytotoxicity = {(sample - medium control) / (cell control - medium control)}*100%

[1055] (% Cytotoxicity={(Sample-medium control) / (cell con trol-medium control)}*100%)

[1056] Cell Control: Average Lum of cell control wells

[1057] Medium control: the average Lum of medium control wells

[1058] min: y-axis lower limit, max: y-axis upper limit, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max

[1059] The compounds of the invention were tested essentially as described above. The results are shown below.

[1060] Fumaric acid cocrystal type I of the compound represented by formula (IB): EC 50 5.53 μM, CC 50 >200μM

[1061] Experimental Example 9: Effects of human serum and mouse serum on anti-SARS-CoV-2 activity

[1062] <Operation steps>

[1063] Dilution and injection of test samples

[1064] The test sample was diluted to an appropriate concentration with DMSO in advance, and a 3-fold serial dilution series was prepared and dispensed into a 96-well plate.

[1065] ·Addition of serum-supplemented culture medium

[1066] The solution was adjusted with a culture medium (MEM, 2% FBS, penicillin-streptomycin) to 0, 6.25%, 12.5%, 25%, or 50% human serum or 0, 3.125%, 6.25%, 12.5%, or 25% mouse serum, dispensed into the wells containing the test samples, and incubated at room temperature for 1 hour.

[1067] Dilution and injection of cells and SARS-CoV-2

[1068] VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 3cells / well) and SARS-CoV-2hCoV-19 / Japan / TY7-501 / 2021 (1000 TCID 50 / well, 10000 TCID in mouse serum 50 / well) were mixed with culture medium (MEM, 2% FBS, penicillin-streptomycin), dispensed equally into wells containing test samples and human serum or mouse serum, and cultured in a CO2 incubator for 3 days.

[1069] · CellTiter-Glo (registered trademark) 2.0 injection and luminescence signal measurement

[1070] After the plate was returned to room temperature after culturing for 3 days, CellTiter-Glo (registered trademark) 2.0 was dispensed into each well and mixed using a plate mixer. After standing for a certain period of time, the luminescence signal (Lum) was measured using a microplate reader.

[1071] <Calculation of each measurement item value>

[1072] 50% SARS-CoV-2 infected cell death inhibition concentration (EC 50 )calculate

[1073] When x is the logarithmic value of the compound concentration and y is the % efficacy, the inhibition curve is fitted by the following Logistic regression equation, and the x value when y=50(%) is calculated as the EC 50 .

[1074] y=min+(max-min) / {1+(X50 / x)^Hill}

[1075] % efficacy = {(sample - virus control) / (cell control - virus control)}*100%

[1076] Cell Control: Average Lum of cell control wells

[1077] Virus control: Average Lum of virus control wells

[1078] min: y-axis lower limit, max: y-axis upper limit, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max

[1079] Protein-Adjusted EC 50 (PA-EC 50 )

[1080] Calculate the EC at each serum concentration 50 Then, the PA-EC under the condition of 100% serum was calculated by linear regression. 50value.

[1081] The compounds of the invention were tested essentially as described above. The results are shown below.

[1082] Fumaric acid cocrystal type I of the compound represented by formula (IB): human serum PA-EC 50 3.02μM, mouse serum PA-EC 50 3.93μM

[1083] Test Example 10-1: Cytopathic effect (CPE) inhibition test using human ACE2 and TMPRSS2 expressing HEK293T cells (HEK293T / ACE2-TMPRSS2 cells)

[1084] <Operation steps>

[1085] Dilution and injection of test samples

[1086] The test sample was diluted to an appropriate concentration with DMSO in advance, and a 3-fold serial dilution series was prepared and dispensed into a 96-well plate.

[1087] Dilution and injection of cells and SARS-CoV-2

[1088] HEK293T / ACE2-TMPRSS2 cells (SL222, 1.5×10 4 cells / well) and SARS-CoV-2 hCoV-19 / Japan / TY / WK-521 / 2020, hCoV-19 / Japan / QK002 / 2020, hCoV-19 / Japan / TY7-501 / 2021, hCoV-19 / Japan / TY8-612 / 2021, hCoV-19 / Japan / TY11-927-P1 / 2021 (3000-9000 TCID 50 / well) were mixed in a culture medium (MEM, 2% FBS, penicillin-streptomycin), dispensed into the wells containing the test samples, and cultured in a CO2 incubator for 3 days.

[1089] · CellTiter-Glo (registered trademark) 2.0 injection and luminescence signal measurement

[1090] After the plate was returned to room temperature after culturing for 3 days, CellTiter-Glo (registered trademark) 2.0 was dispensed into each well and mixed using a plate mixer. After standing for a certain period of time, the luminescence signal (Lum) was measured using a microplate reader.

[1091] <Calculation of each measurement item value>

[1092] 50% SARS-CoV-2 infected cell death inhibition concentration (EC 50 )calculate

[1093] When x is the logarithmic value of the compound concentration and y is the % efficacy, the inhibition curve is fitted by the following Logistic regression equation, and the x value when y=50(%) is calculated as the EC 50 .

[1094] y=min+(max-min) / {1+(X50 / x)^Hill}

[1095] % efficacy = {(sample - virus control) / (cell control - virus control)}*100%

[1096] Cell Control: Average Lum of cell control wells

[1097] Virus control: Average Lum of virus control wells

[1098] min: y-axis lower limit, max: y-axis upper limit, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max

[1099] The compounds of the invention were tested essentially as described above. The results are shown below.

[1100] Fumaric acid cocrystal type I of the compound represented by formula (IB): 0.026-0.058 μM

[1101] Test Example 10-2: Cytopathic effect (CPE) inhibition test using human ACE2 and TMPRSS2 expressing HEK293T cells (HEK293T / ACE2-TMPRSS2 cells)

[1102] <Operation steps>

[1103] Dilution and injection of test samples

[1104] The test sample was diluted to an appropriate concentration with DMSO in advance, and a 3-fold serial dilution series was prepared and dispensed into a 96-well plate.

[1105] Dilution and injection of cells and SARS-CoV-2

[1106] HEK293T / ACE2-TMPRSS2 cells (SL222, 1.5×10 4 cells / well) and SARS-CoV-2hCoV-19 / Japan / TY38-873 / 2021 (9000TCID 50 / well) were mixed in a culture medium (MEM, 2% FBS, penicillin-streptomycin), dispensed into the wells containing the test samples, and cultured in a CO2 incubator for 3 days.

[1107] · CellTiter-Glo (registered trademark) 2.0 injection and luminescence signal measurement

[1108] After the plate was returned to room temperature after culturing for 3 days, CellTiter-Glo (registered trademark) 2.0 was dispensed into each well and mixed using a plate mixer. After standing for a certain period of time, the luminescence signal (Lum) was measured using a microplate reader.

[1109] <Calculation of each measurement item value>

[1110] 50% SARS-CoV-2 infected cell death inhibition concentration (EC 50 )calculate

[1111] When x is the logarithmic value of the compound concentration and y is the % efficacy, the inhibition curve is fitted by the following Logistic regression equation, and the x value when y=50(%) is calculated as the EC 50 .

[1112] y=min+(max-min) / {1+(X50 / x)^Hill}

[1113] % efficacy = {(sample - virus control) / (cell control - virus control)}*100%

[1114] Cell Control: Average Lum of cell control wells

[1115] Virus control: Average Lum of virus control wells

[1116] min: y-axis lower limit, max: y-axis upper limit, X50: x-coordinate of the inflection point, Hill: slope of the curve at the midpoint between min and max

[1117] The compounds of the invention were tested essentially as described above. The results are shown below.

[1118] Fumaric acid cocrystal type I of the compound represented by formula (IB): 0.083 μM

[1119] Test Example 11: Test on the inhibition of virus transmission from SARS-CoV-2 infected animals to non-infected animals by preventive administration of fumaric acid cocrystal type I crystal of the compound represented by formula (IB)

[1120] <Materials and Methods>

[1121] Compounds

[1122] Fumaric acid cocrystal type I crystal of the compound represented by formula (IB) was prepared using a 0.5% methylcellulose (0.5% MC) solution. The administration volume was set to 10 mL / kg. The administration amount represents the amount obtained by converting the free body.

[1123] ·Virus

[1124] The SARS-CoV-2hCoV19 / Japan / TY11-927 / 2021 strain isolated by the National Institute of Infectious Diseases was used.

[1125] Hamster lung infection, drug administration, and lung recovery

[1126] In this study, five-week-old male Syrian hamsters (Japan SLC, Inc.) free of specific pathogens were used. One infected hamster inoculated with the virus was housed in the same cage with three non-inoculated hamsters to test the virus transmission to the hamsters living with the infected hamsters. When inoculating the virus, the hamsters were anesthetized by inhalation of isoflurane. Under anesthesia, 200 μL of hCoV19 / Japan / TY11-927 / 2021 (5.00×10 3 PFU) was inoculated into hamsters through the nose. For hamsters that were not inoculated with the virus, the time of virus inoculation into infected hamsters was used as the starting point, and the fumaric acid cocrystal type I crystal of the compound represented by formula (IB) was orally administered at a dosage of 30 and 200 mg / kg twice a day. For control hamsters, 0.5% MC was orally administered twice a day. Compound administration was set to 6 days from the start of administration. Hamster lungs were recovered 6 days after the start of administration, 5 mL of PBS was added and homogenized, and the supernatant after centrifugation was recovered.

[1127] Determination of viral titer in lungs

[1128] The lung homogenate was diluted 10-fold in a culture medium (MEM, 2% FBS, penicillin-streptomycin) and inoculated onto VeroE6 / TMPRSS2 cells (JCRB1819, 2.0×10 5 After culturing in a CO2 incubator for 2 days, virus plaques were observed and the virus titer contained in the lung homogenate was calculated.

[1129] The compounds of the invention were tested essentially as described above. The results are shown below.

[1130] Six days after cohabitation with infected hamsters, the viral titer in the lungs of the hamsters administered with 0.5% MC was 6.12-log 10PFU / mL, in the fumaric acid co-crystal type I crystal of the compound represented by formula (IB) 30, 200 mg / kg administration group, respectively, showed 3.48-log 10 PFU 50 / mL, below the detection limit ( Fig. 9 In the group administered with the fumaric acid cocrystal type I crystal of the compound represented by formula (IB), the viral titer in the lungs was lower than that in the group administered with 0.5% MC, suggesting that it has an effect of reducing viral transmission from infected hamsters.

[1131] Test Example 12: Virus transmission inhibition test from SARS-CoV-2 infected animals to non-infected animals by administering fumaric acid cocrystal type I crystal of the compound represented by formula (IB) immediately after infection

[1132] <Materials and Methods>

[1133] Compounds

[1134] Fumaric acid cocrystal type I crystal of the compound represented by formula (IB) was prepared using a 0.5% methylcellulose (0.5% MC) solution. The administration volume was set to 10 mL / kg. The administration amount represents the amount obtained by converting the free body.

[1135] ·Virus

[1136] The SARS-CoV-2hCoV19 / Japan / TY11-927 / 2021 strain isolated by the National Institute of Infectious Diseases was used.

[1137] Hamster lung infection, drug administration, and lung recovery

[1138] In this study, five-week-old male Syrian hamsters (Japan SLC, Inc.) free of specific pathogens were used. One infected hamster that was inoculated with the virus was housed in the same cage with two non-infected hamsters that were not inoculated with the virus, and the virus transmission to the non-infected hamsters living with the infected hamsters was tested. When inoculating the virus, the hamsters were anesthetized by inhalation of isoflurane and inoculated intranasally with 200 μL of hCoV-19 / Japan / TY11-927 / 2021 (5.00×10 3 PF U). For infected hamsters, the fumaric acid cocrystal type I crystal of the compound represented by formula (IB) was orally administered at a dose of 200 mg / kg twice a day, starting from the time of virus inoculation. For control infected hamsters, 0.5% MC was orally administered twice a day. Compound administration was set to 4 days from the start of administration. Non-infected hamster lungs were collected 6 days after infection, 5 mL of PBS was added and homogenized, and the supernatant after centrifugation was collected.

[1139] Determination of viral titer in lungs

[1140] The lung homogenate was diluted 10-fold in a culture medium (MEM, 2% FBS, penicillin-streptomycin) and inoculated onto VeroE6 / TMPRSS2 cells (JCRB1819, 2.0×10 5 After culturing in a CO2 incubator for 2 days, virus plaques were observed and the virus titer contained in the lung homogenate was calculated.

[1141] The compounds of the invention were tested essentially as described above. The results are shown below.

[1142] Six days after cohabitation with infected hamsters, the viral titer in the lungs of non-infected hamsters was 6.36-log in the 0.5% MC administration group. 10 PFU / mL, and in the group administered with 200 mg / kg of the fumaric acid co-crystal type I crystal of the compound represented by formula (IB), it was below the detection limit ( Fig.10 In the group administered with the fumaric acid cocrystal type I crystal of the compound represented by formula (IB), the viral titer in the lungs was lower than that in the group administered with 0.5% MC, suggesting that it has an effect of reducing viral transmission from infected hamsters.

[1143] Test Example 13: Virus transmission inhibition test from SARS-CoV-2 infected animals to non-infected animals by delayed administration of fumaric acid cocrystal type I crystal of the compound represented by formula (IB) after infection

[1144] <Materials and Methods>

[1145] Compounds

[1146] Fumaric acid cocrystal type I crystal of the compound represented by formula (IB) was prepared using a 0.5% methylcellulose (0.5% MC) solution. The administration volume was set to 5 mL / kg. The administration amount represents the amount obtained by converting the free body.

[1147] ·Virus

[1148] The SARS-CoV-2hCoV19 / Japan / TY11-927 / 2021 strain isolated by the National Institute of Infectious Diseases was used.

[1149] Hamster lung infection, drug administration, lung and nasal concha recovery

[1150] In this study, 6-week-old male Syrian hamsters (Japan SLC, Inc.) free of specific pathogens were used. One infected hamster inoculated with the virus and one non-infected hamster not inoculated with the virus were kept in the same cage starting from 2 days after infection, and the virus transmission to the non-infected hamster living with the infected hamster was tested. The experiment was carried out with n=3 in each group. When inoculating the virus, the hamsters were anesthetized by subcutaneous administration of 200μL of anesthetic solution containing 0.07mg / mL of medetomidine hydrochloride, 6.98mg / mL of alfaxalone, and 1.16mg / mL of butorphine tartrate, and 100μL of hCoV-19 / Japan / TY11-927 / 2021 (1.00×10 2 TCID 50 ). For infected hamsters, one day after virus inoculation was used as the starting point, and the fumaric acid cocrystal type I crystal of the compound represented by formula (IB) was orally administered at a dosage of 50 mg / kg twice a day. For the control infected hamsters, 0.5% MC was orally administered twice a day. Compound administration was set to 4 days from the start of administration. Five days after infection, the lungs and nasal conchae of infected and non-infected hamsters were recovered, 5 mL or 1 mL of PBS was added and homogenized, and the supernatant after centrifugation was recovered.

[1151] Determination of virus titer in lungs and nasal concha

[1152] The lung or nasal concha homogenate was diluted 10-fold in a culture medium (MEM, 2% FBS, penicillin-streptomycin) and inoculated into VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4 After culturing in a CO2 incubator for 5 days, the cytopathic effect (CPE) was observed and the virus titer contained in the lung or nasal concha homogenate was calculated.

[1153] The compounds of the invention were tested essentially as described above. The results are shown below.

[1154] Three days after cohabitation with infected hamsters, the viral titer in the lungs of non-infected hamsters was 5.30-log in the 0.5% MC administration group. 10 TCID 50 / mL, and 2.97-10 g in the group administered with 50 mg / kg of the fumaric acid co-crystal type I of the compound represented by formula (IB) 10 TCID 50 / mL( Fig.11A ). In addition, 3 days after cohabitation with infected hamsters, the viral titer in the nasal concha of non-infected hamsters was 6.06-log in the 0.5% MC administration group. 10 TCID50 / mL, and in the group administered with 50 mg / kg of the fumaric acid co-crystal type I of the compound represented by formula (IB), it showed a 2.89-log 10 TCID 50 / mL( Fig. 11B In the group administered with the fumaric acid cocrystal type I crystal of the compound represented by formula (IB), the viral titers in the lungs and nasal concha were lower than those in the group administered with 0.5% MC, suggesting that it has an effect of reducing viral transmission from infected hamsters.

[1155] Test Example 14: Lethality and weight loss inhibition test of SA RS-CoV-2 infected mice by preventive administration of the compound represented by formula (IB)

[1156] <Materials and Methods>

[1157] Compounds

[1158] The compound of the present invention (compound represented by formula (IB)) was prepared using a 0.5% methylcellulose (0.5% MC) solution. The administration volume was set to 10 mL / kg x 2 sites.

[1159] ·Virus

[1160] The mouse-adapted strain MA-P10 of SARS-CoV-2hCoV19 / Japan / TY / WK-521 / 2020 isolated by Hokkaido University was used.

[1161] Mouse lung infection, drug administration, and body weight measurement

[1162] In this study, retired female BAL B / c mice aged 37-57 weeks without specific pathogens (CLEA Japan, Inc.) were used. At the time of virus inoculation, mice were anesthetized by intramuscular administration of 100 μL of anesthetic solution containing 0.03 mg / mL of medetomidine hydrochloride, 0.4 mg / mL of midazolam, and 0.5 mg / mL of butorphine tartrate in PBS. Under anesthesia, mice were inoculated intranasally with 50 μL of MA-P10 (3.00×10 2 TCID 50 ). One day before virus infection, the compound represented by formula (IB) was subcutaneously administered to mice at 32, 64, 96, and 128 mg / kg. For control mice, 0.5% MC was subcutaneously administered once. Body weight was monitored once a day, and in the evaluation of survival rate, death was considered when the body weight immediately before infection was less than 80%.

[1163] The compounds of the invention were tested essentially as described above. The results are shown below.

[1164] In the aged 37-57 weeks old retired mice, 3.00×10 2 TCID 50 In the case of , the body weight decreased from the next day of infection, and deaths were observed in some individuals 4 days after infection. All test cases died 8 days after infection. At this time, in the groups administered with 32, 64, 96, and 128 mg / kg of the compound represented by formula (IB), the survival rates until 14 days after infection were 6.7%, 60%, 80%, and 100%, respectively. In the groups administered with 32 mg / kg or more, the body weight loss was suppressed ( Fig. 12A , B). It should be noted that the plasma concentrations were measured just before infection and one day after administration, and the results were 1740, 2990, 3110, and 3370 μg / mL, respectively.

[1165] The above results indicate that, in retired mice aged 37-57 weeks, subcutaneous administration of 64 mg / kg or more of the compound represented by formula (IB) one day before infection has the effect of inhibiting lethality and weight loss when the plasma concentration during viral infection is 2990 μg / mL or more.

[1166] The above test results show that the compound represented by formula (IB) can suppress the severity of symptoms caused by viral infection when administered subcutaneously for prevention, supporting the usefulness of the compound represented by formula (IB) as a preventive drug for infectious diseases caused by SARS-CoV-2.

[1167] Test Example 15: Virus proliferation inhibitory effect confirmation test using human airway epithelial cells

[1168] <Operation steps>

[1169] Dilution and injection of test samples

[1170] The test sample was diluted to an appropriate concentration with DMSO in advance, and a 3-fold serial dilution series was prepared. TM The culture medium was diluted 200 times and dispensed into a 24-well plate.

[1171] SARS-CoV-2 infection

[1172] MucilAir inoculated in Transwell TM (Nasal cavity, about 5.0×10 5cells / well) were infected with SARS-CoV-2hCoV-19 / Japan / TY11-927-P1 / 2021, hCoV-19 / Japan / TY38-873 / 2021 (5000TCID 50 / well) and incubated 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. For hCoV-19 / Japan / TY11-927-P1 / 2021, MucilAir was added to the transwell 2 or 3 days after infection. TM For hCoV-19 / Japan / TY38-873 / 2021, add MucilAir to the transwell 1 or 2 days after infection TM The culture medium was collected and the supernatant was recovered.

[1173] Determination of virus titer in supernatant

[1174] The recovered supernatant was diluted 10-fold in a culture medium (MEM, 2% FBS, penicillin-streptomycin) and then incubated with VeroE6 / TMPRSS2 cells (JCRB1819, 1.5×10 4 The cells were mixed with 100 cells / well and inoculated into 96-well plates. After culturing in a CO2 incubator for 4 days, the cytopathic effect (CPE) was observed and the virus titer contained in the supernatant was calculated.

[1175] <Calculation of each measurement item value>

[1176] 90% SARS-CoV-2 virus production inhibition concentration (EC 90 )calculate

[1177] Let x be the logarithmic value of the compound concentration and y be the virus titer (Log 10 TCID 50 / mL) concentration dependence curve, compared with 1log relative to the virus control 10 The value equivalent to reduction was calculated by the two-point method based on the virus titers of the two concentrations before and after it.

[1178] [Mathematical formula 1]

[1179] X = the lowest concentration at the average of reduction from control viral titer of <-1

[1180] x = the highest concentration at the average of reduction from control viral titer of ≥ -1

[1181] Y = the average reduction from control viral titer at x

[1182] y = the average reduction from control viral titer at X

[1183] EC was calculated by 90 value.

[1184] EC 90 =10 [log(x)+(-1Y)×(log(x)-log(X)) / (Y-y)]

[1185] The compounds of the invention were tested essentially as described above. The results are shown below.

[1186] [Table 15]

[1187]

[1188] The formulation examples shown below are merely illustrative and are not intended to limit the scope of the invention in any way.

[1189] The compounds of the present invention can be administered as a pharmaceutical composition by any conventional route, in particular, enterally, for example, orally (in the form of, for example, tablets or capsules), or parenterally (in the form of, for example, injection solutions or suspensions), topically in the form of, for example, lotions, gels, ointments or creams, or in the form of nasal or suppositories. The pharmaceutical composition of the compound of the present invention in the form of a free form or a pharmaceutically acceptable salt together with at least one pharmaceutically acceptable carrier or diluent can be manufactured by mixing, granulating or coating methods using conventional methods. For example, as an oral composition, tablets, granules, and capsules containing excipients, disintegrants, binders, lubricants, etc. and active ingredients can be prepared. In addition, as an injectable composition, a solution or suspension can be prepared, which can be sterilized, and can also contain preservatives, stabilizers, buffers, etc.

[1190] Industrial Applicability

[1191] The compounds of the present invention have inhibitory activity against coronavirus 3CL protease, and pharmaceutical compositions containing the compounds of the present invention are useful as therapeutic and / or preventive agents for coronavirus infectious diseases.

Claims

1. Use of a pharmaceutical composition containing a compound represented by the following formula or a pharmaceutically acceptable salt thereof in the manufacture of a drug for inhibiting the spread of SARS-CoV-2 virus, 2. The use according to claim 1, wherein the pharmaceutical composition is used in the following manner: administration is started after the symptoms of the infectious disease caused by SARS-CoV-2 appear, or within 72 hours from the time of determination of SARS-CoV-2 positivity.

3. The use according to claim 1, wherein the pharmaceutical composition is used in the following manner: administration is started after the symptoms of the infectious disease caused by SARS-CoV-2 appear, or within 24 hours from the time of determination of SARS-CoV-2 positivity.

4. The use according to claim 1, wherein the pharmaceutical composition is used in the following manner: administration is started after the symptoms of the infectious disease caused by SARS-CoV-2 appear, or within 120 hours from the time of determination of SARS-CoV-2 positivity.

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