Compounds with physiological activity such as antiviral activity

Abstract

Provided is a compound represented by general formula (1), which exhibits antiviral activity and inhibitory activity against a virus protease, and which has low toxicity to a host cell.

Description

[Technical Field]

[0001] The present invention relates to a compound exhibiting physiological activity such as antiviral activity, and an antiviral agent containing the compound as an active ingredient. [Background technology]

[0002] The novel coronavirus (SARS-CoV-2) infection (COVID-19), which is believed to have originated in Wuhan, China, in December 2019, quickly spread throughout the world. As of July 2021, the total number of infected people worldwide has exceeded 180 million and is still increasing. While most COVID-19 cases are asymptomatic or mild, severe cases require treatment using devices such as respirators and extracorporeal membrane oxygenation (ECMO). Furthermore, the mortality rate is high, with the total death toll well exceeding 4 million. Furthermore, SARS-CoV-2 has already mutated repeatedly (Non-Patent Document 1), raising concerns about further explosive outbreaks and increased mortality rates.

[0003] To improve this situation, vaccinations with mRNA and DNA vaccines, which are said to prevent the onset of COVID-19 by more than 90%, have begun worldwide. However, it is unclear whether these vaccines can prevent SARS-CoV-2 infection itself. Furthermore, although the protective effect has been reported to last for four months, it is unclear whether the effect will be observed for a longer period (Non-Patent Document 2).

[0004] Furthermore, because vaccines are generally preventative medicines, there is a strong need for effective treatments as quickly as possible to overcome the pandemic, and efforts to develop treatments for COVID-19 are underway. However, treatments administered to asymptomatic or mild cases have been mistakenly deemed effective, resulting in inconsistent efficacy assessments across clinical trials. Furthermore, development has become increasingly confused, as many of these studies have been observational and not double-blind. Furthermore, most compounds identified as "candidate treatments" have been administered before basic knowledge has been accumulated through laboratory or animal testing. Furthermore, unfounded expectations, such as "repurposing," have exacerbated this trend.

[0005] In addition, functional inhibitors of the SPIKE protein, ACE2, RNA-dependent RNA polymerase, and viral protease are being targeted in the development of COVID-19 treatments. Of these, only the RNA-dependent RNA polymerase inhibitor remdesivir has been approved. Remdesivir was developed as a treatment for the 2014 Ebola hemorrhagic fever epidemic and has been approved for use in COVID-19 through drug repositioning. However, hepatotoxicity and nephrotoxicity have been reported, and its use is limited to severely ill patients. Furthermore, the satisfaction rate with monotherapy is low. Therefore, there is a need for compounds that exhibit high antiviral activity against SARS-CoV-2 while maintaining low toxicity to patients. Furthermore, no COVID-19 treatment targeting viral protease has yet been developed. [Prior art documents] [Patent documents]

[0006] [Non-Patent Document 1] Francesco Ortuso,a,b et al. "Structural genetics of circulating variants affecting the SARS-CoV-2 spike / human ACE2 complex", J Biomol Struct Dyn.2021, pages 1-11 [Non-patent document 2] Polack et al. "Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine," NEJM, December 31, 2020, Vol. 383, pp. 2603-2615 [Non-patent document 3] Dafydd Owen, "Oral Inhibitors of the SARS-CoV-2 Main Protease for the Treatment of COVID-19," presented at the American Chemical Society Spring 2021 meeting, April 8, 2021. [Non-patent document 4] Pillaiyar Thanigaimalai et al. "Development of potent dipeptide-type SARS-CoV 3CL protease inhibitors with novel P3 scaffolds: Design, synthesis, biological evaluation, and docking studies", Euro J Med Chem. 2013, vol. 68, pp. 372-384 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in consideration of the problems associated with the prior art, and aims to provide compounds that have activity in inhibiting the proteases of viruses such as SARS-CoV-2 and further have antiviral activity, while exhibiting low toxicity to host cells. [Means for solving the problem]

[0008] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, Mitsuya et al., one of the inventors, screened a group of main protease inhibitors that may also be effective against SARS-CoV-2 from a compound library of main protease inhibitors of SARS-CoV (SARS-CoV-1), which was prevalent in 2003, which they had independently accumulated. Using these inhibitors as lead compounds, they then produced and evaluated various derivatives.

[0009] As a result, it was revealed that the compound represented by the following general formula (1) has the activity of inhibiting the main protease of SARS-CoV-2 and further exhibits excellent antiviral activity against the virus.

[0010] [ka]

[0011] On the other hand, the compound showed low toxicity to the host cells of the virus and no acute or subacute toxicity in small animals such as mice.The compound was also found to have a good in vivo half-life and a high bioavailability (BA) calculated from AUC, demonstrating its usefulness as an oral agent.

[0012] In addition, when compared with a Pfizer compound (compound PF-7321332) currently undergoing clinical trials, which is a main protease inhibitor of SARS-CoV-1, the antiviral activity (EC 50 and EC 99 ) the compounds of the present invention were more than 10 times more potent.

[0013] Furthermore, the compounds of the present invention were compared with another SARS-CoV-1 main protease inhibitor (compound 2420 (YH-53), non-patent document 4), and it was found that the compounds of the present invention were significantly superior in terms of antiviral activity and in vivo half-life.

[0014] The present invention is based on these research results. That is, the present invention relates to compounds that exhibit physiological activities such as viral protease inhibitory activity and antiviral activity, and antiviral agents containing such compounds as active ingredients, and more specifically provides the following: <1> The compound represented by the general formula (1) [In the above formula, X1 represents one or more substituents that can be introduced into the benzothiazole group, and the substituents are each independently selected from a halogen element, a cyclic alkyl group having 3 to 6 carbon atoms, a linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group having 3 to 6 carbon atoms, an aminosulfonyl group, and a methoxy group which may have a substituent.] Z1 represents a carbonyl group or a thiocarbonyl group. R1 is

[0015] [ka]

[0016] represents one of the following: X2 represents one or more substituents that can be introduced into the indole group, and the substituents are each independently selected from a halogen atom and an optionally substituted methoxy group. X3 is

[0017] [ka]

[0018] represents any one of the functional groups. X4 is

[0019] [ka]

[0020] represents any one of the functional groups]. <2> <1> An antiviral agent comprising the compound described in 1 above as an active ingredient. <3> It is an anti-SARS-CoV-2 virus agent, <2> The antiviral agent according to claim 1. [Effects of the Invention]

[0021] According to the present invention, the protease activity of viruses such as SARS-CoV-2 is inhibited, thereby exerting antiviral activity, making it possible to treat or prevent such viral infections. Meanwhile, the compounds of the present invention have low toxicity to host cells, making the above-mentioned treatments possible with minimal side effects. In particular, they do not exhibit acute or subacute toxicity, even in small animals such as rodents. Thus, the safety margin of the compounds of the present invention is wide. Furthermore, the compounds of the present invention may also have favorable pharmacokinetic parameters, such as in vivo half-life and BA. Therefore, they can be effectively administered orally three times a day or less. [Brief explanation of the drawings]

[0022] [Figure 1] This is a graph showing the time course of blood concentration of the compound identified as number 245 in Table 1. "IV," "PO," and "IP" indicate the time course of blood concentration in mice administered the compound intravenously, orally, and intraperitoneally, respectively. Furthermore, "#1" and "#2" indicate that each compound was administered to different mice (two mice in this case) (the notations in the figure are the same as those in Figures 2 and 3). [Figure 2] 1 is a graph showing the time course of blood concentration of the compound shown as No. 245 in Table 1. [Figure 3] 1 is a graph showing the time course of blood concentration of the compound shown as No. 272 ​​in Table 1. DETAILED DESCRIPTION OF THE INVENTION

[0023] (compound) As shown in the Examples below, it has been revealed that the compound represented by the following formula has activity to inhibit SARS-CoV-2 protease and further has antiviral activity, while exhibiting low toxicity to host cells. Therefore, the present invention provides a compound represented by the following general formula (1):

[0024] [ka]

[0025] In the above formula, X1 represents one or more substituents that can be introduced into the benzothiazole group, and the substituents are each independently selected from a halogen element, a cyclic alkyl group having 3 to 6 carbon atoms, a linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group having 3 to 6 carbon atoms, an aminosulfonyl group, and a methoxy group which may have a substituent. Z1 represents a carbonyl group or a thiocarbonyl group. R1 is

[0026] [ka]

[0027] represents one of the following: X2 represents one or more substituents that can be introduced into the indole group, and the substituents are each independently selected from a halogen atom and an optionally substituted methoxy group. X3 is

[0028] [ka]

[0029] represents any one of the functional groups. X4 is

[0030] [ka]

[0031] represents any one of the functional groups.

[0032] In the present invention, the term "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, with a fluorine atom or a chlorine atom being preferred, and a fluorine atom being more preferred.

[0033] Examples of the "cyclic alkyl group having 3 to 6 carbon atoms" include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, and a cyclohexyl group, with a cyclopropyl group being preferred.

[0034] Examples of the "straight-chain alkyl group having 1 to 6 carbon atoms" include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, and an n-hexyl group, with a methyl group, an ethyl group, and a propyl group being preferred.

[0035] Examples of the "branched alkyl group having 3 to 6 carbon atoms" include a 1-methylethyl group (i-propyl group), a 1-methylpropyl group (s-butyl group), a 1,1-dimethylethyl group (t-butyl group), a 2-methylpropyl group (i-butyl group), a 1-methylbutyl group, a 2-methylbutyl group, a 3-methylbutyl group, a 1-ethylpropyl group, a 1,1-dimethylpropyl group, a 1,2-dimethylpropyl group, a 2,2-dimethylpropyl group, a 1-methylpentyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 4-methylpentyl group, Examples include 1-ethylbutyl group, 2-ethylbutyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2,2-dimethylbutyl group, 2,3-dimethylbutyl group, 3,3-dimethylbutyl group, 1,1,2-trimethylpropyl group, 1-ethyl-1-methylpropyl group, and 1-ethyl-2-methylpropyl group, but preferred are branched alkyl groups having 3 to 4 carbon atoms (i-propyl group, s-butyl group, t-butyl group, i-butyl group), and more preferred is i-butyl group.

[0036] The term "carbon number" refers to the number of carbon atoms in the group that serves as the mother nucleus. In other words, this number of carbon atoms does not include the number of carbon atoms in the substituent. The same meaning is used hereinafter in this specification.

[0037] The "aminosulfonyl group" is not particularly limited, and examples thereof include an aminosulfonyl group, a methylaminosulfonyl group, a cyclopropylaminosulfonyl group, a dimethylaminosulfonyl group, and a dicyclopropylaminosulfonyl group.

[0038] In the present invention, the "substituent" that may be possessed by a methoxy group, or a linear alkyl group having 1 to 6 carbon atoms or a branched alkyl group having 3 to 6 carbon atoms, as described below, is not particularly limited as long as it is chemically permissible and can exhibit the activity described below. Examples of the "substituent" include halogen atoms such as chlorine atoms, fluorine atoms, bromine atoms, and iodine atoms (halogeno groups such as chloro groups, fluoro groups, bromo groups, and iodo groups); hydroxy groups; methoxy groups, ethoxy groups, n-propyl groups, ... Examples of the "substituted methoxy group" include alkoxy groups having 1 to 6 carbon atoms, such as propoxy, i-propoxy, n-butoxy, s-butoxy, i-butoxy, and t-butoxy; cyano; amino; and alkyl groups having 1 to 10 carbon atoms, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. These "substituents" may have any hydrogen atom substituted with a group having a different structure. The "substituent" that the methoxy group may have is preferably a halogen element (more preferably, fluorine). A preferred example of a "substituted methoxy group" is a trifluoromethoxy group.

[0039] The number of substituents (X1) introduced into the benzothiazole group is not particularly limited as long as they can be introduced, and may be one or more (e.g., two, three, or four). When more than one substituent is introduced, the types of the substituents may all be the same, or some or all of them may be different. The site at which the substituent is introduced is not particularly limited, but at least one site selected from the 4th, 5th, 6th, and 7th positions is preferred. More specifically, the introduction of a substituent into one site or two sites selected from the 4th, 5th, 6th, and 7th positions is preferred. The introduction of one substituent into the 4th, 5th, or 6th position, or the introduction of two substituents into the 4th and 7th positions is more preferred. The numbering of the sites in the benzothiazole group is as shown in the chemical formula below.

[0040] The substituents (X1) introduced into the benzothiazole group are each independently selected from a cyclic alkyl group having 3 to 6 carbon atoms, a linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group having 3 to 6 carbon atoms, an aminosulfonyl group, a methoxy group which may have a substituent, and a halogen element, and specific examples thereof are as described above. Preferably, at least one halogen element is introduced, more preferably at least one fluorine atom or chlorine atom is introduced, and even more preferably at least one fluorine atom is introduced.

[0041] [ka]

[0042] In the compound of the present invention represented by general formula (1), R1 may be an indole group which may have a substituent, as described above. The number of substituents (X2) introduced into the indole group is not particularly limited as long as it can be introduced, and may be one or more (e.g., two, three, or four). When multiple substituents are introduced, the types of the substituents may all be the same, or some or all may be different. The site of introduction is not particularly limited, but at least one site selected from the 4th, 5th, 6th, and 7th positions is preferred. More specifically, introduction of one substituent into the 4th position, or introduction of two substituents into the 4th and 5th positions, the 4th and 6th positions, or the 4th and 7th positions is mentioned, with introduction of two substituents into the 4th and 7th positions being more preferred. The numbering of the sites in the indole group is as shown in the chemical formula below.

[0043] The substituents (X2) introduced into the indole group are each independently selected from an optionally substituted methoxy group and a halogen atom, and specific examples thereof are as described above, but it is preferable that at least two substituents are introduced, more preferably two halogen atoms, or one halogen atom and one optionally substituted methoxy group, and even more preferably two fluorine atoms, or one fluorine atom and one methoxy group. In particular, in the compounds of the present invention, it is desirable that at least two substituents are introduced into the indole group in this manner.

[0044] [ka]

[0045] In the present invention, in addition to the indole group which may have the above-mentioned substituent, as described above, R1 can also take the form of an amidomethyl group having the following X3 and X4.

[0046] [ka]

[0047] Here, X3 represents a branched alkyl group having 3 to 6 carbon atoms which may have a substituent, and more specifically, is selected from the following functional groups, of which preferred is any one of a t-butyl group, a 1-methyl-1-fluoroethyl group, and a 2,2,2-trifluoroethyl group.

[0048] [ka]

[0049] X4 represents a linear alkyl group having 1 to 6 carbon atoms which may have a substituent, or a branched alkyl group having 3 to 6 carbon atoms which may have a substituent, and more specifically, is selected from the following functional groups, and among these, a trifluoromethyl group is preferred.

[0050] [ka]

[0051] Examples of functional groups in the compound of the present invention have been described above. From the viewpoint of further reducing cytotoxicity while exhibiting higher protease inhibitory activity and antiviral activity, which will be described later, in the compound of the present invention, it is preferable that the functional groups take the combinations shown below.

[0052] X1 is a substituent introduced at the 4-position of the benzothiazole group (more preferably a halogen atom, even more preferably a fluorine atom), Z1 is a carbonyl group or a thiocarbonyl group, R1 is an indole group having X2, and X2 is a substituent introduced at the 4- and 7-positions of the indole group (more preferably a halogen atom at both the 4- and 7-positions (even more preferably a fluorine atom at both), or more preferably a halogen atom at either the 4- or 7-position and a methoxy group at the other (even more preferably a halogen atom at the 4-position and a methoxy group at the 7-position, particularly preferably a fluorine atom at the 4-position and a methoxy group at the 7-position)); or A combination in which X1 is a substituent (more preferably a halogen atom, and even more preferably a fluorine atom) introduced at the 4-, 5-, or 6-position of the benzothiazole group, Z1 is a carbonyl group or a thiocarbonyl group, R1 is an amidomethyl group having X3 and X4, X3 is a t-butyl group, and X4 is a trifluoromethyl group.

[0053] Specific embodiments of such combinations include the following compounds shown in the examples below.

[0054] [ka]

[0055] [ka]

[0056] [ka]

[0057] [ka]

[0058] [ka]

[0059] [ka]

[0060] The compounds of the present invention also include pharmacologically acceptable salts, hydrates, or solvates. Such pharmacologically acceptable salts are not particularly limited and can be appropriately selected depending on the structure of the compound, etc. Examples of such pharmacologically acceptable salts include acid addition salts (hydrochloride, sulfate, hydrobromide, nitrate, hydrogensulfate, phosphate, acetate, lactate, succinate, citrate, maleate, hydroxymaleate, tartrate, fumarate, methanesulfonate, p-toluenesulfonate, camphorsulfonate, sulfamate, mandelate, propionate, glycolate, stearate, malate, ascorbate, pamoate, phenylacetate, glutamate, benzoate, salicylate, sulfanilate, 2-acetoxybenzoate, ethanedisulfate, benzoic acid salt, salicylate, sulfanilic acid salt, 2-acetoxybenzoate, ethanedisulfate, benzoic acid salt, 2-acetoxybenzoate ... Examples of suitable salts include sulfonates, oxalates, isethionates, formates, trifluoroacetates, ethylsuccinates, lactobionates, gluconates, glucoheptonates, 2-hydroxyethanesulfonates, benzenesulfonates, lauryl sulfates, aspartates, adipates, hydroiodides, nicotinates, oxalates, picrates, thiocyanates, and undecanoates, as well as base addition salts (sodium salts, potassium salts, zinc salts, calcium salts, bismuth salts, barium salts, magnesium salts, aluminum salts, copper salts, cobalt salts, nickel salts, cadmium salts, ammonium salts, ethylenediamine salts, and N-dibenzylethylenediamine salts). Hydrates and solvates are not particularly limited, and examples include those in which 0.1 to 3 molecules of water or solvent are added to one molecule of the compound or a salt thereof.

[0061] The compounds of the present invention include all isomers and mixtures of isomers, such as tautomers, geometric isomers, optical isomers based on asymmetric carbons, and stereoisomers. Furthermore, the present invention also encompasses compounds that undergo metabolism in vivo, such as oxidation, reduction, hydrolysis, amination, deamination, hydroxylation, phosphorylation, dehydroxylation, alkylation, dealkylation, and conjugation, and still exhibit the desired activity. The present invention also encompasses compounds that undergo metabolism in vivo, such as oxidation, reduction, and hydrolysis, to produce the compounds of the present invention (so-called prodrug forms). Furthermore, the compounds of the present invention can be formulated by known pharmaceutical methods, as described below.

[0062] Furthermore, since the synthesis methods of the compounds of the present invention are shown in detail in the Examples below, a person skilled in the art can synthesize the compounds of the present invention by appropriately selecting reaction raw materials, reaction reagents, reaction conditions (e.g., solvent, reaction temperature, catalyst, reaction time), etc., while referring to the description of the Examples, and appropriately modifying or altering these methods as necessary. Furthermore, the compounds synthesized in this manner can be separated and purified by methods commonly used for isolating and purifying compounds (e.g., reverse phase chromatography, ion exchange chromatography, adsorption chromatography, recrystallization, etc.), either alone or in combination.

[0063] The compounds of the present invention also have protease inhibitory activity and antiviral activity.

[0064] In the present invention, "protease inhibitory activity" refers to the activity of inhibiting the activity of a protease produced by a virus such as SARS-CoV-2 (for example, SARS main protease (3CL protease, an enzyme specified by EC3.4.22.69)). Such activity is expressed as the IC20 calculated using the degree of inhibition of the binding of the protease to its substrate as an index, as shown in Test Example 1 in the Examples below. 50 The compounds of the present invention can be evaluated by IC values ​​of antiprotease inhibitory activity. 50 Preferably, the value is less than 1 μM, more preferably less than 0.3 μM.

[0065] In the present invention, "antiviral activity" means the activity of eliminating or suppressing the proliferation of a virus such as SARS-CoV-2 in cells (host cells) infected with the virus, and includes, for example, the activity of suppressing viral replication in host cells. Such activity can be measured using the EC 50For example, anti-SARS-CoV-2 activity can be evaluated by the measured value after administration of the test compound, obtained by the method described in Test Example 2 in the Examples below. The compounds of the present invention have an EC 50 The EC value showing nearly 100% antiviral activity is preferably less than 1 μM, more preferably less than 0.4 μM, even more preferably less than 0.1 μM, more preferably less than 0.05 μM, and even more preferably less than 0.01 μM. 99 Preferably, the value is less than 5 μM, more preferably less than 1 μM.

[0066] In the present invention, "suppression" means not only partial suppression but also complete suppression (inhibition).

[0067] Furthermore, the compounds of the present invention have low cytotoxicity. In the present invention, "cytotoxicity" means the activity of killing cells, inhibiting their functions, or suppressing their proliferation. Such activity can be measured by CC, which is calculated using as an index the number of surviving host cells (e.g., VeroE6 cells) that can be infected by a virus, as shown in Test Example 3 in the Examples below. 50 The compounds of the present invention can be evaluated by CC 50 The value is preferably 10 μM or more, more preferably 50 μM or more, even more preferably 80 μM or more, and even more preferably 100 μM or more.

[0068] Furthermore, the compound of the present invention preferably has good pharmacokinetic parameters. Here, "pharmacokinetic parameters" refers to, for example, the in vivo half-life (T 1 / 2 ) and bioavailability (BA = bioavailability: an index showing how much of an administered drug circulates throughout the body). In addition, "good pharmacokinetic parameters" means, for example, that the T of the compound of the present invention is preferably high enough to limit the administration to three times a day or less in order to reduce the burden on the subject of administration (i.e., patient, etc.). 1 / 2In order to limit the administration to three times a day or less, the T 1 / 2 Similarly, from the viewpoint of reducing the number of oral administrations per day, the BA is preferably 20% or more, more preferably 30% or more, even more preferably 40% or more, more preferably 50% or more, even more preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and even more preferably 90% or more.

[0069] (Antiviral agents, methods for preventing and treating viral infections) As will be shown in the Examples below, the compounds of the present invention have antiviral activity, etc. Therefore, it is possible to provide an antiviral agent containing the compounds of the present invention as an active ingredient.

[0070] The virus targeted by the present invention is not particularly limited and may be an RNA virus or a DNA virus. However, as described above, it is preferably a virus having a main protease (3CL protease, an enzyme identified by EC3.4.22.69), more preferably SARS-CoV-2. Note that SARS-CoV-2 has already undergone repeated mutations, as shown in Non-Patent Document 1. The present invention desirably targets at least the original genotype of SARS-CoV-2, but may also target more mutant types.

[0071] Furthermore, there are no particular limitations on the infectious diseases that are the subject of the present invention, and examples include SARS-CoV-2 infections (novel coronavirus infection, COVID-19), and all SARS-CoV-2 infections, whether mild, moderate, or severe, regardless of the severity of the symptoms, can be subject to the present invention.

[0072] The compound of the present invention can be formulated by known pharmaceutical methods, and can be used orally or parenterally in the form of, for example, capsules, tablets, pills, liquids, powders, granules, fine granules, film-coated preparations, pellets, troches, sublingual tablets, chewable tablets, buccal tablets, pastes, syrups, suspensions, elixirs, emulsions, liniments, ointments, plasters, poultices, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc.

[0073] In these formulations, the pharmaceutical composition may be appropriately combined with a pharmacologically acceptable carrier or medium, specifically, sterilized water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, isotonic agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, sweetener, thickener, flavoring agent, solubilizing agent, or other additives, etc. More specifically, examples of the carrier include solid carriers such as lactose, kaolin, sucrose, crystalline cellulose, corn starch, talc, agar, pectin, stearic acid, magnesium stearate, lecithin, sodium chloride, etc., and liquid carriers such as glycerin, peanut oil, polyvinylpyrrolidone, olive oil, ethanol, benzyl alcohol, propylene glycol, water, etc.

[0074] The compounds of the present invention may also be used in combination with other known antiviral agents. When the target disease is SARS-CoV-2 infection, such known antiviral agents include, for example, RNA-dependent RNA polymerase inhibitors (e.g., remdesivir), DNA-dependent RNA polymerase inhibitors (reverse transcriptase inhibitors), SPIKE function inhibitors, ACE2 antagonists, and other SARS-CoV-2 protease inhibitors different from the compounds of the present invention.

[0075] Preferred administration forms of the antiviral agent of the present invention are not particularly limited and include oral administration and parenteral administration. More specific examples of parenteral administration include intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration, intramuscular administration, and administration by infusion.

[0076] The antiviral agent of the present invention can be used primarily for humans, but can also be used for animals other than humans, such as laboratory animals.

[0077] When administering the antiviral agent of the present invention, the dosage is appropriately selected depending on the subject's age, body weight, symptoms, health condition, serious condition, drug tolerance, dosage form, etc. The daily dosage of the antiviral agent of the present invention is typically 0.00001 to 1000 mg / kg body weight, and preferably 0.0001 to 100 mg / kg body weight, in terms of the amount of the compound as an active ingredient, and is administered to the subject in a single dose or in divided doses.

[0078] The product or instructions for the antiviral agent of the present invention may be labeled to the effect that the agent is used to treat or prevent viral infections. Here, "labeled on the product or instructions" means that the label is attached to the product itself, container, packaging, etc., or to instructions, package inserts, promotional materials, other printed materials, etc. that disclose product information. In addition, the labeling to the effect that the agent is used to treat viral infections may include, as information regarding the mechanism of action of the antiviral agent of the present invention, that administration of the compound of the present invention can suppress viral protease activity and thereby suppress replication of the virus.

[0079] Thus, the present invention enables the prevention or treatment of infectious diseases by administering the antiviral agent of the present invention to a subject. Accordingly, the present invention also provides a method for preventing or treating viral infections, which comprises administering the compound of the present invention.

[0080] The subjects to which the compounds of the present invention are administered are not particularly limited, and include patients with viral infections, virus carriers before the onset of the infection, and those before infection. For example, the subjects may not only be those infected with SARS-CoV-2, but also those who are not infected (uninfected), or those who have already recovered from SARS-CoV-2 infection. More specifically, the compounds of the present invention can be administered when symptoms of SARS-CoV-2 infection (fever, cough, abnormal taste, abnormal smell, etc.) are observed, or when close contact with a SARS-CoV-2 infected person is confirmed.

[0081] Furthermore, in the present invention, "treatment" includes not only complete recovery from an infectious disease, but also alleviating or ameliorating the symptoms and inhibiting its progression. "Prevention" includes inhibiting or delaying infection, or inhibiting or delaying the onset of the disease. [Example]

[0082] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples. In these examples, the compounds shown below were synthesized. These compounds were then subjected to the tests described below to evaluate various activities.

[0083] [Table 1]

[0084] Each functional group shown in Table 1 corresponds to the following general formula:

[0085] [ka]

[0086] Furthermore, R1 represents an indole group which may have one or more substituents (X2) or an amidomethyl group which has X3 and X4, as shown below.

[0087] [ka]

[0088] Representative examples of synthetic methods for these compounds are shown below. Unless otherwise noted, reactions using air- or moisture-sensitive reagents were carried out in dry glassware under a nitrogen or argon atmosphere using commercially available solvents and reagents.

[0089] In addition, for the analysis of the obtained compounds, thin layer chromatography (TLC) was carried out by developing on Merck 60F254 precoated silica gel, and visualization was carried out by fluorescence quenching with ultraviolet light or by staining with molybdenum phosphate, para-anisaldehyde, or ninhydrin.

[0090] Flash column chromatography was performed using silica gel 60N (Kanto Chemical) or an Isolera One (Biotage) equipped with a SNAP Ultra Silica Cartridge.

[0091] 1 H NMR (400 or 500MHz) and 13 C NMR (100 or 125 MHz) spectra were measured using a Bruker Avance II spectrometer, a Bruker AVANCE 500 spectrometer, or a JEOL JNM-ECA500. Chemical shifts were measured using CDCl3 ( 1 H 7.26 ppm, 13 C 77.16 ppm)MeOD( 1 H 3.31 ppm) or dimethyl sulfoxide (DMSO)-d6 ( 1 H 2.50 ppm, 13 C 39.52 ppm) and was detected as δ (ppm).

[0092] Low- and high-resolution mass spectra (LRMS, HRMS) were recorded on a Bruker Daltonics microTOF focus (electrospray ionization mass spectrometry) spectrometer in either positive or negative ion detection mode.

[0093] Reverse-phase high-performance liquid chromatography (HPLC) was performed using Cosmosil 5C 18 -ARII preparative column (20 × 250 mm, Nacalai Tesque) (flow rate 10.0 mL / min) and Cosmosil 5C 18 An AR II analytical column (4.6 x 250 mm, Nacalai Tesque) was used (flow rate: 1.0 mL / min), and the effluent was detected by UV light at 220 nm. A solvent system containing 0.1% (v / v) TFA / HO (solvent A) and 0.1% (v / v) TFA / MeCN (solvent B) was used for reversed-phase HPLC elution.

[0094] Normal phase HPLC was performed using a CHIRALPAK IC analytical column (4.6 x 250 mm, Daicel) and a CHIRALPAK IC semi-preparative column (10 x 250 mm, Daicel). Elution was performed with hexane and isopropanol, and the effluent was detected by ultraviolet light at 220 nm.

[0095] (Synthesis Example 1) Synthesis of (1R,2S,5S)-3-(4,7-difluoro-1H-indole-2-carbonyl)-N―((S)-1-(4-fluorobenzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (No. 243) To synthesize the compound (TKB0243) represented by the formula below and designated as number 243 in Table 1, first, methyl (1R,2S,5S)-3-(4,7-difluoro-1H-indole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (HNK0502) was synthesized according to the steps shown below.

[0096] [ka]

[0097] [ka]

[0098] Thionyl chloride (0.290 mL, 4.00 mmol) was added dropwise to a solution of (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid hydrochloride (191 mg, 1.00 mmol) in methanol (3.3 mL) at 0 °C. The mixture was stirred at room temperature for 28 hours and then concentrated under reduced pressure to obtain a crude compound in the form of a methyl ester. The crude compound obtained was used in the next reaction without further purification.

[0099] To a solution of the methyl ester in dichloromethane (10 mL) at 0 °C, 4,7-difluoro-1H-indole-2-carboxylic acid (197 mg, 1.00 mmol), 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholino)]uronium hexafluorophosphate (COMU) (514 mg, 1.20 mmol), and N,N-diisopropylethylamine (DIPEA) (0.510 mL, 3.00 mmol) were added and stirred at room temperature for 17 hours. The reaction mixture was quenched with saturated aqueous ammonium chloride, extracted with dichloromethane, and the organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography using hexane-ethyl acetate (4:1) as a developing solvent to give HNK0502 (180 mg, 54%, mixture of rotamers) as a pale yellow solid.

[0100] 1 H NMR(400MHz,CDCl3) δ 10.51(s,0.1H),10.23(s,0.9H),7.05-7.01(m,1H),6.89-6.78(m,1H),6.68-6.59(m,1H),4.84(s,0.1H),4.77(s,0.9H),4.22(dd,J=10.1Hz and 5.4Hz,1H),4.07(d,J=10.1Hz,0.1H),3.99(d,J=10.1Hz,0.9H),3.76(s,2.7H),3.75(s, 0.3H),1.69-1.62(m,1H),1.56-1.48(m,1H),1.08(s,3H),0.99(s,0.3H),0.96(s,2.7H); 13C NMR(125MHz,CDCl3) δ 171.9,159.8,152.9(dd,J=245.0Hz and 2.7Hz),145.9(dd,J=242.2Hz and 3.2Hz),130.8,126.1(dd,J=16.3Hz and 10.5Hz),120.2,(dd,J=24.6Hz and 5.2Hz)109.0(dd,J=19.0Hz and 8.4Hz),104.2(dd,J=21.6Hz and 6.3Hz),102.8,61.0,52.6,48.6,29.9,28.2,26.2,19.7,12.7; HRMS(ESI), m / z calculated for C 18 H 19 F2N2O3[M+H] + 349.1358, found 349.1355.

[0101] On the other hand, tert-Butyl((S)-1-(4-fluorobenzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (CAZ1333) was synthesized by the following reaction.

[0102] [ka]

[0103] n-Butyllithium (1.60 M hexane solution, 2.81 mL, 4.50 mmol) was added dropwise over 15 minutes to a solution of 4-fluorobenzothiazole (766 mg, 5.00 mmol) in tetrahydrofuran (9.0 mL) at -78 °C. After stirring for 1 hour, a solution of methyl ester compound 1 (286 mg, 1.00 mmol) in tetrahydrofuran (1.0 mL) was added dropwise over 20 minutes, and the mixture was stirred for an additional 3 hours. Saturated aqueous ammonium chloride was added to the reaction mixture to quench the reaction, and the mixture was stirred for an additional 20 minutes at 0 °C. The mixture was concentrated under reduced pressure, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography using a 40:1 chloroform-methanol eluent to obtain CAZ1333 (338 mg, 83%) as a brown solid.

[0104] 1 H NMR(500MHz,CDCl3) δ 7.76-7.74(m,1H),7.53-7.49(m,1H),7.27-7.24(m,1H),6.03(s,1H),5.84-5.82(m,1 H),5.61(m,1H),3.42-3.39(m,2H),2.70-2.62(m,2H),2.23-2.08(m,3H),1.44(s,9H); 13 C NMR{1H}(125MHz,CDCl3) δ 193.1,179.8,164.3,157.4(d,J=261.1Hz),142.9(d,J=14.0Hz),139.8(d,J=2.4Hz),129.1(d,J =7.1Hz),118.3(d,J=4.5Hz),112.6(d,J=17.5Hz),80.2,55.6,40.6,38.7,34.7,28.4(3C),28.0; HRMS(ESI), m / z calculated for C 19 H 23 FN3O4S [M+H] + 408.1388, found 408.1384.

[0105] Next, TKB0243 was synthesized from the compounds HNK0502 and CAZ1333 obtained above by the reaction shown below.

[0106] [ka]

[0107] A 2M aqueous solution of lithium hydroxide (0.500 mL, 1.00 mmol) was added to a solution of the methyl ester compound HNK0502 (174 mg, 0.500 mmol) in tetrahydrofuran (5.0 mL) at 0°C. After stirring at room temperature for 3 hours, the reaction mixture was acidified with 1M aqueous hydrochloric acid. The mixture was concentrated under reduced pressure to obtain a crude carboxylic acid compound, which was used in the next reaction without further purification.

[0108] Tetrafluoroboric acid·diethyl ether (0.240 mL, 1.75 mmol) was added dropwise to a solution of Boc-protected amine compound CAZ1333 (203 mg, 0.500 mmol) in dichloromethane (5.0 mL) at 0°C under a nitrogen atmosphere, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and the residue was washed with 2% methanol-containing diethyl ether. The crude tetrafluoroborate salt was used in the next reaction without further purification.

[0109] The crude tetrafluoroborate salt (0.500 mmol) was dissolved in acetonitrile (5.0 mL) at 0 °C. The crude carboxylic acid salt (0.500 mmol), COMU (257 mg, 0.600 mmol), and DIPEA (0.170 mL, 1.00 mmol) were added and stirred at 0 °C for 21 hours. The reaction mixture was quenched by adding saturated aqueous ammonium chloride, extracted with dichloromethane, and the organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was roughly purified by automated silica gel flash column chromatography using a chloroform-methanol (100:0 to 94:6) mixture as a developing solvent to give crude compound TKB0243. Further purification by preparative reverse-phase HPLC and semi-preparative HPLC (CHIRALPAK IC semi-preparative column) gave TKB0243 (39.8 mg, 13%, mixture of rotamers) as a white to pale yellow powder.

[0110] analytical reversed-phase HPLC (linear gradient 40–70% (B / A+B) / 30 min, elution time = 22.7 min); 1 H NMR(500MHz,CDCl3) δ 10.02(s,0.8H),9.89(S,0.2H),9.29-9.28(m,0.2H),8.50-8.49(m,0.8H),7.69-7.65(m,1H),7.46-7.3 9(m,1H),7.25-7.14(m,1H),6.99(s,0.8H),6.90(s,0.2H),6.85-6.79(m,1H),6.65-6.58(m,1H),6.40( m,0.2H),6.28(m,0.8H),5.73-5.72(m,0.8H),5.56-5.54(m,0.2H),4.74-4.72(m,1H),4.29-3.93(m,2H) ),3.38-3.37(m,2H),2.78-2.43(m,4H),2.26-2.11(m,2H),2.08-1.91(m,1H),1.07(s,3H),0.93(m,3H); 13C NMR{1H}(126MHz,CDCl3) δ 191.8,191.7,180.7,180.5,172.0,171.7,164.6,164.6,160.7,160.2,158.2,156.1,153.9,152.0,146. 8,144.9,142.7,142.5,139.7,139.6,130.9,130.9,129.0,128.9,126.2-125.8(m),120.2(dd,J=24.1Hz and 4.8Hz),118.1,112.4(d,J=18.1Hz),109.0-108.7(m),104.3-104.0(m),102.9,101.9,62.4,56.1,55.4,49.2,4 8.7,41.1,41.0,39.7,39.3,34.0,33.3,32.1,30.4,29.0,28.5,28.3,26.3,26.2,25.0,19.7,19.6,12.9,12.8; HRMS(ESI), m / z calculated for C 31 H 28 F3N5O4S [M+H] + 624.1887, found 624.1885.

[0111] (Synthesis Example 2) Synthesis of (1R,2S,5S)-3-(7-fluoro-4-methoxy-1H-indole-2-carbonyl)-N-((S)-1-(4-fluorobenzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (No. 244) To synthesize the compound (TKB0244) represented by the formula below and designated as number 244 in Table 2, first, methyl (1R,2S,5S)-3-(7-fluoro-4-methoxy-1H-indole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (HNK0437) was synthesized according to the following steps.

[0112] [ka]

[0113] [ka]

[0114] Thionyl chloride (0.290 mL, 4.00 mmol) was added dropwise to a solution of (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid hydrochloride (191 mg, 1.00 mmol) in methanol (3.3 mL) at 0 °C. The mixture was stirred at room temperature for 28 hours and then concentrated under reduced pressure to obtain a crude compound in the form of a methyl ester. The crude compound obtained was used in the next reaction without further purification.

[0115] 7-Fluoro-4-methoxy-1H-indole-2-carboxylic acid (209 mg, 1.00 mmol), COMU (514 mg, 1.20 mmol), and DIPEA (0.510 mL, 3.00 mmol) were added to a solution of the methyl ester in dichloromethane (10 mL) at 0 °C and stirred at room temperature for 16 hours. The reaction mixture was quenched by adding saturated aqueous ammonium chloride, extracted with dichloromethane, and the organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography using hexane-ethyl acetate (3:1) as a developing solvent to give HNK0437 (307 mg, 85%, mixture of rotamers) as a white solid.

[0116] 1H NMR(400MHz,CDCl3) δ 10.28(s,0.1H),10.00(s,0.9H),7.08-7.07(m,1H),6.87-6.80(m,1H),6.30-6.24(m,1H),4.87(s,0.1H),4.76(s,0.9H),4.18(dd,J=10.2Hz and 5.4Hz,1H),4.07-4.04(m,0.1H),4.00-3.96(m,0.9H),3.89(s,2.7H),3.86(s, 0.3H),3.75(s,2.7H),3.72(s,0.3H),1.65(d,J=7.5Hz,0.1H)1.59(dd,J=7.4Hz and 5.4Hz,0.9H),1.51-1.45(m,1H),1.05(s,3H),0.96(s,0.3H)0.93(s,2.7H); 13 C NMR(100MHz,CDCl3) δ 172.2,172.0,160.5,160.1,150.3(d,J=2.2Hz),144.6(d,J=238.7Hz),129.6 ,129.5,125.6(d,J=16.1Hz),121.8(d,J=4.9Hz),109.1(d,J=17.7Hz),108.9( d,J=18.3Hz),104.4,104.4,102.7,98.4(d,J=6.1Hz),60.9,60.7,55.6,52.8 ,52.5,48.5,48.2,33.2,29.9,28.2,26.3,26.2,24.9,19.7,19.6,12.6,12.6; HRMS(ESI), m / z calculated for C 19 H 22 FN2O4[M+H] + 361.1558, found 361.1556.

[0117] On the other hand, tert-Butyl((S)-1-(5-fluorobenzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (CAZ1333) was synthesized by the above reaction described in Synthesis Example 1.

[0118] Next, TKB0244 was synthesized from the compounds HNK0437 and CAZ1333 obtained above by the reaction shown below.

[0119] [ka]

[0120] A 2M aqueous solution of lithium hydroxide (0.851 mL, 1.70 mmol) was added to a solution of the methyl ester compound HNK0437 (307 mg, 0.851 mmol) in tetrahydrofuran (8.5 mL) at 0°C. After stirring at room temperature for 4.5 hours, the reaction mixture was acidified with 1M aqueous hydrochloric acid. The mixture was concentrated under reduced pressure to obtain a crude carboxylic acid compound, which was used in the next reaction without further purification.

[0121] Tetrafluoroboric acid·diethyl ether (0.411 mL, 2.97 mmol) was added dropwise to a solution of Boc-protected amine compound CAZ1333 (346 mg, 0.851 mmol) in dichloromethane (8.5 mL) at 0°C under a nitrogen atmosphere, and the mixture was stirred at room temperature for 4.5 hours. The reaction mixture was concentrated under reduced pressure, and the residue was washed with 2% methanol in diethyl ether. The crude tetrafluoroborate salt was used in the next reaction without further purification.

[0122] The crude tetrafluoroborate salt (0.851 mmol) was dissolved in acetonitrile (8.5 mL) at 0 °C. The crude carboxylic acid salt (0.851 mmol), COMU (428 mg, 1.02 mmol), and DIPEA (0.289 mL, 1.70 mmol) were added and stirred at 0 °C for 48 h. The reaction mixture was quenched by the addition of saturated aqueous ammonium chloride, extracted with dichloromethane, and the organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was roughly purified by automated silica gel flash column chromatography using a chloroform-methanol (100:0 to 94:6) mixture as a developing solvent to give crude compound TKB0244. Further purification by preparative reverse-phase HPLC and semi-preparative HPLC (CHIRALPAK IC semi-preparative column) gave TKB0244 (23.4 mg, 4% mixture of rotamers) as a pale yellow solid.

[0123] analytical reversed-phase HPLC (linear gradient 40–70% (B / A+B) / 30 min, elution time = 18.6 min); 1H NMR(500MHz,CDCl3) δ 9.68(s,0.8H),9.60(s,0.2H),8.65(d,J=6.3Hz,0.2H),8.44(d,J=6.3Hz,0.8H),7.67(d,J=7.4Hz,0.8H),7.63(d,J=8.0Hz,0.2H),7.47-7.38(m,1H),7.22-7.18(m,0.2H),7.16-7.12(m,0.8H),7.05-7.03(m,0.8H),6.89-6.88(m,0.2H),6.85(dd,J=10.6Hz and 8.3Hz,0.8H),6.79(dd,J=10.6Hz and 8.3Hz,0.2H),6.30(dd,J=8.6Hz and 2.9Hz,0.8H),6.23(dd,J=8.6Hz and 2.9Hz,0.2H),6.20(brs,0.2H),6.05(brs,0.8H),5.75-5.71(m,0.8H),5.57-5.53(m,0.2H),4.77(s,0.2H),4.72(s,0.8H),4.28-4.22(m,1H),4.07-4.06(m,0.2H),3.98(d,J=10.3Hz,0.8H),3.92(s,2.4H),3.87(s,0.6H),3.38-3.29(m,1.8H),3.21-3.16(m,0.2H),2.79-2.72(m,0.8H),2.70-2.64(m,0.2H),2.58-2.52(m,0.8H),2.35-2.22(m,1.2H),2.22-2.10(m,1H),2.07-1.99(m,1H),1.97-1.82(m,1H),1.66-1.66(m,1H),1.08(s,2.4H),1.06(s,0.6H),0.92-0.92(m,3H); 13C NMR{1H}(126MHz,CDCl3) δ 191.9,191.8,180.1,172.2,171.8,164.7,164.4,161.0,160.5,157.1(d,J=262.0Hz),150.5,150.4,144.6(d,J=239.1Hz),144.5(d,J=2 39.1Hz), 142.6(d,J=13.2Hz),142.6(d,J=14.5Hz),139.6,129.6,129.4,129.0-128.8(m),125.5-125.3(m),121.9(d,J=4.8Hz),118.1-1 18.0(m), 112.4(d,J=16.8Hz),109.1(d,J=18.0Hz),109.0(d,J=18.0Hz),104.5,103.6,98.6(d,J=6.0Hz),98.4(d,J=6.0Hz),62.5,62.3 ,55.8,55.8,55.7,55.4,49.2,48.5,40.8,40.7,39.0,38.9,34.0,33 .3,32.9,30.4,28.6,28.4,26.4,26.3,24.9,19.7,19.5,12.9,12.8; HRMS(ESI), m / z calcd 32 H 31 F2N5O5S [M+H] + 636.2087, found

[0124] (Round 3) (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-N-((S)-1-(4-fluorobenzo[d]thiazol-2-yl)- Structure of 1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (solvent:245). To synthesize the compound (TKB0245) represented by the formula below and designated as number 245 in Table 1, first, (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (TIS1038) was synthesized by the steps shown below.

[0125] [ka]

[0126] [ka]

[0127] (1R,2S,5S)-3-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester (TIS1035) was synthesized according to the method described by Latha G. Nair et al., "P4 capped amides and lactams as HCV NS3 protease inhibitors with improved potency and DMPK profile," Bioorg Med Chem Lett. 2010, Vol. 20, pp. 567-570. Lithium hydroxide monohydrate (116 mg, 2.76 mmol) was then added to a solution of TIS1035 (705 mg, 1.84 mmol) in methanol (12 mL) and HO (6.0 mL) at room temperature. After stirring at room temperature for 3 hours, lithium hydroxide monohydrate (38.6 mg, 0.921 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours. The mixture was then cooled to 0°C, and the reaction mixture was acidified with 2M aqueous hydrochloric acid. Saturated brine was added to the resulting reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate and then concentrated under reduced pressure to obtain a crude carboxylic acid compound. The resulting crude compound was used in the next reaction without further purification.

[0128] The carboxylic acid compound (1.84 mmol) was treated with 4 M hydrochloric acid / dioxane solution (9.2 mL) at 0° C. and stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude compound of the hydrochloride salt of the amine compound, which was used in the next reaction without further purification.

[0129] DIPEA (0.936 mL, 5.52 mmol) and trifluoroacetic anhydride (TFAA) (0.764 mL, 5.52 mmol) were added to a solution of the amine hydrochloride (1.84 mmol) in dichloromethane (18 mL) at 0 °C. The mixture was stirred at room temperature for 10 hours, after which HO was added and the mixture was extracted with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give the crude carboxylic acid TIS1038. The crude compound was purified by silica gel flash column chromatography using chloroform-methanol (100:0 to 10:1) as the developing solvent to give TIS1038 (245 mg, 35% yield over three steps, mixture of rotamers) as a yellow solid.

[0130] 1 H NMR(500MHz,CDCl3) δ 7.49 (d,J=9.7Hz,0.9H),7.42(d,J=9.7Hz,0.1H),6.43(s,1H),4.61(d,J=9.7Hz,0.9H),4.46-4.44(m,1H),4.34(d,J=9.2Hz,0.1H),3.93(dd,J=10.3Hz and 5.2Hz,0.9H),3.86(d,J=10.3Hz,0.9H),3.77(dd,J=12.9 and 5.4 Hz,0.1H),3.55(d,J=12.6Hz,0.1H),1.68(d,J=7.4Hz,0.1H),1.61(d,J=7.4Hz,0.9H),1.54-1.47(m,1H),1.08-0.98(m,12.3H),0.89(s,2.7H); 13C NMR(126MHz,CDCl3) δ 175.3,175.0,169.6,169.4,157.4(q,J=37.6Hz),115.9(q,J=288.5Hz),61.1,59.8,58.4,58.0 ,48.3,47.6,36.9,36.1,32.8,30.2,27.4,26.4,26.4,26.3,26.2,25.7,20.2,19.7,13.3,12.5; HRMS(ESI), m / z calculated for C 18 H 19 F2N2O3[M+H] + 363.1537, found 363.1534.

[0131] On the other hand, tert-Butyl((S)-1-(5-fluorobenzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (CAZ1333) was synthesized by the above reaction described in Synthesis Example 1.

[0132] Next, TKB0245 was synthesized from the compounds TIS1038 and CAZ1333 obtained above by the reaction shown below.

[0133] [ka]

[0134] Tetrafluoroboric acid·diethyl ether (0.0961 mL, 0.700 mmol) was added dropwise to a solution of Boc-protected amine compound CAZ1333 (143 mg, 0.350 mmol) in dichloromethane (3.5 mL) at 0 °C under a nitrogen atmosphere, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was washed with 2% methanol in diethyl ether. The crude tetrafluoroborate salt was used in the next reaction without further purification.

[0135] The crude tetrafluoroborate salt (0.180 mmol) was dissolved in acetonitrile (1.5 mL) at 0 °C, and the crude carboxylic acid TIS1038 (0.150 mmol), COMU (77.1 mg, 0.180 mmol), and DIPEA (0.0765 mL, 0.450 mmol) were added. The mixture was stirred at room temperature for 15 hours. The reaction mixture was quenched with saturated aqueous ammonium chloride, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was roughly purified by automated silica gel flash column chromatography using a chloroform-methanol (100:0 to 94:6) mixture as a developing solvent to give crude compound TKB0245. Further purification by preparative reverse-phase HPLC and semi-preparative HPLC (CHIRALPAK IC semi-preparative column) gave TKB0245 (2.8 mg, 5%, mixture of rotamers) as a white solid.

[0136] analytical reversed-phase HPLC (linear gradient 40–70% (B / A+B) / 30 min, elution time = 23.2 min); 1H NMR(400MHz,CDCl3) δ 8.91(d,J=5.0Hz,0.2H),7.89(d,J=7.3Hz,0.8H),7.76-7.71(m,1H),7.54-7.46(m,1H),7.28-7.22(m,1H),7.09(d,J=9.2Hz,0.8H),7.00(d,J=9.2Hz,0.2H),6.55(s,0.8H),6.32(s,0.2H),5.88-5.82(m,0.8H),5.64-5.59(m,0.2H),4.57(d,J=9.4Hz,0.8H),4.40(s,1H),4.29(d,J=9.2Hz,0.2H),3.98(dd,J=10.2Hz and 5.3Hz,1H),3.83-3.78(m,1H),3.52-3.35(m,2H),3.29-3.21(m,0.2H),2.82-2.65(m,1H),2.64-2.55(m,0.8H),2.38-2.24(m,1H),2.19-2.00(m,2H),1.67(d,J=7.7Hz,0.2H),1.60-1.58(m,0.8H),1.55-1.52(m,0.8H),1.47(dd,J=7.6Hz and 5.5Hz,0.2H),1.07-1.03(m,11H),0.95(s,1.5H),0.88(s,2.5H); 13C NMR{1H}(101MHz,CDCl3) δ 192.2,192.0,181.9,180.8,171.2,171.0,169.2,168.8,164.4,164.2,157.4(d,J=261.1Hz),157.3(d,J=260.3Hz),157.1(q,J=37.4Hz),1 56.8-156.5(m), 142.9(d,J=13.9Hz),142.8(d,J=14.0Hz),139.8(d,J=2.4Hz),129.2(d,J=7.3Hz),129.0(d,J=7.1Hz),118.3(d,J=4.5Hz), 118.1(d,J=4.5Hz),116.0(q,J=288.0Hz),115.9(q,J=287.7Hz),112.7(d,J=17.5Hz),112.5(d,J=17.4Hz),62.0,61.3,58.3,58.0,56.0.54 .7,48.7,48.0,41.3,41.0,39.7,39.1,37.2,36.2,33.8,33.8,32.5,3 0.7,29.3,28.2,27.9,26.6,26.5,26.4,26.2,20.2,19.5,13.5,12.7; HRMS(ESI), m / z calcd 32 H 31 F2N5O5S [M+H] + 654.2368, found

[0137] (Round 4) 2,2,2-trifluoro-N-((S)-1-((1R,2S,5S)-2-(((S)-1-(4-fluorobenzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrrolidine-3-yl)prop The formulation of an-2-yl)carbamothioyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-yl)-3,3-dimethyl-1-oxobutan-2-yl)acetamide(compound;245S). To synthesize the compound represented by the formula below (compound 245S, TKB0245S), which is designated as number 245S in Table 1, tert-butyl (1R,2S,5S)-2-((2-amino-5-nitrophenyl)carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carboxylate (TIS1110) was first synthesized by the steps shown below.

[0138] [ka]

[0139] [ka]

[0140] N-Methylmorpholine (0.550 mL, 5.00 mmol) and isobutyl chloroformate (0.361 mL, 2.75 mmol) were added to a solution of (1R,2S,5S)-3-(tert-butoxycarbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (638 mg, 2.50 mmol) in anhydrous tetrahydrofuran (25 mL) at -10 °C under an argon atmosphere. The reaction mixture was stirred for 10 minutes, and then 4-nitro-1,2-phenylenediamine (422 mg, 2.76 mmol) was added at the same temperature. The mixture was stirred at -10 °C for 2 hours and then at room temperature for an additional 16.5 hours. Saturated aqueous ammonium chloride was added, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by automated silica gel flash column chromatography (Isolera One) using a developing solvent of hexane-ethyl acetate (23:2 to 3:2) to give TIS1110 (881 mg, 2.26 mmol, 90%, mixture of rotamers) as a yellow solid.

[0141] 1H NMR(500MHz,MeOD) δ 8.19(d,J=2.3Hz,0.4H),8.03(d,J=2.3Hz,0.6H),7.95(dt,J=9.2Hz and 2.9Hz,1H),7.92-7.85(m,1H),6.84(d,J=8.6Hz,0.4H),6.80-6.74(m,0.7H),4.24(s,0.4H),4.20(s,0.6H),3.75(dd,J=11.2Hz and 4.6Hz,1H),3.44(t,J=10.9Hz,1H),1.66-1.51(m,2H),1.47(s,5.6H),1.47(s,3.7H),1.11(s,3H),1.04(s,1.0H),1.03(s,1.7H); 13 C NMR(126MHz,CDCl3) δ 170.5,155.6,147.4,138.7.123.8,122.7,121.4,114.7,81.6,61.4,47.3,30.3,28.6(3C),27.7,26.3,19.1,12.7; HRMS(ESI), m / z calculated for C 19 H 27 N4O5[M+H] + 391.1976,found 391.1972.

[0142] Next, tert-butyl (1R,2S,5S)-2-((2-amino-5-nitrophenyl)carbamothioyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carboxylate (TIS1111) was synthesized from the obtained TIS1110 by the following steps.

[0143] [ka]

[0144] Diphosphorus pentasulfide (335 mg, 1.51 mmol) was added to a solution of anhydrous sodium carbonate (160 mg, 1.51 mmol) in anhydrous tetrahydrofuran (15 mL) at room temperature under an argon atmosphere. The mixture was stirred for 2.5 hours until a pale yellow, transparent solution was obtained. TIS1110 (586 mg, 1.50 mmol) was then added to the reaction mixture and stirred for 25 hours. After completion of the reaction, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography using chloroform as a developing solvent to obtain TIS1111 (533 mg, 1.31 mmol, 87%, mixture of rotamers) as a yellow solid.

[0145] 1 H NMR(500MHz,MeOD) δ 8.05-7.94(m,2H),7.92-7.85(m,1H),6.86(d,J=8.6Hz,0.3H),6.78(d,J=9.2Hz,0.7H),4.59(s,0.3H),4.56(s,0.7H),3.86(dd,J=10.9Hz and 5.2Hz,1H),3.49(d,J=10.9Hz,0.3H),3.44(d,J=10.9Hz,0.7H),1.66-1.49(m, 2H),1.46(s,2.5H),1.45(s,6.6H),1.11(s,3H),1.06(s,1.7H),1.05(s,0.9H); 13 C NMR(126MHz,CDCl3) δ 205.6,155.2,148.9,138.2,125.8,125.4,122.0,114.8,81.7,68.7,47.7,34.4,28.7(3C),27.9,26.3,20.0,13.1; HRMS(ESI), m / z calculated for C 19 H 27 N4O4S[M+H] + 407.1748,found 407.1748.

[0146] Next, tert-butyl (1R,2S,5S)-6,6-dimethyl-2-(6-nitro-1H-benzo[d][1,2,3]triazole-1-carbonothioyl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (TIS1123) was synthesized from the obtained TIS1111 by the following steps.

[0147] [ka]

[0148] TIS1111 (276 mg, 0.679 mmol) was dissolved in 95% aqueous acetic acid (7.0 mL) at 40 °C, cooled to 0 °C, and sodium nitrite (69.9 mg, 1.01 mmol) was added portionwise with stirring. The mixture was stirred at room temperature for 30 minutes. Ice and water (~40 mL) were then added, and the precipitate was collected by filtration and washed with ice water. The residue was dried under vacuum at room temperature overnight to obtain TIS1123 as an orange solid, which was subjected to the following reaction without further purification to synthesize tert-butyl (1R,2S,5S)-2-(((S)-1-(4-fluorobenzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamothioyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carboxylate (TIS1123).

[0149] [ka]

[0150] Tetrafluoroboric acid·diethyl ether (0.240 mL, 1.75 mmol) was added dropwise to a solution of CAZ1333 (206 mg, 0.506 mmol) synthesized in the above reaction described in Synthesis Example 1 in dichloromethane (5.0 mL) at 0°C under an argon atmosphere, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was then concentrated under reduced pressure, and the residue was washed with diethyl ether containing 2% methanol. The crude tetrafluoroborate salt was used in the next reaction without further purification.

[0151] At room temperature under an argon atmosphere, N,N-diisopropylethylamine (DIPEA) (0.0850 mL, 0.500 mmol) was added to a solution of the crude tetrafluoroborate salt (0.506 mmol) in anhydrous tetrahydrofuran (5.0 mL). At 0 °C, a solution of TIS1123 (178 mg, 0.426 mmol) in anhydrous tetrahydrofuran (5.0 mL) and DIPEA (0.255 mL, 1.50 mmol) were added to the solution, and the mixture was stirred at room temperature for 6 hours. After completion of the reaction, the solvent was evaporated under reduced pressure, and the residue was crudely purified by automated silica gel flash column chromatography using a chloroform-methanol (100:0 to 19:1) mixture as a developing solvent to obtain crude compound TIS1125.

[0152] Next, as shown below, tetrafluoroboric acid·diethyl ether (0.210 mL, 1.53 mmol) was added dropwise to a solution of TIS1125 (0.426 mmol) in dichloromethane (4.0 mL) at 0 °C under an argon atmosphere, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, and the crude compound was washed with 2% (v / v) methanol-containing diethyl ether. The crude tetrafluoroborate salt thus obtained was used in the next reaction without further purification.

[0153] [ka]

[0154] At 0 °C under an argon atmosphere, a solution of the crude tetrafluoroborate (0.426 mmol) in anhydrous acetonitrile (2.0 mL) was added to a solution of (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid (109 mg, 0.480 mmol) in anhydrous acetonitrile (2.0 mL), COMU (206 mg, 0.481 mmol), and DIPEA (0.289 mL, 1.70 mmol). The reaction was allowed to proceed at room temperature for 22 hours, after which the reaction was quenched by the addition of saturated aqueous sodium bicarbonate. The mixture was extracted with dichloromethane, the organic phase was dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by automated silica gel flash column chromatography using a chloroform-methanol (100:0 to 19:1) mixture as a developing solvent to obtain crude compound TKB0245S. Further purification by semi-preparative HPLC (CHIRALPAK IC semi-preparative column, Daicel) using an elution solvent of hexane-isopropanol (1:1 to 1:9 / 30 min) gave TKB0245S (3.0 mg, 0.0044 mmol, 1%, mixture of rotamers) as a pale yellow solid.

[0155] analytical reversed-phase HPLC (linear gradient 50–70% (B / A+B) / 30 min, elution time = 20.2 min); 11H NMR (500 MHz, DMSO-d6) δ 7.75 (d, J = 8.0 Hz, 1H), 7.52 - 7.48 (m, 1H), 7.27 - 7.24 (m, 1H), 7.07 - 7.02 (m, 1H), 6.20 (brs, 0.3H), 6.16 (t, J = 4.9 Hz, 0.7H), 5.94 (s, 0.3H), 5.80 (s, 0.7H), 4.92 (s, 0.3H), 4.83 (s, 0.7H), 4.58 (d, J = 9.7 Hz, 0.7H), 4.42 (d, J = 9.7 Hz, 0.3H), 4.18 (dd, J = 10.3 Hz and 5.2 Hz, 0.7H), 4.07 (dd, J = 12.9 Hz and 6.0 Hz, 0.7H), 3.80 - 3.70 (m, 1H), 3.38 - 3.30 (m, 2H), 2.78 - 2.72 (m, 0.7H), 2.62 - 2.55 (m, 0.3H), 2.48 - 2.43 (m, 2H), 2.32 - 2.25 (m, 1H), 2.23 - 2.16 (m, 0.3H), 1.96 - 1.87 (m, 0.7H), 1.84 - 1.74 (m, 0.3H), 1.58 - 1.53 (m, 1H), 1.51 - 1.48 (m, 0.3H), 1.38 - 1.30 (m, 1H), 1.04 - 1.03 (m, 9H), 1.00 (s, 4.1H), 0.87 (s, 1.9H); 13 13C NMR (125 MHz, DMSO-d6) δ ; HRMS (ESI), m / z calcd for C 30 H 36 F4N5O4S2 [M + H] + 670.2139, found 670.2141。

[0156] (Synthesis Example 5) Synthesis of (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-N-((S)-1-(5-fluorobenzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (No.: 272) To synthesize the compound (TKB0272) represented by the formula below and identified as number 272 in Table 1, first, (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (TIS1038) was synthesized by the steps shown below.

[0157] [ka]

[0158] [ka]

[0159] Lithium hydroxide monohydrate (116 mg, 2.76 mmol) was added to a solution of (1R,2S,5S)-3-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester (705 mg, 1.84 mmol) synthesized as described above in methanol (12 mL) and HO (6.0 mL) at room temperature. After stirring at room temperature for 3 hours, lithium hydroxide monohydrate (38.6 mg, 0.921 mmol) was added and stirred at room temperature for 1.5 hours. The mixture was then cooled to 0°C and acidified with 2 M aqueous hydrochloric acid. Saturated brine was added to the resulting reaction mixture, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain the crude carboxylic acid compound. The crude compound was used in the next reaction without further purification.

[0160] The carboxylic acid compound (1.84 mmol) was treated with 4 M hydrochloric acid / dioxane solution (9.2 mL) at 0° C. and stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude compound of the hydrochloride salt of the amine compound, which was used in the next reaction without further purification.

[0161] DIPEA (0.936 mL, 5.52 mmol) and trifluoroacetic anhydride (TFAA) (0.764 mL, 5.52 mmol) were added to a solution of the amine hydrochloride (1.84 mmol) in dichloromethane (18 mL) at 0 °C. The mixture was stirred at room temperature for 10 hours, after which HO was added and the mixture was extracted with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give the crude carboxylic acid TIS1038. The crude compound was purified by silica gel flash column chromatography using chloroform-methanol (100:0 to 10:1) as the developing solvent to give TIS1038 (245 mg, 35% yield over three steps, mixture of rotamers) as a yellow solid.

[0162] 1 H NMR(500MHz,CDCl3) δ ;7.49 (d,J=9.7Hz,0.9H),7.42(d,J=9.7Hz,0.1H),6.43(s,1H),4.61(d,J=9.7Hz,0.9H),4.46-4.44(m,1H),4.34(d,J=9.2Hz,0.1H),3.93(dd,J=10.3Hz and 5.2Hz,0.9H),3.86(d,J=10.3Hz,0.9H),3.77(dd,J=12.9 and 5.4 Hz,0.1H),3.55(d,J=12.6Hz,0.1H),1.68(d,J=7.4Hz,0.1H),1.61(d,J=7.4Hz,0.9H),1.54-1.47(m,1H),1.08-0.98(m,12.3H),0.89(s,2.7H); 13 C NMR(126MHz,CDCl3) δ ;175.3,175.0,169.6,169.4,157.4(q,J=37.6Hz),115.9(q,J=288.5Hz),61.1,59.8,58.4,58.0 ,48.3,47.6,36.9,36.1,32.8,30.2,27.4,26.4,26.4,26.3,26.2,25.7,20.2,19.7,13.3,12.5; HRMS(ESI),m / z calcd for C16H22F3N2O4 [MH]- 363.1537, found 363.1534.

[0163] On the other hand, tert-Butyl((S)-1-(5-fluorobenzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (CAZ1235) was synthesized by the following steps.

[0164] [ka]

[0165] n-Butyllithium (1.60 M hexane solution, 1.18 mL, 1.9 mmol) was added dropwise over 15 minutes to a solution of 5-fluorobenzothiazole (322 mg, 2.10 mmol) in tetrahydrofuran (3.2 mL) at −78°C. After stirring for 1 hour, a solution of methyl ester compound 1 (121 mg, 0.421 mmol) in tetrahydrofuran (1.0 mL) was added dropwise over 20 minutes at −78°C, and stirring was continued at −78°C for 3 hours. Saturated aqueous ammonium chloride was added to the reaction mixture to quench the reaction, and the mixture was stirred at 0°C for 20 minutes. The mixture was concentrated under reduced pressure, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography using a 40:1 chloroform / methanol eluent to obtain CAZ1235 (CAZ1417) (129 mg, 75%) as a reddish-brown solid.

[0166] 1 H NMR(500MHz,CDCl3) δ 7.93(dd,J=8.9Hz and 5.0Hz,1H),7.84(dd,J=9.0Hz and 2.4Hz,1H),7.33(ddd,J=8.8Hz,8.8Hz, and 2.5Hz,1H),5.84-5.56(m,3H),3.42-3.40(m,2H),2.70-2.58(m,2H),2.15-2.06(m,3H),1.44(s,9H); 13C NMR(125MHz,CDCl3) δ 193.2,179.6,166.3,162.3(d,J=245.8Hz),155.9,154.4(d,J=12.0Hz),133.0,123.5(d,J=9. 8Hz),117.4(d,J=25.4Hz),111.3(d,J=23.4Hz),80.2,55.5,40.5,38.5,34.5,28.4(3C),28.2; HRMS(ESI),m / z calcd for C19H22FN3NaO4S[M+Na]+ 430.1207, found 430.1212.

[0167] Next, TKB0272 was synthesized from the compounds TIS1038 and CAZ1235 obtained above by the reaction shown below.

[0168] [ka]

[0169] Tetrafluoroboric acid·diethyl ether (0.274 mL, 2.00 mmol) was added dropwise to a solution of Boc-protected amine compound CAZ1235 (163 mg, 0.400 mmol) in dichloromethane (4.0 mL) at 0 °C under a nitrogen atmosphere, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was washed with 2% methanol in diethyl ether. The crude tetrafluoroborate salt was used in the next reaction without further purification.

[0170] The carboxylic acid TIS1038 (160 mg, 0.440 mmol) and COMU (188 mg, 0.439 mmol) were dissolved in acetonitrile (2.0 mL) at 0 °C, and DIPEA (0.135 mL, 0.800 mmol) was added. A solution of the crude tetrafluoroborate salt (0.400 mmol) and DIPEA (0.135 mL, 0.800 mmol) in acetonitrile (2.0 mL) was added to the reaction mixture at 0 °C, and the mixture was stirred at room temperature for 23 hours. The reaction mixture was quenched by adding saturated aqueous sodium bicarbonate, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was crudely purified by automated silica gel flash column chromatography using a chloroform-methanol (100:0 to 19:1) mixture as a developing solvent to obtain crude compound TKB0272. Further purification was achieved by preparative reverse-phase HPLC and semi-preparative HPLC (CHIRALPAK IC semi-preparative column) to give TKB0272 (22.1 mg, 9%, mixture of rotamers) as a white solid.

[0171] analytical reversed-phase HPLC (linear gradient 40–70% (B / A+B) / 30 min, elution time = 22.5 min); 1H NMR(500MHz,CDCl3)δ;9.28(d,J=4.0Hz,0.2H),7.95-7.83(m,2.8H),7.36-7.29(m,1H),7.02(d,J=9.2Hz,0.8H),6.93(d,J=8.6Hz,0.2H),6.22(s,0.8H),5.99(s,0.2H),5.83-5.79(m,0.8H),5.64-5.60(m,0.2H),4.57(d,J=9.7Hz,0.8H),4.40(s,0.8H),4.37(s,0.2H),4.29(d,J=9.2Hz,0.2H),3.98(dd,J=10.0Hz and 5.4Hz,0.8H),3.83-3.79(m,1H),3.52-3.47(m,0.4H),3.43-3.36(m,1.8H),3.33-3.26(m,0.2H),2.79-2.73(m,0.8H),2.70-2.64(m,0.2H),2.62-2.56(m,0.8H),2.33-2.29(m, 0.2H),2.26-2.21(m,0.8H),2.19-2.12(m,0.8H),2.10-1.94(m,1.2H), 1.66(d,J=7.4Hz,0.2H),1.58(d,J=8.3Hz,0.8H),1.54(dd,J=7.4Hz and 5.2Hz,0.8H),1.48(dd,J=7.7Hz and 5.4Hz,0.2H),1.08-1.03(m,11H),0.96(s,1.8H),0.89(s,2.2H); 13C NMR{1H}(125MHz,CDCl3)δ;192.3,192.1,181.7,180.4,171.2,170.9,169.1,168.8,166.4,166.2,162.3(d,J=246.3Hz),162.2(d,J=246.5H). z), 157.1(q,J=37.6Hz),156.6(q,J=37.3Hz),154.5(d,J=12.1Hz),154.4(d,J=12.1Hz),133.0,123.5(d,J=9.6Hz),123.3(d,J=9.7Hz),117. 6-117.4(m), 116.0(q,J=288.4Hz),115.9(q,J=288.0Hz),111.4(d,J=22.8Hz),111.3(d,J=24.1Hz),61.9,61.3,58.3,57.9,56.1,54.8,48.6 ,48.1,41.3,40.9,39.7,38.9,37.2,36.2,33.9,33.6,32.0,30.8,29. 9.28.6,27.9,26.6,26.5(3C),26.4,26.3(3C),20.2,19.5,13.6,12.7; HRMS(ESI), m / z calcd for C30H36F4N5O5S[M+H]+ 654.2368, found 654.2367.

[0172] (Chapter 6) (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-N-((S)-1-(6-fluorobenzo[d]thiazol-2-yl)- Structure of 1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (structure:273). To synthesize the compound (TKB0273) represented by the following formula and shown as number 273 in Table 1, (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (TIS1038) was synthesized by the following reaction.

[0173] On the other hand, tert-butyl ((S)-1-(5-fluorobenzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (CAZ1240) was synthesized according to the following steps.

[0174] [ka]

[0175] To a solution of 6-fluorobenzothiazole (766 mg, 5.00 mmol) in tetrahydrofuran (9.0 mL) at −78°C, n-butyllithium (1.60 M hexane solution, 2.80 mL, 4.48 mmol) was added dropwise over 15 minutes. After stirring for 1 hour, a solution of methyl ester compound 1 (315 mg, 1.10 mmol) in tetrahydrofuran (1.0 mL) was added dropwise over 20 minutes at −78°C, and stirring was continued at −78°C for 3 hours. Saturated aqueous ammonium chloride was added to the reaction mixture to terminate the reaction. The mixture was concentrated under reduced pressure, extracted with dichloromethane, and the organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography using a 40:1 chloroform / methanol eluent to obtain CAZ1240 (99.9 mg, 22%) as a reddish-brown solid.

[0176] 1H NMR(500MHz,CDCl3)δ;8.13(dd,J=9.1Hz and 4.8Hz,1H),7.65(dd,J=8.0Hz and 2.4Hz,1H),7.33(ddd,J=8.9Hz,8.9 Hz, and 2.4Hz,1H),5.85(d,J=7.7Hz,1H),5.68(s,1H),5.57-5.54(m,1H),3.41-3.39(m,2H),2.68-2.58(m,2H),2.18-2.02(m,3H),1.44(s, 9H); 13 C NMR(125MHz,CDCl3)δ;193.0,179.6,163.9,162.3(d,J=250.8Hz),155.9,150.3,138.7(d,J=11.4Hz),127 .2(d,J=9.9Hz),116.7(d,J=25.4Hz),108.5(d,J=26.7Hz),80.2,55.5,40.5,38.5,34.6,28.4(3C),28.3; HRMS(ESI),m / z calcd for C19H22FN3NaO4S[M+Na]+ 430.1207, found 430.1206.

[0177] Next, TKB0273 was synthesized from the compounds TIS1038 and CAZ1240 obtained above by the reaction shown below.

[0178] [ka]

[0179] Tetrafluoroboric acid·diethyl ether (0.343 mL, 2.50 mmol) was added dropwise to a solution of Boc-protected amine compound CAZ1240 (204 mg, 0.501 mmol) in dichloromethane (5.0 mL) at 0°C under a nitrogen atmosphere, and the mixture was stirred at room temperature for 1.5 hours. The reaction mixture was concentrated under reduced pressure, and the residue was washed with 2% methanol in diethyl ether. The crude tetrafluoroborate salt was used in the next reaction without further purification.

[0180] The carboxylic acid TIS1038 (202 mg, 0.554 mmol) and COMU (236 mg, 0.551 mmol) were dissolved in acetonitrile (4.0 mL) at 0 °C, and DIPEA (0.169 mL, 1.00 mmol) was added. A solution of the crude tetrafluoroborate salt (0.501 mmol) and DIPEA (0.169 mL, 1.00 mmol) in acetonitrile (2.0 mL) was added to the reaction mixture at 0 °C, and the mixture was stirred at room temperature for 1.5 hours. The reaction mixture was quenched by adding saturated aqueous sodium bicarbonate, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was crudely purified by automated silica gel flash column chromatography using a chloroform-methanol (100:0 to 47:3) mixture as a developing solvent to obtain crude compound TKB0273. Further purification was achieved by preparative reverse-phase HPLC and semi-preparative HPLC (CHIRALPAK IC semi-preparative column) to give TKB0273 (30.6 mg, 9%, mixture of rotamers) as a white solid.

[0181] analytical reversed-phase HPLC (linear gradient 40–70% (B / A+B) / 30 min, elution time = 22.5 min); 1H NMR(500MHz,CDCl3)δ;9.42(d,J=4.2Hz,0.2H),8.17-8.12(m,1H),8.02(d,J=7.1Hz,0.8H),7.65-7.61(m,1H),7.35-7.29(m,1H),7.02(d,J=9.0Hz,0.8H),6.93(d,J=8.8Hz,0.2H),5.97(s,0.8H),5.86(s,0.2H),5.81-5.76(m,0.8H),5.63-5.59(m,0.2H),4.57(d,J=9.4Hz,0.8H),4.40(s,0.8H),4.36(s,0.2H),4.29(d,J=9.0Hz,0.2H),3.97(dd,J=10.2Hz and 5.4Hz,0.8H),3.83-3.78(m,1H),3.51-3.45(m,0.4H),3.41-3.25(m,1.8H),2.75-2.61(m,1H),2.60-2.54(m,0.8H),2.32-2.28(m,0.2H),2.24-2.11(m,1.6H),2.07-1.91(m,1.4H),1.66(d,J=7.7Hz,0.2H),1.58(d,J=7.7Hz,0.8H),1.53(dd,J=7.5Hz and 5.3Hz,0.8H),1.48(dd,J=7.5Hz and 5.6Hz,0.2H),1.08-1.02(m,11.5H),0.96(s,2H),0.88(s,2.5H); 13C NMR{1H}(125MHz,CDCl3)δ;192.2,192.0,181.5,180.0,171.2,170.8,169. 1,168.7,164.0(d,J=3.9Hz),163.8(d,J=3.6Hz),162.3(d,J=251.1Hz),16 2.2(d,J=251.3Hz),157.1(q,J=37.2Hz),156.5(q,J=37.0Hz),150.4,150. 3,138.7(d,J=11.7Hz),127.3(d,J=10.3Hz),127.2(d,J=9.9Hz),116.7(d,J =25.3Hz),116.5(d,J=25.5Hz),116.0(q,J=288.0Hz),116.0(q,J=287.8Hz ),108.5(d,J=26.8Hz),108.4(d,J=26.4Hz),61.9,61.2,58.3,57.9,56.1,5 4.9,48.6,48.1,41.1,40.6,39.6,38.8,37.2,36.2,33.9,33.5,32.0,30.8 ,30.0,28.8,27.8,26.6,26.5(3C),26.4,26.3(3C),20.2,19.5,13.6,12.7; HRMS(ESI),m / z calcd for C30H36F4N5O5S[M+H]+ 654.2368, found 654.2364.

[0182] Although no specific method is shown, the compound represented by the following formula was also synthesized in the same manner as the above compound.

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[0199] The resulting compounds were tested for viral protease inhibitory activity (IC 50 ), antiviral activity (EC 50 ), cytotoxicity (CC 50 ), in vivo half-life (T 1 / 2 ) and bioavailability (BA) were evaluated.

[0200] (Test Example 1) Evaluation of viral protease (main protease) inhibitory activity The Mpro / 3CL protease inhibitory activity of the above compounds (test compounds) was evaluated using a FRET-based assay kit (product name: 3CL Protease Untagged Assay Kit, BPS Bioscience, CA, USA). Specifically, serially diluted test compounds were dispensed into a 96-well plate at 10 μl / well, followed by the addition of 3CL protease at 15 ng / 30 μL / well and incubation at room temperature for 30 minutes. Immediately thereafter, 200 μM 3CL protease fluorogenic substrate was added at 10 μL / well. After incubation at 25°C for up to 4 hours, the fluorescence intensity (excitation 360 nm / emission 460 nm) was quantified using a Cytation5 microplate reader (BioTek). The IC was calculated from the quantified values. 50 The values ​​were calculated to evaluate the anti-SARS-CoV-2 enzyme inhibitory activity of each test compound. The results are shown in Table 2.

[0201] (Test Example 2) Evaluation of anti-SARS-CoV-2 activity The SARS-CoV-2 strain (SARS-CoV-2WK-521) provided by the National Institute of Infectious Diseases (Japan) was used as the source of infection. VeroE6 cells were used as the infected cells, and were continuously cultured and maintained in DMEM medium containing 10% FCS and antibiotics (penicillin, streptomycin, each 100 μg / ml). VeroE6 cells were plated at 1x10 in a 96-well plate. 4 The cells were seeded at 1000 cells / well. The following day, the SARS-CoV-2WK-521 virus was exposed to VeroE6 cells at a multiplicity of infection (MOI) of 0.05 for 1 hour, and the virus solution was removed by washing. Then, medium containing serially diluted test compounds at various concentrations was added, and the cells were cultured under standard culture conditions of 37°C and 5% CO2 for 3 days. The supernatant from each well was then collected, SARS-CoV-2 RNA was extracted using the QIAamp Viral RNA Mini Kit, and the virus copy number was quantified by RT-PCR. EC 50 The EC values ​​were calculated to evaluate the anti-SARS-CoV-2 activity of each test compound. 75 Furthermore, the EC value was calculated as an index of the concentration showing 100% activity. 99 The values ​​were calculated, and the results are shown in Tables 2, 3 and 5.

[0202] (Test Example 3) Cytotoxicity test The cytotoxicity test of the test compound was carried out using VeroE6 cells. Specifically, VeroE6 cells were cultured in a 96-well plate at 1x10 4 The cells were seeded at 1000 cells / well and incubated with medium containing serially diluted test compounds at various concentrations for 3 days under standard culture conditions of 37°C and 5% CO2. The number of viable cells in each well was then quantified using the MTT assay. 50 The results are shown in Tables 2, 3 and 5.

[0203] (Test Example 4) Measurement of pharmacokinetic parameters Measurement of the in vivo half-life of the test compounds was carried out using mice. Specifically, each test compound was administered at 2 mg / kg via the tail vein (intravenous administration), and blood samples were taken 15, 30, 60, 120, 240, and 480 minutes after administration. The compound concentration in the blood was measured by LC / MS / MS, and the in vivo half-life (T 1 / 2 ) was calculated (n=2).

[0204] The bioavailability (BA) of the test compounds was also determined. Specifically, each test compound was administered at 2 mg / kg intravenously (IV), orally (PO), or intraperitoneally (IP). Blood samples were taken 15, 30, 60, 120, 240, and 480 minutes after administration. Blood compound concentrations were measured by LC / MS / MS, and graphs showing the time course of drug blood concentrations were created (the results are shown in Figures 1 to 3). The area enclosed by the blood concentration and time axis for each administration form (area under the drug blood concentration-time curve: AUC) was then calculated. BA was calculated based on the AUC obtained in this way using the following formula: BA(%)=AUC P.O. / AUC I.V. ×D I.V. / D P.O. ×100 (D represents the drug dose in each dosage form).

[0205] [Table 2]

[0206] [Table 3]

[0207] In the table, EC 50 An EC99 value of "<0.2" or "<0.3" indicates that the compound exhibits more than 50% antiviral activity at a concentration of 0.2 μM or 0.3 μM. An EC99 value of "<1.0" indicates that the compound exhibits more than 99% antiviral activity at a concentration of 1.0 μM. CC 50 Values ​​'>100' and '>80' represent no cytotoxicity at 100 μM and 80 μM concentrations, respectively.

[0208] As is clear from the results shown in Tables 2 and 3, all of the test compounds had an EC 50 It also showed significant inhibitory activity (IC) against the main protease of SARS-CoV-2. 50 On the other hand, the toxicity of these compounds to the host cells of the virus (CC 50 ) was low. In addition, in the case of administering the compound to mice to calculate BA, etc., no acute or subacute toxicity was observed.

[0209] Among these test compounds, the compounds designated by numbers 245 and 245S were found to have particularly excellent inhibitory activity against viral proteases, and also to have particularly excellent antiviral activity.

[0210] Furthermore, in (Test Example 2), instead of SARS-CoV-2 WK-521, its mutant strains (all provided by the National Institute of Infectious Diseases (Japan) and shown in Table 4 below) were used to evaluate the anti-SARS-CoV-2 activity of the compounds designated by numbers 245 and 245S. As a result, although not shown in the table, all of the mutant strains tested exhibited excellent antiviral activity, similar to that of the parent strain.

[0211] [Table 4]

[0212] (Comparative Example 2) Comparison with PF-7321332 compound The compound PF-7321332 shown below is disclosed in Non-Patent Document 4 as a compound that exhibits inhibitory activity against the main protease of SARS-CoV-2 and exhibits antiviral activity.

[0213] [ka]

[0214] Therefore, this compound was subjected to the above (Test Example 2) to (Test Example 4) under the same conditions, and the EC 50 , E.C. 75、 EC 99、 and CC 50 The results are shown in Table 5.

[0215] [Table 5]

[0216] As a result, the EC values ​​of the compounds indicated by numbers 245 and 245S were 50 are 0.08 μM and 0.31 μM as shown in Table 5, whereas the EC 50 The EC values ​​of the compounds designated by numbers 245 and 245S were 1.9 μM. 99 are 0.95 μM and 0.98 μM as shown in Table 5, whereas the EC 99 was 9.68 μM.

[0217] From the above results, at least the compounds indicated by numbers 245 and 245S have EC values ​​20 times and 6 times higher than those of the PF-7321332 compound, respectively, as drug candidates. 50 The values ​​are also shown. 99 The results showed that the values ​​were about 10 times better, demonstrating a strong antiviral effect at lower concentrations. Notably, both compounds almost completely inhibited the proliferation of SARS-CoV-2 at a concentration of 1 μM.

[0218] In addition, for the compound shown in No. 245, T calculated from the graph shown in Figure 1 1 / 2The BA was 45 minutes or more. Furthermore, the BA calculated from the AUC of the graph was 31% or more, suggesting that oral administration of the compound up to three times a day would provide sufficient therapeutic effects. The same experiment was conducted again for the same compound, and the BA was calculated from the AUC of the graph shown in Figure 2. As a result, the BA of the compound shown by number 245 was 47.7%. Furthermore, the same experiment was conducted for the compound shown by number 272, and the BA was calculated from the AUC of the graph shown in Figure 3. As a result, the BA of the compound shown by number 272 was 99.3%.

[0219] (Comparative Example 2) Comparison with 2420 (YH-53) compound Compound 2420 (YH-53) shown below is disclosed in Non-Patent Document 4 as compound 5h that exhibits inhibitory activity against the main protease of SARS-CoV (SARS-CoV-1), which was prevalent in 2003.

[0220] [ka]

[0221] Therefore, this compound was subjected to the above (Test Example 2) and (Test Example 4) and the EC 50 and T 1 / 2 was calculated and compared with those for the above compounds.

[0222] As a result, the EC values ​​of the compounds indicated by numbers 245 and 245S were 50 The EC values ​​of 2420 compounds were 0.08 μM and 0.31 μM, respectively. 50 The T 1 / 2 Regarding compound No. 245, it took more than 45 minutes, while compound No. 2420 took about 10 minutes.

[0223] From the above results, it was revealed that at least the compounds represented by numbers 245 and 245S have superior antiviral activity as drug candidates, even compared to compound 2420. [Industrial Applicability]

[0224] As described above, the present invention makes it possible to provide compounds that have excellent antiviral activity against viruses such as SARS-CoV-2 and low toxicity to host cells. Furthermore, such compounds of the present invention also have good pharmacokinetics. Furthermore, they exhibit similar levels of antiviral activity against existing mutant strains. Therefore, the present invention is extremely useful in preventing or treating pandemic SARS-CoV-2 infections and other diseases.

Claims

1. A compound represented by any one of the following formulas: 【Chemistry 1】 【Chemistry 2】 【Transformation 3】

2. An antiviral agent comprising the compound according to claim 1 as an active ingredient.

3. The antiviral agent according to claim 2, which is an anti-SARS-CoV-2 virus agent.

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