WO2022244821A1 - Compound exhibiting physiological activity such as antiviral activity - Google Patents

Compound exhibiting physiological activity such as antiviral activity Download PDF

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WO2022244821A1
WO2022244821A1 PCT/JP2022/020735 JP2022020735W WO2022244821A1 WO 2022244821 A1 WO2022244821 A1 WO 2022244821A1 JP 2022020735 W JP2022020735 W JP 2022020735W WO 2022244821 A1 WO2022244821 A1 WO 2022244821A1
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裕明 満屋
哲和 玉村
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国立研究開発法人国立国際医療研究センター
国立大学法人東京医科歯科大学
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Publication of WO2022244821A1 publication Critical patent/WO2022244821A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/428—Thiazoles condensed with carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

  • 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.
  • SARS-CoV-2 The novel coronavirus (SARS-CoV-2) infectious disease (COVID-19), which is believed to have originated in Wuhan, China in December 2019, has rapidly spread around the world, and as of April 2021, there have been no infections worldwide.
  • the total number of people is over 100 million.
  • most cases of COVID-19 are asymptomatic or mild, but in severe cases, treatment using a respirator or extracorporeal membrane oxygenation (ECMO) is required.
  • ECMO extracorporeal membrane oxygenation
  • the mortality rate is high, with the total number of deaths well over 3 million.
  • SARS-CoV-2 has already undergone repeated mutations about one year after its emergence (Non-Patent Document 1), and there are concerns about further explosive epidemics, increased mortality, and the like.
  • Non-Patent Document 2 In order to improve this situation, vaccination with mRNA and DNA vaccines, which are said to prevent the onset of COVID-19 by more than 90%, has started worldwide. However, it is unclear whether such vaccines can prevent SARS-CoV-2 infection per se. Moreover, although it has been reported that the protective effect lasts for 4 months, it is unknown whether the effect can be seen over a longer period of time (Non-Patent Document 2).
  • the present invention has been made in view of the problems of the prior art, and has an activity to suppress the protease of viruses such as SARS-CoV-2, and has antiviral activity.
  • the object is to provide compounds with low toxicity.
  • the compound has low toxicity to the host cells of the virus, and did not exhibit acute or subacute toxicity to small animals such as mice. It was also found that the compound has a good in vivo half-life.
  • the compound of the present invention was found to have the same antiviral activity as That was it. Furthermore, compared with compound 2420, the in vivo half-life of the compound of the present invention was also good.
  • the present invention is based on such research results. That is, the present invention relates to a compound exhibiting physiological activities such as viral protease inhibitory activity and antiviral activity, and an antiviral agent containing the compound as an active ingredient, and more specifically provides the following.
  • X 1 represents one or more substituents that can be introduced into the benzothiazole group, and the substituents are a halogen element, an optionally substituted C 3-6 cyclic alkyl group, a substituted an optionally substituted linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group having 3 to 6 carbon atoms which may be substituted, an aminosulfonyl group which may be substituted and a methoxy group which may have a substituent.
  • the indole group may or may not have a substituent.
  • X2 represents one or more substituents that can be introduced into the indole group, and the substituents are each independently selected from a halogen element and an optionally substituted methoxy group.
  • Z 1 represents a carbonyl group or an optionally substituted alkylene group having 1 to 6 carbon atoms.
  • R 1 represents a pyrrolidonyl group or a pyridinyl group.
  • Z2 represents a carbonyl group or a thiocarbonyl group.
  • R 2 represents an optionally substituted C 3-6 branched alkyl group or an optionally substituted C 1-6 linear alkyl group.
  • R 3 represents a hydrogen atom or a methyl group].
  • An antiviral agent comprising the compound according to ⁇ 1> as an active ingredient.
  • the antiviral agent according to ⁇ 2> which is an anti-SARS-CoV-2 viral agent.
  • ⁇ 4> An anti-SARS-CoV-2 virus agent comprising the compound according to ⁇ 1> and a SARS-CoV-2 viral RNA-dependent RNA polymerase inhibitor as active ingredients.
  • the present invention it is possible to treat or prevent viral infections by suppressing the protease activity of viruses such as SARS-CoV-2 and thereby exhibiting antiviral activity.
  • viruses such as SARS-CoV-2
  • the compounds of the present invention have low toxicity to host cells, the above-mentioned treatments can be performed with few side effects. In particular, it does not exhibit acute or subacute toxicity to small animals such as rodents. Thus, the safety margin for the compounds of the invention is wide.
  • the compounds of the invention may also exhibit favorable pharmacokinetic parameters such as half-life in vivo. Therefore, it is also possible to obtain an effect with administration of 3 times or less per day.
  • the compound represented by the following formula has an activity to suppress the protease of SARS-CoV-2, and has antiviral activity, while exhibiting low toxicity to host cells. It was revealed. Accordingly, the present invention provides compounds represented by the following general formula (1).
  • the benzothiazole group may or may not have a substituent.
  • X 1 represents one or more substituents that can be introduced into the benzothiazole group, and the substituents are a halogen element, an optionally substituted C 3-6 cyclic alkyl group, a substituted an optionally substituted linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group having 3 to 6 carbon atoms which may be substituted, an aminosulfonyl group which may be substituted and a methoxy group which may have a substituent.
  • the indole group may or may not have a substituent.
  • X2 represents one or more substituents that can be introduced into the indole group, and the substituents are each independently selected from a halogen element and an optionally substituted methoxy group.
  • Z 1 represents a carbonyl group or an optionally substituted alkylene group having 1 to 6 carbon atoms.
  • R 1 represents a pyrrolidonyl group or a pyridinyl group.
  • Z2 represents a carbonyl group or a thiocarbonyl group.
  • R 2 represents an optionally substituted C 3-6 branched alkyl group or an optionally substituted C 1-6 linear alkyl group.
  • R3 represents a hydrogen atom or a methyl group.
  • the "substituent" that can be introduced into the alkyl group, aminosulfonyl group, methoxy group, or alkylene group described later is not particularly limited as long as it is chemically acceptable and can exhibit the activity described later.
  • Number of carbon atoms indicates the number of carbon atoms in the mother nucleus group. That is, the number of carbon atoms does not include the number of carbon atoms in substituent groups.
  • the number of carbon atoms does not include the number of carbon atoms in substituent groups.
  • the cyclic alkyl group in the "optionally substituted cyclic alkyl group having 3 to 6 carbon atoms" is not particularly limited, and examples thereof include cyclopropyl group, cyclobutyl group, cyclopentyl group and cyclohexyl group. , preferably a cyclopropyl group.
  • the straight-chain alkyl group in the "optionally substituted straight-chain alkyl group having 1 to 6 carbon atoms” is not particularly limited, but methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group and n-hexyl group, preferably methyl group, ethyl group and n-propyl group.
  • the "substituent” that such a linear alkyl group may have is not particularly limited, and can be exemplified by the various functional groups described above, preferably a hydroxy group and a halogen element (more preferably fluorine). .
  • substituted linear alkyl group having 1 to 6 carbon atoms include a hydroxymethyl group, a 2,2,2-trifluoroethyl group, a 2,2-difluoroethyl group, a 2- fluoroethyl group, 3,3,3-trifluoropropyl group, 3,3-difluoropropyl group and 3-fluoropropyl group.
  • the branched alkyl group in the "optionally substituted C 3-6 branched alkyl group” is not particularly limited, but 1-methylethyl group (i-propyl group), 1- methylpropyl group (s-butyl group), 1,1-dimethylethyl group (t-butyl group), 2-methylpropyl group (i-butyl group), 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group , 1-ethylpropyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-methylpentyl group, 2-methylpentyl group, 3-methylpentyl group, 4 -methylpentyl group, 1-ethylbutyl group, 2-ethylbutyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2,2-dimethylbutyl group,
  • the "aminosulfonyl group optionally having substituents” is not particularly limited, and examples thereof include an aminosulfonyl group, a methylaminosulfonyl group, a cyclopropylaminosulfonyl group, a dimethylaminosulfonyl group, and a dicyclopropylaminosulfonyl group. is mentioned.
  • the "substituent” that the methoxy group may have is not particularly limited, and can be exemplified by the above various functional groups, preferably a halogen element ( more preferably fluorine).
  • a preferred example of the "substituted methoxy group” is a trifluoromethoxy group.
  • Halogen atom means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, preferably a fluorine atom or a chlorine atom, more preferably a fluorine atom.
  • the alkylene group in the "optionally substituted alkylene group having 1 to 6 carbon atoms” is not particularly limited, but examples include methylene group, ethylene group, n-propylene group, n-butylene group, n- Examples include pentylene group and n-hexylene group, preferably methylene group.
  • the "substituent” that such an alkylene group may have is not particularly limited, and can be exemplified by the various functional groups described above, preferably a linear alkyl group having 1 to 6 carbon atoms, 6 cyclic alkyl groups and branched chain alkyl groups having 3 to 6 carbon atoms. Specific examples of these alkyl groups are as described above.
  • the compound of the present invention includes benzothiazole It is preferred that the group and the indole group have a substituent. Preferred aspects of the substituent are as described above. Furthermore, from the same point of view, in the compound of the present invention, each functional group preferably has the following combinations.
  • the preferred dosage form of the antiviral agent of the present invention is not particularly limited, and includes oral administration and parenteral administration.
  • Parenteral administration includes, more specifically, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration, intramuscular administration, and administration by infusion.
  • infectious diseases can be prevented or treated by administering the antiviral agent of the present invention to a subject.
  • the present invention also provides a method for preventing or treating viral infections, comprising administering a compound of the present invention.
  • a 2M lithium hydroxide aqueous solution (0.200 mL, 0.400 mmol) was added to a methyl ester compound CAZ1514 (72.5 mg, 0.200 mmol)/tetrahydrofuran (2.0 mL) solution at 0°C. After stirring at room temperature for 30 minutes, the reaction solution was acidified with 1M hydrochloric acid aqueous solution. The mixed solution was concentrated under reduced pressure to obtain a crude carboxylic acid compound. Used for next reaction without further purification.
  • the crude tetrafluoroborate compound (0.200 mmol) was dissolved in acetonitrile (2.0 mL) at 0° C., and the crude carboxylic acid compound (0.200 mmol) and COMU (85.7 mg, 0.200 mmol) were dissolved.
  • DIPEA (0.0675 mL, 0.400 mmol) was added and stirred at 0° C. for 2 hours.
  • Saturated aqueous ammonium chloride was added to the reaction mixture to stop the reaction, and the mixture was extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure.
  • the crude compound NWD-2-4 (0.500 mmol) of the above carboxylic acid form was dissolved in acetonitrile (5.0 mL), and COMU (278 mg, 0.650 mmol) was added at 0°C.
  • the crude compound NWD-2-6 (0.500 mmol) and DIPEA (0.170 mL, 1.00 mmol) were added to the mixed solution and stirred overnight at room temperature.
  • Saturated aqueous ammonium chloride was added to the reaction mixture to stop the reaction, dichloromethane was added for extraction, and the organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure.
  • TKB0187 was synthesized in the steps shown below.
  • a 2M lithium hydroxide aqueous solution (0.200 mL, 0.400 mmol) was added to a methyl ester compound TON-0404 (67.7 mg, 0.200 mmol)/tetrahydrofuran (2.0 mL) solution, and the mixture was stirred at room temperature for 40 minutes.
  • a 1 M hydrochloric acid aqueous solution was added to the reaction solution to make it acidic.
  • the mixed solution was concentrated under reduced pressure to obtain a crude carboxylic acid compound. Used for next reaction without further purification.
  • tetrafluoroboric acid/diethyl ether (0.0961 mL, 0.700 mmol) was added to a Boc-protected amine compound CAZ1235 (81.5 mg, 0.200 mmol)/dichloromethane (2.0 mL) solution under a nitrogen atmosphere at 0°C. It was added dropwise and stirred at room temperature for 1 hour. The reaction was concentrated under reduced pressure, the residue was washed with 2% methanol in diethyl ether, and the crude tetrafluoroborate salt was used in the next reaction without further purification.
  • the crude tetrafluoroborate compound (0.200 mmol) was dissolved in acetonitrile (2.0 mL), and the crude carboxylic acid compound (0.200 mmol) and COMU (85.7 mg, 0.200 mmol) were dissolved at 0°C. , DIPEA (0.0675 mL, 0.400 mmol) was added and stirred at room temperature for 2 hours. Saturated aqueous ammonium chloride was added to the reaction solution to stop the reaction, the mixture was extracted with dichloromethane, and the organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was crudely purified by silica gel flash column chromatography using a developing solvent of chloroform-methanol (95:5). Further purification by preparative reverse-phase HPLC gave TKB0187 (4.06 mg, 3.3%) as a pale yellow powder.
  • TKB0198 was synthesized in the steps shown below.
  • tetrafluoroboric acid/diethyl ether (0.0960 mL, 0.700 mmol) was added dropwise to a Boc-protected amine compound CAZ1333 (81.5 mg, 0.200 mmol)/dichloromethane (2.0 mL) solution under a nitrogen atmosphere at 0°C. and stirred at room temperature for 30 minutes.
  • the reaction was concentrated under reduced pressure, the residue was washed with 2% methanol in diethyl ether, and the crude tetrafluoroborate salt was used in the next reaction without further purification.
  • Viral protease inhibitory activity (IC 50 ), antiviral activity (EC 50 ), cytotoxicity (CC 50 ) and in vivo half-life (T 1/2 ) of the obtained compounds were measured by the methods described below. evaluated.
  • Test compound Evaluation of viral protease (main protease) inhibitory activity Evaluation of the Mpr/3CL protease enzyme inhibitory activity of the above compound (test compound) was performed using a FRET-based assay kit (product name: 3CL Protease Untagged Assay Kit, BPS Bioscience , CA, USA). Specifically, first, each concentration of the test compound after serial dilution was dispensed into a 96-well plate at 10 ⁇ l/well, then 3CL protease was added at 15 ng/30 ⁇ l/well, stirred and incubated at room temperature for 30 minutes. . Immediately thereafter, 200 ⁇ M of 3CL Protease fluorogenic substrate was added at 10 ⁇ L/well.
  • Test Example 2 Evaluation of anti-SARS-CoV-2 activity
  • SARS-CoV-2WK-521 provided by the National Institute of Infectious Diseases (Japan) was used as an infection source.
  • VeroE6 cells were used as infected cells, and the cells were continuously cultured and maintained in a DMEM medium containing 10% FCS and supplemented with antibiotics (100 ⁇ g/ml each of penicillin and streptomycin). VeroE6 cells were seeded on a 96-well plate at 1 ⁇ 10 4 cells/well.
  • 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. Thereafter, a medium supplemented with each concentration of the test compound after serial dilution was added, and cultured under standard culture conditions of 37° C. and 5% CO 2 for 3 days. 2 RNA was extracted with the QIAamp Viral RNA Mini Kit and viral copy number was quantified by RT-PCR. EC50 values were calculated from the quantified values to evaluate the anti-SARS-CoV-2 activity of each test compound. Table 3 shows the results obtained.
  • MOI multiplicity of infection
  • Test Example 3 Cytotoxicity test A cytotoxicity test of the test compound was performed using VeroE6 cells. Specifically, VeroE6 cells were seeded in a 96-well plate at 1 ⁇ 10 4 cells/well, and standard culture was performed at 37° C. and 5% CO 2 for 3 days with medium supplemented with each test compound at each concentration after serial dilution. After culturing under the conditions, the number of viable cells in each well was quantified by MTT assay and the 50 % cytotoxic concentration (CC50) was calculated. Table 3 shows the results obtained.
  • this compound was subjected to the above (Test Example 1) and (Test Example 4), and the EC 50 and T 1/2 for SARS-CoV-2 were calculated and compared with those of the above compounds.
  • the EC50 of compounds numbered 198 and 199 are 0.13-0.26 ⁇ M and 0.87 ⁇ M, respectively, while the EC50 of compound 2420 is 2.7-3 , as shown in Table 3. 0.2 ⁇ M.
  • the present invention it is possible to provide compounds that have excellent antiviral activity against viruses such as SARS-CoV-2 and have low toxicity to host cells.
  • such compounds of the invention have good pharmacokinetics and may also exert significant synergistic effects against SARS-CoV-2 in combination with the only licensed remdesivir. Therefore, the present invention is extremely useful in the prevention or treatment of pandemic SARS-CoV-2 infectious diseases and the like.

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Abstract

The present invention relates to a compound represented by general formula (1) and an antiviral agent employing said compound as the active ingredient.　

Description

抗ウイルス活性等の生理活性を有する化合物Compounds with physiological activity such as antiviral activity
　本発明は、抗ウイルス活性等の生理活性を示す化合物、及び該化合物を有効成分とする抗ウイルス剤に関する。 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.
　２０１９年１２月に中国・武漢で起こったとみられる新型コロナウイルス（ＳＡＲＳ－ＣｏＶ－２）感染症（ＣＯＶＩＤ－１９）は忽ちにして世界中に拡散し、２０２１年４月現在で世界中の感染者総数は１億人を超えている。また、ＣＯＶＩＤ－１９の症例の多くは無症状又は軽症だが、重症となる場合には、人工呼吸器（レスピレーター）や体外式膜型人工肺（ＥＣＭＯ）等を用いた治療が必要となる。さらに、死亡率も高く、死者総数は３００万人を優に超している。また、ＳＡＲＳ－ＣｏＶ－２は、発生約１年後の現在、既に変異を繰り返しており（非特許文献１）、更なる爆発的流行、死亡率の増加等が懸念されている。 The novel coronavirus (SARS-CoV-2) infectious disease (COVID-19), which is believed to have originated in Wuhan, China in December 2019, has rapidly spread around the world, and as of April 2021, there have been no infections worldwide. The total number of people is over 100 million. In addition, most cases of COVID-19 are asymptomatic or mild, but in severe cases, treatment using a respirator or extracorporeal membrane oxygenation (ECMO) is required. In addition, the mortality rate is high, with the total number of deaths well over 3 million. In addition, SARS-CoV-2 has already undergone repeated mutations about one year after its emergence (Non-Patent Document 1), and there are concerns about further explosive epidemics, increased mortality, and the like.
　かかる状況を改善すべく、ＣＯＶＩＤ－１９の発症を９０％以上阻止するとされるｍＲＮＡワクチンやＤＮＡワクチンの接種が世界的に開始された。しかしながら、そうしたワクチンがＳＡＲＳ－ＣｏＶ－２感染そのものを阻止し得るかは不明である。また、その防御効果が４ヶ月持続することが報告されているものの、それ以上の長期に渡って効果が見られるかも不明である（非特許文献２）。 In order to improve this situation, vaccination with mRNA and DNA vaccines, which are said to prevent the onset of COVID-19 by more than 90%, has started worldwide. However, it is unclear whether such vaccines can prevent SARS-CoV-2 infection per se. Moreover, although it has been reported that the protective effect lasts for 4 months, it is unknown whether the effect can be seen over a longer period of time (Non-Patent Document 2).
　さらに、ワクチンは一般的には予防薬であるため、パンデミック状態を乗り切るために、可及的速やかに有用な治療薬が渇望されており、ＣＯＶＩＤ－１９の治療薬の開発が鋭意進められている。しかしながら、無症状～軽症例に投与された治療薬が希望的に「効いた」と間違えて判断され、臨床試験ごとに効果判定が異なる。しかも、多くが観察研究に過ぎず、二重盲検臨床試験でなかったこともあって、開発は混迷を深めている。更に「治療候補薬」とされた化合物の殆どが実験室や動物実験での基礎的知見が蓄積される以前に投薬されている。加えて「ｒｅ－ｐｕｒｐｏｓｉｎｇ」等の確固とした根拠のない期待がその傾向を助長した。 Furthermore, since vaccines are generally preventative medicines, there is a strong need for effective therapeutic agents to survive the pandemic as soon as possible, and the development of therapeutic agents for COVID-19 is being actively pursued. . However, therapeutic drugs administered to asymptomatic to mild cases are erroneously determined to be hopefully “effective,” and efficacy determinations vary from clinical trial to clinical trial. What's more, most of them were only observational studies, not double-blind clinical trials, and the development process has become even more confusing. Furthermore, most of the compounds identified as “therapeutic candidates” were administered before basic knowledge was accumulated in laboratory and animal experiments. In addition, firm and unfounded expectations such as “re-purposing” have encouraged this trend.
　また、ＣＯＶＩＤ－１９治療薬の開発において、「ＳＰＩＫＥタンパク質」、「ＡＣＥ２」「ＲＮＡ依存性ＲＮＡポリメラーゼ」及び「ウイルスプロテアーゼ」の機能阻害剤が対象とされている。この中で、現在、ＲＮＡ依存性ＲＮＡポリメラーゼ阻害剤であるレムデシビルのみが承認されている。レムデシビルは２０１４年に流行したエボラ出血熱治療薬として開発されており、ドラッグリポジショニングとしてＣＯＶＩＤ－１９への適用が承認されている。しかしながら、肝毒性・腎毒性が報告され、また重症患者のみと制限されている。さらに、単剤での治療満足度は低い。そのため、ＳＡＲＳ－ＣｏＶ－２に対して高い抗ウイルス活性を示す一方で、患者に対しては毒性の低い化合物が希求されている。また、ウイルスプロテアーゼを標的としたＣＯＶＩＤ－１９の治療薬はまだ開発されていない。 In addition, in the development of COVID-19 therapeutic agents, functional inhibitors of "SPIKE protein", "ACE2", "RNA-dependent RNA polymerase" and "viral protease" are targeted. Of these, currently only remdesivir, an inhibitor of RNA-dependent RNA polymerase, is approved. Remdesivir is being developed as a treatment for the Ebola epidemic in 2014, and has been approved for COVID-19 as a drug repositioning agent. However, hepatotoxicity and nephrotoxicity have been reported, and it is restricted to critically ill patients only. Furthermore, satisfaction with treatment with single agents is low. Therefore, there is a need for compounds that exhibit high antiviral activity against SARS-CoV-2 while exhibiting low toxicity to patients. Also, no therapeutic drug for COVID-19 targeting viral protease has been developed yet.
　本発明は、前記従来技術の有する課題に鑑みてなされたものであり、ＳＡＲＳ－ＣｏＶ－２等のウイルスのプロテアーゼを抑制する活性を有し、更には抗ウイルス活性を有する一方で、宿主細胞に対する毒性が低い化合物を提供することを目的とする。 The present invention has been made in view of the problems of the prior art, and has an activity to suppress the protease of viruses such as SARS-CoV-2, and has antiviral activity. The object is to provide compounds with low toxicity.
　本発明者らは、前記課題を解決すべく鋭意研究を重ねた結果、発明者の一人である満屋らが、独自に蓄積した２００３年に流行したＳＡＲＳ－ＣｏＶ（ＳＡＲＳ－ＣｏＶ－１）のメインプロテアーゼ阻害剤の化合物ライブラリーから、ＳＡＲＳ－ＣｏＶ－２にも効果を奏し得るリード化合物をスクリーニングアップした。そして、当該阻害剤についての様々な誘導体を作製して評価した。その結果、下記一般式（１）で表される化合物は、ＳＡＲＳ－ＣｏＶ－２のメインプロテアーゼを抑制する活性を有し、さらには当該ウイルスに対して優れた抗ウイルス活性を発揮することを明らかにした。さらにまた、ＳＡＲＳ－ＣｏＶ－２の様々な変異株に対しても優れた抗ウイルス活性を発揮することも明らかにした。 As a result of extensive research to solve the above problems, the present inventors found that Mitsuya et al. A compound library of protease inhibitors was screened up for lead compounds that could also be effective against SARS-CoV-2. Various derivatives of the inhibitor were then prepared and evaluated. As a result, it was found that the compound represented by the following general formula (1) has activity to inhibit the main protease of SARS-CoV-2 and exhibits excellent antiviral activity against the virus. made it Furthermore, it has also been clarified that it exhibits excellent antiviral activity against various mutant strains of SARS-CoV-2.
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
　その一方で、前記化合物は、ウイルスの宿主となる細胞に対する毒性が低く、さらには、マウス等の小動物に対しても急性・亜急性毒性を示さなかった。また、前記化合物は、生体内半減期が良好であることも見出した。 On the other hand, the compound has low toxicity to the host cells of the virus, and did not exhibit acute or subacute toxicity to small animals such as mice. It was also found that the compound has a good in vivo half-life.
　また、公知のＳＡＲＳ－ＣｏＶ－１のメインプロテアーゼ阻害剤　化合物２４２０（非特許文献３に記載の化合物５ｈ、ＹＨ－５３）と比較した結果、抗ウイルス活性において、前記本発明の化合物は同等又はそれ以上であった。さらに、生体内半減期においても化合物２４２０と比較し、前記本発明の化合物は良好であった。 In addition, as a result of comparison with the known SARS-CoV-1 main protease inhibitor compound 2420 (compound 5h, YH-53 described in Non-Patent Document 3), the compound of the present invention was found to have the same antiviral activity as That was it. Furthermore, compared with compound 2420, the in vivo half-life of the compound of the present invention was also good.
　本発明は、かかる研究成果に基づくものである。すなわち、本発明は、ウイルスプロテアーゼ抑制活性、抗ウイルス活性等の生理活性を示す化合物、及び該化合物を有効成分とする抗ウイルス剤に関し、より具体的には以下を提供する。
＜１＞　前記一般式（１）で表される化合物
［前記式中、ベンゾチアゾール基は、置換基を有していてもよく、置換基を有していなくともよい。Ｘ１はベンゾチアゾール基に導入され得る１又は複数の置換基を表し、当該置換基は、ハロゲン元素、置換基を有していてもよい炭素数３～６の環状アルキル基、置換基を有していてもよい炭素数１～６の直鎖状アルキル基、置換基を有していてもよい炭素数３～６の分岐鎖状アルキル基、置換基を有していてもよいアミノスルホニル基及び置換基を有していてもよいメトキシ基から各々独立して選択される。
インドール基は、置換基を有していてもよく、置換基を有していなくともよい。Ｘ２はインドール基に導入され得る１又は複数の置換基を表し、当該置換基は、ハロゲン元素及び置換基を有していてもよいメトキシ基から各々独立して選択される。
Ｚ１はカルボニル基又は置換基を有していてもよい炭素数１～６のアルキレン基を表す。
Ｒ１はピロリドニル基又はピリジニル基を表す。
Ｚ２はカルボニル基又はチオカルボニル基を表す。
Ｒ２は置換基を有していてもよい炭素数３～６の分岐鎖状アルキル基又は置換基を有していてもよい炭素数１～６の直鎖状アルキル基を表す。
Ｒ３は水素原子又はメチル基を表す］。
＜２＞　＜１＞に記載の化合物を有効成分とする、抗ウイルス剤。
＜３＞　抗ＳＡＲＳ－ＣｏＶ－２ウイルス剤である、＜２＞に記載の抗ウイルス剤。
＜４＞　＜１＞に記載の化合物と、ＳＡＲＳ－ＣｏＶ－２ウイルスＲＮＡ依存性ＲＮＡポリメラーゼ阻害剤とを有効成分とする、抗ＳＡＲＳ－ＣｏＶ－２ウイルス剤。 The present invention is based on such research results. That is, the present invention relates to a compound exhibiting physiological activities such as viral protease inhibitory activity and antiviral activity, and an antiviral agent containing the compound as an active ingredient, and more specifically provides the following.
<1> A compound represented by the general formula (1) [wherein the benzothiazole group may or may not have a substituent. X 1 represents one or more substituents that can be introduced into the benzothiazole group, and the substituents are a halogen element, an optionally substituted C 3-6 cyclic alkyl group, a substituted an optionally substituted linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group having 3 to 6 carbon atoms which may be substituted, an aminosulfonyl group which may be substituted and a methoxy group which may have a substituent.
The indole group may or may not have a substituent. X2 represents one or more substituents that can be introduced into the indole group, and the substituents are each independently selected from a halogen element and an optionally substituted methoxy group.
Z 1 represents a carbonyl group or an optionally substituted alkylene group having 1 to 6 carbon atoms.
R 1 represents a pyrrolidonyl group or a pyridinyl group.
Z2 represents a carbonyl group or a thiocarbonyl group.
R 2 represents an optionally substituted C 3-6 branched alkyl group or an optionally substituted C 1-6 linear alkyl group.
R 3 represents a hydrogen atom or a methyl group].
<2> An antiviral agent comprising the compound according to <1> as an active ingredient.
<3> The antiviral agent according to <2>, which is an anti-SARS-CoV-2 viral agent.
<4> An anti-SARS-CoV-2 virus agent comprising the compound according to <1> and a SARS-CoV-2 viral RNA-dependent RNA polymerase inhibitor as active ingredients.
　本発明によれば、ＳＡＲＳ－ＣｏＶ－２等のウイルスのプロテアーゼ活性を抑制し、ひいては抗ウイルス活性を奏することにより、当該ウイルス感染症の治療又は予防が可能となる。その一方で、本発明の化合物は、宿主細胞に対する毒性が低いため、副作用少なく、前記治療等が可能となる。特に、げっ歯類等の小動物に対しても急性・亜急性毒性を示さない。このように、本発明の化合物の安全域は広い。さらにまた、本発明の化合物は、生体内半減期等の薬物動態パラメーターも良好となり得る。そのため、１日３回以下の投与にて効果を奏することも可能となる。 According to the present invention, it is possible to treat or prevent viral infections by suppressing the protease activity of viruses such as SARS-CoV-2 and thereby exhibiting antiviral activity. On the other hand, since the compounds of the present invention have low toxicity to host cells, the above-mentioned treatments can be performed with few side effects. In particular, it does not exhibit acute or subacute toxicity to small animals such as rodents. Thus, the safety margin for the compounds of the invention is wide. Furthermore, the compounds of the invention may also exhibit favorable pharmacokinetic parameters such as half-life in vivo. Therefore, it is also possible to obtain an effect with administration of 3 times or less per day.
各化合物の血中濃度の時間経過を示すグラフである。図中、「１９８」、「１９９」及び「１２８」は、表１に示す化合物の番号に対応する。また、「２４２０（ＹＨ－５３），１」及び「２４２０（ＹＨ－５３），２」は、化合物２４２０について独立して２回行なった試験結果を各々示す。It is a graph showing the time course of the blood concentration of each compound. In the figure, "198", "199" and "128" correspond to the compound numbers shown in Table 1. "2420 (YH-53), 1" and "2420 (YH-53), 2" respectively indicate the results of two independent tests of compound 2420.
　（化合物）
　後述の実施例において示すとおり、下記式で表される化合物は、ＳＡＲＳ－ＣｏＶ－２のプロテアーゼを抑制する活性を有し、更には抗ウイルス活性を有する一方で、宿主細胞に対する毒性が低いことが明らかになった。したがって、本発明は、下記一般式（１）で表される化合物を提供するものである。 (Compound)
As shown in the examples below, the compound represented by the following formula has an activity to suppress the protease of SARS-CoV-2, and has antiviral activity, while exhibiting low toxicity to host cells. It was revealed. Accordingly, the present invention provides compounds represented by the following general formula (1).
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
　前記式中、ベンゾチアゾール基は、置換基を有していてもよく、置換基を有していなくともよい。Ｘ１はベンゾチアゾール基に導入され得る１又は複数の置換基を表し、当該置換基は、ハロゲン元素、置換基を有していてもよい炭素数３～６の環状アルキル基、置換基を有していてもよい炭素数１～６の直鎖状アルキル基、置換基を有していてもよい炭素数３～６の分岐鎖状アルキル基、置換基を有していてもよいアミノスルホニル基及び置換基を有していてもよいメトキシ基から各々独立して選択される。
インドール基は、置換基を有していてもよく、置換基を有していなくともよい。Ｘ２はインドール基に導入され得る１又は複数の置換基を表し、当該置換基は、ハロゲン元素及び置換基を有していてもよいメトキシ基から各々独立して選択される。
Ｚ１はカルボニル基又は置換基を有していてもよい炭素数１～６のアルキレン基を表す。
Ｒ１はピロリドニル基又はピリジニル基を表す。
Ｚ２はカルボニル基又はチオカルボニル基を表す。
Ｒ２は置換基を有していてもよい炭素数３～６の分岐鎖状アルキル基又は置換基を有していてもよい炭素数１～６の直鎖状アルキル基を表す。
Ｒ３は水素原子又はメチル基を表す。 In the above formula, the benzothiazole group may or may not have a substituent. X 1 represents one or more substituents that can be introduced into the benzothiazole group, and the substituents are a halogen element, an optionally substituted C 3-6 cyclic alkyl group, a substituted an optionally substituted linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group having 3 to 6 carbon atoms which may be substituted, an aminosulfonyl group which may be substituted and a methoxy group which may have a substituent.
The indole group may or may not have a substituent. X2 represents one or more substituents that can be introduced into the indole group, and the substituents are each independently selected from a halogen element and an optionally substituted methoxy group.
Z 1 represents a carbonyl group or an optionally substituted alkylene group having 1 to 6 carbon atoms.
R 1 represents a pyrrolidonyl group or a pyridinyl group.
Z2 represents a carbonyl group or a thiocarbonyl group.
R 2 represents an optionally substituted C 3-6 branched alkyl group or an optionally substituted C 1-6 linear alkyl group.
R3 represents a hydrogen atom or a methyl group.
　本発明において、後述のアルキル基、アミノスルホニル基、メトキシ基又はアルキレン基に導入され得る「置換基」は特に制限はなく、化学的に許容され、後述の活性を奏し得る限り特に制限はないが、例えば、塩素原子、フッ素原子、臭素原子、ヨウ素原子のハロゲン原子（クロロ基、フルオロ基、ブロモ基、アイオド基のハロゲノ基）；ヒドロキシ基；メトキシ基、エトキシ基、ｎ－プロポキシ基、ｉ－プロポキシ基、ｎ－ブトキシ基、ｓ－ブトキシ基、ｉ－ブトキシ基、ｔ－ブトキシ基等の炭素数１～６のアルコキシ基；シアノ基；アミノ基；メチル基、エチル基、ｎ－プロピル基、ｉ－プロピル基、ｎ－ブチル基、ｓ－ブチル基、ｉ－ブチル基、ｔ－ブチル基、ｎ－ペンチル基、ｎ－ヘキシル基、ｎ－ヘプチル基、ｎ－オクチル基、ｎ－ノニル基、ｎ－デシル基等の炭素数１～１０のアルキル基が挙げられる。また、これらの「置換基」は、当該置換基中のいずれかの水素原子が、異なる構造の基で置換されていてもよい。 In the present invention, the "substituent" that can be introduced into the alkyl group, aminosulfonyl group, methoxy group, or alkylene group described later is not particularly limited as long as it is chemically acceptable and can exhibit the activity described later. , for example, chlorine atom, fluorine atom, bromine atom, halogen atom of iodine atom (chloro group, fluoro group, bromo group, halogeno group of iodine group); hydroxy group; methoxy group, ethoxy group, n-propoxy group, i- alkoxy groups having 1 to 6 carbon atoms such as propoxy, n-butoxy, s-butoxy, i-butoxy and t-butoxy; cyano; amino; methyl, ethyl, n-propyl; i-propyl group, n-butyl group, s-butyl group, i-butyl group, t-butyl group, n-pentyl group, n-hexyl group, n-heptyl group, n-octyl group, n-nonyl group, An alkyl group having 1 to 10 carbon atoms such as an n-decyl group can be mentioned. In addition, any hydrogen atom in these "substituents" may be substituted with a group having a different structure.
　なお、「炭素数」は、母核となる基の炭素原子数を表している。すなわち、この炭素原子数には、置換基の中に在る炭素原子の数を含まない。以下、本明細書において、同様の意味で用いる。 "Number of carbon atoms" indicates the number of carbon atoms in the mother nucleus group. That is, the number of carbon atoms does not include the number of carbon atoms in substituent groups. Hereinafter, in this specification, it is used with the same meaning.
　「置換基を有していてもよい炭素数３～６の環状アルキル基」における環状アルキル基としては特に制限はないが、シクロプロピル基、シクロブチル基、シクロペンチル基、シクロへキシル基が挙げられるが、好ましくはシクロプロピル基である。 The cyclic alkyl group in the "optionally substituted cyclic alkyl group having 3 to 6 carbon atoms" is not particularly limited, and examples thereof include cyclopropyl group, cyclobutyl group, cyclopentyl group and cyclohexyl group. , preferably a cyclopropyl group.
　「置換基を有していてもよい炭素数１～６の直鎖状アルキル基」における直鎖状アルキル基としては特に制限はないが、メチル基、エチル基、ｎ－プロピル基、ｎ－ブチル基、ｎ－ペンチル基、ｎ－ヘキシル基が挙げられるが、メチル基、エチル基、ｎ－プロピル基が好ましい。かかる直鎖状アルキル基が有し得る「置換基」としては、特に制限はなく、上記各種官能基を例示することができるが、好ましくはヒドロキシ基、ハロゲン元素（より好ましくは、フッ素）である。また、「置換基を有する炭素数１～６の直鎖状アルキル基」の好適な例として、ヒドロキシメチル基、２，２，２－トリフルオロエチル基、２，２－ジフルオロエチル基、２－フルオロエチル基、３，３，３－トリフルオロプロピル基、３，３－ジフルオロプロピル基、３－フルオロプロピル基が挙げられる。 The straight-chain alkyl group in the "optionally substituted straight-chain alkyl group having 1 to 6 carbon atoms" is not particularly limited, but methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group and n-hexyl group, preferably methyl group, ethyl group and n-propyl group. The "substituent" that such a linear alkyl group may have is not particularly limited, and can be exemplified by the various functional groups described above, preferably a hydroxy group and a halogen element (more preferably fluorine). . Preferable examples of the "substituted linear alkyl group having 1 to 6 carbon atoms" include a hydroxymethyl group, a 2,2,2-trifluoroethyl group, a 2,2-difluoroethyl group, a 2- fluoroethyl group, 3,3,3-trifluoropropyl group, 3,3-difluoropropyl group and 3-fluoropropyl group.
　「置換基を有していてもよい炭素数３～６の分岐鎖状アルキル基」における分岐鎖状アルキル基としては特に制限はないが、１－メチルエチル基（ｉ－プロピル基）、１－メチルプロピル基（ｓ－ブチル基）、１，１－ジメチルエチル基（ｔ－ブチル基）、２－メチルプロピル基（ｉ－ブチル基）、１－メチルブチル基、２－メチルブチル基、３－メチルブチル基、１－エチルプロピル基、１，１－ジメチルプロピル基、１，２－ジメチルプロピル基、２，２－ジメチルプロピル基、１－メチルペンチル基、２－メチルペンチル基、３－メチルペンチル基、４－メチルペンチル基、１－エチルブチル基、２－エチルブチル基、１，１－ジメチルブチル基、１，２－ジメチルブチル基、１，３－ジメチルブチル基、２，２－ジメチルブチル基、２，３－ジメチルブチル基、３，３－ジメチルブチル基、１，１，２－トリメチルプロピル基、１－エチル－１－メチルプロピル基、１－エチル－２－メチルプロピル基が挙げられるが、好ましくは炭素数３～４の分岐鎖状アルキル基（ｉ－プロピル基、ｓ－ブチル基、ｔ－ブチル基、ｉ－ブチル基）であり、より好ましくはｉ－ブチル基である。 The branched alkyl group in the "optionally substituted C 3-6 branched alkyl group" is not particularly limited, but 1-methylethyl group (i-propyl group), 1- methylpropyl group (s-butyl group), 1,1-dimethylethyl group (t-butyl group), 2-methylpropyl group (i-butyl group), 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group , 1-ethylpropyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-methylpentyl group, 2-methylpentyl group, 3-methylpentyl group, 4 -methylpentyl group, 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, 1-ethyl-2-methylpropyl group, preferably carbon A branched alkyl group having a number of 3 to 4 (i-propyl group, s-butyl group, t-butyl group, i-butyl group), more preferably i-butyl group.
　「置換基を有していてもよいアミノスルホニル基」としては特に制限はないが、例えば、アミノスルホニル基、メチルアミノスルホニル基、シクロプロピルアミノスルホニル基、ジメチルアミノスルホニル基、ジシクロプロピルアミノスルホニル基が挙げられる。 The "aminosulfonyl group optionally having substituents" is not particularly limited, and examples thereof include an aminosulfonyl group, a methylaminosulfonyl group, a cyclopropylaminosulfonyl group, a dimethylaminosulfonyl group, and a dicyclopropylaminosulfonyl group. is mentioned.
　「置換基を有していてもよいメトキシ基」において、メトキシ基が有し得る「置換基」としては、特に制限はなく、上記各種官能基を例示することができるが、好ましくはハロゲン元素（より好ましくは、フッ素）である。また、「置換基を有するメトキシ基」の好適な例として、トリフルオロメトキシ基が挙げられる。 In the "optionally substituted methoxy group", the "substituent" that the methoxy group may have is not particularly limited, and can be exemplified by the above various functional groups, preferably a halogen element ( more preferably fluorine). A preferred example of the "substituted methoxy group" is a trifluoromethoxy group.
　「ハロゲン原子」とは、フッ素原子、塩素原子、臭素原子、ヨウ素原子を意味するが、フッ素原子又は塩素原子が好ましく、フッ素原子がより好ましい。 "Halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, preferably a fluorine atom or a chlorine atom, more preferably a fluorine atom.
　「置換基を有していてもよい炭素数１～６のアルキレン基」におけるアルキレン基としては特に制限はないが、例えば、メチレン基、エチレン基、ｎ－プロピレン基、ｎ－ブチレン基、ｎ－ペンチレン基、ｎ－ヘキシレン基が挙げられるが、好ましくはメチレン基である。かかるアルキレン基が有し得る「置換基」としては、特に制限はなく、上記各種官能基を例示することができるが、好ましくは、炭素数１～６の直鎖状アルキル基、炭素数３～６の環状アルキル基、炭素数３～６の分岐鎖状アルキル基が挙げられる。なお、これらアルキル基の具体的な例については上述のとおりである。 The alkylene group in the "optionally substituted alkylene group having 1 to 6 carbon atoms" is not particularly limited, but examples include methylene group, ethylene group, n-propylene group, n-butylene group, n- Examples include pentylene group and n-hexylene group, preferably methylene group. The "substituent" that such an alkylene group may have is not particularly limited, and can be exemplified by the various functional groups described above, preferably a linear alkyl group having 1 to 6 carbon atoms, 6 cyclic alkyl groups and branched chain alkyl groups having 3 to 6 carbon atoms. Specific examples of these alkyl groups are as described above.
　「ピロリドニル基」としては、例えば、２－オキソピロリジン－３－イル基、５－オキソピロリジン－２－イル基、２－オキソピロリジン－１－イル基、３－オキソピロリジン－１－イル基、２，５－ジオキソピロリジン－１－イル基が挙げられるが、好ましくは２－オキソピロリジン－３－イル基である。 The "pyrrolidonyl group" includes, for example, 2-oxopyrrolidin-3-yl group, 5-oxopyrrolidin-2-yl group, 2-oxopyrrolidin-1-yl group, 3-oxopyrrolidin-1-yl group, 2 ,5-dioxopyrrolidin-1-yl group, preferably 2-oxopyrrolidin-3-yl group.
　「ピリジニル基」としては、例えば、４－ピリジニル基、３－ピリジニル基、２－ピリジニル基が挙げられるが、好ましくは４－ピリジニル基である。 The "pyridinyl group" includes, for example, a 4-pyridinyl group, a 3-pyridinyl group and a 2-pyridinyl group, preferably a 4-pyridinyl group.
　ベンゾチアゾール基に導入される置換基（Ｘ１）の数としては、導入し得る限り特に制限はなく、１個であってもよく、複数（例えば、２個、３個、４個）であってもよい。また、複数導入される場合、置換基の種類は全て同一であってもよく、一部又は全て異なっていてもよい。導入される部位としては、特に制限はないが、４位、５位、６位及び７位から選択される少なくとも１カ所の部位が好ましい。より具体的には、４位、５位、６位若しくは７位への１個の置換基の導入、又は、４位及び５位若しくは４位及び７位への２個の置換基の導入が挙げられるが、４位への１個の置換基の導入がより好ましい。なお、ベンゾチアゾール基における部位の番号については、下記化学式に示すとおりである。 The number of substituents (X 1 ) to be introduced into the benzothiazole group is not particularly limited as long as it can be introduced, and may be one or plural (eg, 2, 3, 4). may When a plurality of substituents are introduced, the types of substituents may all be the same, or may be partially or entirely different. The site to be introduced is not particularly limited, but at least one site selected from positions 4, 5, 6 and 7 is preferred. More specifically, the introduction of one substituent at the 4-, 5-, 6- or 7-position, or the introduction of two substituents at the 4- and 5-positions or the 4- and 7-positions but introduction of one substituent at the 4-position is more preferred. Incidentally, the numbers of the sites in the benzothiazole group are as shown in the chemical formulas below.
　また、ベンゾチアゾール基に導入される置換基（Ｘ１）は、置換基を有していてもよい炭素数３～６の環状アルキル基、置換基を有していてもよい炭素数１～６の直鎖状アルキル基、置換基を有していてもよい炭素数３～６の分岐鎖状アルキル基、置換基を有していてもよいアミノスルホニル基、置換基を有していてもよいメトキシ基及びハロゲン元素から各々独立して選択されるものであり、それらの具体的な例については上述のとおりであるが、少なくとも１のハロゲン元素が導入されていることが好ましく、少なくとも１のフッ素原子又は塩素原子が導入されていることがより好ましく、少なくとも１のフッ素原子が導入されていることがさらに好ましい。 Further, the substituent (X 1 ) to be introduced into the benzothiazole group is an optionally substituted C 3-6 cyclic alkyl group, an optionally substituted C 1-6 A straight-chain alkyl group, an optionally substituted C 3-6 branched alkyl group, an optionally substituted aminosulfonyl group, an optionally substituted It is independently selected from a methoxy group and a halogen element, and specific examples thereof are as described above. It is preferable that at least one halogen element is introduced, and at least one fluorine It is more preferred that an atom or a chlorine atom is introduced, and it is even more preferred that at least one fluorine atom is introduced.
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000004
　インドール基に導入される置換基（Ｘ２）の数としては、導入し得る限り特に制限はなく、１個であってもよく、複数（例えば、２個、３個、４個）であってもよい。また、複数導入される場合、置換基の種類は全て同一であってもよく、一部又は全て異なっていてもよい。導入される部位としては、特に制限はないが、４位、５位、６位及び７位から選択される少なくとも１の部位が好ましい。より具体的には、４位への１個の置換基の導入、又は、４位及び５位、４位及び６位若しくは４位及び７位への２個の置換基の導入が挙げられるが、４位及び７位への２個の置換基の導入がより好ましい。なお、インドール基における部位の番号については、下記化学式に示すとおりである。 The number of substituents (X 2 ) to be introduced into the indole group is not particularly limited as long as it can be introduced, and may be one or more (eg, 2, 3, 4). good too. When a plurality of substituents are introduced, the types of substituents may all be the same, or may be partially or entirely different. The site to be introduced is not particularly limited, but at least one site selected from positions 4, 5, 6 and 7 is preferred. More specifically, introduction of one substituent at the 4-position, or introduction of two substituents at the 4-position and the 5-position, the 4-position and the 6-position, or the 4-position and the 7-position. , the introduction of two substituents at the 4- and 7-positions is more preferred. The numbers of the sites in the indole group are as shown in the chemical formulas below.
　また、インドール基に導入される置換基（Ｘ２）は、置換基を有していてもよいメトキシ基及びハロゲン元素から各々独立して選択され、その具体的な例については上述のとおりであるが、少なくとも２つの置換基が導入されていることが好ましく、２つのハロゲン元素が導入、又は、１のハロゲン元素及び１の置換基を有していてもよいメトキシ基が導入されていることがより好ましく、２つのフッ素原子が導入、又は、１のフッ素原子及び１のメトキシ基が導入されていることがさらに好ましい。特に、本発明の化合物において、ベンゾチアゾール基が置換基を有さない場合、このようにインドール基は少なくとも２つの置換基が導入されていることが望ましい。 Further, the substituent (X 2 ) introduced into the indole group is independently selected from an optionally substituted methoxy group and a halogen element, and specific examples thereof are as described above. However, it is preferable that at least two substituents are introduced, and two halogen elements are introduced, or one halogen element and one methoxy group optionally having a substituent are introduced. More preferably, two fluorine atoms are introduced, or one fluorine atom and one methoxy group are further preferably introduced. In particular, in the compound of the present invention, when the benzothiazole group has no substituents, it is desirable that at least two substituents are introduced into the indole group.
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000005
　以上、本発明の化合物における官能基の例について説明したが、より高い後述のプロテアーゼ抑制活性及び抗ウイルス活性を発揮させつつ、細胞毒性をより低下させるという観点から、本発明の化合物において、ベンゾチアゾール基及びインドール基は置換基を有していることが好ましい。なお、当該置換基の好適な態様については上述のとおりである。さらに、同観点から、本発明の化合物において、各官能基は、以下に示す組み合わせをとることが好ましい。 Examples of the functional group 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, the compound of the present invention includes benzothiazole It is preferred that the group and the indole group have a substituent. Preferred aspects of the substituent are as described above. Furthermore, from the same point of view, in the compound of the present invention, each functional group preferably has the following combinations.
　Ｘ１は、ベンゾチアゾール基の４位に導入される置換基（より好ましくはハロゲン元素、さらに好ましくはフッ素原子）であり、Ｚ１はカルボニル基であり、Ｒ１は２－オキソピロリジン－３－イル基であり、Ｚ２はカルボニル基であり、Ｒ２は炭素数３～６の分岐鎖状アルキル基（より好ましくは炭素数４の分岐鎖状アルキル基、さらに好ましくはｉ－ブチル基）であり、Ｒ３は水素原子であり、かつＸ２は、インドール基の４位及び７位に導入される置換基（より好ましくは４位及び７位が共にハロゲン元素（さらに好ましくは共にフッ素原子））、又は、より好ましくは４位及び７位のいずれかがハロゲン元素でありかつ他方がメトキシ基（さらに好ましくは４位がメトキシ基でありかつ７位がハロゲン元素、特に好ましくは４位がメトキシ基でありかつ７位がフッ素原子）である組合わせ。 X 1 is a substituent introduced at the 4-position of the benzothiazole group (more preferably a halogen atom, still more preferably a fluorine atom), Z 1 is a carbonyl group, and R 1 is 2-oxopyrrolidine-3- is an yl group, Z 2 is a carbonyl group, and R 2 is a branched-chain alkyl group having 3 to 6 carbon atoms (more preferably a branched-chain alkyl group having 4 carbon atoms, still more preferably an i-butyl group); is, R 3 is a hydrogen atom, and X 2 is a substituent introduced at the 4- and 7-positions of the indole group (more preferably, both the 4- and 7-positions are halogen atoms (more preferably, both are fluorine atoms) ), or more preferably one of the 4-position and the 7-position is a halogen element and the other is a methoxy group (more preferably the 4-position is a methoxy group and the 7-position is a halogen element, particularly preferably the 4-position is a methoxy group and the 7-position is a fluorine atom).
　かかる組合わせの具体的な態様としては、後述の実施例に示す以下の化合物が挙げられる。 Specific embodiments of such combinations include the following compounds shown in Examples below.
Figure JPOXMLDOC01-appb-C000006
Figure JPOXMLDOC01-appb-C000006
Figure JPOXMLDOC01-appb-C000007
Figure JPOXMLDOC01-appb-C000007
　また、本発明の化合物には、薬理学上許容される塩、水和物又は溶媒和物も含まれる。このような薬理学上許容される塩としては、特に制限はなく、化合物の構造等に応じて適宜選択することができ、例えば、酸付加塩（塩酸塩、硫酸塩、臭化水素塩、硝酸塩、硫酸水素酸塩、リン酸塩、酢酸塩、乳酸塩、コハク酸塩、クエン酸塩、マレイン酸塩、ヒドロキシマレイン酸塩、酒石酸塩、フマル酸塩、メタンスルホン酸塩、ｐ－トルエンスルホン酸塩、樟脳スルホン酸塩、スルファミン酸塩、マンデル酸塩、プロピオン酸塩、グリコール酸塩、ステアリン酸塩、リンゴ酸塩、アスコルビン酸塩、パモン酸塩、フェニル酢酸塩、グルタミン酸塩、安息香酸塩、サリチル酸塩、スルファニル酸塩、２－アセトキシ安息香酸塩、エタンジスルホン酸塩、シュウ酸塩、イセチオン酸塩、ギ酸塩、トリフルオロ酢酸塩、エチルコハク酸塩、ラクトビオン酸塩、グルコン酸塩、グルコヘプトン酸塩、２－ヒドロキシエタンスルホン酸塩、ベンゼンスルホン酸塩、ラウリル硫酸塩、アスパラギン酸塩、アジピン酸塩、ヨウ化水素酸塩、ニコチン酸塩、シュウ酸塩、ピクリン酸塩、チオシアン酸塩、ウンデカン酸塩等）、塩基付加塩（ナトリウム塩、カリウム塩、亜鉛塩、カルシウム塩、ビスマス塩、バリウム塩、マグネシウム塩、アルミニウム塩、銅塩、コバルト塩、ニッケル塩、カドミウム塩、アンモニウム塩、エチレンジアミン塩、Ｎ－ジベンジルエチレンジアミン塩等）が挙げられる。また、水和物又は溶媒和物としては、特に制限はなく、例えば、化合物又はその塩１分子に対し、０．１～３分子の水又は溶媒が付加したものが挙げられる。 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 according to the structure of the compound. , hydrogen sulfate, phosphate, acetate, lactate, succinate, citrate, maleate, hydroxymaleate, tartrate, fumarate, methanesulfonate, p-toluenesulfonic acid salt, camphor sulfonate, sulfamate, mandelate, propionate, glycolate, stearate, malate, ascorbate, pamonate, phenylacetate, glutamate, benzoate, Salicylate, Sulfanilate, 2-Acetoxybenzoate, Ethanedisulfonate, Oxalate, Isethionate, Formate, Trifluoroacetate, Ethylsuccinate, Lactobionate, Gluconate, Glucoheptonate , 2-hydroxyethanesulfonate, benzenesulfonate, lauryl sulfate, aspartate, adipate, hydroiodide, nicotinate, oxalate, picrate, thiocyanate, undecanoic acid salt, etc.), base addition salts (sodium salt, potassium salt, zinc salt, calcium salt, bismuth salt, barium salt, magnesium salt, aluminum salt, copper salt, cobalt salt, nickel salt, cadmium salt, ammonium salt, ethylenediamine salt, N-dibenzylethylenediamine salt, etc.). The hydrate or solvate is not particularly limited, and examples thereof include those obtained by adding 0.1 to 3 molecules of water or a solvent to 1 molecule of the compound or salt thereof.
　本発明の化合物には、互変異性体、幾何異性体、不斉炭素に基づく光学異性体、立体異性体等の総ての異性体及び異性体混合物が含まれる。さらに、本発明の化合物が生体内で酸化、還元、加水分解、アミノ化、脱アミノ化、水酸化、リン酸化、脱水酸化、アルキル化、脱アルキル化、抱合等の代謝を受けてなお所望の活性を示す化合物をも包含し、また本発明は生体内で酸化、還元、加水分解等の代謝を受けて本発明の化合物を生成する化合物（所謂、プロドラッグの形態）をも包含する。さらに、本発明の化合物は、後述のとおり、公知の製剤学的方法により製剤化することができる。 The compounds of the present invention include all isomers and isomer mixtures such as tautomers, geometric isomers, optical isomers based on asymmetric carbons, and stereoisomers. Furthermore, the compounds of the present invention undergo metabolism such as oxidation, reduction, hydrolysis, amination, deamination, hydroxylation, phosphorylation, dehydration-oxidation, alkylation, dealkylation, conjugation, etc. in vivo to produce the desired Compounds exhibiting activity are also included, and the present invention also includes compounds that undergo metabolism such as oxidation, reduction, hydrolysis, etc. in vivo 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.
　また、本発明の化合物の合成に関し、その方法は後述の実施例において詳細に示されているので、当業者であれば、実施例の記載を参照しつつ、反応原料、反応試薬、反応条件（例えば、溶媒、反応温度、触媒、反応時間）等を適宜選択しつつ、必要に応じてこれらの方法に適宜、修飾ないし改変を加えることにより、本発明の化合物を合成することは可能である。また、このようにして合成された化合物は、一般の化合物の単離・精製に使用されている方法（逆相クロマトグラフィー、イオン交換クロマトグラフィー、吸着クロマトグラフィー、再結晶法等）を適宜単独又は組み合わせて用いることにより、分離、精製することができる。 In addition, with respect to the synthesis of the compound of the present invention, the method is described in detail in the examples below. For example, the compound of the present invention can be synthesized by appropriately selecting the solvent, reaction temperature, catalyst, reaction time, etc., and modifying or altering these methods as necessary. In addition, the compounds synthesized in this way can be isolated and purified by methods commonly used for isolation and purification of compounds (reverse phase chromatography, ion exchange chromatography, adsorption chromatography, recrystallization method, etc.) as appropriate. By using them in combination, they can be separated and purified.
　また、本発明の化合物は、プロテアーゼ抑制活性及び抗ウイルス活性を有するものである。 In addition, the compounds of the present invention have protease inhibitory activity and antiviral activity.
　本発明において「プロテアーゼ抑制活性」とは、ＳＡＲＳ－ＣｏＶ－２等のウイルスが産生するプロテアーゼ（例えば、ＳＡＲＳメインプロテアーゼ（３ＣＬプロテアーゼ、ＥＣ３．４．２２．６９によって特定される酵素））の活性を抑制する活性を意味する。かかる活性は、後述の実施例の試験例１に示すように、前記プロテアーゼとその基質との結合に対する抑制の程度を指標として算出されるＩＣ５０値にて評価することができる。本発明の化合物は、抗プロテアーゼ抑制活性のＩＣ５０値が１μＭ未満であることが好ましく、０．５μＭ未満であることがより好ましく、０．１μＭ未満であることがさらに好ましく、０．０５μＭ未満であることがより好ましい。 In the present invention, "protease inhibitory activity" refers to 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)). Inhibitory activity is meant. Such activity can be evaluated by an IC50 value calculated using the degree of inhibition of binding between the protease and its substrate as an index, as shown in Test Example 1 of the Examples below. Compounds of the invention preferably have an IC50 value for antiprotease inhibitory activity of less than 1 μM, more preferably less than 0.5 μM, even more preferably less than 0.1 μM, even less than 0.05 μM. It is more preferable to have
　本発明において「抗ウイルス活性」とは、ＳＡＲＳ－ＣｏＶ－２等のウイルスが感染した細胞（宿主細胞）において、当該ウイルスを消滅させる又はその増殖を抑制する活性を意味し、例えば、宿主細胞におけるウイルス複製を抑制する活性が挙げられる。かかる活性は、宿主細胞におけるウイルスのコピー数等を指標として算出されるＥＣ５０値にて評価することができ、例えば、抗ＳＡＲＳ－ＣｏＶ－２活性については、後述の実施例に示す試験例２に記載の方法により得られる、被験化合物投与後の測定値により評価することができる。本発明の化合物は、抗ウイルス活性のＥＣ５０値が１０μＭ未満であることが好ましく、５μＭ未満であることがより好ましく、１μＭ未満であることがさらに好ましく、０．５μＭ未満であることがより好ましい。 In the present invention, the term "antiviral activity" means an activity that eliminates or suppresses the growth of a virus such as SARS-CoV-2 in a cell (host cell) infected with the virus. An activity that suppresses viral replication can be mentioned. Such activity can be evaluated by the EC50 value calculated using the number of virus copies in host cells as an index. It can be evaluated by the measured value after administration of the test compound, which is obtained by the method described in . Compounds of the invention preferably have an antiviral activity EC50 value of less than 10 μM, more preferably less than 5 μM, even more preferably less than 1 μM, more preferably less than 0.5 μM. .
　なお、本発明において「抑制」とは、部分的な抑制のみならず、完全な抑制（阻害）も意味する。 In the present invention, "suppression" means not only partial suppression but also complete suppression (inhibition).
　また、本発明の化合物は、細胞毒性が低い。本発明において「細胞毒性」とは、細胞を殺傷する、その機能を阻害する、またはその増殖を抑制する活性を意味する。かかる活性は、後述の実施例の試験例３に示すように、ウイルスが感染し得る宿主細胞（例えば、ＶｅｒｏＥ６細胞）の生存数等を指標として算出されるＣＣ５０値にて評価することができる。本発明の化合物は、ＣＣ５０値が、１０μＭ以上であることが好ましく、５０μＭ以上であることがより好ましく、１００μＭ以上であることがさらに好ましい。 Also, the compounds of the present invention have low cytotoxicity. In the present invention, "cytotoxicity" means an activity that kills cells, inhibits their function, or suppresses their proliferation. Such activity can be evaluated by a CC50 value calculated using the survival number of virus-infectable host cells (e.g., VeroE6 cells) as an index, as shown in Test Example 3 of the Examples below. . The compounds of the present invention preferably have a CC 50 value of 10 μM or higher, more preferably 50 μM or higher, even more preferably 100 μM or higher.
　さらに、本発明の化合物は、薬物動態パラメーターが良好であることが好ましい。ここで「薬物動態パラメーター」とは、例えば、後述の実施例の試験例４に示すような、生体内半減期（Ｔ１／２）が挙げられる。また「薬物動態パラメーターが良好」とは、例えば、投与対象（所謂、患者等）の負担を軽減すべく、１日３回以下の投与に抑えるという観点から、本発明の化合物の生体内半減期が、２０分以上であることが挙げられる。 Furthermore, the compounds of the invention preferably have good pharmacokinetic parameters. As used herein, the term "pharmacokinetic parameter" includes, for example, in vivo half-life (T 1/2 ) as shown in Test Example 4 of Examples below. In addition, "favorable pharmacokinetic parameters" means, for example, the in vivo half-life of the compound of the present invention from the viewpoint of suppressing administration to 3 times or less per day in order to reduce the burden on the administration subject (so-called patient, etc.) is 20 minutes or longer.
　（抗ウイルス剤、ウイルス感染症の予防方法、治療方法）
　後述の実施例において示すとおり、本発明の化合物は、抗ウイルス活性等を有する。したがって、本発明の化合物を有効成分とする抗ウイルス剤を提供することができる。 (Antiviral agents, methods for preventing and treating viral infections)
As shown in the examples below, the compounds of the present invention have antiviral activity and the like. Therefore, an antiviral agent containing the compound of the present invention as an active ingredient can be provided.
　本発明が対象とするウイルスとしては、特に制限はなく、ＲＮＡウイルスであっても、ＤＮＡウイルスであってもよいが、上述のとおり、好ましくは、メインプロテアーゼ（３ＣＬプロテアーゼ、ＥＣ３．４．２２．６９によって特定される酵素）を有するウイルスであり、より好ましくは、ＳＡＲＳ－ＣｏＶ－２である。なお、ＳＡＲＳ－ＣｏＶ－２は、非特許文献１に示されているように、既に変異を繰り返している。本発明は、少なくとも最初の遺伝子型のＳＡＲＳ－ＣｏＶ－２を対象とすることが望ましいが、後述の実施例に示すとおり、より多くの変異型も対象にし得る。 Viruses targeted by the present invention are not particularly limited, and may be either RNA viruses or DNA viruses. 69), more preferably SARS-CoV-2. SARS-CoV-2 has already undergone repeated mutations, as shown in Non-Patent Document 1. The present invention is preferably directed to at least the first genotype of SARS-CoV-2, but may also be directed to more variants, as shown in the Examples below.
　また、本発明が対象とする感染症としては特に制限はなく、例えば、ＳＡＲＳ－ＣｏＶ－２感染症（新型コロナウイルス感染症、ＣＯＶＩＤ－１９）が挙げられ、その症状の程度に関わらず、軽度、中程度、重症化したＳＡＲＳ－ＣｏＶ－２感染症の全てが本発明の対象となり得る。 In addition, the infectious disease targeted by the present invention is not particularly limited. , moderate and severe SARS-CoV-2 infections can all be covered by the present invention.
　本発明の化合物は、公知の製剤学的方法により製剤化することができる。例えば、カプセル剤、錠剤、丸剤、液剤、散剤、顆粒剤、細粒剤、フィルムコーティング剤、ペレット剤、トローチ剤、舌下剤、咀嚼剤、バッカル剤、ペースト剤、シロップ剤、懸濁剤、エリキシル剤、乳剤、塗布剤、軟膏剤、硬膏剤、パップ剤、経皮吸収型製剤、ローション剤、吸引剤、エアゾール剤、注射剤、坐剤等として、経口的又は非経口的に使用することができる。 The compounds of the present invention can be formulated by known pharmaceutical methods. For example, capsules, tablets, pills, liquids, powders, granules, fine granules, film coating agents, pellets, lozenges, sublingual agents, chewing agents, buccal agents, pastes, syrups, suspensions, Use orally or parenterally as elixirs, emulsions, ointments, ointments, plasters, poultices, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc. can be done.
　これら製剤化においては、薬理学上許容される担体又は媒体、具体的には、滅菌水や生理食塩水、植物油、溶剤、基剤、乳化剤、懸濁剤、界面活性剤、安定剤、香味剤、芳香剤、賦形剤、ベヒクル、防腐剤、結合剤、希釈剤、等張化剤、無痛化剤、増量剤、崩壊剤、緩衝剤、コーティング剤、滑沢剤、着色剤、甘味剤、粘稠剤、矯味矯臭剤、溶解補助剤、あるいはその他の添加剤等と適宜組み合わせることができる。より具体的には、担体として、乳糖、カオリン、ショ糖、結晶セルロース、コーンスターチ、タルク、寒天、ペクチン、ステアリン酸、ステアリン酸マグネシウム、レシチン、塩化ナトリウム等の固体状担体、グリセリン、落花生油、ポリビニルピロリドン、オリーブ油、エタノール、ベンジルアルコール、プロピレングリコール、水等の液状担体も挙げられる。 In these formulations, pharmacologically acceptable carriers or media, specifically sterile water, physiological saline, vegetable oils, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents , flavoring agents, excipients, vehicles, preservatives, binders, diluents, tonicity agents, soothing agents, bulking agents, disintegrants, buffering agents, coating agents, lubricants, coloring agents, sweetening agents, It can be appropriately combined with a thickening agent, a flavoring agent, a solubilizing agent, or other additives. More specifically, solid carriers such as lactose, kaolin, sucrose, crystalline cellulose, cornstarch, talc, agar, pectin, stearic acid, magnesium stearate, lecithin, sodium chloride, glycerin, peanut oil, polyvinyl Liquid carriers such as pyrrolidone, olive oil, ethanol, benzyl alcohol, propylene glycol, water and the like are also included.
　また、本発明の化合物は、公知の他の抗ウイルス剤と併用してもよい。このような公知の抗ウイルス剤としては、対象疾患がＳＡＲＳ－ＣｏＶ－２感染症である場合には、例えば、ＲＮＡ依存性ＲＮＡポリメラーゼ阻害剤（レムデシビル等）、ＤＮＡ依存性ＲＮＡポリメラーゼ阻害剤（逆転写酵素阻害剤）、ＳＰＩＫＥ機能阻害剤、ＡＣＥ２アンタゴニスト、本発明の化合物とは異なる他のＳＡＲＳ－ＣｏＶ－２プロテアーゼ阻害剤が挙げられる。 In addition, the compound of the present invention may 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 (remdesivir, etc.), DNA-dependent RNA polymerase inhibitors (reverse transcriptase inhibitors), SPIKE function inhibitors, ACE2 antagonists, other SARS-CoV-2 protease inhibitors different from the compounds of the present invention.
　本発明の抗ウイルス剤の好ましい投与形態としては特に制限はなく、経口投与又は非経口投与が挙げられる。また、非経口投与について、より具体的には、静脈内投与、動脈内投与、腹腔内投与、皮下投与、皮内投与、気道内投与、直腸投与、筋肉内投与、輸液による投与が挙げられる。 The preferred dosage form of the antiviral agent of the present invention is not particularly limited, and includes oral administration and parenteral administration. Parenteral administration includes, more specifically, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration, intramuscular administration, and administration by infusion.
　本発明の抗ウイルス剤は、主にヒトを対象として使用することができるが、実験用動物等のヒト以外の動物も対象とすることができる。 The antiviral agent of the present invention can be used mainly for humans, but can also be used for animals other than humans, such as experimental animals.
　本発明の抗ウイルス剤を投与する場合、その投与量は、対象の年齢、体重、症状、健康状態、重篤状態、薬物に対する忍容性、投与形態等に応じて、適宜選択される。１日当たりの本発明の抗ウイルス剤の投与量は、有効成分である化合物の量として、通常０．００００１～１０００ｍｇ／ｋｇ体重、好ましくは０．０００１～１００ｍｇ／ｋｇ体重であり、１回又は複数回に分けて対象に投与される。 When administering the antiviral agent of the present invention, the dosage is appropriately selected according to the subject's age, body weight, symptoms, health condition, serious condition, drug tolerance, dosage form, and the like. The daily dose of the antiviral agent of the present invention is usually 0.00001 to 1000 mg/kg body weight, preferably 0.0001 to 100 mg/kg body weight, in terms of the amount of the compound as an active ingredient. It is administered to the subject in divided doses.
　本発明の抗ウイルス剤の製品又はその説明書は、ウイルス感染症を治療又は予防するために用いられる旨の表示を付したものであり得る。ここで「製品又は説明書に表示を付した」とは、製品の本体、容器、包装等に表示を付したこと、又は製品の情報を開示する説明書、添付文書、宣伝物、その他の印刷物等に表示を付したことを意味する。また、ウイルス感染症を治療するために用いられる旨の表示においては、本発明の化合物を投与することにより、ウイルスのプロテアーゼ活性を抑制し、当該ウイルスの複製を抑制できることを、本発明の抗ウイルス剤の作用機序に関する情報として含むことができる。 The product of the antiviral agent of the present invention or its instructions may be labeled as being used to treat or prevent viral infections. Here, "labeled on the product or instruction manual" means that the label is attached to the body, container, packaging, etc. of the product, or instruction manuals, attached documents, advertising materials, and other printed materials that disclose product information It means that the display is attached to etc. In addition, in the label to the effect that the compound of the present invention is used for treating viral infections, the antiviral protease activity of the present invention can be suppressed by administering the compound of the present invention, thereby suppressing the replication of the virus. It can be included as information on the mechanism of action of the agent.
　このように本発明は、本発明の抗ウイルス剤を対象に投与することによって、感染症を予防又は治療することができる。したがって、本発明は、本発明の化合物を投与することを特徴とする、ウイルス感染症を予防又は治療するための方法をも提供するものである。 Thus, according to the present invention, infectious diseases can be prevented or treated 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, comprising administering a compound of the present invention.
　本発明の化合物を投与する対象としては特に制限はなく、ウイルス感染症の罹患者、当該感染症が発症する前のウイルス保有者、感染する前の者等が挙げられる。例えば、ＳＡＲＳ－ＣｏＶ－２感染症を罹患しているもののみならず、罹患していないもの（未感染）であってもよく、ＳＡＲＳ－ＣｏＶ－２感染症から既に回復しているものであってもよい。より具体的に、本発明の化合物は、ＳＡＲＳ－ＣｏＶ－２感染症の症状（発熱、咳嗽、味覚異常、嗅覚異常等）が認められた時点、ＳＡＲＳ－ＣｏＶ－２感染者との濃厚接触が確認された時点において、投与され得る。 There are no particular restrictions on the subjects to whom the compounds of the present invention are administered, and examples include patients with viral infections, viral carriers before the onset of the infectious disease, and those who have not yet been infected. For example, not only those suffering from SARS-CoV-2 infection, but also those not suffering from it (uninfected), those who have already recovered from SARS-CoV-2 infection may More specifically, when symptoms of SARS-CoV-2 infection (fever, cough, dysgeusia, dysosmia, etc.) are observed, the compound of the present invention has close contact with a SARS-CoV-2 infected person. It can be administered at a confirmed time.
　また、本発明において、「治療」には、感染症からの完全な回復のみならず、その症状を緩和又は改善し、その進行を抑制することが含まれる。「予防」には、感染の抑制若しくは遅延、又は発症の抑制若しくは遅延が含まれる。 In addition, in the present invention, "treatment" includes not only complete recovery from an infectious disease, but also alleviation or improvement of its symptoms and suppression of its progression. "Prevention" includes control or delay of infection or control or delay of onset.
　以下、実施例及び比較例に基づいて本発明をより具体的に説明するが、本発明は以下の実施例に限定されるものではない。本実施例においては、下記表１及び２に示される化合物を合成した。そして、それら化合物を、後述の試験に供して各種活性を評価した。 The present invention will be described in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples. In this example, the compounds shown in Tables 1 and 2 below were synthesized. Then, these compounds were subjected to tests described later to evaluate various activities.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
　なお、表１及び２に示す各官能基及び結合は、以下の一般式に対応する。 The functional groups and bonds shown in Tables 1 and 2 correspond to the following general formulas.
Figure JPOXMLDOC01-appb-C000010
Figure JPOXMLDOC01-appb-C000010
　先ず、これら化合物の合成方法の代表例を以下に示す。なお、空気や湿気に過敏な試薬を使用する反応は、注釈が無い限り市販の溶媒と試薬を用いて窒素あるいはアルゴン雰囲気下、乾燥ガラス器具中で行った。 First, representative examples of methods for synthesizing these compounds are shown below. Reactions using reagents sensitive to air or moisture were carried out in dry glassware under a nitrogen or argon atmosphere using commercially available solvents and reagents unless otherwise noted.
　また、得られた化合物の分析において、薄層クロマトグラフィー（ＴＬＣ）は、メルク６０Ｆ２５４プレコートシリカゲル上で展開し、紫外光による蛍光消光、あるいは、リン酸モリブデン、パラアニスアルデヒド、ニンヒドリンによる染色で可視化した。 In addition, for the analysis of the compounds obtained, thin layer chromatography (TLC) was developed on Merck 60F254 precoated silica gel and visualized by fluorescence quenching with ultraviolet light or staining with molybdenum phosphate, para-anisaldehyde and ninhydrin. .
　フラッシュカラムクロマトグフラフィーは、シリカゲル６０Ｎ（関東化学）、あるいは、ＳＮＡＰ　Ｕｌｔｒａ　Ｓｉｌｉｃａ　Ｃａｒｔｒｉｄｇｅを備えたＩｓｏｌｅｒａ　Ｏｎｅ（バイオタージ）により行った。 Flash column chromatography was performed with silica gel 60N (Kanto Kagaku) or Isolera One (Biotage) equipped with a SNAP Ultra Silica Cartridge.
　１Ｈ　ＮＭＲ（４００　ｏｒ　５００ＭＨｚ）と１３Ｃ　ＮＭＲ（１００　ｏｒ　１２５ＭＨｚ）スペクトルは、Ｂｒｕｋｅｒ　Ａｖａｎｃｅ　II　ｓｐｅｃｔｒｏｍｅｔｅｒあるいはＢｒｕｋｅｒ　ＡＶＡＮＣＥ　５００　ｓｐｅｃｔｒｏｍｅｔｅｒを使用して測定した。ケミカルシフトは、ＣＤＣｌ３（１Ｈ　７．２６ｐｐｍ，１３Ｃ　７７．１６ｐｐｍ）あるいはｄｉｍｅｔｈｙｌ　ｓｕｌｆｏｘｉｄｅ（ＤＭＳＯ）－ｄ６（１Ｈ　２．５０ｐｐｍ，１３Ｃ　３９．５２ｐｐｍ）に対するδ（ｐｐｍ）で検出した。 1 H NMR (400 or 500 MHz) and 13 C NMR (100 or 125 MHz) spectra were measured using a Bruker Avance II spectrometer or a Bruker AVANCE 500 spectrometer. Chemical shifts were detected by δ (ppm) relative to CDCl 3 ( 1 H 7.26 ppm, 13 C 77.16 ppm) or dimethyl sulfoxide (DMSO)-d6 ( 1 H 2.50 ppm, 13 C 39.52 ppm).
　低分解と高分解の質量分析スペクトル（ＬＲＭＳ，ＨＲＭＳ）は、Ｂｒｕｋｅｒ　Ｄａｌｔｏｎｉｃｓ　ｍｉｃｒＯＴＯＦ　ｆｏｃｕｓ（エレクトロスプレーイオン化質量分析）ｓｐｅｃｔｒｏｍｅｔｅｒを用い、その陽イオンあるいは陰イオン検出モードで記録した。 Low-resolution and high-resolution mass spectrometry spectra (LRMS, HRMS) were recorded using a Bruker Daltonics microOTOF focus (electrospray ionization mass spectrometry) spectrometer in its positive or negative ion detection mode.
　逆相高速液体クロマトグラフィー（ＨＰＬＣ）は、Ｃｏｓｍｏｓｉｌ　５Ｃ１８－ＡＲII　分取用カラム（２０×２５０ｍｍ，ナカライテスク）（流速　１０．０ｍＬ／ｍｉｎ）とＣｏｓｍｏｓｉｌ　５Ｃ１８－ＡＲ　II　分析用カラム（４．６ｘ２５０ｍｍ，ナカライテスク）（流速　１．０　ｍＬ／ｍｉｎ）を使用し、流出液を２２０ｎｍの紫外光で検出した。０．１％（ｖ／ｖ）　ＴＦＡ／Ｈ２Ｏ（溶媒Ａ）と０．１％（ｖ／ｖ）　ＴＦＡ／ＭｅＣＮ（溶媒Ｂ）を含む溶媒システムを逆相ＨＰＬＣの溶出に用いた。 Reversed-phase high-performance liquid chromatography (HPLC) was performed using a Cosmosil 5C 18 -AR II preparative column (20 x 250 mm, Nacalai Tesque) (flow rate 10.0 mL/min) and a Cosmosil 5C 18 -AR II analytical column (4.6 x 250 mm). , Nacalai Tesque) (flow rate 1.0 mL/min), and the effluent was detected with ultraviolet light at 220 nm. A solvent system containing 0.1% (v/v) TFA/H 2 O (solvent A) and 0.1% (v/v) TFA/MeCN (solvent B) was used for reverse phase HPLC elution.
　順相ＨＰＬＣは、ＣＨＩＲＡＬＰＡＫ　ＩＣ　分析用カラム（４．６ｘ２５０ｍｍ，ダイセル）とＣＨＩＲＡＬＰＡＫ　ＩＣ　ｓｅｍｉ－分取用カラム（１０ｘ２５０ｍｍ，ダイセル）を使用し、ヘキサンとイソプロパノールで溶出し、流出液を２２０ｎｍの紫外光で検出した。 For normal-phase HPLC, a CHIRALPAK IC analytical column (4.6 x 250 mm, Daicel) and a CHIRALPAK IC semi-preparative column (10 x 250 mm, Daicel) were used, eluted with hexane and isopropanol, and the effluent was subjected to ultraviolet light at 220 nm. Detected.
　（合成例１）　ｔｅｒｔ－Ｂｕｔｙｌ（（Ｓ）－１－（（（Ｓ）－１－（ｂｅｎｚｏ［ｄ］ｔｈｉａｚｏｌ－２－ｙｌ）－１－ｏｘｏ－３－（（Ｓ）－２－ｏｘｏｐｙｒｒｏｌｉｄｉｎ－３－ｙｌ）ｐｒｏｐａｎ－２－ｙｌ）ａｍｉｎｏ）－４－ｍｅｔｈｙｌ－１－ｔｈｉｏｘｏｐｅｎｔａｎ－２－ｙｌ）ｃａｒｂａｍａｔｅ（番号：１４１）の合成
　表１において番号：１４１として示され、下記式で表される化合物（化合物９，ＴＫＢ０１４１）を合成するために、先ず、以下に示す工程にて、ｔｅｒｔ－Ｂｕｔｙｌ（Ｓ）－（１－（（２－ａｍｉｎｏ－５－ｎｉｔｒｏｐｈｅｎｙｌ）ａｍｉｎｏ）－４－ｍｅｔｈｙｌ－１－ｏｘｏｐｅｎｔａｎ－２－ｙｌ）ｃａｒｂａｍａｔｅ（化合物２，ＴＩＳ０９１４）を合成した。 (Synthesis Example 1) tert-Butyl ((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrrolidin- Synthesis of 3-yl)propan-2-yl)amino)-4-methyl-1-thioxopentan-2-yl)carbamate (No.: 141) The compound shown as No.: 141 in Table 1 and represented by the following formula In order to synthesize (compound 9, TKB0141), first, tert-Butyl(S)-(1-((2-amino-5-nitrophenyl)amino)-4-methyl-1- oxopentan-2-yl)carbamate (compound 2, TIS0914) was synthesized.
Figure JPOXMLDOC01-appb-C000011
Figure JPOXMLDOC01-appb-C000011
Figure JPOXMLDOC01-appb-C000012
Figure JPOXMLDOC01-appb-C000012
　－１０℃でアルゴン雰囲気下、化合物１　Ｎ－Ｂｏｃ－Ｌ－ロイシン水和物（４９９ｍｇ，２．００ｍｍｏｌ）の無水テトラヒドロフラン（２０ｍＬ）溶液に、Ｎ－メチルモルホリン（０．４４０ｍＬ，４．００ｍｍｏｌ）とイソブチルクロロフォルメート（０．２８９ｍＬ，２．２０ｍｍｏｌ）を加えた。反応液を２０分撹拌し、同温度で４－ニトロ－１，２－フェニレンジアミン（３３７ｍｇ，２．２０ｍｍｏｌ）を加えた。－１０℃で２時間撹拌し、さらに室温で２２．５時間撹拌した。飽和塩化アンモニウム水を加え、酢酸エチルで抽出し、有機相を無水硫酸マグネシウムで乾燥し、減圧濃縮した。残渣を自動シリカゲルフラッシュカラムクロマトグラフィー（Ｉｓｏｌｅｒａ　Ｏｎｅ）によりクロロホルム－メタノール（１００：０から４７：３）の展開溶媒で精製し、化合物２（６５０ｍｇ，１．７７ｍｍｏｌ，８９％）を黄色の固体として得た。 Under an argon atmosphere at -10°C, N-methylmorpholine (0.440 mL, 4.00 mmol) and Isobutyl chloroformate (0.289 mL, 2.20 mmol) was added. The reaction solution was stirred for 20 minutes, and 4-nitro-1,2-phenylenediamine (337 mg, 2.20 mmol) was added at the same temperature. The mixture was stirred at -10°C for 2 hours and further stirred at room temperature for 22.5 hours. Saturated aqueous ammonium chloride was added, the mixture was extracted with ethyl acetate, and 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) with a developing solvent of chloroform-methanol (100:0 to 47:3) to give compound 2 (650 mg, 1.77 mmol, 89%) as a yellow solid. rice field.
　１Ｈ　ＮＭＲ（５００ＭＨｚ，ＣＤＣｌ３）：　δ　８．１３（ｓ，１Ｈ），８．０１－７．８９（ｍ，２Ｈ），６．７１（ｄ，Ｊ＝８．８Ｈｚ，１Ｈ），５．０４（ｄ，Ｊ＝５．６Ｈｚ，１Ｈ），４．２２－４．１１（ｍ，１Ｈ），３．０５（ｂｒｓ，２Ｈ），１．８７－１．７０（ｍ，２Ｈ），１．６８－１．５５（ｍ，１Ｈ），１．４６（ｓ，９Ｈ），１．０１（ｄ，Ｊ＝６．２Ｈｚ，３Ｈ），１．００（ｄ，Ｊ＝６．１Ｈｚ，３Ｈ）；
　１３Ｃ　ＮＭＲ（１２５ＭＨｚ，ＣＤＣｌ３）：　δ　１７１．８，１５７．０，１４８．０，１３９．０，１２４．３，１２３．５，１２１．５，１１５．２，８１．５，５４．３，４０．２，２８．５（３Ｃ），２５．１，２３．１，２２．３；
　ＨＲＭＳ（ＥＳＩ），ｍ／ｚ　ｃａｌｃｄ　ｆｏｒ　Ｃ１７Ｈ２７Ｎ４Ｏ５　［Ｍ＋Ｈ］＋　３６７．１９７６，ｆｏｕｎｄ　３６７．１９７２。 1 H NMR (500 MHz, CDCl 3 ): δ 8.13 (s, 1H), 8.01-7.89 (m, 2H), 6.71 (d, J=8.8Hz, 1H), 5. 04 (d, J=5.6Hz, 1H), 4.22-4.11 (m, 1H), 3.05 (brs, 2H), 1.87-1.70 (m, 2H), 1. 68-1.55 (m, 1H), 1.46 (s, 9H), 1.01 (d, J = 6.2Hz, 3H), 1.00 (d, J = 6.1Hz, 3H);
13 C NMR (125 MHz, CDCl 3 ): δ 171.8, 157.0, 148.0, 139.0, 124.3, 123.5, 121.5, 115.2, 81.5, 54.3 , 40.2, 28.5(3C), 25.1, 23.1, 22.3;
HRMS (ESI), m/z calcd for C17H27N4O5 [M+H] + 367.1976 , found 367.1972 .
　次に、得られた化合物２から、以下に示す工程にて、ｔｅｒｔ－Ｂｕｔｙｌ（Ｓ）－（１－（（２－ａｍｉｎｏ－５－ｎｉｔｒｏｐｈｅｎｙｌ）ａｍｉｎｏ）－４－ｍｅｔｈｙｌ－１－ｔｈｉｏｘｏｐｅｎｔａｎ－２－ｙｌ）ｃａｒｂａｍａｔｅ（化合物３）を合成した。 Next, from the obtained compound 2, tert-Butyl(S)-(1-((2-amino-5-nitrophenyl)amino)-4-methyl-1-thioxopentan-2- yl) carbamate (compound 3) was synthesized.
Figure JPOXMLDOC01-appb-C000013
Figure JPOXMLDOC01-appb-C000013
　室温でアルゴン雰囲気下、無水炭酸ナトリウム（１４４ｍｇ，１．３６ｍｍｏｌ）／無水テトラヒドロフラン（１４ｍＬ）溶液に五硫化二リン（３０２ｍｇ，１．３６ｍｍｏｌ）を加えた。混合物が淡黄色の透明溶液になるまで１時間撹拌した。その後、反応混合物に化合物２（５００ｍｇ，１．３７ｍｍｏｌ）を加え、２２時間撹拌した。反応終了後、溶媒を減圧留去し、残渣をシリカゲルカラムクロマトグラフィーによりクロロホルムを展開溶媒として精製し、化合物３（４３２ｍｇ，１．１３ｍｍｏｌ，８３％）を黄色の固体として得た。 Diphosphorus pentasulfide (302 mg, 1.36 mmol) was added to a solution of anhydrous sodium carbonate (144 mg, 1.36 mmol)/anhydrous tetrahydrofuran (14 mL) under an argon atmosphere at room temperature. The mixture was stirred for 1 hour until it became a pale yellow clear solution. After that, compound 2 (500 mg, 1.37 mmol) was added to the reaction mixture and stirred for 22 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography using chloroform as a developing solvent to obtain compound 3 (432 mg, 1.13 mmol, 83%) as a yellow solid.
　１Ｈ　ＮＭＲ（５００ＭＨｚ，ＣＤＣｌ３）　δ　９．２５（ｓ，１Ｈ），８．１０（ｄ，Ｊ＝２．６Ｈｚ，１Ｈ），８．０６（ｄｄ，Ｊ＝９．０Ｈｚ　ａｎｄ　２．６Ｈｚ，１Ｈ），６．７４（ｄ，Ｊ＝８．５Ｈｚ，１Ｈ），５．１９（ｄ，Ｊ＝６．４Ｈｚ，１Ｈ），４．５２－４．４３（ｍ，１Ｈ），３．４９（ｂｒｓ，２Ｈ），１．９５－１．８５（ｍ，１Ｈ），１．８５－１．６６（ｍ，２Ｈ），１．４３（ｓ，９Ｈ），１．０３（ｄ，Ｊ＝６．３Ｈｚ，３Ｈ），１．０２（ｄ，Ｊ＝６．３Ｈｚ，３Ｈ）；
　１３Ｃ　ＮＭＲ（１２５ＭＨｚ，ＣＤＣｌ３）　δ　２０７．３，１５７．１，１４８．９，１３８．５，１２５．８，１２５．３，１２２．３，１１５．２，８１．６，６１．１，４３．８，２８．５（３Ｃ），２５．２，２３．１，２２．４；
　ＨＲＭＳ（ＥＳＩ），ｍ／ｚ　ｃａｌｃｄ　ｆｏｒ　Ｃ１７Ｈ２７Ｎ４Ｏ４Ｓ　［Ｍ＋Ｈ］＋　３８３．１７４８，　ｆｏｕｎｄ　３８３．１７４３。 1 H NMR (500 MHz, CDCl 3 ) δ 9.25 (s, 1 H), 8.10 (d, J = 2.6 Hz, 1 H), 8.06 (dd, J = 9.0 Hz and 2.6 Hz, 1H), 6.74 (d, J = 8.5Hz, 1H), 5.19 (d, J = 6.4Hz, 1H), 4.52-4.43 (m, 1H), 3.49 ( brs, 2H), 1.95-1.85 (m, 1H), 1.85-1.66 (m, 2H), 1.43 (s, 9H), 1.03 (d, J=6. 3 Hz, 3 H), 1.02 (d, J = 6.3 Hz, 3 H);
13C NMR (125MHz, CDCl3 ) ? 43.8, 28.5 (3C), 25.2, 23.1, 22.4;
HRMS ( ESI), m/z calcd for C17H27N4O4S [M+H] <+ > 383.1748 , found 383.1743 .
　次に、得られた化合物３から、以下に示す工程にて、ｔｅｒｔ－Ｂｕｔｙｌ（Ｓ）－（４－ｍｅｔｈｙｌ－１－（６－ｎｉｔｒｏ－１Ｈ－ｂｅｎｚｏ［ｄ］［１，２，３］ｔｒｉａｚｏｌ－１－ｙｌ）－１－ｔｈｉｏｘｏｐｅｎｔａｎ－２－ｙｌ）ｃａｒｂａｍａｔｅ（化合物４）を合成した。 Next, from the obtained compound 3, tert-Butyl(S)-(4-methyl-1-(6-nitro-1H-benzo[d][1,2,3]triazol -1-yl)-1-thioxopentan-2-yl)carbamate (compound 4) was synthesized.
Figure JPOXMLDOC01-appb-C000014
Figure JPOXMLDOC01-appb-C000014
　化合物３（１５４ｍｇ，０．４０２ｍｍｏｌ）を４０℃で９５％酢酸水溶液（４．０ｍＬ）に溶かし、０℃に冷やして撹拌しながら亜硝酸ナトリウム（４２．１ｍｇ，０．６１０ｍｍｏｌ）を少しずつ加え、室温で３０分間撹拌した。その後、氷と水（～４０ｍＬ）を加え、析出する沈殿物を濾取し、氷水で洗った。残渣を室温で一晩真空乾燥し、化合物４をオレンジ色の固体として得、さらなる精製をせずに、以下に示す反応に供し、ｔｅｒｔ－Ｂｕｔｙｌ（（Ｓ）－１－（（（Ｓ）－１－（ｂｅｎｚｏ［ｄ］ｔｈｉａｚｏｌ－２－ｙｌ）－１－ｏｘｏ－３－（（Ｓ）－２－ｏｘｏｐｙｒｒｏｌｉｄｉｎ－３－ｙｌ）ｐｒｏｐａｎ－２－ｙｌ）ａｍｉｎｏ）－４－ｍｅｔｈｙｌ－１－ｔｈｉｏｘｏｐｅｎｔａｎ－２－ｙｌ）ｃａｒｂａｍａｔｅ（化合物７）を合成した。 Compound 3 (154 mg, 0.402 mmol) was dissolved in 95% aqueous acetic acid (4.0 mL) at 40° C., cooled to 0° C. and sodium nitrite (42.1 mg, 0.610 mmol) was added portionwise with stirring. Stir at room temperature for 30 minutes. Ice and water (~40 mL) were then added, and the deposited precipitate was collected by filtration and washed with ice water. The residue was dried in vacuo at room temperature overnight to give compound 4 as an orange solid, which was subjected without further purification to the reaction shown below, tert-Butyl ((S)-1-(((S)- 1-(benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-thioxopentan- 2-yl) carbamate (compound 7) was synthesized.
Figure JPOXMLDOC01-appb-C000015
Figure JPOXMLDOC01-appb-C000015
　０℃でアルゴン雰囲気下、化合物５（Ｄｒａｇｏｖｉｃｈ，Ｐ．Ｓ．ｅｔ　ａｌ．ＰＣＴ　Ｉｎｔ．Ａｐｐｌ．，２００１０１０８９４，１５　Ｆｅｂ　２００１　参照）（１９９ｍｇ，０．５１２ｍｍｏｌ）／ジクロロメタン（５．１ｍＬ）溶液にテトラフルオロホウ酸・ジエチルエーテル（０．２４６ｍＬ，１．７９ｍｍｏｌ）を滴加し、室温で３０分間撹拌した。その後、反応液を減圧濃縮し、粗化合物６をさらなる精製をせずに次の反応に用いた。 Compound 5 (see Dragovich, P.S. et al. PCT Int. Appl., 2001010894, 15 Feb 2001) (199 mg, 0.512 mmol)/dichloromethane (5.1 mL) solution was added with tetrafluoro Boric acid/diethyl ether (0.246 mL, 1.79 mmol) was added dropwise and stirred at room temperature for 30 minutes. The reaction was then concentrated under reduced pressure and the crude compound 6 was used in the next reaction without further purification.
　室温でアルゴン雰囲気下、粗化合物６（０．５１２ｍｍｏｌ）／無水テトラヒドロフラン（４．０ｍＬ）溶液にＮ，Ｎ－ジイソプロピルエチルアミン（ＤＩＰＥＡ）（０．０８６４ｍＬ，０．５１２ｍｍｏｌ）を加えた。０℃で化合物４（１５９ｍｇ，０．４０５ｍｍｏｌ）／無水テトラヒドロフラン（６．０ｍＬ）溶液とＤＩＰＥＡ（０．２５９ｍＬ，１．５４ｍｍｏｌ）を溶液に加え、室温で８時間撹拌した。反応終了後、溶媒を減圧留去し、残渣を自動シリカゲルフラッシュカラムクロマトグラフィーによりクロロホルム－メタノール（１００：０から４７：３）の展開溶媒で粗精製し、粗化合物７を得た。 N,N-diisopropylethylamine (DIPEA) (0.0864 mL, 0.512 mmol) was added to a solution of crude compound 6 (0.512 mmol)/anhydrous tetrahydrofuran (4.0 mL) under an argon atmosphere at room temperature. Compound 4 (159 mg, 0.405 mmol)/anhydrous tetrahydrofuran (6.0 mL) solution and DIPEA (0.259 mL, 1.54 mmol) were added to the solution at 0° C. and stirred at room temperature for 8 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was crudely purified by automatic silica gel flash column chromatography using a developing solvent of chloroform-methanol (100:0 to 47:3) to obtain crude Compound 7.
　次に、以下に示すとおり、０℃でアルゴン雰囲気下、化合物７（０．４０５ｍｍｏｌ）／ジクロロメタン（４．１ｍＬ）溶液にテトラフルオロホウ酸・ジエチルエーテル（０．１９５ｍＬ，１．４２ｍｍｏｌ）を滴加し、室温で３０分間撹拌した。反応液を減圧濃縮し、粗化合物を２％（ｖ／ｖ）メタノール含有ジエチルエーテルで洗った。このようにして得られた粗化合物８は、さらなる精製をせずに次の反応に用いた。 Then, tetrafluoroborate diethyl ether (0.195 mL, 1.42 mmol) was added dropwise to a solution of compound 7 (0.405 mmol)/dichloromethane (4.1 mL) under an argon atmosphere at 0° C. as shown below. and 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 compound 8 thus obtained was used in the next reaction without further purification.
Figure JPOXMLDOC01-appb-C000016
Figure JPOXMLDOC01-appb-C000016
　０℃でアルゴン雰囲気下、粗化合物８（０．４０５ｍｍｏｌ）／無水アセトニトリル（４．１ｍＬ）溶液に、４－メトキシ－１Ｈ－インドール－２－カルボン酸（７７．６ｍｇ，０．４０６ｍｍｏｌ）と１－［（１－（シアノ－２－エトキシ－２－オキソエチリデンアミノオキシ）ジメチルアミノモルホリノ）］ウロニウム　ヘキサフルオロホスフェイト（ＣＯＭＵ）（１７４ｍｇ，０．４０６ｍｍｏｌ）とＤＩＰＥＡ（０．２７３ｍＬ，１．６２ｍｍｏｌ）を加えた。室温で６．５時間反応し、飽和塩化アンモニウム水を加え反応を止め、ジクロロメタンで抽出し、有機相を無水硫酸マグネシウムで乾燥し、減圧濃縮した。残渣を自動シリカゲルフラッシュカラムクロマトグラフィーによりクロロホルム－メタノール（１００：０から１９：１）の展開溶媒で精製し、化合物９（ＴＫＢ０１４１）を粗化合物として得た。ヘキサン－イソプロパノール（１：１から１：９／３０分）の溶出溶媒を用いるｓｅｍｉ－分取ＨＰＬＣ（ＣＨＩＲＡＬＰＡＫ　ＩＣ　ｓｅｍｉ－分取用カラム，ダイセル）でのさらなる精製により、化合物９（２．３８ｍｇ，４．０２ｍｉｃｒｏｍｏｌ，１．０％）を黄色固体として得た。 4-Methoxy-1H-indole-2-carboxylic acid (77.6 mg, 0.406 mmol) and 1- [(1-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholino)]uronium hexafluorophosphate (COMU) (174 mg, 0.406 mmol) and DIPEA (0.273 mL, 1.62 mmol) added. The mixture was reacted at room temperature for 6.5 hours, saturated aqueous ammonium chloride was added to stop the reaction, extracted with dichloromethane, and 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 developing solvent of chloroform-methanol (100:0 to 19:1) to obtain compound 9 (TKB0141) as a crude compound. 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 compound 9 (2.38 mg, 4.02 micromol, 1.0%) as a yellow solid.
　分析用の逆相ＨＰＬＣ（直線的グラジェント勾配　４５－７５％（Ｂ／Ａ＋Ｂ）／３０分、溶出時間＝２１．６分）；
　１Ｈ　ＮＭＲ（５００ＭＨｚ，ＤＭＳＯ－ｄ６）　δ　１１．５７（ｄ，Ｊ＝１．８Ｈｚ，１Ｈ），１１．０３（ｄ，Ｊ＝６．０Ｈｚ，１Ｈ），８．４４（ｄ，Ｊ＝８．１Ｈｚ，１Ｈ），８．３０－８．１９（ｍ，２Ｈ），７．８３（ｓ，１Ｈ），７．７０－７．６０（ｍ，２Ｈ），７．３７（ｄ，Ｊ＝１．７Ｈｚ，１Ｈ）７．１０（ｔ，Ｊ＝８．０Ｈｚ，１Ｈ），７．００（ｄ，Ｊ＝８．３Ｈｚ，１Ｈ），６．５０（ｄ，Ｊ＝７．７Ｈｚ，１Ｈ），６．０９－５．９９（ｍ，１Ｈ），５．０５－４．９５（ｍ，１Ｈ），３．８８（ｓ，３Ｈ），３．２６－３．１５（ｍ，２Ｈ），２．７０－２．６０（ｍ，１Ｈ），２．４１－２．３２（ｍ，１Ｈ），２．３２－２．２１（ｍ，１Ｈ），２．０９－１．９７（ｍ，１Ｈ），１．９７－１．８５（ｍ，１Ｈ），１．８１－１．６９（ｍ，２Ｈ），１．６３－１．５２（ｍ，１Ｈ），０．９４（ｄ，Ｊ＝６．３Ｈｚ，３Ｈ），０．９２（ｄ，Ｊ＝６．４Ｈｚ，３Ｈ）；
　１３Ｃ　ＮＭＲ（１２５ＭＨｚ，ＤＭＳＯ－ｄ６）　δ　２０７．１，１９０．９，１７８．１，１６４．４，１６０．８，１５３．６，１５２．８，１３７．８，１３６．３，１３０．０，１２８．２，１２７．５，１２５．３，１２４．５，１２３．２，１１８．１，１０５．４，１０１．３，９９．２，６０．０，５６．９，５５．１，４３．５，３８．４，３１．７，２７．６，２５．５，２４．４，２３．０，２１．７；
　ＨＲＭＳ（ＥＳＩ），ｍ／ｚ　ｃａｌｃｄ　ｆｏｒ　Ｃ３０Ｈ３４Ｎ５Ｏ４Ｓ２　［Ｍ＋Ｈ］＋　５９２．２０４７，　ｆｏｕｎｄ　５９２．２０４４。 Analytical reverse-phase HPLC (linear gradient 45-75% (B/A+B)/30 min, elution time = 21.6 min);
1 H NMR (500 MHz, DMSO-d6) δ 11.57 (d, J=1.8 Hz, 1 H), 11.03 (d, J=6.0 Hz, 1 H), 8.44 (d, J=8 .1Hz, 1H), 8.30-8.19 (m, 2H), 7.83 (s, 1H), 7.70-7.60 (m, 2H), 7.37 (d, J = 1 .7 Hz, 1 H) 7.10 (t, J = 8.0 Hz, 1 H), 7.00 (d, J = 8.3 Hz, 1 H), 6.50 (d, J = 7.7 Hz, 1 H), 6.09-5.99 (m, 1H), 5.05-4.95 (m, 1H), 3.88 (s, 3H), 3.26-3.15 (m, 2H), 2. 70-2.60 (m, 1H), 2.41-2.32 (m, 1H), 2.32-2.21 (m, 1H), 2.09-1.97 (m, 1H), 1.97-1.85 (m, 1H), 1.81-1.69 (m, 2H), 1.63-1.52 (m, 1H), 0.94 (d, J=6.3Hz , 3H), 0.92 (d, J = 6.4 Hz, 3H);
13 C NMR (125 MHz, DMSO-d6) δ 207.1, 190.9, 178.1, 164.4, 160.8, 153.6, 152.8, 137.8, 136.3, 130.0 , 128.2, 127.5, 125.3, 124.5, 123.2, 118.1, 105.4, 101.3, 99.2, 60.0, 56.9, 55.1, 43 .5, 38.4, 31.7, 27.6, 25.5, 24.4, 23.0, 21.7;
HRMS (ESI), m/z calcd for C30H34N5O4S2 [M + H] + 592.2047 , found 592.2044 .
　（合成例２）　７－Ｆｌｕｏｒｏ－４－ｍｅｔｈｏｘｙ－Ｎ－（（Ｓ）－４，４，４－ｔｒｉｆｌｕｏｒｏ－１－（（（Ｓ）－１－（５－ｆｌｕｏｒｏｂｅｎｚｏ［ｄ］ｔｈｉａｚｏｌ－２－ｙｌ）－１－ｏｘｏ－３－（（Ｓ）－２－ｏｘｏｐｙｒｒｏｌｉｄｉｎ－３－ｙｌ）ｐｒｏｐａｎ－２－ｙｌ）ａｍｉｎｏ）－１－ｏｘｏｂｕｔａｎ－２－ｙｌ）－１Ｈ－ｉｎｄｏｌｅ－２－ｃａｒｂｏｘａｍｉｄｅ（番号：１５８）の合成
　表１において番号：１５８として示され、下記式で表される化合物（ＴＫＢ０１５８）を合成するために、先ず、以下に示す工程にて、Ｍｅｔｈｙｌ（Ｓ）－４，４，４－ｔｒｉｆｌｕｏｒｏ－２－（７－ｆｌｕｏｒｏ－４－ｍｅｔｈｏｘｙ－１Ｈ－ｉｎｄｏｌｅ－２－ｃａｒｂｏｘａｍｉｄｏ）ｂｕｔａｎｏａｔｅ（ＣＡＺ１５１４）を合成した。 (Synthesis Example 2) 7-Fluoro-4-methoxy-N-((S)-4,4,4-trifluoro-1-(((S)-1-(5-fluorobenzo[d]thiazol-2-yl )-1-oxo-3-((S)-2-oxopyrrolidin-3-yl) propan-2-yl)amino)-1-oxobutan-2-yl)-1H-indole-2-carboxamide (number: 158 ) In order to synthesize the compound (TKB0158) shown as number: 158 in Table 1 and represented by the following formula, first, Methyl (S) -4,4,4-trifluoro -2-(7-fluoro-4-methoxy-1H-indole-2-carboxamido)butanoate (CAZ1514) was synthesized.
Figure JPOXMLDOC01-appb-C000017
Figure JPOXMLDOC01-appb-C000017
Figure JPOXMLDOC01-appb-C000018
Figure JPOXMLDOC01-appb-C000018
　０℃で（Ｓ）－２－（（ターシャル－ブトキシカルボニル）アミノ）－４，４，４－トリフルオロブタン酸（９００ｍｇ，３．５０ｍｍｏｌ）／メタノール（１４ｍＬ）溶液にチオニルクロリド（０．７６２ｍＬ，１０．５ｍｍｏｌ）を滴加した。室温で１８時間攪拌し、その後減圧濃縮し、メチルエステル体の粗化合物を得た。得られた粗化合物はさらなる精製をせずに次の反応に用いた。 Thionyl chloride (0.762 mL, 10.5 mmol) was added dropwise. The mixture was stirred at room temperature for 18 hours and then concentrated under reduced pressure to obtain a crude methyl ester compound. The crude compound obtained was used for the next reaction without further purification.
　０℃でメチルエステル体のジクロロメタン（１４ｍＬ）溶液に７－フルオロ－４－メトキシ－１Ｈ－インドール－２－カルボン酸（７３２ｍｇ，３．５０ｍｍｏｌ）とＣＯＭＵ（１．６５ｇ，３．８５ｍｍｏｌ）、ＤＩＰＥＡ（１．２８ｍＬ，７．３５ｍｍｏｌ）を加え、室温で３時間撹拌した。反応液に飽和塩化アンモニウム水を加え反応を止め、ジクロロメタンで抽出し、有機相を飽和食塩水で洗った後に無水硫酸マグネシウムで乾燥し、減圧濃縮した。残渣をシリカゲルフラッシュカラムクロマトグラフィーによりクロロホルムを展開溶媒として精製し、ＣＡＺ１５１４（６９２ｍｇ，５５％）をオレンジ色の固体として得た。 7-fluoro-4-methoxy-1H-indole-2-carboxylic acid (732 mg, 3.50 mmol) and COMU (1.65 g, 3.85 mmol), DIPEA ( 1.28 mL, 7.35 mmol) was added and stirred at room temperature for 3 hours. Saturated aqueous ammonium chloride was added to the reaction mixture to stop the reaction, and the mixture was extracted with dichloromethane. 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 chloroform as a developing solvent to obtain CAZ1514 (692 mg, 55%) as an orange solid.
　１Ｈ　ＮＭＲ（５００ＭＨｚ，ＣＤＣｌ３）　δ　１０．９（ｓ，１Ｈ），７．５４（ｄ，Ｊ＝７．９Ｈｚ，１Ｈ），７．２２－７．２１（ｍ，１Ｈ），６．８５（ｄｄ，Ｊ＝１０．７Ｈｚ　ａｎｄ　８．４Ｈｚ，１Ｈ），６．２６（ｄｄ，Ｊ＝８．５Ｈｚ　ａｎｄ　２．８Ｈｚ，１Ｈ），５．２９－５．２５（ｍ，１Ｈ），３．８５（ｓ，３Ｈ），３．８４（ｓ，３Ｈ），３．０８－２．９９（ｍ，１Ｈ），２．９３－２．８２（ｍ，１Ｈ）；
　１３Ｃ　ＮＭＲ（１２５ＭＨｚ，ＣＤＣｌ３）　δ　１７０．８，１６１．３，１５０．２（ｄ，Ｊ＝１．７Ｈｚ），１４４．８（ｄ，Ｊ＝２３８．４Ｈｚ），１２９．４，１２６．７（ｄ，Ｊ＝１５．９Ｈｚ），１２５．８（ｑ，Ｊ＝２７７．６Ｈｚ），１２１．４（ｄ，Ｊ＝４．７Ｈｚ），１０９．０（ｄ，Ｊ＝１８．１Ｈｚ），１０２．２，９８．４（ｄ，Ｊ＝６．１Ｈｚ），５５．４，５３．２，４７．６－４７．５（ｍ），３５．４（ｑ，Ｊ＝２８．４Ｈｚ）；
　ＨＲＭＳ（ＥＳＩ），ｍ／ｚ　ｃａｌｃｄ　ｆｏｒ　Ｃ１５Ｈ１５Ｆ４Ｎ２Ｏ４　［Ｍ＋Ｈ］＋　３６３．０９６２，　ｆｏｕｎｄ　３６３．０９６５。 1 H NMR (500 MHz, CDCl 3 ) δ 10.9 (s, 1H), 7.54 (d, J = 7.9 Hz, 1H), 7.22-7.21 (m, 1H), 6.85 (dd, J=10.7 Hz and 8.4 Hz, 1 H), 6.26 (dd, J=8.5 Hz and 2.8 Hz, 1 H), 5.29-5.25 (m, 1 H), 3. 85 (s, 3H), 3.84 (s, 3H), 3.08-2.99 (m, 1H), 2.93-2.82 (m, 1H);
13 C NMR (125 MHz, CDCl 3 ) δ 170.8, 161.3, 150.2 (d, J=1.7 Hz), 144.8 (d, J=238.4 Hz), 129.4, 126. 7 (d, J = 15.9 Hz), 125.8 (q, J = 277.6 Hz), 121.4 (d, J = 4.7 Hz), 109.0 (d, J = 18.1 Hz), 102.2, 98.4 (d, J=6.1 Hz), 55.4, 53.2, 47.6-47.5 (m), 35.4 (q, J=28.4 Hz);
HRMS ( ESI), m/z calcd for C15H15F4N2O4 [M + H] + 363.0962 , found 363.0965 .
　一方、ｔｅｒｔ－Ｂｕｔｙｌ（（Ｓ）－１－（５－ｆｌｕｏｒｏｂｅｎｚｏ［ｄ］ｔｈｉａｚｏｌ－２－ｙｌ）－１－ｏｘｏ－３－（（Ｓ）－２－ｏｘｏｐｙｒｒｏｌｉｄｉｎ－３－ｙｌ）ｐｒｏｐａｎ－２－ｙｌ）ｃａｒｂａｍａｔｅ（ＣＡＺ１２３５）を以下に示す反応にて合成した。 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 reaction shown below.
Figure JPOXMLDOC01-appb-C000019
Figure JPOXMLDOC01-appb-C000019
　－７８℃で５－フルオロベンゾチアゾール（３２２ｍｇ，２．１０ｍｍｏｌ）／テトラヒドロフラン（３．２ｍＬ）溶液にノルマル－ブチルリチウム（１．６０Ｍヘキサン溶液，１．１８ｍＬ，１．９ｍｍｏｌ）を１５分かけて滴加した。１時間撹拌後、－７８℃でメチルエステル化合物１（１２１ｍｇ，０．４２１ｍｍｏｌ）／テトラヒドロフラン（１．０ｍＬ）溶液を２０分かけて滴加し、－７８℃で３時間撹拌を続けた。反応液に飽和塩化アンモニウム水を加え反応を止め、０℃で２０分間撹拌した。混合液を減圧濃縮し、酢酸エチルで抽出し、有機相を飽和食塩水で洗った後に無水硫酸マグネシウムで乾燥し、減圧濃縮した。残渣をシリカゲルフラッシュカラムクロマトグラフィーによりクロロホルム／メタノール（４０：１）の展開溶媒で精製し、ＣＡＺ１２３５（ＣＡＺ１４１７）（１２９ｍｇ，７５％）を赤茶色の固体として得た。 Normal-butyllithium (1.60 M hexane solution, 1.18 mL, 1.9 mmol) was added dropwise to a solution of 5-fluorobenzothiazole (322 mg, 2.10 mmol)/tetrahydrofuran (3.2 mL) at −78° C. over 15 minutes. added. After stirring for 1 hour, a solution of methyl ester compound 1 (121 mg, 0.421 mmol)/tetrahydrofuran (1.0 mL) was added dropwise at -78°C over 20 minutes, and stirring was continued at -78°C for 3 hours. Saturated aqueous ammonium chloride was added to the reaction mixture to stop the reaction, and the mixture was stirred at 0°C for 20 minutes. The mixture was concentrated under reduced pressure and extracted with ethyl acetate. 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 chloroform/methanol (40:1) as a developing solvent to give CAZ1235 (CAZ1417) (129 mg, 75%) as a reddish brown solid.
　１Ｈ　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。 1 H NMR (500 MHz, CDCl 3 ) δ 7.93 (dd, J=8.9 Hz and 5.0 Hz, 1 H), 7.84 (dd, J=9.0 Hz and 2.4 Hz, 1 H), 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);
13 C NMR (125 MHz, CDCl 3 ) δ 193.2, 179.6, 166.3, 162.3 (d, J = 245.8 Hz), 155.9, 154.4 (d, J = 12.0 Hz ), 133.0, 123.5 (d, J = 9.8 Hz), 117.4 (d, J = 25.4 Hz), 111.3 (d, J = 23.4 Hz), 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 .
　次に、上記にて得られた化合物　ＣＡＺ１５１４及びＣＡＺ１４１７（ＣＡＺ１２３５）から、以下に示す反応にて、ＴＫＢ０１５８を合成した。 Next, TKB0158 was synthesized from the compounds CAZ1514 and CAZ1417 (CAZ1235) obtained above through the reaction shown below.
Figure JPOXMLDOC01-appb-C000020
Figure JPOXMLDOC01-appb-C000020
　０℃でメチルエステル化合物　ＣＡＺ１５１４（７２．５ｍｇ，０．２００ｍｍｏｌ）／テトラヒドロフラン（２．０ｍＬ）溶液に２Ｍ水酸化リチウム水溶液（０．２００ｍＬ，０．４００ｍｍｏｌ）を加えた。室温で３０分撹拌した後に、反応液に１Ｍ塩酸水溶液を加えて酸性にした。混合溶液を減圧濃縮し、カルボン酸体の粗化合物を得た。さらなる精製をせずに次の反応に用いた。 A 2M lithium hydroxide aqueous solution (0.200 mL, 0.400 mmol) was added to a methyl ester compound CAZ1514 (72.5 mg, 0.200 mmol)/tetrahydrofuran (2.0 mL) solution at 0°C. After stirring at room temperature for 30 minutes, the reaction solution was acidified with 1M hydrochloric acid aqueous solution. The mixed solution was concentrated under reduced pressure to obtain a crude carboxylic acid compound. Used for next reaction without further purification.
　０℃で窒素雰囲気下、Ｂｏｃ保護アミン化合物　ＣＡＺ１４１７（８１．５ｍｇ，０．２００ｍｍｏｌ）／ジクロロメタン（２．０ｍＬ）溶液にテトラフルオロホウ酸・ジエチルエーテル（０．０９６１ｍＬ，０．７００ｍｍｏｌ）を滴加し、室温で３０分撹拌した。反応液を減圧濃縮し、残渣を２％メタノール含有ジエチルエーテルで洗った。テトラフルオロホウ酸塩の粗化合物を、さらなる精製をせずに次の反応に用いた。 Tetrafluoroboric acid/diethyl ether (0.0961 mL, 0.700 mmol) was added dropwise to a Boc-protected amine compound CAZ1417 (81.5 mg, 0.200 mmol)/dichloromethane (2.0 mL) solution at 0°C under a nitrogen atmosphere. , and stirred at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure, and the residue was washed with diethyl ether containing 2% methanol. The crude compound of tetrafluoroborate was used for the next reaction without further purification.
　０℃で当該テトラフルオロホウ酸塩の粗化合物（０．２００ｍｍｏｌ）をアセトニトリル（２．０ｍＬ）に溶かし、上記カルボン酸体の粗化合物（０．２００ｍｍｏｌ）とＣＯＭＵ（８５．７ｍｇ，０．２００ｍｍｏｌ）、ＤＩＰＥＡ（０．０６７５ｍＬ，０．４００ｍｍｏｌ）を加え、０℃で２時間撹拌した。反応液に飽和塩化アンモニウム水を加え反応を止め、ジクロロメタンで抽出し、有機相を飽和食塩水で洗った後、無水硫酸マグネシウムで乾燥し、減圧濃縮した。残渣を自動シリカゲルフラッシュカラムクロマトグラフィーによりクロロホルム－メタノール（１００：０から９４：６）の展開溶媒で粗精製し、粗化合物　ＴＫＢ０１５８を得た。分取用の逆相ＨＰＬＣおよびｓｅｍｉ－分取ＨＰＬＣ（ＣＨＩＲＡＬＰＡＫ　ＩＣ　ｓｅｍｉ－分取用カラム）によりさらなる精製を行い、ＴＫＢ０１５８（３．８９ｍｇ，３．１％）を淡黄色の固体として得た。 The crude tetrafluoroborate compound (0.200 mmol) was dissolved in acetonitrile (2.0 mL) at 0° C., and the crude carboxylic acid compound (0.200 mmol) and COMU (85.7 mg, 0.200 mmol) were dissolved. , DIPEA (0.0675 mL, 0.400 mmol) was added and stirred at 0° C. for 2 hours. Saturated aqueous ammonium chloride was added to the reaction mixture to stop the reaction, and the mixture was extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was crudely purified by automated silica gel flash column chromatography with a developing solvent of chloroform-methanol (100:0 to 94:6) to give crude compound TKB0158. Further purification by preparative reverse-phase HPLC and semi-preparative HPLC (CHIRALPAK IC semi-preparative column) gave TKB0158 (3.89 mg, 3.1%) as a pale yellow solid.
　分析用の逆相ＨＰＬＣ（直線的グラジェント勾配　４０‐７０％（Ｂ／Ａ＋Ｂ）／３０分、溶出時間＝１８．３分）；
　１Ｈ　ＮＭＲ（５００ＭＨｚ，ＤＭＳＯ－ｄ６）　δ　１２．１（ｓ，１Ｈ），９．１７（ｄ，Ｊ＝６．９Ｈｚ，１Ｈ），８．８４（ｄ，Ｊ＝８．５Ｈｚ，１Ｈ），８．２４（ｄｄ，Ｊ＝９．１Ｈｚ　ａｎｄ　４．９Ｈｚ，１Ｈ），８．１７（ｄｄ，Ｊ＝８．７Ｈｚ　ａｎｄ　２．６Ｈｚ，１Ｈ），７．６８（ｓ，１Ｈ），７．５１（ｄｄｄ，Ｊ＝９．０Ｈｚ，９．０Ｈｚ，　ａｎｄ　２．７Ｈｚ，１Ｈ），７．２９－７．２９（ｍ，１Ｈ），６．９３（ｄｄ，Ｊ＝１１．０Ｈｚ　ａｎｄ　８．５Ｈｚ，１Ｈ），６．４２（ｄｄ，Ｊ＝８．５Ｈｚ　ａｎｄ　２．８Ｈｚ，１Ｈ），５．４８－５．４４（ｍ，１Ｈ），４．９４－４．９０（ｍ，１Ｈ），３．８７（ｓ，３Ｈ），３．３５－３．２９（ｍ，１Ｈ），３．２０－３．１２（ｍ，２Ｈ），２．８５－２．７２（ｍ，２Ｈ），２．３０－２．２４（ｍ，１Ｈ），２．１７－２．１１（ｍ，１Ｈ），１．９０－１．７８（ｍ，２Ｈ）；
　１３Ｃ　ＮＭＲ｛１Ｈ｝（１２５ＭＨｚ，ＤＭＳＯ－ｄ６）　δ　１９２．３，１７８．０，１６９．９，１６４．５（ｄ，Ｊ＝３．５Ｈｚ），１６１．４（ｄ，Ｊ＝２４７．５Ｈｚ），１６０．３，１４９．８，１４９．７（ｄ，Ｊ＝１．６Ｈｚ），１４４．２（ｄ，Ｊ＝２３７．８Ｈｚ），１３７．９（ｄ，Ｊ＝１２．６Ｈｚ），１３１．１，１２６．９（ｄ，Ｊ＝１０．０Ｈｚ），１２６．３（ｑ，２７７．４Ｈｚ），１２５．７（ｄ，Ｊ＝１５．８Ｈｚ），１２０．８（ｄ，Ｊ＝５．３Ｈｚ），１１６．６（ｄ，Ｊ＝２５．５Ｈｚ），１０９．３（ｄ，Ｊ＝２７．４Ｈｚ），１０８．２（ｄ，Ｊ＝１７．８Ｈｚ），１０２．６，９８．４（ｄ，Ｊ＝５．９Ｈｚ），５５．５，５４．０，４７．２，３７．９，３４．７（ｑ，Ｊ＝２７．７Ｈｚ），３１．９，２８．０，２７．４；
　ＨＲＭＳ（ＥＳＩ），ｍ／ｚ　ｃａｌｃｄ　ｆｏｒ　Ｃ２８Ｈ２５Ｆ５Ｎ５Ｏ５Ｓ　［Ｍ＋Ｈ］＋　６３８．１４９１，　ｆｏｕｎｄ　６３８．１４９３。 Analytical reverse phase HPLC (linear gradient 40-70% (B/A+B)/30 min, elution time = 18.3 min);
1 H NMR (500 MHz, DMSO-d6) δ 12.1 (s, 1 H), 9.17 (d, J = 6.9 Hz, 1 H), 8.84 (d, J = 8.5 Hz, 1 H), 8.24 (dd, J=9.1 Hz and 4.9 Hz, 1 H), 8.17 (dd, J=8.7 Hz and 2.6 Hz, 1 H), 7.68 (s, 1 H), 7.51 (ddd, J = 9.0Hz, 9.0Hz, and 2.7Hz, 1H), 7.29-7.29 (m, 1H), 6.93 (dd, J = 11.0Hz and 8.5Hz, 1H), 6.42 (dd, J=8.5Hz and 2.8Hz, 1H), 5.48-5.44 (m, 1H), 4.94-4.90 (m, 1H), 3. 87 (s, 3H), 3.35-3.29 (m, 1H), 3.20-3.12 (m, 2H), 2.85-2.72 (m, 2H), 2.30- 2.24 (m, 1H), 2.17-2.11 (m, 1H), 1.90-1.78 (m, 2H);
13 C NMR {1H} (125 MHz, DMSO-d6) δ 192.3, 178.0, 169.9, 164.5 (d, J = 3.5 Hz), 161.4 (d, J = 247.5 Hz ), 160.3, 149.8, 149.7 (d, J = 1.6 Hz), 144.2 (d, J = 237.8 Hz), 137.9 (d, J = 12.6 Hz), 131 .1, 126.9 (d, J=10.0 Hz), 126.3 (q, 277.4 Hz), 125.7 (d, J=15.8 Hz), 120.8 (d, J=5. 3 Hz), 116.6 (d, J = 25.5 Hz), 109.3 (d, J = 27.4 Hz), 108.2 (d, J = 17.8 Hz), 102.6, 98.4 ( d, J = 5.9 Hz), 55.5, 54.0, 47.2, 37.9, 34.7 (q, J = 27.7 Hz), 31.9, 28.0, 27.4;
HRMS ( ESI), m/z calcd for C28H25F5N5O5S [M+H] + 638.1491 , found 638.1493 .
　（合成例３）　Ｎ－（（Ｓ）－１－（（（Ｓ）－１－（Ｂｅｎｚｏ［ｄ］ｔｈｉａｚｏｌ－２－ｙｌ）－１－ｏｘｏ－３－（ｐｙｒｉｄｉｎ－４－ｙｌ）ｐｒｏｐａｎ－２－ｙｌ）ａｍｉｎｏ）－４－ｍｅｔｈｙｌ－１－ｏｘｏｐｅｎｔａｎ－２－ｙｌ）－４－ｍｅｔｈｏｘｙ－１Ｈ－ｉｎｄｏｌｅ－２－ｃａｒｂｏｘａｍｉｄｅ（番号：１８３）の合成
　表１において番号：１８３として示され、下記式で表される化合物（ＴＫＢ０１８３）を合成するために、先ず、以下に示す工程にて、ｔｅｒｔ－Ｂｕｔｙｌ（Ｓ）－（１－（ｂｅｎｚｏ［ｄ］ｔｈｉａｚｏｌ－２－ｙｌ）－１－ｏｘｏ－３－（ｐｙｒｉｄｉｎ－４－ｙｌ）ｐｒｏｐａｎ－２－ｙｌ）ｃａｒｂａｍａｔｅ（ＮＷＤ－２－２）を合成した。 (Synthesis Example 3) N-((S)-1-(((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-(pyridin-4-yl)propan-2) Synthesis of -yl)amino)-4-methyl-1-oxopentan-2-yl)-4-methoxy-1H-indole-2-carboxamide (number: 183) In Table 1, number: 183, represented by the following formula To synthesize the represented compound (TKB0183), first, tert-Butyl(S)-(1-(benzo[d]thiazol-2-yl)-1-oxo-3- (pyridin-4-yl)propan-2-yl)carbamate (NWD-2-2) was synthesized.
Figure JPOXMLDOC01-appb-C000021
Figure JPOXMLDOC01-appb-C000021
Figure JPOXMLDOC01-appb-C000022
Figure JPOXMLDOC01-appb-C000022
　－７８℃でベンゾチアゾール（１．０３ｍＬ，９．３９ｍｍｏｌ）／テトラヒドロフラン（１５ｍＬ）溶液にノルマル－ブチルリチウム（１．５８Ｍヘキサン溶液，５．３５ｍＬ，８．４５ｍｍｏｌ）を１５分かけて滴加した。１時間撹拌後、ターシャル－ブチル（Ｓ）－（１－（メトキシ（メチル）アミノ）－１－オキソ－３－（ピリジン－４－イル）プロパン－２－イル）カルバメート（５８１ｍｇ，１．８８ｍｍｏｌ）／テトラヒドロフラ（３．８ｍＬ）溶液を滴加し、さらに３時間攪拌した。反応液に飽和塩化アンモニウム水を加え反応を止め、さらに室温で２０分攪拌した。混合液にジクロロメタンを加えて抽出し、有機相を無水硫酸マグネシウムで乾燥し、減圧濃縮した。残渣をシリカゲルフラッシュカラムクロマトグラフィーによりクロロホルム－メタノール（４４：１）の展開溶媒で精製し、ＮＷＤ－２－２（６８５ｍｇ，９５％）を白色の固体として得た。 To a solution of benzothiazole (1.03 mL, 9.39 mmol)/tetrahydrofuran (15 mL) at -78°C, n-butyllithium (1.58 M hexane solution, 5.35 mL, 8.45 mmol) was added dropwise over 15 minutes. After stirring for 1 hour, tert-butyl (S)-(1-(methoxy(methyl)amino)-1-oxo-3-(pyridin-4-yl)propan-2-yl)carbamate (581mg, 1.88mmol) /tetrahydrofuran (3.8 mL) solution was added dropwise and stirred for an additional 3 hours. Saturated aqueous ammonium chloride was added to the reaction solution to stop the reaction, and the mixture was further stirred at room temperature for 20 minutes. Dichloromethane was added to the mixture for extraction, and the organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography using chloroform-methanol (44:1) as a developing solvent to give NWD-2-2 (685 mg, 95%) as a white solid.
　１Ｈ　ＮＭＲ（５００ＭＨｚ，ＣＤＣｌ３）　δ　８．４９－８．４８（ｍ，２Ｈ），８．２１（ｄ，Ｊ＝７．９Ｈｚ，１Ｈ），８．００－７．９８（ｍ，１Ｈ），７．６２－７．５３（ｍ，２Ｈ），７．１１（ｂｒ，２Ｈ），５．９２－５．９１（ｍ，１Ｈ），５．４９－５．４７（ｍ，１Ｈ），３．５３－３．４３（ｍ，１Ｈ），３．１７－３．１２（ｍ，１Ｈ），１．３９（ｓ，９Ｈ）；
　１３Ｃ　ＮＭＲ（１２５ＭＨｚ，ＣＤＣｌ３）　δ　１９２．４，１６３．５，１５５．１，１５３．６，１４９．７，１４５．７，１３７．４，１２８．４，１２７．４，１２５．９，１２４．９，１２２．５，８０．３，５６．９，３８．４，２８．３（３Ｃ）；
　ＨＲＭＳ（ＥＳＩ），ｍ／ｚ　ｃａｌｃｄ　ｆｏｒ　Ｃ２０Ｈ２２Ｎ３Ｏ３Ｓ　［Ｍ＋Ｈ］＋　３８４．１３７６，　ｆｏｕｎｄ　３８４．１３７８。 1 H NMR (500 MHz, CDCl 3 ) δ 8.49-8.48 (m, 2H), 8.21 (d, J=7.9 Hz, 1H), 8.00-7.98 (m, 1H) , 7.62-7.53 (m, 2H), 7.11 (br, 2H), 5.92-5.91 (m, 1H), 5.49-5.47 (m, 1H), 3 .53-3.43 (m, 1H), 3.17-3.12 (m, 1H), 1.39 (s, 9H);
13C NMR (125MHz, CDCl3 ) ? 124.9, 122.5, 80.3, 56.9, 38.4, 28.3 (3C);
HRMS (ESI) , m/z calcd for C20H22N3O3S [M+H] <+ > 384.1376 , found 384.1378 .
　次に、以下に示すとおり、非特許文献３に記載の方法に従い合成したターシャル－ブチルエステル化合物　ＣＡＺ１２３６（１８０ｍｇ，０．５００ｍｍｏｌ）を４Ｍ塩酸／ジオキサン溶液（５．０ｍＬ）で処理し、室温で１８時間撹拌した。反応液を減圧濃縮し、カルボン酸体の粗化合物　ＮＷＤ－２－４を得、さらなる精製をせずに次の反応に用いた。 Next, as shown below, the tert-butyl ester compound CAZ1236 (180 mg, 0.500 mmol) synthesized according to the method described in Non-Patent Document 3 was treated with a 4 M hydrochloric acid/dioxane solution (5.0 mL), and Stirred for an hour. The reaction solution was concentrated under reduced pressure to obtain a crude carboxylic acid compound NWD-2-4, which was used in the next reaction without further purification.
Figure JPOXMLDOC01-appb-C000023
Figure JPOXMLDOC01-appb-C000023
　一方、０℃で窒素雰囲気下、上記にて得られたＢｏｃ保護アミン化合物　ＮＷＤ－２－２（１９２ｍｇ，０．５００ｍｍｏｌ）のジクロロメタン（５．０ｍＬ）溶液に、テトラフルオロホウ酸・ジエチルエーテル（０．２４０ｍＬ，１．７５ｍｍｏｌ）を滴加し、室温で６時間攪拌した。反応液を減圧濃縮し、残渣を２％メタノール含有ジエチルエーテルで洗い、粗化合物　ＮＷＤ－２－６を得、さらなる精製をせずに次の反応に用いた。 On the other hand, in a nitrogen atmosphere at 0° C., a solution of the Boc-protected amine compound NWD-2-2 (192 mg, 0.500 mmol) obtained above in dichloromethane (5.0 mL) was added with tetrafluoroboric acid/diethyl ether (0 .240 mL, 1.75 mmol) was added dropwise and stirred at room temperature for 6 hours. The reaction mixture was concentrated under reduced pressure, and the residue was washed with 2% methanol in diethyl ether to give crude compound NWD-2-6, which was used in the next reaction without further purification.
　上記カルボン酸体の粗化合物　ＮＷＤ－２－４（０．５００ｍｍｏｌ）をアセトニトリル（５．０ｍＬ）に溶かし、０℃でＣＯＭＵ（２７８ｍｇ，０．６５０ｍｍｏｌ）を加えた。その混合溶液に、上記粗化合物　ＮＷＤ－２－６（０．５００ｍｍｏｌ）とＤＩＰＥＡ（０．１７０ｍＬ，１．００ｍｍｏｌ）を加え、室温で一晩撹拌した。反応液に飽和塩化アンモニウム水を加え反応を止め、ジクロロメタンを加えて抽出し、有機相を無水硫酸マグネシウムで乾燥し、減圧濃縮した。残渣をシリカゲルフラッシュカラムクロマトグラフィーによりクロロホルム－メタノール（１００：０から９４：６）の展開溶媒で粗精製し、ＴＫＢ０１８３を得た。分取用の逆相ＨＰＬＣ及びｓｅｍｉ－分取ＨＰＬＣ（ＣＨＩＲＡＬＰＡＫ　ＩＣ　ｓｅｍｉ－分取用カラム）によりさらなる精製を行い、ＴＫＢ０１８３（４．１８ｍｇ，１．２％）をトリフルオロ酢酸塩の淡黄色粉末として得た。 The crude compound NWD-2-4 (0.500 mmol) of the above carboxylic acid form was dissolved in acetonitrile (5.0 mL), and COMU (278 mg, 0.650 mmol) was added at 0°C. The crude compound NWD-2-6 (0.500 mmol) and DIPEA (0.170 mL, 1.00 mmol) were added to the mixed solution and stirred overnight at room temperature. Saturated aqueous ammonium chloride was added to the reaction mixture to stop the reaction, dichloromethane was added for extraction, and the organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was crudely purified by silica gel flash column chromatography with a developing solvent of chloroform-methanol (100:0 to 94:6) to give TKB0183. Further purification by preparative reverse-phase HPLC and semi-preparative HPLC (CHIRALPAK IC semi-preparative column) yielded TKB0183 (4.18 mg, 1.2%) as a pale yellow powder of the trifluoroacetate salt. Obtained.
　分析用の逆相ＨＰＬＣ（直線的グラジェント勾配　３０‐６０％（Ｂ／Ａ＋Ｂ）／３０分、溶出時間＝１９．５分）；
　１Ｈ　ＮＭＲ（５００ＭＨｚ，ＣＤＣｌ３）　δ　９．６１（ｓ，１Ｈ），８．２７－８．２６（ｍ，２Ｈ），８．１８－８．１５（ｍ，１Ｈ），８．０２－７．９８（ｍ，１Ｈ），７．６７（ｄ，Ｊ＝８．２Ｈｚ，１Ｈ），７．５９－７．５５（ｍ，２Ｈ），７．２１（ｔ，Ｊ＝１６．０Ｈｚ，１Ｈ），７．０９（ｄ，Ｊ＝１．６Ｈｚ，１Ｈ），７．０５（ｄ，Ｊ＝８．３Ｈｚ，１Ｈ），７．０１（ｄ，Ｊ＝５．２Ｈｚ，２Ｈ），６．７６（ｄ，Ｊ＝８．３Ｈｚ，１Ｈ），６．５０（ｄ，Ｊ＝７．８Ｈｚ，１Ｈ），６．２１－６．１７（ｍ，１Ｈ），４．８６－４．８１（ｍ，１Ｈ），３．９３（ｓ，３Ｈ），３．４８（ｄｄ，Ｊ＝１４．０Ｈｚ　ａｎｄ　５．１Ｈｚ，１Ｈ），３．２１（ｄｄ，Ｊ＝３．２３Ｈｚ　ａｎｄ　３．１９Ｈｚ，１Ｈ），１．７８－１．６１（ｍ，３Ｈ），０．９１（ｄ，Ｊ＝６．３Ｈｚ，３Ｈ），０．８９（ｄ，Ｊ＝６．２Ｈｚ，３Ｈ）；
　１３Ｃ　ＮＭＲ（１２５ＭＨｚ，ＣＤＣｌ３）　δ　１９２．０，１７２．０，１６３．４，１６１．８，１５４．４，１５３．６，１４９．５，１４５．４，１３８．１，１３７．４，１２８．６，１２８．５，１２７．５，１２６．０，１２６．０，１２４．９，１２２．６，１１９．０，１０５．３，１０１．１，９９．９，５５．９，５５．４，５１．９，４０．９，３８．０，２５．０，２３．０，２２．３；
　ＨＲＭＳ（ＥＳＩ），ｍ／ｚ　ｃａｌｃｄ　ｆｏｒ　Ｃ３１Ｈ３２Ｎ５Ｏ４Ｓ　［Ｍ＋Ｈ］＋　５７０．２１７０，　ｆｏｕｎｄ　５７０．２１７５。 Analytical reverse phase HPLC (linear gradient 30-60% (B/A+B)/30 min, elution time = 19.5 min);
1 H NMR (500 MHz, CDCl 3 ) δ 9.61 (s, 1H), 8.27-8.26 (m, 2H), 8.18-8.15 (m, 1H), 8.02-7 .98 (m, 1H), 7.67 (d, J=8.2Hz, 1H), 7.59-7.55 (m, 2H), 7.21 (t, J=16.0Hz, 1H) , 7.09 (d, J = 1.6 Hz, 1 H), 7.05 (d, J = 8.3 Hz, 1 H), 7.01 (d, J = 5.2 Hz, 2 H), 6.76 ( d, J = 8.3Hz, 1H), 6.50 (d, J = 7.8Hz, 1H), 6.21-6.17 (m, 1H), 4.86-4.81 (m, 1H) ), 3.93 (s, 3H), 3.48 (dd, J = 14.0 Hz and 5.1 Hz, 1 H), 3.21 (dd, J = 3.23 Hz and 3.19 Hz, 1 H), 1 .78-1.61 (m, 3H), 0.91 (d, J = 6.3Hz, 3H), 0.89 (d, J = 6.2Hz, 3H);
13 C NMR (125 MHz, CDCl 3 ) δ 192.0, 172.0, 163.4, 161.8, 154.4, 153.6, 149.5, 145.4, 138.1, 137.4, 128.6, 128.5, 127.5, 126.0, 126.0, 124.9, 122.6, 119.0, 105.3, 101.1, 99.9, 55.9, 55. 4, 51.9, 40.9, 38.0, 25.0, 23.0, 22.3;
HRMS (ESI), m/z calcd for C31H32N5O4S [M+H] + 570.2170 , found 570.2175 .
　（合成例４）　４，７－Ｄｉｆｌｕｏｒｏ－Ｎ－（（Ｓ）－１－（（（Ｓ）－１－（５－ｆｌｕｏｒｏｂｅｎｚｏ［ｄ］ｔｈｉａｚｏｌ－２－ｙｌ）－１－ｏｘｏ－３－（（Ｓ）－２－ｏｘｏｐｙｒｒｏｌｉｄｉｎ－３－ｙｌ）ｐｒｏｐａｎ－２－ｙｌ）ａｍｉｎｏ）－４－ｍｅｔｈｙｌ－１－ｏｘｏｐｅｎｔａｎ－２－ｙｌ）－Ｎ－ｍｅｔｈｙｌ－１Ｈ－ｉｎｄｏｌｅ－２－ｃａｒｂｏｘａｍｉｄｅ（番号：１８７）の合成
　表１において番号：１８７として示され、下記式で表される化合物（ＴＫＢ－０１８７）を合成するために、先ず、以下に示す工程にて、Ｍｅｔｈｙｌ　Ｎ－（４，７－ｄｉｆｌｕｏｒｏ－１Ｈ－ｉｎｄｏｌｅ－２－ｃａｒｂｏｎｙｌ）－Ｎ－ｍｅｔｈｙｌ－Ｌ－ｌｅｕｃｉｎａｔｅ（ＴＯＮ－０４０４）を合成した。 (Synthesis Example 4) 4,7-Difluoro-N-((S)-1-(((S)-1-(5-fluorobenzo[d]thiazol-2-yl)-1-oxo-3-(( S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)-N-methyl-1H-indole-2-carboxymide (number: 187) Synthesis In order to synthesize the compound (TKB-0187) shown as No.: 187 in Table 1 and represented by the following formula, first, Methyl N-(4,7-difluoro-1H- indole-2-carbonyl)-N-methyl-L-leucinate (TON-0404) was synthesized.
Figure JPOXMLDOC01-appb-C000024
Figure JPOXMLDOC01-appb-C000024
Figure JPOXMLDOC01-appb-C000025
Figure JPOXMLDOC01-appb-C000025
　０℃でＮ－（ターシャル－ブトキシカルボニル）－Ｎ－メチル－Ｌ－ロイシン（４９１ｍｇ，２．００ｍｍｏｌ）／メタノール（８．０ｍＬ）溶液にチオニルクロリド（０．４３５ｍＬ，６．００ｍｍｏｌ）を滴加し、室温で１８時間撹拌した。反応溶液を減圧濃縮後、残渣のメチルエステルをさらなる精製せずに次の反応に用いた。 Thionyl chloride (0.435 mL, 6.00 mmol) was added dropwise to a solution of N-(tert-butoxycarbonyl)-N-methyl-L-leucine (491 mg, 2.00 mmol) in methanol (8.0 mL) at 0°C. , and stirred at room temperature for 18 hours. After concentrating the reaction solution under reduced pressure, the residual methyl ester was used for the next reaction without further purification.
　０℃で上記メチルエステル／ジクロロメタン（８．０ｍＬ）溶液に４，７－ジフルオロ－１Ｈ－インドール－２－カルボン酸（３９４ｍｇ，２．００ｍｍｏｌ）とＣＯＭＵ（９４２ｍｇ，２．２０ｍｍｏｌ）、ＤＩＰＥＡ（０．７３２ｍＬ，４．２０ｍｍｏｌ）を加え、室温で１８時間撹拌した。反応液に飽和塩化アンモニウム水を加え反応を止め、ジクロロメタンを加えて抽出し、有機相を無水硫酸マグネシウムで乾燥し、減圧濃縮した。残渣をシリカゲルフラッシュカラムクロマトグラフィーによりクロロホルムを展開溶媒として精製し、ＴＯＮ－０４０４（３９９ｍｇ，５９％）をオレンジ色の固体として得た。 4,7-Difluoro-1H-indole-2-carboxylic acid (394 mg, 2.00 mmol), COMU (942 mg, 2.20 mmol) and DIPEA (0.00 mmol) were added to the above methyl ester/dichloromethane (8.0 mL) solution at 0°C. 732 mL, 4.20 mmol) was added and stirred at room temperature for 18 hours. Saturated aqueous ammonium chloride was added to the reaction mixture to stop the reaction, dichloromethane was added for extraction, and the organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography using chloroform as a developing solvent to obtain TON-0404 (399 mg, 59%) as an orange solid.
　１Ｈ　ＮＭＲ（５００ＭＨｚ，ＣＤＣｌ３）　δ　１０．８（ｓ，１Ｈ），６．９８（ｓ，１Ｈ），６．８４－６．７９（ｍ，１Ｈ），６．６５－６．５９（ｍ，１Ｈ），５．６３－５．６０（ｍ，１Ｈ），３．６５（ｓ，３Ｈ），３．３６（ｓ，３Ｈ），１．８８－１．８５（ｍ，２Ｈ），１．６６－１．５６（ｍ，１Ｈ），０．９９－０．９５（ｍ，６Ｈ）；
　１３Ｃ　ＮＭＲ（１２５ＭＨｚ，ＣＤＣｌ３）δ１７２．２，１６３．３，１５２．７（ｄｄ，Ｊ＝２４４．５Ｈｚ　ａｎｄ　１．８Ｈｚ），１４６．０（ｄｄ，Ｊ＝２４２．２Ｈｚ　ａｎｄ　３．１Ｈｚ），１３０．６，１２６．３（ｄｄ，Ｊ＝１６．２Ｈｚ　ａｎｄ　１０．６Ｈｚ），１１９．８（ｄｄ，Ｊ＝２５．０Ｈｚ　ａｎｄ　４．４Ｈｚ），１０８．５（ｄｄ，Ｊ＝１９．２Ｈｚ　ａｎｄ　８．２Ｈｚ），１０３．９（ｄｄ，Ｊ＝２１．７Ｈｚ　ａｎｄ　６．２Ｈｚ），１０２．９，５５．８，５２．３，３７．６，３３．９，２５．３，２３．３，２１．５；
　ＨＲＭＳ（ＥＳＩ），ｍ／ｚ　ｃａｌｃｄ　ｆｏｒ　Ｃ１７Ｈ２１Ｆ２Ｎ２Ｏ３　［Ｍ＋Ｈ］＋　３３９．１５１５，　ｆｏｕｎｄ　３３９．１５２０。 1 H NMR (500 MHz, CDCl 3 ) δ 10.8 (s, 1H), 6.98 (s, 1H), 6.84-6.79 (m, 1H), 6.65-6.59 (m , 1H), 5.63-5.60 (m, 1H), 3.65 (s, 3H), 3.36 (s, 3H), 1.88-1.85 (m, 2H), 1. 66-1.56 (m, 1H), 0.99-0.95 (m, 6H);
13 C NMR (125 MHz, CDCl 3 ) δ 172.2, 163.3, 152.7 (dd, J=244.5 Hz and 1.8 Hz), 146.0 (dd, J=242.2 Hz and 3.1 Hz) , 130.6, 126.3 (dd, J=16.2 Hz and 10.6 Hz), 119.8 (dd, J=25.0 Hz and 4.4 Hz), 108.5 (dd, J=19.2 Hz and 8.2 Hz), 103.9 (dd, J = 21.7 Hz and 6.2 Hz), 102.9, 55.8, 52.3, 37.6, 33.9, 25.3, 23.3 , 21.5;
HRMS (ESI) , m /z calcd for C17H21F2N2O3 [M + H] + 339.1515 , found 339.1520.
　次に、上記にて合成したＴＯＮ－０４０４と、合成例２で合成したＣＡＺ１２３５とを用いて、以下に示す工程にて、ＴＫＢ０１８７を合成した。 Next, using TON-0404 synthesized above and CAZ1235 synthesized in Synthesis Example 2, TKB0187 was synthesized in the steps shown below.
Figure JPOXMLDOC01-appb-C000026
Figure JPOXMLDOC01-appb-C000026
　メチルエステル化合物　ＴＯＮ－０４０４（６７．７ｍｇ，０．２００ｍｍｏｌ）／テトラヒドロフラン（２．０ｍＬ）溶液に２Ｍ水酸化リチウム水溶液（０．２００ｍＬ，０．４００ｍｍｏｌ）を加え、室温で４０分攪拌した。反応液に１Ｍ塩酸水溶液を加えて酸性にした。混合溶液を減圧濃縮し、カルボン酸体の粗化合物を得た。さらなる精製をせずに次の反応に用いた。 A 2M lithium hydroxide aqueous solution (0.200 mL, 0.400 mmol) was added to a methyl ester compound TON-0404 (67.7 mg, 0.200 mmol)/tetrahydrofuran (2.0 mL) solution, and the mixture was stirred at room temperature for 40 minutes. A 1 M hydrochloric acid aqueous solution was added to the reaction solution to make it acidic. The mixed solution was concentrated under reduced pressure to obtain a crude carboxylic acid compound. Used for next reaction without further purification.
　一方、０℃で窒素雰囲気下、Ｂｏｃ保護アミン化合物　ＣＡＺ１２３５（８１．５ｍｇ，０．２００ｍｍｏｌ）／ジクロロメタン（２．０ｍＬ）溶液に、テトラフルオロホウ酸・ジエチルエーテル（０．０９６１ｍＬ，０．７００ｍｍｏｌ）を滴加し、室温で１時間攪拌した。反応液を減圧濃縮し、残渣を２％メタノール含有ジエチルエーテルで洗い、テトラフルオロホウ酸塩の粗化合物を、さらなる精製をせずに次の反応に用いた。 Meanwhile, tetrafluoroboric acid/diethyl ether (0.0961 mL, 0.700 mmol) was added to a Boc-protected amine compound CAZ1235 (81.5 mg, 0.200 mmol)/dichloromethane (2.0 mL) solution under a nitrogen atmosphere at 0°C. It was added dropwise and stirred at room temperature for 1 hour. The reaction was concentrated under reduced pressure, the residue was washed with 2% methanol in diethyl ether, and the crude tetrafluoroborate salt was used in the next reaction without further purification.
　当該テトラフルオロホウ酸塩の粗化合物（０．２００ｍｍｏｌ）をアセトニトリル（２．０ｍＬ）に溶かし、０℃で上記カルボン酸体の粗化合物（０．２００ｍｍｏｌ）とＣＯＭＵ（８５．７ｍｇ，０．２００ｍｍｏｌ）、ＤＩＰＥＡ（０．０６７５ｍＬ，０．４００ｍｍｏｌ）を加え、室温で２時間撹拌した。反応液に飽和塩化アンモニウム水を加え反応を止め、ジクロロメタンで抽出し、有機相を無水硫酸マグネシウムで乾燥し、減圧濃縮した。残渣をシリカゲルフラッシュカラムクロマトグラフィーによりクロロホルム－メタノール（９５：５）の展開溶媒で粗精製した。分取用の逆相ＨＰＬＣによりさらなる精製を行い、ＴＫＢ０１８７（４．０６ｍｇ，３．３％）を淡黄色の粉末として得た。 The crude tetrafluoroborate compound (0.200 mmol) was dissolved in acetonitrile (2.0 mL), and the crude carboxylic acid compound (0.200 mmol) and COMU (85.7 mg, 0.200 mmol) were dissolved at 0°C. , DIPEA (0.0675 mL, 0.400 mmol) was added and stirred at room temperature for 2 hours. Saturated aqueous ammonium chloride was added to the reaction solution to stop the reaction, the mixture was extracted with dichloromethane, and the organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was crudely purified by silica gel flash column chromatography using a developing solvent of chloroform-methanol (95:5). Further purification by preparative reverse-phase HPLC gave TKB0187 (4.06 mg, 3.3%) as a pale yellow powder.
　分析用の逆相ＨＰＬＣ（直線的グラジェント勾配　４０‐７０％（Ｂ／Ａ＋Ｂ）／３０分、溶出時間＝２５．８分）；
　１Ｈ　ＮＭＲ（５００ＭＨｚ，ＣＤＣｌ３）　δ　１０．３７（ｂｒｓ，１Ｈ），８．５９（ｂｒｓ，１Ｈ），７．９２（ｄｄ，Ｊ＝８．８Ｈｚ　ａｎｄ　４．９Ｈｚ，１Ｈ），７．８０（ｄ，Ｊ＝８．４Ｈｚ，１Ｈ），７．３４－７．３０（ｍ，１Ｈ），６．９５（ｂｒｓ，１Ｈ），６．８８－６．８４（ｍ，１Ｈ），６．６７－６．６４（ｍ，１Ｈ），６．２１（ｂｒｓ，１Ｈ），５．６９－５．６８（ｍ，１Ｈ），５．４２（ｂｒｓ，１Ｈ），３．３９（ｂｒ，２Ｈ），３．２５（ｂｒ，２Ｈ），２．６７（ｂｒ，１Ｈ），２．５２（ｂｒ，１Ｈ），２．１４（ｂｒ，２Ｈ），２．０１－１．９７（ｍ，１Ｈ），１．９３－１．８９（ｍ，１Ｈ），１．８０（ｂｒ，１Ｈ），１．５７（ｂｒ，１Ｈ），０．９７（ｂｒ，６Ｈ）；
　１３Ｃ　ＮＭＲ（１２５ＭＨｚ，ＣＤＣｌ３）　δ　１９１．９，１８０．４，１７１．４，１６６．６，１６４．０，１６２．２（ｄ，Ｊ＝２４５．６Ｈｚ），１５４．４（ｄ，Ｊ＝１２．０Ｈｚ），１５３．９－１５１．９（ｍ），１４７．０－１４５．０（ｍ），１３３．０，１３０．９，１２６．２，１２３．５（ｄ，Ｊ＝９．８Ｈｚ），１２０．０－１１９．８（ｍ），１１７．３（ｄ，Ｊ＝２５．５Ｈｚ），１１１．２（２３．２Ｈｚ），１０８．６（ｄｄ，Ｊ＝１９．１Ｈｚ　ａｎｄ　８．３Ｈｚ），１０４．３（ｄｄ，２１．９Ｈｚ　ａｎｄ　６．３Ｈｚ），１０２．６，５６．２，５５．５，４１．１，３９．８，３６．９，３３．５，３２．６，２９．０，２５．１，２３．３，２２．１；
　ＨＲＭＳ（ＥＳＩ），ｍ／ｚ　ｃａｌｃｄ　ｆｏｒ　Ｃ３０Ｈ３０Ｆ３Ｎ５Ｏ４Ｓ　［Ｍ＋Ｈ］＋　６１４．２０４３，　ｆｏｕｎｄ　６１４．２０３８。 Analytical reverse phase HPLC (linear gradient 40-70% (B/A+B)/30 min, elution time = 25.8 min);
1 H NMR (500 MHz, CDCl 3 ) δ 10.37 (brs, 1H), 8.59 (brs, 1H), 7.92 (dd, J = 8.8Hz and 4.9Hz, 1H), 7.80 (d, J = 8.4Hz, 1H), 7.34-7.30 (m, 1H), 6.95 (brs, 1H), 6.88-6.84 (m, 1H), 6.67 -6.64 (m, 1H), 6.21 (brs, 1H), 5.69-5.68 (m, 1H), 5.42 (brs, 1H), 3.39 (br, 2H), 3.25 (br, 2H), 2.67 (br, 1H), 2.52 (br, 1H), 2.14 (br, 2H), 2.01-1.97 (m, 1H), 1 .93-1.89 (m, 1H), 1.80 (br, 1H), 1.57 (br, 1H), 0.97 (br, 6H);
13 C NMR (125 MHz, CDCl 3 ) δ 191.9, 180.4, 171.4, 166.6, 164.0, 162.2 (d, J = 245.6 Hz), 154.4 (d, J = 12.0 Hz), 153.9-151.9 (m), 147.0-145.0 (m), 133.0, 130.9, 126.2, 123.5 (d, J=9. 8 Hz), 120.0-119.8 (m), 117.3 (d, J = 25.5 Hz), 111.2 (23.2 Hz), 108.6 (dd, J = 19.1 Hz and 8. 3 Hz), 104.3 (dd, 21.9 Hz and 6.3 Hz), 102.6, 56.2, 55.5, 41.1, 39.8, 36.9, 33.5, 32.6, 29.0, 25.1, 23.3, 22.1;
HRMS (ESI), m/z calcd for C30H30F3N5O4S [M+H] + 614.2043 , found 614.2038 .
　（合成例５）　７－Ｆｌｕｏｒｏ－Ｎ－（（Ｓ）－１－（（（Ｓ）－１－（４－ｆｌｕｏｒｏｂｅｎｚｏ［ｄ］ｔｈｉａｚｏｌ－２－ｙｌ）－１－ｏｘｏ－３－（（Ｓ）－２－ｏｘｏｐｙｒｒｏｌｉｄｉｎ－３－ｙｌ）ｐｒｏｐａｎ－２－ｙｌ）ａｍｉｎｏ）－４－ｍｅｔｈｙｌ－１－ｏｘｏｐｅｎｔａｎ－２－ｙｌ）－４－ｍｅｔｈｏｘｙ－１Ｈ－ｉｎｄｏｌｅ－２－ｃａｒｂｏｘａｍｉｄｅ（番号：１９８）の合成
　表１において番号：１９８として示され、下記式で表される化合物（化合物ＴＫＢ０１９８）を合成するために、先ず、以下に示す工程にて、ｔｅｒｔ－Ｂｕｔｙｌ（７－ｆｌｕｏｒｏ－４－ｍｅｔｈｏｘｙ－１Ｈ－ｉｎｄｏｌｅ－２－ｃａｒｂｏｎｙｌ）－Ｌ－ｌｅｕｃｉｎａｔｅ（ＣＡＺ１３１０）を合成した。 (Synthesis Example 5) 7-Fluoro-N-((S)-1-(((S)-1-(4-fluorobenzo[d]thiazol-2-yl)-1-oxo-3-((S) -2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)-4-methoxy-1H-indole-2-carboxamide (No.: 198) Synthesis Table 1 as number: 198 and represented by the following formula (compound TKB0198), first, tert-Butyl (7-fluoro-4-methoxy-1H-indole -2-carbonyl)-L-leucinate (CAZ1310) was synthesized.
Figure JPOXMLDOC01-appb-C000027
Figure JPOXMLDOC01-appb-C000027
Figure JPOXMLDOC01-appb-C000028
Figure JPOXMLDOC01-appb-C000028
　０℃で７－フルオロ－４－メトキシ－１Ｈ－インドール－２－カルボン酸（２．０９ｇ，１０．０ｍｍｏｌ）／ジクロロメタン（１００ｍＬ）溶液にＬ－ロイシンターシャル－ブチルエステル塩酸塩（２．４６ｇ，１１．０ｍｍｏｌ）と１－ヒドキシベンゾトリアゾール水和物（ＨＯＢｔ・Ｈ２Ｏ）（１．６２ｇ，１２．０ｍｍｏｌ）、１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド塩酸塩（ＥＤＣＩ・ＨＣｌ）（２．３０ｇ，１２．０ｍｍｏｌ）、ＤＩＰＥＡ（５．９０ｍＬ，３５．０ｍｍｏｌ）を加え、室温で１８時間攪拌した。反応液に飽和塩化アンモニウム水を加え反応を止め、ジクロロメタンを加えて抽出し、有機相を飽和食塩水で洗った後に無水硫酸マグネシウムで乾燥し、減圧濃縮した。残渣をシリカゲルフラッシュカラムクロマトグラフィーによりヘキサン－酢酸エチル（３：１）の展開溶媒で精製し、ＣＡＺ１３１０（２．７３ｇ，７２％）を黄色の固体として得た。 L-leucine tert-butyl ester hydrochloride (2.46 g, 11.0 mmol), 1-hydroxybenzotriazole hydrate (HOBt.H 2 O) (1.62 g, 12.0 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI. HCl) (2.30 g, 12.0 mmol) and DIPEA (5.90 mL, 35.0 mmol) were added and stirred at room temperature for 18 hours. Saturated aqueous ammonium chloride was added to the reaction solution to stop the reaction, dichloromethane was added for extraction, 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 obtain CAZ1310 (2.73 g, 72%) as a yellow solid.
　１Ｈ　ＮＭＲ（５００ＭＨｚ，ＣＤＣｌ３）　δ　９．３３（ｓ，１Ｈ），７．０４－７．０３（ｍ，１Ｈ），６．８７（ｄｄ，Ｊ＝１０．５Ｈｚ　ａｎｄ　８．５Ｈｚ，１Ｈ），６．６４（ｄ，Ｊ＝８．３Ｈｚ，１Ｈ），６．３３（ｄｄ，Ｊ＝８．５Ｈｚ　ａｎｄ　２．９Ｈｚ，１Ｈ），４．７３（ｄｔ，Ｊ＝８．５Ｈｚ　ａｎｄ　５．１Ｈｚ，１Ｈ），３．９２（ｓ，３Ｈ），１．７９－１．７０（ｍ，２Ｈ），１．４９（ｓ，９Ｈ），０．９９（ｄ，Ｊ＝６．４Ｈｚ，３Ｈ），０．９８（ｄ，Ｊ＝６．６Ｈｚ，３Ｈ）；
　１３Ｃ　ＮＭＲ（１２５ＭＨｚ，ＣＤＣｌ３）　δ　１７２．３，１６０．７，１５０．３（ｄ，Ｊ＝１．９Ｈｚ），１４４．７（ｄ，Ｊ＝２３８．３Ｈｚ），１３０．１，１２６．１（ｄ，Ｊ＝１６．３Ｈｚ），１２１．６（ｄ，Ｊ＝４．６Ｈｚ），１０９．０（ｄ，Ｊ＝１７．７Ｈｚ），１０１．０，９８．９（ｄ，Ｊ＝６．２Ｈｚ），８２．５，５５．８，５１．６，４２．３，２８．２（３Ｃ），２５．２，２３．０，２２．３；
　ＨＲＭＳ（ＥＳＩ），ｍ／ｚ　ｃａｌｃｄ　ｆｏｒ　Ｃ２０Ｈ２８ＦＮ２Ｏ４　［Ｍ＋Ｈ］＋　３７９．２０２８，　ｆｏｕｎｄ　３７９．２０３１。 1 H NMR (500 MHz, CDCl 3 ) δ 9.33 (s, 1H), 7.04-7.03 (m, 1H), 6.87 (dd, J=10.5Hz and 8.5Hz, 1H) , 6.64 (d, J=8.3 Hz, 1 H), 6.33 (dd, J=8.5 Hz and 2.9 Hz, 1 H), 4.73 (dt, J=8.5 Hz and 5.1 Hz , 1H), 3.92 (s, 3H), 1.79-1.70 (m, 2H), 1.49 (s, 9H), 0.99 (d, J = 6.4Hz, 3H), 0.98 (d, J = 6.6 Hz, 3H);
13 C NMR (125 MHz, CDCl 3 ) δ 172.3, 160.7, 150.3 (d, J = 1.9 Hz), 144.7 (d, J = 238.3 Hz), 130.1, 126. 1 (d, J = 16.3 Hz), 121.6 (d, J = 4.6 Hz), 109.0 (d, J = 17.7 Hz), 101.0, 98.9 (d, J = 6 .2 Hz), 82.5, 55.8, 51.6, 42.3, 28.2 (3C), 25.2, 23.0, 22.3;
HRMS (ESI), m/z calcd for C20H28FN2O4 [M + H] <+ > 379.2028 , found 379.2031 .
　一方、下記に示すとおり、ｔｅｒｔ－Ｂｕｔｙｌ（（Ｓ）－１－（４－ｆｌｕｏｒｏｂｅｎｚｏ［ｄ］ｔｈｉａｚｏｌ－２－ｙｌ）－１－ｏｘｏ－３－（（Ｓ）－２－ｏｘｏｐｙｒｒｏｌｉｄｉｎ－３－ｙｌ）ｐｒｏｐａｎ－２－ｙｌ）ｃａｒｂａｍａｔｅ（ＣＡＺ１３３３）を合成した。 On the other hand, as shown below, 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.
Figure JPOXMLDOC01-appb-C000029
Figure JPOXMLDOC01-appb-C000029
　－７８℃で４－フルオロベンゾチアゾール（７６６ｍｇ，５．００ｍｍｏｌ）／テトラヒドロフラン（９．０ｍＬ）溶液にノルマル－ブチルリチウム（１．６０Ｍヘキサン溶液，２．８１ｍＬ，４．５０ｍｍｏｌ）を１５分かけて滴加した。１時間撹拌後、メチルエステル化合物１（２８６ｍｇ，１．００ｍｍｏｌ）／テトラヒドロフラン（１．０ｍＬ）溶液を２０分かけて滴加し、さらに３時間攪拌した。反応液に飽和塩化アンモニウム水を加え反応を止め、さらに０℃で２０分攪拌した。混合液を減圧濃縮後、酢酸エチルを加えて抽出し、有機相を飽和食塩水で洗い、無水硫酸マグネシウムで乾燥し、減圧濃縮した。残渣をシリカゲルフラッシュカラムクロマトグラフィーによりクロロホルム－メタノール（４０：１）の展開溶媒で精製し、ＣＡＺ１３３３（３３８ｍｇ，８３％）を茶褐色の固体として得た。 Normal-butyllithium (1.60 M hexane solution, 2.81 mL, 4.50 mmol) was added dropwise to a solution of 4-fluorobenzothiazole (766 mg, 5.00 mmol)/tetrahydrofuran (9.0 mL) at −78° C. over 15 minutes. added. 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 further stirred for 3 hours. Saturated aqueous ammonium chloride was added to the reaction mixture to stop the reaction, and the mixture was further stirred at 0°C for 20 minutes. After the mixture was concentrated under reduced pressure, ethyl acetate was added for extraction, 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 chloroform-methanol (40:1) as a developing solvent to obtain CAZ1333 (338 mg, 83%) as a brown solid.
　１Ｈ　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，１Ｈ），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）；
　１３Ｃ　ＮＭＲ（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　ｃａｌｃｄ　ｆｏｒ　Ｃ１９Ｈ２３ＦＮ３Ｏ４Ｓ　［Ｍ＋Ｈ］＋　４０８．１３８８，　ｆｏｕｎｄ　４０８．１３８４。 1 H NMR (500 MHz, CDCl 3 ) 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, 1H), 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 (125 MHz, CDCl 3 ) δ 193.1, 179.8, 164.3, 157.4 (d, J=261.1 Hz), 142.9 (d, J=14.0 Hz), 139. 8 (d, J = 2.4 Hz), 129.1 (d, J = 7.1 Hz), 118.3 (d, J = 4.5 Hz), 112.6 (d, J = 17.5 Hz), 80.2, 55.6, 40.6, 38.7, 34.7, 28.4 (3C), 28.0;
HRMS (ESI), m/z calcd for C19H23FN3O4S [M+H] + 408.1388 , found 408.1384 .
　次に、上記にて合成したＣＡＺ１３１０及びＣＡＺ１３３３を用いて、以下に示す工程にて、ＴＫＢ０１９８を合成した。 Next, using CAZ1310 and CAZ1333 synthesized above, TKB0198 was synthesized in the steps shown below.
Figure JPOXMLDOC01-appb-C000030
Figure JPOXMLDOC01-appb-C000030
　ターシャル－ブチルエステル化合物　ＣＡＺ１３１０（７５．７ｍｇ，０．２００ｍｍｏｌ）を４Ｍ塩酸／ジオキサン溶液（２．０ｍＬ）で処理し、室温で１８時間撹拌した。反応液を減圧濃縮し、カルボン酸体の粗化合物を得、さらなる精製をせずに次の反応に用いた。 Tertiary-butyl ester compound CAZ1310 (75.7 mg, 0.200 mmol) was treated with 4 M hydrochloric acid/dioxane solution (2.0 mL) and stirred at room temperature for 18 hours. The reaction solution was concentrated under reduced pressure to obtain a crude carboxylic acid compound, which was used in the next reaction without further purification.
　一方、０℃で窒素雰囲気下、Ｂｏｃ保護アミン化合物　ＣＡＺ１３３３（８１．５ｍｇ，０．２００ｍｍｏｌ）／ジクロロメタン（２．０ｍＬ）溶液にテトラフルオロホウ酸・ジエチルエーテル（０．０９６０ｍＬ，０．７００ｍｍｏｌ）を滴加し、室温で３０分攪拌した。反応液を減圧濃縮し、残渣を２％メタノール含有ジエチルエーテルで洗い、テトラフルオロホウ酸塩の粗化合物を、さらなる精製をせずに次の反応に用いた。 Meanwhile, tetrafluoroboric acid/diethyl ether (0.0960 mL, 0.700 mmol) was added dropwise to a Boc-protected amine compound CAZ1333 (81.5 mg, 0.200 mmol)/dichloromethane (2.0 mL) solution under a nitrogen atmosphere at 0°C. and stirred at room temperature for 30 minutes. The reaction was concentrated under reduced pressure, the residue was washed with 2% methanol in diethyl ether, and the crude tetrafluoroborate salt was used in the next reaction without further purification.
　当該テトラフルオロホウ酸塩の粗化合物（０．２００ｍｍｏｌ）をアセトニトリル（２．０ｍＬ）に溶かし、０℃で上記カルボン酸体の粗化合物（０．２００ｍｍｏｌ）、ＣＯＭＵ（８５．７ｍｇ，０．２００ｍｍｏｌ）及びＤＩＰＥＡ（０．０６７５ｍＬ，０．４００ｍｍｏｌ）を加え、０℃で２時間撹拌した。反応液に飽和塩化アンモニウム水を加え反応を止め、ジクロロメタンで抽出し、有機相を飽和食塩水で洗った後に、無水硫酸マグネシウムで乾燥し、減圧濃縮した。残渣を自動シリカゲルフラッシュカラムクロマトグラフィーによりクロロホルム－メタノール（１００：０から９４：６）の展開溶媒で粗精製し、ＴＫＢ０１９８を粗化合物として得た。分取用の逆相ＨＰＬＣ及びｓｅｍｉ－分取ＨＰＬＣ（ＣＨＩＲＡＬＰＡＫ　ＩＣ　ｓｅｍｉ－分取用カラム）によりさらなる精製を行い、ＴＫＢ０１９８（３．９３ｍｇ，３．２％）を黄色の固体として得た。 The crude compound of the tetrafluoroborate (0.200 mmol) was dissolved in acetonitrile (2.0 mL), and the crude compound of the carboxylic acid form (0.200 mmol) and COMU (85.7 mg, 0.200 mmol) were dissolved at 0°C. and DIPEA (0.0675 mL, 0.400 mmol) were added and stirred at 0° C. for 2 hours. Saturated aqueous ammonium chloride was added to the reaction mixture to stop the reaction, and the mixture was extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was crudely purified by automated silica gel flash column chromatography using a developing solvent of chloroform-methanol (100:0 to 94:6) to obtain TKB0198 as a crude compound. Further purification by preparative reverse-phase HPLC and semi-preparative HPLC (CHIRALPAK IC semi-preparative column) gave TKB0198 (3.93 mg, 3.2%) as a yellow solid.
　分析用の逆相ＨＰＬＣ（直線的グラジェント勾配　４０‐７０％（Ｂ／Ａ＋Ｂ）／３０分、溶出時間＝１９．９分）；
　１Ｈ　ＮＭＲ（５００ＭＨｚ，ＣＤＣｌ３）　δ　９．９２（ｓ，１Ｈ），８．７２－８．７１（ｍ，１Ｈ），７．７３－７．７２（ｍ，１Ｈ），７．５０－７．４６（ｍ，１Ｈ），７．２３－７．１９（ｍ，１Ｈ），７．１２－７．０７（ｍ，２Ｈ），６．８３－６．７９（ｍ，１Ｈ），６．５６（ｓ，１Ｈ），６．２８－６．２５（ｍ，１Ｈ），５．７３－５．７１（ｍ，１Ｈ），５．０１－４．９７（ｍ，１Ｈ），３．８７（ｓ，３Ｈ），３．３８－３．３７（ｍ，２Ｈ），２．６９（ｓ，１Ｈ），２．５２（ｓ，１Ｈ），２．２９－２．２３（ｍ，２Ｈ），２．１１－２．０４（ｍ，１Ｈ），１．８５－１．６６（ｍ，４Ｈ），０．９８（ｄ，Ｊ＝６．２Ｈｚ，３Ｈ），０．９７（ｄ，Ｊ＝６．２Ｈｚ，３Ｈ）；
　１３Ｃ　ＮＭＲ（１２５ＭＨｚ，ＣＤＣｌ３）　δ　１９１．８，１８０．１，１７２．９，１６４．６，１６１．０，１５７．３（ｄ，Ｊ＝２６０．５Ｈｚ），１５０．３（ｄ，Ｊ＝２．０Ｈｚ），１４４．８（ｄ，Ｊ＝２３７．７Ｈｚ），１４２．８（ｄ，Ｊ＝１３．７Ｈｚ），１３９．８（ｄ，Ｊ＝２．２Ｈｚ），１３０．２，１２９．０（ｄ，Ｊ＝６．９Ｈｚ），１２６．２（ｄ，Ｊ＝１５．８Ｈｚ），１２１．５（ｄ，Ｊ＝５．７Ｈｚ），１１８．２（ｄ，Ｊ＝４．３Ｈｚ），１１２．５（ｄ，Ｊ＝１７．６Ｈｚ），１０８．８（ｄ，Ｊ＝１７．９Ｈｚ），１０２．３，９８．７（ｄ，Ｊ＝６．１Ｈｚ），５５．８，５５．３，５１．７，４２．７，４０．９，３９．４，３２．９，２８．５，２５．０，２３．０，２２．３；
　ＨＲＭＳ（ＥＳＩ），ｍ／ｚ　ｃａｌｃｄ　ｆｏｒ　Ｃ３０Ｈ３２Ｆ２Ｎ５Ｏ５Ｓ　［Ｍ＋Ｈ］＋　６１２．２０８７，　ｆｏｕｎｄ　６１２．２０９０。 Analytical reverse phase HPLC (linear gradient 40-70% (B/A+B)/30 min, elution time = 19.9 min);
1 H NMR (500 MHz, CDCl 3 ) δ 9.92 (s, 1H), 8.72-8.71 (m, 1H), 7.73-7.72 (m, 1H), 7.50-7 .46 (m, 1H), 7.23-7.19 (m, 1H), 7.12-7.07 (m, 2H), 6.83-6.79 (m, 1H), 6.56 (s, 1H), 6.28-6.25 (m, 1H), 5.73-5.71 (m, 1H), 5.01-4.97 (m, 1H), 3.87 (s , 3H), 3.38-3.37 (m, 2H), 2.69 (s, 1H), 2.52 (s, 1H), 2.29-2.23 (m, 2H), 2. 11-2.04 (m, 1H), 1.85-1.66 (m, 4H), 0.98 (d, J = 6.2Hz, 3H), 0.97 (d, J = 6.2Hz , 3H);
13 C NMR (125 MHz, CDCl 3 ) δ 191.8, 180.1, 172.9, 164.6, 161.0, 157.3 (d, J = 260.5 Hz), 150.3 (d, J = 2.0 Hz), 144.8 (d, J = 237.7 Hz), 142.8 (d, J = 13.7 Hz), 139.8 (d, J = 2.2 Hz), 130.2, 129 .0 (d, J = 6.9 Hz), 126.2 (d, J = 15.8 Hz), 121.5 (d, J = 5.7 Hz), 118.2 (d, J = 4.3 Hz) , 112.5 (d, J=17.6 Hz), 108.8 (d, J=17.9 Hz), 102.3, 98.7 (d, J=6.1 Hz), 55.8, 55. 3, 51.7, 42.7, 40.9, 39.4, 32.9, 28.5, 25.0, 23.0, 22.3;
HRMS (ESI), m/z calcd for C30H32F2N5O5S [M + H] + 612.2087 , found 612.2090 .
　また、具体的な方法は示さないが、表１及び２に示すその他の化合物（下記式で表される化合物）についても、上記化合物同様に合成した。 Although no specific method is shown, other compounds shown in Tables 1 and 2 (compounds represented by the following formulas) were also synthesized in the same manner as the above compounds.
Figure JPOXMLDOC01-appb-C000031
Figure JPOXMLDOC01-appb-C000031
Figure JPOXMLDOC01-appb-C000032
Figure JPOXMLDOC01-appb-C000032
Figure JPOXMLDOC01-appb-C000033
Figure JPOXMLDOC01-appb-C000033
Figure JPOXMLDOC01-appb-C000034
Figure JPOXMLDOC01-appb-C000034
Figure JPOXMLDOC01-appb-C000035
Figure JPOXMLDOC01-appb-C000035
Figure JPOXMLDOC01-appb-C000036
Figure JPOXMLDOC01-appb-C000036
Figure JPOXMLDOC01-appb-C000037
Figure JPOXMLDOC01-appb-C000037
Figure JPOXMLDOC01-appb-C000038
Figure JPOXMLDOC01-appb-C000038
Figure JPOXMLDOC01-appb-C000039
Figure JPOXMLDOC01-appb-C000039
Figure JPOXMLDOC01-appb-C000040
Figure JPOXMLDOC01-appb-C000040
Figure JPOXMLDOC01-appb-C000041
Figure JPOXMLDOC01-appb-C000041
Figure JPOXMLDOC01-appb-C000042
Figure JPOXMLDOC01-appb-C000042
Figure JPOXMLDOC01-appb-C000043
Figure JPOXMLDOC01-appb-C000043
Figure JPOXMLDOC01-appb-C000044
Figure JPOXMLDOC01-appb-C000044
Figure JPOXMLDOC01-appb-C000045
Figure JPOXMLDOC01-appb-C000045
Figure JPOXMLDOC01-appb-C000046
Figure JPOXMLDOC01-appb-C000046
Figure JPOXMLDOC01-appb-C000047
Figure JPOXMLDOC01-appb-C000047
Figure JPOXMLDOC01-appb-C000048
Figure JPOXMLDOC01-appb-C000048
Figure JPOXMLDOC01-appb-C000049
Figure JPOXMLDOC01-appb-C000049
Figure JPOXMLDOC01-appb-C000050
Figure JPOXMLDOC01-appb-C000050
Figure JPOXMLDOC01-appb-C000051
Figure JPOXMLDOC01-appb-C000051
Figure JPOXMLDOC01-appb-C000052
Figure JPOXMLDOC01-appb-C000052
Figure JPOXMLDOC01-appb-C000053
Figure JPOXMLDOC01-appb-C000053
Figure JPOXMLDOC01-appb-C000054
Figure JPOXMLDOC01-appb-C000054
Figure JPOXMLDOC01-appb-C000055
Figure JPOXMLDOC01-appb-C000055
Figure JPOXMLDOC01-appb-C000056
Figure JPOXMLDOC01-appb-C000056
Figure JPOXMLDOC01-appb-C000057
Figure JPOXMLDOC01-appb-C000057
Figure JPOXMLDOC01-appb-C000058
Figure JPOXMLDOC01-appb-C000058
Figure JPOXMLDOC01-appb-C000059
Figure JPOXMLDOC01-appb-C000059
Figure JPOXMLDOC01-appb-C000060
Figure JPOXMLDOC01-appb-C000060
Figure JPOXMLDOC01-appb-C000061
Figure JPOXMLDOC01-appb-C000061
Figure JPOXMLDOC01-appb-C000062
Figure JPOXMLDOC01-appb-C000062
Figure JPOXMLDOC01-appb-C000063
Figure JPOXMLDOC01-appb-C000063
Figure JPOXMLDOC01-appb-C000064
Figure JPOXMLDOC01-appb-C000064
Figure JPOXMLDOC01-appb-C000065
Figure JPOXMLDOC01-appb-C000065
Figure JPOXMLDOC01-appb-C000066
Figure JPOXMLDOC01-appb-C000066
　そして、得られた化合物に関し、以下に示す方法にて、ウイルスプロテアーゼ阻害活性（ＩＣ５０）、抗ウイルス活性（ＥＣ５０）、細胞毒性（ＣＣ５０）及び生体内半減期（Ｔ１／２）を評価した。 Viral protease inhibitory activity (IC 50 ), antiviral activity (EC 50 ), cytotoxicity (CC 50 ) and in vivo half-life (T 1/2 ) of the obtained compounds were measured by the methods described below. evaluated.
　（試験例１）　ウイルスプロテアーゼ（メインプロテアーゼ）阻害活性の評価
　上記化合物（被験化合物）のＭｐｒ／３ＣＬプロテアーゼ酵素阻害活性の評価は、ＦＲＥＴに基づくアッセイキット（製品名：３ＣＬ　Ｐｒｏｔｅａｓｅ　Ｕｎｔａｇｇｅｄ　Ａｓｓａｙ　Ｋｉｔ、ＢＰＳ　Ｂｉｏｓｃｅｉｅｎｃｅ，ＣＡ，ＵＳＡ）を用いて実施した。具体的には先ず、段階希釈後の各濃度の被験化合物を９６ｗｅｌｌ　ｐｌａｔｅに１０μｌ／ｗｅｌｌで分注したのち、３ＣＬプロテアーゼを１５ｎｇ／３０μＬ／ｗｅｌｌにて添加し、３０分間室温にて攪拌、インキュベートした。その後直ちに３ＣＬ　Ｐｒｏｔｅａｓｅ　ｆｌｕｏｒｏｇｅｎｉｃ　ｓｕｂｓｔｒａｔｅ　２００μＭを１０μＬ／ｗｅｌｌで添加した。２５℃の条件下にて最長４時間インキュベートしたのち、蛍光発光強度（励起３６０ｎｍ／発光４６０ｎｍ）をマイクロプレートリーダー　Ｃｙｔａｔｉｏｎ５（ＢｉｏＴｅｋ）を用いて定量した。定量値からＩＣ５０値を算出し、各被験化合物の抗ＳＡＲＳ－ＣｏＶ－２酵素阻害活性を評価した。得られた結果を表３に示す。 (Test Example 1) Evaluation of viral protease (main protease) inhibitory activity Evaluation of the Mpr/3CL protease enzyme inhibitory activity of the above compound (test compound) was performed using a FRET-based assay kit (product name: 3CL Protease Untagged Assay Kit, BPS Bioscience , CA, USA). Specifically, first, each concentration of the test compound after serial dilution was dispensed into a 96-well plate at 10 μl/well, then 3CL protease was added at 15 ng/30 μl/well, stirred and incubated at room temperature for 30 minutes. . Immediately thereafter, 200 µM of 3CL Protease fluorogenic substrate was added at 10 µL/well. After incubating for up to 4 hours under conditions of 25° C., fluorescence emission intensity (excitation 360 nm/emission 460 nm) was quantified using a microplate reader Cytation 5 (BioTek). IC 50 values were calculated from the quantified values to evaluate the anti-SARS-CoV-2 enzyme inhibitory activity of each test compound. Table 3 shows the results obtained.
　（試験例２）　抗ＳＡＲＳ－ＣｏＶ－２活性の評価
　国立感染症研究所(日本）より譲渡されたＳＡＲＳ－ＣｏＶ－２株（ＳＡＲＳ－ＣｏＶ－２ＷＫ－５２１）を感染源として用いた。被感染細胞は、ＶｅｒｏＥ６細胞を用い、当該細胞を１０％ＦＣＳ含有、抗生剤（ペニシリン，ストレプトマイシン　各１００μｇ／ｍｌ）添加ＤＭＥＭ培地にて継続培養し、維持した。ＶｅｒｏＥ６細胞を９６ｗｅｌｌプレートに１ｘ１０４ｃｅｌｌｓ／ｗｅｌｌになるよう播種した。その翌日に、ＳＡＲＳ－ＣｏＶ－２ＷＫ－５２１ウイルスを、感染多重度（ＭＯＩ）０．０５にて、ＶｅｒｏＥ６細胞に１時間暴露しウイルス液を洗浄により除去した。その後、段階希釈後の各濃度の被験化合物を添加した培地を加え、３７℃、５％ＣＯ２の標準培養条件にて、３日間培養後、各ウェルの上清を回収し、ＳＡＲＳ－ＣｏＶ－２　ＲＮＡをＱＩＡａｍｐ　Ｖｉｒａｌ　ＲＮＡ　Ｍｉｎｉ　Ｋｉｔにて抽出し、ウイルスコピー数をＲＴ－ＰＣＲで定量化した。定量値からＥＣ５０値を算出し、各被験化合物の抗ＳＡＲＳ－ＣｏＶ－２活性を評価した。得られた結果を表３に示す。 (Test Example 2) Evaluation of anti-SARS-CoV-2 activity A SARS-CoV-2 strain (SARS-CoV-2WK-521) provided by the National Institute of Infectious Diseases (Japan) was used as an infection source. VeroE6 cells were used as infected cells, and the cells were continuously cultured and maintained in a DMEM medium containing 10% FCS and supplemented with antibiotics (100 μg/ml each of penicillin and streptomycin). VeroE6 cells were seeded on a 96-well plate at 1×10 4 cells/well. The next 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. Thereafter, a medium supplemented with each concentration of the test compound after serial dilution was added, and cultured under standard culture conditions of 37° C. and 5% CO 2 for 3 days. 2 RNA was extracted with the QIAamp Viral RNA Mini Kit and viral copy number was quantified by RT-PCR. EC50 values were calculated from the quantified values to evaluate the anti-SARS-CoV-2 activity of each test compound. Table 3 shows the results obtained.
　（試験例３）　細胞毒性試験
　被験化合物の細胞毒性試験はＶｅｒｏＥ６細胞を用いて行った。具体的には、ＶｅｒｏＥ６細胞を９６ｗｅｌｌプレートに１ｘ１０４ｃｅｌｌｓ／ｗｅｌｌになるよう播種し、段階希釈後の各濃度の各被験化合物を添加した培地と共に３日間、３７℃、５％ＣＯ２の標準培養条件で培養した後、各ウェルの生存細胞数をＭＴＴアッセイで定量化し、５０％細胞毒性濃度（ＣＣ５０）を算出した。得られた結果を表３に示す。 (Test Example 3) Cytotoxicity test A cytotoxicity test of the test compound was performed using VeroE6 cells. Specifically, VeroE6 cells were seeded in a 96-well plate at 1×10 4 cells/well, and standard culture was performed at 37° C. and 5% CO 2 for 3 days with medium supplemented with each test compound at each concentration after serial dilution. After culturing under the conditions, the number of viable cells in each well was quantified by MTT assay and the 50 % cytotoxic concentration (CC50) was calculated. Table 3 shows the results obtained.
　（試験例４）　生体内半減期の測定
　被験化合物の生体内半減期の測定はマウスを用いて実施した。具体的には、各被験化合物を尾静脈から２ｍｇ／ｋｇにて投与し、投与後１５，３０，６０，１２０，２４０分後に採血し、ＬＣ／ＭＳ／ＭＳにて血中化合物濃度を測定し、化合物の生体内半減期（Ｔ１／２）を算出した（ｎ＝２）。さらに、表１において番号：１９８及び１９９として各々示される化合物については、Ｃｏｂｉｃｉｓｔａｔ（体内における医薬品代謝に関与するＣＹＰ３Ａ等の肝臓酵素の阻害剤）の存在下（被験物質と同時に同じ投与経路で５ｍｇ／ｋｇを投与）においても、生体内半減期を前記同様に算出した。得られた結果を表３及び図１に示す。 (Test Example 4) Measurement of in vivo half-life The in vivo half-life of the test compound was measured using mice. Specifically, each test compound was administered at 2 mg/kg through the tail vein, blood was collected 15, 30, 60, 120, and 240 minutes after administration, and the blood compound concentration was measured by LC/MS/MS. , the in vivo half-life (T 1/2 ) of the compound was calculated (n=2). Furthermore, for the compounds shown in Table 1 as numbers: 198 and 199, respectively, in the presence of Cobicistat (inhibitor of liver enzymes such as CYP3A involved in drug metabolism in the body) (5 mg / kg), the in vivo half-life was calculated in the same manner as described above. The results obtained are shown in Table 3 and FIG.
Figure JPOXMLDOC01-appb-T000067
Figure JPOXMLDOC01-appb-T000067
　表３に示した結果から明らかなように、被験化合物はいずれも、ＳＡＲＳ－ＣｏＶ－２に対するＥＣ５０が１０μＭ未満であるという、優れた抗ウイルス活性を示した。また同様に、ＳＡＲＳ－ＣｏＶ－２のメインプロテアーゼに対して顕著な阻害活性（ＩＣ５０）も示した、一方、これら化合物の、ウイルスの宿主となる細胞に対する毒性（ＣＣ５０）は低かった。また、生体内半減期を測定するために行った化合物の投与例において、マウスに対する急性・亜急性毒性が認められなかった。さらに、医薬品候補化合物として良好な、生体内半減期が２０分以上を示す化合物も認められた。 As can be seen from the results shown in Table 3, all of the tested compounds exhibited excellent antiviral activity with an EC50 of less than 10 μM against SARS-CoV-2. Similarly, they also exhibited significant inhibitory activity (IC 50 ) against the main protease of SARS-CoV-2, while the toxicity (CC 50 ) of these compounds to the viral host cells was low. In addition, acute/subacute toxicity to mice was not observed in the administration examples of the compounds that were performed to measure the in vivo half-lives. In addition, some compounds exhibiting an in vivo half-life of 20 minutes or more, which are favorable as drug candidate compounds, were also found.
　また、これら被験化合物の中で、番号：１２５、１９８及び１９９の化合物において、ウイルスプロテアーゼに対する特に優れた阻害活性が認められた。さらには、番号：１９８及び１９９の化合物において、特に優れた抗ウイルス活性も認められた。なお、これら２化合物について、ＣＹＰ３Ａ等の代謝を受けるプロテアーゼ阻害剤の影響を懸念して試験を行ったが、当該化合物はＣＹＰ３Ａ等の代謝を受けていない、あるいは受け難いことが示された。 In addition, among these test compounds, compounds Nos. 125, 198 and 199 were found to have particularly excellent inhibitory activity against viral proteases. Furthermore, particularly excellent antiviral activity was also observed in compounds Nos. 198 and 199. In addition, these two compounds were tested due to concerns about the influence of protease inhibitors metabolized by CYP3A and the like, but it was shown that the compounds were not metabolized by CYP3A or hardly metabolized.
　更に、（試験例２）において、ＳＡＲＳ－ＣｏＶ－２ＷＫ－５２１の代わりに、その変異株（全て国立感染症研究所(日本）より譲受）を対象とし、番号：１９８の化合物について抗ＳＡＲＳ－ＣｏＶ－２活性を評価した。その結果、下記表４に示すとおり、試験した変異株のいずれに対しても、その親株同様に優れた抗ウイルス活性を示した。 Furthermore, in (Test Example 2), instead of SARS-CoV-2WK-521, the mutant strain (all transferred from the National Institute of Infectious Diseases (Japan)) was targeted, and number: anti-SARS-CoV for the compound of 198 -2 activity was assessed. As a result, as shown in Table 4 below, all of the tested mutant strains exhibited excellent antiviral activity similar to the parent strain.
Figure JPOXMLDOC01-appb-T000068
Figure JPOXMLDOC01-appb-T000068
　（比較例）　化合物２４２０（ＹＨ－５３）との比較
　下記に示す化合物２４２０（ＹＨ－５３）は、非特許文献３において、２００３年に流行したＳＡＲＳ－ＣｏＶ（ＳＡＲＳ－ＣｏＶ－１）のメインプロテアーゼに対して阻害活性を示す化合物５ｈとして開示されている。 (Comparative example) Comparison with compound 2420 (YH-53) Compound 2420 (YH-53) shown below is the main protease of SARS-CoV (SARS-CoV-1) prevalent in 2003 in Non-Patent Document 3 is disclosed as compound 5h, which exhibits inhibitory activity against
Figure JPOXMLDOC01-appb-C000069
Figure JPOXMLDOC01-appb-C000069
　そこで、この化合物を上記（試験例１）及び（試験例４）に供し、ＳＡＲＳ－ＣｏＶ－２に関するＥＣ５０及びＴ１／２を算出し、上記化合物におけるそれらと比較した。 Therefore, this compound was subjected to the above (Test Example 1) and (Test Example 4), and the EC 50 and T 1/2 for SARS-CoV-2 were calculated and compared with those of the above compounds.
　その結果、番号：１９８及び１９９の化合物のＥＣ５０は、表３に示すとおり、各々０．１３～０．２６μＭ及び０．８７μＭであるのに対し、化合物２４２０のＥＣ５０は２．７～３．２μＭであった。 As a result, the EC50 of compounds numbered 198 and 199 are 0.13-0.26 μM and 0.87 μM, respectively, while the EC50 of compound 2420 is 2.7-3 , as shown in Table 3. 0.2 μM.
　また、Ｔ１／２に関しては、番号：１９８及び１９９の化合物は、表３に示すとおり、各々２２分及び２５．１分であるのに対し、化合物２４２０は約１０分であった。さらに、図１に示す、生体内半減期を測定するためのＡＵＣ（Ａｒｅａ　Ｕｎｄｅｒ　ｔｈｅ　Ｃｕｒｖｅ）から明らかなように、前述のＴ１／２以上に、番号：１９８及び１９９の化合物と化合物２４２０との間で顕著な差が認められた。例えば、２４０分後の被験物質濃度の差が４０倍程と大きかった。 In terms of T 1/2 , compounds numbered 198 and 199 were 22 minutes and 25.1 minutes, respectively, as shown in Table 3, while compound 2420 was about 10 minutes. Furthermore, as is clear from the AUC (Area Under the Curve) for measuring the in vivo half -life shown in FIG. A significant difference was observed between For example, the difference in test substance concentration after 240 minutes was as large as 40 times.
　以上の結果から、少なくとも番号：１９８及び１９９の化合物は、化合物２４２０と比較して、医薬品候補として優れた動力学的パラメーターを有することが明らかになった。 From the above results, it was clarified that at least the compounds of numbers: 198 and 199 have superior kinetic parameters as drug candidates compared to compound 2420.
　以上説明したように、本発明によれば、ＳＡＲＳ－ＣｏＶ－２等のウイルスに対して優れた抗ウイルス活性を有し、宿主細胞に対する毒性が低い化合物を提供することが可能となる。さらに、このような本発明の化合物は、薬物動態においても良好であり、さらに、唯一許認可されているレムデシビルと併用することによって、ＳＡＲＳ－ＣｏＶ－２に対して顕著な相乗効果も発揮し得る。したがって、本発明は、パンデミックとなっているＳＡＲＳ－ＣｏＶ－２感染症等の予防又は治療において極めて有用である。

  As described above, according to the present invention, it is possible to provide compounds that have excellent antiviral activity against viruses such as SARS-CoV-2 and have low toxicity to host cells. In addition, such compounds of the invention have good pharmacokinetics and may also exert significant synergistic effects against SARS-CoV-2 in combination with the only licensed remdesivir. Therefore, the present invention is extremely useful in the prevention or treatment of pandemic SARS-CoV-2 infectious diseases and the like.

Claims (4)

  1. 　下記一般式（１）で表される化合物
    Figure JPOXMLDOC01-appb-C000001
     ［前記式中、ベンゾチアゾール基は、置換基を有していてもよく、置換基を有していなくともよい。Ｘ１はベンゾチアゾール基に導入され得る１又は複数の置換基を表し、当該置換基は、ハロゲン元素、置換基を有していてもよい炭素数３～６の環状アルキル基、置換基を有していてもよい炭素数１～６の直鎖状アルキル基、置換基を有していてもよい炭素数３～６の分岐鎖状アルキル基、置換基を有していてもよいアミノスルホニル基及び置換基を有していてもよいメトキシ基から各々独立して選択される。
    インドール基は、置換基を有していてもよく、置換基を有していなくともよい。Ｘ２はインドール基に導入され得る１又は複数の置換基を表し、当該置換基は、ハロゲン元素及び置換基を有していてもよいメトキシ基から各々独立して選択される。
    Ｚ１はカルボニル基又は置換基を有していてもよい炭素数１～６のアルキレン基を表す。
    Ｒ１はピロリドニル基又はピリジニル基を表す。
    Ｚ２はカルボニル基又はチオカルボニル基を表す。
    Ｒ２は置換基を有していてもよい炭素数３～６の分岐鎖状アルキル基又は置換基を有していてもよい炭素数１～６の直鎖状アルキル基を表す。
    Ｒ３は水素原子又はメチル基を表す］。     A compound represented by the following general formula (1)
    Figure JPOXMLDOC01-appb-C000001
     [In the above formula, the benzothiazole group may or may not have a substituent. X 1 represents one or more substituents that can be introduced into the benzothiazole group, and the substituents are a halogen element, an optionally substituted C 3-6 cyclic alkyl group, a substituted an optionally substituted linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group having 3 to 6 carbon atoms which may be substituted, an aminosulfonyl group which may be substituted and a methoxy group which may have a substituent.
    The indole group may or may not have a substituent. X2 represents one or more substituents that can be introduced into the indole group, and the substituents are each independently selected from a halogen element and an optionally substituted methoxy group.
    Z 1 represents a carbonyl group or an optionally substituted alkylene group having 1 to 6 carbon atoms.
    R 1 represents a pyrrolidonyl group or a pyridinyl group.
    Z2 represents a carbonyl group or a thiocarbonyl group.
    R 2 represents an optionally substituted C 3-6 branched alkyl group or an optionally substituted C 1-6 linear alkyl group.
    R 3 represents a hydrogen atom or a methyl group].
  2. 　請求項１に記載の化合物を有効成分とする、抗ウイルス剤。 An antiviral agent comprising the compound according to claim 1 as an active ingredient.
  3. 　抗ＳＡＲＳ－ＣｏＶ－２ウイルス剤である、請求項２に記載の抗ウイルス剤。 The antiviral agent according to claim 2, which is an anti-SARS-CoV-2 viral agent.
  4. 　請求項１に記載の化合物と、ＳＡＲＳ－ＣｏＶ－２ウイルスＲＮＡ依存性ＲＮＡポリメラーゼ阻害剤とを有効成分とする、抗ＳＡＲＳ－ＣｏＶ－２ウイルス剤。 An anti-SARS-CoV-2 viral agent comprising the compound according to claim 1 and a SARS-CoV-2 viral RNA-dependent RNA polymerase inhibitor as active ingredients.
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