WO2022065424A1 - Carcinostatic agent, carcinogenesis inhibitor and life-prolonging agent, comprising novel compound with mtorc1 inhibitory effect - Google Patents

Carcinostatic agent, carcinogenesis inhibitor and life-prolonging agent, comprising novel compound with mtorc1 inhibitory effect Download PDF

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WO2022065424A1
WO2022065424A1 PCT/JP2021/035038 JP2021035038W WO2022065424A1 WO 2022065424 A1 WO2022065424 A1 WO 2022065424A1 JP 2021035038 W JP2021035038 W JP 2021035038W WO 2022065424 A1 WO2022065424 A1 WO 2022065424A1
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lower alkyl
compound
carboxy
same
amino
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PCT/JP2021/035038
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French (fr)
Japanese (ja)
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秀行 宮武
嘉浩 伊藤
ラエフ ソリマン アメッド シェームス
昭博 松川
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国立研究開発法人理化学研究所
国立大学法人 岡山大学
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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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention is useful as an anticancer agent, a carcinogenic inhibitor and / or a life-prolonging agent, which contains a compound having an mTORC1 inhibitory effect.
  • mTOR (mammalian target of rapamycin) is a serine / threonine kinase identified as a target molecule of rapamycin, and was named mTOR (taget of rapamycin) because it plays a central role in the regulation of cell division and survival. ..
  • PI3 kinase Phosphoinositide 3-kinase: PI3K
  • Akt serine / threonine phosphorylating enzyme
  • Akt kinase phosphorylated
  • Cancer cells are cells that have abnormal cell cycle control due to gene mutations and have continued cell division, and mTORC1 activation is not possible for cancer cells that have an advanced cell cycle. It will promote cell proliferation.
  • mTORC1 activity inhibitors such as rapamycin can suppress both cell carcinogenesis (anticancer and carcinogenesis) and aging.
  • mTOR inhibitors have the drawback of immunosuppression, they are highly effective as anticancer agents because mTOR is activated in many cancer cells and sarcoma cells, and some mTOR inhibitors have already been introduced. It has been developed and used as an anticancer agent. Rapamycin was originally an immunosuppressive drug that prevented organ transplant rejection, and immunosuppressive drugs usually increase the incidence of cancer, whereas rapamycin, on the contrary, treats patients who have undergone kidney or liver transplants.
  • follow-up studies have shown that patients taking rapamycin have a very low incidence of cancer.
  • Some of the compounds of the present invention are known compounds. However, the specific biological activity of these compounds is unknown. On the other hand, as described above, the development of mTOR inhibitors based on rapamycin has been diligently carried out, but none of them have a skeleton similar to the compound of the present invention, and are smaller molecules and cancer cells. There are no known compounds that are specifically toxic to.
  • aging causes the onset and progression of various diseases, which are affected by the mTORC1 signaling system, and among various aging-related diseases, cognitive decline, Alzheimer's disease, malignant tumors, etc.
  • the mTORC1 inhibitor of the present invention acts in an improving direction for many diseases such as kidney disease, heart disease, and autoimmune disease (Non-Patent Documents 1 and 2).
  • An object of the present invention is to provide an anticancer agent, a carcinogenic suppressant and a life-prolonging agent containing a compound having an mTORC1 inhibitory action.
  • An anticancer agent and / or a carcinogenic inhibitor comprising the following partial structures (I), (II), and (III), a compound having an mTORC1 inhibitory action or a pharmaceutically acceptable salt thereof.
  • R 1 , R 2 , R 3 and R 4 are the same or different, H, lower alkyl, halo lower alkyl, lower alkoxy, lower alkyl amino, OH, protected OH, amino, protected amino, carboxy, Represents a protected carboxy or halogen atom
  • R 5 and R 6 indicate the same or different protective groups for H or OH, or R 5 and R 6 together indicate a methylene or dimethyl methylene group.
  • A indicates O or S. ).
  • X and Y are the same or different, and may be intervened by-(CH 2 ) 1 to 6- , oxygen atom- (CH 2 ) 1 to 6- , NH. It may be described in one of ⁇ 1> to ⁇ 2>, which indicates a linker of-(CH 2 ) 1 to 6 -or substituted with the following substituent-(CH 2 ) 1 to 6- .
  • ⁇ 4> The anticancer agent and / or carcinogenic inhibitor according to item 1 of ⁇ 1> to ⁇ 3>, wherein L, X and Y are the same or different and indicate-(CH 2 ) 1 to 6- . .. ⁇ 5>
  • R 1 , R 2 , R 3 and R 4 represent H, lower alkyl, halo lower alkyl, lower alkoxy, lower alkyl amino, carboxy, protected carboxy or halogen atom, identical or different, ⁇
  • ⁇ 6> The anticancer agent and / or carcinogenic inhibitor according to item 1 of ⁇ 1> to ⁇ 5>, wherein R1 and R2 are hydrogen atoms.
  • R 3 is H and R 4 is lower alkyl, halo lower alkyl, lower alkoxy, carboxy, protected carboxy or halogen atom, or R 3 and R 4 are the same or different.
  • Lower alkyl, halo lower alkyl, lower alkoxy, carboxy, protected carboxy or halogen atom the anticancer agent and / or carcinogenic inhibitor according to item 1 of ⁇ 1> to ⁇ 6>.
  • R 3 is H and R 4 is lower alkyl, halo lower alkyl, lower alkoxy, protected carboxy or halogen atom, or R 3 and R 4 are the same or different and lower.
  • R 3 is H and R 4 represents a lower alkyl, halo lower alkyl, or halogen atom, or R 3 and R 4 are the same or different, lower alkyl, halo lower alkyl, or.
  • ⁇ 10> The anticancer agent and / or carcinogenic suppressant according to item 1 of ⁇ 1> to ⁇ 9>, wherein R3 or / and R4 are substituted at the ortho position.
  • ⁇ 11> The anticancer agent and / or carcinogenic inhibitor according to any one of ⁇ 1 > to ⁇ 10 >, wherein R5 and R6 together represent a methylene or dimethylmethylene group.
  • ⁇ 12> The anticancer agent and / or carcinogenic suppressant according to any one of ⁇ 1> to ⁇ 11>, wherein A indicates O.
  • ⁇ 14> The anticancer agent and / or carcinogenic inhibitor according to ⁇ 1>, wherein the compound is one compound selected from the following compounds.
  • ⁇ 15> The anticancer agent and / or carcinogenic inhibitor according to ⁇ 1>, wherein the compound is one compound selected from the following compounds.
  • R 1 , R 2 , R 3 and R 4 are the same or different, H, lower alkyl, halo lower alkyl, lower alkoxy, lower alkyl amino, OH, protected OH, amino, protected amino, carboxy, Represents a protected carboxy or halogen atom
  • R 5 and R 6 indicate the same or different protective groups for H or OH, or R 5 and R 6 together indicate a methylene or dimethyl methylene group.
  • A indicates O or S. ).
  • R 1 , R 2 , R 3 and R 4 represent H, lower alkyl, halo lower alkyl, lower alkoxy, lower alkyl amino, carboxy, protected carboxy or halogen atom, identical or different, ⁇
  • R 3 is H
  • R 4 is lower alkyl, halo lower alkyl, lower alkoxy, carboxy, protected carboxy or halogen atom, or R 3 and R 4 are the same or different.
  • the life extender according to item 1 of ⁇ 16> to ⁇ 19> which represents a lower alkyl, a halo lower alkyl, a lower alkoxy or a halogen atom.
  • ⁇ 21> The life extender according to any one of ⁇ 16 > to ⁇ 20>, wherein R5 and R6 together represent a methylene or dimethylmethylene group.
  • ⁇ 22> The life extender according to ⁇ 16>, wherein the compound is one compound selected from the following compounds (where A is O or S).
  • the molecular weight of the compound included in the present invention is 450 to 850, more preferably 500 to 800.
  • ⁇ A> A compound having an mTORC1 inhibitory effect or a pharmaceutically acceptable salt thereof, which comprises the above partial structures (I), (II), and (III), is administered to a subject (preferably a mammal including a human).
  • Methods of controlling cancer and / or carcinogenesis including: ⁇ B>
  • a compound having an mTORC1 inhibitory effect or a pharmaceutically acceptable salt thereof, which comprises the above partial structures (I), (II), and (III) is administered to a subject (preferably a mammal including a human). How to extend life, including doing.
  • ⁇ C> A compound having an mTORC1 inhibitory effect or pharmacologically acceptable thereof, which comprises the above-mentioned partial structures (I), (II), and (III) for use in a treatment for suppressing cancer and / or carcinogenesis. salt.
  • ⁇ D> A compound having an mTORC1 inhibitory effect or a pharmacologically acceptable salt thereof, which comprises the above-mentioned partial structures (I), (II), and (III) for use in a life-prolonging treatment.
  • ⁇ E> A compound having an mTORC1 inhibitory effect or a pharmacologically acceptable salt thereof, which comprises the partial structures (I), (II), and (III) described above for the production of an anticancer agent and / or a carcinogenic inhibitor. use.
  • ⁇ F> Use of a compound having an mTORC1 inhibitory effect or a pharmaceutically acceptable salt thereof, which comprises the partial structures (I), (II), and (III) described above, for producing a life-prolonging agent.
  • lower alkyl is, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, etc.
  • n-hexyl isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2 -Represents a linear or branched alkyl group having 1 to 6 carbon atoms such as dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl and 2-ethylbutyl, preferably 1 to 4 carbon atoms. It is a linear or branched alkyl group.
  • halo lower alkyl refers to a group in which the "halogen atom” described later is bonded to the “lower alkyl", for example, trifluoromethyl, trichloromethyl, difluoromethyl, dichloromethyl, dibromomethyl, fluoromethyl, 2,2. , 2-Trichloroethyl, 2,2,2-trifluoroethyl, 2-bromoethyl, 2-chloroethyl, 2-fluoroethyl, 2,2-dibromoethyl and the like can be mentioned, preferably tri. It is fluoromethyl.
  • lower alkoxy refers to a group in which the "lower alkyl” is bonded to an oxygen atom, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, s-butoxy, tert-butoxy, n.
  • -Pentoxy Isopentoxy, 2-Methylbutoxy, Neopentoxy, n-Hexyloxy, 4-Methylpentoxy, 3-Methylpentoxy, 2-Methylpentoxy, 3,3-dimethylbutoxy, 2,2-dimethylbutoxy, 1 , 1-dimethylbutoxy, 1,2-dimethylbutoxy, 1,3-dimethylbutoxy, 2,3-dimethylbutoxy, etc. show linear or branched alkoxy groups having 1 to 6 carbon atoms, preferably It is a linear or branched alkoxy group having 1 to 4 carbon atoms.
  • lower alkylamino refers to a group to which the “lower alkyl” is bonded to an amino, for example, methylamino, ethylamino, n-propylamino, isopylamino, n-butylamino, isobutylamino, s-butylamino, and the like.
  • tert-Butylamino n-pentylamino, isopentylamino, 2-methylbutylamino, neopentylamino, n-hexylamino, 3,3-dimethylbutylamino, 2,2-dimethylbutylamino, 1,1-dimethyl It exhibits a linear or branched amino group having 1 to 6 carbon atoms such as butylamino, 1,2-dimethylbutylamino, 1,3-dimethylbutylamino and 2,3-dimethylbutylamino, preferably It is a linear or branched amino group having 1 to 4 carbon atoms.
  • the "protecting group” and / or “protecting group of OH” of “protected OH” is a “protecting group in reaction” that can be cleaved by a chemical method such as hydrogenolysis, hydrolysis, electrolysis, photolysis. Is shown.
  • Such “protective groups in the reaction” include, for example, formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl.
  • allylcarbonyl group such as benzoyl, ⁇ -naphthoyl, ⁇ -naphthoyl, halogenoallyl carbonyl group such as 2-bromobenzoyl, 4-chlorobenzoyl, 2,4,6-trimethyl Lower alkylated allylcarbonyl groups such as benzoyl, 4-toluoil, lower alkoxylated allylcarbonyl groups such as 4-anisoil, 2-carboxybenzoyl, 3-carboxybenzoyl, carboxy such as 4-carboxybenzoyl Carbonylated allylcarbonyl groups such as allylcarbonyl groups, 4-nitrobenzoyl, 2-nitrobenzoyl, lower alkoxycarbonylated allylcarbonyl groups such as 2- (methoxycarbonyl) benzoyl, 4-phenyl "Aromatic acyl groups” such as allylated allylcarbonyl groups such as
  • Alkenyloxy such as oxycarbonyl, allyloxycarbonyl Carbonyl group "; one or two lower alkoxy or nitro such as benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl.
  • alkenyloxy such as oxycarbonyl, allyloxycarbonyl Carbonyl group "; one or two lower alkoxy or nitro such as benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl.
  • Examples thereof include an “aralkyloxycarbonyl group” in which the allyl ring may be substituted with the group.
  • the "protective group” of the "protected amino” means a group in which the following protective group protects one or two amino groups, and the protective group is usually used as a protective group for an amino group. If not limited, preferred are alkylcarbonyl groups such as formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, lauroyl, myristol, tridecanoyl, palmitoyl, stearoyl, chloroacetyl.
  • alkylcarbonyl groups such as formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, lauroyl, myristol, tridecanoyl, palmitoyl, stearoyl, chloroacet
  • Harogeno lower alkylcarbonyl groups such as dichloroacetyl, trichloroacetyl, trifluoroacetyl, lower alkoxy lower alkylcarbonyl groups such as methoxyacetyl, unsaturated alkylcarbonyl groups such as (E) -2-methyl-2-butenoyl.
  • Harogeno lower alkylcarbonyl groups such as dichloroacetyl, trichloroacetyl, trifluoroacetyl, lower alkoxy lower alkylcarbonyl groups such as methoxyacetyl, unsaturated alkylcarbonyl groups such as (E) -2-methyl-2-butenoyl.
  • allylcarbonyl groups such as benzoyl, ⁇ -naphthoyl, ⁇ -naphthoyl, 2-bromobenzoyl, halogenoallyl carbonyl groups such as 4-chlorobenzoyl, 2,4,6-
  • Lower alkylated allylcarbonyl groups such as benzoyl, 4-toluoil, lower alkoxylated allylcarbonyl groups such as 4-anisoil, 4-nitrobenzoyl, nitrylated allylcarbonyl such as 2-nitrobenzoyl
  • Aromatic acyl groups such as groups, lower alkoxycarbonylated allylcarbonyl groups such as 2- (methoxycarbonyl) benzoyl, allylylated allylcarbonyl groups such as 4-phenylbenzoyl; methoxycarbonyl, ethoxycarbonyl.
  • T-Butoxycarbonyl lower alkoxycarbonyl groups such as isobutoxycarbonyl, halogens such as 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethoxycarbonyl or lower alkoxycarbonyl groups substituted with tri-lower alkylsilyl groups.
  • Ekoxycarbonyl groups such as; vinyloxycarbonyl, alkenyloxycarbonyl groups such as allyloxycarbonyl; benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 4- An aralkyloxycarbonyl group whose allyl ring may be substituted with one or two lower alkoxy or nitro groups, such as nitrobenzyloxycarbonyl; trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsiri Or, a trilower alkylsilyl group such as methyldiisopropylsilyl, methyldi-t-butylsilyl, triisopropylsilyl, one or two aryls such as diphenylmethylsilyl, diphenylbutylsilyl, dipheny
  • Silyl groups such as tri-lower alkylsilyl groups substituted with groups; benzyl, phenethyl, 3-phenylpropyl, ⁇ -naphthylmethyl, ⁇ -naphthylmethyl, diphenylmethyl, triphenylmethyl, ⁇ -naphthyldiphenylmethyl, 9-an.
  • Lower alkyl groups substituted with 1 to 3 allyl groups such as thrillmethyl, 4-methylbenzyl, 2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 4-cyanobenzyl, 4-cyanobenzyldiphenylmethyl, bis (2-nitrophenyl) methyl, piperonyl
  • An aralkyl group such as a lower alkyl group substituted with 1 to 3 aryl groups in which the aryl ring is substituted with a lower alkyl, lower alkoxy, nitro, halogen, cyano group; or N, N-dimethyl.
  • a shift base such as aminomethylene, benzylidene, 4-methoxybenzylidene, 4-nitrobenzylidene, salicylidene, 5-chlorosalicylidene, diphenylmethylene, (5-chloro-2-hydroxyphenyl) phenylmethylene It is a methylene group, more preferably an aliphatic acyl group.
  • the "protecting group” of a “protected carboxy” refers to a “protecting group in a reaction” that can be cleaved by chemical methods such as hydrogenolysis, hydrolysis, electrolysis, photolysis.
  • Such “protective groups in the reaction” are preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl.
  • Alkyl groups such as lower alkyl groups substituted with 1 to 3 allyl groups substituted with a ru ring; trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, tert-butyldimethylsilyl, methyldiisopropylsilyl, methyldi Examples thereof include “silyl groups” such as tert-butyl silyl, triisopropyl silyl, methyl diphenyl silyl, isopropyl diphenyl silyl, butyl diphenyl silyl and phenyl diisopropyl silyl, and preferred are "lower alkyl groups”.
  • halogen atom is a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, and is preferably a fluorine atom or a chlorine atom.
  • May be intervened by oxygen atom- (CH 2 ) 1 to 6- means a group in which "-(CH 2 ) 1 to 6- " is intervened by one or two oxygen atoms. Shown, for example, methyleneoxymethylene.
  • the compound (I) of the present invention may form a salt with a base or an acid depending on the type of the substituent.
  • the salt is a pharmaceutically acceptable salt, and is preferably an inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, or phosphoric acid, formic acid, acetic acid, propionic acid, or shu.
  • Acid addition salts with organic acids such as acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, benzenesulfonic acid, aspartic acid, glutamate, sodium, potassium
  • organic acids such as acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, benzenesulfonic acid, aspartic acid, glutamate, sodium, potassium
  • inorganic bases such as magnesium, calcium and aluminum
  • organic bases such as methylamine, ethylamine, ethanolamine, lysine and ornithine, and ammonium salts.
  • compound C inhibited as much as RAP.
  • Compound C showed a higher tumor growth inhibitory effect than RAP.
  • Compound C showed a tumor lung metastasis promoting activity weaker than RAP. Discontinued administration at D11 and investigated tumor recurrence at D20. Compound C showed lower tumor relapse than RAP. Blood such as tumor photographs that have spread to the lungs, blood urea nitrogen, creatinine, blood urea nitrogen / creatin, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and / or glucose concentration. The results of the test and other animal experiments are shown. Shows the effect of Compounds C and RAP on body weight loss, with no significant reduction. No significant weight loss was observed with Compound C during the experimental period, and no obvious toxicity was detected in the blood.
  • R 2 and X have the same meaning as described above.
  • the carboxy group was protected in an aqueous solution of, for example, 150 ml of TEA (51.8 mmol) and the amino acid (2) (51.8 mmol) whose carboxy group may be protected by conventional protective groups.
  • a solution of isothiocyanate (1) (eg, a solution of an inert organic solvent such as i-PrOH (eg 200 ml)) was added and the reaction mixture was refluxed for example for 30 minutes. ..
  • the precipitate was filtered and dried to give the desired product (3), for example in 80% yield, which was used in step B without further purification.
  • the "protecting group for a carboxy group" has the same meaning as described above.
  • Step B In the above figure, R 2 and X have the same meaning as described above.
  • a solution of NaOH (3 eq, 2.27 g) dissolved in 100 ml of water and an acid (3) (18.9 mmol, 1 eq) whose carboxy group may be protected by conventional protecting groups. was heated to 50 ° C. The mixture was then cooled, H 2 O 2 (10 eq, 35% in water) was added and stirred.
  • the reaction temperature is ⁇ 10 ° C. to 170 ° C., preferably 30 ° C. to 100 ° C.
  • the reaction time varies mainly depending on the reaction temperature, the raw material compound, the reaction reagent or the type of the solvent used, but is usually 10 minutes to 2 hours, preferably 30 minutes.
  • an acid such as hydrochloric acid or phosphoric acid
  • It can be a blended acid such as an organic acid such as acetic acid, formic acid, oxalic acid, methanesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, preferably an organic acid, and further.
  • acetic acid was added, the precipitate was filtered and dried.
  • the desired product (4) (eg, NMR purity 95%) was obtained, for example in 96% yield, and used in step C without further purification.
  • Step C In the above figure, R 1 , R 2 , R 5 , R 6 , L and X have the same meaning as described above. If necessary, the protecting group of the carboxy group is removed, the acid (4) (eg, 18.4 mmol, 1 eq) is dissolved in, for example, dioxane (60 ml) and the CDI (1.1 eq) is at room temperature. I added it little by little. The mixture was stirred at 50 ° C. for 3 hours and then amine (5) (18.4 mmol, 1 eq) was added.
  • the reaction is carried out by condensing carboxy with an amine compound in a solvent.
  • the solvent used is not particularly limited as long as it is inert, but for example, methylene chloride, halogenated monovalent hydrogens such as chloroform, dioxane, ethers, ethers such as tetrahydrofuran, dimethylformamide and dimethylacetamide.
  • amides can be mentioned.
  • the reaction is carried out, for example, azodicarboxylic acid dilower alkyl-triphenylphosphines such as diethyl-triphenylphosphine azodicarboxylic acid, N-such as N-ethyl-5-phenylisooxazolium-3'-sulfonate.
  • N', N'-dicycloalkylcarbodiimides such as N', N'-dicyclohexylcarbodiimide (DCC), di-2- Diheteroaryl diselenides such as pyridyl diselenides, triarylphosphines such as triphenylphosphine, arylsulfonyltriazolides such as p-nitrobenzenesulfonyltriazolides, 2-chlor-1-methyl 2-Halo-1-lower alkylpyridinium halides such as pyridinium iodide and diallylphosphoryl azides such as diphenylphosphoryl azide (DPPA), imidazole derivatives such as N, N'-carbodiimidazole (CDI), 1-hydroxy Bentotriazole derivatives such as benzotriazole (HOBT), dicarboxyimide
  • DCC N', N'-dicyclohexylcarbodiimide
  • DCC di
  • the reaction temperature is ⁇ 10 ° C. to 25 ° C.
  • the reaction between the active ester compound and the amine is near room temperature
  • the reaction time is 30 minutes to 10 hours.
  • Removal of the protecting group of the carboxy group varies depending on the type, but is generally carried out by a method well known in the art in the art as follows.
  • a lower alkyl group or an aryl group is used as the protecting group for the carboxy group, it can be removed by treating with an acid or a base.
  • the acid hydrochloric acid, sulfuric acid, phosphoric acid, and hydrobromic acid are used, and the base is not particularly limited as long as it does not affect other parts of the compound, but sodium carbonate and potassium carbonate are preferable.
  • Alkali metal carbonates such as, sodium hydroxide, alkali metal hydroxides such as potassium hydroxide or concentrated ammonia-methanol solutions are used. It should be noted that hydrolysis with a base may cause isomerization.
  • the solvent used is not particularly limited as long as it is used for a normal hydrolysis reaction and does not inhibit the reaction, such as water or methanol, ethanol, n-propanol and the like.
  • a mixed solvent of water and an organic solvent such as alcohols or ethers such as tetrahydrofuran and dioxane is suitable.
  • the reaction temperature and reaction time vary depending on the starting material, solvent, reagent used and the like, and are not particularly limited, but are usually carried out at 0 ° C. to 150 ° C.
  • the protecting group of the carboxy group is a dially substituted methyl group such as diphenylmethyl, it is usually removed by treatment with an acid in a solvent.
  • a solvent aromatic hydrocarbons such as anisole are preferable, and as the acid used, a fluoride organic acid such as trifluoroacetic acid is used.
  • the reaction temperature and reaction time will vary depending on the starting material, solvent, acid used, etc., but are usually carried out at room temperature for 30 minutes to 10 hours.
  • the protecting group of the carboxy group is an aralkyl group or a halogeno lower alkyl group, it is usually removed by reduction in a solvent.
  • the reducing method when the protecting group of the carboxy group is a halogeno lower alkyl group, a method by chemical reduction such as zinc-acetic acid is preferable, and when it is an aralkyl group, palladium carbon or platinum can be used. It is carried out by a catalytic reduction method using such a catalyst, or by a chemical reduction method using an alkali metal sulfide such as potassium sulfide and sodium sulfide.
  • the solvent used is not particularly limited as long as it is not involved in this reaction, but is limited to alcohols such as metanol and etanol; ethers such as tetrahydrofuran and dioxane; acetic acid.
  • Such fatty acids or a mixed solvent of these organic solvents and water are suitable.
  • the reaction temperature and reaction time vary depending on the starting material, solvent, reduction method, etc., but are usually carried out at around 0 ° C. to room temperature (for example, 25 ° C.) for 5 minutes to 12 hours.
  • the protecting group of the carboxy group is an alkoxymethyl group, it is usually removed by treatment with an acid in a solvent.
  • the acid used is not particularly limited as long as it is usually used as a blended acid, but is preferably an inorganic acid such as hydrochloric acid or sulfuric acid or an organic acid such as acetic acid or paratoluenesulfonic acid. ..
  • the solvent used is not particularly limited as long as it does not participate in this reaction, but alcohols such as metanol and etanol; ethers such as tetrahydrofuran and dioxane, or these.
  • a mixed solvent of an organic solvent and water is suitable.
  • the reaction temperature and reaction time vary depending on the starting material, the solvent, the type of acid used, and the like, but are usually carried out at 0 ° C. to 50 ° C. for 10 minutes to 18 hours.
  • Step D In the above figure, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , L, Y, and X have the same meanings as described above.
  • Z indicates a leaving group.
  • the "desorbing group” is usually not particularly limited as long as it is a group desorbed as a nucleophilic residue, but is preferably a halogen atom such as chlorine, bromine, or nitrous; methoxycarbonyloxy, ethoxycarbonyloxy.
  • Lower alkoxycarbonyloxy groups such as; alkylcarbonyloxy groups such as acetoxy, propionyloxy; halogenated alkylcarbonyloxy groups such as chloroacetyloxy, dichloroacetyloxy, trichloroacetyloxy, trifluoroacetyloxy, methoxyacetyloxy
  • Lower alkoxyalkylcarbonyloxy groups such as (E) aliphatic acyloxy groups such as unsaturated alkylcarbonyloxy groups such as (E) -2-methyl-2-butenoyloxy; allylcarbonyloxy groups such as benzoyloxy, 2 -Halogenized allylcarbonyloxy groups such as bromobenzoyloxy, 4-chlorobenzoyloxy, lower alkylated allylcarbonyloxy groups such as 2,4,6-trimethylbenzoyloxy and 4-toluoleoxy, Aromatic acyloxy groups such as lower alkoxylated
  • the base is not particularly limited as long as it is used as a base in a normal reaction, but is preferably alkali metal carbonates such as sodium carbonate, potassium carbonate and lithium carbonate; sodium hydrogen carbonate and hydrogen carbonate. Alkali metal hydrogen carbonates such as potassium and lithium hydrogen carbonate, most preferably potassium carbonate.
  • Cold water eg 100 ml was added and the precipitate was filtered.
  • the white precipitate was dried and recrystallized from DMF (eg 8 ml).
  • the precipitate was filtered, washed with i-PrOH (eg 15 ml) and dried to dry the white precipitate to give the compound (8) of interest, for example in 40% yield (95% purity by NMR, by LCMS). Purity 96%).
  • the protecting group was deprotected according to a conventional method. Removal of protecting groups varies by type, but is generally carried out by methods well known in the art in the art as follows.
  • the protecting group of the carboxy group can be removed as described above.
  • a silyl group When a silyl group is used as the protecting group for the amino group, it is usually removed by treatment with a compound that produces a fluorine anion, such as tetrabutylammonium fluoride.
  • the reaction solvent is not particularly limited as long as it does not inhibit the reaction, but ethers such as tetrahydrofuran and dioxane are suitable.
  • the reaction temperature and reaction time are not particularly limited, but the reaction is usually carried out at room temperature for 10 to 18 hours. If the protecting group for the amino group is an aliphatic acyl group, an aromatic acyl group, an alkoxycarbonyl group or a substituted methylene group forming a Schiff base, it is treated with an acid or a base in the presence of an aqueous solvent.
  • the acid used is usually used as an acid and is not particularly limited as long as it does not inhibit the reaction, but is preferably an inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid and hydrobromic acid.
  • Is used, and the base used is not particularly limited as long as it does not affect other parts of the compound, but preferably metal alkoxides such as sodium methoxydo, sodium carbonate, and potassium carbonate.
  • Alkaline metal carbonates such as lithium carbonate, alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, lithium hydroxide or ammonia water, ammonia such as concentrated ammonia-methanol are used.
  • the solvent used is not particularly limited as long as it is used for a normal hydrolysis reaction, and water; alcohols such as metanol, etanol, n-propanol; tetrahydrofuran, An organic solvent such as ethylene such as dioxane or a mixed solvent of water and the above organic solvent is suitable.
  • the reaction temperature and reaction time vary depending on the starting material, solvent, acid or base used, etc., and are not particularly limited, but are usually 0 ° C. to 150 ° C. for 1 hour to 10 hours in order to suppress side reactions. Will be implemented.
  • the protecting group of the amino group is an aralkyl group or an aralkyloxycarbonyl group
  • it is usually removed by contacting with a reducing agent in a solvent (preferably, catalytic reduction under a catalyst at room temperature).
  • a method of removing with an oxidizing agent is preferable.
  • the solvent used in the removal by catalytic reduction is not particularly limited as long as it does not participate in this reaction, but is limited to hydrocarbons such as methanol, ethane, isopropanol, diethyl ester, and the like.
  • Solvents such as dioxane, aromatic hydrocarbons such as toluene, benzene and xylene, aliphatic hydrocarbons such as hexane and cyclohexane, esters such as ethyl acetate and propyl acetate, acetic acid.
  • fatty acids or a mixed solvent of these organic solvents and water are suitable.
  • the catalyst used is not particularly limited as long as it is usually used for a catalytic reduction reaction, but is preferably palladium carbon, lane-nickel, platinum oxide, platinum black, rhodium-aluminum oxide, or tri.
  • Triphenylphosphine-rhodium chloride and palladium-barium sulfate are used.
  • the pressure is not particularly limited, but is usually 1 to 10 atm.
  • the reaction temperature and reaction time vary depending on the type of starting material, solvent, catalyst, etc., but are usually carried out at 0 ° C. to 100 ° C. for 5 minutes to 24 hours.
  • the solvent used for removal by oxidation is not particularly limited as long as it does not participate in this reaction, but is preferably a water-containing organic solvent.
  • Suitable such organic solvents are ketones such as acetone, methylene chloride, chloroform, halogenated hydrocarbons such as carbon tetrachloride, nitriles such as acetonitrile, diethyl ether, tetrahydrofuran, dioxane and the like. Etels, amides such as dimethylformamide, dimethylacetamide, hexamethylphosphorotriamide and sulfoxides such as dimethylsulfoxide can be mentioned.
  • the oxidizing agent used is not particularly limited as long as it is a compound used for oxidation, but preferably potassium persulfate, sodium persulfate, ammonium cerium nitrate (CAN), 2,3-dichloro-5.
  • 6-Dicyano-p-benzoquinone DDQ
  • the reaction temperature and reaction time vary depending on the type of starting material, solvent, catalyst, etc., but are usually carried out at 0 ° C. to 150 ° C. for 10 minutes to 24 hours.
  • the protecting group for the amino group is usually an alkenyloxycarbonyl group
  • the protecting group for the amino group is usually a substituted methylene group forming the aliphatic acyl group, aromatic acyl group, alkoxycarbonyl group or Schiff base described above. It is achieved by treating with a base, similar to the conditions of the removal reaction in some cases.
  • a method of removing palladium and triphenylphosphine or nickel tetracarbonyl is particularly convenient and can be carried out with few side reactions.
  • a silyl group When a silyl group is used as the protective group for the hydroxyl group, it is usually treated with a compound that produces a fluorinated anion such as tetrabutylammonium fluoride, hydrofluoric acid, hydrofluoric acid-pyridine, and potassium fluoride. Alternatively, it can be removed by treatment with an organic acid such as acetic acid, methanesulfonic acid, paratoluenesulfonic acid, trifluoroacetic acid, trifluoromethanesulfonic acid or an inorganic acid such as hydrochloric acid.
  • a compound that produces a fluorinated anion such as tetrabutylammonium fluoride, hydrofluoric acid, hydrofluoric acid-pyridine, and potassium fluoride.
  • an organic acid such as acetic acid, methanesulfonic acid, paratoluenesulfonic acid, trifluoroacetic acid, trifluoromethane
  • the solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, but is preferably diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, or diethylene glycol.
  • Ethers such as dimethyl ether; acetonitrile, nitriles such as isobutyronitrile; water; organic acids such as acetic acid and mixed solvents thereof can be mentioned.
  • the reaction temperature and reaction time are not particularly limited, but are usually carried out at 0 ° C. to 100 ° C. (preferably 10 ° C. to 30 ° C.) for 1 hour to 24 hours.
  • the protecting group of the hydroxyl group is an aralkyl group or an aralkyloxycarbonyl group
  • it is usually removed by contacting it with a reducing agent in a solvent (preferably, catalytically reducing it under a catalyst at room temperature) or oxidation.
  • a reducing agent preferably, catalytically reducing it under a catalyst at room temperature
  • a method of removing with an agent is preferable.
  • the solvent used in the removal by catalytic reduction is not particularly limited as long as it does not participate in this reaction, but alcohols such as metanol, etanol, isopropanol, diethyl ether, and the like.
  • Ethyls such as tetrahydrofuran, dioxane, aromatic hydrocarbons such as toluene, benzene, xylene, hexane, aliphatic hydrocarbons such as cyclohexane, esters such as ethyl acetate, propyl acetate, formamides, Amidos such as dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone, hexamethylphosphorotriamide, fatty acids such as formic acid and acetic acid, water, or a mixed solvent thereof are suitable, and more preferably.
  • the catalyst used is not particularly limited as long as it is usually used for a catalytic reduction reaction, but is preferably palladium carbon, palladium black, lane-nickel, platinum oxide, platinum black, or rhodium-oxidation.
  • Aluminum, triphenylphosphine-rhodium chloride, palladium-barium sulfate are used.
  • the pressure is not particularly limited, but is usually 1 to 10 atm.
  • the reaction temperature and reaction time vary depending on the type of starting material, solvent, catalyst, etc., but are usually 0 ° C to 100 ° C (preferably 20 ° C to 70 ° C), 5 minutes to 48 hours (preferably 1). Hours to 24 hours).
  • the solvent used for removal by oxidation is not particularly limited as long as it does not participate in this reaction, but is preferably a water-containing organic solvent. Suitable such organic solvents are ketones such as acetone, methylene chloride, chloroform, halogenated hydrocarbons such as carbon tetrachloride, nitriles such as acetonitrile, diethyl ether, tetrahydrofuran, dioxane and the like.
  • the oxidizing agent used is not particularly limited as long as it is a compound used for oxidation, but preferably potassium persulfate, sodium persulfate, ammonium cerium nitrate (CAN), 2,3-dichloro-5. , 6-Dicyano-p-benzoquinone (DDQ) is used.
  • the reaction temperature and reaction time vary depending on the type of starting material, solvent, catalyst, etc., but are usually carried out at 0 ° C. to 150 ° C. for 10 minutes to 24 hours.
  • alkali metals such as metallic lithium and metallic sodium
  • it can be removed by using an aluminum chloride-sodium iodide or an alkylsilyl halide such as trimethylsilyliodide in a solvent.
  • the solvent used is not particularly limited as long as it does not participate in this reaction, but preferably nitriles such as acetonitrile, methylene chloride, halogenated hydrocarbons such as chloroform, or a mixed solvent thereof. Is used.
  • the reaction temperature and reaction time vary depending on the starting material, solvent, etc., but are usually carried out at 0 ° C to 50 ° C for 5 minutes to 3 days.
  • the protecting group of the hydroxyl group is an aliphatic acyl group, an aromatic acyl group or an alkoxycarbonyl group
  • it is removed by treatment with a base in a solvent.
  • the base used is not particularly limited as long as it does not affect other parts of the compound, but is preferably metal alkoxides such as sodium methoxydo; sodium carbonate, potassium carbonate, lithium carbonate and the like.
  • Alkali metal carbonates alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, lithium hydroxide, barium hydroxide or aqueous ammonia, ammonia such as concentrated ammonia-methanol are used.
  • the solvent used is not particularly limited as long as it is used for a normal hydrolysis reaction, and water; alcohols such as metanol, etanol, n-propanol, tetrahydrofuran, and the like.
  • An organic solvent such as ethylene such as dioxane or a mixed solvent of water and the above organic solvent is suitable.
  • the reaction temperature and reaction time vary depending on the starting material, solvent, base used, etc., and are not particularly limited, but are usually carried out at 0 ° C.
  • the protecting group for the hydroxyl group is an alkoxymethyl group, a tetrahydropyranyl group, a tetrahydrothiopyranyl group, a tetrahydrofuranyl group, a tetrahydrothiofuranyl group or a substituted ethyl group
  • it is usually treated with an acid in a solvent. It is removed by doing.
  • the acid used is not particularly limited as long as it is usually used as Bronsted acid or Lewis acid, and is preferably hydrogen chloride; an inorganic acid such as hydrochloric acid, sulfuric acid or nitric acid; or acetic acid or tri.
  • Blended acids such as organic acids such as fluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid: Lewis acids such as boron trifluoride, but also strongly acidic cation exchange resins such as Dawex 50W. Can be used.
  • the solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, but preferably aliphatic hydrocarbons such as hexane, heptane, ligroin, and petroleum ether; Aromatic hydrocarbons such as benzene, toluene and xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, dichlorobenzene; ethyl formate, ethyl acetate, propyl acetate, butyl acetate, Esters such as diethyl carbonate; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol dimethyl ether; metall, etanol, n-propanol, isopropano- Alcoles
  • Isophorone, ketones such as cyclohexanone; water or a mixed solvent thereof is suitable, and more preferably halogenated hydrocarbons, esters or ethers.
  • the reaction temperature and reaction time vary depending on the type and concentration of the starting material, solvent and acid used, but are usually -10 ° C to 100 ° C (preferably -5 ° C to 50 ° C) for 5 minutes to 5 minutes. 48 hours (preferably 30 minutes to 10 hours).
  • the protecting group of the hydroxyl group is an alkenyloxycarbonyl group
  • the conditions for the removal reaction when the protecting group of the hydroxyl group is an aliphatic acyl group, an aromatic acyl group or an alkoxycarbonyl group are usually the same.
  • the protecting group of the hydroxyl group may be removed at the same time by the operation of removing the protecting group of the amino group as described above.
  • the desired removal reaction can be sequentially carried out in no particular order.
  • step C the compound in which A is S is similarly obtained by directly performing step C on the compound produced in step A (4: A indicates S) without depending on step B, as described below. Can be manufactured to.
  • the target compound (I) of this reaction is collected from the reaction mixture according to a conventional method.
  • the reaction mixture is appropriately neutralized, and if insoluble matter is present, it is removed by filtration, an immiscible organic solvent such as ethyl acetate is added to water, and the mixture is washed with water or the like to contain the target compound. It is obtained by separating the organic layer, drying it with anhydrous magnesium sulfate or the like, and then distilling off the solvent.
  • the obtained target compound can be obtained by a conventional method such as recrystallization, reprecipitation, or a method commonly used for separation and purification of an organic compound, for example, silica gel, alumina, magnesium-silicalate-based fluorodyl.
  • Adsorption column chromatography method using various carriers Cefadex LH-20 (manufactured by Pharmacia), Amberlite XAD-11 (manufactured by Roam & Haas), Diaion HP-20 (manufactured by Mitsubishi Kasei), etc.
  • a method using a synthetic adsorbent such as partition column chromatography using a carrier, a method using ion exchange chromatography, or a normal phase / reverse phase column chromatography method using silica gel or alkylated silica gel (preferably, high-speed liquid). It can be separated and purified by appropriately combining (chromatographic)) and eluting with an appropriate eluent. When it is necessary to separate the isomers, it can be carried out by the separation and purification means at an appropriate time after the reaction of each of the above steps is completed or after the desired step is completed.
  • a compound having an mTORC1 inhibitory action as an active ingredient or a pharmaceutically acceptable salt thereof may be blended alone, but pharmaceutically acceptable. It may be provided in the form of a pharmaceutical product containing an acceptable carrier or pharmaceutical additive.
  • the active ingredient, a compound having an inhibitory effect on mTORC1, or a pharmaceutically acceptable salt thereof can be contained, for example, in the formulation in an amount of 0.1 to 99.9% by mass.
  • the pharmaceutically acceptable carrier or pharmaceutical additive is not particularly limited, and is, for example, an excipient, a disintegrant, a disintegrant, a binder, a lubricant, a coating agent, a dye, a diluent, and a solubilizer. , Dissolution aids, tonicity agents, pH adjusters, stabilizers and the like.
  • preparation suitable for oral administration examples include powders, powders, tablets, capsules, fine granules, granules, liquids, syrups and the like.
  • various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate, glycine, starch, preferably corn, potato or tapioca starch, and alginic acid and certain types. May be used with various disintegrants such as alginic acid compound salt and granule-forming binders such as polyvinylpyrrolidone, starch, gelatin and gum arabic.
  • disintegrants such as alginic acid compound salt and granule-forming binders such as polyvinylpyrrolidone, starch, gelatin and gum arabic.
  • tablets may be formed using lubricants such as magnesium stearate, sodium lauryl sulfate, and talc. It can also be an aqueous suspension and / or an elixir for oral administration.
  • Examples of the preparation suitable for parenteral administration include injections, suppositories, paps, nasal drops, eye drops and the like.
  • the injection can be administered, for example, by intravenous injection, intra-articular injection, intramuscular injection or subcutaneous injection.
  • the dose of the anticancer agent, carcinogenic inhibitor and / or life-prolonging agent of the present invention is not particularly limited, and various conditions such as the type of cancer, the age and symptoms of the patient, the route of administration, the purpose of treatment, and the presence or absence of concomitant agents. It is possible to select an appropriate dose according to the patient.
  • the daily dose of the active ingredient for an adult for example, body weight 60 kg
  • the daily dose of the active ingredient for an adult is about 1 ⁇ g to 10 g, preferably 100 ⁇ g to 1 g, and more preferably 1 mg to 500 mg.
  • These daily doses may be administered in 2 to 4 divided doses.
  • VL virtual ligands
  • rapamycin was used as a template for the docking guide.
  • the atomic property field (APF) of rapamycin was taken into consideration.
  • the docking score selected the highest ligand for each group (L # 1 to L # 3) to obtain the following three types of VL.
  • FIG. 2 shows the crystal structure of FKBP12-RAP-FRB (PDB ID: 1FAP) and the docking model of FKBP12-compound C-FRB (FIG. 2).
  • FKBP12 and FRB genes are cloned from the FKBP-SmBiT and FRB-LgBiT control vectors included in the NanoBit PPI system (Promega, USA) kit and incorporated into a pET15b vector (Novagen, USA).
  • In-Fusion cloning kit (Takara Bio, Japan) was used for insertion.
  • the constructed plasmid was transformed into DH5 ⁇ Escherichia coli (Nippon Gene, Japan), applied onto an LB agar plate containing 0.1 mM ampicillin, and the colonies that appeared were sequenced by colony PCR.
  • Bacterial cells were collected by centrifugation (4 ° C., 8000 rpm, 15 minutes) and stored at ⁇ 80 ° C. 5 g cells (FRB or FKBP12), 100 mL lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.04 mg / ml lysozyme, 0.16 mg / ml DNase I, 1 tablet x It was dissolved in a compacte TM protease inhibitor (Sigma-Aldrich) and ultrasonically disrupted (Soniffier (R) (Branson, USA), 5.0 W, 30-40% cycle / sec, 5 min, one ice).
  • a compacte TM protease inhibitor Sigma-Aldrich
  • Roniffier (R) Branson, USA
  • the pellet was washed 3 times with wash buffer A (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 4 urea, pH 8.0). Then, it was washed three times with washing buffer B (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA). In addition, the pellet was dissolved in 20 mL lysis buffer (50 mM Tris-HCl, 8 urea, 100 mM NaH 2 PO 4 , 10 mM 2-mercaptoethanol, pH 8.0) and stirred with a rotator (2 hours, room temperature). ..
  • Refolding buffer 50 mM, Tris-HCl, 40 mM NaCl, 1 mM EDTA, 1 M L-arginine, 10% (v / v) glycerol, pH 8.0) (4). °C, stirring.
  • Refolding buffer was concentrated in 400 mL Amicon Stillred Cell (Millipore, USA) and Amicon Ultra-15 (MWCO: 3 kDa, Millipore, Germany) and bound buffer A (50 mM Tris-HCl, pH 8.0). The buffer was exchanged with (v / v) glycerol) to obtain a concentrated FRB solution.
  • the reaction was preliminarily equilibrated with a gel filtration buffer (50 mM Tris-HCl, 150 mM NaCl, 10% (v / v) glycerol) on a HiRoad (R) 26/60 Superdex-200 column (Cytiva, Japan). After gel filtration and the obtained sample was concentrated, it was stored at ⁇ 80 ° C.
  • a gel filtration buffer 50 mM Tris-HCl, 150 mM NaCl, 10% (v / v) glycerol
  • rapamycin (Funakoshi Co., Ltd., Japan) or a solution of the selected compound was added to the wells, sealed again, and incubated in the dark at room temperature for 1 hour or longer.
  • Alpha sinal generated from the well was measured with an EnSpire multimode plate reader (PerkinElmer, USA) with AlphaLISA (R) measurement mode.
  • the equilibrium dissociation constant (Kd) was obtained by varying the concentration of 6xHis-FRB, biotin-FKBP12 or ligand (0.01-1000 nM) to create multiple wells and measuring Alpha signal.
  • Competitive inhibition experiments on ligands (compounds) or rapamycin were obtained by varying their concentrations (0.18 nM-18 ⁇ M) and incubating with the Fed.
  • Biotinylated FKBP12 was immobilized on donor beads and 6xHis-FRB was immobilized on acceptor beads, and the binding of 1 ⁇ M compound A, compound B, compound C, and rapamycin (RAP: positive control) was measured.
  • RAP positive control
  • Compound C was found to have a binding force comparable to that of rapamycin (RAP).
  • the binding force of compound A and compound B was weak.
  • Cell line Human metastatic breast cancer cells human metastatic breast cancer cells (MCF-7)), human cervical cancer cells (HeLa, # CCL-2), human liver cancer cells (HepG-2), human embryonic Fibroblasts HEK293 and rat renal stromal fibroblasts (NRK-49F) were obtained from RIKEN cell bank (RIKEN, Japan). Cells were cultured in high-glucose Gibco DMEM (Thermo Fisher Scientific, USA) with 10% FBS and 1% Penicillin-Streptomycin (P / S) (Thermo Fisher Scientific, USA).
  • NanoBiT PPI Starter Systems (Promega, USA) was used. First, 104 HEK293, HeLa or MCF- 7 cells were placed in B & W Isoplatate-96 TC (PerkinElmer, USA) and cultured overnight in DMED (10% FBS, 1% P / S) (5% CO). 2 , 37 ° C). The next day, the FKBP12-SmBiT (4360 bp) and FRB-LgBiT (4762 bp) control vectors enclosed in the NanoBiT PPI Starter System were loaded with FuGene HD (Promega, USA) to a concentration of 50 ng / well.
  • Each cell was co-transfected and incubated for 24-48 hours (5% CO 2 , 37 ° C.) using a 1: 1 (v / w) ratio.
  • the medium was then replaced with Opti-MEM rediculated serum medium (Thermo Fisher Scientific, USA), and 20 ⁇ L 20-fold diluted Nano-Glo luciferase assay substrate (Promega, USA) was added to each well.
  • the baseline was measured using the emission measurement mode of the EnSpire multimode plate reader (PerkinElmer, USA).
  • ligand (0.1 pM-10 ⁇ M) + rapamycin (1 ⁇ M) or ligand (0.1 pM-10 ⁇ M) was added to each well, and luminescence by PPI was measured with a plate reader.
  • Cell Viability Assay For the cell viability assay, CellTiter-Blue Cell Viability assay kit (Promega, USA) was used. Cells ( 104 cells / well) were seeded on 96-well plates and cultured overnight (5% CO 2 , 37 ° C.). The cells were cultured under starvation conditions for an additional 18 hours. Then, each concentration of ligand was added to the cells and cultured for 72 hours, then 20 ⁇ L of CellTiter-Blue Cell Viability solution was added to each well and incubated for 1-4 hours (5% CO 2, 37 ° C.). Fluorescence at 560/590 nm was measured with an EnSpire multimode plate reader. Alternatively, cell viability was assayed by directly counting the number of cells.
  • Seed 105 cells / well on a 6 -well plate perform the same operation as above, and finally exfoliate the cells with trypsin / EDTA, add a 5% trypan blue solution, and add TC10 automated cell counter (TC10 automated cell counter). The number of cells was counted by Bio-Rad, USA). Cellular photographs were taken with an Olympus IX71 microscope + Olympus DP74 camera, and image analysis was performed with ImageJ software.
  • mTORC1 Kinase Assay The kinase activity of mTORC1 is as follows: AlphaLISA (R) SureFire Ultra TM HV p-p70 S6K (T389) assay kit (TGR Biosciences, Australia, Australia, and Perkin E Detected by measurement. HEK293 cells or NRK-49F cells ( 104 cells / well) were incubated overnight in 96-well plates using high-glucose DMEM (10% FBS, 1% P / S) (5% CO 2 , 37 ° C). The next day, the cells were starved for 18 hours, with (or without) various concentrations of ligand added and cultured for 1-3 hours.
  • Cells were disrupted with the time-prepared 50 ⁇ L 1x cell lysis buffer included in the kit and stirred with a plate shaker for 10 minutes. 6 ⁇ L cell disruption solution was dispensed into 384-well OptiPlate (PerkinElmer, USA), 3 ⁇ L anti-S6K coated Donor was added under dark conditions, the wells were sealed and then incubated (2-4 hours, room temperature). Alpha signal was measured in Alpha mode of EnSpire.
  • the nitrocellulose membrane was incubated with the following antibody solution (1: 1000) (overnight or 1 hour, 4 ° C.), respectively.
  • Primary antibody rabbit monoclonal antibody against S6 (# 2217), S235 / 236 p-S6 (# 4856), 4E-BP1 (# 9452), T37 / 46 p-4E-BP1 (# 2855), m ), And ⁇ -tubulin (# 2146) (all Cell Signaling Tech., USA).
  • a secondary antibody goat anti-rabbit secondary antibody (# A16104, Novex, Thermo Fisher Scientifics, USA) was incubated for 2 hours at room temperature. Western blotting images were taken with WSE-6100 LuminoGraph I (ATTO, Japan).
  • compound C exerts cell damage to cancer cells (HeLa) more than RAP under both activation conditions and starvation conditions, while it exerts cell damage to non-cancer cells (NRK-49F). It was found that they showed almost no cell damage. On the other hand, RAP also exhibited cell damage to non-cancer cells (NRK-49F). This indicates that compound C exerts more cell-damaging properties specifically for cancer cells than RAP.
  • the phosphorylation inhibitory activity of T389 p-70S6K1 of Compounds C and RAP was 3.5 nM and 0.4 nM, respectively (FIG. 13).
  • compound C is a compound that inhibits the phosphorylation activity of mTORC1 specifically for cancer cells, and as a result, exhibits cell-damaging activity specifically for cancer cells, and is more than rapamycin.
  • mice Female, 7-9 weeks old were used to perform xenograft studies. Mice were randomly grouped (5 / cage) and irradiated with light / dark (12-hour cycle) illumination in an air-conditioned environment. Free access to water and feeding areas.
  • a human breast cancer model stage IV model with metastasis
  • 107 4T1 cells were xenografted into mice under the subcutaneous mammary gland. After the tumor size reached 100 mm 3 (about 5 days later), drug administration was started. 100 ⁇ L / day PBS (peritoneal administration), 25 mg / kg / day Compound C (oral administration), or 25 mg / kg / day rapamycin (oral administration) was administered for 10 days.
  • Tumor size was measured every 2 days for 20 days by the microCT method.
  • Tumor size V (width 2 x length) / 2.
  • the body weight of the mice was measured every two days.
  • the metastatic activity of the 4T1 tumor was measured by slaughtering the mice, immersing the lungs in a tissue-fixing reagent (Bouin fixative) to fix them, and then counting the number of white colonies on the lung surface (FIG. 16). Blood samples were taken from the compound C-administered group, the non-administered group (positive control), and the non-tumor transplanted group (negative control), respectively.
  • ALT Serum alanine transaminase
  • BUN blood urea nitrogen
  • CRE creatinine
  • glucose were quantified by standard methods, respectively.
  • A Compound C showed a higher tumor growth inhibitory effect than RAP.
  • B Compound C showed a tumor lung metastasis promoting activity weaker than RAP.
  • C Administration was discontinued at D11, and tumor recurrence at D20 was investigated. Compound C showed lower tumor relapse than RAP.
  • Life extension effect The life extension effect of the compound of the present invention is described in David E. et al. Harrison et al., "Rapamycin fed late in life extends lifeespan in genetically heterogeneous meeting", nature, Vol 460
  • Compounds D to N shown in FIG. 18 were purchased from Enamine (Enami Ltd, Kyiv, Ukraine), ASINEX Corporation (NC, USA), and ChemDiv (ChemDiv, CA, USA), respectively.
  • NanoBiT PPI Starter Systems (Promega, USA) were used for in vivo ligand selection.
  • 104 HeLa cells / wel were placed in B & W Isolate-96 TC (PerkinElmer, USA) and cultured overnight in DMED (10% FBS, 1% P / S) (5% CO 2 , 37 ° C.). ).
  • DMED 10% FBS, 1% P / S
  • the FKBP12-SmBiT (4360bp) and FRB-LgBiT (4762bp) control vectors enclosed in the NanoBiT PPI Starter System were loaded with FuGene HD (Promega, USA) at a concentration of 50 ng / well.
  • Each cell was co-transfected and incubated for 24-48 hours (5% CO 2 , 37 ° C.) using a v / w) ratio.
  • the medium was replaced with Opti-MEM rediculated serum medium (Thermo Fisher Scientific, USA) (39 ⁇ L / well), and 20 ⁇ L 20-fold diluted Nano-Glo luciferase assay was added to each assay (Promega).
  • the baseline was measured using the emission measurement mode of the EnSpire multimode plate reader (PerkinElmer, USA).
  • Ligand (1 ⁇ M or 10 ⁇ M) was then added to each well, 10 ⁇ L of 20xFurimazine / well was added, and luminescence by PPI was measured with a plate reader.
  • mTORC1 Kinase Assay The kinase activity of mTORC1 is as follows: AlphaLISA (R) SureFire Ultra TM HV p-p70 S6K (T389) assay kit (TGR Biosciences, Australia, Australia, and Perkin E Detected by measurement. HEK293 cells ( 104 cells / well) were incubated overnight in 96-well plates using high-glucose DMEM (10% FBS, 1% P / S) (5% CO 2 , 37 ° C). The next day, the cells were starved overnight (99 ⁇ l OptiMEM media / well), then 1 ⁇ M Ligan was added and cultured for 1-2 hours.
  • the derivative of the present invention inhibits the phosphorylation activity of mTORC1 specifically for cancer cells without causing toxicity to normal cells, and as a result, cell damage is specific for cancer cells. It was found that it is a sexually active compound and further inhibits T37 / 46 p-4E-BP1 more efficiently than rapamycin. These indicate that this compound has a low possibility of causing side effects and has high potential performance as an anticancer agent, a carcinogenic inhibitor and / or a life-prolonging agent.

Abstract

A carcinostatic agent, a carcinogenesis inhibitor and/or a life-prolonging agent each comprising a compound with mTORC1 inhibitory effect, said compound having partial structures (I), (II) and (III), or a pharmacologically acceptable salt thereof. (In the formulae: R1, R2, R3 and R4 are the same or different and represent an H, a lower alkyl, a lower haloalkyl, a lower alkoxy, a lower alkylamino, an OH, a protected OH, an amino, a protected amino, a carboxy, a protected carboxy or a halogen atom; R5 and R6 are the same or different and represent an H or a protecting group of an OH, or R5 and R6 form together a methylene or dimethylmethylene group; and A represents O or S.)

Description

新規なｍＴＯＲＣ１阻害作用を有する化合物を含む、制癌剤、発がん抑制剤および寿命延長剤Anti-cancer agents, carcinogenic inhibitors and life-prolonging agents, including new compounds with mTORC1 inhibitory activity
　本発明は、ｍＴＯＲＣ１阻害作用を有する化合物を含む、制癌剤、発がん抑制剤および／または寿命延長剤として有用である。 The present invention is useful as an anticancer agent, a carcinogenic inhibitor and / or a life-prolonging agent, which contains a compound having an mTORC1 inhibitory effect.
　ｍＴＯＲ（ｍａｍｍａｌｉａｎ　ｔａｒｇｅｔ　ｏｆ　ｒａｐａｍｙｃｉｎ）はラパマイシンの標的分子として同定されたセリン・スレオニンキナーゼで、細胞の***や生存などの調節に中心的な役割を果たすことから、ｍＴＯＲ（ｔａｒｇｅｔ　ｏｆ　ｒａｐａｍｙｃｉｎ）と命名された。
　細胞が増殖因子などで刺激を受けるとＰＩ３キナーゼ（Ｐｈｏｓｐｈｏｉｎｏｓｉｔｉｄｅ　３－ｋｉｎａｓｅ：ＰＩ３Ｋ）というリン酸化酵素が活性化され、これがＡｋｔというセリン・スレオニンリン酸化酵素をリン酸化して活性化し、細胞内のシグナル伝達に関与する様々な蛋白質の活性を調節することによって細胞の増殖や生存（死）の調節を行うが、このＡｋｔのターゲットの一つがｍＴＯＲであり、Ａｋｔによってリン酸化（活性化）されたｍＴＯＲは、細胞***や細胞死や血管新生やエネルギー産生などに作用してがん細胞の増殖を促進（ＰＩ３Ｋ／Ａｋｔ／ｍＴＯＲ経路）し、がん細胞や肉腫細胞の増殖を促進するメカニズムとして極めて重要である。
すなわち、ＰＩ３Ｋ／Ａｋｔ／ｍＴＯＲ経路の阻害はがん細胞や肉腫細胞の増殖を抑制し、細胞死（アポトーシス）を誘導でき、がん治療、発がん抑制のターゲットとして注目され、また、抗がん剤や放射線治療の効き目を高める効果も報告されている。 mTOR (mammalian target of rapamycin) is a serine / threonine kinase identified as a target molecule of rapamycin, and was named mTOR (taget of rapamycin) because it plays a central role in the regulation of cell division and survival. ..
When a cell is stimulated by a growth factor or the like, a phosphorylating enzyme called PI3 kinase (Phosphoinositide 3-kinase: PI3K) is activated, which phosphorylates and activates a serine / threonine phosphorylating enzyme called Akt, resulting in an intracellular signal. It regulates cell proliferation and survival (death) by regulating the activity of various proteins involved in transmission. One of the targets of this Akt is mTOR, which is a kinase phosphorylated (activated) by Akt. Is extremely important as a mechanism that promotes the growth of cancer cells (PI3K / Akt / mTOR pathway) by acting on cell division, cell death, angiogenesis, energy production, etc., and promotes the growth of cancer cells and sarcoma cells. Is.
That is, inhibition of the PI3K / Akt / mTOR pathway suppresses the growth of cancer cells and sarcoma cells, induces cell death (apoptosis), and is attracting attention as a target for cancer treatment and carcinogenesis suppression. And the effect of enhancing the efficacy of radiation therapy has also been reported.
　また、栄養素（グルコースやアミノ酸など）、インスリン、インスリン様成長因子、その他の増殖因子はｍＴＯＲＣ１シグナル伝達系を活性化し、細胞の成長や細胞周期（細胞***した細胞が再び***を起こすまでの過程）の進行を促進する。遺伝子変異などによって細胞周期の制御に異常が起こり、止めどなく細胞***を行うようになった細胞ががん細胞であり、細胞周期が進行しているがん細胞に対してはｍＴＯＲＣ１の活性化は細胞増殖を促進することになる。 In addition, nutrients (such as glucose and amino acids), insulin, insulin-like growth factors, and other growth factors activate the mTORC1 signaling system, resulting in cell growth and cell cycle (the process until cell division occurs again). Promote the progress of. Cancer cells are cells that have abnormal cell cycle control due to gene mutations and have continued cell division, and mTORC1 activation is not possible for cancer cells that have an advanced cell cycle. It will promote cell proliferation.
　一方、細胞が古くなり、細胞周期が停止している細胞（Ｇ０期）にｍＴＯＲＣ１活性が亢進すると細胞は老化した状態（完全に細胞増殖能を喪失し細胞機能も低下する）に移行する。 On the other hand, when mTORC1 activity is enhanced in cells whose cell cycle is stopped (G0 phase) as the cells become old, the cells shift to an aged state (the cell proliferation ability is completely lost and the cell function is also reduced).
　したがって、ラパマイシンのようなｍＴＯＲＣ１の活性阻害剤は、細胞のがん化（制癌及び発がん）と老化の両方を抑制することができる。
　なお、ｍＴＯＲ阻害剤は免疫抑制という欠点を持つが、がん細胞や肉腫細胞の多くにおいてｍＴＯＲが活性化されているため、抗がん剤として有効性が高く、すでに幾つかのｍＴＯＲ阻害剤が開発され、抗がん剤として使用されている。
　ラパマイシンは、元々、臓器移植の拒絶反応を予防する免疫抑制剤であり、通常、免疫抑制剤はがんの発生率を高めるが、ラパマイシンは逆に、腎臓移植や肝臓移植を受けた患者さんを追跡調査するとラパマイシンを服用している患者のがんの発生率は極めて低いことが多くの研究で明らかになっている。 Therefore, mTORC1 activity inhibitors such as rapamycin can suppress both cell carcinogenesis (anticancer and carcinogenesis) and aging.
Although mTOR inhibitors have the drawback of immunosuppression, they are highly effective as anticancer agents because mTOR is activated in many cancer cells and sarcoma cells, and some mTOR inhibitors have already been introduced. It has been developed and used as an anticancer agent.
Rapamycin was originally an immunosuppressive drug that prevented organ transplant rejection, and immunosuppressive drugs usually increase the incidence of cancer, whereas rapamycin, on the contrary, treats patients who have undergone kidney or liver transplants. Follow-up studies have shown that patients taking rapamycin have a very low incidence of cancer.
　本願発明の化合物の一部は公知の化合物である。しかし、これら化合物について、具体的な生物活性は知られていない。
　他方、上記のように、ラパマイシンを基にした、ｍＴＯＲ阻害剤の開発は、鋭意行われているが、本願発明の化合物に類似する骨格を有するものはなく、より低分子で、かつ、癌細胞に特異的に毒性を示す化合物は知られていない。 Some of the compounds of the present invention are known compounds. However, the specific biological activity of these compounds is unknown.
On the other hand, as described above, the development of mTOR inhibitors based on rapamycin has been diligently carried out, but none of them have a skeleton similar to the compound of the present invention, and are smaller molecules and cancer cells. There are no known compounds that are specifically toxic to.
　また、老化は様々な疾患の発症と進展の原因になるが、それらはｍＴＯＲＣ１シグナル伝達系の影響を受けており、老化が関連する様々な疾患のうち、認知機能低下、アルツハイマー病、悪性腫瘍、腎臓疾患、心臓疾患、自己免疫疾患など多くの疾患に対して、本発明のｍＴＯＲＣ１阻害剤は改善する方向で作用する（非特許文献１および２）。 In addition, aging causes the onset and progression of various diseases, which are affected by the mTORC1 signaling system, and among various aging-related diseases, cognitive decline, Alzheimer's disease, malignant tumors, etc. The mTORC1 inhibitor of the present invention acts in an improving direction for many diseases such as kidney disease, heart disease, and autoimmune disease (Non-Patent Documents 1 and 2).
　本発明の課題は、ｍＴＯＲＣ１阻害作用を有する化合物を含む、制癌剤、発がん抑制剤および寿命延長剤を提供することである。 An object of the present invention is to provide an anticancer agent, a carcinogenic suppressant and a life-prolonging agent containing a compound having an mTORC1 inhibitory action.
　本発明によれば、以下の発明が提供される。
＜１＞　下記の部分構造（Ｉ）、（ＩＩ）、および（ＩＩＩ）を含む、ｍＴＯＲＣ１阻害作用を有する化合物またはその薬理上許容される塩を含む、制癌剤および／または発がん抑制剤。
Figure JPOXMLDOC01-appb-C000013
 （式中、
　Ｒ１、Ｒ２、Ｒ３およびＲ４は、同一又は異なって、Ｈ、低級アルキル、ハロ低級アルキル、低級アルコキシ、低級アルキルアミノ、ＯＨ、保護されたＯＨ、アミノ、保護されたアミノ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示し、
　Ｒ５およびＲ６は、同一又は異なって、ＨまたはＯＨの保護基を示すか、或いは、Ｒ５およびＲ６が一緒になって、メチレンまたはジメチルメチレン基を示し、
　Ａは、ＯまたはＳを示す。）。 According to the present invention, the following inventions are provided.
<1> An anticancer agent and / or a carcinogenic inhibitor comprising the following partial structures (I), (II), and (III), a compound having an mTORC1 inhibitory action or a pharmaceutically acceptable salt thereof.
Figure JPOXMLDOC01-appb-C000013
 (During the ceremony,
R 1 , R 2 , R 3 and R 4 are the same or different, H, lower alkyl, halo lower alkyl, lower alkoxy, lower alkyl amino, OH, protected OH, amino, protected amino, carboxy, Represents a protected carboxy or halogen atom,
R 5 and R 6 indicate the same or different protective groups for H or OH, or R 5 and R 6 together indicate a methylene or dimethyl methylene group.
A indicates O or S. ).
＜２＞　　式（ＩＶ）の化合物またはその薬理上許容される塩
Figure JPOXMLDOC01-appb-C000014
 （式中、
　Ｌ、ＸおよびＹは、同一又は異なって、リンカーを示し、
　Ｒ１、Ｒ２、Ｒ３およびＲ４は、同一又は異なって、Ｈ、低級アルキル、ハロ低級アルキル、低級アルコキシ、低級アルキルアミノ、ＯＨ、保護されたＯＨ、アミノ、保護されたアミノ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示し、
　Ｒ５およびＲ６は、同一又は異なって、ＨまたはＯＨの保護基を示すか、或いは、Ｒ５およびＲ６が一緒になって、メチレンまたはジメチルメチレン基を示し、
　Ａは、ＯまたはＳを示す。）を含む、＜１＞に記載された制癌剤および／または発がん抑制剤。 <2> The compound of formula (IV) or a pharmacologically acceptable salt thereof.
Figure JPOXMLDOC01-appb-C000014
 (During the ceremony,
L, X and Y indicate the same or different linkers,
R 1 , R 2 , R 3 and R 4 are the same or different, H, lower alkyl, halo lower alkyl, lower alkoxy, lower alkyl amino, OH, protected OH, amino, protected amino, carboxy, Represents a protected carboxy or halogen atom,
R 5 and R 6 indicate the same or different protective groups for H or OH, or R 5 and R 6 together indicate a methylene or dimethyl methylene group.
A indicates O or S. ), The anticancer agent and / or the carcinogenic suppressant according to <1>.
＜３＞　Ｌ、ＸおよびＹが、同一又は異なって、－（ＣＨ２）１～６－、酸素原子で介在されていてもよい－（ＣＨ２）１～６－、ＮＨで介在されていてもよい－（ＣＨ２）１～６－、または下記置換基で置換された－（ＣＨ２）１～６－のリンカーを示す、＜１＞～＜2＞のうちの１項に記載された制癌剤および／または発がん抑制剤。
　置換基：低級アルキル、ハロ低級アルキル、低級アルコキシ、ハロゲン、アミノおよびＯＨ基。
＜４＞　Ｌ、ＸおよびＹが、同一又は異なって、－（ＣＨ２）１～６－を示す、＜１＞～＜3＞のうちの１項に記載された制癌剤および／または発がん抑制剤。
＜５＞　Ｒ１、Ｒ２、Ｒ３およびＲ４は、同一又は異なって、Ｈ、低級アルキル、ハロ低級アルキル、低級アルコキシ、低級アルキルアミノ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示す、＜１＞～＜4＞のうちの１項に記載された制癌剤および／または発がん抑制剤。
＜６＞　Ｒ１およびＲ２が、水素原子である、＜１＞～＜5＞のうちの１項に記載された制癌剤および／または発がん抑制剤。
＜７＞　Ｒ３が、Ｈで、Ｒ４が、低級アルキル、ハロ低級アルキル、低級アルコキシ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示すか、あるいは、Ｒ３およびＲ４が、同一又は異なって、低級アルキル、ハロ低級アルキル、低級アルコキシ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示す、＜１＞～＜6＞のうちの１項に記載された制癌剤および／または発がん抑制剤。
＜８＞　Ｒ３が、Ｈで、Ｒ４が、低級アルキル、ハロ低級アルキル、低級アルコキシ、保護されたカルボキシまたはハロゲン原子を示すか、あるいは、Ｒ３およびＲ４が、同一又は異なって、低級アルキル、ハロ低級アルキル、低級アルコキシまたはハロゲン原子を示す、＜１＞～＜7＞のうちの１項に記載された制癌剤および／または発がん抑制剤。
＜９＞　Ｒ３が、Ｈで、Ｒ４が、低級アルキル、ハロ低級アルキル、またはハロゲン原子を示すか、あるいは、Ｒ３およびＲ４が、同一又は異なって、低級アルキル、ハロ低級アルキル、またはハロゲン原子を示す、＜１＞～＜8＞のうちの１項に記載された制癌剤および／または発がん抑制剤。
＜１０＞　Ｒ３または／およびＲ４が、オルト位に置換している、＜１＞～＜9＞のうちの１項に記載された制癌剤および／または発がん抑制剤。
＜１１＞　Ｒ５およびＲ６が、一緒になって、メチレンまたはジメチルメチレン基を示す、＜１＞～＜１0＞のいずれか１項に記載された制癌剤および／または発がん抑制剤。
＜１２＞　Ａが、Ｏを示す、＜１＞～＜１１＞のいずれか１項に記載された制癌剤および／または発がん抑制剤。 <3> L, X and Y are the same or different, and may be intervened by-(CH 2 ) 1 to 6- , oxygen atom- (CH 2 ) 1 to 6- , NH. It may be described in one of <1> to <2>, which indicates a linker of-(CH 2 ) 1 to 6 -or substituted with the following substituent-(CH 2 ) 1 to 6- . Anticancer agents and / or carcinogens.
Substituents: lower alkyl, halo lower alkyl, lower alkoxy, halogen, amino and OH groups.
<4> The anticancer agent and / or carcinogenic inhibitor according to item 1 of <1> to <3>, wherein L, X and Y are the same or different and indicate-(CH 2 ) 1 to 6- . ..
<5> R 1 , R 2 , R 3 and R 4 represent H, lower alkyl, halo lower alkyl, lower alkoxy, lower alkyl amino, carboxy, protected carboxy or halogen atom, identical or different, < The anticancer agent and / or the carcinogenic suppressant according to item 1 of 1> to <4>.
<6> The anticancer agent and / or carcinogenic inhibitor according to item 1 of <1> to <5>, wherein R1 and R2 are hydrogen atoms.
<7> R 3 is H and R 4 is lower alkyl, halo lower alkyl, lower alkoxy, carboxy, protected carboxy or halogen atom, or R 3 and R 4 are the same or different. , Lower alkyl, halo lower alkyl, lower alkoxy, carboxy, protected carboxy or halogen atom, the anticancer agent and / or carcinogenic inhibitor according to item 1 of <1> to <6>.
<8> R 3 is H and R 4 is lower alkyl, halo lower alkyl, lower alkoxy, protected carboxy or halogen atom, or R 3 and R 4 are the same or different and lower. The anticancer agent and / or carcinogenic inhibitor according to item 1 of <1> to <7>, which represents an alkyl, a halo lower alkyl, a lower alkoxy or a halogen atom.
<9> R 3 is H and R 4 represents a lower alkyl, halo lower alkyl, or halogen atom, or R 3 and R 4 are the same or different, lower alkyl, halo lower alkyl, or. The anticancer agent and / or carcinogenic suppressant according to item 1 of <1> to <8>, which indicates a halogen atom.
<10> The anticancer agent and / or carcinogenic suppressant according to item 1 of <1> to <9>, wherein R3 or / and R4 are substituted at the ortho position.
<11> The anticancer agent and / or carcinogenic inhibitor according to any one of < 1 > to < 10 >, wherein R5 and R6 together represent a methylene or dimethylmethylene group.
<12> The anticancer agent and / or carcinogenic suppressant according to any one of <1> to <11>, wherein A indicates O.
＜１３＞　化合物が以下の化合物から選択される１の化合物である（但し、Ａは、ＯまたはＳである。）、＜１＞に記載された制癌剤および／または発がん抑制剤。
Figure JPOXMLDOC01-appb-C000015
Figure JPOXMLDOC01-appb-I000016
<13> The anticancer agent and / or carcinogenic inhibitor according to <1>, wherein the compound is one compound selected from the following compounds (where A is O or S).
Figure JPOXMLDOC01-appb-C000015
Figure JPOXMLDOC01-appb-I000016
＜１４＞　化合物が以下の化合物から選択される１の化合物である、＜１＞に記載された制癌剤および／または発がん抑制剤。
Figure JPOXMLDOC01-appb-C000017
 <14> The anticancer agent and / or carcinogenic inhibitor according to <1>, wherein the compound is one compound selected from the following compounds.
Figure JPOXMLDOC01-appb-C000017
＜１５＞　化合物が以下の化合物から選択される１の化合物である、＜１＞に記載された制癌剤および／または発がん抑制剤。
Figure JPOXMLDOC01-appb-C000018
 <15> The anticancer agent and / or carcinogenic inhibitor according to <1>, wherein the compound is one compound selected from the following compounds.
Figure JPOXMLDOC01-appb-C000018
＜１６＞
　下記の部分構造（Ｉ）、（ＩＩ）、および（ＩＩＩ）を含む、ｍＴＯＲＣ１阻害作用を有する化合物またはその薬理上許容される塩を含む、寿命延長剤
Figure JPOXMLDOC01-appb-C000019
 （式中、
　Ｒ１、Ｒ２、Ｒ３およびＲ４は、同一又は異なって、Ｈ、低級アルキル、ハロ低級アルキル、低級アルコキシ、低級アルキルアミノ、ＯＨ、保護されたＯＨ、アミノ、保護されたアミノ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示し、
　Ｒ５およびＲ６は、同一又は異なって、ＨまたはＯＨの保護基を示すか、或いは、Ｒ５およびＲ６が一緒になって、メチレンまたはジメチルメチレン基を示し、
　Ａは、ＯまたはＳを示す。）。 <16>
A life-prolonging agent containing a compound having an mTORC1 inhibitory effect or a pharmaceutically acceptable salt thereof, which comprises the following partial structures (I), (II), and (III).
Figure JPOXMLDOC01-appb-C000019
 (During the ceremony,
R 1 , R 2 , R 3 and R 4 are the same or different, H, lower alkyl, halo lower alkyl, lower alkoxy, lower alkyl amino, OH, protected OH, amino, protected amino, carboxy, Represents a protected carboxy or halogen atom,
R 5 and R 6 indicate the same or different protective groups for H or OH, or R 5 and R 6 together indicate a methylene or dimethyl methylene group.
A indicates O or S. ).
＜１７＞
　式（ＩＶ）の化合物またはその薬理上許容される塩
Figure JPOXMLDOC01-appb-C000020
 （式中、
　Ｌ、ＸおよびＹは、同一又は異なって、リンカーを示し、
　Ｒ１、Ｒ２、Ｒ３およびＲ４は、同一又は異なって、Ｈ、低級アルキル、ハロ低級アルキル、低級アルコキシ、低級アルキルアミノ、ＯＨ、保護されたＯＨ、アミノ、保護されたアミノ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示し、
　Ｒ５およびＲ６は、同一又は異なって、ＨまたはＯＨの保護基を示すか、或いは、Ｒ５およびＲ６が一緒になって、メチレンまたはジメチルメチレン基を示し、
　Ａは、ＯまたはＳを示す。）を含む、＜１６＞に記載された寿命延長剤。 <17>
Compound of formula (IV) or pharmacologically acceptable salt thereof
Figure JPOXMLDOC01-appb-C000020
 (During the ceremony,
L, X and Y indicate the same or different linkers,
R 1 , R 2 , R 3 and R 4 are the same or different, H, lower alkyl, halo lower alkyl, lower alkoxy, lower alkyl amino, OH, protected OH, amino, protected amino, carboxy, Represents a protected carboxy or halogen atom,
R 5 and R 6 indicate the same or different protective groups for H or OH, or R 5 and R 6 together indicate a methylene or dimethyl methylene group.
A indicates O or S. ), The life extension agent according to <16>.
＜１８＞　Ｌ、ＸおよびＹが、同一又は異なって、－（ＣＨ２）１～６－を示す、＜１６＞～＜１７＞のうちの１項に記載された寿命延長剤。
＜１９＞　Ｒ１、Ｒ２、Ｒ３およびＲ４は、同一又は異なって、Ｈ、低級アルキル、ハロ低級アルキル、低級アルコキシ、低級アルキルアミノ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示す、＜１６＞～＜１８＞のうちの１項に記載された寿命延長剤。
＜２０＞　Ｒ３が、Ｈで、Ｒ４が、低級アルキル、ハロ低級アルキル、低級アルコキシ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示すか、あるいは、Ｒ３およびＲ４が、同一又は異なって、低級アルキル、ハロ低級アルキル、低級アルコキシまたはハロゲン原子を示す、＜１６＞～＜１９＞のうちの１項に記載された寿命延長剤。
＜２１＞　Ｒ５およびＲ６が、一緒になって、メチレンまたはジメチルメチレン基を示す、＜１６＞～＜２０＞のいずれか１項に記載された寿命延長剤。
＜２２＞　化合物が以下の化合物から選択される１の化合物である（但し、Ａは、ＯまたはＳである。）、＜１６＞に記載された寿命延長剤。
Figure JPOXMLDOC01-appb-C000021
Figure JPOXMLDOC01-appb-I000022
<18> The life extending agent according to item 1 of <16> to <17>, wherein L, X and Y are the same or different and indicate − (CH 2 ) 1 to 6 −.
<19> R 1 , R 2 , R 3 and R 4 represent H, lower alkyl, halo lower alkyl, lower alkoxy, lower alkyl amino, carboxy, protected carboxy or halogen atom, identical or different, < The life extending agent according to item 1 of 16> to <18>.
<20> R 3 is H, R 4 is lower alkyl, halo lower alkyl, lower alkoxy, carboxy, protected carboxy or halogen atom, or R 3 and R 4 are the same or different. , The life extender according to item 1 of <16> to <19>, which represents a lower alkyl, a halo lower alkyl, a lower alkoxy or a halogen atom.
<21> The life extender according to any one of < 16 > to <20>, wherein R5 and R6 together represent a methylene or dimethylmethylene group.
<22> The life extender according to <16>, wherein the compound is one compound selected from the following compounds (where A is O or S).
Figure JPOXMLDOC01-appb-C000021
Figure JPOXMLDOC01-appb-I000022
＜２３＞　化合物が以下の化合物から選択される１の化合物である、＜１６＞に記載された寿命延長剤。
Figure JPOXMLDOC01-appb-C000023
 <23> The life extender according to <16>, wherein the compound is one compound selected from the following compounds.
Figure JPOXMLDOC01-appb-C000023
＜２４＞　化合物が以下の化合物から選択される１の化合物である、＜１６＞に記載された寿命延長剤。
Figure JPOXMLDOC01-appb-C000024
 <24> The life extender according to <16>, wherein the compound is one compound selected from the following compounds.
Figure JPOXMLDOC01-appb-C000024
　好適には、本発明に包含される化合物の分子量は、４５０～８５０、より好ましくは、５００～８００である。 Preferably, the molecular weight of the compound included in the present invention is 450 to 850, more preferably 500 to 800.
＜Ａ＞　上記の部分構造（Ｉ）、（ＩＩ）、および（ＩＩＩ）を含む、ｍＴＯＲＣ１阻害作用を有する化合物またはその薬理上許容される塩を、対象（好ましくはヒトを含む哺乳動物）に投与することを含む、癌および/または発がんを抑制する方法。
＜Ｂ＞　上記の部分構造（Ｉ）、（ＩＩ）、および（ＩＩＩ）を含む、ｍＴＯＲＣ１阻害作用を有する化合物またはその薬理上許容される塩を、対象（好ましくはヒトを含む哺乳動物）に投与することを含む、寿命を延長する方法。
＜Ｃ＞　癌および/または発がんを抑制する処置において使用するための、上記の部分構造（Ｉ）、（ＩＩ）、および（ＩＩＩ）を含む、ｍＴＯＲＣ１阻害作用を有する化合物またはその薬理上許容される塩。
＜Ｄ＞　寿命を延長する処置において使用するための、上記の部分構造（Ｉ）、（ＩＩ）、および（ＩＩＩ）を含む、ｍＴＯＲＣ１阻害作用を有する化合物またはその薬理上許容される塩。
＜Ｅ＞　制癌剤および／または発がん抑制剤の製造のための、記の部分構造（Ｉ）、（ＩＩ）、および（ＩＩＩ）を含む、ｍＴＯＲＣ１阻害作用を有する化合物またはその薬理上許容される塩の使用。
＜Ｆ＞　寿命延長剤の製造のための、記の部分構造（Ｉ）、（ＩＩ）、および（ＩＩＩ）を含む、ｍＴＯＲＣ１阻害作用を有する化合物またはその薬理上許容される塩の使用。 <A> A compound having an mTORC1 inhibitory effect or a pharmaceutically acceptable salt thereof, which comprises the above partial structures (I), (II), and (III), is administered to a subject (preferably a mammal including a human). Methods of controlling cancer and / or carcinogenesis, including:
<B> A compound having an mTORC1 inhibitory effect or a pharmaceutically acceptable salt thereof, which comprises the above partial structures (I), (II), and (III), is administered to a subject (preferably a mammal including a human). How to extend life, including doing.
<C> A compound having an mTORC1 inhibitory effect or pharmacologically acceptable thereof, which comprises the above-mentioned partial structures (I), (II), and (III) for use in a treatment for suppressing cancer and / or carcinogenesis. salt.
<D> A compound having an mTORC1 inhibitory effect or a pharmacologically acceptable salt thereof, which comprises the above-mentioned partial structures (I), (II), and (III) for use in a life-prolonging treatment.
<E> A compound having an mTORC1 inhibitory effect or a pharmacologically acceptable salt thereof, which comprises the partial structures (I), (II), and (III) described above for the production of an anticancer agent and / or a carcinogenic inhibitor. use.
<F> Use of a compound having an mTORC1 inhibitory effect or a pharmaceutically acceptable salt thereof, which comprises the partial structures (I), (II), and (III) described above, for producing a life-prolonging agent.
　本発明において、
　「低級アルキル」とは、例えば、メチル、エチル、ｎ－プロピル、イソプロピル、ｎ－ブチル、イソブチル、ｓ－ブチル、ｔｅｒｔ－ブチル、ｎ－ペンチル、イソペンチル、２－メチルブチル、ネオペンチル、１－エチルプロピル、ｎ－ヘキシル、イソヘキシル、４－メチルペンチル、３－メチルペンチル、２－メチルペンチル、１－メチルペンチル、３，３－ジメチルブチル、２，２－ジメチルブチル、１，１－ジメチルブチル、１，２－ジメチルブチル、１，３－ジメチルブチル、２，３－ジメチルブチル、２－エチルブチルのような炭素数１乃至６個の直鎖又は分枝鎖アルキル基を示し、好適には炭素数１乃至４個の直鎖又は分枝鎖アルキル基である。 In the present invention
The "lower alkyl" is, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, etc. n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2 -Represents a linear or branched alkyl group having 1 to 6 carbon atoms such as dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl and 2-ethylbutyl, preferably 1 to 4 carbon atoms. It is a linear or branched alkyl group.
　「ハロ低級アルキル」とは、後記「ハロゲン原子」が前記「低級アルキル」に結合した基をいい、例えば、トリフルオロメチル、トリクロロメチル、ジフルオロメチル、ジクロロメチル、ジブロモメチル、フルオロメチル、２，２，２－トリクロロエチル、２，２，２－トリフルオロエチル、２－ブロモエチル、２－クロロエチル、２－フルオロエチル、２，２－ジブロモエチルのような基を挙げることができ、好適には、トリフルオロメチルである。 The "halo lower alkyl" refers to a group in which the "halogen atom" described later is bonded to the "lower alkyl", for example, trifluoromethyl, trichloromethyl, difluoromethyl, dichloromethyl, dibromomethyl, fluoromethyl, 2,2. , 2-Trichloroethyl, 2,2,2-trifluoroethyl, 2-bromoethyl, 2-chloroethyl, 2-fluoroethyl, 2,2-dibromoethyl and the like can be mentioned, preferably tri. It is fluoromethyl.
　「低級アルコキシ」とは、前記「低級アルキル」が酸素原子に結合した基をいい、例えば、メトキシ、エトキシ、ｎ－プロポキシ、イソプロポキシ、ｎ－ブトキシ、イソブトキシ、ｓ－ブトキシ、ｔｅｒｔ－ブトキシ、ｎ－ペントキシ、イソペントキシ、２－メチルブトキシ、ネオペントキシ、ｎ－ヘキシルオキシ、４－メチルペントキシ、３－メチルペントキシ、２－メチルペントキシ、３，３－ジメチルブトキシ、２，２－ジメチルブトキシ、１，１－ジメチルブトキシ、１，２－ジメチルブトキシ、１，３－ジメチルブトキシ、２，３－ジメチルブトキシのような炭素数１乃至６個の直鎖又は分枝鎖アルコキシ基を示し、好適には炭素数１乃至４個の直鎖又は分枝鎖アルコキシ基である。 The "lower alkoxy" refers to a group in which the "lower alkyl" is bonded to an oxygen atom, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, s-butoxy, tert-butoxy, n. -Pentoxy, Isopentoxy, 2-Methylbutoxy, Neopentoxy, n-Hexyloxy, 4-Methylpentoxy, 3-Methylpentoxy, 2-Methylpentoxy, 3,3-dimethylbutoxy, 2,2-dimethylbutoxy, 1 , 1-dimethylbutoxy, 1,2-dimethylbutoxy, 1,3-dimethylbutoxy, 2,3-dimethylbutoxy, etc. show linear or branched alkoxy groups having 1 to 6 carbon atoms, preferably It is a linear or branched alkoxy group having 1 to 4 carbon atoms.
　「低級アルキルアミノ」とは、前記「低級アルキル」がアミノに結合した基をいい、例えば、メチルアミノ、エチルアミノ、ｎ－プロピルアミノ、イソピルアミノ、ｎ－ブチルアミノ、イソブチルアミノ、ｓ－ブチルアミノ、ｔｅｒｔ－ブチルアミノ、ｎ－ペンチルアミノ、イソペンチルアミノ、２－メチルブチルアミノ、ネオペンチルアミノ、ｎ－ヘキシルアミノ、３，３－ジメチルブチルアミノ、２，２－ジメチルブチルアミノ、１，１－ジメチルブチルアミノ、１，２－ジメチルブチルアミノ、１，３－ジメチルブチルアミノ、２，３－ジメチルブチルアミノのような炭素数１乃至６個の直鎖又は分枝鎖アミノ基を示し、好適には炭素数１乃至４個の直鎖又は分枝鎖アミノ基である。 The "lower alkylamino" refers to a group to which the "lower alkyl" is bonded to an amino, for example, methylamino, ethylamino, n-propylamino, isopylamino, n-butylamino, isobutylamino, s-butylamino, and the like. tert-Butylamino, n-pentylamino, isopentylamino, 2-methylbutylamino, neopentylamino, n-hexylamino, 3,3-dimethylbutylamino, 2,2-dimethylbutylamino, 1,1-dimethyl It exhibits a linear or branched amino group having 1 to 6 carbon atoms such as butylamino, 1,2-dimethylbutylamino, 1,3-dimethylbutylamino and 2,3-dimethylbutylamino, preferably It is a linear or branched amino group having 1 to 4 carbon atoms.
　「保護されたＯＨ」の「保護基」および／または「ＯＨの保護基」とは、加水素分解、加水分解、電気分解、光分解のような化学的方法により開裂し得る「反応における保護基」を示す。
　斯かる「反応における保護基」としては、例えば、ホルミル、アセチル、プロピオニル、ブチリル、イソブチリル、ペンタノイル、ピバロイル、バレリル、イソバレリル、オクタノイル、ノナノイル、デカノイル、３－メチルノナノイル、８－メチルノナノイル、３－エチルオクタノイル、３，７－ジメチルオクタノイル、ウンデカノイル、ドデカノイル、トリデカノイル、テトラデカノイル、ペンタデカノイル、ヘキサデカノイル、１－メチルペンタデカノイル、１４－メチルペンタデカノイル、１３，１３－ジメチルテトラデカノイル、ヘプタデカノイル、１５－メチルヘキサデカノイル、オクタデカノイル、１－メチルヘプタデカノイル、ノナデカノイル、アイコサノイル及びヘナイコサノイルのようなアルキルカルボニル基、スクシノイル、グルタロイル、アジポイルのようなカルボキシ化アルキルカルボニル基、クロロアセチル、ジクロロアセチル、トリクロロアセチル、トリフルオロアセチルのようなハロゲノ低級アルキルカルボニル基、メトキシアセチルのような低級アルコキシ低級アルキルカルボニル基、（Ｅ）－２－メチル－２－ブテノイルのような不飽和アルキルカルボニル基等の「脂肪族アシル基」；ベンゾイル、α－ナフトイル、β－ナフトイルのようなアリ－ルカルボニル基、２－ブロモベンゾイル、４－クロロベンゾイルのようなハロゲノアリ－ルカルボニル基、２，４，６－トリメチルベンゾイル、４－トルオイルのような低級アルキル化アリ－ルカルボニル基、４－アニソイルのような低級アルコキシ化アリ－ルカルボニル基、２－カルボキシベンゾイル、３－カルボキシベンゾイル、４－カルボキシベンゾイルのようなカルボキシ化アリ－ルカルボニル基、４－ニトロベンゾイル、２－ニトロベンゾイルのようなニトロ化アリ－ルカルボニル基、２－（メトキシカルボニル）ベンゾイルのような低級アルコキシカルボニル化アリ－ルカルボニル基、４－フェニルベンゾイルのようなアリ－ル化アリ－ルカルボニル基等の「芳香族アシル基」；テトラヒドロピラン－２－イル、３－ブロモテトラヒドロピラン－２－イル、４－メトキシテトラヒドロピラン－４－イル、テトラヒドロチオピラン－２－イル、４－メトキシテトラヒドロチオピラン－４－イルのような「テトラヒドロピラニル又はテトラヒドロチオピラニル基」；テトラヒドロフラン－２－イル、テトラヒドロチオフラン－２－イルのような「テトラヒドロフラニル又はテトラヒドロチオフラニル基」；トリメチルシリル、トリエチルシリル、イソプロピルジメチルシリル、ｔ－ブチルジメチルシリル、メチルジイソプロピルシリル、メチルジ－ｔ－ブチルシリル、トリイソプロピルシリルのようなトリ低級アルキルシリル基、ジフェニルメチルシリル、ジフェニルブチルシリル、ジフェニルイソプロピルシリル、フェニルジイソプロピルシリルのような１乃至２個のアリ－ル基で置換されたトリ低級アルキルシリル基等の「シリル基」；メトキシメチル、１，１－ジメチル－１－メトキシメチル、エトキシメチル、プロポキシメチル、イソプロポキシメチル、ブトキシメチル、ｔ－ブトキシメチルのような低級アルコキシメチル基、２－メトキシエトキシメチルのような低級アルコキシ化低級アルコキシメチル基、２，２，２－トリクロロエトキシメチル、ビス（２－クロロエトキシ）メチルのようなハロゲノ低級アルコキシメチル等の「アルコキシメチル基」；１－エトキシエチル、１－（イソプロポキシ）エチルのような低級アルコキシ化エチル基、２，２，２－トリクロロエチルのようなハロゲン化エチル基等の「置換エチル基」；ベンジル、α－ナフチルメチル、β－ナフチルメチル、ジフェニルメチル、トリフェニルメチル、α－ナフチルジフェニルメチル、９－アンスリルメチルのような１乃至３個のアリ－ル基で置換された低級アルキル基、４－メチルベンジル、２，４，６－トリメチルベンジル、３，４，５－トリメチルベンジル、４－メトキシベンジル、４－メトキシフェニルジフェニルメチル、２－ニトロベンジル、４－ニトロベンジル、４－クロロベンジル、４－ブロモベンジル、４－シアノベンジル、メチル、ピペロニルのような低級アルキル、低級アルコキシ、ハロゲン、シアノ基でアリ－ル環が置換された１乃至３個のアリ－ル基で置換された低級アルキル基等の「アラルキル基」；メトキシカルボニル、エトキシカルボニル、ｔ－ブトキシカルボニル、イソブトキシカルボニルのような低級アルコキシカルボニル基、２，２，２－トリクロロエトキシカルボニル、２－トリメチルシリルエトキシカルボニルのようなハロゲン又はトリ低級アルキルシリル基で置換された低級アルコキシカルボニル基等の「アルコキシカルボニル基」；ビニルオキシカルボニル、アリルオキシカルボニルのような「アルケニルオキシカルボニル基」；ベンジルオキシカルボニル、４－メトキシベンジルオキシカルボニル、３，４－ジメトキシベンジルオキシカルボニル、２－ニトロベンジルオキシカルボニル、４－ニトロベンジルオキシカルボニルのような、１乃至２個の低級アルコキシ又はニトロ基でアリ－ル環が置換されていてもよい「アラルキルオキシカルボニル基」を挙げることができる。 The "protecting group" and / or "protecting group of OH" of "protected OH" is a "protecting group in reaction" that can be cleaved by a chemical method such as hydrogenolysis, hydrolysis, electrolysis, photolysis. Is shown.
Such "protective groups in the reaction" include, for example, formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl. , 3,7-dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl, 14-methylpentadecanoyl, 13,13-dimethyltetradecanoyl, Alkylcarbonyl groups such as heptadecanoyl, 15-methylhexadecanoyl, octadecanoyl, 1-methylheptadecanoyl, nonadecanoyl, aicosanoyl and henaicosanoyl, carboxylated alkylcarbonyl groups such as succinoyl, glutaroyl, adipoyl, chloroacetyl, dichloro Harogeno lower alkylcarbonyl groups such as acetyl, trichloroacetyl, trifluoroacetyl, lower alkoxy lower alkylcarbonyl groups such as methoxyacetyl, unsaturated alkylcarbonyl groups such as (E) -2-methyl-2-butenoyl, etc. "Adioxyacyl group"; allylcarbonyl group such as benzoyl, α-naphthoyl, β-naphthoyl, halogenoallyl carbonyl group such as 2-bromobenzoyl, 4-chlorobenzoyl, 2,4,6-trimethyl Lower alkylated allylcarbonyl groups such as benzoyl, 4-toluoil, lower alkoxylated allylcarbonyl groups such as 4-anisoil, 2-carboxybenzoyl, 3-carboxybenzoyl, carboxy such as 4-carboxybenzoyl Carbonylated allylcarbonyl groups such as allylcarbonyl groups, 4-nitrobenzoyl, 2-nitrobenzoyl, lower alkoxycarbonylated allylcarbonyl groups such as 2- (methoxycarbonyl) benzoyl, 4-phenyl "Aromatic acyl groups" such as allylated allylcarbonyl groups such as benzoyl; tetrahydropyran-2-yl, 3-bromotetrahydropyran-2-yl, 4-methoxytetrahydropyran-4-yl, tetrahydro "Tetrahydropyranyl or tetrahydrothiopyranyl groups" such as thiopyran-2-yl, 4-methoxytetrahydrothiopyran-4-yl; tetrahydrofuran-2-yl, tetrahydrothiofuran-2 -"Tetrahydrofuranyl or tetrahydrothiofuranyl group" such as yl; trilower alkyl such as trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, methyldiisopropylsilyl, methyldi-t-butylsilyl, triisopropylsilyl A "silyl group" such as a tri-lower alkylsilyl group substituted with one or two allyl groups such as silyl group, diphenylmethylsilyl, diphenylbutylsilyl, diphenylisopropylsilyl, phenyldiisopropylsilyl; methoxymethyl, 1 , 1-dimethyl-1-methoxymethyl, ethoxymethyl, propoxymethyl, isopropoxymethyl, butoxymethyl, lower alkoxymethyl groups such as t-butoxymethyl, lower alkoxylated lower alkoxymethyl groups such as 2-methoxyethoxymethyl , 2,2,2-Trichloroethoxymethyl, "alkoxymethyl group" such as halogeno lower alkoxymethyl such as bis (2-chloroethoxy) methyl; lower such as 1-ethoxyethyl, 1- (isopropoxy) ethyl "Substituted ethyl groups" such as ethyl alkoxylated groups, ethyl halide groups such as 2,2,2-trichloroethyl; benzyl, α-naphthylmethyl, β-naphthylmethyl, diphenylmethyl, triphenylmethyl, α-naphthyl Lower alkyl groups substituted with 1 to 3 aller groups such as diphenylmethyl, 9-anthrylmethyl, 4-methylbenzyl, 2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl , 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 4-cyanobenzyl, methyl, lower alkyl such as piperonyl, lower alkoxy, "Aralkyl groups" such as lower alkyl groups substituted with 1 to 3 allyl groups substituted with halogen, cyano groups; methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, isobutoxycarbonyl An "alkoxycarbonyl group" such as a lower alkoxycarbonyl group such as, a lower alkoxycarbonyl group substituted with a halogen or trilower alkylsilyl group such as 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethoxycarbonyl; vinyl. "Alkenyloxy" such as oxycarbonyl, allyloxycarbonyl Carbonyl group "; one or two lower alkoxy or nitro such as benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl. Examples thereof include an "aralkyloxycarbonyl group" in which the allyl ring may be substituted with the group.
　「保護されたアミノ」の「保護基」とは、下記の保護基が１又は２個アミノ基を保護している基を示し、該保護基としては、通常アミノ基の保護基として使用するものであれば限定はないが、好適には、ホルミル、アセチル、プロピオニル、ブチリル、イソブチリル、ペンタノイル、ピバロイル、バレリル、イソバレリル、オクタノイル、ラウロイル、ミリストイル、トリデカノイル、パルミトイル、ステアロイルのようなアルキルカルボニル基、クロロアセチル、ジクロロアセチル、トリクロロアセチル、トリフルオロアセチルのようなハロゲノ低級アルキルカルボニル基、メトキシアセチルのような低級アルコキシ低級アルキルカルボニル基、（Ｅ）－２－メチル－２－ブテノイルのような不飽和アルキルカルボニル基等の脂肪族アシル基；ベンゾイル、α－ナフトイル、β－ナフトイルのようなアリ－ルカルボニル基、２－ブロモベンゾイル、４－クロロベンゾイルのようなハロゲノアリ－ルカルボニル基、２，４，６－トリメチルベンゾイル、４－トルオイルのような低級アルキル化アリ－ルカルボニル基、４－アニソイルのような低級アルコキシ化アリ－ルカルボニル基、４－ニトロベンゾイル、２－ニトロベンゾイルのようなニトロ化アリ－ルカルボニル基、２－（メトキシカルボニル）ベンゾイルのような低級アルコキシカルボニル化アリ－ルカルボニル基、４－フェニルベンゾイルのようなアリ－ル化アリ－ルカルボニル基等の芳香族アシル基；メトキシカルボニル、エトキシカルボニル、ｔ－ブトキシカルボニル、イソブトキシカルボニルのような低級アルコキシカルボニル基、２，２，２－トリクロロエトキシカルボニル、２－トリメチルシリルエトキシカルボニルのようなハロゲン又はトリ低級アルキルシリル基で置換された低級アルコキシカルボニル基等のアルコキシカルボニル基；ビニルオキシカルボニル、アリルオキシカルボニルのようなアルケニルオキシカルボニル基；ベンジルオキシカルボニル、４－メトキシベンジルオキシカルボニル、３，４－ジメトキシベンジルオキシカルボニル、２－ニトロベンジルオキシカルボニル、４－ニトロベンジルオキシカルボニルのような、１乃至２個の低級アルコキシ又はニトロ基でアリ－ル環が置換されていてもよいアラルキルオキシカルボニル基；トリメチルシリル、トリエチルシリル、イソプロピルジメチルシリル、ｔ－ブチルジメチルシリル、メチルジイソプロピルシリル、メチルジ－ｔ－ブチルシリル、トリイソプロピルシリルのようなトリ低級アルキルシリル基、ジフェニルメチルシリル、ジフェニルブチルシリル、ジフェニルイソプロピルシリル、フェニルジイソプロピルシリルのような１乃至２個のアリ－ル基で置換されたトリ低級アルキルシリル基等のシリル基；ベンジル、フェネチル、３－フェニルプロピル、α－ナフチルメチル、β－ナフチルメチル、ジフェニルメチル、トリフェニルメチル、α－ナフチルジフェニルメチル、９－アンスリルメチルのような１乃至３個のアリ－ル基で置換された低級アルキル基、４－メチルベンジル、２，４，６－トリメチルベンジル、３，４，５－トリメチルベンジル、４－メトキシベンジル、４－メトキシフェニルジフェニルメチル、２－ニトロベンジル、４－ニトロベンジル、４－クロロベンジル、４－ブロモベンジル、４－シアノベンジル、４－シアノベンジルジフェニルメチル、ビス（２－ニトロフェニル）メチル、ピペロニルのような低級アルキル、低級アルコキシ、ニトロ、ハロゲン、シアノ基でアリ－ル環が置換された１乃至３個のアリ－ル基で置換された低級アルキル基等のアラルキル基；又はＮ，Ｎ－ジメチルアミノメチレン、ベンジリデン、４－メトキシベンジリデン、４－ニトロベンジリデン、サリシリデン、５－クロロサリシリデン、ジフェニルメチレン、（５－クロロ－２－ヒドロキシフェニル）フェニルメチレンのようなシッフ塩基を形成する置換されたメチレン基であり、更に好適には、脂肪族アシル基である。 The "protective group" of the "protected amino" means a group in which the following protective group protects one or two amino groups, and the protective group is usually used as a protective group for an amino group. If not limited, preferred are alkylcarbonyl groups such as formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, lauroyl, myristol, tridecanoyl, palmitoyl, stearoyl, chloroacetyl. , Harogeno lower alkylcarbonyl groups such as dichloroacetyl, trichloroacetyl, trifluoroacetyl, lower alkoxy lower alkylcarbonyl groups such as methoxyacetyl, unsaturated alkylcarbonyl groups such as (E) -2-methyl-2-butenoyl. And other aliphatic acyl groups; allylcarbonyl groups such as benzoyl, α-naphthoyl, β-naphthoyl, 2-bromobenzoyl, halogenoallyl carbonyl groups such as 4-chlorobenzoyl, 2,4,6-trimethyl. Lower alkylated allylcarbonyl groups such as benzoyl, 4-toluoil, lower alkoxylated allylcarbonyl groups such as 4-anisoil, 4-nitrobenzoyl, nitrylated allylcarbonyl such as 2-nitrobenzoyl Aromatic acyl groups such as groups, lower alkoxycarbonylated allylcarbonyl groups such as 2- (methoxycarbonyl) benzoyl, allylylated allylcarbonyl groups such as 4-phenylbenzoyl; methoxycarbonyl, ethoxycarbonyl. , T-Butoxycarbonyl, lower alkoxycarbonyl groups such as isobutoxycarbonyl, halogens such as 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethoxycarbonyl or lower alkoxycarbonyl groups substituted with tri-lower alkylsilyl groups. Ekoxycarbonyl groups such as; vinyloxycarbonyl, alkenyloxycarbonyl groups such as allyloxycarbonyl; benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 4- An aralkyloxycarbonyl group whose allyl ring may be substituted with one or two lower alkoxy or nitro groups, such as nitrobenzyloxycarbonyl; trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsiri Or, a trilower alkylsilyl group such as methyldiisopropylsilyl, methyldi-t-butylsilyl, triisopropylsilyl, one or two aryls such as diphenylmethylsilyl, diphenylbutylsilyl, diphenylisopropylsilyl, phenyldiisopropylsilyl. Silyl groups such as tri-lower alkylsilyl groups substituted with groups; benzyl, phenethyl, 3-phenylpropyl, α-naphthylmethyl, β-naphthylmethyl, diphenylmethyl, triphenylmethyl, α-naphthyldiphenylmethyl, 9-an. Lower alkyl groups substituted with 1 to 3 allyl groups such as thrillmethyl, 4-methylbenzyl, 2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 4-cyanobenzyl, 4-cyanobenzyldiphenylmethyl, bis (2-nitrophenyl) methyl, piperonyl An aralkyl group such as a lower alkyl group substituted with 1 to 3 aryl groups in which the aryl ring is substituted with a lower alkyl, lower alkoxy, nitro, halogen, cyano group; or N, N-dimethyl. Substituted to form a shift base such as aminomethylene, benzylidene, 4-methoxybenzylidene, 4-nitrobenzylidene, salicylidene, 5-chlorosalicylidene, diphenylmethylene, (5-chloro-2-hydroxyphenyl) phenylmethylene It is a methylene group, more preferably an aliphatic acyl group.
　「保護されたカルボキシ」の「保護基」とは、加水素分解、加水分解、電気分解、光分解のような化学的方法により開裂し得る「反応における保護基」を示す。
　斯かる「反応における保護基」としては、好適には、メチル、エチル、ｎ－プロピル、イソプロピル、ｎ－ブチル、イソブチル、ｓ－ブチル、ｔｅｒｔ－ブチル、ｎ－ペンチル、イソペンチル、２－メチルブチル、ネオペンチル、１－エチルプロピル、ｎ－ヘキシル、イソヘキシル、４－メチルペンチル、３－メチルペンチル、２－メチルペンチル、１－メチルペンチル、３，３－ジメチルブチル、２，２－ジメチルブチル、１，１－ジメチルブチル、１，２－ジメチルブチル、１，３－ジメチルブチル、２，３－ジメチルブチル、２－エチルブチルのような「低級アルキル基」；
　エテニル、１－プロペニル、２－プロペニル、１－メチル－２－プロペニル、１－メチル－１－プロペニル、２－メチル－１－プロペニル、２－メチル－２－プロペニル、２－エチル－２－プロペニル、１－ブテニル、２－ブテニル、１－メチル－２－ブテニル、１－メチル－１－ブテニル、３－メチル－２－ブテニル、１－エチル－２－ブテニル、３－ブテニル、１－メチル－３－ブテニル、２－メチル－３－ブテニル、１－エチル－３－ブテニル、１－ペンテニル、２－ペンテニル、１－メチル－２－ペンテニル、２－メチル－２－ペンテニル、３－ペンテニル、１－メチル－３－ペンテニル、２－メチル－３－ペンテニル、４－ペンテニル、１－メチル－４－ペンテニル、２－メチル－４－ペンテニル、１－ヘキセニル、２－ヘキセニル、３－ヘキセニル、４－ヘキセニル、５－ヘキセニルのような「アルケニル基」；エチニル、２－プロピニル、１－メチル－２－プロピニル、２－メチル－２－プロピニル、２－エチル－２－プロピニル、２－ブチニル、１－メチル－２－ブチニル、２－メチル－２－ブチニル、１－エチル－２－ブチニル、３－ブチニル、１－メチル－３－ブチニル、２－メチル－３－ブチニル、１－エチル－３－ブチニル、２－ペンチニル、１－メチル－２－ペンチニル、２－メチル－２－ペンチニル、３－ペンチニル、１－メチル－３－ペンチニル、２－メチル－３－ペンチニル、４－ペンチニル、１－メチル－４－ペンチニル、２－メチル－４－ペンチニル、２－ヘキシニル、３－ヘキシニル、４－ヘキシニル、５－ヘキシニルのような「アルキニル基」；トリフルオロメチル、トリクロロメチル、ジフルオロメチル、ジクロロメチル、ジブロモメチル、フルオロメチル、２，２，２－トリフルオロエチル、２，２，２－トリクロロエチル、２－ブロモエチル、２－クロロエチル、２－フルオロエチル、２－ヨードエチル、３－クロロプロピル、４－フルオロブチル、６－ヨードヘキシル、２，２－ジブロモエチルのような「ハロゲノ低級アルキル基」；２－ヒドロキシエチル、２，３－ジヒドロキシプロピル、３－ヒドロキシプロピル、３，４－ジヒドロキシブチル、４－ヒドロキシブチルのようなヒドロキシ「低級アルキル基」；アセチルメチルのような「脂肪族アシル」－「低級アルキル基」；ベンジル、フェネチル、３－フェニルプロピル、α－ナフチルメチル、β－ナフチルメチル、ジフェニルメチル、トリフェニルメチル、６－フェニルヘキシル、α－ナフチルジフェニルメチル、９－アンスリルメチルのような１乃至３個のアリ－ル基で置換された「低級アルキル基」、４－メチルベンジル、２，４，６－トリメチルベンジル、３，４，５－トリメチルベンジル、４－メトキシベンジル、４－メトキシフェニルジフェニルメチル、２－ニトロベンジル、４－ニトロベンジル、４－クロロベンジル、４－ブロモベンジル、４－シアノベンジル、４－シアノベンジルジフェニルメチル、ビス（２－ニトロフェニル）メチル、ピペロニル、４－メトキシカルボニルベンジルのような低級アルキル、低級アルコキシ、ニトロ、ハロゲン、シアノ、アルコキシカルボニル基でアリ－ル環が置換された１乃至３個のアリ－ル基で置換された低級アルキル基等の「アラルキル基」；トリメチルシリル、トリエチルシリル、イソプロピルジメチルシリル、ｔｅｒｔ－ブチルジメチルシリル、メチルジイソプロピルシリル、メチルジｔｅｒｔ－ブチルシリル、トリイソプロピルシリル、メチルジフェニルシリル、イソプロピルジフェニルシリル、ブチルジフェニルシリル、フェニルジイソプロピルシリルのような「シリル基」を挙げることができ、好適には、「低級アルキル基」である。 The "protecting group" of a "protected carboxy" refers to a "protecting group in a reaction" that can be cleaved by chemical methods such as hydrogenolysis, hydrolysis, electrolysis, photolysis.
Such "protective groups in the reaction" are preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl. , 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1- "Lower alkyl groups" such as dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl;
Ethenyl, 1-propenyl, 2-propenyl, 1-methyl-2-propenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 2-ethyl-2-propenyl, 1-butenyl, 2-butenyl, 1-methyl-2-butenyl, 1-methyl-1-butenyl, 3-methyl-2-butenyl, 1-ethyl-2-butenyl, 3-butenyl, 1-methyl-3- Butenyl, 2-methyl-3-butenyl, 1-ethyl-3-butenyl, 1-pentenyl, 2-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-pentenyl, 1-methyl- 3-Pentenyl, 2-methyl-3-pentenyl, 4-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5- "Alkenyl groups" such as hexenyl; ethynyl, 2-propynyl, 1-methyl-2-propynyl, 2-methyl-2-propynyl, 2-ethyl-2-propynyl, 2-butynyl, 1-methyl-2-butynyl , 2-Methyl-2-butynyl, 1-ethyl-2-butynyl, 3-butynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1-ethyl-3-butynyl, 2-pentynyl, 1 -Methyl-2-pentynyl, 2-methyl-2-pentynyl, 3-pentynyl, 1-methyl-3-pentynyl, 2-methyl-3-pentynyl, 4-pentynyl, 1-methyl-4-pentynyl, 2-methyl "Alkinyl groups" such as -4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl; trifluoromethyl, trichloromethyl, difluoromethyl, dichloromethyl, dibromomethyl, fluoromethyl, 2,2 , 2-Trifluoroethyl, 2,2,2-trichloroethyl, 2-bromoethyl, 2-chloroethyl, 2-fluoroethyl, 2-iodoethyl, 3-chloropropyl, 4-fluorobutyl, 6-iodohexyl, 2, "Halogeno lower alkyl groups" such as 2-dibromoethyl; hydroxy "lower alkyl groups" such as 2-hydroxyethyl, 2,3-dihydroxypropyl, 3-hydroxypropyl, 3,4-dihydroxybutyl, 4-hydroxybutyl "Alipid acyl" such as acetylmethyl- "lower alkyl group"; benzyl, phenethyl, 3-phenylpropyl, α-naphthylmethyl, β-naphthylmethy "Lower alkyl group" substituted with 1 to 3 allyl groups such as le, diphenylmethyl, triphenylmethyl, 6-phenylhexyl, α-naphthyldiphenylmethyl, 9-anthrylmethyl, 4-methyl Benzyl, 2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromo Ali on lower alkyl, lower alkoxy, nitro, halogen, cyano, alkoxycarbonyl groups such as benzyl, 4-cyanobenzyl, 4-cyanobenzyldiphenylmethyl, bis (2-nitrophenyl) methyl, piperonyl, 4-methoxycarbonylbenzyl. "Aralkyl groups" such as lower alkyl groups substituted with 1 to 3 allyl groups substituted with a ru ring; trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, tert-butyldimethylsilyl, methyldiisopropylsilyl, methyldi Examples thereof include "silyl groups" such as tert-butyl silyl, triisopropyl silyl, methyl diphenyl silyl, isopropyl diphenyl silyl, butyl diphenyl silyl and phenyl diisopropyl silyl, and preferred are "lower alkyl groups".
　「ハロゲン原子」とは、弗素原子、塩素原子、臭素原子又は沃素原子であり、好適には、弗素原子、塩素原子である。 The "halogen atom" is a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, and is preferably a fluorine atom or a chlorine atom.
　「酸素原子で介在されていてもよい－（ＣＨ２）１～６－」とは、「－（ＣＨ２）１～６－」が、１または２個の酸素原子で介在されている基を示し、たとえば、メチレンオキシメチレンである。 "May be intervened by oxygen atom- (CH 2 ) 1 to 6- " means a group in which "-(CH 2 ) 1 to 6- " is intervened by one or two oxygen atoms. Shown, for example, methyleneoxymethylene.
　「ＮＨで介在されていてもよい－（ＣＨ２）１～６－」とは、「－（ＣＨ２）１～６－」が、１または２個のＮＨで介在されている基を示し、たとえば、メチレンアミノメチレンである。 "May be intervened by NH- (CH 2) 1 to 6-" indicates a group in which "-(CH 2 ) 1 to 6- " is intervened by one or two NHs. For example, methyleneaminomethylene.
　本発明化合物（Ｉ）は、置換基の種類によっては塩基または酸との塩を形成する場合もある。かかる塩としては、製薬学的に許容される塩であり、好ましくは、塩酸、臭化水素酸、ヨウ化水素酸、硫酸、硝酸、リン酸等の無機酸、ギ酸、酢酸、プロピオン酸、シュウ酸、マロン酸、コハク酸、フマル酸、マレイン酸、乳酸、リンゴ酸、酒石酸、クエン酸、メタンスルホン酸、ベンゼンスルホン酸、アスパラギン酸、グルタミン酸等の有機酸との酸付加塩、ナトリウム、カリウム、マグネシウム、カルシウム、アルミニウム等の無機塩基、メチルアミン、エチルアミン、エタノールアミン、リジン、オルニチン等の有機塩基との塩やアンモニウム塩等が挙げられる。 The compound (I) of the present invention may form a salt with a base or an acid depending on the type of the substituent. The salt is a pharmaceutically acceptable salt, and is preferably an inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, or phosphoric acid, formic acid, acetic acid, propionic acid, or shu. Acid addition salts with organic acids such as acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, benzenesulfonic acid, aspartic acid, glutamate, sodium, potassium, Examples thereof include salts with inorganic bases such as magnesium, calcium and aluminum, and organic bases such as methylamine, ethylamine, ethanolamine, lysine and ornithine, and ammonium salts.
は、Ｉｎ　ｓｉｌｉｃｏにより選択された、３種類の化合物を示す。Shows three compounds selected by In silico. は、ＦＫＢＰ１２－ＲＡＰ－ＦＲＢおよびＦＫＢＰ１２－化合物Ｃ－ＲＡＰの構造モデルを示し、Shows structural models of FKBP12-RAP-FRB and FKBP12-Compound C-RAP. は、ＦＫＢＰ１２－ＲＡＰ－ＦＲＢの、全体図および拡大した結晶構造（ＰＤＢ　ＩＤ：１ＦＡＰ）を示し、Shows the overall view and enlarged crystal structure (PDB ID: 1FAP) of FKBP12-RAP-FRB. は、ＦＫＢＰ１２－化合物Ｃ－ＦＲＢの全体図および拡大したドッキングシミュレーションモデルを示す。Shows an overview of FKBP12-Compound C-FRB and an enlarged docking simulation model. は、ＦＫＢＰ１２およびＦＲＢの調製を示し、Shows the preparation of FKBP12 and FRB. は、６ｘＨｉｓ－ＦＲＢのゲルろ過クロマトグラムとＳＤＳ－ＰＡＧＥを、6xHis-FRB gel filtration chromatogram and SDS-PAGE. は、６ｘＨｉｓ－ＦＫＢＰ１２のゲルろ過クロマトグラムとＳＤＳ－ＰＡＧＥを、6xHis-FKBP12 gel filtration chromatogram and SDS-PAGE. は、ビオチン化ＦＫＢＰ１２のゲルろ過クロマトグラムとＳＤＳ－ＰＡＧＥを示す。　青矢印のピークを結合実験には使用した。Shows the gel filtration chromatogram of biotinylated FKBP12 and SDS-PAGE. The peak of the blue arrow was used for the binding experiment. は、ＡｌｐｈａＬＩＳＡ(R)によるｉｎ　ｖｉｔｒｏ結合アッセイを示し、化合物Ｃが、ラパマイシン（ＲＡＰ）と同程度の結合力を持つこと、一方、化合物Ａおよび化合物Ｂの結合力は弱いことが分かった。Showed an in vitro binding assay with AlphaLISA (R) and found that compound C had similar binding strength to rapamycin (RAP), while compound A and compound B had weak binding strength. は、ＡｌｐｈａＬＩＳＡ(R)によるＫＤ測定を示し、化合物ＣおよびＲＡＰはそれぞれ、４　ｎＭ、２．２　ｎＭの平衡解離定数を示した。Showed KD measurements by AlphaLISA (R) , where compounds C and RAP showed equilibrium dissociation constants of 4 nM and 2.2 nM, respectively. は、ＮａｎｏＢｉＴ(R)によるＨＥＫ２９３細胞内での親和性測定を示し、化合物ＣおよびＲＡＰは同等のシグナルを示したが、化合物Ａおよび化合物Ｂのシグナルは弱かった。Showed an affinity measurement in HEK293 cells by NanoBiT (R) , where Compounds C and RAP showed equivalent signals, but the signals of Compounds A and B were weak. は、ＮａｎｏＢｉＴ（登録商標）アッセイ系に、化合物Ｃの濃度を変えて投与した結果を示す。化合物Ｃの濃度依存的に、発光強度が増加していることから、化合物ＣがＦＫＢＰ１２－ＦＲＢに特異的に結合していることを示す。Shows the results of administration to the NanoBiT® assay system at varying concentrations of Compound C. Since the emission intensity increases depending on the concentration of compound C, it is shown that compound C is specifically bound to FKBP12-FRB. は、細胞損傷性アッセイを示し、化合物Ｃは、非がん細胞（ＮＲＫ－４９Ｆ）の損傷性（ＩＣ５０＞１０　μＭ）と比較して、がん細胞をより損傷（ＭＣＦ－７のＩＣ５０＝２８．３　ｎＭ、ＨｅｐＧ２のＩＣ５０＝１７．９　ｎＭ）することが分かった。Shows a cell damaging assay, where compound C is more damaging to cancer cells ( IC50 of MCF-7) compared to the damaging (IC 50 > 10 μM) of non-cancer cells (NRK-49F). = 28.3 nM, IC50 of HepG2 = 17.9 nM). は、化合物ＣとＲＡＰのがん細胞（ＨｅＬａ）に対する細胞損傷性の比較を示し、がん細胞（ＨｅＬａ）に対して、化合物Ｃとラパマイシンの細胞損傷性（ＩＣ５０）を比較したところ、化合物Ｃの方がより強い細胞損傷性（化合物Ｃ：ＩＣ５０＝１．７　ｎＭ，ＲＡＰ：ＩＣ５０＝２４．７　ｎＭ）を示した。Shows a comparison of the cell-damaging properties of compound C and RAP against cancer cells (HeLa), and compares the cell-damaging properties of compound C and rapamycin (IC 50 ) with respect to cancer cells (HeLa). C showed stronger cell damage (Compound C: IC 50 = 1.7 nM, RAP: IC 50 = 24.7 nM). は、化合物ＣとＲＡＰの、活性化条件（＋／＋　Ｇｌｕｃｏｓｅ／ＦＢＳ）および飢餓条件（－／－　Ｇｌｕｃｏｓｅ／ＦＢＳ）での細胞損傷性の比較を示す。Shows a comparison of cell damage properties of Compounds C and RAP under activation conditions (+ / + Glucose / FBS) and starvation conditions (-/-Glucose / FBS). は、化合物ＣとＲＡＰの非がん細胞（ＨＥＫ２９３）に対する細胞損傷性の比較を示す。Shows a comparison of the cell damaging properties of Compound C and RAP against non-cancer cells (HEK293). は、ＡｌｐｈａＬＩＳＡ　ＳｕｒｅＦｉｒｅ　Ｕｌｔｒａ　ＨＶ　ｐ－ｐ７０　Ｓ６Ｋ　（Ｔ３８９）　ａｓｓａｙ　ｋｉｔによる、がん細胞（ＨｅＬａ）のリン酸化阻害活性測定を示し、化合物Ａおよび化合物Ｂは、ほとんどＴ３８９ｐ－７０Ｓ６Ｋ１のリン酸化を阻害しなかったが、化合物ＣはＲＡＰと同程度に阻害した。Shows measurements of phosphorylation inhibitory activity of cancer cells (HeLa) by AlphaLISA SureFire Ultra HV p-p70 S6K (T389) assay kit, where Compound A and Compound B mostly inhibit the phosphorylation of T 389 p-70S6K1. However, compound C inhibited as much as RAP. は、ＡｌｐｈａＬＩＳＡ　ＳｕｒｅＦｉｒｅ　Ｕｌｔｒａ　ＨＶ　ｐ－ｐ７０　Ｓ６Ｋ　（Ｔ３８９）　ａｓｓａｙ　ｋｉｔによる、がん細胞（ＨｅＬａ）のリン酸化阻害活性の定量測定を示す。Shows quantitative measurement of phosphorylation inhibitory activity of cancer cells (HeLa) by AlphaLISA SureFire Ultra HV p-p70 S6K (T389) assay kit. は、がん細胞（ＨｅＬａ）を使用した、Ｗｅｓｔｅｒｎブロッティングによる、Ｔ３７／４６ｐ－４Ｅ－ＢＰ１およびＳ２３５／２３６ｐ－Ｓ６のリン酸化阻害活性アッセイを示す。Shows a phosphorylation inhibitory activity assay of T37 / 46 p-4E-BP1 and S235 / 236 p-S6 by Western blotting using cancer cells (HeLa). は、非がん細胞（ＨＥＫ２９３）使用した、Ｗｅｓｔｅｒｎブロッティングによる、化合物ＣおよびＲＡＰの、Ｓ２３５／２３６ｐ－Ｓ６のリン酸化阻害活性アッセイを示す。Shows the S235 / 236 p-S6 phosphorylation inhibitory activity assay of compounds C and RAP by Western blotting using non-cancer cells (HEK293). は、化合物ＣおよびＲＡＰの動物実験の結果を示す。Shows the results of animal experiments with compounds C and RAP. は、化合物ＣはＲＡＰよりも、高い腫瘍成長抑制効果を示した。Compound C showed a higher tumor growth inhibitory effect than RAP. は、化合物Ｃは、ＲＡＰよりも弱い、腫瘍肺転移促進活性を示した。Compound C showed a tumor lung metastasis promoting activity weaker than RAP. は、Ｄ１１で投与を停止し、Ｄ２０での腫瘍再発を調べた。化合物Ｃは、ＲＡＰよりも低い腫瘍再発性を示した。Discontinued administration at D11 and investigated tumor recurrence at D20. Compound C showed lower tumor relapse than RAP. は、肺に転移した腫瘍写真、血中尿素窒素、クレアチニン、血中尿素窒素／クレアチン、アラニンアミノ基転移酵素、アスパラギン酸アミノ基転移酵素、乳酸脱水素酵素および／またはグルコース濃度のような、血液試験及びその他の動物実験の結果を示す。Blood such as tumor photographs that have spread to the lungs, blood urea nitrogen, creatinine, blood urea nitrogen / creatin, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and / or glucose concentration. The results of the test and other animal experiments are shown. は、化合物ＣおよびＲＡＰの体重減への影響を示し、顕著な減少は見られない。なお、実験期間中、化合物Ｃにより顕著な体重減少は見られず、また、血液から明らかな毒性は検出されなかった。Shows the effect of Compounds C and RAP on body weight loss, with no significant reduction. No significant weight loss was observed with Compound C during the experimental period, and no obvious toxicity was detected in the blood. は、実施例で評価した１１種類の化合物を示す。 Shows 11 kinds of compounds evaluated in Examples. は、ＮａｎｏＢｉＴ(R)によるＨｅＬａ細胞内での親和性測定を示す。Shows the measurement of affinity in HeLa cells by NanoBiT (R) . は、ＮａｎｏＢｉＴ(R)によるＨｅＬａ細胞内での親和性測定を示す。Shows the measurement of affinity in HeLa cells by NanoBiT (R) . は、ＡｌｐｈａＬＩＳＡ　ＳｕｒｅＦｉｒｅ　Ｕｌｔｒａ　ＨＶ　ｐ－ｐ７０　Ｓ６Ｋ　（Ｔ３８９）　ａｓｓａｙ　ｋｉｔによる、がん細胞（ＨｅＬａ）のリン酸化阻害活性測定を示す。Shows the measurement of phosphorylation inhibitory activity of cancer cells (HeLa) by AlphaLISA SureFire Ultra HV p-p70 S6K (T389) assay kit.
[本発明の一般的製法]
　本件発明の化合物の一部は、公知化合物であり、たとえば、Ｓｔｅｒｌｉｎｇ　ａｎｄ　Ｉｒｗｉｎ， Ｊ． Ｃｈｅｍ． Ｉｎｆ． Ｍｏｄｅｌ，　２０１５　ｈｔｔｐ：／／ｐｕｂｓ．ａｃｓ．ｏｒｇ／ｄｏｉ／ａｂｓ／１０．１０２１／ａｃｓ．ｊｃｉｍ．５ｂ００５５９で検索ずることによって供給元から入手することができる。
　その他、公知の化合物以外の化合物は、以下に準じて同様に合成できる。 [General manufacturing method of the present invention]
Some of the compounds of the present invention are known compounds, for example, Stalling and Irwin, J. Mol. Chem. Inf. Model, 2015 http: // pubs. acs. org / doi / abs / 10.1102 / acs. jsim. It can be obtained from the supplier by searching for 5b00559.
In addition, compounds other than known compounds can be similarly synthesized according to the following.
一般製法
ステップＡ：
Figure JPOXMLDOC01-appb-C000025
 　上図において、Ｒ２およびＸは前記と同意義を示す。
　たとえば、ＴＥＡ（５１．８ミリモル）と、そのカルボキシ基が、通常の保護基で保護されてもよいアミノ酸（２）（５１．８ミリモル）のたとえば１５０　ｍｌの水溶液に、カルボキシ基が保護されたイソチオシアネート（１）（５１．８ミリモル、１ｅｑ）の溶液（たとえば、ｉ－ＰｒＯＨ（たとえば２００　ｍｌ）のような不活性の有機溶媒の溶液）を加えて、反応混合物を、たとえば３０分間還流した。次に、混合物を冷却し、酸性化（たとえば、ＨＣｌ水溶液）してｐＨ＝２にした。
　沈殿物を濾過し、乾燥させ、所望の生成物（３）を、たとえば、収率８０％で得、さらに精製することなく、ステップＢに使用した。
　なお、「カルボキシ基の保護基」は、前記と同意義である。 General manufacturing method
Step A:
Figure JPOXMLDOC01-appb-C000025
 In the above figure, R 2 and X have the same meaning as described above.
For example, the carboxy group was protected in an aqueous solution of, for example, 150 ml of TEA (51.8 mmol) and the amino acid (2) (51.8 mmol) whose carboxy group may be protected by conventional protective groups. A solution of isothiocyanate (1) (51.8 mmol, 1eq) (eg, a solution of an inert organic solvent such as i-PrOH (eg 200 ml)) was added and the reaction mixture was refluxed for example for 30 minutes. .. The mixture was then cooled and acidified (eg, aqueous HCl) to pH = 2.
The precipitate was filtered and dried to give the desired product (3), for example in 80% yield, which was used in step B without further purification.
The "protecting group for a carboxy group" has the same meaning as described above.
ステップＢ：
Figure JPOXMLDOC01-appb-C000026
 　上図において、Ｒ２およびＸは前記と同意義を示す。
　たとえば、１００　ｍｌの水に溶解したＮａＯＨ（３ｅｑ、２．２７ｇ）と、そのカルボキシ基が、通常の保護基で保護されていてもよい酸（３）（１８．９　ｍｍｏｌ、１　ｅｑ）の溶液を５０℃に加熱した。次に、混合物を冷却し、Ｈ２Ｏ２（１０当量、水中３５％）を加え撹拌した。
　反応温度は－１０℃乃至１７０℃で行なわれるが、好適には、３０℃乃至１００℃である。
　反応時間は、主に反応温度、原料化合物、反応試薬又は使用される溶媒の種類によって異なるが、通常、１０分乃至２時間であり、好適には、３０分間である。
　その後、たとえば、反応混合物を冷却し、ｐＨ＝２になるまで、酸（通常の反応において酸として使用されるものであれば特に限定はないが、好適には塩酸、燐酸のような無機酸又は酢酸、蟻酸、蓚酸、メタンスルホン酸、ｐ－トルエンスルホン酸、カンファースルホン酸、トリフルオロ酢酸、トリフルオロメタンスルホン酸のような有機酸等のブレンステッド酸でありえ、好適には有機酸であり、更に好適には酢酸である。）を加え、沈殿物を濾過し、乾燥させた。
　所望の生成物（４）（たとえばＮＭＲによる純度９５％）を、たとえば収率９６％で得、さらに精製することなく、ステップＣに使用した。 Step B:
Figure JPOXMLDOC01-appb-C000026
 In the above figure, R 2 and X have the same meaning as described above.
For example, a solution of NaOH (3 eq, 2.27 g) dissolved in 100 ml of water and an acid (3) (18.9 mmol, 1 eq) whose carboxy group may be protected by conventional protecting groups. Was heated to 50 ° C. The mixture was then cooled, H 2 O 2 (10 eq, 35% in water) was added and stirred.
The reaction temperature is −10 ° C. to 170 ° C., preferably 30 ° C. to 100 ° C.
The reaction time varies mainly depending on the reaction temperature, the raw material compound, the reaction reagent or the type of the solvent used, but is usually 10 minutes to 2 hours, preferably 30 minutes.
Then, for example, the reaction mixture is cooled and an acid (not particularly limited as long as it is used as an acid in a normal reaction, but preferably an inorganic acid such as hydrochloric acid or phosphoric acid) is used until pH = 2. It can be a blended acid such as an organic acid such as acetic acid, formic acid, oxalic acid, methanesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, preferably an organic acid, and further. (Preferably acetic acid) was added, the precipitate was filtered and dried.
The desired product (4) (eg, NMR purity 95%) was obtained, for example in 96% yield, and used in step C without further purification.
ステップＣ：
Figure JPOXMLDOC01-appb-C000027
 　上図において、Ｒ１、Ｒ２、Ｒ５、Ｒ６、ＬおよびＸは前記と同意義を示す。
　必要に応じて、カルボキシ基の保護基を除去し、酸（４）（たとえば、１８．４ミリモル、１当量）を、たとえば、ジオキサン（６０ｍｌ）に溶解し、ＣＤＩ（１．１当量）を室温で少しずつ加えた。
　混合物を、５０℃で、３時間撹拌してから、アミン（５）（１８．４ミリモル、１当量）を加えた。
　次に、混合物を室温で１２時間撹拌した後、冷水（１００ｍｌ）を加え、沈殿物を濾過し、ｉ－ＰｒＯＨ（１０ｍｌ）で洗浄し、白色の沈殿物を乾燥させた。
　スペクトルは、所望の生成物（６）（たとえば、ＮＭＲによる純度９５％）、たとえば収率８１％を示し、これはさらに精製することなく、ステップＤに使用した。
　反応は、溶媒中、カルボキシを、アミン化合物と縮合させることによって行われる。
　使用される溶媒としては、不活性であれば特に限定はないが、例えば、メチレンクロリド、クロロホルムのようなハロゲン化単価水素類、ジオキサン、エーテル、テトラヒドロフランのようなエーテル類、ジメチルホルムアミド、ジメチルアセトアミドのようなアミド類を挙げることができる。
　反応は、例えば、アゾジカルボン酸ジエチル－トリフェニルホスフィンのようなアゾジカルボン酸ジ低級アルキル－トリフェニルホスフィン類、Ｎ－エチル－５－フェニルイソオキサゾリウム－３’－スルホナ－トのようなＮ－低級アルキル－５－アリールイソオキサゾリウム－３’－スルホナ－ト類、Ｎ’，Ｎ’－ジシクロヘキシルカルボジイミド（ＤＣＣ）のようなＮ’，Ｎ’－ジシクロアルキルカルボジイミド類、ジ－２－ピリジルジセレニドのようなジヘテロアリールジセレニド類、トリフェニルホスフィンのようなトリアリールホスフィン類、ｐ－ニトロベンゼンスルホニルトリアゾリドのようなアリールスルホニルトリアゾリド類、２－クロル－１－メチルピリジニウム　ヨーダイドのような２－ハロ－１－低級アルキルピリジニウム　ハライド及びジフェニルホスホリルアジド（ＤＰＰＡ）のようなジアリールホスホリルアジド類、Ｎ，Ｎ’－カルボジイミダゾール（ＣＤＩ）のようなイミダゾール誘導体、１－ヒドロキシベンゾトリアゾール（ＨＯＢＴ）のようなベンゾトリアゾール誘導体、Ｎ－ヒドロキシ－５－ノルボルネン－２，３－ジカルボキシイミド（ＨＯＮＢ）のようなジカルボキシイミド誘導体、１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド（ＥＤＡＰＣ）のようなカルボジイミド誘導体のような縮合剤の存在下に好適に行われる。
　反応温度は、－１０℃乃至２５℃であり、活性エステル化合物とアミンとの反応では室温付近であり、反応時間は、３０分乃至１０時間である。
　カルボキシ基の保護基の除去はその種類によって異なるが、一般にこの分野の技術において周知の方法によって以下の様に実施される。
　カルボキシ基の保護基として、低級アルキル基又はアリ－ル基を使用した場合には、酸又は塩基で処理することにより除去することができる。
　酸としては、塩酸、硫酸、燐酸、臭化水素酸が用いられ、塩基としては、化合物の他の部分に影響を与えないものであれば特に限定はないが、好適には炭酸ナトリウム、炭酸カリウムのようなアルカリ金属炭酸塩、水酸化ナトリウム、水酸化カリウムのようなアルカリ金属水酸化物又は濃アンモニア－メタノ－ル溶液が用いられる。
　尚、塩基による加水分解では異性化が起こることがある。
　使用される溶媒としては、通常の加水分解反応に使用されるもので、反応を阻害しないものであれば特に限定はなく、水又はメタノ－ル、エタノ－ル、ｎ－プロパノ－ルのようなアルコ－ル類若しくはテトラヒドロフラン、ジオキサンのようなエ－テル類のような有機溶媒と水との混合溶媒が好適である。
　反応温度及び反応時間は、出発物質、溶媒及び用いる試薬等により異なり、特に限定はないが、副反応を抑制するために、通常は０℃乃至１５０℃で、１時間乃至１０時間実施される。
　カルボキシ基の保護基がジフェニルメチルのようなジアリ－ル置換メチル基である場合には、通常、溶媒中、酸で処理することにより除去される。
　使用される溶媒としては、アニソ－ルのような芳香族炭化水素類が好ましく、使用される酸としては、トリフルオロ酢酸のような弗化有機酸が用いられる。
　反応温度及び反応時間は、出発物質、溶媒、使用される酸等により異なるが、通常は室温で３０分乃至１０時間実施される。
　カルボキシ基の保護基がアラルキル基又はハロゲノ低級アルキル基である場合には、通常、溶媒中、還元により除去される。
　還元方法としては、カルボキシ基の保護基がハロゲノ低級アルキル基である場合には、亜鉛－酢酸のような化学的還元による方法が好適であり、アラルキル基である場合には、パラジウム炭素、白金のような触媒を用い接触還元による方法を行なうか、又は硫化カリウム、硫化ナトリウムのようなアルカリ金属硫化物を用いて、化学的還元による方法により実施される。
　使用される溶媒としては、本反応に関与しないものであれば特に限定はないが、メタノ－ル、エタノ－ルのようなアルコ－ル類；テトラヒドロフラン、ジオキサンのようなエ－テル類；酢酸のような脂肪酸又はこれらの有機溶媒と水との混合溶媒が好適である。
　反応温度及び反応時間は、出発物質、溶媒及び還元方法等により異なるが、通常は０℃乃至室温（例えば、２５℃）付近で、５分乃至１２時間実施される。
　カルボキシ基の保護基がアルコキシメチル基である場合には、通常、溶媒中、酸で処理することにより除去される。
　使用される酸としては、通常ブレンステッド酸として使用されるものであれば特に限定はないが、好適には塩酸、硫酸のような無機酸又は酢酸、パラトルエンスルホン酸のような有機酸である。
　使用される溶媒としては、本反応に関与しないものであれば特に限定はないが、メタノ－ル、エタノ－ルのようなアルコ－ル類；テトラヒドロフラン、ジオキサンのようなエ－テル類又はこれらの有機溶媒と水との混合溶媒が好適である。
　反応温度及び反応時間は、出発物質、溶媒及び使用される酸の種類等により異なるが、通常は０℃乃至５０℃で、１０分乃至１８時間実施される。 Step C:
Figure JPOXMLDOC01-appb-C000027
 In the above figure, R 1 , R 2 , R 5 , R 6 , L and X have the same meaning as described above.
If necessary, the protecting group of the carboxy group is removed, the acid (4) (eg, 18.4 mmol, 1 eq) is dissolved in, for example, dioxane (60 ml) and the CDI (1.1 eq) is at room temperature. I added it little by little.
The mixture was stirred at 50 ° C. for 3 hours and then amine (5) (18.4 mmol, 1 eq) was added.
The mixture was then stirred at room temperature for 12 hours, then cold water (100 ml) was added, the precipitate was filtered, washed with i-PrOH (10 ml) and the white precipitate was dried.
The spectrum showed the desired product (6) (eg, NMR purity 95%), eg 81% yield, which was used in step D without further purification.
The reaction is carried out by condensing carboxy with an amine compound in a solvent.
The solvent used is not particularly limited as long as it is inert, but for example, methylene chloride, halogenated monovalent hydrogens such as chloroform, dioxane, ethers, ethers such as tetrahydrofuran, dimethylformamide and dimethylacetamide. Such amides can be mentioned.
The reaction is carried out, for example, azodicarboxylic acid dilower alkyl-triphenylphosphines such as diethyl-triphenylphosphine azodicarboxylic acid, N-such as N-ethyl-5-phenylisooxazolium-3'-sulfonate. -Lower alkyl-5-arylisooxazolium-3'-sulfonates, N', N'-dicycloalkylcarbodiimides such as N', N'-dicyclohexylcarbodiimide (DCC), di-2- Diheteroaryl diselenides such as pyridyl diselenides, triarylphosphines such as triphenylphosphine, arylsulfonyltriazolides such as p-nitrobenzenesulfonyltriazolides, 2-chlor-1-methyl 2-Halo-1-lower alkylpyridinium halides such as pyridinium iodide and diallylphosphoryl azides such as diphenylphosphoryl azide (DPPA), imidazole derivatives such as N, N'-carbodiimidazole (CDI), 1-hydroxy Bentotriazole derivatives such as benzotriazole (HOBT), dicarboxyimide derivatives such as N-hydroxy-5-norbornen-2,3-dicarboxyimide (HONB), 1-ethyl-3- (3-dimethylaminopropyl) ) It is preferably carried out in the presence of a condensing agent such as a carbodiimide derivative such as carbodiimide (EDAPC).
The reaction temperature is −10 ° C. to 25 ° C., the reaction between the active ester compound and the amine is near room temperature, and the reaction time is 30 minutes to 10 hours.
Removal of the protecting group of the carboxy group varies depending on the type, but is generally carried out by a method well known in the art in the art as follows.
When a lower alkyl group or an aryl group is used as the protecting group for the carboxy group, it can be removed by treating with an acid or a base.
As the acid, hydrochloric acid, sulfuric acid, phosphoric acid, and hydrobromic acid are used, and the base is not particularly limited as long as it does not affect other parts of the compound, but sodium carbonate and potassium carbonate are preferable. Alkali metal carbonates such as, sodium hydroxide, alkali metal hydroxides such as potassium hydroxide or concentrated ammonia-methanol solutions are used.
It should be noted that hydrolysis with a base may cause isomerization.
The solvent used is not particularly limited as long as it is used for a normal hydrolysis reaction and does not inhibit the reaction, such as water or methanol, ethanol, n-propanol and the like. A mixed solvent of water and an organic solvent such as alcohols or ethers such as tetrahydrofuran and dioxane is suitable.
The reaction temperature and reaction time vary depending on the starting material, solvent, reagent used and the like, and are not particularly limited, but are usually carried out at 0 ° C. to 150 ° C. for 1 hour to 10 hours in order to suppress side reactions.
When the protecting group of the carboxy group is a dially substituted methyl group such as diphenylmethyl, it is usually removed by treatment with an acid in a solvent.
As the solvent used, aromatic hydrocarbons such as anisole are preferable, and as the acid used, a fluoride organic acid such as trifluoroacetic acid is used.
The reaction temperature and reaction time will vary depending on the starting material, solvent, acid used, etc., but are usually carried out at room temperature for 30 minutes to 10 hours.
When the protecting group of the carboxy group is an aralkyl group or a halogeno lower alkyl group, it is usually removed by reduction in a solvent.
As the reducing method, when the protecting group of the carboxy group is a halogeno lower alkyl group, a method by chemical reduction such as zinc-acetic acid is preferable, and when it is an aralkyl group, palladium carbon or platinum can be used. It is carried out by a catalytic reduction method using such a catalyst, or by a chemical reduction method using an alkali metal sulfide such as potassium sulfide and sodium sulfide.
The solvent used is not particularly limited as long as it is not involved in this reaction, but is limited to alcohols such as metanol and etanol; ethers such as tetrahydrofuran and dioxane; acetic acid. Such fatty acids or a mixed solvent of these organic solvents and water are suitable.
The reaction temperature and reaction time vary depending on the starting material, solvent, reduction method, etc., but are usually carried out at around 0 ° C. to room temperature (for example, 25 ° C.) for 5 minutes to 12 hours.
When the protecting group of the carboxy group is an alkoxymethyl group, it is usually removed by treatment with an acid in a solvent.
The acid used is not particularly limited as long as it is usually used as a blended acid, but is preferably an inorganic acid such as hydrochloric acid or sulfuric acid or an organic acid such as acetic acid or paratoluenesulfonic acid. ..
The solvent used is not particularly limited as long as it does not participate in this reaction, but alcohols such as metanol and etanol; ethers such as tetrahydrofuran and dioxane, or these. A mixed solvent of an organic solvent and water is suitable.
The reaction temperature and reaction time vary depending on the starting material, the solvent, the type of acid used, and the like, but are usually carried out at 0 ° C. to 50 ° C. for 10 minutes to 18 hours.
ステップＤ：
Figure JPOXMLDOC01-appb-C000028
 　上図において、Ｒ１、Ｒ２、Ｒ３、Ｒ４、Ｒ５、Ｒ６、Ｌ、ＹおよびＸは前記と同意義を示す。
　Ｚは、脱離基を示す。
　「脱離基」とは、通常、求核残基として脱離する基であれば特に限定はないが、好適には、塩素、臭素、沃素のようなハロゲン原子；メトキシカルボニルオキシ、エトキシカルボニルオキシのような低級アルコキシカルボニルオキシ基；アセトキシ、プロピオニルオキシのようなアルキルカルボニルオキシ基；クロロアセチルオキシ、ジクロロアセチルオキシ、トリクロロアセチルオキシ、トリフルオロアセチルオキシのようなハロゲン化アルキルカルボニルオキシ基、メトキシアセチルオキシのような低級アルコキシアルキルカルボニルオキシ基、（Ｅ）－２－メチル－２－ブテノイルオキシのような不飽和アルキルカルボニルオキシ基等の脂肪族アシルオキシ基；ベンゾイルオキシのようなアリ－ルカルボニルオキシ基、２－ブロモベンゾイルオキシ、４－クロロベンゾイルオキシのようなハロゲン化アリ－ルカルボニルオキシ基、２，４，６－トリメチルベンゾイルオキシ、４－トルオイルオキシのような低級アルキル化アリ－ルカルボニルオキシ基、４－アニソイルオキシのような低級アルコキシ化アリ－ルカルボニルオキシ基、４－ニトロベンゾイルオキシ、２－ニトロベンゾイルオキシのようなニトロ化アリ－ルカルボニルオキシ基等の芳香族アシルオキシ基；トリクロロメチルオキシのようなトリハロゲノメチルオキシ基；メタンスルホニルオキシ、エタンスルホニルオキシのような低級アルカンスルホニルオキシ基；トリフルオロメタンスルホニルオキシ、ペンタフルオロエタンスルホニルオキシのようなハロゲノ低級アルカンスルホニルオキシ基；ベンゼンスルホニルオキシ、ｐ－トルエンスルホニルオキシ、ｐ－ニトロベンゼンスルホニルオキシのようなアリ－ルスルホニルオキシ基を挙げることができ、更に好適には、ハロゲン原子である。 Step D:
Figure JPOXMLDOC01-appb-C000028
 In the above figure, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , L, Y, and X have the same meanings as described above.
Z indicates a leaving group.
The "desorbing group" is usually not particularly limited as long as it is a group desorbed as a nucleophilic residue, but is preferably a halogen atom such as chlorine, bromine, or nitrous; methoxycarbonyloxy, ethoxycarbonyloxy. Lower alkoxycarbonyloxy groups such as; alkylcarbonyloxy groups such as acetoxy, propionyloxy; halogenated alkylcarbonyloxy groups such as chloroacetyloxy, dichloroacetyloxy, trichloroacetyloxy, trifluoroacetyloxy, methoxyacetyloxy Lower alkoxyalkylcarbonyloxy groups such as (E) aliphatic acyloxy groups such as unsaturated alkylcarbonyloxy groups such as (E) -2-methyl-2-butenoyloxy; allylcarbonyloxy groups such as benzoyloxy, 2 -Halogenized allylcarbonyloxy groups such as bromobenzoyloxy, 4-chlorobenzoyloxy, lower alkylated allylcarbonyloxy groups such as 2,4,6-trimethylbenzoyloxy and 4-toluoleoxy, Aromatic acyloxy groups such as lower alkoxylated allylcarbonyloxy groups such as 4-anisoyleoxy, nitrated allylcarbonyloxy groups such as 4-nitrobenzoyloxy and 2-nitrobenzoyloxy; trichloromethyloxy Trihalogenomethyloxy groups such as; lower alkanesulfonyloxy groups such as methanesulfonyloxy, ethanesulfonyloxy; halogeno lower alkanesulfonyloxy groups such as trifluoromethanesulfonyloxy, pentafluoroethanesulfonyloxy; benzenesulfonyloxy, p. -Alkane sulfonyloxy groups such as toluenesulfonyloxy and p-nitrobenzenesulfonyloxy can be mentioned, with more preferred halogen atoms.
　化合物（６）（たとえば、９．４ミリモル、１当量）、化合物（７）（９．９ミリモル、１．０５当量）およびＫ２ＣＯ３（１４．１５ミリモル、１．５当量）を１０ｍｌのＤＭＦに溶解し、混合物を、８０Ｃで、１８時間撹拌した。
　塩基としては、通常の反応において塩基として使用されるものであれば、特に限定はないが、好適には、炭酸ナトリウム、炭酸カリウム、炭酸リチウムのようなアルカリ金属炭酸塩類；炭酸水素ナトリウム、炭酸水素カリウム、炭酸水素リチウムのようなアルカリ金属炭酸水素塩類で、最も好適には、炭酸カリウムである。
　冷水（たとえば１００ｍｌ）を加え、沈殿物を濾過した。白色沈殿物を乾燥させ、ＤＭＦ（たとえば８ｍｌ）から再結晶させた。沈殿物を濾過し、ｉ－ＰｒＯＨ（たとえば１５ｍｌ）で洗浄し、白色の沈殿物を乾燥させ、目的化合物（８）が、たとえば収率４０％で得られた（ＮＭＲによる純度９５％、ＬＣＭＳによる純度９６％）。
　なお、所望により、保護基を定法に従って脱保護した。
　保護基の除去はその種類によって異なるが、一般にこの分野の技術において周知の方法によって以下の様に実施される。 10 ml of compound (6) (eg, 9.4 mmol, 1 eq), compound (7) (9.9 mmol, 1.05 eq) and K2 CO 3 ( 14.15 mmol, 1.5 eq). Dissolved in DMF, the mixture was stirred at 80 C for 18 hours.
The base is not particularly limited as long as it is used as a base in a normal reaction, but is preferably alkali metal carbonates such as sodium carbonate, potassium carbonate and lithium carbonate; sodium hydrogen carbonate and hydrogen carbonate. Alkali metal hydrogen carbonates such as potassium and lithium hydrogen carbonate, most preferably potassium carbonate.
Cold water (eg 100 ml) was added and the precipitate was filtered. The white precipitate was dried and recrystallized from DMF (eg 8 ml). The precipitate was filtered, washed with i-PrOH (eg 15 ml) and dried to dry the white precipitate to give the compound (8) of interest, for example in 40% yield (95% purity by NMR, by LCMS). Purity 96%).
If desired, the protecting group was deprotected according to a conventional method.
Removal of protecting groups varies by type, but is generally carried out by methods well known in the art in the art as follows.
　カルボキシ基の保護基の除去は、前述の通り行うことができる。 The protecting group of the carboxy group can be removed as described above.
　アミノ基の保護基として、シリル基を使用した場合には、通常、弗化テトラブチルアンモニウムのような弗素アニオンを生成する化合物で処理することにより除去される。
　反応溶媒は、反応を阻害しないものであれば特に限定はないが、テトラヒドロフラン、ジオキサンのようなエ－テル類が好適である。
　反応温度及び反応時間は、特に限定はないが、通常、室温で１０時間乃至１８時間反応させる。
　アミノ基の保護基が、脂肪族アシル基、芳香族アシル基、アルコキシカルボニル基又はシッフ塩基を形成する置換されたメチレン基である場合には、水性溶媒の存在下に、酸又は塩基で処理することにより除去することができる。
　使用される酸としては、通常酸として使用されるもので、反応を阻害しないものであれば特に限定はないが、好適には、塩酸、硫酸、リン酸、臭化水素酸のような無機酸が用いられ、使用される塩基としては、化合物の他の部分に影響を与えないものであれば特に限定はないが、好適には、ナトリウムメトキシドのような金属アルコキシド類、炭酸ナトリウム、炭酸カリウム、炭酸リチウムのようなアルカリ金属炭酸塩類、水酸化ナトリウム、水酸化カリウム、水酸化リチウムのようなアルカリ金属水酸化物類又はアンモニア水、濃アンモニア－メタノ－ルのようなアンモニア類が用いられる。
　使用される溶媒としては、通常の加水分解反応に使用されるものであれば特に限定はなく、水；メタノ－ル、エタノ－ル、ｎ－プロパノ－ルのようなアルコ－ル類；テトラヒドロフラン、ジオキサンのようなエ－テル類等の有機溶媒又は水と上記有機溶媒との混合溶媒が好適である。
　反応温度及び反応時間は、出発物質、溶媒及び使用される酸若しくは塩基等により異なり、特に限定はないが、副反応を抑制するために、通常は０℃乃至１５０℃で、１時間乃至１０時間実施される。
　アミノ基の保護基が、アラルキル基又はアラルキルオキシカルボニル基である場合には、通常、溶媒中で、還元剤と接触させることにより（好適には、触媒下に常温にて接触還元）除去する方法又は酸化剤を用いて除去する方法が好適である。
　接触還元による除去において使用される溶媒としては、本反応に関与しないものであれば特に限定はないが、メタノ－ル、エタノ－ル、イソプロパノ－ルのようなアルコ－ル類、ジエチルエ－テル、テトラヒドロフラン、ジオキサンのようなエ－テル類、トルエン、ベンゼン、キシレンのような芳香族炭化水素類、ヘキサン、シクロヘキサンのような脂肪族炭化水素類、酢酸エチル、酢酸プロピルのようなエステル類、酢酸のような脂肪酸類又はこれらの有機溶媒と水との混合溶媒が好適である。
　使用される触媒としては、通常、接触還元反応に使用されるものであれば、特に限定はないが、好適には、パラジウム炭素、ラネ－ニッケル、酸化白金、白金黒、ロジウム－酸化アルミニウム、トリフェニルホスフィン－塩化ロジウム、パラジウム－硫酸バリウムが用いられる。
　圧力は、特に限定はないが、通常１気圧乃至１０気圧で行なわれる。
　反応温度及び反応時間は、出発物質、溶媒及び触媒の種類等により異なるが、通常、０℃乃至１００℃で、５分乃至２４時間実施される。
　酸化による除去において使用される溶媒としては、本反応に関与しないものであれば特に限定はないが、好適には、含水有機溶媒である。
　このような有機溶媒として好適には、アセトンのようなケトン類、メチレンクロリド、クロロホルム、四塩化炭素のようなハロゲン化炭化水素類、アセトニトリルのようなニトリル類、ジエチルエ－テル、テトラヒドロフラン、ジオキサンのようなエ－テル類、ジメチルホルムアミド、ジメチルアセトアミド、ヘキサメチルホスホロトリアミドのようなアミド類及びジメチルスルホキシドのようなスルホキシド類を挙げることができる。
　使用される酸化剤としては、酸化に使用される化合物であれば特に限定はないが、好適には、過硫酸カリウム、過硫酸ナトリウム、アンモニウムセリウムナイトレイト（ＣＡＮ）、２，３－ジクロロ－５，６－ジシアノ－ｐ－ベンゾキノン（ＤＤＱ）が用いられる。
　反応温度及び反応時間は、出発物質、溶媒及び触媒の種類等により異なるが、通常、０℃乃至１５０℃で、１０分乃至２４時間実施される。
　アミノ基の保護基がアルケニルオキシカルボニル基である場合は、通常、アミノ基の保護基が前記の脂肪族アシル基、芳香族アシル基、アルコキシカルボニル基又はシッフ塩基を形成する置換されたメチレン基である場合の除去反応の条件と同様にして、塩基と処理することにより達成される。
　尚、アリルオキシカルボニルの場合は、特に、パラジウム、及びトリフェニルホスフィン若しくはニッケルテトラカルボニルを使用して除去する方法が簡便で、副反応が少なく実施することができる。 When a silyl group is used as the protecting group for the amino group, it is usually removed by treatment with a compound that produces a fluorine anion, such as tetrabutylammonium fluoride.
The reaction solvent is not particularly limited as long as it does not inhibit the reaction, but ethers such as tetrahydrofuran and dioxane are suitable.
The reaction temperature and reaction time are not particularly limited, but the reaction is usually carried out at room temperature for 10 to 18 hours.
If the protecting group for the amino group is an aliphatic acyl group, an aromatic acyl group, an alkoxycarbonyl group or a substituted methylene group forming a Schiff base, it is treated with an acid or a base in the presence of an aqueous solvent. It can be removed by this.
The acid used is usually used as an acid and is not particularly limited as long as it does not inhibit the reaction, but is preferably an inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid and hydrobromic acid. Is used, and the base used is not particularly limited as long as it does not affect other parts of the compound, but preferably metal alkoxides such as sodium methoxydo, sodium carbonate, and potassium carbonate. , Alkaline metal carbonates such as lithium carbonate, alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, lithium hydroxide or ammonia water, ammonia such as concentrated ammonia-methanol are used.
The solvent used is not particularly limited as long as it is used for a normal hydrolysis reaction, and water; alcohols such as metanol, etanol, n-propanol; tetrahydrofuran, An organic solvent such as ethylene such as dioxane or a mixed solvent of water and the above organic solvent is suitable.
The reaction temperature and reaction time vary depending on the starting material, solvent, acid or base used, etc., and are not particularly limited, but are usually 0 ° C. to 150 ° C. for 1 hour to 10 hours in order to suppress side reactions. Will be implemented.
When the protecting group of the amino group is an aralkyl group or an aralkyloxycarbonyl group, it is usually removed by contacting with a reducing agent in a solvent (preferably, catalytic reduction under a catalyst at room temperature). Alternatively, a method of removing with an oxidizing agent is preferable.
The solvent used in the removal by catalytic reduction is not particularly limited as long as it does not participate in this reaction, but is limited to hydrocarbons such as methanol, ethane, isopropanol, diethyl ester, and the like. Solvents, ethers such as dioxane, aromatic hydrocarbons such as toluene, benzene and xylene, aliphatic hydrocarbons such as hexane and cyclohexane, esters such as ethyl acetate and propyl acetate, acetic acid. Such fatty acids or a mixed solvent of these organic solvents and water are suitable.
The catalyst used is not particularly limited as long as it is usually used for a catalytic reduction reaction, but is preferably palladium carbon, lane-nickel, platinum oxide, platinum black, rhodium-aluminum oxide, or tri. Triphenylphosphine-rhodium chloride and palladium-barium sulfate are used.
The pressure is not particularly limited, but is usually 1 to 10 atm.
The reaction temperature and reaction time vary depending on the type of starting material, solvent, catalyst, etc., but are usually carried out at 0 ° C. to 100 ° C. for 5 minutes to 24 hours.
The solvent used for removal by oxidation is not particularly limited as long as it does not participate in this reaction, but is preferably a water-containing organic solvent.
Suitable such organic solvents are ketones such as acetone, methylene chloride, chloroform, halogenated hydrocarbons such as carbon tetrachloride, nitriles such as acetonitrile, diethyl ether, tetrahydrofuran, dioxane and the like. Etels, amides such as dimethylformamide, dimethylacetamide, hexamethylphosphorotriamide and sulfoxides such as dimethylsulfoxide can be mentioned.
The oxidizing agent used is not particularly limited as long as it is a compound used for oxidation, but preferably potassium persulfate, sodium persulfate, ammonium cerium nitrate (CAN), 2,3-dichloro-5. , 6-Dicyano-p-benzoquinone (DDQ) is used.
The reaction temperature and reaction time vary depending on the type of starting material, solvent, catalyst, etc., but are usually carried out at 0 ° C. to 150 ° C. for 10 minutes to 24 hours.
When the protecting group for the amino group is an alkenyloxycarbonyl group, the protecting group for the amino group is usually a substituted methylene group forming the aliphatic acyl group, aromatic acyl group, alkoxycarbonyl group or Schiff base described above. It is achieved by treating with a base, similar to the conditions of the removal reaction in some cases.
In the case of allyloxycarbonyl, a method of removing palladium and triphenylphosphine or nickel tetracarbonyl is particularly convenient and can be carried out with few side reactions.
　水酸基の保護基として、シリル基を使用した場合には、通常、弗化テトラブチルアンモニウム、弗化水素酸、弗化水素酸－ピリジン、弗化カリウムのような弗素アニオンを生成する化合物で処理するか、又は、酢酸、メタンスルホン酸、パラトルエンスルホン酸、トリフルオロ酢酸、トリフルオロメタンスルホン酸のような有機酸又は塩酸のような無機酸で処理することにより除去できる。
　使用される溶媒としては、反応を阻害せず、出発物質をある程度溶解するものであれば特に限定はないが、好適には、ジエチルエ－テル、ジイソプロピルエ－テル、テトラヒドロフラン、ジオキサン、ジメトキシエタン、ジエチレングリコールジメチルエーテルのようなエ－テル類；アセトニトリル、イソブチロニトリルのようなニトリル類；水；酢酸のような有機酸及びこれらの混合溶媒を挙げることができる。
　反応温度及び反応時間は、特に限定はないが、通常、０℃乃至１００℃（好適には、１０℃乃至３０℃）で、１時間乃至２４時間実施される。
　水酸基の保護基が、アラルキル基又はアラルキルオキシカルボニル基である場合には、通常、溶媒中、還元剤と接触させることにより（好適には、触媒下に常温にて接触還元）除去する方法又は酸化剤を用いて除去する方法が好適である。
　接触還元による除去において使用される溶媒としては、本反応に関与しないものであれば特に限定はないが、メタノ－ル、エタノ－ル、イソプロパノ－ルのようなアルコ－ル類、ジエチルエ－テル、テトラヒドロフラン、ジオキサンのようなエ－テル類、トルエン、ベンゼン、キシレンのような芳香族炭化水素類、ヘキサン、シクロヘキサンのような脂肪族炭化水素類、酢酸エチル、酢酸プロピルのようなエステル類、ホルムアミド、ジメチルホルムアミド、ジメチルアセトアミド、Ｎ－メチル－２－ピロリドン、ヘキサメチルホスホロトリアミドのようなアミド類、蟻酸、酢酸のような脂肪酸類、水、又はこれらの混合溶媒が好適であり、更に好適には、アルコ－ル類、脂肪酸類、アルコ－ル類とエーテル類との混合溶媒、アルコ－ル類と水との混合溶媒、又は、脂肪酸類と水との混合溶媒である。
　使用される触媒としては、通常、接触還元反応に使用されるものであれば、特に限定はないが、好適には、パラジウム炭素、パラジウム黒、ラネ－ニッケル、酸化白金、白金黒、ロジウム－酸化アルミニウム、トリフェニルホスフィン－塩化ロジウム、パラジウム－硫酸バリウムが用いられる。
　圧力は、特に限定はないが、通常１気圧乃至１０気圧で行なわれる。
　反応温度及び反応時間は、出発物質、溶媒及び触媒の種類等により異なるが、通常、０℃乃至１００℃（好適には、２０℃乃至７０℃）、５分乃至４８時間（好適には、１時間乃至２４時間）である。
　酸化による除去において使用される溶媒としては、本反応に関与しないものであれば特に限定はないが、好適には、含水有機溶媒である。
　このような有機溶媒として好適には、アセトンのようなケトン類、メチレンクロリド、クロロホルム、四塩化炭素のようなハロゲン化炭化水素類、アセトニトリルのようなニトリル類、ジエチルエ－テル、テトラヒドロフラン、ジオキサンのようなエ－テル類、ジメチルホルムアミド、ジメチルアセトアミド、ヘキサメチルホスホロトリアミドのようなアミド類及びジメチルスルホキシドのようなスルホキシド類を挙げることができる。
　使用される酸化剤としては、酸化に使用される化合物であれば特に限定はないが、好適には、過硫酸カリウム、過硫酸ナトリウム、アンモニウムセリウムナイトレイト（ＣＡＮ）、２，３－ジクロロ－５，６－ジシアノ－ｐ－ベンゾキノン（ＤＤＱ）が用いられる。
　反応温度及び反応時間は、出発物質、溶媒及び触媒の種類等により異なるが、通常、０℃乃至１５０℃で、１０分乃至２４時間実施される。
　又、液体アンモニア中若しくはメタノ－ル、エタノ－ルのようなアルコ－ル中において、－７８℃乃至－２０℃で、金属リチウム、金属ナトリウムのようなアルカリ金属類を作用させることによっても除去できる。
　更に、溶媒中、塩化アルミニウム－沃化ナトリウム、又はトリメチルシリルイオダイドのようなアルキルシリルハライド類を用いても除去することができる。
　使用される溶媒としては、本反応に関与しないものであれば特に限定はないが、好適には、アセトニトリルのようなニトリル類、メチレンクロリド、クロロホルムのようなハロゲン化炭化水素類又はこれらの混合溶媒が使用される。
　反応温度及び反応時間は、出発物質、溶媒等により異なるが、通常は０℃乃至５０℃で、５分乃至３日間実施される。
　水酸基の保護基が、脂肪族アシル基、芳香族アシル基又はアルコキシカルボニル基である場合には、溶媒中、塩基で処理することにより除去される。
　使用される塩基としては、化合物の他の部分に影響を与えないものであれば特に限定はないが、好適にはナトリウムメトキシドのような金属アルコキシド類；炭酸ナトリウム、炭酸カリウム、炭酸リチウムのようなアルカリ金属炭酸塩；水酸化ナトリウム、水酸化カリウム、水酸化リチウム、水酸化バリウムのようなアルカリ金属水酸化物又はアンモニア水、濃アンモニア－メタノ－ルのようなアンモニア類が用いられる。
　使用される溶媒としては、通常の加水分解反応に使用されるものであれば特に限定はなく、水；メタノ－ル、エタノ－ル、ｎ－プロパノ－ルのようなアルコ－ル類、テトラヒドロフラン、ジオキサンのようなエ－テル類等の有機溶媒又は水と上記有機溶媒との混合溶媒が好適である。
　反応温度及び反応時間は、出発物質、溶媒及び使用される塩基等により異なり特に限定はないが、副反応を抑制するために、通常は０℃乃至１５０℃で、１時間乃至１０時間実施される。
　水酸基の保護基が、アルコキシメチル基、テトラヒドロピラニル基、テトラヒドロチオピラニル基、テトラヒドロフラニル基、テトラヒドロチオフラニル基又は置換されたエチル基である場合には、通常、溶媒中、酸で処理することにより除去される。
　使用される酸としては、通常、ブレンステッド酸又はルイス酸として使用されるものであれば特に限定はなく、好適には、塩化水素；塩酸、硫酸、硝酸のような無機酸；又は酢酸、トリフルオロ酢酸、メタンスルホン酸、ｐ－トルエンスルホン酸のような有機酸等のブレンステッド酸：三弗化ホウ素のようなルイス酸であるが、ダウエックス５０Ｗのような強酸性の陽イオン交換樹脂も使用することができる。
　使用される溶媒としては、反応を阻害せず、出発物質をある程度溶解するものであれば特に限定はないが、好適には、ヘキサン、ヘプタン、リグロイン、石油エーテルのような脂肪族炭化水素類；ベンゼン、トルエン、キシレンのような芳香族炭化水素類；メチレンクロリド、クロロホルム、四塩化炭素、ジクロロエタン、クロロベンゼン、ジクロロベンゼンのようなハロゲン化炭化水素類；蟻酸エチル、酢酸エチル、酢酸プロピル、酢酸ブチル、炭酸ジエチルのようなエステル類；ジエチルエ－テル、ジイソプロピルエ－テル、テトラヒドロフラン、ジオキサン、ジメトキシエタン、ジエチレングリコールジメチルエーテルのようなエ－テル類；メタノ－ル、エタノ－ル、ｎ－プロパノ－ル、イソプロパノ－ル、ｎ－ブタノ－ル、イソブタノ－ル、ｔｅｒｔ－ブタノ－ル、イソアミルアルコ－ル、ジエチレングリコール、グリセリン、オクタノール、シクロヘキサノール、メチルセロソルブ、のようなアルコ－ル類；アセトン、メチルエチルケトン、メチルイソブチルケトン、イソホロン、シクロヘキサノンのようなケトン類；水、又は、これらの混合溶媒が好適であり、更に好適には、ハロゲン化炭化水素類、エステル類又はエ－テル類である。
　反応温度及び反応時間は、出発物質、溶媒及び使用される酸の種類・濃度等により異なるが、通常は－１０℃乃至１００℃（好適には、－５℃乃至５０℃）で、５分乃至４８時間（好適には、３０分乃至１０時間）である。
　水酸基の保護基が、アルケニルオキシカルボニル基である場合は、通常、水酸基の保護基が前記の脂肪族アシル基、芳香族アシル基又はアルコキシカルボニル基である場合の除去反応の条件と同様にして、塩基と処理することにより達成される。
　尚、アリルオキシカルボニルの場合は、特にパラジウム、及びトリフェニルホ
　スフィン、又はビス（メチルジフェニルホスフィン）（１，５－シクロオクタジエン）イリジウム（Ｉ）・ヘキサフルオロホスフェ－トを使用して除去する方法が簡便で、副反応が少なく実施することができる。 When a silyl group is used as the protective group for the hydroxyl group, it is usually treated with a compound that produces a fluorinated anion such as tetrabutylammonium fluoride, hydrofluoric acid, hydrofluoric acid-pyridine, and potassium fluoride. Alternatively, it can be removed by treatment with an organic acid such as acetic acid, methanesulfonic acid, paratoluenesulfonic acid, trifluoroacetic acid, trifluoromethanesulfonic acid or an inorganic acid such as hydrochloric acid.
The solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, but is preferably diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, or diethylene glycol. Ethers such as dimethyl ether; acetonitrile, nitriles such as isobutyronitrile; water; organic acids such as acetic acid and mixed solvents thereof can be mentioned.
The reaction temperature and reaction time are not particularly limited, but are usually carried out at 0 ° C. to 100 ° C. (preferably 10 ° C. to 30 ° C.) for 1 hour to 24 hours.
When the protecting group of the hydroxyl group is an aralkyl group or an aralkyloxycarbonyl group, it is usually removed by contacting it with a reducing agent in a solvent (preferably, catalytically reducing it under a catalyst at room temperature) or oxidation. A method of removing with an agent is preferable.
The solvent used in the removal by catalytic reduction is not particularly limited as long as it does not participate in this reaction, but alcohols such as metanol, etanol, isopropanol, diethyl ether, and the like. Ethyls such as tetrahydrofuran, dioxane, aromatic hydrocarbons such as toluene, benzene, xylene, hexane, aliphatic hydrocarbons such as cyclohexane, esters such as ethyl acetate, propyl acetate, formamides, Amidos such as dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone, hexamethylphosphorotriamide, fatty acids such as formic acid and acetic acid, water, or a mixed solvent thereof are suitable, and more preferably. , Alcohols, fatty acids, mixed solvents of alcohols and ethers, mixed solvents of alcohols and water, or mixed solvents of fatty acids and water.
The catalyst used is not particularly limited as long as it is usually used for a catalytic reduction reaction, but is preferably palladium carbon, palladium black, lane-nickel, platinum oxide, platinum black, or rhodium-oxidation. Aluminum, triphenylphosphine-rhodium chloride, palladium-barium sulfate are used.
The pressure is not particularly limited, but is usually 1 to 10 atm.
The reaction temperature and reaction time vary depending on the type of starting material, solvent, catalyst, etc., but are usually 0 ° C to 100 ° C (preferably 20 ° C to 70 ° C), 5 minutes to 48 hours (preferably 1). Hours to 24 hours).
The solvent used for removal by oxidation is not particularly limited as long as it does not participate in this reaction, but is preferably a water-containing organic solvent.
Suitable such organic solvents are ketones such as acetone, methylene chloride, chloroform, halogenated hydrocarbons such as carbon tetrachloride, nitriles such as acetonitrile, diethyl ether, tetrahydrofuran, dioxane and the like. Etels, amides such as dimethylformamide, dimethylacetamide, hexamethylphosphorotriamide and sulfoxides such as dimethylsulfoxide can be mentioned.
The oxidizing agent used is not particularly limited as long as it is a compound used for oxidation, but preferably potassium persulfate, sodium persulfate, ammonium cerium nitrate (CAN), 2,3-dichloro-5. , 6-Dicyano-p-benzoquinone (DDQ) is used.
The reaction temperature and reaction time vary depending on the type of starting material, solvent, catalyst, etc., but are usually carried out at 0 ° C. to 150 ° C. for 10 minutes to 24 hours.
It can also be removed by allowing alkali metals such as metallic lithium and metallic sodium to act at −78 ° C. to −20 ° C. in liquid ammonia or in alcohols such as metanol and ethanol. ..
Further, it can be removed by using an aluminum chloride-sodium iodide or an alkylsilyl halide such as trimethylsilyliodide in a solvent.
The solvent used is not particularly limited as long as it does not participate in this reaction, but preferably nitriles such as acetonitrile, methylene chloride, halogenated hydrocarbons such as chloroform, or a mixed solvent thereof. Is used.
The reaction temperature and reaction time vary depending on the starting material, solvent, etc., but are usually carried out at 0 ° C to 50 ° C for 5 minutes to 3 days.
When the protecting group of the hydroxyl group is an aliphatic acyl group, an aromatic acyl group or an alkoxycarbonyl group, it is removed by treatment with a base in a solvent.
The base used is not particularly limited as long as it does not affect other parts of the compound, but is preferably metal alkoxides such as sodium methoxydo; sodium carbonate, potassium carbonate, lithium carbonate and the like. Alkali metal carbonates; alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, lithium hydroxide, barium hydroxide or aqueous ammonia, ammonia such as concentrated ammonia-methanol are used.
The solvent used is not particularly limited as long as it is used for a normal hydrolysis reaction, and water; alcohols such as metanol, etanol, n-propanol, tetrahydrofuran, and the like. An organic solvent such as ethylene such as dioxane or a mixed solvent of water and the above organic solvent is suitable.
The reaction temperature and reaction time vary depending on the starting material, solvent, base used, etc., and are not particularly limited, but are usually carried out at 0 ° C. to 150 ° C. for 1 hour to 10 hours in order to suppress side reactions. ..
When the protecting group for the hydroxyl group is an alkoxymethyl group, a tetrahydropyranyl group, a tetrahydrothiopyranyl group, a tetrahydrofuranyl group, a tetrahydrothiofuranyl group or a substituted ethyl group, it is usually treated with an acid in a solvent. It is removed by doing.
The acid used is not particularly limited as long as it is usually used as Bronsted acid or Lewis acid, and is preferably hydrogen chloride; an inorganic acid such as hydrochloric acid, sulfuric acid or nitric acid; or acetic acid or tri. Blended acids such as organic acids such as fluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid: Lewis acids such as boron trifluoride, but also strongly acidic cation exchange resins such as Dawex 50W. Can be used.
The solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, but preferably aliphatic hydrocarbons such as hexane, heptane, ligroin, and petroleum ether; Aromatic hydrocarbons such as benzene, toluene and xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, dichlorobenzene; ethyl formate, ethyl acetate, propyl acetate, butyl acetate, Esters such as diethyl carbonate; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol dimethyl ether; metall, etanol, n-propanol, isopropano- Alcoles such as le, n-butanol, isobutanol, tert-butanol, isoamyl alcohol, diethylene glycol, glycerin, octanol, cyclohexanol, methyl cellosolve; acetone, methyl ethyl ketone, methyl isobutyl ketone. , Isophorone, ketones such as cyclohexanone; water or a mixed solvent thereof is suitable, and more preferably halogenated hydrocarbons, esters or ethers.
The reaction temperature and reaction time vary depending on the type and concentration of the starting material, solvent and acid used, but are usually -10 ° C to 100 ° C (preferably -5 ° C to 50 ° C) for 5 minutes to 5 minutes. 48 hours (preferably 30 minutes to 10 hours).
When the protecting group of the hydroxyl group is an alkenyloxycarbonyl group, the conditions for the removal reaction when the protecting group of the hydroxyl group is an aliphatic acyl group, an aromatic acyl group or an alkoxycarbonyl group are usually the same. Achieved by treating with a base.
In the case of allyloxycarbonyl, a method of removing it using palladium, triphenylphosphine, or bis (methyldiphenylphosphine) (1,5-cyclooctadiene) iridium (I) / hexafluorophosphate. Is simple and can be carried out with few side reactions.
　尚、上記のようなアミノ基の保護基を除去する操作によって、水酸基の保護基が同時に除去されることもある。 In addition, the protecting group of the hydroxyl group may be removed at the same time by the operation of removing the protecting group of the amino group as described above.
　上記の水酸基の保護基の除去反応及びアミノ基の保護基の除去反応は、順不同で希望する除去反応を順次実施することができる。 As for the above-mentioned removal reaction of the protecting group of the hydroxyl group and the removal reaction of the protecting group of the amino group, the desired removal reaction can be sequentially carried out in no particular order.
　また、ＡがＳである化合物は、下記のように、ステップＡで製造した化合物（４：ＡはＳを示す。）を、ステップＢによらずに、直接、ステップＣを行うことにより、同様に製造することができる。
Figure JPOXMLDOC01-appb-C000029
 Further, the compound in which A is S is similarly obtained by directly performing step C on the compound produced in step A (4: A indicates S) without depending on step B, as described below. Can be manufactured to.
Figure JPOXMLDOC01-appb-C000029
　なお、上記化合物（５）および（７）のベンゼン環の置換基を交換して、上記の工程を行うことにより、部分構造（ＩＩ）について、逆の配置の構造の化合物を製造することができる。
　また、アミド結合を考慮することにより、部分構造（Ｉ）、（ＩＩ）および（ＩＩＩ）が任意順番でリンカーに結合している本発明の化合物を製造することができる。 By exchanging the substituents of the benzene rings of the above compounds (5) and (7) and performing the above steps, a compound having the reverse arrangement of the partial structure (II) can be produced. ..
Further, by considering the amide bond, the compound of the present invention in which the partial structures (I), (II) and (III) are bonded to the linker in an arbitrary order can be produced.
　反応終了後、本反応の目的化合物（Ｉ）は常法に従って、反応混合物から採取される。
　例えば、反応混合物を適宜中和し、又、不溶物が存在する場合には濾過により除去した後、水と酢酸エチルのような混和しない有機溶媒を加え、水等で洗浄後、目的化合物を含む有機層を分離し、無水硫酸マグネシウム等で乾燥後、溶剤を留去することによって得られる。
　得られた目的化合物は必要ならば、常法、例えば再結晶、再沈殿、又は、通常、有機化合物の分離精製に慣用されている方法、例えば、シリカゲル、アルミナ、マグネシウムーシリカゲル系のフロリジルのような担体を用いた吸着カラムクロマトグラフィー法；セファデックスＬＨ－２０（ファルマシア社製）、アンバーライトＸＡＤ－１１（ローム・アンド・ハース社製）、ダイヤイオンＨＰ－２０（三菱化成社製）などの担体を用いた分配カラムクロマトグラフィー等の合成吸着剤を使用する方法、イオン交換クロマトを使用する方法、又は、シリカゲル若しくはアルキル化シリカゲルによる順相・逆相カラムクロマトグラフィー法（好適には、高速液体クロマトグラフィーである。）を適宜組合せ、適切な溶離剤で溶出することによって分離、精製することができる。
　尚、異性体を分離する必要がある場合には、上記各工程の反応終了後、又は、所望工程の終了後の適切な時期に、上記分離精製手段によって行なうことができる。 After completion of the reaction, the target compound (I) of this reaction is collected from the reaction mixture according to a conventional method.
For example, the reaction mixture is appropriately neutralized, and if insoluble matter is present, it is removed by filtration, an immiscible organic solvent such as ethyl acetate is added to water, and the mixture is washed with water or the like to contain the target compound. It is obtained by separating the organic layer, drying it with anhydrous magnesium sulfate or the like, and then distilling off the solvent.
If necessary, the obtained target compound can be obtained by a conventional method such as recrystallization, reprecipitation, or a method commonly used for separation and purification of an organic compound, for example, silica gel, alumina, magnesium-silicalate-based fluorodyl. Adsorption column chromatography method using various carriers; Cefadex LH-20 (manufactured by Pharmacia), Amberlite XAD-11 (manufactured by Roam & Haas), Diaion HP-20 (manufactured by Mitsubishi Kasei), etc. A method using a synthetic adsorbent such as partition column chromatography using a carrier, a method using ion exchange chromatography, or a normal phase / reverse phase column chromatography method using silica gel or alkylated silica gel (preferably, high-speed liquid). It can be separated and purified by appropriately combining (chromatographic)) and eluting with an appropriate eluent.
When it is necessary to separate the isomers, it can be carried out by the separation and purification means at an appropriate time after the reaction of each of the above steps is completed or after the desired step is completed.
　具体的には、たとえば、化合物（１）（＝化合物Ｃ）は、以下のように、製造することができる。
Figure JPOXMLDOC01-appb-C000030
 　化合物１の製造
（１）ＴＥＡ（５．２３　ｇ、５１．８ミリモル）とアミノ酸（５．３５　ｇ、５１．８ミリモル）の１５０　ｍｌの水溶液に、イソチオシアネート１（１０　ｇ、５１．８０ミリモル、１ｅｑ）のｉ－ＰｒＯＨ（２００　ｍｌ）。反応混合物を３０分間還流した。次に、混合物を冷却し、酸性化（ＨＣｌ）してｐＨ＝２にした。沈殿物を濾過し、乾燥させた。所望の生成物（１１ｇ、ＮＭＲによる純度９５％）を、さらに精製することなく使用した。収率８０％。
（２）１００　ｍｌの水に溶解したＮａＯＨ（３ｅｑ、２．２７ｇ）と酸３（５ｇ、１８．９　ｍｍｏｌ、１　ｅｑ）の溶液を５０℃に加熱しました。次に、混合物を冷却し、Ｈ２Ｏ２（１０当量、水中３５％）を加えた。反応混合物を５０℃で３０分間撹拌した。反応混合物を冷却し、ｐＨ２になるまでＨＯＡｃを加えた。沈殿物を濾過し、乾燥させた。所望の生成物（４．５ｇ、ＮＭＲによる純度９５％）を、さらに精製することなく使用した。収率９６％。
（３）酸４（４．５ｇ、１８．４ミリモル、１当量）をジオキサン（６０ｍＬ）に溶解し、ＣＤＩ（１．１当量、３．２３ｇ）を室温で少しずつ加えた。混合物を５０℃で３時間撹拌してから、アミン６（２．７４ｇ、１８．４ミリモル、１当量）を加えた。次に、混合物を室温で１２時間撹拌した後、冷水（１００ｍｌ）を加え、沈殿物を濾過し、ｉ－ＰｒＯＨ（１０ｍｌ）で洗浄した。白色の沈殿物を乾燥させた。スペクトルは、所望の生成物（５．６ｇ、ＮＭＲによる純度９５％）を示し、これはさらに精製することなく使用された。収率８１％。
（４）化合物６（３．６ｇ、９．４ミリモル、１当量）、化合物７（２．０２ｇ、９．９ミリモル、１．０５当量）およびＫ２ＣＯ３（１．９５ｇ、１４．１５ミリモル、１．５当量）を１０ｍｌのＤＭＦに溶解し、混合物を撹拌した。８０Ｃで１８時間。冷水（１００ｍｌ）を加え、沈殿物を濾過した。白色沈殿物を乾燥させ、ＤＭＦ（８ｍｌ）から再結晶させた。沈殿物を濾過し、ｉ－ＰｒＯＨ（１５ｍｌ）で洗浄した。白色の沈殿物を乾燥させた。標的化合物が２．１ｇの量で得られた（ＮＭＲによる純度９５％、ＬＣＭＳによる純度９６％）、収率４０％。 Specifically, for example, compound (1) (= compound C) can be produced as follows.
Figure JPOXMLDOC01-appb-C000030
 Preparation of Compound 1 (1) Isothiocyanate 1 (10 g, 51.80 mmol) in a 150 ml aqueous solution of TEA (5.23 g, 51.8 mmol) and amino acid (5.35 g, 51.8 mmol). 1, eq) i-PrOH (200 ml). The reaction mixture was refluxed for 30 minutes. The mixture was then cooled and acidified (HCl) to pH = 2. The precipitate was filtered and dried. The desired product (11 g, 95% purity by NMR) was used without further purification. Yield 80%.
(2) A solution of NaOH (3 eq, 2.27 g) and acid 3 (5 g, 18.9 mmol, 1 eq) dissolved in 100 ml of water was heated to 50 ° C. The mixture was then cooled and H2O2 (10 eq, 35% in water) was added. The reaction mixture was stirred at 50 ° C. for 30 minutes. The reaction mixture was cooled and HOAc was added until pH 2 was reached. The precipitate was filtered and dried. The desired product (4.5 g, 95% purity by NMR) was used without further purification. Yield 96%.
(3) Acid 4 (4.5 g, 18.4 mmol, 1 equivalent) was dissolved in dioxane (60 mL), and CDI (1.1 equivalent, 3.23 g) was added little by little at room temperature. The mixture was stirred at 50 ° C. for 3 hours and then amine 6 (2.74 g, 18.4 mmol, 1 eq) was added. The mixture was then stirred at room temperature for 12 hours, then cold water (100 ml) was added, the precipitate was filtered and washed with i-PrOH (10 ml). The white precipitate was dried. The spectrum showed the desired product (5.6 g, 95% purity by NMR), which was used without further purification. Yield 81%.
(4) Compound 6 (3.6 g, 9.4 mmol, 1 equivalent), Compound 7 (2.02 g, 9.9 mmol, 1.05 equivalent) and K2CO3 (1.95 g, 14.15 mmol, 1. 5 eq) was dissolved in 10 ml DMF and the mixture was stirred. 18 hours at 80C. Cold water (100 ml) was added and the precipitate was filtered. The white precipitate was dried and recrystallized from DMF (8 ml). The precipitate was filtered and washed with i-PrOH (15 ml). The white precipitate was dried. The target compound was obtained in an amount of 2.1 g (purity by NMR 95%, purity 96% by LCMS), and the yield was 40%.
　本発明の制癌剤、発がん抑制剤および／または寿命延長剤としては、有効成分であるｍＴＯＲＣ１阻害作用を有する化合物またはその薬理上許容される塩を単独で配合されたものでもよいが、製薬学的に許容しうる担体あるいは製剤用添加物を配合した製剤の形態として提供してもよい。この場合、有効成分であるｍＴＯＲＣ１阻害作用を有する化合物またはその薬理上許容される塩は、例えば、製剤中において０．１～９９．９質量％含有することができる。 As the anticancer agent, carcinogenesis inhibitor and / or life prolonging agent of the present invention, a compound having an mTORC1 inhibitory action as an active ingredient or a pharmaceutically acceptable salt thereof may be blended alone, but pharmaceutically acceptable. It may be provided in the form of a pharmaceutical product containing an acceptable carrier or pharmaceutical additive. In this case, the active ingredient, a compound having an inhibitory effect on mTORC1, or a pharmaceutically acceptable salt thereof, can be contained, for example, in the formulation in an amount of 0.1 to 99.9% by mass.
  製薬学的に許容しうる担体あるいは製剤用添加物としては、特に限定されないが、例えば賦形剤、崩壊剤、崩壊補助剤、結合剤、滑沢剤、コーティング剤、色素、希釈剤、溶解剤、溶解補助剤、等張化剤、ｐH調整剤、安定化剤等が挙げられる。 The pharmaceutically acceptable carrier or pharmaceutical additive is not particularly limited, and is, for example, an excipient, a disintegrant, a disintegrant, a binder, a lubricant, a coating agent, a dye, a diluent, and a solubilizer. , Dissolution aids, tonicity agents, pH adjusters, stabilizers and the like.
  経口投与に適する製剤としては、例えば散剤、粉剤、錠剤、カプセル剤、細粒剤、顆粒剤、液剤またはシロップ剤等を挙げることができる。 Examples of the preparation suitable for oral administration include powders, powders, tablets, capsules, fine granules, granules, liquids, syrups and the like.
　経口投与の場合、微晶質セルロース、クエン酸ナトリウム、炭酸カルシウム、リン酸ジカリウム、グリシンのような種々の賦形剤を、澱粉、好適にはとうもろこし、じゃがいもまたはタピオカの澱粉、およびアルギン酸やある種のケイ酸複塩のような種々の崩壊剤、およびポリビニルピロリドン、蔗糖、ゼラチン、アラビアゴムのような顆粒形成結合剤と共に使用してもよい。また、ステアリン酸マグネシウム、ラウリル硫酸ナトリウム、タルク等の滑沢剤を使用して錠剤を形成してもよい。経口投与用として水性懸濁液および／またはエリキシルにすることもできる。 For oral administration, various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate, glycine, starch, preferably corn, potato or tapioca starch, and alginic acid and certain types. May be used with various disintegrants such as alginic acid compound salt and granule-forming binders such as polyvinylpyrrolidone, starch, gelatin and gum arabic. In addition, tablets may be formed using lubricants such as magnesium stearate, sodium lauryl sulfate, and talc. It can also be an aqueous suspension and / or an elixir for oral administration.
  非経口投与に適する製剤としては、例えば注射剤、坐剤、パップ剤、点鼻剤、点眼剤等を挙げることができる。注射剤は、例えば、静脈内注射、関節内注射、筋肉注射又は皮下注射により投与することができる。 Examples of the preparation suitable for parenteral administration include injections, suppositories, paps, nasal drops, eye drops and the like. The injection can be administered, for example, by intravenous injection, intra-articular injection, intramuscular injection or subcutaneous injection.
　本発明の制癌剤、発がん抑制剤および／または寿命延長剤の投与量は特に限定されず、がんの種類、患者の年齢や症状、投与経路、治療の目的、併用薬剤の有無等の種々の条件に応じて適切な投与量を選択することが可能である。例えば、経口投与の場合には成人（例えば、体重６０ｋｇ）１日当たり有効成分の投与量として、１μｇから１０ｇ程度、好ましくは１００μｇ～１ｇ、より好ましくは１ｍｇから５００ｍｇである。これらの１日投与量は２から４回に分けて投与されてもよい。 The dose of the anticancer agent, carcinogenic inhibitor and / or life-prolonging agent of the present invention is not particularly limited, and various conditions such as the type of cancer, the age and symptoms of the patient, the route of administration, the purpose of treatment, and the presence or absence of concomitant agents. It is possible to select an appropriate dose according to the patient. For example, in the case of oral administration, the daily dose of the active ingredient for an adult (for example, body weight 60 kg) is about 1 μg to 10 g, preferably 100 μg to 1 g, and more preferably 1 mg to 500 mg. These daily doses may be administered in 2 to 4 divided doses.
実験方法
[実施例１] experimental method
[Example 1]
　Ｉｎ　ｓｉｌｉｃｏによる小分子（ｓｍａｌｌ　ｍｏｌｅｃｕｌｅ）の選択（ｉｎ　ｓｉｌｉｃｏ　ｓｅｌｅｃｔｉｏｎ）
　（１）ＺＩＮＣ１５データベース（ｈｔｔｐｓ：／／ｚｉｎｃ１５．ｄｏｃｋｉｎｇ．ｏｒｇ／）から、約７００，０００個のバーチャルリガンド（ＶＬ）を、表１に示す基準で抽出した。
　ＶＬは、Ｌ＃１～Ｌ＃３の３つのグループに分けた。
Figure JPOXMLDOC01-appb-T000031
 　バーチャルリガンド選択（Ｖｉｒｔｕａｌ　Ｌｉｇａｎｄ　Ｓｅｌｅｃｔｉｏｎ：ＶＬＳ）は、ＩＣＭ－Ｐｒｏ３．８ソフトウエア（Ｍｏｌｓｏｆｔ　Ｌ．Ｌ．Ｃ．）により行った。
　ＦＫＢＰ１２－ラパマイシン－ＦＲＢヘテロ３量体複合体結晶構造（ＰＤＢ　ＩＤ：１ＦＡＰ）をＩＣＭ－Ｐｒｏ３．８に読み込ませ、ラパマイシンを中心とした周辺に、結合ｂｏｘを自動的に生成した。
　次に、すべてのＶＬが結合ボックス内に含まれるように、マニュアルで結合ボックスサイズを調整した。結合ボックス内に含まれるＦＫＢＰ１２およびＦＲＢのアミノ酸側鎖には、ＳＣＡＲＥ法により柔軟性を導入した。ＶＬのドッキングの際には、ラパマイシンをドッキングガイドのためのテンプレートとして用いた。その際、ラパマイシンのａｔｏｍｉｃ　ｐｒｏｐｅｒｔｙ　ｆｉｅｌｄ（ＡＰＦ）を考慮した。
　すべてのＶＬのドッキング後、ドッキングスコアーが、それぞれのグループ（Ｌ＃１～Ｌ＃３）の最上位のリガンドを選択し、以下の３種類のＶＬを得た。
　＊Ｌ＃１：ジメチル　５－（２－（（２，５－ジメチル－４－スルファモイルフラン－３－カルボニル）オキシ）アセトアミド）イソフタレート（化合物Ａ）；
　＊Ｌ＃２：（１Ｒ，２Ｓ，３Ｓ）－Ｎ１，Ｎ２－ビス（３，５－ジメトキシベンジル）－３－メチル－３－（２－（メチルアミノ）ベンゾイル）シクロプロパン－１，２－ジカルボキサミド（化合物Ｂ）；
　＊Ｌ＃３：Ｎ－（１，３－ベンゾジオキソール－５－イルメチル）－４－［１－［２－［（３－クロロフェニル）アミノ］－２－オキソエチル］－２，４－ジオキソキナゾリン－３－イル］ブタンアミド（化合物Ｃ） Selection of small molecules by in silico (in silico selection)
(1) About 700,000 virtual ligands (VL) were extracted from the ZINC15 database (https://zinc15.docking.org/) according to the criteria shown in Table 1.
VL was divided into three groups, L # 1 to L # 3.
Figure JPOXMLDOC01-appb-T000031
 Virtual Ligand Selection (VLS) was performed by ICM-Pro3.8 software (Molsoft LLC).
The FKBP12-rapamycin-FRB heterotrimer complex crystal structure (PDB ID: 1FAP) was loaded into ICM-Pro3.8, and a binding box was automatically generated around rapamycin.
The join box size was then manually adjusted so that all VLs were contained within the join box. Flexibility was introduced into the amino acid side chains of FKBP12 and FRB contained in the binding box by the SCARE method. When docking the VL, rapamycin was used as a template for the docking guide. At that time, the atomic property field (APF) of rapamycin was taken into consideration.
After docking of all VLs, the docking score selected the highest ligand for each group (L # 1 to L # 3) to obtain the following three types of VL.
* L # 1: Dimethyl 5- (2-((2,5-dimethyl-4-sulfamoylfuran-3-carbonyl) oxy) acetamide) isophthalate (Compound A);
* L # 2: (1R, 2S, 3S) -N 1 , N 2 -bis (3,5-dimethoxybenzyl) -3-methyl-3- (2- (methylamino) benzoyl) cyclopropane-1,2 -Dicarboxamide (Compound B);
* L # 3: N- (1,3-benzodioxole-5-ylmethyl) -4- [1- [2-[(3-chlorophenyl) amino] -2-oxoethyl] -2,4-dioxo Quinazoline-3-yl] Butanamide (Compound C)
　（２）ＦＫＢＰ１２－ＦＲＢに結合する少分子のｉｎ　ｓｉｌｉｃｏ選択の結果。
　上記のように、Ｉｎ　ｓｉｌｉｃｏ選択の結果、リガンドライブラリーＬ＃１から化合物Ａ、Ｌ＃３から化合物Ｂおよび化合物Ｃが選択された（図１）。 (2) Results of in silico selection of small molecules that bind to FKBP12-FRB.
As described above, as a result of in silico selection, compound A was selected from the ligand library L # 1, and compound B and C were selected from L # 3 (FIG. 1).
　化合物Ａ、化合物Ｂおよび化合物Ｃは、ラパマイシン結合ポケットの、４８％、６８％および７６％をそれぞれ占めており、ｍＴＯＲＣ１の潜在的なアロステリック阻害剤であることが示唆された。
　図２に、ＦＫＢＰ１２－ＲＡＰ－ＦＲＢの結晶構造（ＰＤＢ　ＩＤ：１ＦＡＰ）および、ＦＫＢＰ１２－化合物Ｃ－ＦＲＢのドッキングモデルを示す（図２）。
[実施例２] Compound A, Compound B and Compound C occupy 48%, 68% and 76% of the rapamycin binding pocket, respectively, suggesting that they are potential allosteric inhibitors of mTORC1.
FIG. 2 shows the crystal structure of FKBP12-RAP-FRB (PDB ID: 1FAP) and the docking model of FKBP12-compound C-FRB (FIG. 2).
[Example 2]
　ＦＫＢＰ１２－ＦＲＢに結合するリガンドのｉｎ　ｖｉｔｒｏ選択性
　化合物Ａ、化合物Ｂおよび化合物Ｃを、それぞれ、エナミン社（Ｅｎａｍｉｎｅ　Ｌｔｄ，　Ｋｙｉｖ，　Ｕｋｒａｉｎｅ）、アシネックス社（ＡＳＩＮＥＸ　Ｃｏｒｐｏｒａｔｉｏｎ，　ＮＣ，　ＵＳＡ）および、ケムディブ社（ＣｈｅｍＤｉｖ，　ＣＡ，　ＵＳＡ）より購入した。 In vitro selectivity compound A, compound B, and compound C of ligands that bind to FKBP12-FRB were prepared by Enamine Ltd. (Enamine Ltd, Kyiv, Ukraine), ASINEX Corporation (NC, USA), and Chemdiv, respectively. Purchased from (ChemDiv, CA, USA).
　（１）タンパク質発現系構築およびタンパク質精製
　ＦＫＢＰ１２およびＦＲＢ遺伝子は、ＮａｎｏＢｉｔ　ＰＰＩ　ｓｙｓｔｅｍ　（Ｐｒｏｍｅｇａ，　ＵＳＡ）キットに含まれる、ＦＫＢＰ－ＳｍＢｉＴおよびＦＲＢ－ＬｇＢｉＴ　ｃｏｎｔｒｏｌ　ベクターからクローニングし、ｐＥＴ１５ｂベクター（Ｎｏｖａｇｅｎ，　ＵＳＡ）に、Ｉｎ－Ｆｕｓｉｏｎ　ｃｌｏｎｉｎｇ　ｋｉｔ（Ｔａｋａｒａ　Ｂｉｏ，　Ｊａｐａｎ）を用いてインサートした。
　構築したプラスミドをＤＨ５α大腸菌（Ｎｉｐｐｏｎ　Ｇｅｎｅ，　Ｊａｐａｎ）にトランスフォームし、０．１　ｍＭアンピシリン含有のＬＢアガープレート上に塗布し、出現したコロニーにコロニーＰＣＲによりシークエンスを行った。
　シークエンス確認したコロニーをＬＢ培地で培養（３７℃、６時間）後、ＮｕｃｌｅｏＳｐｉｎ　Ｐｌａｓｍｉｄ　ＥａｓｙＰｕｒｅ　Ｋｉｔ　（ＭＡＣＨＥＲＥＹ－ＮＡＧＥＬ，　Ｇｅｒｍａｎｙ）でプラスミドを精製した。
　得られたプラスミドを、ＥＣＯＳＴＭコンピテント大腸菌ＢＬ２１（ＤＥ３）（Ｎｉｐｐｏｎ　Ｇｅｎｅ，　Ｊａｐａｎ）にトランスフォームし、５ｍＬ　ＬＢ培地（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ，　ＵＳＡ）で前培養した（３７℃、６時間）。さらに、５Ｌ　改良ＴＢ培地（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ，　ＵＳＡ）で培養した。
　ＯＤ６００＝１に到達時点で、１ｍＭ　ＩＰＴＧ（Ｆｕｊｉｆｉｌｍ　Ｗａｋｏ　Ｐｕｒｅ　Ｃｈｅｍｉｃａｌｓ　Ｃｏｒｐｏｒａｔｉｏｎ，　Ｊａｐａｎ）を加え、さらに一晩培養を継続した。
 
　遠心（４℃、８０００ｒｐｍ，　１５分）により菌体を回収し、－８０℃で保管した。
　５　ｇ菌体（ＦＲＢまたはＦＫＢＰ１２）を、１００　ｍＬ　溶解バッファー（５０ｍＭ　Ｔｒｉｓ－ＨＣｌ，　ｐＨ８．０，　１００　ｍＭ　ＮａＣｌ，　１ｍＭ　ＥＤＴＡ，　０．０４　ｍｇ／ｍｌ　リゾチーム、０．１６ｍｇ／ｍｌ　ＤＮａｓｅＩ、　１錠ｘ　ｃＯｍｐｌｅｔｅＴＭ　プロテアーゼ阻害剤（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ））に溶解し、超音波破砕した（Ｓｏｎｉｆｉｅｒ(R)　（Ｂｒａｎｓｏｎ，　ＵＳＡ），　５．０　Ｗ，　３０－４０％　ｃｙｃｌｅ／ｓｅｃ，　５ｍｉｎ，　ｏｎ　ｉｃｅ）。
　ＦＫＢＰ１２の精製では、破砕液を超遠心（４℃、４０，０００ｒｐｍ、１時間）した後、上清を得た。
 
　上清を、結合バッファー　Ａ（５０　ｍＭ　Ｔｒｉｓ－ＨＣｌ，　ｐＨ　８．０，　１０％　（ｖ／ｖ）　ｇｌｙｃｅｒｏｌ）により予め平衡化した、５ｍＬ　ＨｉｓＴｒａｐ　ＨＰ　（Ｃｙｔｉｖａ，　Ｊａｐａｎ）Ｎｉカラムにロードした。
　精製装置は、ＡＫＴＡ　ｐｒｉｍｅ　ｐｌｕｓ　（Ｃｙｔｉｖａ，　Ｊａｐａｎ）を用いた。溶出バファーＡ（５０　ｍＭ　Ｔｒｉｓ－ＨＣｌ，　１０％　ｖ／ｖ　ｇｌｙｃｅｒｏｌ，　１　Ｍ　ｉｍｉｄａｚｏｌｅ，　ｐＨ　８．０）の濃度を徐々に上昇させ、溶出ピークを得た。
　ＳＤＳ－ＰＡＧＥにより、溶出ピークにＦＫＢＰ１２が含まれていることを確認後、Ａｍｉｃｏｎ　Ｕｌｔｒａ　（ＭＷＣＯ：３ＫＤａ，　Ｍｉｌｌｉｐｏｒｅ，　Ｇｅｒｍａｎｙ）により、結合バッファーＡで溶媒置換した。次に、ゲルろ過用バッファー（５０　ｍＭ　Ｔｒｉｓ－ＨＣｌ，　１５０　ｍＭ　ＮａＣｌ，　ｐＨ　８．０，　１０％（ｖ／ｖ）　ｇｌｙｃｅｒｏｌ）で予め平衡化した、ＨｉＬｏａｄ(R)　２６／６０　Ｓｕｐｅｒｄｅｘ－２００　カラム（Ｃｙｔｉｖａ，　Ｊａｐａｎ）で精製し、５０ｍｇ／ｍＬまで濃縮後、－８０℃で保管した。
 
　ＦＲＢの精製では、超遠心後、ペレットを回収した。
　ペレットを、洗浄バッファーＡ（５０　ｍＭ　Ｔｒｉｓ－ＨＣｌ，　１００　ｍＭ　ＮａＣｌ，　１　ｍＭ　ＥＤＴＡ，　４　Ｍ　ｕｒｅａ，　ｐＨ　８．０）で３回洗浄した。
 
　その後、洗浄バッファーＢ（５０　ｍＭ　Ｔｒｉｓ－ＨＣｌ，　１００　ｍＭ　ＮａＣｌ，　１ｍＭ　ＥＤＴＡ）で３回洗浄した。さらに、ペレットを２０ｍＬ　溶解バッファー（５０　ｍＭ　Ｔｒｉｓ－ＨＣｌ，　８　Ｍ　ｕｒｅａ，　１００　ｍＭ　ＮａＨ２ＰＯ４，　１０　ｍＭ　２－ｍｅｒｃａｐｔｏｅｔｈａｎｏｌ，　ｐＨ　８．０）に溶解し、ローテーターで撹拌した（２時間、室温）。超遠心（４０，０００ｒｐｍ，　１時間、４℃）後、予め平衡化バッファー（５０　ｍＭ　Ｔｒｉｓ－ＨＣｌ，　８　Ｍ　ｕｒｅａ，　１００　ｍＭ　ＮａＨ２ＰＯ４，　５　ｍＭ　２－ｍｅｒｃａｐｔｏｅｔｈａｎｏｌ，　ｐＨ　８．０）で平衡化した、５　ｍＬ　ＨｉｓＴｒａｐ　ＦＦ　ｃｒｕｄｅ　（Ｃｙｔｉｖａ，　Ｊａｐａｎ）　Ｎｉ　カラムにロードした。
 
　溶出バッファー（５０　ｍＭ　Ｔｒｉｓ－ＨＣｌ，　８　Ｍ　ｕｒｅａ，　１００　ｍＭ　ＮａＨ２ＰＯ４，　５　ｍＭ　２－ｍｅｒｃａｐｔｏｅｔｈａｎｏｌ，　１　Ｍ　ｉｍｉｄａｚｏｌｅ，　ｐＨ　８．０）の濃度を徐々に上昇（０→　１００％）させ、得られたピークにＦＲＢが含まれることをＳＤＳ－ＰＡＧＥにより確認した。
 
　得られた溶液を、１Ｌリフォールディングバッファー（５０　ｍＭ，　Ｔｒｉｓ－ＨＣｌ，　４０　ｍＭ　ＮａＣｌ，　１　ｍＭ　ＥＤＴＡ，　１Ｍ　Ｌ－ａｒｇｉｎｉｎｅ，　１０％　（ｖ／ｖ）　ｇｌｙｃｅｒｏｌ，　ｐＨ　８．０）に滴下した（４℃、撹拌）。
 
　リフォールディングバッファーを、４００　ｍＬ　Ａｍｉｃｏｎ　Ｓｔｉｒｒｅｄ　Ｃｅｌｌ　（Ｍｉｌｌｉｐｏｒｅ，　ＵＳＡ）およびＡｍｉｃｏｎ　Ｕｌｔｒａ－１５　（ＭＷＣＯ：　３ｋＤａ，　Ｍｉｌｌｉｐｏｒｅ，　Ｇｅｒｍａｎｙ）で濃縮し、結合バッファーＡ（５０　ｍＭ　Ｔｒｉｓ－ＨＣｌ，　ｐＨ　８．０，　１０％　（ｖ／ｖ）　ｇｌｙｃｅｒｏｌ）でバッファー交換し、濃縮ＦＲＢ溶液を得た。
 
　これを、ゲルろ過用バッファー（５０　ｍＭ　Ｔｒｉｓ－ＨＣｌ，　１５０　ｍＭ　ＮａＣｌ，　ｐＨ　８．０，　１０％（ｖ／ｖ）　ｇｌｙｃｅｒｏｌ）で予め平衡化した、ＨｉＬｏａｄ（Ｒ）　２６／６０　Ｓｕｐｅｒｄｅｘ－２００　カラム（Ｃｙｔｉｖａ，　Ｊａｐａｎ）で精製し、４０　ｍｇ／ｍｌまで濃縮し、－８０℃で保管した。 (1) Protein expression system construction and protein purification The FKBP12 and FRB genes are cloned from the FKBP-SmBiT and FRB-LgBiT control vectors included in the NanoBit PPI system (Promega, USA) kit and incorporated into a pET15b vector (Novagen, USA). , In-Fusion cloning kit (Takara Bio, Japan) was used for insertion.
The constructed plasmid was transformed into DH5α Escherichia coli (Nippon Gene, Japan), applied onto an LB agar plate containing 0.1 mM ampicillin, and the colonies that appeared were sequenced by colony PCR.
After culturing the sequence-confirmed colonies in LB medium (37 ° C., 6 hours), the plasmid was purified with NucleoSpin Plasmamid EasyPure Kit (MACHEREY-NAGEL, Germany).
The resulting plasmid was transformed into ECOS TM competent E. coli BL21 (DE3) (Nippon Gene, Japan) and precultured in 5 mL LB medium (Sigma-Aldrich, USA) (37 ° C., 6 hours). Further, the cells were cultured in 5 L improved TB medium (Sigma-Aldrich, USA).
When OD 600 = 1, 1 mM IPTG (Fujifilm Wako Pure Chemicals Corporation, Japan) was added, and the culture was continued overnight.

Bacterial cells were collected by centrifugation (4 ° C., 8000 rpm, 15 minutes) and stored at −80 ° C.
5 g cells (FRB or FKBP12), 100 mL lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.04 mg / ml lysozyme, 0.16 mg / ml DNase I, 1 tablet x It was dissolved in a compacte TM protease inhibitor (Sigma-Aldrich) and ultrasonically disrupted (Soniffier (R) (Branson, USA), 5.0 W, 30-40% cycle / sec, 5 min, one ice).
In the purification of FKBP12, the crushed solution was ultracentrifuged (4 ° C., 40,000 rpm, 1 hour), and then a supernatant was obtained.

The supernatant was loaded onto a 5 mL HisTrap HP (Cytiva, Japan) Ni column pre-equilibriumed with binding buffer A (50 mM Tris-HCl, pH 8.0, 10% (v / v) glycerol).
As the purification apparatus, AKTA prime plus (Cytiva, Japan) was used. The concentration of elution buffer A (50 mM Tris-HCl, 10% v / v glycerol, 1 imidazole, pH 8.0) was gradually increased to obtain an elution peak.
After confirming that FKBP12 was contained in the elution peak by SDS-PAGE, the solvent was replaced with the binding buffer A by Amicon Ultra (MWCO: 3KDa, Millipore, Germany). Next, a HiRoad (R) 26/60 Superdex-200 column pre-equilibrated with gel filtration buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0, 10% (v / v) glycerol) ( Purified with Cytiva (Japan), concentrated to 50 mg / mL, and stored at −80 ° C.

For Fed purification, pellets were collected after ultracentrifugation.
The pellet was washed 3 times with wash buffer A (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 4 urea, pH 8.0).

Then, it was washed three times with washing buffer B (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA). In addition, the pellet was dissolved in 20 mL lysis buffer (50 mM Tris-HCl, 8 urea, 100 mM NaH 2 PO 4 , 10 mM 2-mercaptoethanol, pH 8.0) and stirred with a rotator (2 hours, room temperature). .. After ultracentrifugation (40,000 rpm, 1 hour, 4 ° C), pre-equilibrium with equilibration buffer (50 mM Tris-HCl, 8 urea, 100 mM NaH 2 PO 4 , 5 mM 2-mercaptoethanol, pH 8.0). It was loaded into a 5 mL HisTrap FF clamp (Cytiva, Japan) Ni column.

Gradually increase the concentration (0 → 100%) of the elution buffer (50 mM Tris-HCl, 8 Murea, 100 mM NaH 2 PO 4 , 5 mM 2-mercaptoethanol, 1 Mimidazole, pH 8.0). It was confirmed by SDS-PAGE that FRB was contained in the peaks obtained.

The resulting solution was added dropwise to 1 L refolding buffer (50 mM, Tris-HCl, 40 mM NaCl, 1 mM EDTA, 1 M L-arginine, 10% (v / v) glycerol, pH 8.0) (4). ℃, stirring).

Refolding buffer was concentrated in 400 mL Amicon Stillred Cell (Millipore, USA) and Amicon Ultra-15 (MWCO: 3 kDa, Millipore, Germany) and bound buffer A (50 mM Tris-HCl, pH 8.0). The buffer was exchanged with (v / v) glycerol) to obtain a concentrated FRB solution.

This was pre-equilibrated with a gel filtration buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0, 10% (v / v) glycerol) on a HiRoad (R) 26/60 Superdex-200 column ( Purified with Cytiva, Japan), concentrated to 40 mg / ml, and stored at −80 ° C.
　（２）ＦＫＢＰ１２の６ｘＨｉｓ－タグ削除およびビオチン化
　ＰＢＳに溶解した１ｍｇ　ＦＫＢＰ１２に、１０　ｕｎｉｔ　トロンビン（ＴｈｅｒｍｏＦｉｓｈｅｒ，　ＵＳＡ）を加えて撹拌した（１６時間、室温）。
　Ｈｉｓ　ＳｐｉｎＴｒａｐ　ｃｏｌｕｍｎ　（Ｃｙｔｉｖａ，　Ｊａｐａｎ）に反応液をロードし、フロースルー溶液を得た。
　これを、１　ｍＬ　ＨｉｓＴｒａｐ　Ｂｅｎｚａｍｉｄｉｎｅ　ＦＦ　ｃｏｌｕｍｎ　（Ｃｙｔｉｖａ，　Ｊａｐａｎ）にロードし、トロンビンを除去した。
　次に、６ｘＨｉｓ－タグを除去したＦＫＢＰ１２と、Ｂｉｏｔｉｎ－ｄＰＥＧ（Ｒ） ２４－ＮＨＳ　ｅｓｔｅｒ　（Ｑｕａｎｔａ　ＢｉｏＤｅｇｉｇｎ　Ｌｉｍｉｔｅｄ，　ＵＳＡ）を１：２０のモル比で撹拌・反応させた（２時間、室温）。
 
　反応液を、予めゲルろ過バッファ（５０　ｍＭ　Ｔｒｉｓ－ＨＣｌ，　１５０　ｍＭ　ＮａＣｌ，　１０％　（ｖ／ｖ）　ｇｌｙｃｅｒｏｌ）で平衡化した、ＨｉＬｏａｄ（Ｒ）　２６／６０　Ｓｕｐｅｒｄｅｘ－２００　カラム（Ｃｙｔｉｖａ，　Ｊａｐａｎ）でゲルろ過し、得られた試料を濃縮後、－８０℃で保存した。 (2) 6xHis-tag removal of FKBP12 and biotinlation To 1 mg FKBP12 dissolved in PBS, 10 unit thrombin (Thermo Fisher, USA) was added and stirred (16 hours, room temperature).
The reaction solution was loaded into His SpinTrap volume (Cytiva, Japan) to obtain a flow-through solution.
This was loaded into 1 mL HisTrap Benzamidine FF volume (Cytiva, Japan) and thrombin was removed.
Next, FKBP12 from which the 6xHis-tag was removed and Biotin-dPEG (R) 24 -NHS ester (Quanta BioDesign Limited, USA) were stirred and reacted at a molar ratio of 1:20 (2 hours, room temperature).

The reaction was preliminarily equilibrated with a gel filtration buffer (50 mM Tris-HCl, 150 mM NaCl, 10% (v / v) glycerol) on a HiRoad (R) 26/60 Superdex-200 column (Cytiva, Japan). After gel filtration and the obtained sample was concentrated, it was stored at −80 ° C.
　（３）Ｉｎ　ｖｉｔｒｏリガンド選択
　２　μＬ　６ｘＨｉｓ－タグ－ＦＲＢ（２５０　ｎＭ）を、２　μＬ（１００　μｇ／ｍｌ）　ａｎｔｉ－Ｈｉｓ　ｔａｇ　ｃｏａｔｅｄ　ａｌｐｈａ　ａｃｃｅｐｔｏｒ　ｂｅａｄｓ　（ＰｅｒｋｉｎＥｌｍｅｒ，　ＵＳＡ）と３８４－ｗｅｌｌ　ＯｐｔｉＰｌａｔｅ　（ＰｅｒｋｉｎＥｌｍｅｒ，　ＵＳＡ）のウエル中で混合し、シールしてから、インキュベーションした（暗所、１時間、室温）。
　次に、２　μＬ　ビオチン化ＦＫＢＰ１２　（２５０　ｎＭ）と２　μＬ　ｓｔｒｅｐｔａｖｉｄｉｎ－ｃｏａｔｅｄ　ａｌｐｈａ　ｄｏｎｎｅｒ　ｂｅａｄｓ　（１００　μｇ／ｍｌ）を混合し、これをさらにウエルに加えた。
 
　２　μＬ　ラパマイシン（Ｆｕｎａｋｏｓｈｉ　Ｃｏ．，　Ｌｔｄ．，　Ｊａｐａｎ）または、選択した化合物溶液をウエルに加え、再びシールし、暗所・室温で１時間以上インキュベーションした。
 
　ウエルから発生するＡｌｐｈａ　ｓｉｇｎａｌを、ＡｌｐｈａＬＩＳＡ（Ｒ）測定モード付きＥｎＳｐｉｒｅ　マルチモードプレートリーダ（ＰｅｒｋｉｎＥｌｍｅｒ，　ＵＳＡ）で測定した。
　平衡解離定数（Ｋｄ）は、６ｘＨｉｓ－ＦＲＢ、ｂｉｏｔｉｎ－ＦＫＢＰ１２またはリガンドの濃度を変化（０．０１－１０００　ｎＭ）させて、複数のウエルを作成し、Ａｌｐｈａ　ｓｉｇｎａｌを測定することにより得た。
 
　リガンド（化合物）またはラパマイシンの競合阻害実験は、それらの濃度を変化（０．１８　ｎＭ－１８　μＭ）させ、ＦＲＢとインキュベーションすることにより得た。 (3) In vitro ligand selection 2 μL 6xHis-tag-FRB (250 nM), 2 μL (100 μg / ml) anti-His tag coated alpha acceptor beads (PerkinElmer, USA) and 384-well OptiPlate (PerkinElmer, USA) ) Was mixed in the well, sealed, and then incubated (dark place, 1 hour, room temperature).
Next, 2 μL streptavidin-coated alpha donner beads (100 μg / ml) were mixed with 2 μL biotinylated FKBP12 (250 nM) and further added to the wells.

2 μL rapamycin (Funakoshi Co., Ltd., Japan) or a solution of the selected compound was added to the wells, sealed again, and incubated in the dark at room temperature for 1 hour or longer.

Alpha sinal generated from the well was measured with an EnSpire multimode plate reader (PerkinElmer, USA) with AlphaLISA (R) measurement mode.
The equilibrium dissociation constant (Kd) was obtained by varying the concentration of 6xHis-FRB, biotin-FKBP12 or ligand (0.01-1000 nM) to create multiple wells and measuring Alpha signal.

Competitive inhibition experiments on ligands (compounds) or rapamycin were obtained by varying their concentrations (0.18 nM-18 μM) and incubating with the Fed.
　（４）ＦＫＢＰ１２－ＦＲＢに結合するリガンドのｉｎ　ｖｉｔｒｏ選択の結果
　化合物Ａ、化合物Ｂおよび化合物Ｃの、ＦＫＢＰ１２－ＦＲＢに対する親和性を確認するために、ＡｌｐｈａＬＩＳＡ(R)による測定を行った。
　６ｘＨｉｓ－ＦＲＢ、６ｘＨｉｓ－ＦＫＢＰ１２および、ビオチン化ＦＫＢＰ１２は図３に示すようにゲルろ過クロマトグラフィーで精製した（図３）。 (4) Results of in vitro selection of ligand binding to FKBP12-FRB Measurement by AlphaLISA (R) was performed to confirm the affinity of Compound A, Compound B and Compound C for FKBP12-FRB.
The 6xHis-FRB, 6xHis-FKBP12, and biotinylated FKBP12 were purified by gel filtration chromatography as shown in FIG. 3 (FIG. 3).
　ビオチン化ＦＫＢＰ１２はドナービーズに、６ｘＨｉｓ－ＦＲＢはアクセプタービーズに固定し、１　μＭの化合物Ａ、化合物Ｂ、化合物Ｃ、およびラパマイシン（ＲＡＰ：　ポジティブコントロール）の結合を測定したところ、ラパマイシンと化合物Ｃは同程度のＡｌｐｈａシグナルを与えることが分かった（図４）。
　化合物Ｃが、ラパマイシン（ＲＡＰ）と同程度の結合力を持つことが分かった。
　一方、化合物Ａおよび化合物Ｂの結合力は弱かった。 Biotinylated FKBP12 was immobilized on donor beads and 6xHis-FRB was immobilized on acceptor beads, and the binding of 1 μM compound A, compound B, compound C, and rapamycin (RAP: positive control) was measured. Was found to give comparable Alpha signals (Fig. 4).
Compound C was found to have a binding force comparable to that of rapamycin (RAP).
On the other hand, the binding force of compound A and compound B was weak.
　さらに、化合物ＣとＲＡＰのＡｌｐｈａＬＩＳＡによるｔｉｔｒａｔｉｏｎ　結合実験により、それぞれの平衡解離定数ＫＤを求めたところ、化合物ＣはＫＤ　＝　４　ｎＭに対して、ＲＡＰはＫＤ　＝　２．２　ｎＭと、ほぼ同等の親和性を示すことが分かった（図５）。
　化合物ＣおよびＲＡＰはそれぞれ、４　ｎＭ、２．２　ｎＭの平衡解離定数を示した。 Furthermore, when the equilibrium dissociation constant KD of each of the compounds C and RAP was determined by the titration binding experiment by AlphaLISA, the equilibrium dissociation constant KD of compound C was KD = 4 nM, while that of RAP was KD = 2.2 nM. It was found to show equivalent affinity (Fig. 5).
Compounds C and RAP showed equilibrium dissociation constants of 4 nM and 2.2 nM, respectively.
　さらに、ＮａｎｏＢｉＴ(R)およびＨＥＫ２９３細胞を用いて、ｉｎ　ｖｉｖｏ選択を行ったところ、同様に化合物ＣはＲＡＰと同程度の親和性を細胞内でも持つことが確認できた（図６）。
　化合物ＣおよびＲＡＰは同等のシグナルを示したが、化合物Ａおよび化合物Ｂのシグナルは弱かった。 Furthermore, when in vivo selection was performed using NanoBiT (R) and HEK293 cells, it was confirmed that compound C also had the same affinity as RAP in the cells (FIG. 6).
Compounds C and RAP showed comparable signals, but compounds A and B had weaker signals.
　化合物Ｃの濃度依存的に、発光強度が増加していることから、化合物ＣがＦＫＢＰ１２－ＦＲＢに特異的に結合していることを示す（図７）。
 [実施例３] Since the emission intensity increases depending on the concentration of compound C, it is shown that compound C is specifically bound to FKBP12-FRB (FIG. 7).
[Example 3]
　（１）細胞株
　ヒト転移性乳がん細胞（ｈｕｍａｎ　ｍｅｔａｓｔａｔｉｃ　ｍａｍｍａｒｙ　ｃａｒｃｉｎｏｍａ：　ＭＣＦ－７）、ヒト子宮頸がん細胞（ＨｅＬａ，　＃ＣＣＬ－２），ヒト肝がん細胞（ＨｅｐＧ－２）、ヒト胚性線維芽細胞ＨＥＫ２９３、ラット腎間質線維芽細胞　（ＮＲＫ－４９Ｆ）は、理研セルバンク（ＲＩＫＥＮ，　Ｊａｐａｎ）より入手した。
　細胞は、ｈｉｇｈ－ｇｌｕｃｏｓｅ　Ｇｉｂｃｏ　ＤＭＥＭ　（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ，　ＵＳＡ）に１０％　ＦＢＳおよび１％　Ｐｅｎｉｃｉｌｌｉｎ－Ｓｔｒｅｐｔｏｍｙｃｉｎ　（Ｐ／Ｓ）　（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ，　ＵＳＡ）を加えて培養した。 (1) Cell line Human metastatic breast cancer cells (human metastatic breast cancer cells (MCF-7)), human cervical cancer cells (HeLa, # CCL-2), human liver cancer cells (HepG-2), human embryonic Fibroblasts HEK293 and rat renal stromal fibroblasts (NRK-49F) were obtained from RIKEN cell bank (RIKEN, Japan).
Cells were cultured in high-glucose Gibco DMEM (Thermo Fisher Scientific, USA) with 10% FBS and 1% Penicillin-Streptomycin (P / S) (Thermo Fisher Scientific, USA).
　（２）Ｉｎ　ｖｉｖｏ　リガンド選択
　Ｉｎ　ｖｉｖｏリガンド選択には、ＮａｎｏＢｉＴ　ＰＰＩ　Ｓｔａｒｔｅｒ　Ｓｙｓｔｅｍｓ　（Ｐｒｏｍｅｇａ，　ＵＳＡ）を利用した。
　まず、１０４個のＨＥＫ２９３、ＨｅＬａまたはＭＣＦ－７細胞を、Ｂ＆Ｗ　Ｉｓｏｐｌａｔｅ－９６　ＴＣ　（ＰｅｒｋｉｎＥｌｍｅｒ，　ＵＳＡ）に入れ、ＤＭＥＤ　（１０％　ＦＢＳ，　１％　Ｐ／Ｓ）で一晩培養した（５％　ＣＯ２，　３７℃）。
　翌日、ＮａｎｏＢｉＴ　ＰＰＩ　Ｓｔａｒｔｅｒ　Ｓｙｓｔｅｍに同封されている、ＦＫＢＰ１２－ＳｍＢｉＴ　（４３６０　ｂｐ）およびＦＲＢ－ＬｇＢｉＴ　（４７６２　ｂｐ）コントロールベクターを、５０　ｎｇ／ｗｅｌｌ濃度になるように、ＦｕＧｅｎｅ　ＨＤ　（Ｐｒｏｍｅｇａ，　ＵＳＡ）を３：１（ｖ／ｗ）の割合で用いて、各細胞に共トランスフェクションし、２４－４８　時間インキュベーションした（５％　ＣＯ２，　３７℃）。
　次に、培地をＯｐｔｉ－ＭＥＭ　ｒｅｄｕｃｅｄ　ｓｅｒｕｍ　ｍｅｄｉｕｍ　（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ，　ＵＳＡ）で置換し、２０　μＬ　２０倍希釈Ｎａｎｏ－Ｇｌｏ　ｌｕｃｉｆｅｒａｓｅ　ａｓｓａｙ　ｓｕｂｓｔｒａｔｅ　（Ｐｒｏｍｅｇａ，　ＵＳＡ）を各ウエルに加えた。
　ＥｎＳｐｉｒｅ　マルチモードプレートリーダー（ＰｅｒｋｉｎＥｌｍｅｒ，　ＵＳＡ）の発光測定モードを使用し、ベースラインを測定した。
　次に、リガンド（０．１　ｐＭ－１０　μＭ）＋ラパマイシン（１　μＭ）、または、リガンド（０．１　ｐＭ－１０　μＭ）を各ウエルに加え、ＰＰＩによる発光をプレートリーダーで測定した。 (2) In vivo Ligand Selection For in vivo ligand selection, NanoBiT PPI Starter Systems (Promega, USA) was used.
First, 104 HEK293, HeLa or MCF- 7 cells were placed in B & W Isoplatate-96 TC (PerkinElmer, USA) and cultured overnight in DMED (10% FBS, 1% P / S) (5% CO). 2 , 37 ° C).
The next day, the FKBP12-SmBiT (4360 bp) and FRB-LgBiT (4762 bp) control vectors enclosed in the NanoBiT PPI Starter System were loaded with FuGene HD (Promega, USA) to a concentration of 50 ng / well. Each cell was co-transfected and incubated for 24-48 hours (5% CO 2 , 37 ° C.) using a 1: 1 (v / w) ratio.
The medium was then replaced with Opti-MEM rediculated serum medium (Thermo Fisher Scientific, USA), and 20 μL 20-fold diluted Nano-Glo luciferase assay substrate (Promega, USA) was added to each well.
The baseline was measured using the emission measurement mode of the EnSpire multimode plate reader (PerkinElmer, USA).
Next, ligand (0.1 pM-10 μM) + rapamycin (1 μM) or ligand (0.1 pM-10 μM) was added to each well, and luminescence by PPI was measured with a plate reader.
　（３）細胞生存率アッセイ
　細胞生存率アッセイは、ＣｅｌｌＴｉｔｅｒ－Ｂｌｕｅ　Ｃｅｌｌ　Ｖｉａｂｉｌｉｔｙ　ａｓｓａｙ　ｋｉｔ　（Ｐｒｏｍｅｇａ，　ＵＳＡ）を用いた。細胞（１０４個／ウエル）を９６－ｗｅｌｌプレートにｓｅｅｄし、一晩培養した（５％　ＣＯ２，　３７℃）。
　さらに１８時間、飢餓条件下で培養した。その後、各濃度のリガンドを細胞に加えて７２時間培養後、２０　μＬのＣｅｌｌＴｉｔｅｒ－Ｂｌｕｅ　Ｃｅｌｌ　Ｖｉａｂｉｌｉｔｙ溶液を各ウエルに加え、１－４時間インキュベーションした（５％　ＣＯ２，　３７℃）。
　ＥｎＳｐｉｒｅマルチモードプレートリーダーで、５６０／５９０　ｎｍの蛍光を測定した。または、細胞数を直接数えることにより、細胞生存率をアッセイした。
　６－ｗｅｌｌ　プレートに、１０５個／ｗｅｌｌの細胞をｓｅｅｄし、上記と同様な操作を行い、最後に、トリプシン／ＥＤＴＡで細胞を剥離し、５％　ｔｒｙｐａｎ　ｂｌｕｅ溶液を加え、ＴＣ１０　ａｕｔｏｍａｔｅｄ　ｃｅｌｌ　ｃｏｕｎｔｅｒ　（Ｂｉｏ－Ｒａｄ，　ＵＳＡ）で細胞数を数えた。
　Ｏｌｙｍｐｕｓ　ＩＸ７１顕微鏡＋Ｏｌｙｍｐｕｓ　ＤＰ７４カメラで細胞写真を撮影し、ＩｍａｇｅＪ　ｓｏｆｔｗａｒｅで画像解析した。 (3) Cell Viability Assay For the cell viability assay, CellTiter-Blue Cell Viability assay kit (Promega, USA) was used. Cells ( 104 cells / well) were seeded on 96-well plates and cultured overnight (5% CO 2 , 37 ° C.).
The cells were cultured under starvation conditions for an additional 18 hours. Then, each concentration of ligand was added to the cells and cultured for 72 hours, then 20 μL of CellTiter-Blue Cell Viability solution was added to each well and incubated for 1-4 hours (5% CO 2, 37 ° C.).
Fluorescence at 560/590 nm was measured with an EnSpire multimode plate reader. Alternatively, cell viability was assayed by directly counting the number of cells.
Seed 105 cells / well on a 6 -well plate, perform the same operation as above, and finally exfoliate the cells with trypsin / EDTA, add a 5% trypan blue solution, and add TC10 automated cell counter (TC10 automated cell counter). The number of cells was counted by Bio-Rad, USA).
Cellular photographs were taken with an Olympus IX71 microscope + Olympus DP74 camera, and image analysis was performed with ImageJ software.
　（４）ｍＴＯＲＣ１キナーゼアッセイ
　ｍＴＯＲＣ１のキナーゼ活性は、ＡｌｐｈａＬＩＳＡ(R)　ＳｕｒｅＦｉｒｅ　ＵｌｔｒａＴＭ　ＨＶ　ｐ－ｐ７０　Ｓ６Ｋ　（Ｔ３８９）　ａｓｓａｙ　ｋｉｔ　（ＴＧＲ　Ｂｉｏｓｃｉｅｎｃｅｓ，　Ａｕｓｔｒａｌｉａ，　ａｎｄ　ＰｅｒｋｉｎＥｌｍｅｒ，　ＵＳＡ）を用いて、Ｓ６Ｋタンパク質のリン酸化を測定することにより検出した。ＨＥＫ２９３細胞またはＮＲＫ－４９Ｆ細胞（１０４個／ｗｅｌｌ）をｈｉｇｈ－ｇｌｕｃｏｓｅ　ＤＭＥＭ（１０％　ＦＢＳ，　１％　Ｐ／Ｓ）を用いて、９６－ｗｅｌｌ　プレート中で一晩インキュベーションした（５％　ＣＯ２，　３７℃）。
　翌日、細胞を１８時間飢餓状態にし、様々な濃度のリガンドを加え、（または加えず）、１－３時間培養した。キットに含まれる、用時調製した５０　μＬ　１ｘ　ｃｅｌｌ　ｌｙｓｉｓバッファーで細胞を破砕し、プレートシェーカーで１０分間撹拌した。
　６　μＬ　細胞破砕液を３８４－ｗｅｌｌ　ＯｐｔｉＰｌａｔｅ　（ＰｅｒｋｉｎＥｌｍｅｒ，　ＵＳＡ）に分注し、３　μＬ　ａｎｔｉ－Ｓ６Ｋ　ｃｏａｔｅｄ　Ｄｏｎｏｒ　を暗条件下で加え、ウエルをシール後、インキュベーションした（２－４時間、室温）。
　Ａｌｐｈａ　ｓｉｇｎａｌを、ＥｎＳｐｉｒｅのＡｌｐｈａモードで測定した。 (4) mTORC1 Kinase Assay The kinase activity of mTORC1 is as follows: AlphaLISA (R) SureFire Ultra TM HV p-p70 S6K (T389) assay kit (TGR Biosciences, Australia, Australia, and Perkin E Detected by measurement. HEK293 cells or NRK-49F cells ( 104 cells / well) were incubated overnight in 96-well plates using high-glucose DMEM (10% FBS, 1% P / S) (5% CO 2 , 37 ° C).
The next day, the cells were starved for 18 hours, with (or without) various concentrations of ligand added and cultured for 1-3 hours. Cells were disrupted with the time-prepared 50 μL 1x cell lysis buffer included in the kit and stirred with a plate shaker for 10 minutes.
6 μL cell disruption solution was dispensed into 384-well OptiPlate (PerkinElmer, USA), 3 μL anti-S6K coated Donor was added under dark conditions, the wells were sealed and then incubated (2-4 hours, room temperature).
Alpha signal was measured in Alpha mode of EnSpire.
　（５）Ｗｅｓｔｅｒｎ　ブロッティング
　ＨｅＬａ細胞、ＨＥＫ２９３細胞およびＮＲＫ－４９Ｆ細胞は、６－ｗｅｌｌ　ｐｌａｔｅで３－６時間培養し、剥離し、用事調製した１ｘｌｙｓｉｓバッファー（ＡｌｐｈａＬＩＳＡ(R)　ＳｕｒｅＦｉｒｅ　ＵｌｔｒａＴＭ　ＨＶ　ｐ－ｐ７０　Ｓ６Ｋ　（Ｔ３８９）　ａｓｓａｙ　ｋｉｔに同封）で洗浄した。
　タンパク質量はＢＣＡ　ａｓｓａｙ　ｋｉｔ　（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃｓ，　ＵＳＡ）で定量した。
　ＳＤＳ－ＰＡＧＥ後、タンパク質バンドはニトロセルロース膜に、Ｔｒａｎｓ－Ｂｌｏｔ　Ｔｒａｎｓｆｅｒ　Ｓｙｓｔｅｍ　（Ｂｉｏ－Ｒａｄ，　ＵＳＡ）を用いて転写した。
　５％　ｗ／ｖ　ｂｏｖｉｎｅ　ｓｅｒｕｍ　ａｌｂｕｍｉｎ　（ＢＳＡ）　１ｘＴＢＳ溶液で１時間ブロッキング後、ニトロセルロース膜を、それぞれ以下の抗体溶液（１：１０００）でインキュベーションした（一晩、または１時間、４℃）。
　一次抗体：ｒａｂｂｉｔ　ｍｏｎｏｃｌｏｎａｌ　ｐｒｉｍａｒｙ　ａｎｔｉｂｏｄｉｅｓ　ａｇａｉｎｓｔ　Ｓ６　（＃２２１７），　Ｓ２３５／２３６ｐ－Ｓ６　（＃４８５６），　４Ｅ－ＢＰ１　（＃９４５２），　Ｔ３７／４６ｐ－４Ｅ－ＢＰ１　（＃２８５５），　ｍＴＯＲ　（＃２９８３），　そして　β－ｔｕｂｕｌｉｎ　（＃２１４６）　（すべて、Ｃｅｌｌ　Ｓｉｇｎａｌｉｎｇ　Ｔｅｃｈ．，　ＵＳＡ）。　次に、２次抗体：　ｇｏａｔ　ａｎｔｉ－ｒａｂｂｉｔ　ｓｅｃｏｎｄａｒｙ　ａｎｔｉｂｏｄｙ　（＃Ａ１６１０４，　Ｎｏｖｅｘ，　Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃｓ，　ＵＳＡ）　を２時間・室温でインキュベーションした。
Ｗｅｓｔｅｒｎ　ブロッティングイメージは、ＷＳＥ－６１００　ＬｕｍｉｎｏＧｒａｐｈ　Ｉ　（ＡＴＴＯ，　Ｊａｐａｎ）で撮影した。 (5) Western blotting HeLa cells, HEK293 cells and NRK-49F cells were cultured in a 6-well plate for 3-6 hours, exfoliated, and prepared for use in a 1xlysis buffer (AlphaLISA (R) SureFire Ultra TM HV p-p70 S6K. (T389) enclosed in an assay kit).
The amount of protein was quantified by BCA assay kit (Thermo Fisher Scientifics, USA).
After SDS-PAGE, the protein band was transferred to the nitrocellulose membrane using the Trans-Blot Transfer System (Bio-Rad, USA).
After blocking with 5% w / v bovine serum albumin (BSA) 1xTBS solution for 1 hour, the nitrocellulose membrane was incubated with the following antibody solution (1: 1000) (overnight or 1 hour, 4 ° C.), respectively.
Primary antibody: rabbit monoclonal antibody against S6 (# 2217), S235 / 236 p-S6 (# 4856), 4E-BP1 (# 9452), T37 / 46 p-4E-BP1 (# 2855), m ), And β-tubulin (# 2146) (all Cell Signaling Tech., USA). Next, a secondary antibody: goat anti-rabbit secondary antibody (# A16104, Novex, Thermo Fisher Scientifics, USA) was incubated for 2 hours at room temperature.
Western blotting images were taken with WSE-6100 LuminoGraph I (ATTO, Japan).
　（６）実験結果 (6) Experimental results
　（６－１）細胞損傷性アッセイの結果
　３種類のがん細胞（ＭＣＦ－７，　ＨｅｐＧ２）および１種類の非がん細胞（ＮＲＫ－４９Ｆ）に、様々な濃度の化合物Ｃおよびラパマイシンを加えたところ、図８に示す細胞損傷性が観察された。 (6-1) Results of Cell Damage Assay To 3 types of cancer cells (MCF-7, HepG2) and 1 type of non-cancer cells (NRK-49F), various concentrations of compound C and rapamycin were added. However, the cell damaging property shown in FIG. 8 was observed.
　化合物Ｃは、非がん細胞（ＮＲＫ－４９Ｆ）の損傷性（ＩＣ５０＞１０　μＭ）と比較して、がん細胞をより損傷（ＭＣＦ－７のＩＣ５０＝２８．３　ｎＭ、ＨｅｐＧ２のＩＣ５０＝１７．９　ｎＭ）することが分かった。
 
　また、がん細胞（ＨｅＬａ）に対して、化合物Ｃとラパマイシンの細胞損傷性（ＩＣ５０）を比較したところ、化合物Ｃの方がより強い細胞損傷性（化合物Ｃ：　ＩＣ５０＝１．７　ｎＭ，　ＲＡＰ：ＩＣ５０＝２４．７　ｎＭ）を示した（図９）。 Compound C is more damaging to cancer cells (MCF-7 IC 50 = 28.3 nM, HepG2 IC) compared to non-cancer cell (NRK-49F) damaging (IC 50 > 10 μM). It was found that 50 = 17.9 nM).

Moreover, when the cell damage property of compound C and rapamycin (IC 50 ) was compared with respect to cancer cells (HeLa), the cell damage property of compound C was stronger (compound C: IC 50 = 1.7 nM). , RAP: IC 50 = 24.7 nM) (FIG. 9).
　また、非がん細胞（ＮＲＫ－４９Ｆ）と、がん細胞（ＨｅＬａ）の、活性化条件（＋／＋／　Ｇｌｕｃｏｓｅ／ＦＢＳ）および飢餓条件（－／－　Ｇｌｕｃｏｓｅ／ＦＢＳ）下での、化合物ＣおよびＲＡＰの細胞損傷性を測定した（図１０）。 In addition, compound C of non-cancer cells (NRK-49F) and cancer cells (HeLa) under activation conditions (+ / + / Glucose / FBS) and starvation conditions (-/-Glucose / FBS). And the cell damage property of RAP was measured (Fig. 10).
　結果としては、化合物Ｃは、がん細胞（ＨｅＬａ）に対しては、活性化条件・飢餓条件どちらでも細胞損傷性をＲＡＰ以上に発揮する一方で、非がん細胞（ＮＲＫ－４９Ｆ）に対しては、ほとんど細胞損傷性を示さないことが分かった。
　一方、ＲＡＰは、非がん細胞（ＮＲＫ－４９Ｆ）に対しても、細胞損傷性を発揮した。これは、化合物Ｃが、ＲＡＰよりも、がん細胞特異的に細胞損傷性を発揮することを示している。 As a result, compound C exerts cell damage to cancer cells (HeLa) more than RAP under both activation conditions and starvation conditions, while it exerts cell damage to non-cancer cells (NRK-49F). It was found that they showed almost no cell damage.
On the other hand, RAP also exhibited cell damage to non-cancer cells (NRK-49F). This indicates that compound C exerts more cell-damaging properties specifically for cancer cells than RAP.
　さらに、非がん細胞（ＨＥＫ２９３）に対する、化合物Ｃの細胞損傷性を、活性化条件（＋／＋　Ｇｌｕｃｏｓｅ／ＦＢＳ）および飢餓条件（－／－　Ｇｌｕｃｏｓｅ／ＦＢＳ）で定量的に測定した（図１１）。 Furthermore, the cell damage property of compound C to non-cancer cells (HEK293) was quantitatively measured under activation conditions (+ / + Glucose / FBS) and starvation conditions (-/-Glucose / FBS) (FIG. 11). ).
　その結果、化合物ＣはＨＥＫ２９３細胞に対しても、活性化条件ではほとんど細胞損傷性を示さず（ＩＣ５０　＞１０　μＭ）、飢餓条件下でも比較的弱い細胞損傷性しか示さなかった（ＩＣ５０＝１７８　ｎＭ）。
　これは、化合物Ｃは、非がん細胞にはほとんど作用しないことを示す結果である。 As a result, compound C showed almost no cell damage to HEK293 cells under activation conditions (IC 50 > 10 μM), and showed relatively weak cell damage even under starvation conditions (IC 50 =). 178 nM).
This is a result showing that compound C has almost no action on non-cancer cells.
　（６－２）ｍＴＯＲＣ１キナーゼ活性アッセイの結果
　ＡｌｐｈａＬＩＳＡ　ＳｕｒｅＦｉｒｅ　Ｕｌｔｒａ　ＨＶ　ｐ－ｐ７０　Ｓ６Ｋ　（Ｔ３８９）　ａｓｓａｙ　ｋｉｔを用いて、がん細胞（ＨｅＬａ）内で、１　μＭの化合物Ａ、化合物Ｂ、化合物ＣおよびＲＡＰが、リボソームプロテイン６キナーゼ１（ｐ７０Ｓ６Ｋ１）タンパク質のＴ３８９のリン酸化を阻害するか測定した（図１２）。 (6-2) Results of mTORC1 Kinase Activity Assay 1 μM Compound A, Compound B, Compound C and RAP in cancer cells (HeLa) using AlphaLISA SureFire Ultra HV p-p70 S6K (T389) assay kit. Inhibits the phosphorylation of T389 of the ribosome protein 6 kinase 1 (p70S6K1) protein (FIG. 12).
　化合物Ａおよび化合物Ｂは、ほとんどＴ３８９ｐ－７０Ｓ６Ｋ１のリン酸化を阻害しなかったが、化合物ＣはＲＡＰと同程度に阻害した。
　その結果、化合物Ａおよび化合物Ｂは、ほとんどＴ３８９Ｓ６Ｋ１のリン酸化を阻害しなかったが、化合物ＣはＲＡＰと同程度に阻害した。 Compound A and Compound B hardly inhibited the phosphorylation of T 389 p-70S6K1, but Compound C inhibited it to the same extent as RAP.
As a result, compound A and compound B hardly inhibited the phosphorylation of T389 S6K1, but compound C inhibited it to the same extent as RAP.
　また、化合物ＣとＲＡＰの、Ｔ３８９ｐ－７０Ｓ６Ｋ１のリン酸化阻害活性は、それぞれ３．５　ｎＭおよび０．４　ｎＭであった（図１３）。 The phosphorylation inhibitory activity of T389 p-70S6K1 of Compounds C and RAP was 3.5 nM and 0.4 nM, respectively (FIG. 13).
　さらに、Ｗｅｓｔｅｒｎブロッティングにより、がん細胞（ＨｅＬａ）中で、１　μＭの化合物ＣおよびＲＡＰが、Ｓ２３５／２３６ｐ－Ｓ６およびＴ３７／４７ｐ－４Ｅ－ＢＰ１のリン酸化阻害活性を調べた（図１４）。 Furthermore, Western blotting was performed to examine the phosphorylation inhibitory activity of S235 / 236 p-S6 and T37 / 47 p-4E-BP1 in cancer cells (HeLa) with 1 μM compounds C and RAP (FIG. 14). ).
　化合物ＣとＲＡＰは、Ｓ２３５／２３６ｐ－Ｓ６を同程度に阻害したが、Ｔ３７／４６ｐ－４Ｅ－ＢＰ１は、化合物Ｃの方がより効率的に阻害した。
 
　また、非がん細胞（ＨＥＫ２９３）で、化合物ＣとＲＡＰのＳ２３５／２３６ｐ－Ｓ６に対する阻害活性を、それぞれ０．１および１　μＭで測定した（図１５）。 Compound C and RAP inhibited S235 / 236 p-S6 to the same extent, while T37 / 46 p-4E-BP1 inhibited S235 / 236 p-4E-BP1 more efficiently.

In addition, in non-cancer cells (HEK293), the inhibitory activities of Compound C and RAP against S235 / 236 p-S6 were measured at 0.1 and 1 μM, respectively (FIG. 15).
　その結果、非がん細胞でも、ＲＡＰはＳ２３５／２３６ｐ－Ｓ６を阻害したのに対し、化合物Ｃは阻害しないことが示された。 As a result, it was shown that even in non-cancer cells, RAP inhibited S235 / 236p-S6, whereas compound C did not.
　以上の結果から、化合物Ｃはラパマイシンと異なり、がん細胞特異的にｍＴＯＲＣ１のリン酸化活性を阻害し、その結果、がん細胞特異的に細胞損傷性活性を示す化合物であり、さらに、ラパマイシンよりも効率的にＴ３７／４６ｐ－４Ｅ－ＢＰ１を阻害する事がわかった。
　これらは、化合物Ｃの抗がん剤としての潜在性能が高いことを示すものである。
[実施例４] From the above results, unlike rapamycin, compound C is a compound that inhibits the phosphorylation activity of mTORC1 specifically for cancer cells, and as a result, exhibits cell-damaging activity specifically for cancer cells, and is more than rapamycin. Was also found to efficiently inhibit T37 / 46 p-4E-BP1.
These indicate that compound C has high potential as an anticancer agent.
[Example 4]
　動物実験
　ＢＡＬＢ／ｃマウス（メス、７－９週齢）を用いて、異種移植（ｘｅｎｏｇｒａｆｔ）研究を行った。マウスは、ランダムにグループ化し（５匹／ケージ）、空調された環境で、明・暗（１２時間周期）照明を照射した。
　給水および給餌場所には自由にアクセスできるようにした。ヒト乳がんモデル（転移ありステージＩＶモデル）として、１０７　個の４Ｔ１細胞をマウスに皮下の乳腺に異種移植した。腫瘍サイズが１００　ｍｍ３到達後（約５日後）、薬剤投与を開始した。１００　μＬ　／日ＰＢＳ（腹腔投与）、２５　ｍｇ／ｋｇ／日　化合物Ｃ（経口投与）、または２５　ｍｇ／ｋｇ／日　ラパマイシン（経口投与）で、１０日間投与した。
　腫瘍サイズは、ｍｉｃｒｏＣＴ法により、２日毎に２０日間測定した。腫瘍サイズＶ＝（幅２　ｘ　長さ）／２で与えられる。
　また、マウスの体重は２日おきに測定した。
　４Ｔ１腫瘍の転移活性は、マウスを屠殺後、肺を組織固定用試薬（Ｂｏｕｉｎ固定溶液）に浸して固定してから、肺表面の白色コロニー数を数えることにより測定した（図１６）。
　血液サンプルは、化合物Ｃ投与群、非投与群（ｐｏｓｉｔｉｖｅ　ｃｏｎｔｒｏｌ）および腫瘍非移植群（ｎｅｇａｔｉｖｅ　ｃｏｎｔｒｏｌ）からそれぞれ採取した。血清ａｌａｎｉｎｅ　ｔｒａｎｓａｍｉｎａｓｅ　（ＡＬＴ）、血中ｕｒｅａ　ｎｉｔｒｏｇｅｎ　（ＢＵＮ）、ｃｒｅａｔｉｎｉｎｅ　（ＣＲＥ）およびグルコースはそれぞれ標準法により定量した。
（ａ）化合物ＣはＲＡＰよりも、高い腫瘍成長抑制効果を示した。
（ｂ）化合物Ｃは、ＲＡＰよりも弱い、腫瘍肺転移促進活性を示した。
（ｃ）Ｄ１１で投与を停止し、Ｄ２０での腫瘍再発を調べた。化合物Ｃは、ＲＡＰよりも低い腫瘍再発性を示した。 Animal experiments BALB / c mice (female, 7-9 weeks old) were used to perform xenograft studies. Mice were randomly grouped (5 / cage) and irradiated with light / dark (12-hour cycle) illumination in an air-conditioned environment.
Free access to water and feeding areas. As a human breast cancer model (stage IV model with metastasis), 107 4T1 cells were xenografted into mice under the subcutaneous mammary gland. After the tumor size reached 100 mm 3 (about 5 days later), drug administration was started. 100 μL / day PBS (peritoneal administration), 25 mg / kg / day Compound C (oral administration), or 25 mg / kg / day rapamycin (oral administration) was administered for 10 days.
Tumor size was measured every 2 days for 20 days by the microCT method. Tumor size V = (width 2 x length) / 2.
In addition, the body weight of the mice was measured every two days.
The metastatic activity of the 4T1 tumor was measured by slaughtering the mice, immersing the lungs in a tissue-fixing reagent (Bouin fixative) to fix them, and then counting the number of white colonies on the lung surface (FIG. 16).
Blood samples were taken from the compound C-administered group, the non-administered group (positive control), and the non-tumor transplanted group (negative control), respectively. Serum alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine (CRE) and glucose were quantified by standard methods, respectively.
(A) Compound C showed a higher tumor growth inhibitory effect than RAP.
(B) Compound C showed a tumor lung metastasis promoting activity weaker than RAP.
(C) Administration was discontinued at D11, and tumor recurrence at D20 was investigated. Compound C showed lower tumor relapse than RAP.
　血液アッセイおよびその他の結果は、図１７に示す。
　実験期間中、化合物Ｃにより顕著な体重減少は見られず、また、血液から明らかな毒性は検出されなかった。
[実施例５] The blood assay and other results are shown in FIG.
No significant weight loss was observed with Compound C during the experiment, and no apparent toxicity was detected in the blood.
[Example 5]
寿命延長効果
　本発明の化合物の寿命延長効果は、Ｄａｖｉｄ　Ｅ．　Ｈａｒｒｉｓｏｎら、「Ｒａｐａｍｙｃｉｎ　ｆｅｄ　ｌａｔｅ　ｉｎ　ｌｉｆｅ　ｅｘｔｅｎｄｓ　ｌｉｆｅｓｐａｎ　ｉｎ　ｇｅｎｅｔｉｃａｌｌｙ　ｈｅｔｅｒｏｇｅｎｅｏｕｓ　ｍｉｃｅ」、ｎａｔｕｒｅ，　Ｖｏｌ　４６０｜１６　Ｊｕｌｙ　２００９｜ｄｏｉ：１０．１０３８／ｎａｔｕｒｅ０８２２１に記載の方法に準じて、ラパマイシンを本発明の化合物に変えて測定する。
　なお、さらに詳細な方法および参照文献は、この文献のオンライン版において、ｗｗｗ．ｎａｔｕｒｅ．ｃｏｍ／ｎａｔｕｒｅで入手することができる。
[実施例６] Life extension effect The life extension effect of the compound of the present invention is described in David E. et al. Harrison et al., "Rapamycin fed late in life extends lifeespan in genetically heterogeneous meeting", nature, Vol 460 | 16 July 2009 | doi: 10.1038 / n according to the method. Measure.
Further detailed methods and references can be found in the online version of this document at www. nature. It can be obtained at com / nature.
[Example 6]
　図１８に示す化合物Ｄ～Ｎを、それぞれ、エナミン社（Ｅｎａｍｉｎｅ　Ｌｔｄ，　Ｋｙｉｖ，　Ｕｋｒａｉｎｅ）、アシネックス社（ＡＳＩＮＥＸ　Ｃｏｒｐｏｒａｔｉｏｎ，　ＮＣ，　ＵＳＡ）および、ケムディブ社（ＣｈｅｍＤｉｖ，　ＣＡ，ＵＳＡ）より購入した。 Compounds D to N shown in FIG. 18 were purchased from Enamine (Enami Ltd, Kyiv, Ukraine), ASINEX Corporation (NC, USA), and ChemDiv (ChemDiv, CA, USA), respectively.
（１）ＦＫＢＰ１２－ＦＲＢに結合するリガンドのｉｎ　ｖｉｖｏ選択性(1) In vivo selectivity of the ligand that binds to FKBP12-FRB
　Ｉｎ　ｖｉｖｏリガンド選択には、ＮａｎｏＢｉＴ　ＰＰＩ　Ｓｔａｒｔｅｒ　Ｓｙｓｔｅｍｓ　（Ｐｒｏｍｅｇａ，　ＵＳＡ）を利用した。
　まず、１０４個のＨｅＬａ細胞／ｗｅｌを、Ｂ＆Ｗ　Ｉｓｏｐｌａｔｅ－９６　ＴＣ　（ＰｅｒｋｉｎＥｌｍｅｒ，　ＵＳＡ）に入れ、ＤＭＥＤ　（１０％　ＦＢＳ，　１％　Ｐ／Ｓ）で一晩培養した（５％　ＣＯ２，　３７℃）。
　翌日、ＮａｎｏＢｉＴ　ＰＰＩ　Ｓｔａｒｔｅｒ　Ｓｙｓｔｅｍに同封されている、ＦＫＢＰ１２－ＳｍＢｉＴ（４３６０ｂｐ）およびＦＲＢ－ＬｇＢｉＴ（４７６２ｂｐ）コントロールベクターを、５０ｎｇ／ｗｅｌｌ濃度になるように、ＦｕＧｅｎｅ　ＨＤ　（Ｐｒｏｍｅｇａ，　ＵＳＡ）を３：１（ｖ／ｗ）の割合で用いて、各細胞に共トランスフェクションし、２４－４８　時間インキュベーションした（５％ＣＯ２，３７℃）。
　次に、培地をＯｐｔｉ－ＭＥＭ　ｒｅｄｕｃｅｄ　ｓｅｒｕｍ　ｍｅｄｉｕｍ　（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ，　ＵＳＡ）（39μＬ/well）で置換し、２０μＬ　２０倍希釈Ｎａｎｏ－Ｇｌｏ　ｌｕｃｉｆｅｒａｓｅ　ａｓｓａｙ　ｓｕｂｓｔｒａｔｅ　（Ｐｒｏｍｅｇａ，　ＵＳＡ）を各ウエルに加えた。
　ＥｎＳｐｉｒｅ　マルチモードプレートリーダー（ＰｅｒｋｉｎＥｌｍｅｒ，　ＵＳＡ）の発光測定モードを使用し、ベースラインを測定した。
　次に、リガンド（１μＭまたは１０μＭ）を各ウエルに加え、10μＬの 20xFurimazine /wellを加えて、ＰＰＩによる発光をプレートリーダーで測定した。 NanoBiT PPI Starter Systems (Promega, USA) were used for in vivo ligand selection.
First, 104 HeLa cells / wel were placed in B & W Isolate-96 TC (PerkinElmer, USA) and cultured overnight in DMED (10% FBS, 1% P / S) (5% CO 2 , 37 ° C.). ).
The next day, the FKBP12-SmBiT (4360bp) and FRB-LgBiT (4762bp) control vectors enclosed in the NanoBiT PPI Starter System were loaded with FuGene HD (Promega, USA) at a concentration of 50 ng / well. Each cell was co-transfected and incubated for 24-48 hours (5% CO 2 , 37 ° C.) using a v / w) ratio.
Next, the medium was replaced with Opti-MEM rediculated serum medium (Thermo Fisher Scientific, USA) (39 μL / well), and 20 μL 20-fold diluted Nano-Glo luciferase assay was added to each assay (Promega).
The baseline was measured using the emission measurement mode of the EnSpire multimode plate reader (PerkinElmer, USA).
Ligand (1 μM or 10 μM) was then added to each well, 10 μL of 20xFurimazine / well was added, and luminescence by PPI was measured with a plate reader.
　その結果、化合物Ｄ～Ｎは、親和性を細胞内でも持つことが確認できた（図１９）。化合物Ｊ、Ｌ、Ｍは細胞内親和性は弱かった。
　化合物Ｄ、Ｅ、Ｆ、Ｈ及びＫは、濃度依存的に、発光強度が増加していることから、ＦＫＢＰ１２－ＦＲＢに特異的に結合していることを示す（図２０）。 As a result, it was confirmed that the compounds D to N also have an affinity intracellularly (FIG. 19). Compounds J, L and M had a weak intracellular affinity.
The compounds D, E, F, H and K have increased emission intensities in a concentration-dependent manner, indicating that they are specifically bound to FKBP12-FRB (FIG. 20).
（２）ｍＴＯＲＣ１キナーゼアッセイ
　ｍＴＯＲＣ１のキナーゼ活性は、ＡｌｐｈａＬＩＳＡ(R)　ＳｕｒｅＦｉｒｅ　ＵｌｔｒａＴＭ　ＨＶ　ｐ－ｐ７０　Ｓ６Ｋ　（Ｔ３８９）　ａｓｓａｙ　ｋｉｔ　（ＴＧＲ　Ｂｉｏｓｃｉｅｎｃｅｓ，　Ａｕｓｔｒａｌｉａ，　ａｎｄ　ＰｅｒｋｉｎＥｌｍｅｒ，　ＵＳＡ）を用いて、Ｓ６Ｋタンパク質のリン酸化を測定することにより検出した。ＨＥＫ２９３細胞（１０４個／ｗｅｌｌ）をｈｉｇｈ－ｇｌｕｃｏｓｅ　ＤＭＥＭ（１０％　ＦＢＳ，　１％　Ｐ／Ｓ）を用いて、９６－ｗｅｌｌ　プレート中で一晩インキュベーションした（５％　ＣＯ２，　３７℃）。
　翌日、細胞を一晩飢餓状態（９９μｌのＯｐｔｉＭＥＭ　ｍｅｄｉａ／ｗｅｌｌ）にし、その後、１μＭのリガンを加え、１－２時間培養した。キットに含まれる、用時調製した２５μＬ　１０ｘ　ｃｅｌｌ　ｌｙｓｉｓバッファ／ｗｅｌｌで３０分間細胞を破砕した。
　６μＬの細胞破砕液を３８４－ｗｅｌｌ　ＯｐｔｉＰｌａｔｅ　（ＰｅｒｋｉｎＥｌｍｅｒ，　ＵＳＡ）に分注し、３μＬ　ａｎｔｉ－Ｓ６Ｋ　ｃｏａｔｅｄ　Ｄｏｎｏｒを暗条件下で加え、ウエルをシール後、インキュベーションした（２－４時間、室温）。
　Ａｌｐｈａ　ｓｉｇｎａｌを、ＥｎＳｐｉｒｅのＡｌｐｈａモードで測定した。 (2) mTORC1 Kinase Assay The kinase activity of mTORC1 is as follows: AlphaLISA (R) SureFire Ultra TM HV p-p70 S6K (T389) assay kit (TGR Biosciences, Australia, Australia, and Perkin E Detected by measurement. HEK293 cells ( 104 cells / well) were incubated overnight in 96-well plates using high-glucose DMEM (10% FBS, 1% P / S) (5% CO 2 , 37 ° C).
The next day, the cells were starved overnight (99 μl OptiMEM media / well), then 1 μM Ligan was added and cultured for 1-2 hours. Cells were disrupted for 30 minutes in the time-prepared 25 μL 10x cell lysis buffer / well included in the kit.
6 μL of cell disruption solution was dispensed into 384-well OptiPlate (PerkinElmer, USA), 3 μL anti-S6K coated Donor was added under dark conditions, the wells were sealed and then incubated (2-4 hours, room temperature).
Alpha signal was measured in Alpha mode of EnSpire.
　化合物Ｋは、ほとんどＴ３８９ｐＳ６Ｋ１のリン酸化を阻害しなかったが、化合物Ｋ以外の化合物はＴ３８９ｐＳ６Ｋ１のリン酸化をある程度阻害した。 Compound K hardly inhibited the phosphorylation of T 389 pS6K1, but compounds other than compound K inhibited the phosphorylation of T 389 pS6K1 to some extent.
［発明の効果］
　以上の結果から、本発明の誘導体は、ラパマイシンと異なり、正常細胞に毒性を齎すことなく、がん細胞特異的にｍＴＯＲＣ１のリン酸化活性を阻害し、その結果、がん細胞特異的に細胞損傷性活性を示す化合物であり、さらに、ラパマイシンよりも効率的にＴ３７／４６ｐ－４Ｅ－ＢＰ１を阻害する事がわかった。
　これらは、本化合物が、副作用の発現可能性が少なく、抗がん剤、発がん抑制剤および／または寿命延長剤として、潜在性能が高いことを示すものである。
  [The invention's effect]
From the above results, unlike rapamycin, the derivative of the present invention inhibits the phosphorylation activity of mTORC1 specifically for cancer cells without causing toxicity to normal cells, and as a result, cell damage is specific for cancer cells. It was found that it is a sexually active compound and further inhibits T37 / 46 p-4E-BP1 more efficiently than rapamycin.
These indicate that this compound has a low possibility of causing side effects and has high potential performance as an anticancer agent, a carcinogenic inhibitor and / or a life-prolonging agent.

Claims (24)

  1. 　下記の部分構造（Ｉ）、（ＩＩ）、および（ＩＩＩ）を含む、ｍＴＯＲＣ１阻害作用を有する化合物またはその薬理上許容される塩を含む、制癌剤および／または発がん抑制剤：
    Figure JPOXMLDOC01-appb-C000001
     （式中、
    　Ｒ１、Ｒ２、Ｒ３およびＲ４は、同一又は異なって、Ｈ、低級アルキル、ハロ低級アルキル、低級アルコキシ、低級アルキルアミノ、ＯＨ、保護されたＯＨ、アミノ、保護されたアミノ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示し、
    　Ｒ５およびＲ６は、同一又は異なって、ＨまたはＯＨの保護基を示すか、或いは、Ｒ５およびＲ６が一緒になって、メチレンまたはジメチルメチレン基を示し、
    　Ａは、ＯまたはＳを示す。）。     Anticancer agents and / or carcinogens, including compounds having mTORC1 inhibitory activity or pharmacologically acceptable salts thereof, including the following partial structures (I), (II), and (III):
    Figure JPOXMLDOC01-appb-C000001
     (During the ceremony,
    R 1 , R 2 , R 3 and R 4 are the same or different, H, lower alkyl, halo lower alkyl, lower alkoxy, lower alkyl amino, OH, protected OH, amino, protected amino, carboxy, Represents a protected carboxy or halogen atom,
    R 5 and R 6 indicate the same or different protective groups for H or OH, or R 5 and R 6 together indicate a methylene or dimethyl methylene group.
    A indicates O or S. ).
  2. 　式（ＩＶ）の化合物またはその薬理上許容される塩：
    Figure JPOXMLDOC01-appb-C000002
     （式中、
    　Ｌ、ＸおよびＹは、同一又は異なって、リンカーを示し、
    　Ｒ１、Ｒ２、Ｒ３およびＲ４は、同一又は異なって、Ｈ、低級アルキル、ハロ低級アルキル、低級アルコキシ、低級アルキルアミノ、ＯＨ、保護されたＯＨ、アミノ、保護されたアミノ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示し、
    　Ｒ５およびＲ６は、同一又は異なって、ＨまたはＯＨの保護基を示すか、或いは、Ｒ５およびＲ６が一緒になって、メチレンまたはジメチルメチレン基を示し、
    　Ａは、ＯまたはＳを示す。）を含む、請求項１に記載された制癌剤および／または発がん抑制剤。     Compound of formula (IV) or pharmacologically acceptable salt thereof:
    Figure JPOXMLDOC01-appb-C000002
     (During the ceremony,
    L, X and Y indicate the same or different linkers,
    R 1 , R 2 , R 3 and R 4 are the same or different, H, lower alkyl, halo lower alkyl, lower alkoxy, lower alkyl amino, OH, protected OH, amino, protected amino, carboxy, Represents a protected carboxy or halogen atom,
    R 5 and R 6 indicate the same or different protective groups for H or OH, or R 5 and R 6 together indicate a methylene or dimethyl methylene group.
    A indicates O or S. ), The anticancer agent and / or the carcinogenic suppressant according to claim 1.
  3. 　Ｌ、ＸおよびＹが、同一又は異なって、－（ＣＨ２）１～６－、酸素原子で介在されていてもよい－（ＣＨ２）１～６－、ＮＨで介在されていてもよい－（ＣＨ２）１～６－、または下記置換基で置換された－（ＣＨ２）１～６－のリンカーを示す、請求項１～2のうちの１項に記載された制癌剤および／または発がん抑制剤。
    　置換基：低級アルキル、ハロ低級アルキル、低級アルコキシ、ハロゲン、アミノおよびＯＨ基。     L, X and Y are the same or different and may be intervened by-(CH 2 ) 1 to 6- , oxygen atom-(CH 2 ) 1 to 6- , NH. The anticancer agent and / or carcinogenic agent according to claim 1-2, which indicates a linker of (CH 2 ) 1 to 6 -or substituted with the following substituent-(CH 2 ) 1 to 6- . Inhibitor.
    Substituents: lower alkyl, halo lower alkyl, lower alkoxy, halogen, amino and OH groups.
  4. 　Ｌ、ＸおよびＹが、同一又は異なって、－（ＣＨ２）１～６－を示す、請求項１～3のうちの１項に記載された制癌剤および／または発がん抑制剤。 The anticancer agent and / or carcinogenic inhibitor according to claim 1, wherein L, X and Y are the same or different and indicate − (CH 2 ) 1 to 6 −.
  5. 　Ｒ１、Ｒ２、Ｒ３およびＲ４は、同一又は異なって、Ｈ、低級アルキル、ハロ低級アルキル、低級アルコキシ、低級アルキルアミノ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示す、請求項１～4のうちの１項に記載された制癌剤および／または発がん抑制剤。 Claims 1 to 1, wherein R 1 , R 2 , R 3 and R 4 represent the same or different H, lower alkyl, halo lower alkyl, lower alkoxy, lower alkyl amino, carboxy, protected carboxy or halogen atom. The anticancer agent and / or the carcinogenic suppressant according to item 1 of 4.
  6. 　Ｒ１およびＲ２が、水素原子である、請求項１～5のうちの１項に記載された制癌剤および／または発がん抑制剤。 The anticancer agent and / or carcinogenic inhibitor according to claim 1, wherein R1 and R2 are hydrogen atoms.
  7. 　Ｒ３が、Ｈで、Ｒ４が、低級アルキル、ハロ低級アルキル、低級アルコキシ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示すか、あるいは、Ｒ３およびＲ４が、同一又は異なって、低級アルキル、ハロ低級アルキル、低級アルコキシ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示す、請求項１～6のうちの１項に記載された制癌剤および／または発がん抑制剤。 R 3 is H and R 4 is lower alkyl, halo lower alkyl, lower alkoxy, carboxy, protected carboxy or halogen atom, or R 3 and R 4 are the same or different, lower alkyl. , A halo lower alkyl, a lower alkoxy, a carboxy, a protected carboxy or halogen atom, the anticancer agent and / or a carcinogenic inhibitor according to claim 1.
  8. 　Ｒ３が、Ｈで、Ｒ４が、低級アルキル、ハロ低級アルキル、低級アルコキシ、保護されたカルボキシまたはハロゲン原子を示すか、あるいは、Ｒ３およびＲ４が、同一又は異なって、低級アルキル、ハロ低級アルキル、低級アルコキシまたはハロゲン原子を示す、請求項１～7のうちの１項に記載された制癌剤および／または発がん抑制剤。 R 3 is H and R 4 is lower alkyl, halo lower alkyl, lower alkoxy, protected carboxy or halogen atom, or R 3 and R 4 are the same or different, lower alkyl, halo. The anticancer agent and / or carcinogenic inhibitor according to claim 1, which indicates a lower alkyl, lower alkoxy or halogen atom.
  9. 　Ｒ３が、Ｈで、Ｒ４が、低級アルキル、ハロ低級アルキル、またはハロゲン原子を示すか、あるいは、Ｒ３およびＲ４が、同一又は異なって、低級アルキル、ハロ低級アルキル、またはハロゲン原子を示す、請求項１～8のうちの１項に記載された制癌剤および／または発がん抑制剤。 R 3 is H and R 4 represents a lower alkyl, halo lower alkyl, or halogen atom, or R 3 and R 4 are the same or different, lower alkyl, halo lower alkyl, or halogen atom. The anticancer agent and / or the carcinogenic suppressant according to claim 1 of claim 1-8.
  10. 　Ｒ３または／およびＲ４が、オルト位に置換している、請求項１～9のうちの１項に記載された制癌剤および／または発がん抑制剤。 The anticancer agent and / or carcinogenic inhibitor according to claim 1, wherein R3 or / and R4 are substituted at the ortho position.
  11. 　Ｒ５およびＲ６が、一緒になって、メチレンまたはジメチルメチレン基を示す、請求項１～１0のいずれか１項に記載された制癌剤および／または発がん抑制剤。 The anticancer agent and / or carcinogenic inhibitor according to any one of claims 1 to 10 , wherein R5 and R6 together represent a methylene or dimethylmethylene group.
  12. 　Ａが、Ｏを示す、請求項１～１１のいずれか１項に記載された制癌剤および／または発がん抑制剤。 The anticancer agent and / or carcinogenic suppressant according to any one of claims 1 to 11, wherein A indicates O.
  13. 　化合物が以下の化合物から選択される１の化合物である（但し、Ａは、ＯまたはＳである。）、請求項１に記載された制癌剤および／または発がん抑制剤。
    Figure JPOXMLDOC01-appb-C000003
    Figure JPOXMLDOC01-appb-I000004
    The anticancer agent and / or carcinogenic inhibitor according to claim 1, wherein the compound is one compound selected from the following compounds (where A is O or S).
    Figure JPOXMLDOC01-appb-C000003
    Figure JPOXMLDOC01-appb-I000004
  14. 　化合物が以下の化合物から選択される１の化合物である、請求項１に記載された制癌剤および／または発がん抑制剤。
    Figure JPOXMLDOC01-appb-C000005
         The anticancer agent and / or carcinogenic suppressant according to claim 1, wherein the compound is one compound selected from the following compounds.
    Figure JPOXMLDOC01-appb-C000005
  15. 　化合物が以下の化合物から選択される１の化合物である、請求項１に記載された制癌剤および／または発がん抑制剤。
    Figure JPOXMLDOC01-appb-C000006
         The anticancer agent and / or carcinogenic suppressant according to claim 1, wherein the compound is one compound selected from the following compounds.
    Figure JPOXMLDOC01-appb-C000006
  16. 　下記の部分構造（Ｉ）、（ＩＩ）、および（ＩＩＩ）を含む、ｍＴＯＲＣ１阻害作用を有する化合物またはその薬理上許容される塩を含む、寿命延長剤
    Figure JPOXMLDOC01-appb-C000007
     （式中、
    　Ｒ１、Ｒ２、Ｒ３およびＲ４は、同一又は異なって、Ｈ、低級アルキル、ハロ低級アルキル、低級アルコキシ、低級アルキルアミノ、ＯＨ、保護されたＯＨ、アミノ、保護されたアミノ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示し、
    　Ｒ５およびＲ６は、同一又は異なって、ＨまたはＯＨの保護基を示すか、或いは、Ｒ５およびＲ６が一緒になって、メチレンまたはジメチルメチレン基を示し、
    　Ａは、ＯまたはＳを示す。）。     A life-prolonging agent containing a compound having an mTORC1 inhibitory effect or a pharmaceutically acceptable salt thereof, which comprises the following partial structures (I), (II), and (III).
    Figure JPOXMLDOC01-appb-C000007
     (During the ceremony,
    R 1 , R 2 , R 3 and R 4 are the same or different, H, lower alkyl, halo lower alkyl, lower alkoxy, lower alkyl amino, OH, protected OH, amino, protected amino, carboxy, Represents a protected carboxy or halogen atom,
    R 5 and R 6 indicate the same or different protective groups for H or OH, or R 5 and R 6 together indicate a methylene or dimethyl methylene group.
    A indicates O or S. ).
  17. 　式（ＩＶ）の化合物またはその薬理上許容される塩
    Figure JPOXMLDOC01-appb-C000008
     （式中、
    　Ｌ、ＸおよびＹは、同一又は異なって、リンカーを示し、
    　Ｒ１、Ｒ２、Ｒ３およびＲ４は、同一又は異なって、Ｈ、低級アルキル、ハロ低級アルキル、低級アルコキシ、低級アルキルアミノ、ＯＨ、保護されたＯＨ、アミノ、保護されたアミノ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示し、
    　Ｒ５およびＲ６は、同一又は異なって、ＨまたはＯＨの保護基を示すか、或いは、Ｒ５およびＲ６が一緒になって、メチレンまたはジメチルメチレン基を示し、
    　Ａは、ＯまたはＳを示す。）を含む、請求項１６に記載された寿命延長剤。     Compound of formula (IV) or pharmacologically acceptable salt thereof
    Figure JPOXMLDOC01-appb-C000008
     (During the ceremony,
    L, X and Y indicate the same or different linkers,
    R 1 , R 2 , R 3 and R 4 are the same or different, H, lower alkyl, halo lower alkyl, lower alkoxy, lower alkyl amino, OH, protected OH, amino, protected amino, carboxy, Represents a protected carboxy or halogen atom,
    R 5 and R 6 indicate the same or different protective groups for H or OH, or R 5 and R 6 together indicate a methylene or dimethyl methylene group.
    A indicates O or S. ), The life extension agent according to claim 16.
  18. 　Ｌ、ＸおよびＹが、同一又は異なって、－（ＣＨ２）１～６－を示す、請求項１６～１７のうちの１項に記載された寿命延長剤。 The life extension agent according to claim 16, wherein L, X and Y are the same or different and indicate − (CH 2 ) 1 to 6 −.
  19. 　Ｒ１、Ｒ２、Ｒ３およびＲ４は、同一又は異なって、Ｈ、低級アルキル、ハロ低級アルキル、低級アルコキシ、低級アルキルアミノ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示す、請求項１６～１８のうちの１項に記載された寿命延長剤。 16- _ _ _ The life extender according to item 1 of 18.
  20. 　Ｒ３が、Ｈで、Ｒ４が、低級アルキル、ハロ低級アルキル、低級アルコキシ、カルボキシ、保護されたカルボキシまたはハロゲン原子を示すか、あるいは、Ｒ３およびＲ４が、同一又は異なって、低級アルキル、ハロ低級アルキル、低級アルコキシまたはハロゲン原子を示す、請求項１６～１９のうちの１項に記載された寿命延長剤。 R 3 is H and R 4 is lower alkyl, halo lower alkyl, lower alkoxy, carboxy, protected carboxy or halogen atom, or R 3 and R 4 are the same or different, lower alkyl. , A lifetime extender according to claim 16-19, which comprises a halo lower alkyl, lower alkoxy or halogen atom.
  21. 　Ｒ５およびＲ６が、一緒になって、メチレンまたはジメチルメチレン基を示す、請求項１６～２０のいずれか１項に記載された寿命延長剤。 The life extender according to any one of claims 16 to 20, wherein R 5 and R 6 together represent a methylene or dimethylmethylene group.
  22. 　化合物が以下の化合物から選択される１の化合物である（但し、Ａは、ＯまたはＳである。）、請求項１６に記載された寿命延長剤。
    Figure JPOXMLDOC01-appb-C000009
    Figure JPOXMLDOC01-appb-I000010
    The life extender according to claim 16, wherein the compound is one compound selected from the following compounds (where A is O or S).
    Figure JPOXMLDOC01-appb-C000009
    Figure JPOXMLDOC01-appb-I000010
  23. 　化合物が以下の化合物から選択される１の化合物である、請求項１６に記載された寿命延長剤。
    Figure JPOXMLDOC01-appb-C000011
         The life extender according to claim 16, wherein the compound is one compound selected from the following compounds.
    Figure JPOXMLDOC01-appb-C000011
  24. 　化合物が以下の化合物から選択される１の化合物である、請求項１６に記載された寿命延長剤。
    Figure JPOXMLDOC01-appb-C000012
         The life extender according to claim 16, wherein the compound is one compound selected from the following compounds.
    Figure JPOXMLDOC01-appb-C000012
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