Anticancer agents, cancer inhibitors, and life-extending agents containing novel compounds with mTORC1 inhibitory activity.

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

[Technical Field]

[0001] The present invention contains compounds having mTORC1 inhibitory activity and is useful as an anticancer agent, cancer inhibitor, and / or life extension agent. [Background technology]

[0002] mTOR (mammalian target of rapamycin) is a serine / threonine kinase identified as a target molecule of rapamycin. It was named mTOR (target of rapamycin) because it plays a central role in regulating cell division and survival. When cells are stimulated by growth factors, a phosphorylation enzyme called PI3 kinase (Phosphoinositide 3-kinase: PI3K) is activated. This then phosphorylates and activates a serine / threonine phosphorylation enzyme called Akt, which regulates the activity of various proteins involved in intracellular signal transduction, thereby regulating cell proliferation and survival (death). One of Akt's targets is mTOR, and mTOR, phosphorylated (activated) by Akt, acts on cell division, cell death, angiogenesis, and energy production, promoting the proliferation of cancer cells (PI3K / Akt / mTOR pathway). This is an extremely important mechanism for promoting the proliferation of cancer cells and sarcoma cells. In other words, inhibition of the PI3K / Akt / mTOR pathway can suppress the proliferation of cancer and sarcoma cells and induce apoptosis (programmed cell death), making it a target for cancer treatment and cancer prevention. It has also been reported to enhance the effectiveness of anticancer drugs and radiation therapy.

[0003] Furthermore, nutrients (such as glucose and amino acids), insulin, insulin-like growth factors, and other growth factors activate the mTORC1 signaling pathway, promoting cell growth and the progression of the cell cycle (the process from when a cell divides until it divides again). Cancer cells are cells in which abnormalities in the control of the cell cycle occur due to gene mutations or other factors, causing them to divide uncontrollably. In cancer cells where the cell cycle is progressing, activation of mTORC1 promotes cell proliferation.

[0004] On the other hand, when mTORC1 activity is increased in cells that have aged and whose cell cycle has stopped (G0 phase), the cells transition to an aged state (complete loss of cell proliferation ability and decline in cellular function).

[0005] Therefore, mTORC1 activity inhibitors such as rapamycin can suppress both cell carcinogenesis (anti-cancer and carcinogenic) and aging. Although mTOR inhibitors have the drawback of suppressing the immune system, they are highly effective as anticancer agents because mTOR is activated in many cancer and sarcoma cells. Several mTOR inhibitors have already been developed and are being used as anticancer drugs. Rapamycin was originally an immunosuppressant used to prevent organ transplant rejection. While immunosuppressants typically increase the incidence of cancer, numerous studies have shown the opposite effect with rapamycin, revealing that patients who took rapamycin after kidney or liver transplants had an extremely low incidence of cancer.

[0006] 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 mentioned above, although the development of mTOR inhibitors based on rapamycin is being actively pursued, none have a similar structure to the compound of the present invention, and no compound that is smaller in molecular weight and exhibits specific toxicity to cancer cells is known.

[0007] Furthermore, aging is a cause of the onset and progression of various diseases, which are influenced by the mTORC1 signaling pathway. Among the various diseases associated with aging, the mTORC1 inhibitor of the present invention acts in a way that improves many of them, including cognitive decline, Alzheimer's disease, malignant tumors, kidney disease, heart disease, and autoimmune diseases (Non-Patent Documents 1 and 2). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Ginza Tokyo Clinic website (http: / / www.f-gtc.or.jp / mTORC1 / rapamycin.html) "Anti-cancer effects of rapamycin" [Non-Patent Document 2] Ginza Tokyo Clinic website (http: / / www.f-gtc.or.jp / mTOR / mTOR.html) "Cancer treatment targeting the inhibition of mammalian target of rapamycin (mTOR) activity" [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The object of the present invention is to provide an anticancer agent, a cancer inhibitor, and a life-extending agent that contains a compound having mTORC1 inhibitory activity. [Means for solving the problem]

[0010] The present invention provides the following: <1> An anticancer agent and / or cancer inhibitor comprising a compound having mTORC1 inhibitory activity or a pharmaceutically acceptable salt thereof, comprising the following substructures (I), (II), and (III). [ka] (In the formula, R 1 , R 2 , R 3 and R 4is the same or different and represents H, lower alkyl, halo-lower alkyl, lower alkoxy, lower alkylamino, OH, protected OH, amino, protected amino, carboxy, protected carboxy or a halogen atom, R 5 and R 6 is the same or different and represents a protecting group for H or OH, or R 5 and R 6 together represent a methylene or dimethylmethylene group, A represents O or S.).

[0011] <2> A compound of formula (IV) or a pharmaceutically acceptable salt thereof [Chemical formula] (wherein L, X and Y are the same or different and represent a linker, R 1 , R 2 , R 3 and R 4 are the same or different and represent H, lower alkyl, halo-lower alkyl, lower alkoxy, lower alkylamino, OH, protected OH, amino, protected amino, carboxy, protected carboxy or a halogen atom, R 5 and R 6 are the same or different and represent a protecting group for H or OH, or R 5 and R 6 together represent a methylene or dimethylmethylene group, A represents O or S.). The anticancer agent and / or cancer-suppressing agent described in <1>.

[0012] <3> L, X and Y are the same or different and are -(CH2) 1~6 -, -(CH2) which may be intervened by an oxygen atom 1~6 -, -(CH2) which may be intervened by NH 1~6 -, or -(CH2) substituted with the following substituent 1~6- Indicates the linker, <1> ~ <2> An anticancer agent and / or cancer inhibitor as described in item 1 of the above. Substituents: lower alkyl, halolower alkyl, lower alkoxy, halogen, amino, and OH groups. <4> L, X, and Y are the same or different, -(CH2) 1~6 - indicates <1> ~ <3> An anticancer agent and / or cancer inhibitor as described in item 1 of the above. <5> R 1 , R 2 , R 3 and R 4 These represent, either identically or distinctly, H, lower alkyl, halo-lower alkyl, lower alkoxy, lower alkylamino, carboxy, protected carboxy, or halogen atoms. <1> ~ <4> An anticancer agent and / or cancer inhibitor as described in item 1 of the above. <6> R 1 and R 2 However, it is a hydrogen atom. <1> ~ <5> An anticancer agent and / or cancer inhibitor as described in item 1 of the above. <7> R 3 But, H, R 4 However, it may represent a lower alkyl, halolower alkyl, lower alkoxy, carboxy, protected carboxy or halogen atom, or R 3 and R 4 However, they are identical or different in that they represent a lower alkyl, halolower alkyl, lower alkoxy, carboxy, protected carboxy, or halogen atom. <1> ~ <6> An anticancer agent and / or cancer inhibitor as described in item 1 of the above. <8> R 3 But, H, R 4 However, it may represent a lower alkyl, halolower alkyl, lower alkoxy, protected carboxy or halogen atom, or R 3 and R 4 However, they are the same or different, and represent a lower alkyl, halolower alkyl, lower alkoxy, or halogen atom. <1> ~ <7> An anticancer agent and / or cancer inhibitor as described in item 1 of the above. <9> R 3 But, H, R 4However, it may represent a lower alkyl, halolower alkyl, or halogen atom, or R 3 and R 4 However, they are the same or different, and represent lower alkyl, halo-lower alkyl, or halogen atoms. <1> ~ <8> An anticancer agent and / or cancer inhibitor as described in item 1 of the above. <10> R 3 or / and R 4 However, it is being substituted in the ortho position. <1> ~ <9> An anticancer agent and / or cancer inhibitor as described in item 1 of the above. <11> R 5 and R 6 However, together they form a methylene or dimethylmethylene group. <1> ~ <10> An anticancer agent and / or cancer inhibitor as described in any one of the items. <12> A indicates O. <1> ~ <11> An anticancer agent and / or cancer inhibitor as described in any one of the items.

[0013] <13> The compound is one compound selected from the following compounds (where A is either O or S). <1> Anticancer agents and / or cancer inhibitors as described above. [ka] JPEG0007847761000004.jpg199170

[0014] <14> The compound is one compound selected from the following compounds. <1> Anticancer agents and / or cancer inhibitors as described above. [ka]

[0015] <15> The compound is one compound selected from the following compounds. <1> Anticancer agents and / or cancer inhibitors as described above. [ka]

[0016] <16> Life extension agent comprising a compound having mTORC1 inhibitory activity or a pharmaceutically acceptable salt thereof, comprising the following substructures (I), (II), and (III). [ka] (In the formula, R 1 , R 2 , R 3 and R 4 These represent, either identically or distinctly, H, lower alkyl, halo-lower alkyl, lower alkoxy, lower alkylamino, OH, protected OH, amino, protected amino, carboxy, protected carboxy, or halogen atoms. R 5 and R 6 These are identical or different, indicating a protecting group of H or OH, or R 5 and R 6 Together, they form a methylene or dimethylmethylene group. A represents either O or S.

[0017] <17> Compounds of formula (IV) or their pharmaceutically acceptable salts [ka] (In the formula, L, X, and Y may be the same or different, indicating the linker. R 1 , R 2 , R 3 and R 4 These represent, either identically or distinctly, H, lower alkyl, halo-lower alkyl, lower alkoxy, lower alkylamino, OH, protected OH, amino, protected amino, carboxy, protected carboxy, or halogen atoms. R 5 and R 6 These are identical or different, indicating a protecting group of H or OH, or R 5 and R 6 Together, they form a methylene or dimethylmethylene group. A represents O or S. <16> A life-extending agent as described.

[0018] <18> L, X, and Y are the same or different, -(CH2) 1~6 - indicates <16> ~ <17> A life-extending agent as described in item 1 of the list. <19> R 1 , R 2 , R 3 and R 4 These represent, either identically or distinctly, H, lower alkyl, halo-lower alkyl, lower alkoxy, lower alkylamino, carboxy, protected carboxy, or halogen atoms. <16> ~ <18> A life-extending agent as described in item 1 of the list. <20> R 3 But, H, R 4 However, it may represent a lower alkyl, halolower alkyl, lower alkoxy, carboxy, protected carboxy or halogen atom, or R 3 and R 4 However, they are the same or different, and represent a lower alkyl, halolower alkyl, lower alkoxy, or halogen atom. <16> ~ <19> A life-extending agent as described in item 1 of the list. <21> R 5 and R 6 However, together they form a methylene or dimethylmethylene group. <16> ~ <20> A life-extending agent as described in any one of the items. <22> The compound is one compound selected from the following compounds (where A is either O or S). <16> A life-extending agent as described. [ka] JPEG0007847761000010.jpg201170

[0019] <23> The compound is one compound selected from the following compounds. <16> A life-extending agent as described. [ka]

[0020] <24> The compound is one compound selected from the following compounds. <16> A life-extending agent as described. [ka]

[0021] Preferably, the molecular weight of the compounds included in the present invention is 450 to 850, more preferably 500 to 800.

[0022] A method for suppressing cancer and / or carcinogenesis, comprising administering a compound having mTORC1 inhibitory activity or a pharmaceutically acceptable salt thereof, comprising the above substructures (I), (II), and (III), to a subject (preferably a mammal including a human). A method for extending lifespan, comprising administering to a subject (preferably a mammal including a human) a compound having mTORC1 inhibitory activity, comprising the above-described substructures (I), (II), and (III), or a pharmaceutically acceptable salt thereof. <c>Compounds having mTORC1 inhibitory activity, comprising the above substructures (I), (II), and (III), or pharmaceutically acceptable salts thereof, for use in treatments to suppress cancer and / or carcinogenesis. <d>Compounds having mTORC1 inhibitory activity, comprising the above substructures (I), (II), and (III), or pharmaceutically acceptable salts thereof, for use in life-extending treatments. <e>Use of compounds having mTORC1 inhibitory activity or pharmaceutically acceptable salts thereof, comprising the substructures (I), (II), and (III) described herein, for the manufacture of anticancer agents and / or cancer inhibitors. <f>Use of compounds having mTORC1 inhibitory activity or pharmaceutically acceptable salts thereof, comprising the substructures (I), (II), and (III) described below, for the manufacture of life-extending agents.

[0023] In the present invention, "Lower alkyl" refers to linear or branched alkyl groups having 1 to 6 carbon atoms, such as 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-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, and 2-ethylbutyl, and preferably linear or branched alkyl groups having 1 to 4 carbon atoms.

[0024] "Halo-lower alkyl" refers to a group in which a "halogen atom" (described below) is bonded to the "lower alkyl" group. Examples of such groups include trifluoromethyl, trichloromethyl, difluoromethyl, dichloromethyl, dibromomethyl, fluoromethyl, 2,2,2-trichloroethyl, 2,2,2-trifluoroethyl, 2-bromoethyl, 2-chloroethyl, 2-fluoroethyl, and 2,2-dibromoethyl, with trifluoromethyl being preferred.

[0025] "Lower alkoxy" refers to a group in which the "lower alkyl" is bonded to an oxygen atom, and includes, for example, linear or branched alkoxy groups having 1 to 6 carbon atoms, such as 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, and 2,3-dimethylbutoxy, preferably linear or branched alkoxy groups having 1 to 4 carbon atoms.

[0026] "Lower alkylamino" refers to a group in which the "lower alkyl" is bonded to an amino acid, and includes, for example, linear or branched amino groups having 1 to 6 carbon atoms, such as methylamino, ethylamino, n-propylamino, isopyramino, n-butylamino, isobutylamino, s-butylamino, tert-butylamino, n-pentylamino, isopentylamino, 2-methylbutylamino, neopentylamino, n-hexylamino, 3,3-dimethylbutylamino, 2,2-dimethylbutylamino, 1,1-dimethylbutylamino, 1,2-dimethylbutylamino, 1,3-dimethylbutylamino, and 2,3-dimethylbutylamino, preferably linear or branched amino groups having 1 to 4 carbon atoms.

[0027] The "protecting group" of a "protected OH" and / or "protecting group of OH" refers to a "reaction-protecting group" that can be cleaved by chemical methods such as hydrolysis, hydrolysis, electrolysis, and photolysis. Examples of such "protecting groups in reactions" include 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, Alkyl carbonyl groups such as heptadecanoyl, 15-methylhexadecanoyl, octadecanoyl, 1-methylheptadecanoyl, nonadecanoyl, aicosanoyl and henaicosanoyl; carboxylated alkyl carbonyl groups such as succinoyl, glutaloyl and adipoyl; halogenolower alkyl carbonyl groups such as chloroacetyl, dichloroacetyl, trichloroacetyl and trifluoroacetyl; lower alkoxylower alkyl carbonyl groups such as methoxyacetyl; unsaturated alkyl carbonyl groups such as (E)-2-methyl-2-butenoyl Aliphatic acyl groups such as chlorocarbonyl groups; arylcarbonyl groups such as benzoyl, α-naphthoyl, β-naphthoyl; halogenoarylcarbonyl groups such as 2-bromobenzoyl, 4-chlorobenzoyl; lower alkylated arylcarbonyl groups such as 2,4,6-trimethylbenzoyl, 4-toluyl; lower alkoxylated arylcarbonyl groups such as 4-anisoiyl; carboxylated arylcarbonyl groups such as 2-carboxybenzoyl, 3-carboxybenzoyl, 4-carboxybenzoyl; 4-nitrobenzoyl, 2 Aromatic acyl groups such as nitrated allylcarbonyl groups like nitrobenzoyl, lower alkoxycarbonyl allylcarbonyl groups like 2-(methoxycarbonyl)benzoyl, and allylized allylcarbonyl groups like 4-phenylbenzoyl; tetrahydropyran-2-yl, 3-bromotetrahydropyran-2-yl, 4-methoxytetrahydropyran-4-yl, tetrahydrothiopyran-2-yl, and 4-methoxytetrahydrothiopyran-4-yl;"Tetrahydrofuranyl or tetrahydrothiofuranyl groups" such as tetrahydrofuran-2-yl and tetrahydrothiofuran-2-yl; tri-lower alkylsilyl groups such as trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, methyldiisopropylsilyl, methyldi-t-butylsilyl, and triisopropylsilyl; and tri-lower alkylsilyl groups substituted with one or two allyl groups such as diphenylmethylsilyl, diphenylbutylsilyl, diphenylisopropylsilyl, and phenyldiisopropylsilyl. Silyl groups such as methyl groups; lower alkoxymethyl groups such as methoxymethyl, 1,1-dimethyl-1-methoxymethyl, ethoxymethyl, propoxymethyl, isopropoxymethyl, butoxymethyl, t-butoxymethyl; lower alkoxymethyl groups such as 2-methoxyethoxymethyl; halogenolower alkoxymethyl groups such as 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl; lower alkoxyethyl groups such as 1-ethoxyethyl, 1-(isopropoxy)ethyl, 2,2, Substitutable ethyl groups such as halogenated ethyl groups like 2-trichloroethyl; lower alkyl groups substituted with 1 to 3 allyl groups such as benzyl, α-naphthylmethyl, β-naphthylmethyl, diphenylmethyl, triphenylmethyl, α-naphthyldiphenylmethyl, 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-shea "Aralkyl groups" such as lower alkyl groups such as nobenzyl, methyl, and piperonyl, lower alkoxy groups, halogens, and lower alkyl groups substituted with one to three allyl groups on an allyl ring; "Alkoxycarbonyl groups" such as lower alkoxycarbonyl groups such as methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, and isobutoxycarbonyl, and lower alkoxycarbonyl groups substituted with halogens or trilower alkylsilyl groups such as 2,2,2-trichloroethoxycarbonyl and 2-trimethylsilylethoxycarbonyl;Examples include "alkenyloxycarbonyl groups" such as vinyloxycarbonyl and allyloxycarbonyl; and "aralkyloxycarbonyl groups" where the allyl ring may be substituted with one or two lower alkoxy or nitro groups, such as benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, and 4-nitrobenzyloxycarbonyl.

[0028] The "protecting group" in "protected amino" refers to a group in which one or two protecting groups protect an amino group. The protecting group is not limited as long as it is one that is normally used as a protecting group for amino groups, but preferably includes alkylcarbonyl groups such as formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, lauroyl, myristoyl, tridecanoyl, palmitoyl, stearoyl, chloroacetyl, dichloroacetyl, trichloroacetyl, and trifluoroacetyl. Aliphatic acyl groups such as halogeno lower alkylcarbonyl groups, lower alkoxy lower alkylcarbonyl groups such as methoxyacetyl, and unsaturated alkylcarbonyl groups such as (E)-2-methyl-2-butenoyl; arylcarbonyl groups such as benzoyl, α-naphthoyl, and β-naphthoyl; halogenoarylcarbonyl groups such as 2-bromobenzoyl and 4-chlorobenzoyl; lower alkylated arylcarbonyl groups such as 2,4,6-trimethylbenzoyl and 4-toluyl; and lower alkoxylated arylcarbonyl groups such as 4-anisoiyl. Aromatic acyl groups such as -arocarbonyl groups, nitrated allylcarbonyl groups like 4-nitrobenzoyl and 2-nitrobenzoyl, lower alkoxycarbonyl allylcarbonyl groups like 2-(methoxycarbonyl)benzoyl, and allylyl allylcarbonyl groups like 4-phenylbenzoyl; lower alkoxycarbonyl groups like methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, and isobutoxycarbonyl, and 2,2,2-trichloroethoxycarbonyl and 2-trimethylsilylethoxycarbonyl. Alkoxycarbonyl groups such as lower alkoxycarbonyl groups substituted with halogens or trilower alkylsilyl groups; alkenyloxycarbonyl groups such as vinyloxycarbonyl and allyloxycarbonyl; aralkyloxycarbonyl groups in which the allyl ring may be substituted with one or two lower alkoxy or nitro groups, such as benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, and 4-nitrobenzyloxycarbonyl;Silyl groups such as trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, methyldiisopropylsilyl, methyldi-t-butylsilyl, triisopropylsilyl; tri-lower alkylsilyl groups substituted with one or two allyl groups such as diphenylmethylsilyl, diphenylbutylsilyl, diphenylisopropylsilyl, phenyldiisopropylsilyl; lower alkyl groups substituted with one to three allyl groups such as benzyl, phenethyl, 3-phenylpropyl, α-naphthylmethyl, β-naphthylmethyl, diphenylmethyl, triphenylmethyl, α-naphthyldiphenylmethyl, 9-anthurylmethyl; 4-methylbenzyl, 2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl Aralkyl groups such as 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 4-cyanobenzyl, 4-cyanobenzyldiphenylmethyl, bis(2-nitrophenyl)methyl, piperonyl, and other lower alkyl, lower alkoxy, nitro, halogen, or lower alkyl groups substituted with one to three allyl groups in which the allyl ring is substituted with an allyl group; or substituted methylene groups that form a Schiff base, such as N,N-dimethylaminomethylene, benzylidene, 4-methoxybenzylidene, 4-nitrobenzylidene, salicylidene, 5-chlorosalisylidene, diphenylmethylene, and (5-chloro-2-hydroxyphenyl)phenylmethylene, and more preferably aliphatic acyl groups.

[0029] The "protecting group" in "protected carboxyl group" refers to a "reaction-protecting group" that can be cleaved by chemical methods such as hydrolysis, hydrolysis, electrolysis, and photolysis. Preferably, such "protecting groups in the reaction" are "lower alkyl groups" such as 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-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, and 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 -Alkenyl groups such as -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-hexenyl; ethinyl, 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 "Alkynyl groups" such as -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-4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl; trifluoromethyl, trichloromethyl, difluoromethyl, dichloromethyl, dibromomethyl, fluoromethyl "Halogeno lower alkyl groups" such as 2,2,2-trifluoroethyl, 2,2,2-trichloroethyl, 2-bromoethyl, 2-chloroethyl, 2-fluoroethyl, 2-iodoethyl, 3-chloropropyl, 4-fluorobutyl, 6-iodohexyl, 2,2-dibromoethyl; hydroxy "lower alkyl groups" such as 2-hydroxyethyl, 2,3-dihydroxypropyl, 3-hydroxypropyl, 3,4-dihydroxybutyl, 4-hydroxybutyl; "aliphatic acyl" "lower alkyl groups" such as acetylmethyl;"Lower alkyl groups" substituted with one to three allyl groups such as benzyl, phenethyl, 3-phenylpropyl, α-naphthylmethyl, β-naphthylmethyl, diphenylmethyl, triphenylmethyl, 6-phenylhexyl, α-naphthyldiphenylmethyl, 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, 4-cyanobenzyldiphenylmethyl, bis(2-nitrophenyl)methyl, pipero Examples of "aralkyl groups" include lower alkyl groups such as nyl, 4-methoxycarbonylbenzyl, lower alkoxy, nitro, halogen, cyano, and lower alkyl groups substituted with one to three allyl groups on the allyl ring; and "silyl groups" such as trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, tert-butyldimethylsilyl, methyldiisopropylsilyl, methylditert-butylsilyl, triisopropylsilyl, methyldiphenylsilyl, isopropyldiphenylsilyl, butyldiphenylsilyl, and phenyldiisopropylsilyl, preferably "lower alkyl groups".

[0030] A "halogen atom" refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, preferably a fluorine atom or a chlorine atom.

[0031] "It may be interposed by an oxygen atom - (CH2)" 1~6 -" means "-(CH2)" 1~6 The hyphen indicates a group that is interposed by one or two oxygen atoms, such as methyleneoxymethylene.

[0032] "It may be intervened by NH-(CH2)" 1~6 -" means "-(CH2)" 1~6 The hyphen indicates a group interposed by one or two NH groups, such as methyleneaminomethylene.

[0033] Compound (I) of the present invention may form salts with bases or acids depending on the type of substituent. Such salts are pharmaceutically acceptable salts and preferably include acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, benzenesulfonic acid, aspartic acid, and glutamic acid; salts with inorganic bases such as sodium, potassium, magnesium, calcium, and aluminum; and salts with organic bases such as methylamine, ethylamine, ethanolamine, lysine, and ornithine, as well as ammonium salts. [Brief explanation of the drawing]

[0034] < / f> < / e> < / d> < / c> [Figure 1] This shows three compounds selected by in silico analysis. [Figure 2] This shows the structural models of FKBP12-RAP-FRB and FKBP12-compound C-RAP. [Figure 2(A)] This shows the overall diagram and magnified crystal structure (PDB ID: 1FAP) of FKBP12-RAP-FRB. [Figure 2(B)] This shows the overall diagram of the FKBP12-compound C-FRB and an enlarged docking simulation model. [Figure 3] This shows the preparation of FKBP12 and FRB. [Figure 3(a)] This involves 6xHis-FRB gel filtration chromatograms and SDS-PAGE. [Figure 3(b)] This involves gel filtration chromatograms and SDS-PAGEs using 6xHis-FKBP12. [Figure 3(c)] The graph shows the gel filtration chromatogram and SDS-PAGE of biotinylated FKBP12. The peak indicated by the blue arrow was used in the binding experiment. [Figure 4] The results of an in vitro binding assay using AlphaLISA(R) showed that compound C has a binding affinity similar to that of rapamycin (RAP), while compounds A and B have weaker binding affinity. [Figure 5] The results show KD measurements by AlphaLISA(R), where compounds C and RAP exhibited equilibrium dissociation constants of 4 nM and 2.2 nM, respectively. [Figure 6] The results show affinity measurements in HEK293 cells using NanoBiT(R). Compounds C and RAP showed similar signals, while compounds A and B showed weaker signals. [Figure 7] The graph shows the results of administering compound C at varying concentrations to the NanoBiT® assay system. The concentration-dependent increase in luminescence intensity indicates that compound C specifically binds to FKBP12-FRB. [Figure 8] The cell damage assay showed that compound C damaged cancer cells more than non-cancer cells (NRK-49F) (IC50 > 10 μM), compared to MCF-7 (IC50 = 28.3 nM, HepG2 (IC50 = 17.9 nM)). [Figure 9] This study compares the cytotoxic effects of compound C and RAP on cancer cells (HeLa). When comparing the cytotoxic effects (IC50) of compound C and rapamycin on cancer cells (HeLa), compound C showed stronger cytotoxicity (compound C: IC50 = 1.7 nM, RAP: IC50 = 24.7 nM). [Figure 10] This shows a comparison of the cellular damage caused by compounds C and RAP under activation conditions (+ / + Glucose / FBS) and starvation conditions (- / - Glucose / FBS). [Figure 11] This shows a comparison of the cytotoxic effects of compound C and RAP on non-cancer cells (HEK293). [Figure 12] This shows the measurement of phosphorylation inhibitory activity in cancer cells (HeLa) using the AlphaLISA SureFire Ultra HV p-p70 S6K (T389) assay kit. Compounds A and B hardly inhibited T389p-70S6K1 phosphorylation, but compound C inhibited it to a similar extent as RAP. [Figure 13] This shows the quantitative measurement of phosphorylation inhibitory activity in cancer cells (HeLa) using the AlphaLISA SureFire Ultra HV p-p70 S6K (T389) assay kit. [Figure 14] This shows a Western blotting assay of the phosphorylation inhibitory activity of T37 / 46p-4E-BP1 and S235 / 236p-S6 using cancer cells (HeLa). [Figure 15] This shows the assay of the phosphorylation inhibitory activity of compounds C and RAP at S235 / 236p-S6 using Western blotting with non-cancer cells (HEK293). [Figure 16] The results of animal experiments with compounds C and RAP are shown below. [Figure 16(a)] Compound C showed a higher tumor growth inhibitory effect than RAP. [Figure 16(b)] Compound C showed weaker tumor lung metastasis promoting activity than RAP. [Figure 16(c)] Administration was discontinued on D11, and tumor recurrence was examined on D20. Compound C showed a lower tumor recurrence rate than RAP. [Figure 17(b)-(i)] This shows images of tumors that have metastasized to the lungs, and results from blood tests and other animal experiments, such as blood urea nitrogen, creatinine, blood urea nitrogen / creatine, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and / or glucose concentration. [Figure 17(a)] The study showed the effects of compound C and RAP on weight loss, but no significant reduction was observed. Furthermore, no significant weight loss was observed with compound C during the experiment, and no obvious toxicity was detected in the blood. [Figure 18] The following shows the 11 compounds evaluated in the examples. [Figure 19] This shows affinity measurement within HeLa cells using NanoBiT(R). [Figure 20] This shows affinity measurement within HeLa cells using NanoBiT(R). [Figure 21] This shows the measurement of phosphorylation inhibitory activity in cancer cells (HeLa) using the AlphaLISA SureFire Ultra HV p-p70 S6K (T389) assay kit. [Modes for carrying out the invention]

[0035] [General manufacturing method of the present invention] Some of the compounds of the present invention are known compounds and can be obtained from suppliers by searching, for example, Sterling and Irwin, J. Chem. Inf. Model, 2015 http: / / pubs.acs.org / doi / abs / 10.1021 / acs.jcim.5b00559. Other compounds, besides those already known, can be synthesized similarly in accordance with the following.

[0036] General manufacturing method Step A: [ka] In the figure above, R 2 And X has the same meaning as above. For example, to 150 ml of an aqueous solution of TEA (51.8 mmol) and amino acid (2) (51.8 mmol), whose carboxyl group may be protected with a conventional protecting group, a solution of isothiocyanate (1) (51.8 mmol, 1 eq) with a protected carboxyl group (for example, a solution in an inert organic solvent such as i-PrOH (for example, 200 ml)) was added, and the reaction mixture was refluxed for 30 minutes, for example. The mixture was then cooled and acidified (for example, with an aqueous solution of HCl) to pH=2. The precipitate was filtered and dried to obtain the desired product (3) in, for example, 80% yield, which was used in step B without further purification. Note that "protecting group for carboxyl group" has the same meaning as described above.

[0037] Step B: [ka] In the figure above, R 2 And X has the same meaning as above. For example, a solution of NaOH (3 eq, 2.27 g) dissolved in 100 ml of water and acid (3) (18.9 mmol, 1 eq), whose carboxyl group may be protected with a conventional protecting group, was heated to 50°C. The mixture was then cooled, and H2O2 (10 equivalents, 35% in water) was added and stirred. The reaction temperature is typically between -10°C and 170°C, but preferably between 30°C and 100°C. The reaction time varies mainly depending on the reaction temperature, the starting compounds, the reaction reagents, or the type of solvent used, but is usually 10 minutes to 2 hours, preferably 30 minutes. Subsequently, for example, the reaction mixture was cooled and an acid (not particularly limited as long as it is an acid commonly used in reactions, but preferably an inorganic acid such as hydrochloric acid or phosphoric acid, or a Brønsted acid such as an organic acid such as acetic acid, formic acid, oxalic acid, methanesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, trifluoroacetic acid, or trifluoromethanesulfonic acid, preferably an organic acid, and more preferably acetic acid) was added until the pH became 2, the precipitate was filtered, and the mixture was dried. The desired product (4) (e.g., 95% purity as determined by NMR) was obtained, for example, in a yield of 96%, and used in step C without further purification.

[0038] Step C: [ka] In the figure above, 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 carboxyl group was removed, and acid (4) (e.g., 18.4 mmol, 1 equivalent) was dissolved in, for example, dioxane (60 ml), and CDI (1.1 equivalents) was added little by little at room temperature. The mixture was stirred at 50°C for 3 hours, after which amine (5) (18.4 mmol, 1 equivalent) was added. Next, the mixture was stirred at room temperature for 12 hours, then 100 ml of cold water 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) (e.g., 95% purity by NMR), with a yield of 81%, which was used in step D without further purification. The reaction is carried out by condensing carboxyl with an amine compound in a solvent. The solvent used is not particularly limited as long as it is inert, but examples include halogenated hydrogen compounds such as methylene chloride and chloroform, ethers such as dioxane, ethers and tetrahydrofuran, and amides such as dimethylformamide and dimethylacetamide. The reaction involves, for example, azodicarboxylic acid dilow alkyl-triphenylphosphines such as azodicarboxylic acid diethyl-triphenylphosphine, N-lower alkyl-5-arylisoxazolium-3'-sulfonates such as N-ethyl-5-phenylisoxazolium-3'-sulfonate, N',N'-dicycloalkylcarbodiimides such as N',N'-dicyclohexylcarbodiimide (DCC), diheteroaryldiselenides such as di-2-pyridyldiselenide, triarylphosphines such as triphenylphosphine, arylsulfonyl triazolides such as p-nitrobenzenesulfonyl triazolide, and 2-halo-1-lower alkylpyridinium such as 2-chlor-1-methylpyridinium iodide. This process is preferably carried out in the presence of a condensing agent such as a halide and diaryl phosphoryl azides like diphenyl phosphoryl azide (DPPA), imidazole derivatives like N,N'-carbodimidazole (CDI), benzotriazole derivatives like 1-hydroxybenzotriazole (HOBT), dicarboxyimide derivatives like N-hydroxy-5-norbornene-2,3-dicarboximide (HONB), and carbodiimide derivatives like 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAPC). The reaction temperature is -10°C to 25°C, and for the reaction between the active ester compound and the amine, it is around room temperature, with a reaction time of 30 minutes to 10 hours. The removal of protecting groups from carboxyl groups varies depending on the type, but is generally carried out as follows using methods well known in the art of this field. If a lower alkyl group or allyl group is used as a protecting group for the carboxyl group, it can be removed by treatment with an acid or base. As acids, hydrochloric acid, sulfuric acid, phosphoric acid, and hydrobromic acid can be used, and as bases, there are no particular limitations as long as they do not affect other parts of the compound, but preferably alkali metal carbonates such as sodium carbonate and potassium carbonate, alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, or concentrated ammonia-methyl solution can be used. Furthermore, isomerization may occur during hydrolysis with a base. The solvent used is not particularly limited as long as it is one that is normally used in hydrolysis reactions and does not inhibit the reaction. Water or alcohols such as methanol, ethanol, or n-propanol, or a mixed solvent of an organic solvent such as ethers such as tetrahydrofuran or dioxane and water are preferred. The reaction temperature and reaction time vary depending on the starting materials, solvent, and reagents used, and are not particularly limited. However, to suppress side reactions, the reaction is usually carried out at 0°C to 150°C for 1 to 10 hours. When the protecting group of a carboxyl group is a diaryl-substituted methyl group such as diphenylmethyl, it is usually removed by treatment with an acid in a solvent. Aromatic hydrocarbons such as anisole are preferred as solvents, and fluorinated organic acids such as trifluoroacetic acid are used as acids. The reaction temperature and time vary depending on the starting materials, solvent, and acid used, but are usually carried out at room temperature for 30 minutes to 10 hours. When the protecting group of a carboxyl group is an aralkyl group or a halogeno-lower alkyl group, it is usually removed by reduction in a solvent. As for the reduction method, if the protecting group of the carboxyl group is a halogeno lower alkyl group, a chemical reduction method such as zinc-acetic acid is preferred. If it is an aralkyl group, a catalytic reduction method using a catalyst such as palladium-carbon or platinum is used, or a chemical reduction method using an alkali metal sulfide such as potassium sulfide or sodium sulfide is used. The solvent used is not particularly limited as long as it does not participate in this reaction, but alcohols such as methanol and ethanol; ethers such as tetrahydrofuran and dioxane; fatty acids such as acetic acid; or mixed solvents of these organic solvents with water are preferred. The reaction temperature and time vary depending on the starting materials, solvent, and reduction method, but are usually carried out at around 0°C to room temperature (e.g., 25°C) for 5 minutes to 12 hours. When the protecting group of a carboxyl 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 one that is normally used as a Brønsted acid, but preferably it is an inorganic acid such as hydrochloric acid or sulfuric acid, or an organic acid such as acetic acid or p-toluenesulfonic acid. The solvent used is not particularly limited as long as it does not participate in this reaction, but alcohols such as methanol and ethanol; ethers such as tetrahydrofuran and dioxane; or mixed solvents of these organic solvents with water are preferred. The reaction temperature and time vary depending on the starting materials, solvent, and type of acid used, but are usually carried out at 0°C to 50°C for 10 minutes to 18 hours.

[0039] Step D: [ka] In the figure above, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 L, Y, and X have the same meaning as above. Z represents a leaving group. The term "leaving group" is generally not limited to any group that leaves as a nucleophilic residue, but preferably includes halogen atoms such as chlorine, bromine, and iodine; lower alkoxycarbonyloxy groups such as methoxycarbonyloxy and ethoxycarbonyloxy; alkylcarbonyloxy groups such as acetoxy and propionyloxy; halogenated alkylcarbonyloxy groups such as chloroacetyloxy, dichloroacetyloxy, trichloroacetyloxy, and trifluoroacetyloxy; lower alkoxyalkylcarbonyloxy groups such as methoxyacetyloxy; aliphatic acyloxy groups such as unsaturated alkylcarbonyloxy groups such as (E)-2-methyl-2-butenoyloxy; arylcarbonyloxy groups such as benzoyloxy, and halogenated arylcarbonyloxy groups such as 2-bromobenzoyloxy and 4-chlorobenzoyloxy. Examples of aromatic acyloxy groups include lower alkylated allylcarbonyloxy groups such as 2,4,6-trimethylbenzoyloxy and 4-toluyloxy, lower alkoxylated allylcarbonyloxy groups such as 4-anissoyloxy, and nitrated allylcarbonyloxy groups such as 4-nitrobenzoyloxy and 2-nitrobenzoyloxy; trihalogenomethyloxy groups such as trichloromethyloxy; lower alkanesulfonyloxy groups such as methanesulfonyloxy and ethanesulfonyloxy; halogenolower alkanesulfonyloxy groups such as trifluoromethanesulfonyloxy and pentafluoroethanesulfonyloxy; and allylsulfonyloxy groups such as benzenesulfonyloxy, p-toluenesulfonyloxy and p-nitrobenzenesulfonyloxy. More preferably, halogen atoms are used.

[0040] Compound (6) (e.g., 9.4 mmol, 1 equivalent), compound (7) (9.9 mmol, 1.05 equivalent), and K2CO3 (14.15 mmol, 1.5 equivalent) were dissolved in 10 ml of DMF, and the mixture was stirred at 80°C for 18 hours. As for the base, there are no particular limitations as long as it is used as a base in normal reactions, but preferably alkali metal carbonates such as sodium carbonate, potassium carbonate, and lithium carbonate; alkali metal bicarbonates such as sodium bicarbonate, potassium bicarbonate, and lithium bicarbonate, with potassium carbonate being the most preferred. Cold water (e.g., 100 ml) was added, and the precipitate was filtered. The white precipitate was dried and recrystallized from DMF (e.g., 8 ml). The precipitate was filtered, washed with i-PrOH (e.g., 15 ml), and the white precipitate was dried to obtain the target compound (8) in, for example, 40% yield (95% purity by NMR, 96% purity by LC-MS). Furthermore, upon request, the protective base was removed in accordance with established regulations. The removal of the protecting group varies depending on its type, but is generally carried out as follows, using methods well known in the art of this field.

[0041] The protecting group of the carboxyl group can be removed as described above.

[0042] When a silyl group is used as a protecting group for an 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 preferred. There are no particular limitations on the reaction temperature and reaction time, but typically the reaction is carried out at room temperature for 10 to 18 hours. If the protecting group of the amino group is an aliphatic acyl group, an aromatic acyl group, an alkoxycarbonyl group, or a substituted methylene group that forms a Schiff base, it can be removed by treatment with an acid or base in the presence of an aqueous solvent. The acid used is not particularly limited as long as it is a commonly used acid that does not inhibit the reaction, but preferably inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, and hydrobromic acid are used. 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 methoxide, alkali metal carbonates such as sodium carbonate, potassium carbonate, and lithium carbonate, alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, and lithium hydroxide, or ammonia compounds such as aqueous ammonia and concentrated ammonia-methyl. The solvent used is not particularly limited as long as it is one that is normally used in hydrolysis reactions. Water; alcohols such as methanol, ethanol, and n-propanol; ethers such as tetrahydrofuran and dioxane; organic solvents such as water and the above organic solvents are preferred. The reaction temperature and reaction time vary depending on the starting materials, solvent, and the acid or base used, and are not particularly limited. However, to suppress side reactions, the reaction is usually carried out at 0°C to 150°C for 1 to 10 hours. When the protecting group of the amino group is an aralkyl group or an aralkyloxycarbonyl group, it is generally preferable to remove it by contacting it with a reducing agent in a solvent (preferably by catalytic reduction at room temperature under a catalyst) or by using an oxidizing agent. The solvent used in removal by catalytic reduction is not particularly limited as long as it does not participate in this reaction, but preferred solvents include alcohols such as methanol, ethanol, and isopropanol; ethers such as diethyl ether, tetrahydrofuran, and dioxane; aromatic hydrocarbons such as toluene, benzene, and xylene; aliphatic hydrocarbons such as hexane and cyclohexane; esters such as ethyl acetate and propyl acetate; fatty acids such as acetic acid; or mixed solvents of these organic solvents with water. The catalyst used is not particularly limited as long as it is one that is normally used in catalytic reduction reactions, but preferably, palladium-carbon, rane-nickel, platinum oxide, platinum black, rhodium-aluminum oxide, triphenylphosphine-rhodium chloride, and palladium-barium sulfate are used. There are no specific pressure limitations, but it is usually performed at 1 to 10 atmospheres. The reaction temperature and time vary depending on the type of starting material, solvent, and catalyst, but are usually carried out at 0°C to 100°C for 5 minutes to 24 hours. The solvent used in the removal by oxidation is not particularly limited as long as it does not participate in this reaction, but preferably it is an aqueous organic solvent. Suitable organic solvents for this purpose include ketones such as acetone, halogenated hydrocarbons such as methylene chloride, chloroform, and carbon tetrachloride, nitriles such as acetonitrile, ethers such as diethyl ether, tetrahydrofuran, and dioxane, amides such as dimethylformamide, dimethylacetamide, and hexamethylphosphorotriamide, and sulfoxides such as dimethyl sulfoxide. The oxidizing agent used is not particularly limited as long as it is a compound used for oxidation, but preferably potassium persulfate, sodium persulfate, cerium ammonium nitrate (CAN), and 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) are used. The reaction temperature and reaction time vary depending on the type of starting material, solvent, and catalyst, but are usually carried out at 0°C to 150°C for 10 minutes to 24 hours. When the protecting group of the amino group is an alkenyloxycarbonyl group, this is usually achieved by treating it with a base under the same conditions as the removal reaction when the protecting group of the amino group is an aliphatic acyl group, aromatic acyl group, alkoxycarbonyl group, or a substituted methylene group that forms a Schiff base. In the case of allyloxycarbonyl, the removal method using palladium and triphenylphosphine or nickel tetracarbonyl is particularly simple and can be carried out with few side reactions.

[0043] When a silyl group is used as a protecting group for a hydroxyl group, it can usually be removed by treating it with a compound that produces a fluorine anion, such as tetrabutylammonium fluoride, hydrofluoric acid, pyridine hydrofluoric acid, or potassium fluoride, or by treating it with an organic acid such as acetic acid, methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, or 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 materials to some extent, but preferred solvents include ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether; nitriles such as acetonitrile and isobutyronitrile; water; organic acids such as acetic acid and mixtures thereof. There are no particular limitations on the reaction temperature and reaction time, but it is usually carried out at 0°C to 100°C (preferably 10°C to 30°C) for 1 to 24 hours. When the protecting group of the hydroxyl group is an aralkyl group or an aralkyloxycarbonyl group, it is usually preferable to remove it by contacting it with a reducing agent in a solvent (preferably by catalytic reduction at room temperature under a catalyst) or by using an oxidizing agent. The solvent used in removal by catalytic reduction is not particularly limited as long as it does not participate in this reaction, but preferred solvents include alcohols such as methanol, ethanol, and isopropanol; ethers such as diethyl ether, tetrahydrofuran, and dioxane; aromatic hydrocarbons such as toluene, benzene, and xylene; aliphatic hydrocarbons such as hexane and cyclohexane; esters such as ethyl acetate and propyl acetate; amides such as formamide, dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone, and hexamethylphosphorotriamide; fatty acids such as formic acid and acetic acid; water; or mixed solvents thereof. More preferably, the solvents are 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 one that is normally used in catalytic reduction reactions, but preferably, palladium carbon, palladium black, Rane-nickel, platinum oxide, platinum black, rhodium-aluminum oxide, triphenylphosphine-rhodium chloride, and palladium-barium sulfate are used. There are no specific pressure limitations, but it is usually performed at 1 to 10 atmospheres. The reaction temperature and reaction time vary depending on the type of starting material, solvent, and catalyst, but are typically 0°C to 100°C (preferably 20°C to 70°C) and 5 minutes to 48 hours (preferably 1 hour to 24 hours). The solvent used in the removal by oxidation is not particularly limited as long as it does not participate in this reaction, but preferably it is an aqueous organic solvent. Suitable organic solvents for this purpose include ketones such as acetone, halogenated hydrocarbons such as methylene chloride, chloroform, and carbon tetrachloride, nitriles such as acetonitrile, ethers such as diethyl ether, tetrahydrofuran, and dioxane, amides such as dimethylformamide, dimethylacetamide, and hexamethylphosphorotriamide, and sulfoxides such as dimethyl sulfoxide. The oxidizing agent used is not particularly limited as long as it is a compound used for oxidation, but preferably potassium persulfate, sodium persulfate, cerium ammonium nitrate (CAN), and 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) are used. The reaction temperature and reaction time vary depending on the type of starting material, solvent, and catalyst, but are usually carried out at 0°C to 150°C for 10 minutes to 24 hours. Furthermore, it can also be removed by reacting alkali metals such as metallic lithium or metallic sodium in liquid ammonia or alcohol such as methanol or ethanol at -78°C to -20°C. Furthermore, it can also be removed using an aqueous chloride-sodium iodide or alkylsilyl halides such as trimethylsilyl iodide 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, halogenated hydrocarbons such as methylene chloride and chloroform, or mixtures thereof are used. The reaction temperature and time vary depending on the starting materials, solvent, etc., but are usually carried out at 0°C to 50°C for 5 minutes to 3 days. If the protecting group of the hydroxyl group is an aliphatic acyl group, an aromatic acyl group, or an alkoxycarbonyl group, it can be 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 preferably metal alkoxides such as sodium methoxide; alkali metal carbonates such as sodium carbonate, potassium carbonate, and lithium carbonate; alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, lithium hydroxide, and barium hydroxide; or ammonia compounds such as aqueous ammonia and concentrated ammonia-methyl. The solvent used is not particularly limited as long as it is one that is normally used in hydrolysis reactions. Water; organic solvents such as alcohols like methanol, ethanol, and n-propanol, ethers like tetrahydrofuran and dioxane, or mixed solvents of water and the above organic solvents are preferred. The reaction temperature and reaction time vary depending on the starting materials, solvent, and base used, and are not particularly limited. However, to suppress side reactions, the reaction is usually carried out at 0°C to 150°C for 1 to 10 hours. When the protecting group of a 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 removed by treatment with an acid in a solvent. The acid used is not particularly limited as long as it is commonly used as a Brønsted acid or Lewis acid. Preferably, it is a Brønsted acid such as hydrogen chloride; an inorganic acid such as hydrochloric acid, sulfuric acid, or nitric acid; or an organic acid such as acetic acid, trifluoroacetic acid, methanesulfonic acid, or p-toluenesulfonic acid; or a Lewis acid such as boron trifluoride. However, strongly acidic cation exchange resins such as Dowex 50W can also be used. The solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting materials to some extent, but is 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, and dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and diethyl carbonate; diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, and dimethyl ether. Ethers such as thylene and diethylene glycol dimethyl ether; alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, isoamyl alcohol, diethylene glycol, glycerin, octanol, cyclohexanol, and methyl cellosolve; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, and cyclohexanone; water, or mixed solvents thereof are preferred, and more preferably halogenated hydrocarbons, esters, or ethers. The reaction temperature and reaction time vary depending on the starting materials, solvent, and the type and concentration of acid used, but are usually -10°C to 100°C (preferably -5°C to 50°C) and 5 minutes to 48 hours (preferably 30 minutes to 10 hours). When the protecting group of the hydroxyl group is an alkenyloxycarbonyl group, this is usually achieved by treating it with a base under the same conditions as the removal reaction when the protecting group of the hydroxyl group is an aliphatic acyl group, aromatic acyl group, or alkoxycarbonyl group. Furthermore, in the case of allyloxycarbonyl, palladium and triphenylethanolamine are particularly important. Removing it using sphing or bis(methyldiphenylphosphine)(1,5-cyclooctadiene)iridium(I)·hexafluorophosphate is a simple method that can be carried out with few side reactions.

[0044] Furthermore, the process of removing the protecting group from the amino group as described above may also simultaneously remove the protecting group from the hydroxyl group.

[0045] The above-mentioned removal reactions of hydroxyl group protecting groups and amino group protecting groups can be carried out sequentially in any order in which the desired removal reaction occurs.

[0046] Furthermore, compounds in which A is S can also be similarly produced by directly carrying out step C without going through step B, using the compound produced in step A (4: A represents S), as shown below. [ka]

[0047] Furthermore, by exchanging the substituents on the benzene rings of compounds (5) and (7) and performing the above steps, a compound with the reverse configuration of substructure (II) can be produced. Furthermore, by considering amide bonds, it is possible to produce the compounds of the present invention in which substructures (I), (II), and (III) are bonded to the linker in any order.

[0048] After the reaction is complete, the target compound (I) of this reaction is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is neutralized as appropriate, and if insoluble matter is present, it is removed by filtration. Then, water and an immiscible organic solvent such as ethyl acetate are added, and after washing with water, the organic layer containing the target compound is separated. After drying with anhydrous magnesium sulfate or the like, the solvent is removed by distillation to obtain the target compound. The obtained target compound can be separated and purified by conventional methods, such as recrystallization, reprecipitation, or methods commonly used for the separation and purification of organic compounds, such as adsorption column chromatography using a carrier like silica gel, alumina, or magnesium silica gel-based Florisil; partition column chromatography using synthetic adsorbents such as Sephadex LH-20 (Pharmacia), Amberlite XAD-11 (Rohm & Haas), or Diaion HP-20 (Mitsubishi Chemical); ion exchange chromatography; or a combination of normal-phase and reverse-phase column chromatography using silica gel or alkylated silica gel (preferably high-performance liquid chromatography), and elution with an appropriate eluent. Furthermore, if it is necessary to separate isomers, this can be done using the separation and purification means described above at an appropriate time after the completion of each of the above steps, or after the completion of the desired step.

[0049] Specifically, for example, compound (1) (=compound C) can be manufactured as follows. [ka] Manufacturing of Compound 1 (1) To a 150 ml aqueous solution of TEA (5.23 g, 51.8 mmol) and amino acid (5.35 g, 51.8 mmol), i-PrOH (200 ml) of isothiocyanate 1 (10 g, 51.80 mmol, 1 eq) was added. 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) dissolved in 100 ml of water and acid 3 (5 g, 18.9 mmol, 1 eq) was heated to 50°C. The mixture was then cooled and H2O2 (10 equivalents, 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 the pH reached 2. 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 equivalents, 3.23 g) was added gradually at room temperature. The mixture was stirred at 50 °C for 3 hours, and then amine 6 (2.74 g, 18.4 mmol, 1 equivalent) was added. Next, the mixture was 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 equivalent) were dissolved in 10 ml of DMF and the mixture was stirred. Incubated at 80°C for 18 hours. 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. 2.1 g of the target compound was obtained (purity 95% by NMR, purity 96% by LC-MS), yield 40%.

[0050] The anticancer agent, cancer inhibitor, and / or life-extending agent of the present invention may be provided as a compound having mTORC1 inhibitory activity as an active ingredient or a pharmaceutically acceptable salt thereof, alone, or as a formulation containing a pharmaceutically acceptable carrier or pharmaceutical additive. In this case, the compound having mTORC1 inhibitory activity as an active ingredient or a pharmaceutically acceptable salt thereof may be contained in the formulation in an amount of, for example, 0.1 to 99.9% by mass.

[0051] Pharmaceutically acceptable carriers or pharmaceutical additives are not particularly limited, but examples include excipients, disintegrants, disintegration aids, binders, lubricants, coatings, dyes, diluents, solubilizers, solubilizers, isotonic agents, pH adjusters, stabilizers, and the like.

[0052] Suitable formulations for oral administration include, for example, powders, tablets, capsules, granules, liquids, or syrups.

[0053] For oral administration, various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate, and glycine may be used together with starch, preferably corn, potato, or tapioca starch, and various disintegrants such as alginic acid or certain double silicates, and granule-forming binders such as polyvinylpyrrolidone, sucrose, gelatin, and gum arabic. Alternatively, tablets may be formed using lubricants such as magnesium stearate, sodium lauryl sulfate, and talc. For oral administration, aqueous suspensions and / or elixirs may also be used.

[0054] Suitable formulations for parenteral administration include, for example, injections, suppositories, poultices, nasal sprays, and eye drops. Injections can be administered, for example, by intravenous injection, intra-articular injection, intramuscular injection, or subcutaneous injection.

[0055] The dosage of the anticancer agent, cancer inhibitor, and / or life-extending agent of the present invention is not particularly limited, and an appropriate dosage can be selected according to various conditions such as the type of cancer, the patient's age and symptoms, the route of administration, the purpose of treatment, and the presence or absence of concomitant drugs. For example, in the case of oral administration, the daily dose of the active ingredient for an adult (e.g., weighing 60 kg) is approximately 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. [Examples]

[0056] Experimental method [Example 1]

[0057] In silico selection of small molecules. (1) Approximately 700,000 virtual ligands (VLs) 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, L#2, and L#3. [Table 1] Virtual ligand selection (VLS) was performed using ICM-Pro3.8 software (Molsoft LLC). The crystal structure of the FKBP12-rapamycin-FRB heterotrimer complex (PDB ID: 1FAP) was loaded into ICM-Pro3.8, and binding boxes were automatically generated around rapamycin. Next, the binding box size was manually adjusted so that all VLs were contained within the binding box. Flexibility was introduced to the amino acid side chains of FKBP12 and FRB contained within the binding box using the SCARE method. During VL docking, rapamycin was used as a template for the docking guide, taking into account the atomic property field (APF) of rapamycin. After docking all VLs, the docking scores selected the top ligands in each group (L#1 to L#3), resulting in the following three types of VLs. *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-benzodioxol-5-ylmethyl)-4-[1-[2-[(3-chlorophenyl)amino]-2-oxoethyl]-2,4-dioxoquinazoline-3-yl]butanamide (compound C)

[0058] (2) Results of in silico selection of small molecules that bind to FKBP12-FRB. As described above, in silico selection resulted in the selection of compound A from ligand library L#1, and compounds B and C from L#3 (Figure 1).

[0059] Compounds A, B, and C occupied 48%, 68%, and 76% of the rapamycin-binding pocket, respectively, suggesting they are potential allosteric inhibitors of mTORC1. Figure 2 shows the crystal structure of FKBP12-RAP-FRB (PDB ID: 1FAP) and the docking model of FKBP12-compound C-FRB (Figure 2). [Example 2]

[0060] In vitro selectivity of ligands that bind to FKBP12-FRB Compounds A, B, and C were purchased from Enamine Ltd (Kyiv, Ukraine), ASINEX Corporation (NC, USA), and ChemDiv (CA, USA), respectively.

[0061] (1) Construction of a protein expression system and protein purification The FKBP12 and FRB genes were cloned from the FKBP-SmBiT and FRB-LgBiT control vectors included in the NanoBit PPI system (Promega, USA) kit, and inserted into the pET15b vector (Novagen, USA) using the In-Fusion cloning kit (Takara Bio, Japan). The constructed plasmid was transformed into DH5α Escherichia coli (Nippon Gene, Japan) and spread on an LB agar plate containing 0.1 mM ampicillin. The colonies that appeared were sequenced by colony PCR. The colonies confirmed by sequencing were cultured in LB medium (37 °C, 6 hours), and then the plasmid was purified using the NucleoSpin Plasmid EasyPure Kit (MACHEREY-NAGEL, Germany). The obtained plasmid was used for ECOS TM competent Escherichia coli BL21(DE3) (Nippon Gene, Japan) was transformed and pre-cultured in 5 mL LB medium (Sigma-Aldrich, USA) (37 °C, 6 hours). Then, it was cultured in 5 L of modified TB medium (Sigma-Aldrich, USA). OD 600 When OD reached 1, 1 mM IPTG (Fujifilm Wako Pure Chemicals Corporation, Japan) was added, and the overnight culture was continued. The cells were collected by centrifugation (4 °C, 8000 rpm, 15 minutes) and stored at -80 °C. 5 g of cells (FRB or FKBP12) were dissolved in 100 mL of lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.04 mg / ml lysozyme, 0.16 mg / ml DNaseI, 1 tablet of cOmplete TM protease inhibitor (Sigma-Aldrich)) and disrupted by sonication (Sonifier (R) (Branson, USA), 5.0 W, 30 - 40% cycle / sec, 5 min, on ice). For the purification of FKBP12, the disrupted solution was ultracentrifuged (4 °C, 40,000 rpm, 1 hour), and then the supernatant was obtained. The supernatant was loaded onto a 5 mL HisTrap HP (Cytiva, Japan) Ni column that had been pre-equilibrated with binding buffer A (50 mM Tris-HCl, pH 8.0, 10% (v / v) glycerol). An AKTA prime plus (Cytiva, Japan) was used for purification. The concentration of elution buffer A (50 mM Tris-HCl, 10% v / v glycerol, 1 M imidazole, pH 8.0) was gradually increased to obtain the elution peak. After confirming the presence of FKBP12 in the elution peak by SDS-PAGE, the solvent was replaced with binding buffer A using Amicon Ultra (MWCO: 3KDa, Millipore, Germany). Next, the sample was pre-equilibrated with gel filtration buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0, 10% (v / v) glycerol) and then processed using HiLoad. (R) The sample was purified using a 26 / 60 Superdex-200 column (Cytiva, Japan), concentrated to 50 mg / mL, and stored at -80°C. In the purification of FRB, the pellet was recovered after ultracentrifugation. The pellet was washed three times with washing buffer A (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 4 M urea, pH 8.0). The pellet was then washed three times with washing buffer B (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA). Furthermore, the pellet was dissolved in 20 mL of lysis buffer (50 mM Tris-HCl, 8 M urea, 100 mM NaH2PO4, 10 mM 2-mercaptoethanol, pH 8.0) and stirred in a rotator (2 hours, room temperature). After ultracentrifugation (40,000 rpm, 1 hour, 4°C), the pellet was loaded onto a 5 mL HisTrap FF crude (Cytiva, Japan) Ni column that had been pre-equilibrated with equilibration buffer (50 mM Tris-HCl, 8 M urea, 100 mM NaH2PO4, 5 mM 2-mercaptoethanol, pH 8.0). The concentration of the elution buffer (50 mM Tris-HCl, 8 M urea, 100 mM NaH2PO4, 5 mM 2-mercaptoethanol, 1 M imidazole, pH 8.0) was gradually increased (0 to 100%), and the presence of FRB in the resulting peak was confirmed by SDS-PAGE. The resulting solution was added dropwise to 1 L of 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, stirred). The refolding buffer was concentrated with 400 mL of Amicon Stirred Cell (Millipore, USA) and Amicon Ultra-15 (MWCO: 3kDa, Millipore, Germany), and the buffer was exchanged with binding buffer A (50 mM Tris-HCl, pH 8.0, 10% (v / v) glycerol) to obtain a concentrated FRB solution. This was pre-equilibrated with gel filtration buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0, 10% (v / v) glycerol) and then processed using HiLoad. (R) Purified using a 26 / 60 Superdex-200 column (Cytiva, Japan), concentrated up to 40 mg / ml, and stored at -80 °C.

[0062] (2) Deletion of 6xHis-tag and biotinylation of FKBP12 10 units of thrombin (ThermoFisher, USA) was added to 1 mg of FKBP12 dissolved in PBS and stirred (16 hours, room temperature). The reaction solution was loaded onto a His SpinTrap column (Cytiva, Japan) to obtain a flow-through solution. This was loaded onto a 1 mL HisTrap Benzamidine FF column (Cytiva, Japan) to remove thrombin. Next, FKBP12 with the 6xHis-tag removed and Biotin-dPEG (R) 24 -NHS ester (Quanta BioDegign Limited, USA) were stirred and reacted at a molar ratio of 1:20 (2 hours, room temperature). The reaction solution was gel-filtered using a HiLoad 26 / 60 Superdex-200 column (Cytiva, Japan) pre-equilibrated with gel filtration buffer (50 mM Tris-HCl, 150 mM NaCl, 10% (v / v) glycerol). The obtained sample was concentrated and stored at -80 °C. (R) 2 μL of 6xHis-tag-FRB (250 nM) was mixed in a well of a 384-well OptiPlate (PerkinElmer, USA) with 2 μL (100 μg / ml) of anti-His tag coated alpha acceptor beads (PerkinElmer, USA), sealed, and then incubated (in the dark, 1 hour, room temperature).

[0063] (3) In vitro ligand selection 2 μL of 6xHis-tag-FRB (250 nM) was mixed in a well of a 384-well OptiPlate (PerkinElmer, USA) with 2 μL (100 μg / ml) of anti-His tag coated alpha acceptor beads (PerkinElmer, USA), sealed, and then incubated (in the dark, 1 hour, room temperature). Next, 2 μL of biotinylated FKBP12 (250 nM) and 2 μL of streptavidin-coated alpha donner beads (100 μg / ml) were mixed and added to the wells. 2 μL of rapamycin (Funakoshi Co., Ltd., Japan) or the selected compound solution was added to the well, sealed again, and incubated in the dark at room temperature for at least 1 hour. AlphaLISA receives the Alpha signal generated from the well. (R) Measurements were taken using an EnSpire multimode plate reader with measurement mode (PerkinElmer, USA). The equilibrium dissociation constant (Kd) was obtained by creating multiple wells with varying concentrations of 6xHis-FRB, biotin-FKBP12, or ligand (0.01–1000 nM) and measuring the alpha signal. Competitive inhibition experiments with ligands (compounds) or rapamycin were obtained by varying their concentrations (0.18 nM–18 μM) and incubating with FRB.

[0064] (4) Results of in vitro selection of ligands that bind to FKBP12-FRB To confirm the affinity of compounds A, B, and C for FKBP12-FRB, AlphaLISA was used. (R) Measurements were taken using the following method. 6xHis-FRB, 6xHis-FKBP12, and biotinylated FKBP12 were purified by gel filtration chromatography as shown in Figure 3 (Figure 3).

[0065] Biotinylated FKBP12 was immobilized on donor beads and 6xHis-FRB was immobilized on acceptor beads. Binding of 1 μM compounds A, B, C, and rapamycin (RAP: positive control) was measured, and it was found that rapamycin and compound C gave similar Alpha signals (Figure 4). Compound C was found to have a binding affinity similar to that of rapamycin (RAP). On the other hand, the bonding force between compound A and compound B was weak.

[0066] Furthermore, AlphaLISA titration binding experiments of compounds C and RAP revealed their respective equilibrium dissociation constants K D When we determined that compound C is K D For = 4 nM, RAP is K D It was found to exhibit nearly equivalent affinity to 2.2 nM (Figure 5). Compounds C and RAP exhibited equilibrium dissociation constants of 4 nM and 2.2 nM, respectively.

[0067] Furthermore, NanoBiT (R) In vivo selection using HEK293 cells confirmed that compound C also exhibits a similar level of affinity within cells as RAP (Figure 6). Compounds C and RAP showed similar signals, while compounds A and B showed weaker signals.

[0068] The luminescence intensity increases in a concentration-dependent manner with compound C, indicating that compound C specifically binds to FKBP12-FRB (Figure 7). [Example 3]

[0069] (1) Cell line Human metastatic mammary carcinoma (MCF-7), human cervical cancer (HeLa, #CCL-2), human liver cancer (HepG-2), human embryonic fibroblasts (HEK293), and rat renal stromal fibroblasts (NRK-49F) were obtained from the 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).

[0070] (2) In vivo ligand selection NanoBiT PPI Starter Systems (Promega, USA) were used for in vivo ligand selection. First, 10 4 Each HEK293, HeLa, or MCF-7 cell was placed in a B&W Isoplate-96 TC (PerkinElmer, USA) and cultured overnight in DMED (10% FBS, 1% P / S) (5% CO2, 37°C). The following day, the FKBP12-SmBiT (4360 bp) and FRB-LgBiT (4762 bp) control vectors included in the NanoBiT PPI Starter System were co-transfected into each cell using FuGene HD (Promega, USA) in a 3:1 (v / w) ratio to a concentration of 50 ng / well, and incubated for 24-48 hours (5% CO2, 37°C). Next, the culture medium was replaced with Opti-MEM reduced serum medium (Thermo Fisher Scientific, USA), and 20 μL of 20-fold diluted Nano-Glo luciferase assay substrate (Promega, USA) was added to each well. The baseline was measured using the luminescence 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) alone, was added to each well, and the PPI-induced luminescence was measured using a plate reader.

[0071] (3) Cell viability assay The cell viability assay was performed using the CellTiter-Blue Cell Viability assay kit (Promega, USA). 4 The seeds (individual cells / well) were seeded into a 96-well plate and incubated overnight (5% CO2, 37°C). The cells were then cultured under starvation conditions for an additional 18 hours. Subsequently, ligands of each concentration were added to the cells, and after 72 hours of culture, 20 μL of CellTiter-Blue Cell Viability solution was added to each well and incubated for 1–4 hours (5% CO2). 2, 37℃). Fluorescence at 560 / 590 nm was measured using an EnSpire multimode plate reader. Alternatively, cell viability was assayed by directly counting the number of cells. 10 in a 6-well plate 5 Cells were seeded at a rate of 1 / well, and the same procedure as above was performed. Finally, the cells were detached with trypsin / EDTA, 5% trypan blue solution was added, and the number of cells was counted using a TC10 automated cell counter (Bio-Rad, USA). Cell images were taken using an Olympus IX71 microscope and an Olympus DP74 camera, and the images were analyzed using ImageJ software.

[0072] (4) mTORC1 kinase assay The kinase activity of mTORC1 is AlphaLISA (R) SureFire Ultra TM Detection was performed by measuring the phosphorylation of the S6K protein using the HV p-p70 S6K (T389) assay kit (TGR Biosciences, Australia, and PerkinElmer, USA). HEK293 cells or NRK-49F cells (10 4 The samples (1 per well) were incubated overnight in a 96-well plate using high-glucose DMEM (10% FBS, 1% P / S) (5% CO2, 37°C). The following day, the cells were starved for 18 hours, then cultured for 1–3 hours with (or without) various concentrations of ligand. The cells were then lysed with 50 μL of 1x cell lysis buffer, prepared fresh from the kit, and mixed with a plate shaker for 10 minutes. 6 μL of cell lysate was dispensed into 384-well OptiPlate (PerkinElmer, USA), 3 μL of anti-S6K coated donor was added under dark conditions, the wells were sealed, and the mixture was incubated (2-4 hours, room temperature). The alpha signal was measured using the Alpha mode of the EnSpire.

[0073] (5) Western blotting HeLa cells, HEK293 cells, and NRK-49F cells were cultured in a 6-well plate for 3-6 hours, detached, and prepared in a freshly prepared 1x lysis buffer (AlphaLISA). (R) SureFire Ultra TM The sample was washed with HV p-p70 S6K (T389) assay kit (included in the kit). Protein levels were quantified using the BCA assay kit (Thermo Fisher Scientifics, USA). After SDS-PAGE, protein bands were transferred to a nitrocellulose membrane using a Trans-Blot Transfer System (Bio-Rad, USA). After blocking with 5% w / v bovine serum albumin (BSA) 1xTBS solution for 1 hour, the nitrocellulose membranes were incubated with the following antibody solutions (1:1000) (overnight or for 1 hour at 4°C). Primary antibody: rabbit monoclonal primary antibodies against S6 (#2217), S235 / 236 p-S6 (#4856), 4E-BP1 (#9452), T37 / 46 p-4E-BP1 (#2855), mTOR (#2983), and β-tubulin (#2146) (all from Cell Signaling Tech., USA) were used. Next, the 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 captured with a WSE-6100 LuminoGraph I (ATTO, Japan).

[0074] (6) Experimental results

[0075] (6-1) Results of the cell damage assay When compound C and rapamycin were added to three types of cancer cells (MCF-7, HepG2) and one type of non-cancer cell (NRK-49F) at various concentrations, the cell damage shown in Figure 8 was observed.

[0076] Compound C causes damage to non-cancer cells (NRK-49F) (IC 50 Compared to (>10 μM), it damages cancer cells more (MCF-7 IC5). 50 =28.3 nM, IC of HepG2 50 It was found that the result was =17.9 nM. Furthermore, the cytotoxicity of compound C and rapamycin against cancer cells (HeLa) was investigated (IC). 50 When comparing the compounds, compound C showed stronger cell damage (compound C: IC2). 50 =1.7 nM, RAP:IC 50 The result was 24.7 nM (Figure 9).

[0077] Furthermore, the cytotoxic effects of compounds C and RAP on non-cancer cells (NRK-49F) and cancer cells (HeLa) were measured under activation conditions (+ / + / Glucose / FBS) and starvation conditions (- / - Glucose / FBS) (Figure 10).

[0078] The results showed that compound C exhibited greater cytotoxicity than RAP against cancer cells (HeLa) under both activation and starvation conditions, while showing almost no cytotoxicity against non-cancer cells (NRK-49F). On the other hand, RAP also exerted cytotoxic effects against non-cancer cells (NRK-49F). This indicates that compound C exerts cytotoxic effects more specifically on cancer cells than RAP.

[0079] Furthermore, the cytotoxic effect of compound C on non-cancer cells (HEK293) was quantitatively measured under activation conditions (+ / + Glucose / FBS) and starvation conditions (- / - Glucose / FBS) (Figure 11).

[0080] As a result, compound C showed almost no cytotoxicity to HEK293 cells under activation conditions (IC). 50 (>10 μM), it showed relatively weak cell damage even under starvation conditions (IC). 50 =178 nM). This result indicates that compound C has little effect on non-cancer cells.

[0081] (6-2) Results of mTORC1 kinase activity assay Using the AlphaLISA SureFire Ultra HV p-p70 S6K (T389) assay kit, we measured whether 1 μM of compounds A, B, C, and RAP inhibited the phosphorylation of ribosomal protein 6 kinase 1 (p70S6K1) protein at T389 in cancer cells (HeLa) (Figure 12).

[0082] Compounds A and B are almost T 389 Compound C did not inhibit the phosphorylation of p-70S6K1, but it inhibited it to a similar extent as RAP. As a result, compounds A and B are almost T389 Compound C did not inhibit the phosphorylation of S6K1, but it inhibited it to a similar extent as RAP.

[0083] Furthermore, compound C and RAP, T389 The phosphorylation inhibitory activity of p-70S6K1 was 3.5 nM and 0.4 nM, respectively (Figure 13).

[0084] Furthermore, Western blotting revealed that 1 μM of compounds C and RAP were present in cancer cells (HeLa). S235 / 236 p-S6 and T37 / 47 The phosphorylation inhibitory activity of p-4E-BP1 was investigated (Figure 14).

[0085] Compounds C and RAP are, S235 / 236 It inhibited p-S6 to a similar degree, T37 / 46 p-4E-BP1 was inhibited more efficiently by compound C. Furthermore, in non-cancer cells (HEK293), compound C and RAP S235 / 236 The inhibitory activity against p-S6 was measured at 0.1 and 1 μM, respectively (Figure 15).

[0086] The results showed that even in non-cancer cells, RAP inhibited S235 / 236p-S6, while compound C did not.

[0087] Based on these results, compound C, unlike rapamycin, specifically inhibits the phosphorylation activity of mTORC1 in cancer cells, and as a result, exhibits cancer cell-specific cytotoxic activity. Furthermore, it is more efficient than rapamycin. T37 / 46 It was found to inhibit p-4E-BP1. These findings indicate that compound C has high potential as an anticancer agent. [Example 4]

[0088] Animal experiments Xenograft studies were conducted using BALB / c mice (female, 7-9 weeks old). The mice were randomly grouped (5 mice / cage) and exposed to light-dark (12-hour cycle) lighting in a climate-controlled environment. Watering and feeding areas were made freely accessible. As a human breast cancer model (stage IV model with metastasis), 10 7 Four T1 cells were xenotransplanted into the subcutaneous mammary glands of mice. The tumor size was 100 mm. 3 Drug administration was started after arrival (approximately 5 days later). The drugs were administered for 10 days at a dose of 100 μL / day PBS (intraperitoneal administration), 25 mg / kg / day compound C (oral administration), or 25 mg / kg / day rapamycin (oral administration). Tumor size was measured every two days for 20 days using microCT. Tumor size V = (width) 2 It is given by x (length) / 2. Additionally, the mice's weight was measured every two days. The metastatic activity of 4T1 tumors was measured by counting the number of white colonies on the lung surface after immersing the lungs in a tissue fixation reagent (Bouin fixation solution) following the sacrifice of mice (Figure 16). Blood samples were collected from the compound C administration group, the non-administration group (positive control), and the non-tumor transplant group (negative control). Serum alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine (CRE), and glucose were quantified using standard methods. (a) Compound C showed a higher tumor growth inhibitory effect than RAP. (b) Compound C showed weaker tumor lung metastasis promoting activity than RAP. (c) Administration was discontinued on D11, and tumor recurrence was examined on D20. Compound C showed a lower tumor recurrence rate than RAP.

[0089] The results of the blood assay and other analyses are shown in Figure 17. During the experiment, no significant weight loss was observed with compound C, and no obvious toxicity was detected in the blood. [Example 5]

[0090] Life extension effect The life-extending effect of the compound of the present invention is measured by replacing rapamycin with the compound of the present invention, in accordance with the method described by David E. Harrison et al., "Rapamycin fed late in life extends lifespan in genetically heterogeneous mice," Nature, Vol 460|16 July 2009|doi:10.1038 / nature08221. Further details on the methods and references are available in the online version of this document at www.nature.com / nature. [Example 6]

[0091] Compounds D through N shown in Figure 18 were purchased from Enamine Ltd (Kyiv, Ukraine), ASINEX Corporation (NC, USA), and ChemDiv (CA, USA), respectively.

[0092] (1) In vivo selectivity of ligands that bind to FKBP12-FRB

[0093] NanoBiT PPI Starter Systems (Promega, USA) were used for in vivo ligand selection. First, 10 4 HeLa cells were placed in a B&W Isoplate-96 TC (PerkinElmer, USA) and cultured overnight in DMED (10% FBS, 1% P / S) (5% CO2, 37°C). The following day, the FKBP12-SmBiT (4360 bp) and FRB-LgBiT (4762 bp) control vectors included in the NanoBiT PPI Starter System were co-transfected into each cell using FuGene HD (Promega, USA) in a 3:1 (v / w) ratio to a concentration of 50 ng / well, and incubated for 24-48 hours (5% CO2, 37°C). Next, the culture medium was replaced with Opti-MEM reduced serum medium (Thermo Fisher Scientific, USA) (39 μL / well), and 20 μL of 20-fold diluted Nano-Glo luciferase assay substrate (Promega, USA) was added to each well. The baseline was measured using the luminescence measurement mode of the EnSpire multimode plate reader (PerkinElmer, USA). Next, ligand (1 μM or 10 μM) was added to each well, and 10 μL of 20x Furimazine / well was added, and the PPI-induced luminescence was measured using a plate reader.

[0094] As a result, compounds D to N were confirmed to have affinity even inside cells (Figure 19). Compounds J, L, and M had weak intracellular affinity. Compounds D, E, F, H, and K show a concentration-dependent increase in luminescence intensity, indicating that they specifically bind to FKBP12-FRB (Figure 20).

[0095] (2) mTORC1 kinase assay The kinase activity of mTORC1 is AlphaLISA (R) SureFire Ultra TM S6K protein phosphorylation was detected by measuring the phosphorylation of the S6K protein using the HV p-p70 S6K (T389) assay kit (TGR Biosciences, Australia, and PerkinElmer, USA). HEK293 cells (10 4 The samples (1 per well) were incubated overnight in a 96-well plate using high-glucose DMEM (10% FBS, 1% P / S) (5% CO2, 37°C). The following day, the cells were starved overnight (99 μl OptiMEM media / well), then 1 μM Rigant was added and the cells were cultured for 1-2 hours. The cells were then lysed for 30 minutes with 25 μL 10x cell lysis buffer / well, which was prepared fresh as included in the kit. 6 μL of cell lysate was dispensed into 384-well OptiPlate (PerkinElmer, USA), 3 μL of anti-S6K coated donor was added under dark conditions, the wells were sealed, and the mixture was incubated (2-4 hours, room temperature). The alpha signal was measured using the Alpha mode of the EnSpire.

[0096] Compound K is almost T 389 Although it did not inhibit the phosphorylation of pS6K1, compounds other than compound K did not inhibit T 389 It inhibited the phosphorylation of pS6K1 to some extent.

[0097] [Effects of the invention] From the above results, it can be concluded that, unlike rapamycin, the derivative of the present invention inhibits the phosphorylation activity of mTORC1 specifically in cancer cells without causing toxicity to normal cells, and as a result, it is a compound that exhibits cancer cell-specific cytotoxic activity, and furthermore, is more efficient than rapamycin. T37 / 46 It was found to inhibit p-4E-BP1. These findings indicate that this compound has a low likelihood of causing side effects and possesses high potential as an anticancer agent, cancer inhibitor, and / or life extension agent.

Claims

[Claim 1] An anticancer agent and / or cancer inhibitor comprising the following compounds: 【Chemistry 1】

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