JP2016108318A - Novel glutamic acid derivative and use of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a GGT (γ-glutamyl transpeptidase) recognizing prodrug, in which the prodrug is a prodrug recognized by GGT and can liberate an active compound selectively in a tissue with high level expression of GGT and is expected to exhibit an improved therapeutic effect with decreased side effects, and can be used as a medicine that exhibits sufficient stability and effect required as a medicine.SOLUTION: When preparing a prodrug by γ-glutamylating a pharmaceutical, with an aromatic amide linker with a specific structure interposed, a prodrug is stable even in a condition close to physiological environments and is recognized by GGT and liberates a pharmacological active pharmaceutical very rapidly. That is, in a tissue with a high level expression of GGT, a pharmaceutical to which a γ-glutamyl aromatic amide is bonded through an ether bond can rapidly liberates a pharmacologically active pharmaceutical and become a pharmaceutical which has a high therapeutic effect.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a novel glutamic acid derivative which is enzyme-selectively activated at a target site and uses thereof. More specifically, the present invention relates to a novel glutamic acid derivative which is a prodrug activated by γ-glutamyltranspeptidase (GGT, EC 2.3.2.2) and use thereof.

  Prodrugs are those that are metabolized in vivo to an active drug. The purpose of making a drug into a prodrug includes improvement of stability, improvement of solubility, improvement of absorption, reduction of side effects, improvement of action time (sustained action), expression of action at a specific site, etc. . Until now, several drugs have been developed as prodrugs and clinically used as therapeutic drugs for various diseases.

  Many of the anticancer agents used for cancer chemotherapy show a cell growth inhibitory effect not only on cancer cells but also on normal cells, and thus side effects resulting from this are problematic. For this reason, if an anticancer agent can be made to selectively act on cancer cells, an anticancer agent with reduced side effects can be provided. Therefore, if an anticancer drug can be converted into a prodrug and selectively activated at a target site such as a tumor tissue, side effects can be reduced and a therapeutic effect can be greatly improved.

  As a method for selectively converting to an active compound at a target site, a method utilizing an enzyme highly expressed in a target tissue is considered. Regarding prodrug formation using an enzyme-specific reaction at a target site, a method is known in which a self-cleavable linker is interposed between the enzyme recognition site and the drug (Non-patent Document 1). This allows an enzyme recognition site to be cleaved by an enzyme-specific reaction, and in the resulting linker-drug complex, the linker part self-cleaves to release the drug.

  γ-Glutamyltranspeptidase (GGT) is the enzyme responsible for the initial stage of metabolic degradation of glutathione (γ-Glu-Cys-Gly) and glutathione conjugates, and is universally present in almost every organism from higher animals and plants to microorganisms Are known (Non-Patent Document 2, Non-Patent Document 3, and Non-Patent Document 4). GGT is an enzyme that hydrolyzes the γ-glutamyl bond of glutathione, and produces Glu and Cys-Gly. On the other hand, GGT gives a γ-glutamyl transfer product with various amino acids, dipeptides, and amines as receptors.

GGT is known to be highly expressed in a wide variety of cancer cells, and there is also a report pointing out GGT as a drug target for cancer chemotherapy (Non-patent Document 5).
Patent Document 1 discloses a compound obtained by converting an anticancer agent into γ-glutamyl. As a prodrug using a self-cleaving linker, a prodrug that can be cleaved by trypsin is mentioned (Non-patent Document 1). However, there is no mention of prodrugs that can be recognized by GGT and thereby cleaved. Non-Patent Document 6 includes a compound obtained by glutamylating an anticancer agent, and shows cytotoxicity depending on the enzyme. However, the pharmacologically active compound is dissociated at a very high enzyme concentration and cannot function as a prodrug in the in vivo environment. Non-Patent Document 7 includes a prodrug-type compound through an appropriate linker as a compound in which a drug is bonded to the glutamic acid γ position.

J. et al. Med. Chem. , 24, 479-480, (1981) Adv. Enzymol. Relat. Area Mol. Biol. , 72, 239-278 (1998) Methods Enzymol. 113, 400-419 (1985). Methods Enzymol. 113, 419-437 (1985). Biochemical Pharmacology 71, 231-238 (2006) Environmental and Molecular Mutagenesis 32, 377-386 (1998) Chem. Commun. , 49, 1389-1391 (2013)

US7,989,188

  A prodrug recognized by GGT can selectively release an active compound in a GGT-expressing tissue, so that it can be expected to be a drug with reduced side effects and improved therapeutic effects. However, no GDT-recognized prodrug that can be used as a pharmaceutical product is desired because a product that sufficiently exhibits stability and effects required as a pharmaceutical product has not been obtained.

［式中、Ｒ_１及びＲ_２は、それぞれ独立して水素原子、置換基を有していても良いアルキル基及び置換基を有していても良いアルコキシカルボニル基からなる群から選択される基を示し、Ｒ_３は、水素原子又は置換基を有していても良いアルキル基を示し、Ａ_１及びＡ_２は、Ｃ−Ｒ_６、Ｃ−Ｒ_７及び窒素原子からなる群から選択される基であり、該Ｒ_６は、水素原子、ハロゲン原子、ニトロ基、水酸基、置換基を有していても良いアルキル基及び置換基を有していても良いアルコキシ基からなる群から選択される１種以上の基を示し、該Ｒ_７は下記一般式（２）

［式中、Ｒ_４及びＲ_５は、それぞれ独立して水素原子又は置換基を有していても良いアルキル基を示し、Ｘは芳香族性水酸基を有する生理活性物質の結合残基を示す。］であり、ここで、前記Ａ_１及び前記Ａ_２は何れか一方が前記Ｃ−Ｒ_７であって、他方が前記Ｃ−Ｒ_６又は窒素原子であり、Ｂ_１、Ｂ_２及びＢ_３は、それぞれ独立して前記Ｃ−Ｒ_６又は窒素原子である。］で表されるグルタミン酸誘導体又はその薬理学的に許容される塩が、ＧＧＴ認識プロドラッグとして有用であることを見出し、本発明を完成した。 In order to solve the above-mentioned problems, the present inventors have conducted extensive research. As a result, a drug to which an appropriate γ-glutamyl aromatic amide is bound is recognized by GGT and rapidly bioactive while being extremely stable. It has been found that a drug having an action is released. Specifically, the following general formula (1)

[Wherein, R ₁ and R ₂ are each independently selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group and an optionally substituted alkoxycarbonyl group. R ₃ represents a hydrogen atom or an optionally substituted alkyl group, and A ₁ and A ₂ are selected from the group consisting of C—R ₆ , C—R ₇ and a nitrogen atom. And R ₆ is selected from the group consisting of a hydrogen atom, a halogen atom, a nitro group, a hydroxyl group, an optionally substituted alkyl group and an optionally substituted alkoxy group. Represents one or more groups, and R ₇ represents the following general formula (2)

[Wherein, R ₄ and R ₅ each independently represent a hydrogen atom or an alkyl group which may have a substituent, and X represents a binding residue of a physiologically active substance having an aromatic hydroxyl group. Here, _one of the A ₁ and the A ₂ is the C—R ₇ , the other is the C—R ₆ or a nitrogen atom, and B ₁ , B ₂ and B ₃ are Each independently represents the C—R ₆ or nitrogen atom. And the pharmacologically acceptable salt thereof were found to be useful as GGT-recognizing prodrugs, and the present invention was completed.

［式中、Ｒ_１及びＲ_２は、それぞれ独立して水素原子、置換基を有していても良いアルキル基及び置換基を有していても良いアルコキシカルボニル基からなる群から選択される基を示し、Ｒ_３は、水素原子又は置換基を有していても良いアルキル基を示し、Ａ_１及びＡ_２は、Ｃ−Ｒ_６、Ｃ−Ｒ_７及び窒素原子からなる群から選択される基であり、該Ｒ_６は、水素原子、ハロゲン原子、ニトロ基、水酸基、置換基を有していても良いアルキル基及び置換基を有していても良いアルコキシ基からなる群から選択される１種以上の基を示し、該Ｒ_７は下記一般式（２）

［式中、Ｒ_４及びＲ_５は、それぞれ独立して水素原子又は置換基を有していても良いアルキル基を示し、Ｘは芳香族性水酸基を有する生理活性物質の結合残基を示す。］であり、ここで、前記Ａ_１及び前記Ａ_２は何れか一方が前記Ｃ−Ｒ_７であって、他方が前記Ｃ−Ｒ_６又は窒素原子であり、Ｂ_１、Ｂ_２及びＢ_３は、それぞれ独立して前記Ｃ−Ｒ_６又は窒素原子である。］で表されるグルタミン酸誘導体又はその薬理学的に許容される塩。
［２］ Ｘで示される前記生理活性物質の結合残基における該生理活性物質が、７−エチル−１０−ヒドロキシカンプトテシン、ノギテカン及びその誘導体からなる群から選択される１種である前記［１］に記載のグルタミン酸誘導体又はその薬理学的に許容される塩。
［３］ Ｘで示される前記生理活性物質の結合残基における該生理活性物質が、エトポシド、テニポシド及びその誘導体からなる群から選択される１種である前記［１］に記載のグルタミン酸誘導体又はその薬理学的に許容される塩。
［４］ Ｘで示される前記生理活性物質の結合残基における該生理活性物質が、ラロキシフェン及びその誘導体からなる群から選択される１種である前記［１］に記載のグルタミン酸誘導体又はその薬理学的に許容される塩。
［５］ Ｘで示される前記生理活性物質の結合残基における該生理活性物質が、ゴセレリン、リュープリン及びその誘導体である前記［１］に記載のグルタミン酸誘導体又はその薬理学的に許容される塩。
［６］ Ｘで示される前記生理活性物質の結合残基における該生理活性物質が、２−ブチルアミノ−２−デメトキシヒポクレリン及びその誘導体である前記［１］に記載のグルタミン酸誘導体又はその薬理学的に許容される塩。
［７］ Ｘで示される前記生理活性物質の結合残基における該生理活性物質が、コンブレタスタチン及びその誘導体である前記［１］に記載のグルタミン酸誘導体又はその薬理学的に許容される塩。
［８］ Ｘで示される前記生理活性物質の結合残基における該生理活性物質が、ガネテスピブ、マクベシン、ラディシコル及びその誘導体である前記［１］に記載のグルタミン酸誘導体又はその薬理学的に許容される塩。
［９］ Ｘで示される前記生理活性物質の結合残基における該生理活性物質が、ドキソルビシン、ダウノルビシン、エピルビシン、ピラルビシン、イダルビシン、ミトキサントロン、アムルビシン及びその誘導体である前記［１］に記載のグルタミン酸誘導体又はその薬理学的に許容される塩。
［１０］ 一般式（１）において、Ｒ_１、Ｒ_２及びＲ_３は水素原子である前記［１］〜［９］の何れか１項に記載のグルタミン酸誘導体又はその薬理学的に許容される塩。
［１１］ 前記［１］〜［１０］の何れか１項に記載のグルタミン酸誘導体又はその薬理学的に許容される塩を有効成分として含有する医薬。
［１２］ 抗がん剤である前記［１１］に記載の医薬。 That is, the gist of the present application is the invention shown in the following [1] to [12].
[1] General formula (1)

[Wherein, R ₁ and R ₂ are each independently selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group and an optionally substituted alkoxycarbonyl group. R ₃ represents a hydrogen atom or an optionally substituted alkyl group, and A ₁ and A ₂ are selected from the group consisting of C—R ₆ , C—R ₇ and a nitrogen atom. And R ₆ is selected from the group consisting of a hydrogen atom, a halogen atom, a nitro group, a hydroxyl group, an optionally substituted alkyl group and an optionally substituted alkoxy group. Represents one or more groups, and R ₇ represents the following general formula (2)

[Wherein, R ₄ and R ₅ each independently represent a hydrogen atom or an alkyl group which may have a substituent, and X represents a binding residue of a physiologically active substance having an aromatic hydroxyl group. Here, _one of the A ₁ and the A ₂ is the C—R ₇ , the other is the C—R ₆ or a nitrogen atom, and B ₁ , B ₂ and B ₃ are Each independently represents the C—R ₆ or nitrogen atom. Or a pharmacologically acceptable salt thereof.
[2] The above-mentioned [1], wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is one selected from the group consisting of 7-ethyl-10-hydroxycamptothecin, nogitecan and derivatives thereof Or a pharmacologically acceptable salt thereof.
[3] The glutamic acid derivative according to the above [1], wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is one selected from the group consisting of etoposide, teniposide and derivatives thereof Pharmacologically acceptable salt.
[4] The glutamic acid derivative or the pharmacology thereof according to [1], wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is one selected from the group consisting of raloxifene and derivatives thereof Acceptable salt.
[5] The glutamic acid derivative or the pharmaceutically acceptable salt thereof according to [1], wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is goserelin, leupurine and derivatives thereof.
[6] The glutamic acid derivative according to the above [1] or a derivative thereof, wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is 2-butylamino-2-demethoxyhypocreline and its derivative Pharmacologically acceptable salt.
[7] The glutamic acid derivative or the pharmaceutically acceptable salt thereof according to [1], wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is combretastatin and a derivative thereof.
[8] The glutamic acid derivative or the pharmacologically acceptable derivative thereof according to [1], wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is ganetespib, macbecin, radicicol, and derivatives thereof salt.
[9] The glutamic acid according to [1], wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is doxorubicin, daunorubicin, epirubicin, pirarubicin, idarubicin, mitoxantrone, amrubicin and derivatives thereof. Derivatives or pharmacologically acceptable salts thereof.
[10] In the general formula (1), R ₁ , R ₂ and R ₃ are hydrogen atoms, the glutamic acid derivative according to any one of the above [1] to [9] or a pharmacologically acceptable one thereof salt.
[11] A medicament comprising the glutamic acid derivative according to any one of [1] to [10] or a pharmacologically acceptable salt thereof as an active ingredient.
[12] The medicament according to [11] above, which is an anticancer agent.

  The glutamic acid derivative of the present invention or a pharmacologically acceptable salt thereof is recognized by GGT and has a property of rapidly releasing a physiologically active substance having an aromatic hydroxyl group. GGT is known to be highly expressed in many malignant tumors. Therefore, by applying the glutamic acid derivative of the present invention to a compound having an antitumor effect, it is possible to liberate a compound that exhibits antitumor activity selectively in a target tissue, reducing side effects and improving the therapeutic effect. A neoplastic agent can be provided.

It is a result of the cell growth-inhibition test of the compound of Example 1 with respect to OS-RC-2 cells. It is a result of the cell growth-inhibition test of the compound of Example 1 with respect to SK-OV-3 cell. It is a result of the cell growth suppression test of the compound of Example 1 with respect to OS-RC-2 cells in the presence of a GGT inhibitor.

  The present invention relates to a glutamic acid derivative obtained by binding a γ-glutamyl aromatic amide to a physiologically active substance having an aromatic hydroxyl group, or a pharmacologically acceptable salt thereof. The present invention also relates to the use of the compound as a pharmaceutical product. Details of the present invention will be described below.

［式中、Ｒ_１及びＲ_２は、それぞれ独立して水素原子、置換基を有していても良いアルキル基及び置換基を有していても良いアルコキシカルボニル基からなる群から選択される基を示し、Ｒ_３は、水素原子又は置換基を有していても良いアルキル基を示し、Ａ_１及びＡ_２は、Ｃ−Ｒ_６、Ｃ−Ｒ_７及び窒素原子からなる群から選択される基であり、該Ｒ_６は、水素原子、ハロゲン原子、ニトロ基、水酸基、置換基を有していても良いアルキル基及び置換基を有していても良いアルコキシ基からなる群から選択される１種以上の基を示し、該Ｒ_７は下記一般式（２）

［式中、Ｒ_４及びＲ_５は、それぞれ独立して水素原子又は置換基を有していても良いアルキル基を示し、Ｘは芳香族性水酸基を有する生理活性物質の結合残基を示す。］であり、ここで、前記Ａ_１及び前記Ａ_２は何れか一方が前記Ｃ−Ｒ_７であって、他方が前記Ｃ−Ｒ_６又は窒素原子であり、Ｂ_１、Ｂ_２及びＢ_３は、それぞれ独立して前記Ｃ−Ｒ_６又は窒素原子である。］で表されるグルタミン酸誘導体又はその薬理学的に許容される塩である。 The glutamic acid derivative of the present invention or a pharmacologically acceptable salt thereof is represented by the following general formula (1)

[Wherein, R ₁ and R ₂ are each independently selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group and an optionally substituted alkoxycarbonyl group. R ₃ represents a hydrogen atom or an optionally substituted alkyl group, and A ₁ and A ₂ are selected from the group consisting of C—R ₆ , C—R ₇ and a nitrogen atom. And R ₆ is selected from the group consisting of a hydrogen atom, a halogen atom, a nitro group, a hydroxyl group, an optionally substituted alkyl group and an optionally substituted alkoxy group. Represents one or more groups, and R ₇ represents the following general formula (2)

[Wherein, R ₄ and R ₅ each independently represent a hydrogen atom or an alkyl group which may have a substituent, and X represents a binding residue of a physiologically active substance having an aromatic hydroxyl group. Here, _one of the A ₁ and the A ₂ is the C—R ₇ , the other is the C—R ₆ or a nitrogen atom, and B ₁ , B ₂ and B ₃ are Each independently represents the C—R ₆ or nitrogen atom. ] Or a pharmacologically acceptable salt thereof.

R ₁ and R ₂ in the general formula (1) are each independently a hydrogen atom, an alkyl group which may have a substituent, or an alkoxycarbonyl group which may have a substituent.
In the alkyl group which may have the substituent, the alkyl group represents a linear, branched or cyclic alkyl group having 1 to 30 carbon atoms. Examples of the linear alkyl group include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-hexyl group, an n-dodecyl group, an n-tetradecyl group, and an n-hexadecyl group. . Examples of the branched alkyl group include isopropyl group, t-butyl group, 1-methyl-propyl group, 2-methyl-propyl group, 2,2-dimethylpropyl group and the like. Examples of the cyclic alkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, an adamantyl group, and the like.

  The alkoxycarbonyl group in the alkoxycarbonyl group which may have a substituent represents an alkoxycarbonyl group having 1 to 10 carbon atoms. For example, in addition to a methoxycarbonyl group, an ethoxycarbonyl group, etc., a primary alkoxycarbonyl group having an appropriate aromatic substituent, such as a benzyloxycarbonyl group, a 9-fluorenylmethoxycarbonyl group, an isopropoxycarbonyl group, sec- A secondary alkoxycarbonyl group such as a butoxycarbonyl group or a tertiary alkoxycarbonyl group such as a t-butoxycarbonyl group may be mentioned.

  Examples of the substituent that the alkyl group and the alkoxycarbonyl group may have include, for example, a mercapto group, a hydroxyl group, a halogen atom, a nitro group, a cyano group, an alkenyl group having 2 to 10 carbon atoms, and an alkyl group having 2 to 10 carbon atoms. Alkynyl group, carbocyclic or heterocyclic aryl group, alkylthio group having 1 to 8 carbon atoms, arylthio group, alkylsulfinyl group having 1 to 8 carbon atoms, arylsulfinyl group, alkylsulfonyl group having 1 to 8 carbon atoms, arylsulfonyl group , An alkoxy group having 1 to 8 carbon atoms, an aryloxy group, an aliphatic or aromatic amino group, an alkyl group having 1 to 8 carbon atoms substituted with an aliphatic amino group, a formyl group, an acyl group, a carboxy group, or silyl Groups and the like. The substitution position on the aromatic ring may be ortho, meta, or para.

The alkyl group which may have a substituent in R ₁ and R ₂ in the general formula (1) or the alkoxycarbonyl group which may have a substituent is preferably an amino-protecting group. That is, any protecting group for an amino group in an organic synthesis reaction can be used without particular limitation. The alkoxycarbonyl group is particularly preferably a benzyloxycarbonyl group (Cbz group), a t-butoxycarbonyl group (Boc group), a 9-fluorenylmethoxycarbonyl group (Fmoc group), an allyloxycarbonyl group (Aloc group). And the like.
R ₁ and R ₂ are preferably a hydrogen atom and / or an alkoxycarbonyl group which may have a substituent. A case where both R ₁ and R ₂ are hydrogen atoms, or a combination of a hydrogen atom and an alkoxycarbonyl group which may have a substituent is preferable.

Examples of R ₃ in the general formula (1) include a hydrogen atom or an alkyl group which may have a substituent.
In the alkyl group which may have the substituent, the alkyl group represents a linear, branched or cyclic alkyl group having 1 to 30 carbon atoms. Examples of the linear alkyl group include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-hexyl group, an n-dodecyl group, an n-tetradecyl group, and an n-hexadecyl group. . Examples of the branched alkyl group include isopropyl group, t-butyl group, 1-methyl-propyl group, 2-methyl-propyl group, 2,2-dimethylpropyl group and the like. Examples of the cyclic alkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, an adamantyl group, and the like. Moreover, the benzyl group, 9-fluorenylmethyl group, etc. which have a suitable aromatic substituent are mentioned. The substituent in the alkyl group of R _{3 has} the same meaning as described above.
As the alkyl group which may have a substituent for R ₃ , a carboxylic acid protecting group is preferably used. Any protecting group for a carboxylic acid in an organic synthesis reaction can be used without particular limitation. Particularly preferred are methyl group, ethyl group, t-butyl group, allyl group, benzyl group and 9-fluorenylmethyl group.

In the general formula (1), _{A 1} and _{A 2} are from the group consisting of _{C-R 7} and the nitrogen atom is a carbon atom substituted with _C-R 6, _{R 7} is a carbon atom substituted by _{R 6} The group to be selected. _Further, B 1, _{B 2} and _{B 3} are _{C-R 6} or a nitrogen atom is a carbon atom substituted by _{R 6} independently.

R ₆ is one or more selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxyl group, a nitro group, an optionally substituted alkyl group, and an optionally substituted alkoxy group. Is a substituent.
The halogen atom is a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.
The alkyl group in the alkyl group which may have a substituent represents a linear, branched or cyclic alkyl group having 1 to 30 carbon atoms. Examples of the linear alkyl group include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-hexyl group, an n-dodecyl group, an n-tetradecyl group, and an n-hexadecyl group. . Examples of the branched alkyl group include isopropyl group, t-butyl group, 1-methyl-propyl group, 2-methyl-propyl group, 2,2-dimethylpropyl group and the like. Examples of the cyclic alkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, an adamantyl group, and the like.

  The alkoxy group in the alkoxy group which may have a substituent represents an alkoxy group having 1 to 10 carbon atoms. Examples thereof include primary alkoxy groups such as methoxy group, ethoxy group, and benzyloxy group, secondary alkoxy groups such as isopropoxy group and sec-butoxy group, and tertiary alkoxy groups such as t-butoxy group.

  Examples of the substituent that the alkyl group and the alkoxy group may have include, for example, a mercapto group, a hydroxyl group, a halogen atom, a nitro group, a cyano group, an alkenyl group having 2 to 10 carbon atoms, and an alkynyl group having 2 to 10 carbon atoms. Group, carbocyclic or heterocyclic aryl group, alkylthio group having 1 to 8 carbon atoms, arylthio group, alkylsulfinyl group having 1 to 8 carbon atoms, arylsulfinyl group, alkylsulfonyl group having 1 to 8 carbon atoms, arylsulfonyl group, C1-C8 alkoxy group, aryloxy group, aliphatic or aromatic amino group, C1-C8 alkyl group substituted with aliphatic amino group, formyl group, acyl group, carboxy group, or silyl group , Etc. The substitution position on the aromatic ring may be ortho, meta, or para.

A ₁ , A ₂ , B ₁ , B ₂ and B ₃ may be nitrogen atoms. That is, the 6-membered aromatic group composed of A _{1 to} B ₃ may be a nitrogen-containing heterocyclic ring. The nitrogen-containing heterocyclic ring includes a heterocyclic group containing 1 to 3 nitrogen atoms in A _{1 to} B ₃ . For example, a pyridine ring, pyridazine ring, pyrimidine ring, pyrazine ring or triazine ring. The nitrogen-containing heterocycle may have a substituent. The substituent is a substituent defined by R ₆ .

Examples of R ₄ and R ₅ independently include a hydrogen atom or an alkyl group which may have a substituent.
The alkyl group in the alkyl group which may have a substituent represents a linear, branched or cyclic alkyl group having 1 to 30 carbon atoms. Examples of the linear alkyl group include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-hexyl group, an n-dodecyl group, an n-tetradecyl group, and an n-hexadecyl group. . Examples of the branched alkyl group include isopropyl group, t-butyl group, 1-methyl-propyl group, 2-methyl-propyl group, 2,2-dimethylpropyl group and the like. Examples of the cyclic alkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, an adamantyl group, and the like. The substituents that may have are as defined above.

The physiologically active substance in X can be used without particular limitation as long as it is a chemical substance that exhibits a pharmacological function when administered in vivo, and has an aromatic hydroxyl group. That is, the X group is a binding residue that is ether-bonded via an oxygen atom of the aromatic hydroxyl group of the physiologically active substance.
The physiological activity of the physiologically active substance is not particularly limited, but is preferably a pharmacological activity related to disease treatment, and a pharmacologically active compound for disease treatment is preferably used. Since GGT is highly expressed in malignant tumors, the physiologically active substance is preferably an antitumor active substance, and an anticancer agent is preferably applied. That is, the physiologically active substance represented by X in the general formula (1) is preferably an anticancer agent having an aromatic hydroxyl group.

Anticancer agents suitable as the physiologically active substance represented by X in the general formula (1) include DNA topoisomerase inhibitors, hormone therapy agents, photodynamic therapy agents, microtubule polymerization inhibitors, Hsp90 inhibitors and other cells. The anticancer agent which has a division | segmentation inhibitory effect can be mentioned.
Examples of the DNA topoisomerase inhibitor include 7-ethyl-10-hydroxycamptothecin and nogitecan, which are DNA topoisomerase type I inhibitors. Examples of DNA topoisomerase type II inhibitors include etoposide and teniposide. Moreover, anthracycline anticancer agents such as doxorubicin, daunorubicin, epirubicin, pirarubicin, idarubicin, mitoxantrone and amrubicin can be exemplified.
Examples of the hormone therapeutic agent include raloxifene, goserelin, leuprorelin and the like.
Examples of the photochemotherapeutic agent include 2-butylamino-2-demethoxyhypocreline.
Examples of the microtubule polymerization inhibitor include combretastatin and derivatives thereof.
Examples of the Hsp90 inhibitor include ganetespib, macbecin, radicicol and the like.
These anticancer agents are ether-bonded via the oxygen atom of the aromatic hydroxyl group, so that they are excellent in stability in plasma, and can be quickly bonded and dissociated by contact with GGT. A prodrug excellent in responsiveness to be released can be provided. In particular, it is preferable to use 7-ethyl-10-hydroxycamptothecin or nogitecan as a physiologically active substance having an aromatic hydroxyl group related to X, wherein X is the 10-position of 7-ethyl-10-hydroxycamptothecin or nogitecan A bond residue ether-bonded with a hydroxyl group is preferred.

一般式（３）において、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４、Ｒ_５、Ａ_２、Ｂ_１、Ｂ_２、Ｂ_３及びＸは、前述と同義である。 The present invention is characterized in that any _one of A ₁ and A ₂ is C—R ₇ . That is, one aspect of the present invention is a glutamic acid derivative represented by the following general formula (3) in which A ₁ is C—R ₇ or a pharmacologically acceptable salt thereof.

In the general formula (3), R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , A ₂ , B ₁ , B ₂ , B ₃ and X are as defined above.

一般式（４）において、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４、Ｒ_５、Ａ_１、Ｂ_１、Ｂ_２、Ｂ_３及びＸは、前述と同義である。 Another embodiment of the present invention is a glutamic acid derivative represented by the following general formula (4) wherein A ₂ is C—R ₇ or a pharmacologically acceptable salt thereof.

In the general formula (4), R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , A ₁ , B ₁ , B ₂ , B ₃ and X are as defined above.

In the glutamic acid derivative represented by the general formula (1) of the present invention or a pharmacologically acceptable salt thereof, it is necessary that the γ-glutamic acid-binding partial structure is a free amino acid structure in order to be recognized by GGT. is there. That is, in order for the glutamic acid derivative to function as a prodrug that is recognized and activated by GGT, it is necessary that R ₁ , R _2, and R ₃ are all hydrogen atoms. Therefore, when the glutamic acid derivative is used as a medicament for exerting pharmacological activity, it is preferable that all of R ₁ , R ₂ and R ₃ are hydrogen atoms. However, any one of R ₁ , R ₂ and R ₃ is an amino group or carboxy protecting group, and the protecting group is dissociated after administration into a living body, and the R ₁ , R ₂ and R ₃ are released. The case where all of these have a hydrogen atom structure is also included as an aspect of the glutamic acid derivative for pharmaceutical use.
The compound in which R ₁ , R ₂ and R ₃ are an alkyl group or an alkoxycarbonyl group which may have a substituent is a compound useful as an intermediate in the production of a compound for pharmaceutical use, and It is included in the content of the invention.

  The glutamic acid derivative represented by the general formula (1) may exist as a pharmacologically acceptable salt. Examples of the salt include base addition salts, acid addition salts, amino acid salts and the like. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt and magnesium salt, and organic amine salts such as ammonium salt, triethylamine salt, piperidine salt and morpholine salt. Examples of the acid addition salt include mineral acid salts such as hydrochloride, sulfate, and nitrate, and organic acid salts such as methanesulfonate, paratoluenesulfonate, citrate, and oxalate. Examples of amino acid salts include glycine salts. However, the salt of the compound of the present invention is not limited to these.

  The glutamic acid derivative represented by the general formula (1) and a salt thereof may have one or two or more asymmetric carbons depending on the type of the substituent, and may be a stereo such as an optical isomer or a diastereoisomer. Isomers may exist. Pure forms of stereoisomers, any mixture of stereoisomers, racemates, and the like are all within the scope of the present invention.

  The glutamic acid derivative represented by the general formula (1) and a salt thereof may exist as a hydrate or a solvate, and any of these substances is included in the scope of the present invention. Although the kind of solvent which forms a solvate is not specifically limited, For example, solvents, such as ethanol, acetone, isopropanol, can be mentioned. The number of bonds in the hydrate or solvate is not particularly limited as long as it is a stable hydrate or solvate that can be isolated.

  In the examples of the present specification, production methods for typical compounds included in the compounds of the present invention represented by the general formula (1) are specifically shown. Any compound included in the general formula (1) can be easily produced by referring to the disclosure and appropriately selecting starting materials, reagents, reaction conditions and the like as necessary.

スキーム（Ｉ）中、Ｒ_１〜Ｒ_３、Ｘ、Ａ_１、Ｂ_１〜Ｂ_３は、前述と同義であり、Ｒ_１及び／又はＲ_２はアミノ基の保護基であり、Ｒ_３はカルボン酸の保護基である。ＬＧはハロゲン原子、メタンスルホニルオキシ基等の脱離基を表す。以下に各工程を説明する。 The glutamic acid derivative represented by the general formula (1) of the present invention can be produced, for example, as follows when A ₂ is C—R ₇ .
Scheme (I)

In scheme (I), R _{1 to} R ₃ , X, A ₁ , B _{1 to} B ₃ are as defined above, R ₁ and / or R ₂ are amino-protecting groups, and R ₃ is a carboxyl group. Acid protecting group. LG represents a leaving group such as a halogen atom or a methanesulfonyloxy group. Each step will be described below.

  Step A: A step of synthesizing a γ-glutamic acid amide derivative (A-1) by amidating a glutamic acid derivative in which an amino group and an α-carboxy group are protected with an aromatic amine. This step is an amidation condensation reaction and can be carried out using a general condensing agent. For example, 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is used as a condensing agent and is reacted at a temperature of 0 ° C. to 150 ° C., preferably 0 ° C. to 30 ° C., in a solvent such as dichloromethane. Can be implemented. Examples of the condensing agent include carbodiimide condensing agents, imidazole dehydrating condensing agents, triazine condensing agents, phosphonium dehydrating condensing agents, uronium condensing agents, diphenylphosphoric acid azide (DPPA), BOP reagents, 4- (4, 6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride (DMT-MM) or the like can be used. If necessary, an activator such as 1-hydroxybenzotriazole, N, N-dimethyl-4-aminopyridine may be allowed to coexist.

  Step B: A step of synthesizing the derivative represented by the general formula (A-2) from the γ-glutamic acid amide derivative represented by the general formula (A-1). LG in the general formula (A-2) is a leaving group, and examples thereof include a methanesulfonyloxy group and a p-toluenesulfonyloxy group. In this step, for example, when the leaving group is a methanesulfonyloxy group, the methanesulfonyl chloride is -30 ° C to 150 ° C, preferably -10 ° C in a solvent such as dichloromethane in the presence of N, N-diisopropylethylamine. To 30 ° C. for the reaction.

  Step C: A step of synthesizing the physiologically active substance-binding derivative represented by the general formula (A-3) from the derivative represented by the general formula (A-2). In this step, for example, a physiologically active substance having an aromatic hydroxyl group is reacted in a solvent such as N, N-dimethylformamide in the presence of cesium carbonate at a temperature of 0 ° C. to 150 ° C., preferably 0 ° C. to 30 ° C. Can be implemented.

  Process D: a process route for synthesizing a physiologically active substance-binding derivative represented by the general formula (A-3) in one step from the γ-glutamic acid amide derivative represented by the general formula (A-1) . In this step, for example, the compound having an aromatic hydroxyl group is converted to 0 ° C. to 150 ° C., preferably 0 ° C. to 30 ° C. in a solvent such as N, N-dimethylformamide in the presence of triphenylphosphine and diisopropyl azodicarboxylate. It can implement by making it react at the temperature of.

Step E: This is a step of deprotecting the amino group and carboxy group of the γ-glutamic acid binding moiety in the physiologically active substance-binding derivative represented by the general formula (A-3).
When either R ₁ or R ₂ is a t-butoxycarbonyl group (Boc group), the other is a hydrogen atom, and R ₃ is a t-butyl group, the deprotection of Step E is performed under acidic conditions can do. Examples of the acid that can be used include inorganic acids such as hydrochloric acid and sulfuric acid, and carboxylic acids such as acetic acid and trifluoroacetic acid. In addition, any catalyst that is known to be capable of deprotecting a t-butoxycarbonyl group or a t-butyl ester and that does not affect the portion other than the protecting group can be used without particular limitation.
Further, when either _one of R ₁ and R ₂ is a 9-fluorenylmethoxycarbonyl group (Fmoc group), the other is a hydrogen atom, and R ₃ is a fluorenylmethyl group, The step E can be performed. As the base, ammonia or an organic base such as piperidine or morpholine can be used. In addition, any catalyst that is known to be capable of deprotecting a fluorenylmethoxycarbonyl group or a fluorenylmethyl ester and that does not affect parts other than the protecting group can be used under any deprotection reaction conditions. It can be used even if it exists.
In addition, when either _one of R ₁ and R ₂ is an allyloxycarbonyl group (Aloc group), the other is a hydrogen atom, and R ₃ is an allyl group, the step E is performed in the presence of a palladium catalyst. be able to. Tetrakis (triphenylphosphine) palladium (0) or the like can be used as the palladium catalyst. In addition, any catalyst that is known to be capable of deprotecting an allyloxycarbonyl group or allyl ester and that does not affect the portion other than the protecting group can be used under any deprotection reaction conditions. be able to.

The glutamic acid derivative of the present invention or a pharmaceutically acceptable salt thereof can release a physiologically active substance and functions as a prodrug of the physiologically active substance. Therefore, it can be used as a medicament containing the glutamic acid derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
The pharmaceutical containing the glutamic acid derivative of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient may use the glutamic acid or a salt thereof alone, but usually in combination with a pharmaceutically acceptable additive. It is preferable to prepare a pharmaceutical composition and use it as a pharmaceutical preparation. The additives include excipients, disintegrants, binders, lubricants, fluidizing agents, coating agents, suspending agents, emulsifiers, stabilizers, preservatives, flavoring agents, flavoring agents, diluents. And pharmaceutically acceptable additives such as a solubilizing agent, and a pharmaceutical preparation is prepared by mixing with these. The preparation is safely administered orally or parenterally (systemic administration, topical administration, etc.) in the form of powders, granules, tablets, tablets, capsules, injections, suppositories, ointments and the like. The The content of the glutamic acid derivative of the present invention or pharmaceutically acceptable salt in the preparation varies depending on the preparation, but it is usually preferably 0.1 to 100% by weight.

When used as a pharmaceutical containing the glutamic acid derivative or a pharmaceutically acceptable salt thereof as an active ingredient, the dosage varies depending on the administration route, the age of the patient and the actual symptoms to be prevented or treated, and is not particularly limited. Absent. The glutamic acid derivative of the present invention or a pharmaceutically acceptable salt thereof is preferably used as an anticancer agent because it is recognized by GGT highly expressed in malignant tumors and has a physical property of releasing a physiologically active substance. When used as an anticancer agent, for example, when orally administered to an adult, the active ingredient can be 0.01 mg to 2000 mg, preferably 0.1 mg to 1000 mg per day, once or several times a day. Can be administered separately. In addition, when administered parenterally such as intravenous administration, the active ingredient per body surface area can be 0.01 mg to 2000 mg / m ² , preferably 0.1 mg to 1000 mg / m ^2. It is preferable to administer once or several times.

EXAMPLES Next, although an Example demonstrates this invention further more concretely, this invention is not restrict | limited at all by these examples.
LC / MS measurement conditions for detection and identification of Example compounds and purity measurement are as follows.
Model: Shimadzu LCMS-2020
Column: Inertsil ODS-3, 2.1 mm × 100 mm,
Mobile phase A: acetonitrile / formic acid (99.9 / 0.1)
Mobile phase B: water / formic acid (99.9 / 0.1)
Gradient: Time (min) 0.0 5.5 6.5 6.51 10.0 A concentration (%) 20 90 90 20 20
Flow rate: 0.3 mL / min

The LC / MS measurement conditions for detection and identification of the reference example compound and purity measurement are as follows.
Model: Shimadzu LCMS-2010A
Column: Inertsil ODS-3, 2.1 mm × 100 mm,
Mobile phase A: acetonitrile / formic acid (99.9 / 0.1)
Mobile phase B: water / formic acid (99.9 / 0.1)
Gradient: Time (min) 0.0 5.5 6.5 6.51 10.0
A concentration (%) 5 90 90 5 5
Flow rate: 0.3 mL / min

Example 1: Synthesis of 10- (4-((S) 4-amino-4-carboxybutanamido) benzyloxy) -7-ethylcamptothecin 2 trifluoroacetate [Scheme 1]

Example 1-1: Synthesis of 5-((4-hydroxymethyl) benzylamino) -1- (t-butyl) N- (t-butoxycarbonyl) -L-glutamate 1-t-butyl N- (t- N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in a solution of butoxycarbonyl) -L-glutamate (1.00 g) and 4-aminobenzyl alcohol (0.487 g) in dry dichloromethane (15 mL). ) (1.019 g) was added and stirred at room temperature for 18 hours. 1N Hydrochloric acid was added, and the mixture was extracted with dichloromethane. The organic layer was washed with water and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, the residue was washed with diethyl ether, and 5-((4-hydroxymethyl) benzylamino) -1- (t-butyl) N- (t-butoxycarbonyl) -L-glutamate ( 0.890 g) was obtained.

NMR [400 MHz, CDCl ₃ , TMS] ppm: 1.46 (9H, s), 1.47 (9H, s), 1.82-1.89 (1H, m), 2.27-2.30 ( 2H, m), 2.44 (2H, t), 4.21-4.24 (1H, m), 4.66 (2H, s), 5.35-5.37 (1H, m), 7 .33 (2H, d), 7.62 (2H, d), 8.87 (1H, brs).
LC / MS retention time: 5.6 min; m / z (ESI, POS): 431 [M + Na] ⁺

Example 1-2: Synthesis of 5-((4-chloromethyl) benzylamino) -1- (t-butyl) N- (t-butoxycarbonyl) -L-glutamate 5 In a solution of-((4-hydroxymethyl) benzylamino) -1- (t-butyl) N- (t-butoxycarbonyl) -L-glutamate (88 mg, 0.22 mmol) in dry dichloromethane (4.3 mL), Diisopropylethylamine (0.073 mL, 0.43 mmol) and methanesulfonyl chloride (0.025 mL, 0.32 mmol) were added in this order, and the mixture was stirred for 2 hours and 5 minutes under ice cooling. The reaction mixture is diluted with ethyl acetate (40 mL), washed with 0.3 molar aqueous sodium hydrogen carbonate solution (3 mL) -water (10 mL), water (10 mL), and brine (10 mL), then anhydrous sodium sulfate And dried. Sodium sulfate was removed by filtration, the solvent was distilled off under reduced pressure, and 5-((4-chloromethyl) benzylamino) -1- (t-butyl) N- (t-butoxycarbonyl) -L-glutamate (83. 7 mg) was obtained. This was used for the next condensation reaction without purification.

Example 1-3: 10-(((4-((S) -5- (t-butoxy) -4-((t-butoxycarbonyl) amino) -5-oxopentanamido) benzyloxy) -7 Synthesis of ethyl camptothecin In a suspension of 7-ethyl-10-hydroxy-camptothecin (EHC 29 mg, 0.074 mmol) in dry dimethylformamide (1.5 mL) under argon atmosphere and stirring at room temperature, cesium carbonate (24 mg, 0.074 mmol) was added, and the resulting pale yellow suspension was stirred at room temperature for 9 minutes to give an orange homogeneous solution, then crude 5-((4-chloromethyl) benzylamino) with stirring at room temperature. -1- (t-butyl) N- (t-butoxycarbonyl) -L-glutamate (36 mg, 0.084 mmol) in dry dimethylformamide (1.5 mL) The resulting orange solution was stirred at room temperature for 2 hours and 40 minutes, then under ice-cooling for 1.5 hours, and diluted with ethyl acetate (20 mL) while stirring with ice-cooling. The organic layer was separated, washed with water (8 mL, 2 times) and brine (6 mL), and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain a pale yellow solid (61 mg), which was purified by preparative thin layer chromatography (silica gel, ethyl acetate) and purified by 10-(((4-((S)). -5- (t-butoxy) -4-((t-butoxycarbonyl) amino) -5-oxopentanamide) benzyloxy) -7-ethylcamptothecin (19 mg) was obtained NMR [400 MHz, CDCl3, TMS ] P m: 1.033 (3H, t, J = 7.3 Hz), 1.353 (3H, t, J = 7.7 Hz), 1.259 (9H, s), 1.473 (9H, s), 1.80-1.95 (3H, m), 2.289 (1H, m), 2.453 (2H, t, J = 6.5 Hz), 3.108 (2H, q, J = 7.7 Hz) ), 3.828 (1H, s), 4.229 (1H, m), 5.205 (2H, s), 5.220 (2H, s), 5.298 (1H, d, J = 16. 2Hz), 5.389 (1H, d, J = 8.1 Hz), 5.745 (1H, d, J = 16.2 Hz, 7.368 (1H, d, J = 2.6 Hz), 7.463 (2H, d, J = 8.6 Hz), 7.510 (1H, dd, J = 9.2 Hz, 2.6 Hz), 7.600 (1H, s), 7.693 (2H, d J = 8.2Hz), 8.125 (1H, d, J = 9.2Hz), 9.062 (1H, s). LC / MS retention time: 6.9 minutes; m / z (ESI, NEG): 781 [M−H] ⁻

Example 1-4: Synthesis of 10- (4-((S) 4-amino-4-carboxybutanamide) benzyloxy) -7-ethylcamptothecin 2 trifluoroacetate salt -(((4-((S) -5- (t-butoxy) -4-((t-butoxycarbonyl) amino) -5-oxopentanamido) benzyloxy) -7-ethylcamptothecin (33.7 mg , 0.043 mmol) in 1,2-dichloroethane (3 mL), trifluoroacetic acid (1 mL) was added, and the resulting yellow solution was stirred for 5 minutes under ice-cooling and then at room temperature (21 ° C.) for 2 hours. The reaction solution was dried under reduced pressure, 1,2-dichloroethane (8 mL) was added to the residue, and the mixture was again dried under reduced pressure to obtain a yellow solid (41 mg), which was divided into dimethylformamide (1 mL), Cetonitrile (MeCN, 7 mL) and water (4 mL) were added and dissolved, and the resulting solution (12 mL) was separated and purified by preparative liquid chromatography (4 mL, 3 times). The solvents were distilled off under reduced pressure, and 10- (4-((S) 4-amino-4-carboxybutanamido) benzyloxy) -7-ethylcamptothecin 2 trifluoroacetate (Examples 1, 21. NMR [400 MHz, DMSO-d6, TMS] ppm: 0.874 (3H, t, J = 7.3 Hz), 1.272 (3H, t, J = 7.6 Hz), 1. 864 (2H, m), 2.082 (2H, m), 2.566 (overlapped), 3.185 (2H, q, J = 7.3 Hz), 3.980 (overlapped), 5.299, 5 .3 9, 5.429 (each 2H, s), 6.50 (1H, b), 7.269 (1H, s), 7.47-7.65 (6H, m), 8.09 (1H, d) , J = 9.0 Hz), 8.262 (2H, d, J = 4.1 Hz), 10.105 (1H, s), 13.9 (1H, b) LC / MS Retention time: 4.1 Min; m / z (ESI, POS): 628 [M + 2H] ⁺

Reference Example 1: Synthesis of 10-(((((4-((S) 4-amino-4-carboxybutanamido) benzyl) oxy) carbonyl) oxy) -7-ethylcamptothecin [Scheme 2]

Reference Example 1-1: (S) -t-butyl 2-((t-butoxycarbonyl) amino) -5-((4-((((4-nitrophenoxy) carbonyl) oxy) methyl) phenyl) amino) Synthesis of -5-oxopentanoate 5-((4-hydroxymethyl) benzylamino) -1- (t-butyl) N- (t-butoxycarbonyl) -L-glutamate described in Example 1-1 0.10 g) and pyridine (0.0494 mL) in dry tetrahydrofuran (10 mL) were added dropwise a solution of 4-nitrophenyl chloroformate (0.0987 g) in dry tetrahydrofuran (10 mL) at 0 ° C. at room temperature. For 2 hours. Aqueous citric acid solution was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, the residue was purified by silica gel column chromatography, and (S) -t-butyl 2-((t-butoxycarbonyl) amino) -5-((4-((((4- Nitrophenoxy) carbonyl) oxy) methyl) phenyl) amino) -5-oxopentanoate (0.038 g) was obtained.

NMR [400 MHz, CDCl ₃ , TMS] ppm: 1.46 (9H, s), 1.48 (9H, s), 1.79-1.90 (1H, m), 2.6-2.28 ( 1H, m), 2.45 (2H, t), 4.20-4.28 (1H, m), 5.26 (2H, s), 5.38-5.40 (1H, m), 7 .37 (2H, d), 7.41 (2H, d), 7.69 (2H, d), 8.27 (2H, d), 9.15 (1H, brs).
LC / MS retention time: 7.1 minutes; m / z (ESI, POS): 596 [M + Na] ⁺

Reference Example 1-2: (((4-((S) -5- (t-butoxy) -4-((t-butoxycarbonyl) amino) -5-oxopentanamido) benzyl) oxy) carbonyl)- Synthesis of 10-oxy-7-ethylcamptothecin (S) -t-butyl 2-((t-butoxycarbonyl) amino) -5-((4-((((4-nitrophenoxy) carbonyl) oxy) methyl) Phenyl) amino) -5-oxopentanoate (0.125 g) was dissolved in N, N-dimethylformamide (8 mL), 7-ethyl-10-hydroxycamptothecin (EHC 0.0855 g) was added, and diisopropyl Ethylamine (0.37 mL) was added. After stirring at room temperature for 18 hours, water was added and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure to give a crude product (((4-((S) -5- (t-butoxy) -4-((t-butoxycarbonyl) Amino) -5-oxopentanamide) benzyl) oxy) carbonyl) -10-oxy-7-ethylcamptothecin (0.227 g) was obtained.
LC / MS retention time: 6.6 min; m / z (ESI, POS): 827 [M + H] ⁺

Reference Example 1-3: Synthesis of 10-((((4-((S) 4-amino-4-carboxybutanamido) benzyl) oxy) carbonyl) oxy) -7-ethylcamptothecin Crude (((((4 -((S) -5- (t-butoxy) -4-((t-butoxycarbonyl) amino) -5-oxopentanamido) benzyl) oxy) carbonyl) -10-oxy-7-ethylcamptothecin (0 0.02 g) was dissolved in 4N hydrochloric acid dioxane solution (2.0 mL) and stirred for 30 minutes, the solvent was distilled off, water (4 mL) was added, and the mixed solution was purified by preparative HPLC (((4 -((S) 4-Amino-4-carboxybutanamide) benzyl) oxy) carbonyl) -10-oxy-7-ethylcamptothecin (Reference Example 1, 0.0015 g) was obtained.

NMR [400 MHz, DMSO-d ₆ , TMS] ppm: 0.88 (3H, t), 1.29 (3H, t), 1.84-2.11 (6H, m), 3.16-3. 29 (3H, m), 5.28 (2H, s), 5.36 (2H, s), 5.45 (2H, s), 6.55 (1H, s), 7.34 (1H, s) ), 7.44 (2H, d), 7.65 (2H, d), 7.78-7.80 (1H, m), 8.18-8.25 (2H, m), 10.21 ( 1H, brs).
LC / MS retention time: 3.9 min; m / z (ESI, POS): 671 [M + H] ⁺

Reference Example 2: Synthesis of (((4-((S) 4-amino-4-carboxybutanamido) benzyl) oxy) carbonyl) camptothecin [Scheme 3]

Reference Example 2-1: ((((4-((S) -5- (t-butoxy) -4-((t-butoxycarbonyl) amino) -5-oxopentanamido) benzyl) oxy) carbonyl) camptothecin A solution of dimethylaminopyridine (0.0561 g) in dichloromethane (2 mL) was slowly added dropwise to a suspension of camptothecin (0.050 g) and triphosgene (0.0158 g) in dry dichloromethane (6 mL). After stirring for 30 minutes, 5-((4-hydroxymethyl) benzylamino) -1- (t-butyl) N- (t-butoxycarbonyl) -L-glutamate (0.059 g) described in Example 1-1. And stirred at room temperature for 18 hours. 1N Hydrochloric acid (50 mL) was added, and the mixture was extracted twice with dichloromethane. The organic layer was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure to give (((4-((S) -5- (t-butoxy) -4-((t-butoxycarbonyl) amino) -5-oxopentanamide) as a crude product). Benzyl) oxy) carbonyl) camptothecin (0.108 g) was obtained.
LC / MS retention time: 6.8 minutes; m / z (ESI, POS): 805 [M + Na] ⁺

Reference Example 2-2: Synthesis of ((((4-((S) 4-amino-4-carboxybutanamido) benzyl) oxy) carbonyl) camptothecin Crude (((((4-((S) -5- (t -Butoxy) -4-((t-butoxycarbonyl) amino) -5-oxopentanamide) benzyl) oxy) carbonyl) camptothecin (0.028 g) in 4N hydrochloric acid-ethyl acetate solution (3.0 mL) Dissolved at 0 ° C. and stirred for 3 hours. The solvent was distilled off, and the resulting residue was purified by preparative HPLC to give (((4-((S) 4-amino-4-carboxybutanamido) benzyl) oxy) carbonyl) camptothecin (Reference Example 2, 0). .0017g) was obtained.

NMR [400 MHz, DMSO-d ₆ , TMS] ppm: 0.90 (3H, t), 1.90-1.97 (3H, m), 2.13-2.21 (3H, m), 3. 16-3.28 (1H, m), 5.09 (2H, q), 5.33 (2H, s), 5.52 (2H, s), 7.02 (1H, s), 7.27 (2H, d), 7.53 (2H, d), 7.74 (1H, t), 7.89 (1H, t), 8.15-8.21 (2H, m), 8.73 ( 1H, s), 10.29 (1H, brs).
LC / MS retention time: 3.9 min; m / z (ESI, POS): 627 [M + H] ⁺

Test Example 1: Stability test in phosphate buffered saline (PBS) PBS (0.450 mL) was added to the EHC prodrug 0.864 mg / mL DMSO solution (0.050 mL) of Example 1 and 37 The reaction was carried out at ° C. When the residual ratio of the prodrug was determined by HPLC analysis of the reaction solution, 90% of the prodrug remained after 24 hours.
PBS (0.220 mL) was added to a 60 mg / mL DMSO solution (0.005 mL) of the EHC prodrug of Reference Example 1 to prepare an EHC prodrug solution. PBS (0.100 mL) was added to the EHC prodrug solution (0.100 mL) and reacted at 37 ° C. When the residual ratio of the prodrug was determined by HPLC analysis of the reaction solution, the residual ratio of the prodrug was 52% after 3.5 hours.
A camptothecin prodrug solution was prepared by adding PBS (0.294 mL) to the 6.3 mg / mL DMSO solution (0.073 mL) of Reference Example 2. PBS (0.117 mL) was added to the camptothecin prodrug solution (0.117 mL) and reacted at 37 ° C. The prodrug remaining rate was determined by HPLC analysis of the reaction solution, and after 3 hours, the prodrug remaining rate was 74%.
Therefore, it was shown that the EHC prodrug of Example 1 according to the present invention is chemically stable in PBS as compared with Reference Examples 1 and 2.

Test Example 2: Stability test in mouse plasma Mouse plasma (0.180 mL) was added to the EHC prodrug 0.856 mg / mL DMSO solution (0.020 mL) of Example 1 and reacted at 37 ° C.
When the residual ratio of the prodrug was determined by HPLC analysis of the reaction solution, 94% of the prodrug remained after 24 hours.
Therefore, it was shown that the EHC prodrug of Example 1 according to the present invention is chemically stable even in mouse plasma.

Test Example 3: γ-Glutamyltranspeptidase (GGT) enzyme recognition test The EHC prodrug of Example 1 was dissolved in DMSO to prepare a 0.34 mg / mL solution. PBS (0.300 mL) was added to the EHC prodrug solution (0.300 mL) to prepare an EHC prodrug solution (2).
γ-Glutamyltranspeptidase (GGT Sigma-Aldrich) was dissolved in PBS to prepare a 0.227 mg / mL solution.
A GGT solution (0.250 mL) was added to the EHC prodrug solution (2) (0.250 mL) and reacted at 37 ° C.
The reaction solution was subjected to HPLC analysis to determine the prodrug residual rate and EHC production rate.

  As a result of Test Example 3, the EHC prodrug of Example 1 produced a stoichiometric amount of EHC in 1 hour of reaction. This result shows that the compound of Example 1 is recognized by GGT, and can quickly cleave the γ-glutamyl aromatic amide linker via an ether bond to produce EHC as a physiologically active substance.

  From the results of Test Examples 1 and 2, it was shown that the EHC prodrug according to Example 1 has stable physical properties in a PBS solution and a solution in the presence of mouse plasma. On the other hand, Reference Example 1 and Reference Example 2, which are prodrugs of the carbonate ester type binding mode, had a result that the bond was rapidly cleaved with the PBS solution, and the solution stability could not be ensured. On the other hand, it was shown that the EHC prodrug according to Example 1 can rapidly release EHC having pharmacological activity in the presence of GGT. From these results, when preparing a prodrug of a drug having an aromatic hydroxyl group, it is stably present as a prodrug in an in vivo environment such as a solution for administration or in the bloodstream, and by enzyme recognition in the presence of GGT. In order to release a drug by a rapid dissociation reaction, it is necessary to interpose a linker segment between the γ-glutamic acid binding site and the drug in an appropriate manner. By adopting, it became clear that superior solution stability and GGT recognition ability can be exhibited.

Test Example 4: Cell growth inhibitory activity test OS-RC-2 cells (RIKEN BioResource Center) known to have high GGT activity and SK-OV-3 cells (American Type) known to have low GGT activity (Culture Collection) was used to evaluate the cell growth inhibitory activity of the EHC prodrug according to Example 1.
96-well plates were seeded with 4,000 cells / well of OS-RC-2 cells with high GGT activity and SK-OV-3 cells with low GGT activity, respectively, and cultured at 37 ° C. under 5% CO ₂ for 1 day. Thereafter, the EHC prodrug according to Example 1 was added at a final concentration of 0.0001 to 1 μM. After culturing for 6 hours, the well was washed and further cultured for 3 days. After culture, the cells were fixed with methanol and stained with methylene blue dye. After dyeing, the dye was extracted with 0.3% hydrochloric acid and the absorbance at 660 nm was measured. About the obtained light absorbency, cell growth inhibitory activity was evaluated by the decreasing rate with respect to the light absorbency measured from the cell which does not add a compound.
By the same operation, EHC which is an active ingredient of Example 1 was used as it was, and the cell growth inhibitory activity was evaluated from the absorbance.
The results are shown in FIG. 1 (OS-RC-2 cells with high GGT activity) and FIG. 2 (SK-OV-3 cells with low GGT activity).

Test Example 5: Confirmation test of GGT dependence of prodrug GGT activity dependence on cell growth inhibitory activity of EHC prodrug according to Example 1 using OS-RC-2 cells known to have high GGT activity Sex was evaluated. After seeding OS-RC-2 cells in the same manner as in Test Example 4 and culturing for one day, GGsTop (Wako Pure Chemical Industries, Ltd.), a GGT inhibitor, was added at a final concentration of 10 μM and cultured for 1 hour. Thereafter, the EHC prodrug of Example 1 was added at a final concentration of 0.0001-1 μM. After culturing for 6 hours, the inside of the well was washed, and further cultured for 3 days, and then cell growth inhibitory activity was evaluated in the same manner as in Test Example 4. By the same operation, EHC which is an active ingredient of Example 1 was used as it was, and the cell growth inhibitory activity was evaluated from the absorbance. The results are shown in FIG.

As a result of Test Example 4, in the OS-RC-2 cells known to have high GGT activity, when 0.01 μM EHC was added, the cell growth inhibitory activity was 26%. In addition, when 0.01 μM of the EHC prodrug according to Example 1 was added, the cell growth inhibitory activity was 25%.
On the other hand, in the SK-OV-3 cells known to have low GGT activity, when 0.01 μM EHC was added, the cell growth inhibitory activity was 27.7%. On the other hand, when 0.01 μM of the EHC prodrug according to Example 1 was added, the cell growth inhibitory activity was 1.3%.
Comparing the results of cell growth inhibitory activity in cells with high GGT activity and cells with low GGT activity, the EHC prodrug of Example 1 according to the present invention releases EHC by GGT and exhibits cell growth inhibitory activity. In the absence of GGT, it was revealed that the cell growth inhibitory activity was low and the cell-killing property was hardly exhibited. From this, it was shown that the EHC prodrug according to the present invention can exhibit the cell growth inhibitory activity depending on the GGT activity.
In addition, as a result of Test Example 5, in OS-RC-2 cells having high GGT activity, the GGT inhibitor did not affect the cell growth inhibitory activity of EHC, but inhibited the activity of EHC prodrug. From this, it was shown that the EHC prodrug based on this invention is a physical property which exhibits the cell growth inhibitory activity depending on GGT activity.

Test Example 6: Measurement of tissue drug concentration A mouse renal cell carcinoma OS-RC-2 tumor subcultured subcutaneously in mice was made into blocks of about 2 mm square and transplanted subcutaneously into mice using a trocar. On day 17 after tumor implantation, the EHC prodrug of Example 1 according to the present invention was suspended in DMSO / 5% glucose aqueous solution = 1: 9 solution and administered into the tail vein at 20 mg / kg as EHC conversion. As a control drug, irinotecan hydrochloride (CPT-11), which is a prodrug of EHC, was dissolved in a 5% glucose aqueous solution and administered into the tail vein at 20 mg / kg in terms of EHC.
At 5 minutes, 1, 3, and 6 hours after administration, plasma, liver, bone marrow, and tumor tissues were collected under ether anesthesia. The drug concentrations of EHC, the unchanged form of Example 6 and the unchanged form of CPT-11 in each tissue were quantitatively analyzed by the LC-MS / MS method under the following measuring instruments and conditions.
Model: ABSciex API4000
Shimadzu LC-20AD
Column: Waters XBridge, C ₁₈ , 2.1 mm × 50 mm
Mobile phase A: formic acid / water (1/1000)
Mobile phase B: formic acid / acetonitrile (1/1000)
Ionization method: Electrospray ionization (positive ion)
Detection method: Multiple reaction monitoring

From the EHC in the tissue at each time point, the prodrug according to Example 1 and the CPT-11 concentration, AUC _0-6 hr of each component until 6 hours after the start of administration was calculated. As the prodrug activation rate of Example 1 and CPT-11 in each tissue, the AUC _{0-6 hr} ratio of the active substance to the unchanged form (AUC _EHC / AUC _{Example 6} and AUC _EHC / AUC _CPT-11 ) was determined. It was. The results are summarized in Table 1.

[Table 1]

Test Example 7: Anti-tumor effect test In the same manner as in Test Example 6, the mice were transplanted with mouse renal cell carcinoma OS-RC-2, and the EHC prodrug 20 of Example 6 according to the present invention on the 17th day after tumor transplantation, 40 and 80 mg / kg were administered into the tail vein three times every 4 days. After administration, the long diameter (Lmm) and short diameter (Wmm) of the tumor were measured at intervals of 2 to 3 days using a caliber, and the tumor volume was calculated by (L × W ² ) / 2. Tumor volume change rate / volume change rate of the untreated group was determined and shown in Table 2.

[Table 2]

As a result of Test Example 6, the activation rate of the EHC prodrug of Example 1 was about 9.4 times as high as that of CPT-11 in plasma, liver and bone marrow with low GGT expression. On the other hand, among the tumors expressing GGT, the AUC _EHC / AUC _{Example 6} ratio of Example 1 was 61.8 times as high as that of the CPT-11 administration group. From this, it was shown that the EHC prodrug of Example 6 according to the present invention is activated in a GGT-dependent manner in a tissue and has a physical property capable of selectively releasing EHC in a tissue expressing GGT.
In addition, as a result of Test Example 7, the EHC prodrug of Example 1 showed a dose-dependent antitumor effect, showed a remarkable tumor shrinking effect in the 80 mg / kg administration group, and exhibited an excellent antitumor effect. It was shown that this is a physical property.

Claims (12)

General formula (1)

[Wherein, R ₁ and R ₂ are each independently selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group and an optionally substituted alkoxycarbonyl group. R ₃ represents a hydrogen atom or an optionally substituted alkyl group, and A ₁ and A ₂ are selected from the group consisting of C—R ₆ , C—R ₇ and a nitrogen atom. And R ₆ is selected from the group consisting of a hydrogen atom, a halogen atom, a nitro group, a hydroxyl group, an optionally substituted alkyl group and an optionally substituted alkoxy group. Represents one or more groups, and R ₇ represents the following general formula (2)

[Wherein, R ₄ and R ₅ each independently represent a hydrogen atom or an alkyl group which may have a substituent, and X represents a binding residue of a physiologically active substance having an aromatic hydroxyl group. Here, _one of the A ₁ and the A ₂ is the C—R ₇ , the other is the C—R ₆ or a nitrogen atom, and B ₁ , B ₂ and B ₃ are Each independently represents the C—R ₆ or nitrogen atom. Or a pharmacologically acceptable salt thereof.   The glutamic acid according to claim 1, wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is one selected from the group consisting of 7-ethyl-10-hydroxycamptothecin, nogitecan and derivatives thereof. Derivatives or pharmacologically acceptable salts thereof.   2. The glutamic acid derivative according to claim 1, wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is one selected from the group consisting of etoposide, teniposide and derivatives thereof. Acceptable salt.   The glutamic acid derivative or a pharmacologically acceptable salt thereof according to claim 1, wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is one selected from the group consisting of raloxifene and derivatives thereof. Salt.   2. The glutamic acid derivative according to claim 1, wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is one selected from the group consisting of goserelin, leuprin and derivatives thereof. Acceptable salt.   The physiologically active substance in the binding residue of the physiologically active substance represented by X is one selected from the group consisting of 2-butylamino-2-demethoxyhypocrellin and derivatives thereof. Or a pharmacologically acceptable salt thereof.   2. The glutamic acid derivative according to claim 1, wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is one selected from the group consisting of combretastatin and derivatives thereof. Acceptable salt.   The glutamic acid derivative or pharmacology thereof according to claim 1, wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is one selected from the group consisting of ganetespib, macbecin, radicicol and derivatives thereof. Acceptable salt.   The physiologically active substance in the binding residue of the physiologically active substance represented by X is one selected from the group consisting of doxorubicin, daunorubicin, epirubicin, pirarubicin, idarubicin, mitoxantrone, amrubicin and derivatives thereof. 2. The glutamic acid derivative according to 1, or a pharmacologically acceptable salt thereof. In the general formula _{(1), R 1, R} 2 and R ₃ is glutamic acid derivative or a pharmacologically acceptable salt thereof according to any one of claims 1 to 9 is a hydrogen atom.   The pharmaceutical which contains the glutamic acid derivative of any one of Claims 1-10, or its pharmacologically acceptable salt as an active ingredient. The medicament according to claim 11, which is an anticancer agent.

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