WO2015178265A1

WO2015178265A1 - Novel glutamic acid derivative and use thereof

Info

Publication number: WO2015178265A1
Application number: PCT/JP2015/063736
Authority: WO
Inventors: 米田　靖; 啓子関根; 節子新妻
Original assignee: 日本化薬株式会社
Priority date: 2014-05-23
Filing date: 2015-05-13
Publication date: 2015-11-26
Also published as: TW201625313A

Abstract

It was discovered that interposing a suitable aromatic amidation linker in making a prodrug to serve as a γ-glutamylation drug provides stability even in a close to physiological environment, recognition by GGT, and very rapid release of the pharmacologically active drug, and the invention was achieved. Specifically, a drug represented by general formula (1) to which a γ-glutamyl aromatic amide has been bonded releases the pharmacologically active drug rapidly in tissues expressing a high level of GGT and can become a drug having a high therapeutic effect.

Description

Novel glutamic acid derivatives and uses thereof

The present invention relates to a novel glutamic acid derivative that is enzyme-selectively activated at a target site and its use. 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 change to active drugs after being metabolized in vivo. 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 into an active compound at a target site, a method using an enzyme highly expressed in a target tissue is considered. As a method for producing a prodrug of a drug using an enzyme-specific reaction at a target site, a method in which a self-cleavable linker is interposed between the enzyme recognition site and the drug is known (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, a drug can be released by self-cleaving the linker part.

γ-Glutamyltranspeptidase (GGT) is an enzyme that controls 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 γ-glutamylizing an anticancer agent. 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.

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 pharmaceutical 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.

In order to solve the above-mentioned problems, the present inventors have conducted intensive 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 alkyl group which may have a substituent, 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 optionally substituted alkyl group, and L represents a bond selected from the group consisting of an oxygen atom, an oxycarbonyl group and a bond. X represents a binding residue of a physiologically active substance having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group, an amino group, and a carboxy group;
(I) When X is a binding residue of a physiologically active substance having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group and an amino group, the L is an oxycarbonyl group,
(Ii) when X is a binding residue of a physiologically active substance having a carboxy group, L is an oxygen atom;
(Iii) When X is a binding residue of a physiologically active substance having an aromatic hydroxyl group, L is a bond or oxycarbonyl. ],
Here, _one of the A ₁ and the A ₂ is the CR ₇ and the other is the CR ₆ or a nitrogen atom, and B ₁ , B ₂ and B ₃ are each independently C—R ₆ or a nitrogen atom. And the pharmacologically acceptable salt thereof were found to be useful as GGT-recognizing prodrugs, and the present invention was completed.

That is, the gist of the present application is the invention shown in the following [1] to [10].
[1] General formula (1)

[Wherein, R ₄ and R ₅ each independently represent a hydrogen atom or an optionally substituted alkyl group, and L represents a bond selected from the group consisting of an oxygen atom, an oxycarbonyl group and a bond. X represents a binding residue of a physiologically active substance having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group, an amino group, and a carboxy group;
(I) When X is a binding residue of a physiologically active substance having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group and an amino group, the L is an oxycarbonyl group,
(Ii) when X is a binding residue of a physiologically active substance having a carboxy group, L is an oxygen atom;
(Iii) When X is a binding residue of a physiologically active substance having an aromatic hydroxyl group, L is a bond or an oxycarbonyl group. ],
Here, _one of the A ₁ and the A ₂ is the CR ₇ and the other is the CR ₆ or a nitrogen atom, and B ₁ , B ₂ and B ₃ are each independently C—R ₆ or a nitrogen atom. Or a pharmacologically acceptable salt thereof.
[2] R ₇ represents the following general formula (3)

[Wherein, R ₄ and R ₅ are as defined above, and X is a binding of a physiologically active substance having at least one functional group selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group and an amino group. Residue. ] The glutamic acid derivative or its pharmacologically acceptable salt as described in [1] above.
[3] The glutamic acid derivative or the pharmacologically acceptable derivative thereof according to [1] or [2], wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is camptothecin and its derivatives salt.
[4] The above [1] or [2], wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is a physiologically active substance selected from the group consisting of doxorubicin, daunorubicin, epirubicin, pirarubicin and amrubicin. Or a pharmacologically acceptable salt thereof.
[5] The physiologically active substance in the binding residue of the physiologically active substance represented by X is gemcitabine, ethynylcytidine, cytarabine and CNDAC (2′-cyano-2′-deoxy-1-β-D-arabinofurano The glutamic acid derivative or a pharmaceutically acceptable salt thereof according to [1] or [2], which is a physiologically active substance selected from the group consisting of silcytosine).
[6] R ₇ represents the following general formula (4)

[Wherein, R ₄ and R ₅ are as defined above, and X is a binding residue of a physiologically active substance having an aromatic hydroxyl group. ] The glutamic acid derivative or its pharmacologically acceptable salt as described in [1] above.
[7] 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 7-ethyl-10-hydroxycamptothecin, nogitecan and derivatives thereof Or the glutamic acid derivative or its pharmacologically acceptable salt as described in [6].
[8] 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 [7] or a pharmacologically acceptable salt thereof salt.
[9] A medicament comprising the glutamic acid derivative according to any one of [1] to [8] or a pharmacologically acceptable salt thereof as an active ingredient.
[10] The medicament according to [9], 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. It is known that GGT is 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 the result of the cell growth suppression test of Example 1 with respect to OS-RC-2 cells. FIG. 3 shows the results of the cell growth inhibition test of Example 1 for SK-OV-3 cells. FIG. FIG. 4 shows the results of the cell growth inhibition test of Example 1 for OS-RC-2 cells in the presence of a GGT inhibitor. FIG. It is the result of the cell growth inhibitory test of the compound of Example 6 on OS-RC-2 cells. It is the result of the cell growth suppression test of the compound of Example 6 on SK-OV-3 cells. FIG. 6 shows the results of a cell growth inhibition test of the compound of Example 6 on OS-RC-2 cells in the presence of a GGT inhibitor. FIG.

The present invention relates to a glutamic acid derivative obtained by binding 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 medicine. Details of the present invention will be described below.

The glutamic acid derivative of the present invention or a pharmacologically acceptable salt thereof is represented by the following general formula (1)

[Wherein, R ₄ and R ₅ each independently represent a hydrogen atom or an optionally substituted alkyl group, and L represents a bond selected from the group consisting of an oxygen atom, an oxycarbonyl group and a bond. X represents a binding residue of a physiologically active substance having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group, an amino group, and a carboxy group;
(I) When X is a binding residue of a physiologically active substance having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group and an amino group, the L is an oxycarbonyl group,
(Ii) when X is a binding residue of a physiologically active substance having a carboxy group, L is an oxygen atom;
(Iii) When X is a binding residue of a physiologically active substance having an aromatic hydroxyl group, L is a bond or an oxycarbonyl group. Wherein either one of the A ₁ and the A ₂ is the CR ₇ and the other is the CR ₆ or nitrogen atom, and B ₁ , B ₂ and B ₃ Each independently represents the aforementioned C—R ₆ or a 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 a 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.

In the alkoxycarbonyl group which may have a substituent, the alkoxycarbonyl group means 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 which the alkyl group and 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 the benzyloxycarbonyl group (Cbz group), t-butoxycarbonyl group (Boc group), 9-fluorenylmethoxycarbonyl group (Fmoc group), 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 a 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. Further, examples thereof include a benzyl group and a 9-fluorenylmethyl group having an appropriate aromatic substituent. 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 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 heterocycle 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 substituent which may have is the same as the substituent in R ₁ and R ₂ described above.

R ₇ is a group represented by the general formula (2). In the general formula (2), L represents a linking group selected from the group consisting of an oxygen atom, an oxycarbonyl group and a bond, and X represents an aliphatic hydroxyl group, an aromatic hydroxyl group, an amino group and a carboxy group. A binding residue of a physiologically active substance having one or more functional groups selected from the group;
(I) When X is a binding residue of a physiologically active substance having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group and an amino group, the L is an oxycarbonyl group,
(Ii) when X is a binding residue of a physiologically active substance having a carboxy group, L is an oxygen atom;
(Iii) When X is a binding residue of a physiologically active substance having an aromatic hydroxyl group, L is a bond.
Here, when the X group is a binding residue of a physiologically active substance having an aliphatic hydroxyl group or an aromatic hydroxyl group, the hydroxyl group is a binding residue in the form of a carbonate bond with an oxycarbonyl group. . On the other hand, when the X group is a binding residue of a physiologically active substance having an amino group, the amino group is a binding residue in the form of a urethane bond with an oxycarbonyl group.
In addition, when the X group is a binding residue in a physiologically active substance having a carboxy group, a residual bond in the form of an ester bond with a carbonyl group derived from the physiologically active substance via an oxygen atom as the binding group L. It is a group.
Further, when the X group is a binding residue of a physiologically active substance having an aromatic hydroxyl group, the X group is a binding residue ether-bonded via an oxygen atom of the aromatic hydroxyl group of the physiologically active substance.

In one embodiment of the present invention, R ₇ is represented by the following general formula (3):

[Wherein, R ₄ and R ₅ are as defined above, and X is a binding of a physiologically active substance having at least one functional group selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group and an amino group. Residue. ].

In another embodiment of the present invention, R ₇ is represented by the following general formula (4):

[Wherein, R ₄ and R ₅ are as defined above, and X is a binding residue of a physiologically active substance having an aromatic hydroxyl group. ].

The physiologically active substance in X is a chemical substance that exhibits a pharmacological function when administered in vivo, and is selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group, an amino group, and a carboxy group Any compound having the above functional group can be used without any particular limitation.
In the general formula (2), when X is a binding residue of a physiologically active substance having a fatty attribute hydroxyl group and / or amino group, the L uses an oxycarbonyl group as a linking group, and is a carbonate bond and / or a urethane bond. The resulting compound is When X is a binding residue of a physiologically active substance having a carboxy group, L becomes an oxygen atom and becomes a compound ester-bonded with a carbonyl group derived from the physiologically active substance. When X is a binding residue of a physiologically active substance having an aromatic hydroxyl group, L is a bond or an oxycarbonyl group, and becomes a compound having an ether bond or a carbonate bond.

The aliphatic hydroxyl group may be any substituent of primary hydroxyl group, secondary hydroxyl group or tertiary hydroxyl group. The amino group may be any substituent of a primary amino group, a secondary amino group, or a tertiary amino group.

The physiologically active substance in X may be a physiologically active substance in which an aliphatic hydroxyl group and / or an aromatic hydroxyl group and an amino group coexist. In the case of a compound in which an aliphatic hydroxyl group and / or an aromatic hydroxyl group and an amino group coexist, the X group is generally considered to be a binding residue due to an amino group. In consideration of the above, it may be a residue bonded by any active functional group of the aliphatic hydroxyl group and / or aromatic hydroxyl group and the amino group, and may depend on the aliphatic hydroxyl group and / or aromatic hydroxyl group. It may be a mixture of a binding residue and a binding residue due to the amino group.

Further, the physiologically active substance in X may be a physiologically active substance in which an aliphatic hydroxyl group and / or an aromatic hydroxyl group and / or an amino group and a carboxy group coexist. When using a physiologically active substance in which an aliphatic hydroxyl group and / or an aromatic hydroxyl group and / or an amino group and a carboxy group coexist, the binding mode can be appropriately selected depending on the reaction conditions.

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 (2) is an anticancer having one or more functional groups selected from the group consisting of aliphatic hydroxyl groups, aromatic hydroxyl groups, amino groups, and carboxy groups. It is preferable that it is an agent.

As an anticancer agent suitable as a physiologically active substance represented by X in the general formula (2), a sirolimus anticancer agent, an anthracycline anticancer agent, a cytidine anticancer agent, a tyrosine kinase inhibitor, Examples thereof include DNA topoisomerase inhibitors, hormone therapy agents, photodynamic therapy agents, microtubule polymerization inhibitors, Hsp90 inhibitors, and other anticancer agents having cell division inhibitory activity.

Examples of the sirolimus anticancer agent include everolimus, temsirolimus, tacrolimus, rapamycin and the like.

Examples of the cytidine anticancer agent include ethynyl cytidine, CNDAC (2'-cyano-2'-deoxy-1-β-D-arabinofuranosylcytosine), gemcitabine, cytarabine and the like.

The tyrosine kinase inhibitor is a tyrosine kinase inhibitor having a hydroxyl group and / or an amino group, and examples thereof include crizotinib and dasatinib.

Examples of the DNA topoisomerase inhibitor include camptothecin-type anticancer agents which are DNA topoisomerase type I inhibitors such as camptothecin, 7-ethyl-10-hydroxycamptothecin, irinotecan, nogithecan, 9-aminocamptothecin, 9-nitrocamptothecin Is mentioned. 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 photochemotherapy agent include 2-butylamino-2-demethoxyhypocreline.

Examples of the microtubule polymerization inhibitor include taxane anticancer agents such as paclitaxel and docetaxel, combretastatin and derivatives thereof, podophyllotoxin, eribulin, and auristatin.

Examples of the Hsp90 inhibitor include ganetespib, macbecin, radicicol and the like.

In addition, in X of the general formula (2), suitable anticancer agents as physiologically active substances having a carboxy group include, for example, methotrexate, pemetrexed, DMXAA (5,6-dimethylxanthenone-4-acetic acid), bexarotene , Tamibarotene and the like.

Further, in X of the general formula (2), the physiologically active substance having an amino group or a carboxy group may be physiologically active peptides. The physiologically active peptides having the amino group or carboxy group can be bonded using a terminal amino group or carboxy group.
Examples of the physiologically active peptides include ester derivatives such as bestatin and bestatin methyl ester, glutanide, ghrelin, ghrelin, tertomotide, PR1, octreotide, lanreotide, and lanreotide. Examples include pasireotide.

X in the general formula (2) is preferably a binding residue of a physiologically active substance having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group and an amino group. In this case, an oxycarbonyl group is used as the bonding group L in the general formula (2). That is, in this case, R ₇ is a substituent represented by the general formula (3).
Examples of the physiologically active substance having one or more functional groups selected from the group consisting of the aliphatic hydroxyl group, aromatic hydroxyl group and amino group include camptothecin, 7-ethyl-10-hydroxycamptothecin, irinotecan, nogitecan, 9 -A camptothecin derivative such as aminocamptothecin or 9-nitrocamptothecin is preferred. These have a lactone ring tertiary hydroxyl group, an aromatic hydroxyl group, and an amino group, and these substituents form a carbonate bond and / or a urethane bond with the oxycarbonyl group that is the linking group.
Further, as a physiologically active substance having one or more functional groups selected from the group consisting of the aliphatic hydroxyl group, aromatic hydroxyl group and amino group, doxorubicin, daunorubicin, epirubicin, pirarubicin, idarubicin, mitoxantrone, amrubicin Anthracycline anticancer agents such as these are also preferable. More preferred are doxorubicin, daunorubicin, epirubicin, pirarubicin, and amrubicin. These have a hydroxyl group and / or an amino group, and these substituents form a carbonate bond and / or a urethane bond with the oxycarbonyl group, which is the linking group.
Furthermore, gemcitabine, ethynylcytidine, cytarabine, CNDAC (2′-cyano-2) are physiologically active substances having one or more functional groups selected from the group consisting of the aliphatic hydroxyl group, aromatic hydroxyl group and amino group. A cytidine anticancer agent such as' -deoxy-1-β-D-arabinofuranosylcytosine) is preferred. These have a hydroxyl group and / or an amino group of a cytidine base, and these substituents form a carbonate bond and / or a urethane bond with the oxycarbonyl group which is the above-mentioned bonding group.

As another embodiment, X in the general formula (2) is preferably a binding residue of a physiologically active substance having an aromatic hydroxyl group. In this case, a bond is used as the bonding group L in the general formula (2). That is, in this case, R ₇ is a substituent represented by the general formula (4).
As the physiologically active substance having an aromatic hydroxyl group, 7-ethyl-10-hydroxycamptothecin or nogitecan is preferably used, and the X is a bond ether-bonded with the 10-position hydroxyl group of 7-ethyl-10-hydroxycamptothecin or nogitecan It is preferably a residue.

The present invention is characterized in that _one of A ₁ and A ₂ is CR ₇ . That is, one aspect of the present invention is a glutamic acid derivative represented by the following general formula (5) 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 , L and X are as defined above.

Another aspect of the present invention is a glutamic acid derivative represented by the following general formula (6) 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 , L 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, in order to be recognized by GGT, the γ-glutamic acid binding partial structure needs to be a free amino acid structure. 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.

The glutamic acid derivative represented by the general formula (1) of the present invention is a physiologically active substance in which A ₂ is C—R ₇ and X has an aliphatic hydroxyl group and / or an aromatic hydroxyl group and / or an amino group When L is an oxycarbonyl group, for example, it can be produced as follows.
[Scheme (I)]

In the scheme (I), R ₁ to R ₃ , X, A ₁ and B ₁ to B ₃ have the same meanings as described above. Here, R ₁ and / or R ₂ is an amino-protecting group, and R ₃ is a carboxylic acid-protecting group. LG represents a leaving group such as a halogen atom, a p-nitrophenoxy group or a hydroxysuccinimide 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 with an amino group and an α-carboxy group 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 the reaction is performed 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 a carbodiimide condensing agent, an imidazole dehydrating condensing agent, a triazine condensing agent, a phosphonium dehydrating condensing agent, a uronium condensing agent, diphenylphosphoric acid azide (DPPA), BOP reagent, 4- (4, 6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride (DMT-MM) and the like can be used. If necessary, an activator such as 1-hydroxybenzotriazole can coexist.

[Step B]: A step of synthesizing the carbonyl derivative represented by the general formula (A-2) from the γ-glutamic acid amide derivative represented by the general formula (A-1). This step can be performed, for example, by reacting p-nitrophenyl chloroformate in the presence of pyridine in a solvent such as tetrahydrofuran at a temperature of −30 ° C. to 150 ° C., preferably −10 ° C. to 30 ° C.

[Step C]: A step of synthesizing the physiologically active substance-binding derivative represented by the general formula (A-3) from the carbonyl derivative represented by the general formula (A-2). In this step, for example, a physiologically active substance having an aliphatic hydroxyl group and / or an aromatic hydroxyl group and / or an amino group is treated in the presence of diisopropylethylamine in a solvent such as N, N-dimethylformamide at 0 ° C. to 150 ° C. It can be carried out by reacting at a temperature of preferably 0 ° C. to 30 ° C.

[Step D]: Process route for synthesizing the 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) It is. In this step, for example, a carbonylated physiologically active substance derivative represented by the general formula (I-1) is treated in the presence of N, N-dimethylaminopyridine in a solvent such as dichloromethane at 0 ° C. to 150 ° C., preferably 0 It can carry out by making it react at the temperature of 30 to 30 degreeC.
The carbonylated physiologically active substance derivative represented by the general formula (I-1) is a physiologically active substance X having an aliphatic hydroxyl group and / or an aromatic hydroxyl group and / or an amino group, for example, N, N-dimethyl It can be produced by reacting with triphosgene in the presence of aminopyridine in a solvent such as dichloromethane at a temperature of 0 ° C. to 150 ° C., preferably 0 ° C. to 30 ° C. Alternatively, for example, it can be produced by reacting p-nitrophenyl chloroformate in the presence of pyridine in a solvent such as dichloromethane at a temperature of 0 ° C. to 150 ° C., preferably 0 ° C. to 30 ° C.

[Step E]: A step of deprotecting the amino group and carboxyl 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. As the acid, inorganic acids such as hydrochloric acid and sulfuric acid, carboxylic acids such as acetic acid and trifluoroacetic acid, and the like can be used. In addition, any catalyst that is known to be capable of deprotecting a t-butoxycarbonyl group or t-butyl ester and that does not affect the portion other than the protecting group can be used without particular limitation.
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.

When X is a binding residue of a physiologically active substance having an amino group, a derivative represented by the general formula (A-3) can be produced, for example, as follows.
[Scheme (II)]

In the scheme (II), R ₁ to R ₃ , X, A ₁ , B ₁ to B ₃ are as defined above, R ₁ and / or R ₂ is a protecting group for an amino group, and R ₃ is a carboxyl group. Acid protecting group. Each step will be described below.

[Step F]: A step of synthesizing a physiologically active substance-binding derivative represented by the general formula (A-3) from the γ-glutamic acid amide derivative represented by the general formula (A-1). In this step, for example, an isocyanate derivative of a physiologically active substance X having an amino group is heated to 0 ° C. to 150 ° C., preferably 0 ° C. to 30 ° C. in a solvent such as dichloromethane in the presence of N, N-dimethylaminopyridine. It can implement by making it react.
The isocyanate derivative of the physiologically active substance X having an amino group is, for example, the physiologically active substance X having an amino group in a mixed solvent such as an aqueous potassium hydrogen carbonate solution and dichloromethane, preferably 0 ° C to 150 ° C, preferably 0 ° C to 30 ° C. It can manufacture by making it react with triphosgene at the temperature of.

When X is a physiologically active substance having a carboxyl group and L is an oxygen atom, the physiologically active substance-bound derivative represented by the general formula (A-3) can be produced, for example, as follows.
[Scheme (III)]
In the scheme (III), 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 carboxylic acid. Acid protecting group. Each step will be described below.

[Step G]: A step of esterifying the γ-glutamic acid amide derivative represented by the general formula (A-1) and a physiologically active substance having a carboxyl group by a condensation reaction. In this step, a general condensing agent can be used. For example, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is used as the condensing agent, and in a solvent such as N, N-dimethylformamide, The reaction can be carried out at a temperature of 0 ° C. to 150 ° C., preferably 0 ° C. to 30 ° C. Other condensing agents include carbodiimide condensing agents, imidazole dehydrating condensing agents, triazine condensing agents, phosphonium dehydrating condensing agents, uronium condensing agents, diphenyl phosphate azide (DPPA), BOP reagents, 4- (4, 6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride (DMT-MM) and the like can be used. If necessary, an activator such as 1-hydroxybenzotriazole, N, N-dimethyl-4-aminopyridine can be coexisted.

The glutamic acid derivative represented by the general formula (1) of the present invention in which A ₂ is C—R ₇ and X is an aromatic hydroxyl group and L is a bond is, for example, as follows: Can be manufactured.
[Scheme (IV)]

In scheme (IV), R ₁ to R ₃ , X, A ₁ , and B ₁ to B ₃ have the same meanings as described above. Here, R ₁ and / or R ₂ is an amino-protecting group, and R ₃ is a carboxylic 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 the derivative represented by the general formula (A-5) from the γ-glutamic acid amide derivative represented by the general formula (A-1). LG in the general formula (A-5) 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, methanesulfonyl chloride is -30 ° C to 150 ° C in a solvent such as dichloromethane in the presence of N, N-diisopropylethylamine, preferably -10 ° C. To 30 ° C. for the reaction.

[Step B]: A step of synthesizing the physiologically active substance-binding derivative represented by the general formula (A-6) from the derivative represented by the general formula (A-5). 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.

[Step C]: Process route for synthesizing the physiologically active substance-binding derivative represented by the general formula (A-6) in one step from the γ-glutamic acid amide derivative represented by the general formula (A-1) It is. In this step, for example, a 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 D]: 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-6).
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. As the acid, inorganic acids such as hydrochloric acid and sulfuric acid, carboxylic acids such as acetic acid and trifluoroacetic acid, and the like can be used. In addition, any catalyst that is known to be capable of deprotecting a t-butoxycarbonyl group or t-butyl ester and that does not affect the portion other than the protecting group can be used without particular limitation.
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. Such preparations can be 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 adults, 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 the case of parenteral administration 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.

Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

LC / MS measurement conditions for the detection and identification of the compounds of Examples 1 to 3 and Comparative Examples 1 and 2 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

LC / MS measurement conditions for the detection and identification of the compounds of Examples 4 to 6 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

Example 1
Synthesis of (((4-((S) -4-amino-4-carboxybutanamide) benzyl) oxy) carbonyl) doxorubicin [Scheme 1]

Example 1-1
(S)-(9H-fluoren-9-yl) methyl 2-(((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5-((4- (hydroxymethyl) phenyl) amino)- Synthesis of 5-oxopentanoate (S) -5-((9H-fluoren-9-yl) methoxy) -4-((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5 In a solution of oxopentanoic acid (0.182 g) and 4-aminobenzyl alcohol (0.049 g) in dry dichloromethane (5 mL), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (0. 103 g) was added and stirred at room temperature for 18 hours. 1N Hydrochloric acid was added, and the resulting crystals were filtered, and (S)-(9H-fluoren-9-yl) methyl 2-((((9H-fluoren-9-yl) methoxy) carbonyl) amino was obtained as a crude product. ) -5-((4- (hydroxymethyl) phenyl) amino) -5-oxopentanoate (0.173 g) was obtained.
NMR [400 MHz, DMSO-d ₆ , TMS] ppm: 1.81-1.90 (1H, m), 2.01-2.12 (1H, m), 2.40-2.46 (2H, m ), 4.17-4.43 (9H, m), 7.22-7.33 (6H, m), 7.37-7.44 (4H, m), 7.55 (2H, d), 7.68-7.76 (4H, m), 7.86-7.96 (5H, m), 9.89 (1H, brs).
LC / MS retention time: 7.3 minutes; m / z (ESI, POS): 653 [M + H] ⁺

Example 1-2
(S)-(9H-fluoren-9-yl) methyl 2-((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5-((4-((((4-nitrophenoxy) Synthesis of carbonyl) oxy) methyl) phenyl) amino) -5-oxopentanoate (S)-(9H-fluoren-9-yl) methyl 2-(((((9H-fluoren-9-yl) methoxy) carbonyl ) Amino) -5-((4- (hydroxymethyl) phenyl) amino) -5-oxopentanoate (0.145 g) and pyridine (0.0448 mL) in dry tetrahydrofuran (100 mL) at 0 ° C. A solution of 4-nitrophenyl chloroformate (0.089 g) in dry tetrahydrofuran (10 mL) was added dropwise, and the mixture was stirred at room temperature for 18 hours. Water was added and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and (S)-(9H-fluoren-9-yl) methyl 2-(((((9H-fluoren-9-yl) was obtained as a crude product. ) Methoxy) carbonyl) amino) -5-((4-((((4-nitrophenoxy) carbonyl) oxy) methyl) phenyl) amino) -5-oxopentanoate (0.130 g) was obtained.
LC / MS retention time: 8.1 minutes; m / z (ESI, POS): 840 [M + Na] ⁺

Example 1-3
(((((4-((S) -5-((9H-fluoren-9-yl) methoxy) -4-(((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5-oxopenta Synthesis of namid) benzyl) oxy) carbonyl) doxorubicin Crude (S)-(9H-fluoren-9-yl) methyl 2-(((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5 -((4-((((4-nitrophenoxy) carbonyl) oxy) methyl) phenyl) amino) -5-oxopentanoate (0.05 g) was dissolved in N, N-dimethylformamide (3 mL), After adding doxorubicin hydrochloride (0.03 g), diisopropylethylamine (0.13 mL) was added. After stirring at room temperature for 15 hours, water was added and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to give ((((4-((S) -5-((9H-fluoren-9-yl) methoxy) -4 as a crude product). -(((9H-Fluoren-9-yl) methoxy) carbonyl) amino) -5-oxopentanamido) benzyl) oxy) carbonyl) doxorubicin (0.09 g) was obtained.
LC / MS retention time: 7.8 minutes; m / z (ESI, POS): 1245 [M + Na] ⁺

Example 1-4
Synthesis of ((((4-((S) -4-amino-4-carboxybutanamide) benzyl) oxy) carbonyl) doxorubicin Crude (((((((4-((S) -5-((9H-fluorene- 9-yl) methoxy) -4-((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5-oxopentanamido) benzyl) oxy) carbonyl) doxorubicin (0.085 g) N, N -Dissolved in dimethylformamide (1.1725 mL), a solution of piperidine in N, N-dimethylformamide (10%, 0.275 mL) was added dropwise with ice cooling, and the mixture was stirred for 30 minutes. Water (4 mL) was added and the mixed solution was purified by preparative HPLC to obtain (((4- (4-amino-4-carboxybutanamido) benzyl) oxy) carbonyl) doxorubicin (Example 1, 0.025 g). Obtained.
NMR [400 MHz, DMSO-d ₆ , TMS] ppm: 1.11 (3H, d), 1.37-1.51 (2H, m), 1.76-1.92 (4H, m), 2. 07-2.22 (2H, m), 2.62-2.69 (1H, m), 2.98-3.12 (3H, m), 3.19-3.28 (1H, m), 3.67-3.76 (1H, m), 3.98 (3H, s), 4.10-4.17 (1H, m), 4.56 (2H, s), 4.69-4. 73 (1H, m), 4.87-4.98 (5H, m), 5.22 (1H, s), 5.46 (1H, s), 6.84 (1H, d), 7.23 (2H, d), 7.53 (2H, d), 7.63 (1H, d), 7.88-7.95 (2H, m), 10.42 (1H, brs).
LC / MS retention time: 4.0 minutes; m / z (ESI, POS): 822 [M + H] ⁺

Example 2
Synthesis of (((4-((S) -4-amino-4-carboxybutanamide) benzyl) oxy) carbonyl) -10-oxy-7-ethylcamptothecin [Scheme 2]

Example 2-1
Synthesis of t-butyl (S) -2-((t-butoxycarbonyl) amino) -5-((4- (hydroxymethyl) phenyl) amino) -5-oxopentanoate (S) -5- (t In a solution of -butoxy) -4-((t-butoxy) carbonyl) amino) -5-oxopentanoic acid (1.00 g) and 4-aminobenzyl alcohol (0.487 g) in dry dichloromethane (15 mL), N- Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (1.019 g) was added, and the mixture was 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 tert-butyl (S) -2-((t-butoxycarbonyl) amino) -5-((4- (hydroxymethyl) phenyl) amino) -5-oxopentanoate (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: 5.5 minutes; m / z (ESI, POS): 431 [M + Na] ⁺

Example 2-2
t-butyl (S) -2-((t-butoxycarbonyl) amino) -5-((4-((((4-nitrophenoxy) carbonyl) oxy) methyl) phenyl) amino) -5-oxopentano Synthesis of ate t-butyl (S) -2-((t-butoxycarbonyl) amino) -5-((4- (hydroxymethyl) phenyl) amino) -5-oxopentanoate (0.10 g) and pyridine To a solution of (0.0494 mL) in dry tetrahydrofuran (10 mL) was added dropwise a solution of 4-nitrophenyl chloroformate (0.0987 g) in dry tetrahydrofuran (10 mL) at 0 ° C., and the mixture was stirred 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 t-butyl (S) -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), 2.26-2.28 ( 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] ⁺

Example 2-3
(((4-((S) -5- (t-butoxy) -4-((t-butoxycarbonyl) amino) -5-oxopentanamido) benzyl) oxy) carbonyl) -10-oxy-7- Synthesis of ethylcamptothecin t-butyl (S) -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 (0.0855 g) was added, and diisopropylethylamine (0.37 mL) was added. 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 the solvent was evaporated 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] ⁺

Example 2-4
Synthesis of ((((4-((S) -4-amino-4-carboxybutanamide) benzyl) oxy) carbonyl) -10-oxy-7-ethylcamptothecin Crude ((((((4-((S)- 4- (t-butoxy) -4-((t-butoxycarbonyl) amino) -5-oxopentanamido) benzyl) oxy) carbonyl) -10-oxy-7-ethylcamptothecin (0.02 g) The mixture was dissolved in dioxane hydrochloride 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 (Example 2, 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] ⁺

Example 3
Synthesis of (((4-((S) -4-amino-4-carboxybutanamide) benzyl) oxy) carbonyl) camptothecin [Scheme 3]

Example 3-1
Synthesis of (((4-((S) -5- (t-butoxy) -4-((t-butoxycarbonyl) amino) -5-oxopentanamide) benzyl) oxy) carbonyl) camptothecin (0. A solution of dimethylaminopyridine (0.0561 g) in dichloromethane (2 mL) was slowly added dropwise to a suspension of 050 g) and triphosgene (0.0158 g) in dry dichloromethane (6 mL). After stirring for 30 minutes, t-butyl (S) -2-((t-butoxycarbonyl) amino) -5-((4- (hydroxymethyl) phenyl) amino) -5-oxopentanoate (0.059 g) 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 obtain a crude product (((4-((S) -5- (t-butoxy) -4-((t-butoxycarbonyl) amino) -5-oxopentanamide) Benzyl) oxy) carbonyl) camptothecin (0.108 g) was obtained.
LC / MS retention time: 6.8 min; m / z (ESI, POS): 805 [M + Na] ⁺

Example 3-2
Synthesis of (((4-((S) -4-amino-4-carboxybutanamide) benzyl) oxy) carbonyl) camptothecin Crude (((((4-((S) -5- (t-butoxy) -4 -((T-Butoxycarbonyl) amino) -5-oxopentanamide) benzyl) oxy) carbonyl) camptothecin (0.028 g) was dissolved in 4N hydrochloric acid ethyl acetate solution (3.0 mL) at 0 ° C. Stir for 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 (Example 3, 0.0017 g) 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] ⁺

Example 4
Synthesis of (((4-((S) -4-amino-4-carboxybutanamide) benzyl) oxy) carbonyl) epirubicin [Scheme 4]

Example 4-1
Synthesis of allyl (S) -2- (allyloxycarbonylamino) -5-((4-((((4-nitrophenoxy) carbonyl) oxy) methyl) phenyl) amino) -5-oxopentanoate S) -2-((allyloxycarbonyl) amino) -5-((4- (hydroxymethyl) phenyl) amino) -5-oxopentanoate (0.400 g) and pyridine (0.214 mL) in dry tetrahydrofuran A solution of 4-nitrophenyl chloroformate (0.428 g) in dry tetrahydrofuran (1 mL) was added dropwise to the (20 mL) solution at 0 ° C., and the mixture was stirred at room temperature for 18 hours. 1N Hydrochloric acid (10 mL) 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, and the residue was purified by silica gel column chromatography. Allyl (S) -2-((allyloxycarbonyl) amino) -5-((4-(((((4-nitrophenoxy)) Carbonyl) oxy) methyl) phenyl) amino) -5-oxopentanoate (0.633 g) was obtained.
NMR [400 MHz, CDCl ₃ , TMS] ppm: 1.96-2.08 (1H, m), 2.34-2.44 (1H, m), 2.48-2.53 (2H, m), 4.42-4.50 (1H, m), 4.62 (2H, d), 4.70 (2H, d), 5.24-5.39 (6H, m), 5.63 (1H, brd), 5.86-5.97 (2H, m), 7.39 (2H, d), 7.43 (2H, d), 7.65 (2H, d), 8.30 (2H, d) ), 8.41 (1H, brs).
LC / MS retention time: 6.6 min; m / z (ESI, POS): 564 [M + Na] ⁺

Example 4-2
Synthesis of (((4-((S) -5-allyl-4- (allyloxycarbonylamino) -5-oxopentanamido) benzyl) oxy) carbonyl) epirubicin allyl (S) -2- (allyloxycarbonyl Amino) -5-((4-((((4-nitrophenoxy) carbonyl) oxy) methyl) phenyl) amino) -5-oxopentanoate (0.130 g) and epirubicin hydrochloride (0.127 g). Dissolved in N, N-dimethylformamide (10 mL), diisopropylethylamine (0.0928 mL) was added. After stirring at room temperature for 6 hours, the reaction solution was added dropwise to diisopropyl ether (200 mL). The resulting solid was dissolved in chloroform, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (chloroform / methanol = 20/1) and (((4-((S) -5-allyl-4- (allyloxycarbonylamino) -5-oxopentanamide) benzyl). ) Oxy) carbonyl) epirubicin (0.222 g) was obtained.
NMR [400 MHz, DMSO-d ₆ , TMS] ppm: 1.18 (3H, d), 1.53-1.64 (1H, m), 1.79-1.91 (2H, m), 2. 02-2.24 (3H, m), 2.38-2.46 (2H, m), 2.90-3.02 (3H, m), 3.49-3.59 (1H, m), 3.84-3.93 (1H, m), 3.98 (3H, s), 4.06-4.13 (1H, m), 4.45-4.49 (2H, m), 4. 53-4.61 (4H, m), 4.83-4.88 (3H, m), 4.93-4.98 (2H, m), 5.16-5.23 (3H, m), 5.26-5.34 (2H, m), 5.49 (1H, s), 5.83-5.96 (2H, m), 7.02 (1H, d), 7.23 (2H, d), 7.53 (2H, d) 7.65 (1H, t), 7.75 (1H, d), 7.91 (2H, d), 9.93 (1H, s), 13.27 (1H, brs), 14.03 (1H , Brs).
LC / MS retention time: 6.2 minutes; m / z (ESI, POS): 968 [M + Na] ⁺

Example 4-3
Synthesis of (((4-((S) -4-amino-4-carboxybutanamide) benzyl) oxy) carbonyl) epirubicin ((((4-((S) -5-allyl-4- (allyloxycarbonylamino ) -5-oxopentanamide) benzyl) oxy) carbonyl) epirubicin (0.200 g) was dissolved in dichloromethane (4.5 mL) and N, N-dimethylformamide (1.0 mL), and tetrakistriphenylphosphine palladium ( 0.012 g) and phenylsilane (0.026 mL) were added, and the mixture was stirred for 30 minutes under an argon atmosphere. The reaction solution was added dropwise to 10% methanol-containing diisopropyl ether (450 mL). The resulting solid was purified by silica gel column chromatography (chloroform / methanol / water = 13/6/1) and (((4-((S) -4-amino-4-carboxybutanamido) benzyl) oxy) carbonyl ) Epirubicin (Example 4, 0.080 g) was obtained.
NMR [400 MHz, DMSO-d ₆ , TMS] ppm:
1.18 (3H, d), 1.52-1.67 (1H, m), 1.80-1.97 (3H, m), 2.13-2.23 (2H, m), 2. 90-3.05 (3H, m), 3.15-3.23 (2H, m), 3.49-3.61 (1H, m), 3.84-3.93 (1H, m), 3.99 (3H, s), 4.56 (2H, s), 4.87 (2H, s), 4.92-5. 01 (2H, m), 5.21 (1H, s), 5 .51 (1H, s), 7.03 (1H, d), 7.23 (2H, d), 7.54 (2H, d), 7.66 (1H, t), 7.91 (2H, d), 10.36 (1H, s), 14.04 (1H, brs).
LC / MS retention time: 4.4 minutes; m / z (ESI, POS): 822 [M + H] ⁺

Example 5
Synthesis of (((4-((S) -4-amino-4-carboxybutanamide) benzyl) oxy) carbonyl) cytarabine [Scheme 3]

Example 5-1
Synthesis of (((4-((S) -5-allyl-4- (allyloxycarbonylamino) -5-oxopentanamide) benzyl) oxy) carbonyl) cytarabine Allyl (S) -2- (allyloxycarbonyl Amino) -5-((4-((((4-nitrophenoxy) carbonyl) oxy) methyl) phenyl) amino) -5-oxopentanoate (0.030 g) was dissolved in tetrahydrofuran (1.85 mL). After adding cytarabine (0.014 g), 1N aqueous sodium hydroxide solution (0.15 mL) was added. After stirring at room temperature for 3 hours, ethyl acetate was added to obtain an organic layer. The obtained organic layer was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol = 20/1 to 5/1) and (((4- ((S) -5-allyl-4- (allyloxycarbonylamino) -5-oxopentanamide) benzyl) oxy) carbonyl) cytarabine (0.016 g) was obtained.
NMR [400 MHz, DMSO-d ₆ , TMS] ppm: 1.79-1.93 (1H, m), 2.02-2.34 (1H, m), 3.49-3.63 (2H, m ), 4.05-4.16 (1H, m), 4.47 (2H, d), 4.60 (2H, d), 4.92-5.09 (3H, m), 5.16- 5.34 (4H, m), 5.68 (1H, d), 5.83 (1H, d), 5.84-5.97 (2H, m), 6.17 (1H, d), 7 .24 (2H, d), 7.56 (2H, d), 7.63 (1H, d), 7.76 (1H, d), 10.00 (1H, brs).
LC / MS retention time: 3.6 minutes; m / z (ESI, POS): 646 [M + H] ⁺

Example 5-2
Synthesis of (((4-((S) -4-amino-4-carboxybutanamide) benzyl) oxy) carbonyl) cytarabine ((((4-((S) -5-allyl-4- (allyloxycarbonylamino ) -5-oxopentanamide) benzyl) oxy) carbonyl) cytarabine (0.016 g) was dissolved in dichloromethane (2.0 mL) and N, N-dimethylformamide (0.40 mL), and tetrakistri under argon atmosphere. Phenylphosphine palladium (0) (0.0028 g) and phenylsilane (0.0030 mL) were added, and the mixture was stirred at room temperature for 45 minutes and then allowed to stand for 18 hours. The solvent was removed and the residue was purified by preparative HPLC to give (((4-((S) -4-amino-4-carboxybutanamido) benzyl) oxy) carbonyl) cytarabine (Example 5, 0.0015 g )
NMR [400 MHz, DMSO-d ₆ , TMS] ppm: 2.02-2.14 (2H, m), 3.53-3.65 (2H, m), 3.73-3.81 (1H, m ), 3.90-3.99 (1H, m), 4.06-4.12 (1H, m), 4.94-5.11 (4H, m), 5.71-5.75. (1H, m), 5.82-5.85 (1H, m), 6.16 (1H, d), 7.25 (2H, d), 7.57 (2H, d), 7.95 ( 1H, s), 8.05-8.30 (2H, m), 10.08 (1H, brs).
LC / MS retention time: 0.8 min; m / z (ESI, POS): 522 [M + H] ⁺

Example 6: Synthesis of 10- (4-((S) -4-amino-4-carboxybutanamide) benzyloxy) -7-ethylcamptothecin 2 trifluoroacetate [Scheme 6]

Example 6-1: 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 6-2: Synthesis of 5-((4-chloromethyl) benzylamino) -1- (t-butyl) N- (t-butoxycarbonyl) -L-glutamate In an argon atmosphere, with ice-cooling and stirring, 5 In a dry dichloromethane (4.3 mL) solution of-((4-hydroxymethyl) benzylamino) -1- (t-butyl) N- (t-butoxycarbonyl) -L-glutamate (88 mg, 0.22 mmol), 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), and 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 6-3: 10-(((4-((S) -5- (t-butoxy) -4-((t-butoxycarbonyl) amino) -5-oxopentanamide) benzyloxy) -7 -Synthesis of ethylcamptothecin 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, and then crude 5-((4-chloromethyl) benzylamino) was stirred 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 6-4: Synthesis of 10- (4-((S) -4-amino-4-carboxybutanamido) benzyloxy) -7-ethylcamptothecin 2trifluoroacetate salt 10-(((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) was added trifluoroacetic acid (1 mL) and the resulting yellow solution was allowed to cool for 5 minutes under ice-cooling and then at room temperature (21 ° C.) for 2 hours. The reaction mixture was evaporated to dryness, 1,2-dichloroethane (8 mL) was added to the residue, and the mixture was again evaporated to dryness to give a yellow solid (41 mg), which was dimethylformamide (1 mL). , Acetonitrile (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 6 and 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. 09, 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] ⁺

Comparative Example 1
Synthesis of (S)-(4-amino-4-carboxybutanamide) doxorubicin

Comparative Example 1-1
(S) -5-((9H-Fluoren-9-yl) methoxy) -4-((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5-oxopentanoic acid (0.100 g) , Doxorubicin hydrochloride (0.095 g), hydroxybenzotriazole (3 mg) and triethylamine (0.024 mL) were dissolved in N, N-dimethylformamide (2 mL) and 1-ethyl-3- (3-dimethylaminopropyl hydrochloride) was dissolved. ) Carbodiimide (0.100 g) was added and stirred at room temperature for 22 hours. Water (40 mL) was added and the resulting precipitate was filtered. The precipitate was purified by silica gel column chromatography to obtain (4-((S) -5-((9H-fluoren-9-yl) methoxy) -4-(((9H-fluoren-9-yl) methoxy) carbonyl). ) Amino) -5-oxopentanamide) doxorubicin (0.090 g) was obtained.
NMR [400 MHz, DMSO-d ₆ , TMS] ppm: 1.13 (3H, d), 1.42-1.54 (1 H, m), 1.64-1.94 (3H, m), 2. 06-2.25 (4H, m), 2.90-3.04 (2H, m), 3.90-4.38 (10H, m), 4.59 (2H, d), 4.77 ( 1H, d), 4.88-4.99 (2H, m), 5.20-5.26 (1H, m), 5.50 (1H, s), 7.20-7.89 (19H, m), 13.28 (1H, s), 14.07 (1H, s).
LC / MS retention time: 7.6 minutes; m / z (ESI, POS): 1096 [M + Na] ⁺

Comparative Example 1-2
(4-((S) -5-((9H-fluoren-9-yl) methoxy) -4-(((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5-oxopentanamide) Doxorubicin (0.020 g) was dissolved in dichloromethane (0.1365 mL), piperidine in dichloromethane (10% v / v, 0.635 mL) was added at 0 ° C., and the mixture was stirred for 3.5 hours. After dichloromethane (1 mL) was added, piperidine in dichloromethane (10% v / v, 0.0158 mL) was added at 0 ° C. and stirring was continued for 2 hours. The precipitate was filtered and washed with ethyl acetate. The obtained precipitate was purified by preparative HPLC to obtain (S)-(4-amino-4-carboxybutanamide) doxorubicin (Comparative Example 1, 0.0021 g).
NMR [400 MHz, D ₂ O, TMS] ppm: 1.17 (3H, d), 1.61-1.74 (1H, m), 1.76-1.93 (2H, m), 1.94 -2.04 (2H, m), 2.11-2.20 (1H, m), 2.24-2.50 (3H, m), 2.66-2.78 (1H, m) 59 (2H, m), 3.75 (3H, s), 3.99-4.16 (2H, m), 5.25 (1H, m), 7.02-7.15 (2H, m) 7.35-7.42 (1H, m).
LC / MS retention time: 3.8 min; m / z (ESI, POS): 673 [M + H] ⁺

Comparative Example 2
Synthesis of 9-((S) -4-amino-4-carboxybutanamide) camptothecin

Comparative Example 2-1
(S) -5- (Benzyloxy) -4- (benzyloxycarbonyl) amino-5-oxopentanoic acid (0.173 g) was converted to 9-aminocamptothecin (0.090 g) in N, N-dimethylformamide (5 mL). After adding to the solution, hydroxybenzotriazole (0.005 g) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (0.297 g) were added, and the mixture was stirred at room temperature for 2 hours and allowed to stand for 2 days. Water (10 mL) was added and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 1/5 to 0/1) to give 9-((4-((S) -5- (benzyloxy) -4- (benzyloxycarbonyl) amino. -5-Oxo) pentanamide) camptothecin (0.024 g) was obtained.
NMR [400 MHz, DMSO-d ₆ , TMS] ppm: 0.89 (3H, t), 1.81-2.02 (4H, m), 2.13-2.29 (1H, m), 2. 58-2.70 (2H, m), 4.22-4.30 (1H, m), 5.08 (2H, d), 5.17 (2H, s), 5.28 (2H, s) , 5.44 (2H, s), 6.56 (1H, s), 7.23-7.40 (11H, m), 7.76-7.87 (2H, m), 7.92 (1H) , D), 8.03 (1H, d), 8.75 (1H, s), 10.20 (1H, s).
LC / MS retention time: 5.8 minutes; m / z (ESI, POS): 717 [M + H] ⁺

Comparative Example 2-2
9-((4-((S) -5- (benzyloxy) -4- (benzyloxycarbonyl) amino-5-oxo) pentanamide) camptothecin (0.020 g) in N, N-dimethylformamide (1 mL) Dissolved, 10% palladium carbon (0.005 g) was added, and the mixture was stirred at room temperature for 2 hours in a hydrogen atmosphere. The filtrate was purified by preparative HPLC to give 9-((S) -4-amino-4-carboxybutanamide) camptothecin (0.012 g).
NMR [400 MHz, D ₂ O, TMS] ppm: 0.86 (3H, t), 1.85 (2H, q), 2.26 (2H, q), 2.71-2.82 (2H, m ), 3.97 (1H, m), 5.24-5.35 (2H, m), 7.21 (1H, s), 7.39-7.44 (1H, m), 7.49 ( 2H, d), 8.30 (1H, s).
LC / MS retention time: 1.2 minutes; m / z (ESI, POS): 493 [M + H] ⁺

Test Example 1: γ-Glutamyltranspeptidase (GGT) enzyme recognition test The doxorubicin prodrug (0.49 mg) of Example 1 was dissolved in phosphate buffered saline (PBS) (0.298 mL), and 2 mM doxorubicin pro A drug PBS solution was prepared.
0.150 mL of 2 U / mL GGT solution in which GGT (0.22 mg, 8 U / mg) (Sigma-Aldrich) was dissolved in PBS (0.880 mL) was added to 0.150 mL of the doxorubicin prodrug PBS solution. The reaction was carried out at ° C.
The reaction solution was subjected to HPLC analysis to determine the prodrug residual rate and doxorubicin production rate. The results are summarized in Table 1.

Comparative Test Example 1; GGT enzyme recognition test The doxorubicin prodrug (0.17 mg) obtained in Comparative Example 1 was dissolved in PBS (0.1265 mL) to prepare a 2 mM doxorubicin prodrug PBS solution.
To 0.100 mL of the doxorubicin prodrug PBS solution, 0.100 mL of 2 U / mL GGT solution in which GGT (0.38 mg, 8 U / mg) (Sigma-Aldrich) was dissolved in PBS (1.520 mL) was added, and 37 The reaction was carried out at ° C.
Six hours later, the reaction solution was analyzed by HPLC. As a result, 93% of the prodrug remained. From this, it was shown that the doxorubicin prodrug which concerns on the comparative example 1 is not recognized by GGT, and cannot release doxorubicin which is a bioactive substance.

From the results of Test Example 1, it was shown that the doxorubicin prodrug according to Example 1 can rapidly release doxorubicin having pharmacological activity in the presence of GGT. On the other hand, Comparative Test Example 1 showed that the doxorubicin prodrug of Comparative Example 1 in which γ-glutamic acid was directly bonded to doxorubicin hardly caused the dissociation reaction of doxorubicin by GGT. Therefore, a rapid dissociation reaction by GGT recognition requires an appropriate linker segment to be interposed between the γ-glutamic acid binding site and the drug, and the aromatic amide type linker segment of the present invention is advantageous. It was revealed that the GGT recognition ability can be exhibited.

Test Example 2: Phosphate buffered saline (PBS) solution stability test The doxorubicin prodrug (0.49 mg) of Example 1 was dissolved in phosphate buffered saline (PBS) (0.298 mL) to prepare doxorubicin. A prodrug PBS solution was prepared.
0.148 mL of the doxorubicin prodrug PBS solution was dissolved in 0.148 mL of PBS solution and reacted at 37 ° C. After 6.5 hours, the reaction solution was analyzed by HPLC. As a result, no decrease in prodrug was confirmed.
Therefore, it was shown that the doxorubicin prodrug of Example 1 according to the present invention does not release doxorubicin in the PBS solution and is chemically stable.

Test Example 3: Cell growth inhibitory activity test Using OS-RC-2 cells (RIKEN BioResource Center) with high GGT activity and SK-OV-3 cells (American Type Culture Collection) with low GGT activity, according to Example 1 The cell growth inhibitory activity of doxorubicin prodrug was evaluated.
OS-RC-2 cells with high GGT activity and SK-OV-3 cells with low GGT activity are seeded in a 96-well plate at 4,000 cells / well, respectively, and cultured at 37 ° C. under 5% CO ₂ for 1 day. Thereafter, the doxorubicin prodrug according to Example 1 was added at a final concentration of 0.039 to 10 μM. After culturing for 6 hours, the inside of the well was washed. After further culturing for 3 days, 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, cell growth inhibitory activity was evaluated from the absorbance using doxorubicin which was not converted into a prodrug. 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 4 Confirmation Test of Prodrug GGT Dependence Using OS-RC-2 cells having high GGT activity, the GGT activity dependence of the cell growth inhibitory activity of doxorubicin prodrug according to Example 1 was evaluated. After seeding OS-RC-2 cells in the same manner as in Test Example 3 and culturing for 1 day, GGsTop (Wako Pure Chemical Industries, Ltd.), a GGT inhibitor, was added at a final concentration of 10 μM, followed by incubation for 1 hour. The doxorubicin prodrug according to Example 1 was added at a final concentration of 0.039-10 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 3. The cell growth inhibitory activity was evaluated from the absorbance of doxorubicin that was not converted into a prodrug by the same operation. The results are shown in FIG.

As a result of Test Example 3, in the OS-RC-2 cells having high GGT activity, when 10 μM doxorubicin was added, the cell growth inhibitory activity was 77%. On the other hand, when 10 μM of doxorubicin prodrug according to Example 1 was added, the cell growth inhibitory activity was 76%.
On the other hand, when 10 μM doxorubicin was added to SK-OV-3 cells with low GGT activity, the cell growth inhibitory activity was 77%. On the other hand, when 10 μM of doxorubicin prodrug according to Example 1 was added, the cell growth inhibitory activity was 13%.
Comparing the results of cell growth inhibitory activity in cells with high GGT activity and cells with low GGT activity, the doxorubicin prodrug of Example 1 according to the present invention releases doxorubicin 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 doxorubicin prodrug which concerns on this invention is a physical property which exhibits the cell growth inhibitory activity depending on GGT activity.
As a result of Test Example 4, in OS-RC-2 cells having high GGT activity, the GGT inhibitor did not affect the cell growth inhibitory activity of doxorubicin, but completely inhibited the activity of doxorubicin prodrug. From this, it was shown that the doxorubicin prodrug which concerns on this invention is a physical property which exhibits the cell growth inhibitory activity depending on GGT activity.

Test Example 5: GGT enzyme recognition test In the same manner as in Test Example 1, the epirubicin prodrug of Example 4 in 2 mM PBS solution and 2 U / mL GGT solution were reacted at 37 ° C.
HPLC analysis of the reaction solution over time confirmed that the prodrug was decomposed by the enzymatic reaction, and the prodrug residual rate after 6 hours was 5%.
From this, it was shown that the epirubicin prodrug which concerns on Example 4 can release the epirubicin which has pharmacological activity rapidly in presence of GGT.

Test Example 6: GGT Enzyme Recognition Test In the same manner as in Test Example 1, the reaction was carried out at 37 ° C. using 1 mM PBS solution of cytarabine prodrug obtained in Example 5 and 2 U / mL GGT solution.
HPLC analysis of the reaction solution over time confirmed that the prodrug was decomposed by the enzymatic reaction, and the prodrug residual rate after 6.5 hours was 34%.
From this, it was shown that the cytarabine prodrug according to Example 5 can release cytarabine having pharmacological activity quickly in the presence of GGT.

Comparative Test Example 2; GGT enzyme recognition test In the same manner as in Comparative Test Example 1, a 2 mM PBS solution of the 9-aminocamptothecin prodrug obtained in Comparative Example 2 was prepared, and a 2 U / mL GGT solution was used. The reaction was carried out at 37 ° C.
Six hours later, HPLC analysis of the reaction solution showed that 99% of the prodrug remained. This indicates that the 9-aminocamptothecin prodrug according to Comparative Example 2 is not recognized by GGT and cannot release 9-aminocamptothecin, which is a physiologically active substance.
The 9-aminocamptothecin prodrug of Comparative Example 2 had a result that almost no dissociation reaction of 9-aminocamptothecin by GGT occurred. Thus, it was necessary to interpose an appropriate linker segment, and it became clear that the aromatic amide type linker segment of the present invention can exert a superior GGT recognition ability.

Test Example 7: Stability test in PBS PBS (0.220 mL) was added to a 60 mg / mL DMSO solution (0.005 mL) of the EHC prodrug of Example 2 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 a 6.3 mg / mL DMSO solution (0.073 mL) of the camptothecin prodrug of Example 3. 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%.
PBS (0.450 mL) was added to the 0.864 mg / mL DMSO solution (0.050 mL) of the EHC prodrug of Example 6 and reacted at 37 ° 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.

Test Example 8: 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 6 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 6 according to the present invention is chemically stable even in mouse plasma.

Test Example 9: GGT enzyme recognition test The EHC prodrug of Example 6 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 9, the EHC prodrug of Example 6 produced a stoichiometric amount of EHC in 1 hour of reaction. This result shows that the compound of Example 6 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 7 and 8, it was shown that the EHC prodrug according to Example 6 has stable physical properties in a PBS solution and a solution in the presence of mouse plasma. Furthermore, it was shown that the EHC prodrug according to Example 6 can rapidly release EHC having pharmacological activity in the presence of GGT.

Test Example 10: 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 The cell growth inhibitory activity of the EHC prodrug according to Example 6 was evaluated using Culture Collection.
A 96-well plate was 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 6 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 6 was used as it was, and the cell growth inhibitory activity was evaluated from the absorbance.
The results are shown in FIG. 4 (OS-RC-2 cells with high GGT activity) and FIG. 5 (SK-OV-3 cells with low GGT activity).

Test Example 11: Confirmation test of GGT dependence of prodrug GGT activity dependence on cell growth inhibitory activity of EHC prodrug according to Example 6 using OS-RC-2 cells known to have high GGT activity Sex was evaluated. OS-RC-2 cells were seeded in the same manner as in Test Example 10 and cultured for 1 day, and then GGT inhibitor GGsTop (Wako Pure Chemical Industries, Ltd.) was added at a final concentration of 10 μM and cultured for 1 hour. Thereafter, the EHC prodrug of Example 6 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 10. In the same manner, EHC which is an active ingredient of Example 6 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 10, 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 6 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 6 was added, the cell growth inhibitory activity was 1.3%.
Comparing the results of cell growth inhibitory activity in cells having high GGT activity and cells having low GGT activity, the EHC prodrug of Example 6 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 11, 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 12: Measurement of tissue drug concentration 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 6 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 in terms of EHC. 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 using 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 6 and the CPT-11 concentration, AUC _0-6hr of each component until 6 hours after the start of administration was calculated. As the prodrug activation rate of Example 6 and CPT-11 in each tissue, the AUC _0-6hr ratio (AUC _EHC / AUC _{Example 6} and AUC _EHC / AUC _CPT-11 ) of the active substance to the unchanged form was determined. (Table 2).

Test Example 13: Anti-tumor effect test In the same manner as in Test Example 12, mice transplanted with mouse renal cell carcinoma OS-RC-2 were treated with 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 major axis (Lmm) and minor axis (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 3.

As a result of Test Example 12, the activation rate of the EHC prodrug of Example 6 was comparable to 9.4 times 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 6} 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 13, the EHC prodrug of Example 6 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

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 alkyl group which may have a substituent, 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 optionally substituted alkyl group, and L represents a bond selected from the group consisting of an oxygen atom, an oxycarbonyl group and a bond. X represents a binding residue of a physiologically active substance having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group, an amino group, and a carboxy group;
(I) When X is a binding residue of a physiologically active substance having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group and an amino group, the L is an oxycarbonyl group,
(Ii) when X is a binding residue of a physiologically active substance having a carboxy group, L is an oxygen atom;
(Iii) When X is a binding residue of a physiologically active substance having an aromatic hydroxyl group, L is a bond or an oxycarbonyl group. ],
Here, one of the A 1 and the A 2 is the CR 7 and the other is the CR 6 or a nitrogen atom, and B 1 , B 2 and B 3 are each independently C—R 6 or a nitrogen atom. Or a pharmacologically acceptable salt thereof.
R 7 represents the following general formula (3)

[Wherein, R 4 and R 5 are as defined above, and X is a binding of a physiologically active substance having at least one functional group selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group and an amino group. Residue. ] The glutamic acid derivative of Claim 1 represented by these, or its salt accept | permitted pharmacologically.
The glutamic acid derivative or a pharmacologically acceptable salt thereof according to claim 1 or 2, wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is camptothecin and its derivative.
The glutamic acid derivative according to claim 1 or 2, wherein the physiologically active substance in the binding residue of the physiologically active substance represented by X is a physiologically active substance selected from the group consisting of doxorubicin, daunorubicin, epirubicin, pirarubicin and amrubicin. Or a pharmacologically acceptable salt thereof.
The physiologically active substance in the binding residue of the physiologically active substance represented by X is gemcitabine, ethynylcytidine, cytarabine and CNDAC (2′-cyano-2′-deoxy-1-β-D-arabinofuranosylcytosine) The glutamic acid derivative or pharmacologically acceptable salt thereof according to claim 1 or 2, which is a physiologically active substance selected from the group consisting of:
R 7 represents the following general formula (4)

[Wherein, R 4 and R 5 are as defined above, and X is a binding residue of a physiologically active substance having an aromatic hydroxyl group. ] The glutamic acid derivative of Claim 1 represented by these, or its salt accept | permitted pharmacologically.
7. 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.
The glutamic acid derivative or a pharmacologically acceptable salt thereof according to any one of claims 1 to 7, wherein in the general formula (1), R 1 , R 2 and R 3 are hydrogen atoms.
A medicament comprising the glutamic acid derivative according to any one of claims 1 to 8 or a pharmacologically acceptable salt thereof as an active ingredient.
The medicament according to claim 9, which is an anticancer agent.