JP6787564B2 - Glutathione S-transferase inhibitor - Google Patents
Glutathione S-transferase inhibitor Download PDFInfo
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- JP6787564B2 JP6787564B2 JP2016133272A JP2016133272A JP6787564B2 JP 6787564 B2 JP6787564 B2 JP 6787564B2 JP 2016133272 A JP2016133272 A JP 2016133272A JP 2016133272 A JP2016133272 A JP 2016133272A JP 6787564 B2 JP6787564 B2 JP 6787564B2
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Description
本発明は、グルタチオンS−トランスフェラーゼ阻害作用を有する化合物又はその塩ン、それを含有する該酵素の阻害剤、抗がん剤、がん診断薬等に関する。 The present invention relates to a compound having a glutathione S-transferase inhibitory action or a salt thereof, an inhibitor of the enzyme containing the compound, an anticancer agent, a cancer diagnostic agent, and the like.
グルタチオンS−トランスフェラーゼ（本明細書において、GSTと示すこともある。）は、薬物代謝の第II相反応おいてグルタチオン（本明細書において、GSHと示すこともある。）抱合を触媒する酵素である。GSTは、生体異物の解毒や活性酸素の除去などの主な機能によって、生体内の環境変化や酸化ストレスから細胞を守るために重要である。 Glutathione S-transferase (sometimes referred to herein as GST) is an enzyme that catalyzes glutathione (sometimes referred to herein as GSH) conjugation in a phase II reaction of drug metabolism. is there. GST is important for protecting cells from environmental changes and oxidative stress in the body by its main functions such as detoxification of foreign substances and removal of active oxygen.
一方で、GSTは、細胞増殖やアポトーシスの調節にも関与するといわれている。例えば、該酵素には複数のタイプが存在しており（非特許文献１及び２）、π型は、アポトーシスを抑制することによってがん細胞を増殖させることが報告されている（非特許文献３及び４）。具体的には、π型は、JNK（MAPK）との複合体形成によりJNKの活性化を阻害する経路、及びASK（MAPKKK）の活性化を引き起こすタンパク質であるTRAF2との複合体形成によりASKの活性化を阻害する経路の2つの経路により、アポトーシスに対して抑制的に働くことが報告されている。また、π型は、各種組織由来の腫瘍において過剰発現することが報告されている（非特許文献５）。このため、GST（特にπ型）を阻害すれば、アポトーシスが促進されて、抗がん効果が発揮されと考えられている。 On the other hand, GST is also said to be involved in the regulation of cell proliferation and apoptosis. For example, there are multiple types of the enzyme (Non-Patent Documents 1 and 2), and it has been reported that the π type proliferates cancer cells by suppressing apoptosis (Non-Patent Document 3). And 4). Specifically, the π-type is a pathway that inhibits JNK activation by complex formation with JNK (MAPK), and ASK by complex formation with TRAF2, a protein that causes ASK (MAPKKK) activation. It has been reported that two pathways that inhibit activation act in a suppressive manner against apoptosis. In addition, it has been reported that π-type is overexpressed in tumors derived from various tissues (Non-Patent Document 5). Therefore, it is considered that inhibition of GST (particularly π-type) promotes apoptosis and exerts an anticancer effect.
GST阻害剤としては、基質であるGSHと競合的にGSTに作用するGSH誘導体が考えられる。しかしながら、細胞内のGSH濃度は非常に高いことから（1〜10mMといわれている）、既存のGSH誘導体による競合阻害効果は限定的なものであると考えられた。 As a GST inhibitor, a GSH derivative that acts on GST in a competitive manner with the substrate GSH can be considered. However, since the intracellular GSH concentration is very high (it is said to be 1 to 10 mM), it was considered that the competitive inhibitory effect of the existing GSH derivative is limited.
そこで、本発明は、より効率的にGSTを阻害することができるGSH誘導体を提供することを課題とする。 Therefore, an object of the present invention is to provide a GSH derivative capable of inhibiting GST more efficiently.
本発明者らは上記課題に鑑みて鋭意研究した結果、特定の構造を有するGSH誘導体又はその塩が、効率的にGSTを阻害できることを見出した。本発明者らはこのような知見に基づき、さらに研究を進めた結果、本発明を完成させた。即ち、本発明は、下記の態様を包含する。 As a result of diligent research in view of the above problems, the present inventors have found that a GSH derivative having a specific structure or a salt thereof can efficiently inhibit GST. The present inventors have completed the present invention as a result of further research based on such findings. That is, the present invention includes the following aspects.
項１． 一般式（1）： Item 1. General formula (1):
［式中、R1はハロゲン原子又は−R11−R12（R11は酸素原子又は硫黄原子を示す。R12はアルキル基又はアリール基を示す。）で表される基を示す。R2はアルキレン基、−アルキレン基−置換されていてもよいフェニレン基−で表される基、又は置換されていてもよいフェニレン基を示す。R3は水素原子、置換されていてもよいアリール基、又は置換されていてもよいアラルキル基を示す。R4はヒドロキシ基又は加水分解性基を示す。R5はヒドロキシ基、加水分解性基、又は機能分子由来の基を示す。］
で表される化合物又はその塩．
項２． 前記R1がハロゲン原子である、項１に記載の化合物又はその塩．
項３． 前記R1がフッ素原子である、項１又は２に記載の化合物又はその塩．
項４． 前記R2がアルキレン基である、項１〜３のいずれかに記載の化合物又はその塩．
項５． 前記機能性分子がタンパク質タグ又は蛍光分子である、項１〜４のいずれかに記載の化合物又はその塩．
項６． 前記加水分解性基がアルコキシ基、アルコキシアルコキシ基、アシルオキシ基、アシルオキシアルコキシキ基、カーボネート基、カルバメート基、又はアミド基である、項１〜５のいずれかに記載の化合物又はその塩．
項７． 項１〜６のいずれかに記載の化合物又はその塩を含有する医薬組成物．
項８． 項１〜６のいずれかに記載の化合物又はその塩を含有する生物試験用試薬．
項９． 項１〜６のいずれかに記載の化合物又はその塩を含有するグルタチオンS−トランスフェラーゼ阻害剤．
項１０． 前記グルタチオンS−トランスフェラーゼがπ型である、項９に記載の阻害剤．
項１１． 項１〜６のいずれかに記載の化合物又はその塩を含有する抗がん剤．
項１２． 項１〜６のいずれかに記載の化合物又はその塩を含有するがん診断薬．
[In the formula, R 1 indicates a halogen atom or −R 11 −R 12 (R 11 indicates an oxygen atom or a sulfur atom. R 12 indicates an alkyl group or an aryl group). R 2 represents an alkylene group, a-alkylene group-a group represented by an optionally substituted phenylene group-or an optionally substituted phenylene group. R 3 represents a hydrogen atom, an optionally substituted aryl group, or an optionally substituted aralkyl group. R 4 indicates a hydroxy group or a hydrolyzable group. R 5 indicates a hydroxy group, a hydrolyzable group, or a group derived from a functional molecule. ]
A compound represented by or a salt thereof.
Item 2. Item 2. The compound according to Item 1, or a salt thereof, wherein R 1 is a halogen atom.
Item 3. Item 2. The compound according to Item 1 or 2, or a salt thereof, wherein R 1 is a fluorine atom.
Item 4. Item 4. The compound according to any one of Items 1 to 3 or a salt thereof, wherein R 2 is an alkylene group.
Item 5. Item 4. The compound according to any one of Items 1 to 4, or a salt thereof, wherein the functional molecule is a protein tag or a fluorescent molecule.
Item 6. Item 5. The compound according to any one of Items 1 to 5, wherein the hydrolyzable group is an alkoxy group, an alkoxyalkoxy group, an acyloxy group, an acyloxyalkoxy group, a carbonate group, a carbamate group, or an amide group.
Item 7. A pharmaceutical composition containing the compound according to any one of Items 1 to 6 or a salt thereof.
Item 8. A reagent for biological testing containing the compound according to any one of Items 1 to 6 or a salt thereof.
Item 9. A glutathione S-transferase inhibitor containing the compound according to any one of Items 1 to 6 or a salt thereof.
Item 10. Item 9. The inhibitor according to Item 9, wherein the glutathione S-transferase is of type π.
Item 11. An anticancer agent containing the compound according to any one of Items 1 to 6 or a salt thereof.
Item 12. A cancer diagnostic agent containing the compound according to any one of Items 1 to 6 or a salt thereof.
本発明の化合物は、GSHの存在下であっても（特に高濃度に存在する場合であっても）、効率的にGSTを阻害することができる。このため、本発明の化合物又はその塩は、各種生物試験用試薬、医薬組成物等として、特にGST阻害剤、抗がん剤等として有用である。 The compounds of the present invention can efficiently inhibit GST even in the presence of GSH (especially in high concentrations). Therefore, the compound of the present invention or a salt thereof is useful as a reagent for various biological tests, a pharmaceutical composition, etc., particularly as a GST inhibitor, an anticancer agent, and the like.
また、本発明の化合物は、GSTと不可逆的に結合すると考えられ、このため該化合物を用いて組織からGSTを分離することができると考えられる。分離されたGST（特にGSTπ型）の量や型を測定することにより、がんを診断することも可能である。よって、本発明の化合物又はその塩は、GST分離剤、がん診断剤等としても有用である。 In addition, the compound of the present invention is considered to bind irreversibly to GST, and therefore it is considered that GST can be separated from the tissue using the compound. It is also possible to diagnose cancer by measuring the amount and type of isolated GST (particularly GSTπ type). Therefore, the compound of the present invention or a salt thereof is also useful as a GST separating agent, a cancer diagnostic agent, and the like.
本明細書において、「含有」とは、comprise、consist essentially of及びconsist ofのいずれも包含する概念である。また、本明細書において、「置換されていてもよい」とは、「置換及び／又は非置換」を意味する。 As used herein, the term "contains" is a concept that includes all of compute, concept essentially of and const of. Further, in the present specification, "may be substituted" means "replacement and / or non-replacement".
1．化合物又はその塩
本発明は、その一態様として、一般式（1）：
1. 1. Compound or salt thereof The present invention has, as one embodiment, the general formula (1):
［式中、R1はハロゲン原子又は−R11−R12（R11は酸素原子又は硫黄原子を示す。R12はアルキル基又はアリール基を示す。）で表される基を示す。R2はアルキレン基、−アルキレン基−置換されていてもよいフェニレン基−で表される基、又は置換されていてもよいフェニレン基を示す。R3は水素原子、置換されていてもよいアリール基、又は置換されていてもよいアラルキル基を示す。R4はヒドロキシ基又は加水分解性基を示す。R5はヒドロキシ基、加水分解性基、又は機能分子由来の基を示す。］
で表される化合物又はその塩に関する。以下、これについて説明する。
[In the formula, R 1 indicates a halogen atom or −R 11 −R 12 (R 11 indicates an oxygen atom or a sulfur atom. R 12 indicates an alkyl group or an aryl group). R 2 represents an alkylene group, a-alkylene group-a group represented by an optionally substituted phenylene group-or an optionally substituted phenylene group. R 3 represents a hydrogen atom, an optionally substituted aryl group, or an optionally substituted aralkyl group. R 4 indicates a hydroxy group or a hydrolyzable group. R 5 indicates a hydroxy group, a hydrolyzable group, or a group derived from a functional molecule. ]
With respect to a compound represented by or a salt thereof. This will be described below.
本発明においては、限定的な解釈を望むものではないが、一般式（1）におけるR1がGSTの活性残基（例えば、GSTπ型におけるN末端から7番目のアミノ酸（チロシン）残基）と共有結合することにより、GSTを効率的に阻害できると考えられる。よって、R1はこの限りにおいて特に限定されない。 In the present invention, although not limited interpretation is desired, R 1 in the general formula (1) is the active residue of GST (for example, the 7th amino acid (tyrosine) residue from the N-terminal in GST π type). It is considered that GST can be effectively inhibited by covalent bonding. Therefore, R 1 is not particularly limited to this extent.
一般式（1）において、R1で示されるハロゲン原子としては、例えばフッ素原子、塩素原子、臭素原子、ヨウ素原子等が挙げられる。これらの中でも、GST阻害活性の観点から、好ましくはフッ素原子、塩素原子、臭素原子等が挙げられ、より好ましくはフッ素原子が挙げられる。 In the general formula (1), examples of the halogen atom represented by R 1 include a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like. Among these, from the viewpoint of GST inhibitory activity, a fluorine atom, a chlorine atom, a bromine atom and the like are preferably mentioned, and a fluorine atom is more preferable.
一般式（1）において、R12で示されるアルキル基には、直鎖状又は分枝鎖状のいずれのものも包含される。該アルキル基の炭素数は、特に制限されないが、例えば1〜8、好ましくは1〜6、より好ましくは1〜4である。該アルキル基の具体例としては、メチル基、エチル基、n−プロピル基、イソプロピル基、n−ブチル基、イソブチル基、tert−ブチル基、sec−ブチル基、n−ペンチル基、ネオペンチル基、n−ヘキシル基、3−メチルペンチル基等が挙げられる。 In the general formula (1), the alkyl group represented by R 12 includes either a linear or branched chain. The number of carbon atoms of the alkyl group is not particularly limited, but is, for example, 1 to 8, preferably 1 to 6, and more preferably 1 to 4. Specific examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, neopentyl group, n. -Hexyl group, 3-methylpentyl group and the like can be mentioned.
一般式（1）において、R12で示されるアリール基としては、単環アリール基（フェニル基）及び多環（好ましくは2〜3環、より好ましくは2環）アリール基のいずれも採用することができ、例えば、フェニル基、ナフチル基、フルオレニル基、アントリル基、ビフェニリル基、テトラヒドロナフチル基、フェナントリル基等が挙げられる。 In the general formula (1), as the aryl group represented by R 12 , both a monocyclic aryl group (phenyl group) and a polycyclic (preferably 2 to 3 rings, more preferably 2 rings) aryl group shall be adopted. Examples thereof include a phenyl group, a naphthyl group, a fluorenyl group, an anthryl group, a biphenylyl group, a tetrahydronaphthyl group, a phenanthryl group and the like.
一般式（1）において、R1としては、GST阻害活性の観点から、好ましくはハロゲン原子が挙げられる。 In the general formula (1), R 1 is preferably a halogen atom from the viewpoint of GST inhibitory activity.
一般式（1）において、R2で示されるアルキレン基には、直鎖状又は分枝鎖状のいずれのものも包含される。該アルキレン基は、好ましくは直鎖状である。炭素数は、特に制限されないが、GST阻害活性の観点から、例えば1〜8、好ましくは1〜6、より好ましくは2〜6、さらに好ましくは2〜4、よりさらに好ましくは2〜3、特に好ましくは2である。主鎖上の炭素数は、特に制限されないが、GST阻害活性の観点から、例えば1〜8、好ましくは1〜6、より好ましくは2〜6、さらに好ましくは2〜4、よりさらに好ましくは2〜3、特に好ましくは2である。該アルキレン基の具体例としては、メチレン基、エチレン基、ｎ−プロピレン基、イソプロピレン基、ｎ−ブチレン基、イソブチレン基、ｎ−ヘキシレン基等が挙げられる。 In the general formula (1), the alkylene group represented by R 2 includes either a linear group or a branched chain group. The alkylene group is preferably linear. The number of carbon atoms is not particularly limited, but from the viewpoint of GST inhibitory activity, for example, 1 to 8, preferably 1 to 6, more preferably 2 to 6, still more preferably 2 to 4, still more preferably 2 to 3, in particular. It is preferably 2. The number of carbon atoms on the main chain is not particularly limited, but from the viewpoint of GST inhibitory activity, for example, 1 to 8, preferably 1 to 6, more preferably 2 to 6, still more preferably 2 to 4, still more preferably 2. ~ 3, particularly preferably 2. Specific examples of the alkylene group include a methylene group, an ethylene group, an n-propylene group, an isopropylene group, an n-butylene group, an isobutylene group, an n-hexylene group and the like.
一般式（1）において、R2で示されるフェニレン基としては、例えば1,2−フェニレン基、1,3−フェニレン基、1,4−フェニレン基等が挙げられる。これらの中でも、GST阻害活性の観点から、好ましくは1,4−フェニレン基が挙げられる。該フェニレン基は、例えばニトロ基等の置換基を1〜3個（特に1個）有することもできる。 In the general formula (1), examples of the phenylene group represented by R 2 include a 1,2-phenylene group, a 1,3-phenylene group, a 1,4-phenylene group and the like. Among these, 1,4-phenylene group is preferably mentioned from the viewpoint of GST inhibitory activity. The phenylene group may also have 1 to 3 (particularly 1) substituents such as a nitro group.
一般式（1）において、R2で示される−アルキレン基−置換されていてもよいフェニレン基−で表される基とは、上記R2で示されるアルキレン基とR2で示されるフェニレン基が連結してなる二価の基であり、この限りにおいて特に制限されない。 In the general formula (1), represented by R 2 - alkylene group - optionally substituted phenylene group - and a group represented by phenylene group represented by an alkylene group and R 2 represented by the R 2 It is a concatenated divalent group and is not particularly limited as long as this is the case.
一般式（1）において、R2としては、GST阻害活性の観点から、好ましくはアルキレン基が挙げられる。 In the general formula (1), R 2 is preferably an alkylene group from the viewpoint of GST inhibitory activity.
一般式（1）において、R3で示されるアリール基としては、単環アリール基（フェニル基）及び多環（好ましくは2〜3環、より好ましくは2環）アリール基のいずれも採用することができ、例えば、フェニル基、ナフチル基、フルオレニル基、アントリル基、ビフェニリル基、テトラヒドロナフチル基、フェナントリル基等が挙げられる。該アリール基は、例えばフッ素原子、臭素原子等のハロゲン原子やその他の置換基を1〜3個（特に1個）有することもできる。 In the general formula (1), as the aryl group represented by R 3 , either a monocyclic aryl group (phenyl group) or a polycyclic (preferably 2 to 3 rings, more preferably 2 rings) aryl group shall be adopted. Examples thereof include a phenyl group, a naphthyl group, a fluorenyl group, an anthryl group, a biphenylyl group, a tetrahydronaphthyl group, a phenanthryl group and the like. The aryl group may also have 1 to 3 (particularly 1) halogen atoms such as a fluorine atom and a bromine atom and other substituents.
一般式（1）において、R3で示されるアラルキル基としては、例えば1〜3個（特に1個）の上記R3で示されるアリール基で置換されたアルキル基を採用することができる。このアルキル基は、直鎖状又は分枝鎖状のいずれでもよいが、好ましくは直鎖状である。このアルキル基の炭素数は、特に制限されないが、例えば1〜8、好ましくは1〜6、より好ましくは1〜4である。R3で示されるアラルキル基の具体例としては、例えば、ベンジル基、フェネチル基、ナフチルメチル基、フェニルベンジル基等が挙げられる。該アラルキル基は、例えばフッ素原子、臭素原子等のハロゲン原子やその他の置換基を1〜3個（特に1個）有することもできる。 In the general formula (1), as the aralkyl group represented by R 3 , for example, 1 to 3 (particularly 1) alkyl groups substituted with the aryl group represented by R 3 can be adopted. The alkyl group may be linear or branched, but is preferably linear. The number of carbon atoms of this alkyl group is not particularly limited, but is, for example, 1 to 8, preferably 1 to 6, and more preferably 1 to 4. Specific examples of the aralkyl group represented by R 3 include a benzyl group, a phenethyl group, a naphthylmethyl group, a phenylbenzyl group and the like. The aralkyl group may also have 1 to 3 (particularly 1) halogen atoms such as a fluorine atom and a bromine atom and other substituents.
一般式（1）において、R3は、GSTの各タイプへの選択性に関与することが知られている（非特許文献６等）。例えば、R3を、上記置換されていてもよいアリール基や上記置換されていてもよいアラルキル基等の嵩高い基とすることにより、GSTπ型に対する選択性をより高めることが可能である。 In the general formula (1), R 3 is known to be involved in the selectivity of GST to each type (Non-Patent Document 6 etc.). For example, by using R 3 as a bulky group such as the above-mentioned optionally substituted aryl group or the above-mentioned optionally substituted aralkyl group, it is possible to further enhance the selectivity for the GSTπ type.
一般式（1）において、R4は、ヒドロキシ基である場合に、GST中のアミノ酸と相互作用することができる。このため、R4で示される加水分解性基とは、ヒドロキシ基に比べて本発明の化合物の細胞膜透過性をより高めることができ且つ加水分解して（好ましくは生理的条件下で加水分解して）ヒドロキシ基を生じる基である限り特に制限されない。加水分解性基の具体例としては、アルコキシ基、アルコキシアルコキシ基、アシルオキシ基、アシルオキシアルコキシキ基、カーボネート基、カルバメート基、アミド基等が挙げられ、好ましくはアルコキシ基、アルコキシアルコキシ基、アシルオキシ基、アシルオキシアルコキシキ基等が挙げられ、より好ましくはアルコキシ基が挙げられ、さらに好ましくは炭素原子数１〜８（好ましくは１〜５、より好ましくは１〜３）のアルコキシ基が挙げられる。 In general formula (1), R 4 can interact with amino acids in GST when it is a hydroxy group. Therefore, the hydrolyzable group represented by R 4 can further enhance the cell membrane permeability of the compound of the present invention as compared with the hydroxy group and is hydrolyzed (preferably hydrolyzed under physiological conditions). The group is not particularly limited as long as it is a group that produces a hydroxy group. Specific examples of the hydrolyzable group include an alkoxy group, an alkoxyalkoxy group, an acyloxy group, an acyloxyalkoxy group, a carbonate group, a carbamate group, an amide group, and the like, preferably an alkoxy group, an alkoxyalkoxy group, and an acyloxy group. Examples thereof include an acyloxyalkoxy group, more preferably an alkoxy group, and further preferably an alkoxy group having 1 to 8 carbon atoms (preferably 1 to 5, more preferably 1 to 3).
一般式（1）において、R4としては、細胞膜透過性をより向上させることができるという観点から、加水分解性基であることが望ましい。一方で、細胞膜透過を必要としない実施態様の場合であれば、R4はヒドロキシ基が好ましい。 In the general formula (1), R 4 is preferably a hydrolyzable group from the viewpoint of further improving cell membrane permeability. On the other hand, in the case of an embodiment that does not require cell membrane permeation, R 4 is preferably a hydroxy group.
一般式（1）において、R5で示される加水分解性基は、R4で示される加水分解性基と同様である。 In the general formula (1), the hydrolyzable group represented by R 5 is the same as the hydrolyzable group represented by R 4 .
一般式（1）において、R5で示される機能性分子由来の基とは、機能性分子（或いは機能性分子に適当なリンカーが連結してなる分子）から1つの官能基又は原子が除かれてなる基であり、この限りにおいて特に制限されない。 In the general formula (1), the group derived from the functional molecule represented by R 5 is obtained by removing one functional group or atom from the functional molecule (or a molecule formed by linking an appropriate linker to the functional molecule). It is a basic group, and is not particularly limited as long as it is used.
本発明の化合物において、R5の部位は、GSTと直接相互作用する部位（R4）とは反対側にあるので、R5の種類が本発明の化合物の活性に与える影響は少ないと考えられる。ただ、GSTへの認識をより妨げないとの観点から、機能性分子の分子量は比較的低いことが望ましく、例えば２０００以下、好ましくは１０００以下、より好ましくは５００以下である。 In the compound of the present invention, since the site of R 5 is on the opposite side of the site that directly interacts with GST (R 4 ), it is considered that the type of R 5 has little effect on the activity of the compound of the present invention. .. However, from the viewpoint of not hindering the recognition of GST, it is desirable that the molecular weight of the functional molecule is relatively low, for example, 2000 or less, preferably 1000 or less, and more preferably 500 or less.
タンパク質タグとしては、例えばビオチン、Hisタグ、FLAGタグ、Haloタグ、MBPタグ、HAタグ、Mycタグ、V5タグ、PAタグ等が挙げられる。タンパク質タグを採用することにより、本発明の化合物を用いて、公知のアフィニティー精製方法に従って、生体内のGSTを分離することができる。分離されたGST（特にGSTπ型）の量や型を測定することにより、がんを診断することも可能である。 Examples of the protein tag include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag and the like. By adopting a protein tag, the compound of the present invention can be used to separate GST in vivo according to a known affinity purification method. It is also possible to diagnose cancer by measuring the amount and type of isolated GST (particularly GSTπ type).
蛍光分子としては、例えばフルオレセイン系蛍光化合物、ローダミン系蛍光化合物、クマリン系蛍光化合物、ピレン系蛍光化合物、シアニン系蛍光化合物、BODIPY系蛍光化合物、CyDye系蛍光化合物、ダンシル系蛍光化合物、ベンゾオキサジアゾール系蛍光化合物、希土類蛍光標識化合物等が挙げられる。蛍光分子を採用することにより、生体内のGSTが過剰発現している部位（例えばがん組織部位）を蛍光発光させることができる。 Examples of fluorescent molecules include fluorescein-based fluorescent compounds, rhodamine-based fluorescent compounds, coumarin-based fluorescent compounds, pyrene-based fluorescent compounds, cyanine-based fluorescent compounds, BODIPY-based fluorescent compounds, CyDye-based fluorescent compounds, dancil-based fluorescent compounds, and benzoxaziazole. Examples thereof include system fluorescent compounds and rare earth fluorescent labeling compounds. By adopting a fluorescent molecule, it is possible to fluoresce a site in which GST is overexpressed (for example, a cancer tissue site) in the living body.
一般式（1）において、R5としては、細胞膜透過性をより向上させることができるという観点から、加水分解性基であることが望ましい。一方で、細胞膜透過を必要としない実施態様の場合であれば、GST阻害活性の観点から、R5はヒドロキシ基が好ましい。 In the general formula (1), R 5 is preferably a hydrolyzable group from the viewpoint of further improving cell membrane permeability. On the other hand, in the case of an embodiment that does not require cell membrane permeation, a hydroxy group is preferable for R 5 from the viewpoint of GST inhibitory activity.
一般式（1）で表される化合物としては、GST阻害活性の観点から、好ましくは一般式（1A）： The compound represented by the general formula (1) is preferably the general formula (1A): from the viewpoint of GST inhibitory activity.
［式中、R2aはアルキレン基を示す。R3〜R5は前記に同じである。］
で表される化合物が挙げられ、より好ましくは一般式（1B）：
[In the formula, R 2a represents an alkylene group. R 3 to R 5 are the same as described above. ]
Examples thereof include compounds represented by, more preferably the general formula (1B) :.
［式中、R3〜R5は前記に同じである。］
で表される化合物が挙げられる。
[In the formula, R 3 to R 5 are the same as described above. ]
Examples thereof include compounds represented by.
一般式（1）で表される化合物の塩は、薬学的に許容される塩である限り、特に制限されるものではない。該塩としては、酸性塩、塩基性塩のいずれも採用することができる。酸性塩の例としては、塩酸塩、臭化水素酸塩、硫酸塩、硝酸塩、リン酸塩等の無機酸塩； 酢酸塩、プロピオン酸塩、酒石酸塩、フマル酸塩、マレイン酸塩、リンゴ酸塩、クエン酸塩、メタンスルホン酸塩、パラトルエンスルホン酸塩等の有機酸塩が挙げられ、塩基性塩の例としては、ナトリウム塩、及びカリウム塩等のアルカリ金属塩； 並びにカルシウム塩、マグネシウム塩等のアルカリ土類金属塩； アンモニアとの塩； モルホリン、ピペリジン、ピロリジン、モノアルキルアミン、ジアルキルアミン、トリアルキルアミン、モノ（ヒドロキシアルキル）アミン、ジ（ヒドロキシアルキル）アミン、トリ（ヒドロキシアルキル）アミン等の有機アミンとの塩等が挙げられる。 The salt of the compound represented by the general formula (1) is not particularly limited as long as it is a pharmaceutically acceptable salt. As the salt, either an acidic salt or a basic salt can be adopted. Examples of acidic salts are inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, phosphate; acetate, propionate, tartrate, fumarate, maleate, malic acid. Organic acid salts such as salts, citrates, methane sulfonates and paratoluene sulfonates can be mentioned, and examples of basic salts include alkali metal salts such as sodium salt and potassium salt; as well as calcium salt and magnesium. Alkaline earth metal salts such as salts; Salts with ammonia; Morphorine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono (hydroxyalkyl) amine, di (hydroxyalkyl) amine, tri (hydroxyalkyl) Examples thereof include salts with organic amines such as amines.
本発明の化合物は溶媒和物とすることもできる。溶媒としては、例えば、水や、薬学的に許容される有機溶媒（例えばエタノール、グリセロール、酢酸等）等が挙げられる。 The compound of the present invention can also be a solvate. Examples of the solvent include water, a pharmaceutically acceptable organic solvent (for example, ethanol, glycerol, acetic acid, etc.) and the like.
2．製造方法
本発明の化合物は、様々な方法で合成することができる。
2. Production Method The compound of the present invention can be synthesized by various methods.
例えば、一般式（1B）で表される化合物の内、R4及びR5がヒドロキシ基又は加水分解性基である場合の化合物については、例えば以下の反応式： For example, among the compounds represented by the general formula (1B), when R 4 and R 5 are hydroxy groups or hydrolyzable groups, for example, the following reaction formula:
［式中、R4a及びR5aは同一又は異なって加水分解性基を示す。］
に従って合成することができる。
[In the formula, R 4a and R 5a show the same or different hydrolyzable groups. ]
Can be synthesized according to.
（2-1）一般式（2）で表される化合物→一般式（1C）で表される化合物
本工程では、一般式（2）で表される化合物とエタンスルホニルフルオリドとを反応させることで、一般式（1C）で表される化合物を得ることができる。
(2-1) Compound represented by general formula (2) → Compound represented by general formula (1C) In this step, the compound represented by general formula (2) is reacted with ethanesulfonyl fluoride. Then, the compound represented by the general formula (1C) can be obtained.
エタンスルホニルフルオリドの使用量は、収率、合成の容易さ等の観点から、通常、一般式（2）で表される化合物1モルに対して0.1〜1.5モルが好ましく、0.2〜0.8モルがより好ましい。 The amount of ethanesulfonyl fluoride used is usually preferably 0.1 to 1.5 mol, preferably 0.2 to 0.8 mol, based on 1 mol of the compound represented by the general formula (2), from the viewpoints of yield, ease of synthesis and the like. More preferred.
本工程は溶媒を添加して行ってもよい。溶媒としては、例えばアセトニトリル、クロロホルム、ジクロロメタン、ジエチルエーテル、テトラヒドロフラン、酢酸エチル、アセトン、ヘキサン、N,N-ジメチルホルムアミド、ジメチルスルホキシド等の1種又は2種以上を使用することができる。これらの中でも、収率、合成の容易さ等の観点から、アセトニトリルが好ましい。 This step may be carried out by adding a solvent. As the solvent, for example, one kind or two or more kinds such as acetonitrile, chloroform, dichloromethane, diethyl ether, tetrahydrofuran, ethyl acetate, acetone, hexane, N, N-dimethylformamide, dimethyl sulfoxide and the like can be used. Among these, acetonitrile is preferable from the viewpoint of yield, ease of synthesis, and the like.
本工程においては、上記成分以外にも、本発明の効果を損なわない範囲で、適宜添加剤を使用することもできる。 In this step, in addition to the above components, additives may be appropriately used as long as the effects of the present invention are not impaired.
反応雰囲気は、通常、不活性ガス雰囲気（アルゴンガス雰囲気、窒素ガス雰囲気等）を採用し得る。反応温度は、加熱下、常温下及び冷却下のいずれでも行うことができ、通常、0〜100℃で行うことが好ましい。反応時間は特に制限されず、通常、8時間〜48時間、特に16時間〜36時間とすることができる。 As the reaction atmosphere, an inert gas atmosphere (argon gas atmosphere, nitrogen gas atmosphere, etc.) can be usually adopted. The reaction temperature can be any of heating, normal temperature and cooling, and is usually preferably 0 to 100 ° C. The reaction time is not particularly limited, and can be usually 8 hours to 48 hours, particularly 16 hours to 36 hours.
反応終了後は、必要に応じて常法にしたがって精製処理をすることもできる。また、精製処理を施さずに次の工程を行うこともできる。 After completion of the reaction, purification treatment can be carried out according to a conventional method, if necessary. Further, the next step can be performed without performing the purification treatment.
（2-2）一般式（1C）で表される化合物→一般式（1D）で表される化合物
本工程では、一般式（1C）で表される化合物と、R4a−OH及びR5a−OHとを反応させることで、一般式（1D）で表される化合物を得ることができる。
(2-2) Compound represented by general formula (1C) → Compound represented by general formula (1D) In this step, the compound represented by general formula (1C) and R 4a −OH and R 5a − By reacting with OH, a compound represented by the general formula (1D) can be obtained.
R4a−OH及びR5a−OHの使用総量は、収率、合成の容易さ等の観点から、通常、一般式（1C）で表される化合物1モルに対して10〜300モルが好ましく、30〜200モルがより好ましい。 The total amount of R 4a −OH and R 5a −OH used is usually preferably 10 to 300 mol per 1 mol of the compound represented by the general formula (1C) from the viewpoint of yield, ease of synthesis and the like. 30-200 mol is more preferred.
本工程は溶媒を添加して行ってもよい。溶媒としては、例えばアセトニトリル、クロロホルム、ジクロロメタン、ジエチルエーテル、テトラヒドロフラン、酢酸エチル、アセトン、ヘキサン、N,N-ジメチルホルムアミド、ジメチルスルホキシド等の1種又は2種以上を使用することができる。 This step may be carried out by adding a solvent. As the solvent, for example, one kind or two or more kinds such as acetonitrile, chloroform, dichloromethane, diethyl ether, tetrahydrofuran, ethyl acetate, acetone, hexane, N, N-dimethylformamide, dimethyl sulfoxide and the like can be used.
本工程においては、上記成分以外にも、本発明の効果を損なわない範囲で、適宜添加剤を使用することもできる。例えば、塩化チオニル等のハロゲン化剤や、脱水剤、縮合剤等が挙げられる。 In this step, in addition to the above components, additives may be appropriately used as long as the effects of the present invention are not impaired. For example, a halogenating agent such as thionyl chloride, a dehydrating agent, a condensing agent and the like can be mentioned.
或いは、R4a及びR5aがメトキシ基である場合であれば、本工程は、一般式（1C）で表される化合物と、トリメチルシリルジアゾメタンやジアゾメタン等のメチル化剤とを反応させることによっても行うことができる。この際、メタノールを添加することにより、効率的に目的化合物を得ることが可能である。 Alternatively, if R 4a and R 5a are methoxy groups, this step is also carried out by reacting the compound represented by the general formula (1C) with a methylating agent such as trimethylsilyldiazomethane or diazomethane. be able to. At this time, the target compound can be efficiently obtained by adding methanol.
反応雰囲気は、通常、不活性ガス雰囲気（アルゴンガス雰囲気、窒素ガス雰囲気等）を採用し得る。反応温度は、加熱下、常温下及び冷却下のいずれでも行うことができ、通常、0〜100℃（特に15〜50℃）で行うことが好ましい。反応時間は特に制限されず、通常、30分間〜12時間、特に2時間〜8時間とすることができる。 As the reaction atmosphere, an inert gas atmosphere (argon gas atmosphere, nitrogen gas atmosphere, etc.) can be usually adopted. The reaction temperature can be any of heating, normal temperature and cooling, and is usually preferably 0 to 100 ° C. (particularly 15 to 50 ° C.). The reaction time is not particularly limited, and can be usually 30 minutes to 12 hours, particularly 2 hours to 8 hours.
反応終了後は、必要に応じて常法にしたがって精製処理をすることもできる。また、精製処理を施さずに次の工程を行うこともできる。 After completion of the reaction, purification treatment can be carried out according to a conventional method, if necessary. Further, the next step can be performed without performing the purification treatment.
（2-3）その他の化合物の合成方法
上記した以外の一般式（1）で表される化合物についても、公知の方法に従って又は準じて合成することができる。
(2-3) Method for synthesizing other compounds Compounds represented by the general formula (1) other than the above can also be synthesized according to or according to a known method.
例えば、R4及び／又はR5が機能性分子由来の基である場合の化合物については、必要に応じてアミノ基等を保護した上で、R4及び／又はR5がヒドロキシ基である化合物の該ヒドロキシ基と、機能性分子（或いは機能性分子に適当なリンカーが連結してなる分子）のアミノ基とをペプチド結合させることにより合成することができる。この際、必要に応じて縮合剤を添加することが望ましい。 For example, for a compound in which R 4 and / or R 5 is a group derived from a functional molecule, a compound in which R 4 and / or R 5 is a hydroxy group after protecting an amino group or the like as necessary. It can be synthesized by peptide-bonding the hydroxy group of the above to the amino group of a functional molecule (or a molecule formed by linking an appropriate linker to the functional molecule). At this time, it is desirable to add a condensing agent as needed.
例えば、R4及び／又はR5がアシルオキシアルコキシキ基である場合の化合物については、以下の反応式： For example, for a compound in which R 4 and / or R 5 is an acyloxyalkoxy group, the following reaction formula:
に従って又は準じて合成することができる。 Can be synthesized according to or according to.
例えば、R1が塩素原子又は臭素原子である場合の化合物については、以下の反応式： For example, for a compound where R 1 is a chlorine atom or a bromine atom, the following reaction formula:
に従って又は準じて合成することができる。 Can be synthesized according to or according to.
例えば、R2が−アルキレン基−置換されていてもよいフェニレン基−で表される基、又は置換されていてもよいフェニレン基である場合の化合物は、以下の反応式： For example, when R 2 is a-alkylene group-a group represented by an optionally substituted phenylene group-or a optionally substituted phenylene group, the compound has the following reaction formula:
又は以下の反応式： Or the following reaction formula:
に従って又は準じて合成することができる。 Can be synthesized according to or according to.
例えば、R3が置換されていてもよいアリール基、又は置換されていてもよいアラルキル基である場合の化合物は、以下の反応式： For example, when R 3 is an optionally substituted aryl group or an optionally substituted aralkyl group, the compound has the following reaction formula:
又は以下の反応式： Or the following reaction formula:
又は以下の反応式： Or the following reaction formula:
に従って又は準じて合成することができる。 Can be synthesized according to or according to.
例えば、R2が各種炭素原子数のアルキレン基である場合の化合物は、以下の反応式： For example, when R 2 is an alkylene group having various carbon atoms, the compound has the following reaction formula:
に従って又は準じて合成することができる。 Can be synthesized according to or according to.
例えば、R1が−O−R12である場合の化合物は、以下の反応式： For example, when R 1 is −O−R 12 , the compound has the following reaction formula:
に従って又は準じて合成することができる。 Can be synthesized according to or according to.
例えば、R1が−S−R12である場合の化合物は、以下の反応式： For example, when R 1 is −S−R 12 , the compound has the following reaction formula:
に従って又は準じて合成することができる。 Can be synthesized according to or according to.
3．用途
本発明の化合物は、GSHの存在下であっても（特に高濃度に存在する場合であっても）、効率的にGSTを阻害することができる。このため、本発明の化合物又はその塩は、各種生物試験用試薬、医薬組成物等として、特にGST阻害剤、抗がん剤等として有用である。また、本発明の化合物は、GSTと不可逆的に結合すると考えられ、このため該化合物を用いて公知のアフィニティー精製方法に従えば組織からGSTを分離することができると考えられる。分離されたGST（特にGSTπ型）の量や型を測定することにより、がんを診断することも可能である。よって、本発明の化合物又はその塩は、GST分離剤、がん診断剤等としても有用である。以下、これらを総称して「本発明の剤」と示すこともある。
3. 3. Applications The compounds of the present invention can efficiently inhibit GST even in the presence of GSH (especially in high concentrations). Therefore, the compound of the present invention or a salt thereof is useful as a reagent for various biological tests, a pharmaceutical composition, etc., particularly as a GST inhibitor, an anticancer agent, and the like. In addition, the compound of the present invention is considered to irreversibly bind to GST, and therefore it is considered that GST can be separated from the tissue according to a known affinity purification method using the compound. It is also possible to diagnose cancer by measuring the amount and type of isolated GST (particularly GSTπ type). Therefore, the compound of the present invention or a salt thereof is also useful as a GST separating agent, a cancer diagnostic agent, and the like. Hereinafter, these may be collectively referred to as "the agent of the present invention".
本発明の剤が阻害する（或いは共有結合する）標的となるGSTの型は特に制限されない。GSTの型としては、例えばヒトの場合であれば、α型、μ型、π型、θ型、ζ型、ω型、σ型等が挙げられ、好ましくはπ型が挙げられる。 The type of GST targeted by the agent of the present invention to inhibit (or covalently bind) is not particularly limited. Examples of the GST type include α-type, μ-type, π-type, θ-type, ζ-type, ω-type, and σ-type in the case of humans, and π-type is preferable.
本発明の剤は、本発明の化合物を含有する限りにおいて特に制限されず、必要に応じてさらに他の成分を含んでいてもよい。他の成分としては、薬学的に許容される成分であれば特に限定されるものではないが、例えば基剤、担体、溶剤、分散剤、乳化剤、緩衝剤、安定剤、賦形剤、結合剤、崩壊剤、滑沢剤、増粘剤、保湿剤、着色料、香料、キレート剤等が挙げられる。 The agent of the present invention is not particularly limited as long as it contains the compound of the present invention, and may further contain other components if necessary. The other components are not particularly limited as long as they are pharmaceutically acceptable components, but are, for example, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, and binders. , Disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents and the like.
本発明の剤の使用態様は、特に制限されず、その種類に応じて適切な使用態様を採ることができる。本発明の剤は、例えばin vitroで使用する（例えば、培養細胞の培地に添加する）こともできるし、in vivoで使用する（例えば、動物に投与する）こともできる。 The mode of use of the agent of the present invention is not particularly limited, and an appropriate mode of use can be adopted according to the type of the agent. The agent of the present invention can be used, for example, in vitro (for example, added to the medium of cultured cells) or in vivo (for example, administered to an animal).
本発明の剤の適用対象は特に限定されず、例えば、ヒト、サル、マウス、ラット、イヌ、ネコ、ウサギ等の種々の哺乳類動物； 動物細胞等が挙げられる。細胞の種類も特に制限されず、例えば血液細胞、造血幹細胞・前駆細胞、配偶子（精子、卵子）、線維芽細胞、上皮細胞、血管内皮細胞、神経細胞、肝細胞、ケラチン生成細胞、筋細胞、表皮細胞、内分泌細胞、ES細胞、iPS細胞、組織幹細胞、がん細胞等が挙げられる。 The application target of the agent of the present invention is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits; animal cells and the like. The type of cell is not particularly limited, and for example, blood cell, hematopoietic stem cell / precursor cell, sperm (sperm, egg), fibroblast, epithelial cell, vascular endothelial cell, nerve cell, hepatocyte, keratin-producing cell, muscle cell , Epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells and the like.
本発明の剤を抗がん剤として用いる場合、そのがん細胞の種類は特に限定されず、例えば腎臓がん細胞、白血病細胞、食道がん細胞、胃がん細胞、大腸がん細胞、肝臓がん細胞、すい臓がん細胞、肺がん細胞、前立腺がん細胞、皮膚がん細胞、乳がん細胞、子宮頚がん細胞等が挙げられる。 When the agent of the present invention is used as an anticancer agent, the type of cancer cells is not particularly limited, and for example, kidney cancer cells, leukemia cells, esophageal cancer cells, gastric cancer cells, colon cancer cells, liver cancer. Examples thereof include cells, pancreatic cancer cells, lung cancer cells, prostate cancer cells, skin cancer cells, breast cancer cells, cervical cancer cells and the like.
本発明の剤の剤形は特に制限されず、その使用態様に応じて適切な剤形を採ることができる。例えば、動物に投与する場合であれば、例えば錠剤、カプセル剤、顆粒剤、散剤、細粒剤、シロップ剤、腸溶剤、徐方性カプセル剤、咀嚼錠、ドロップ、丸剤、内用液剤、菓子錠剤、徐放剤、徐放性顆粒剤等の経口剤； 点鼻剤、吸入剤、肛門坐剤、挿入剤、浣腸剤、ゼリー剤等の外用剤等が挙げられる。また、本発明の剤は、固形剤、半固形剤、液剤のいずれでもよい。 The dosage form of the agent of the present invention is not particularly limited, and an appropriate dosage form can be taken according to the mode of use. For example, when administered to animals, for example, tablets, capsules, granules, powders, fine granules, syrups, enema, slow-moving capsules, chewing tablets, drops, pills, internal solutions, Oral preparations such as confectionery tablets, sustained-release agents, sustained-release granules; external preparations such as nasal drops, inhalants, anal suppositories, inserts, enemas, and jelly agents can be mentioned. Further, the agent of the present invention may be a solid agent, a semi-solid agent, or a liquid agent.
本発明の剤中の本発明の化合物の含有量は、使用態様、適用対象、適用対象の状態等に左右されるものであり、限定はされないが、例えば0.0001〜100重量％、好ましくは0.001〜50重量％とすることができる。 The content of the compound of the present invention in the agent of the present invention depends on the mode of use, the object of application, the state of the object of application, etc., and is not limited, but is, for example, 0.0001 to 100% by weight, preferably 0.001 to 0.001. It can be 50% by weight.
本発明の剤を動物に投与する場合の投与量は、薬効を発現する有効量であれば特に限定されず、通常は、有効成分である本発明の化合物の重量として、一般に経口投与の場合には一日あたり0.1〜1000 mg/kg体重、好ましくは一日あたり0.5〜50 mg/kg体重であり、非経口投与の場合には一日あたり0.01〜100 mg/kg体重、好ましくは0.1〜10 mg/kg体重である。上記投与量は1日1回又は2〜3回に分けて投与するのが好ましく、年齢、病態、症状により適宜増減することもできる。 The dose of the agent of the present invention when administered to an animal is not particularly limited as long as it is an effective amount that exerts a medicinal effect, and is usually the weight of the compound of the present invention as an active ingredient, generally in the case of oral administration. Is 0.1 to 1000 mg / kg body weight per day, preferably 0.5 to 50 mg / kg body weight per day, and 0.01 to 100 mg / kg body weight per day, preferably 0.1 to 10 for parenteral administration. It is mg / kg body weight. The above dose is preferably administered once a day or divided into 2 to 3 times, and can be appropriately increased or decreased depending on the age, pathological condition, and symptoms.
以下に、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例によって限定されるものではない。 Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.
実施例1：GS-ESFの合成Example 1: Synthesis of GS-ESF
丸底フラスコにグルタチオン (1.53 g, 4.97 mmol) 及び水 (18.0 mL) を加え、アルゴンバブリングを5分間行った。攪拌しながらエタンスルホニルフルオリド (148.4 μL, 1.79 mmol) のアセトニトリル溶液 (2.0 mL) を添加し、そのまま室温で24時間攪拌した。メンブランフィルター (0.45 μm) でろ過し、分取RP-HPLCクロマトグラフィー [Hydosphere C18, 250 × 20 mm, 5 μm, UV 220 nm, 10.0 mL/min, 水 (1‰ トリフルオロ酢酸)/アセトニトリル (1‰トリフルオロ酢酸)] で精製して、目的化合物を白色固体として得た (1.05 g, 51%) 。1H NMR (400 MHz, D2O) δ: 4.64 (1H, dd, J= 8.8, 8.6 Hz), 4.07―3.98 (5H, m), 3.17―3.10 (3H, m), 2.97 (1H, dd, J= 14.4, 8.8 Hz), 2.62―2.55 (2H, m), 2.26―2.20 (2H, m); 13C NMR (100 MHz, D2O) δ: 174.5, 173.0, 172.5, 172.0, 52.9, 52.6, 50.4, 50.3, 41.2, 32.9, 31.0, 25.6, 24.6。 Glutathione (1.53 g, 4.97 mmol) and water (18.0 mL) were added to a round bottom flask, and argon bubbling was performed for 5 minutes. Acetonitrile solution (2.0 mL) of ethanesulfonyl fluoride (148.4 μL, 1.79 mmol) was added with stirring, and the mixture was stirred as it was at room temperature for 24 hours. Filter through a membrane filter (0.45 μm) and perform preparative RP-HPLC chromatography [Hydosphere C18, 250 × 20 mm, 5 μm, UV 220 nm, 10.0 mL / min, water (1 ‰ trifluoroacetic acid) / acetonitrile (1) Purification with ‰ trifluoroacetic acid)] gave the target compound as a white solid (1.05 g, 51%). 1 H NMR (400 MHz, D 2 O) δ: 4.64 (1H, dd, J = 8.8, 8.6 Hz), 4.07-3.98 (5H, m), 3.17-3.10 (3H, m), 2.97 (1H, dd) , J = 14.4, 8.8 Hz), 2.62-2.55 (2H, m), 2.26-2.20 (2H, m); 13 C NMR (100 MHz, D 2 O) δ: 174.5, 173.0, 172.5, 172.0, 52.9, 52.6, 50.4, 50.3, 41.2, 32.9, 31.0, 25.6, 24.6.
実施例2：GS-ESF methyl esterの合成Example 2: Synthesis of GS-ESF methyl ester
GS-ESF (28.0 mg, 0.0671 mmol) のジクロロホルム:メタノール (4:1, 0.7 mL)溶液を、0℃、アルゴン雰囲気下で攪拌した。そこへ、トリメチルシリルジアゾメタン (80.6 μL, 2.0 M in hexane) を滴下して、得られた溶液を25℃で4時間攪拌した。薄層クロマトグラフィーにより反応を終了させた。濃縮後、得られた残渣を分取RP-HPLCクロマトグラフィー [Hydosphere C18, 250 × 20 mm, 5 μm, UV 220 nm, 10.0 mL/min, 水 (1‰トリフルオロ酢酸)/アセトニトリル (1‰トリフルオロ酢酸)] で精製して、目的化合物を白色固体として得た (16.3 mg, 55%) 。1H NMR (400 MHz, CD3OD) δ: 4.63 (1H, dd, J = 8.6, 8.4 Hz), 4.12 (1H, t, J= 6.8 Hz), 3.98―3.92 (4H, m), 3.85 (3H, s), 3.73 (3H, s), 3.12―3.05 (3H, m), 2.86 (1H, dd, J = 14.4, 8.4 Hz), 2.57―2.53 (2H, m), 2.24―2.16 (2H, m); 13C NMR (100 MHz, CD3OD) δ: 172.8, 171.5, 170.3, 169.3, 52.4, 52.2, 51.4, 50.4, 50.2, 40.5, 33.4, 30.8, 25.6, 24.7。 A solution of GS-ESF (28.0 mg, 0.0671 mmol) in dichloroform: methanol (4: 1, 0.7 mL) was stirred at 0 ° C. under an argon atmosphere. Trimethylsilyldiazomethane (80.6 μL, 2.0 M in hexane) was added dropwise thereto, and the obtained solution was stirred at 25 ° C. for 4 hours. The reaction was terminated by thin layer chromatography. After concentration, the obtained residue was collected by preparative RP-HPLC chromatography [Hydosphere C18, 250 × 20 mm, 5 μm, UV 220 nm, 10.0 mL / min, water (1 ‰ trifluoroacetic acid) / acetonitrile (1 ‰ tri). Purification with (fluoroacetic acid)] gave the target compound as a white solid (16.3 mg, 55%). 1 H NMR (400 MHz, CD 3 OD) δ: 4.63 (1H, dd, J = 8.6, 8.4 Hz), 4.12 (1H, t, J = 6.8 Hz), 3.98-3.92 (4H, m), 3.85 ( 3H, s), 3.73 (3H, s), 3.12-3.05 (3H, m), 2.86 (1H, dd, J = 14.4, 8.4 Hz), 2.57−2.53 (2H, m), 2.24-2.16 (2H, s) m); 13 C NMR (100 MHz, CD 3 OD) δ: 172.8, 171.5, 170.3, 169.3, 52.4, 52.2, 51.4, 50.4, 50.2, 40.5, 33.4, 30.8, 25.6, 24.7.
実施例3：GS-ESF ethyl esterの合成Example 3: Synthesis of GS-ESF ethyl ester
GS-ESF (44.7 mg, 0.107 mmol) のエタノール (1.1 mL) 溶液に、0℃、不活性雰囲気下で塩化チオニル (23.3 μL, 0.321 mmol) を滴下した。得られた溶液を還流しながら2時間攪拌し、濃縮した。残渣を分取RP-HPLCクロマトグラフィー [Hydosphere C18, 250 × 20 mm, 5 μm, UV 220 nm, 10.0 mL/min, 水 (1‰トリフルオロ酢酸)/アセトニトリル (1‰トリフルオロ酢酸)] で精製して、目的化合物を白色固体として得た (43.0 mg, 68%) 。1H NMR (400 MHz, CD3OD) δ: 4.63 (1H, dd, J = 8.8, 8.6 Hz), 4.31 (2H, q, J= 7.2 Hz), 4.18 (2H, q, J = 7.2 Hz), 4.10 (1H, t, J = 6.8 Hz), 3.97―3.91 (4H, m), 3.13―3.04 (3H, m), 2.86 (1H, dd, J = 14.4, 8.8 Hz), 2.56 (2H, t, J = 6.8 Hz), 2.25―2.16 (2H, m), 1.33 (3H, t, J = 7.2 Hz), 1.27 (3H, t, J = 7.2 Hz); 13C NMR (100 MHz, CD3OD) δ: 172.2, 170.9, 169.1, 168.2, 61.8, 60.4, 52.0, 51.6, 49.7, 49.6, 40.1, 32.7, 30.1, 25.0, 24.1, 12.4, 12.3。 Thionyl chloride (23.3 μL, 0.321 mmol) was added dropwise to a solution of GS-ESF (44.7 mg, 0.107 mmol) in ethanol (1.1 mL) at 0 ° C. under an inert atmosphere. The resulting solution was stirred with reflux for 2 hours and concentrated. Residues purified by preparative RP-HPLC chromatography [Hydosphere C18, 250 x 20 mm, 5 μm, UV 220 nm, 10.0 mL / min, water (1 ‰ trifluoroacetic acid) / acetonitrile (1 ‰ trifluoroacetic acid)] The target compound was obtained as a white solid (43.0 mg, 68%). 1 H NMR (400 MHz, CD 3 OD) δ: 4.63 (1H, dd, J = 8.8, 8.6 Hz), 4.31 (2H, q, J = 7.2 Hz), 4.18 (2H, q, J = 7.2 Hz) , 4.10 (1H, t, J = 6.8 Hz), 3.97-3.91 (4H, m), 3.13-3.04 (3H, m), 2.86 (1H, dd, J = 14.4, 8.8 Hz), 2.56 (2H, t) , J = 6.8 Hz), 2.25-2.16 (2H, m), 1.33 (3H, t, J = 7.2 Hz), 1.27 (3H, t, J = 7.2 Hz); 13 C NMR (100 MHz, CD 3 OD) ) δ: 172.2, 170.9, 169.1, 168.2, 61.8, 60.4, 52.0, 51.6, 49.7, 49.6, 40.1, 32.7, 30.1, 25.0, 24.1, 12.4, 12.3.
合成例1：9-BBN protected GS-ESFの合成Synthesis example 1: Synthesis of 9-BBN protected GS-ESF
GS-ESF (613.6 mg, 1.47 mmol) を、アルゴン雰囲気下でメタノール (9.8 mL) に懸濁した。そこへ、9-ボラビシクロ[3.3.1]ノナン (3.2 mL, 0.5 M in テトラヒドロフラン) を滴下した。得られた溶液を還流しながら3時間攪拌した。ニンヒドリン染色型薄層クロマトグラフィー (4:1:1 ブタノール/酢酸/水) による評価の結果、両方が保護されたアミノ酸への変換はほぼ定量的であった。残渣を分取RP-HPLCクロマトグラフィー [Hydosphere C18, 250 × 20 mm, 5 μm, UV 220 nm, 10.0 mL/min, 水 (1‰トリフルオロ酢酸)/アセトニトリル (1‰トリフルオロ酢酸)] で精製して、目的化合物を白色固体として得た (616.8 mg, 78%) 。1H NMR (400 MHz, CD3OD) δ: 6.39―6.29 (2H, m), 4.64 (1H, dd, J = 8.8, 8.8 Hz), 3.99―3.88 (4H, m), 3.80―3.74 (1H, m), 3.14―3.01 (3H, m), 2.84 (1H, dd, J = 14.4, 8.8 Hz), 2.65―2.55 (2H, m), 2.26―2.19 (1H, m), 2.14―2.07 (1H, m), 1.91―1.65 (10H, m), 1.48―1.45 (2H, m), 0.56 (2H, s); 13C NMR (100 MHz, CD3OD) δ: 176.9, 175.3, 172.8, 56.2, 53.8, 51.7, 51.6, 41.9, 34.7, 33.1, 32.5, 32.4, 32.3, 27.3, 26.0, 25.7, 25.2。 GS-ESF (613.6 mg, 1.47 mmol) was suspended in methanol (9.8 mL) under an argon atmosphere. 9-Borabicyclo [3.3.1] nonane (3.2 mL, 0.5 M in tetrahydrofuran) was added dropwise thereto. The resulting solution was stirred with reflux for 3 hours. As a result of evaluation by ninhydrin-stained thin layer chromatography (4: 1: 1 butanol / acetic acid / water), conversion to amino acids in which both were protected was almost quantitative. Residues purified by preparative RP-HPLC chromatography [Hydosphere C18, 250 x 20 mm, 5 μm, UV 220 nm, 10.0 mL / min, water (1 ‰ trifluoroacetic acid) / acetonitrile (1 ‰ trifluoroacetic acid)] The target compound was obtained as a white solid (616.8 mg, 78%). 1 H NMR (400 MHz, CD 3 OD) δ: 6.39-6.29 (2H, m), 4.64 (1H, dd, J = 8.8, 8.8 Hz), 3.99-3.88 (4H, m), 3.80-3.74 (1H) , m), 3.14-3.01 (3H, m), 2.84 (1H, dd, J = 14.4, 8.8 Hz), 2.65-2.55 (2H, m), 2.26-2.19 (1H, m), 2.14-2.07 (1H) , m), 1.91-1.65 (10H, m), 1.48-1.45 (2H, m), 0.56 (2H, s); 13 C NMR (100 MHz, CD 3 OD) δ: 176.9, 175.3, 172.8, 56.2, 53.8, 51.7, 51.6, 41.9, 34.7, 33.1, 32.5, 32.4, 32.3, 27.3, 26.0, 25.7, 25.2.
実施例4：GS-ESF biotinの合成Example 4: Synthesis of GS-ESF biotin
N-ビオチニル-3,6-ジオキサオクタン-1,8-ジアミンのトリフルオロ酢酸塩 (281.5 mg, 0.752 mmol) をN,N-ジメチルホルムアミド (5.0 mL) に溶解して、トリメチルアミン (85.0 μL, 0.610 mmol)を用いてpHを8に調整した。得られた溶液に、9-BBN protected GS-ESF (269.3 mg, 0.501 mmol), 1-ヒドロキシベンゾトリアゾール (101.6 mg, 0.752 mmol) 及び 1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩 (144.1 mg, 0.752 mmol) を添加し、室温下で40時間攪拌した。反応混合物を真空下で濃縮し、フラッシュカラムクロマトグラフィーで精製して、中間体化合物（9-BBN protected GS-ESF biotin）を白色固体として得た (191.0 mg, 43%) 。1H NMR (400 MHz, CD3OD) δ: 8.55―8.48 (1H, m), 6.39―6.25 (2H, m), 4.59 (1H, dd, J = 7.2, 7.2 Hz), 4.50 (1H, dd, J = 7.8, 7.6 Hz), 4.32 (1H, dd, J = 7.8, 7.6 Hz), 3.99―3.94 (2H, m), 3.89 (2H, s), 3.84―3.77 (1H, m), 3.62 (4H, s), 3.56 (4H, ddd, J = 5.2 Hz), 3.41―3.37 (4H, m), 3.21 (1H, dt, J = 4.8, 4.8, 4.4, 4.4 Hz), 3.13―3.04 (3H, m), 2.96―2.86 (2H, m), 2.72 (1H, d, J= 12.8 Hz), 2.63―2.59 (2H, m), 2.27―2.21 (3H, m), 2.14―2.07 (1H, m), 1.89―1.85 (4H, m), 1.79―1.56 (10H, m), 1.48―1.41 (4H, m), 0.57 (2H, s); 13C NMR (100 MHz, CD3OD) δ: 174.9, 174.1, 173.4, 170.9, 169.2, 164.0, 69.3, 69.2, 68.6, 68.4, 61.3, 59.6, 55.0, 54.1, 52.2, 49.7, 49.5, 41.5, 39.1, 38.4, 38.3, 34.8, 32.4, 31.0, 30.5, 30.4, 30.3, 27.7, 27.5, 25.4, 24.9, 24.0, 23.7, 23.2。 Trifluoroacetic acid salt (281.5 mg, 0.752 mmol) of N-biotinyl-3,6-dioxaoctane-1,8-diamine was dissolved in N, N-dimethylformamide (5.0 mL) and trimethylamine (85.0 μL, 0.610 mmol) was used to adjust the pH to 8. In the resulting solution, 9-BBN protected GS-ESF (269.3 mg, 0.501 mmol), 1-hydroxybenzotriazole (101.6 mg, 0.752 mmol) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (144.1 mg, 0.752 mmol) was added, and the mixture was stirred at room temperature for 40 hours. The reaction mixture was concentrated under vacuum and purified by flash column chromatography to give the intermediate compound (9-BBN protected GS-ESF biotin) as a white solid (191.0 mg, 43%). 1 H NMR (400 MHz, CD 3 OD) δ: 8.55-8.48 (1H, m), 6.39-6.25 (2H, m), 4.59 (1H, dd, J = 7.2, 7.2 Hz), 4.50 (1H, dd) , J = 7.8, 7.6 Hz), 4.32 (1H, dd, J = 7.8, 7.6 Hz), 3.99-3.94 (2H, m), 3.89 (2H, s), 3.84-3.77 (1H, m), 3.62 ( 4H, s), 3.56 (4H, ddd, J = 5.2 Hz), 3.41-3.37 (4H, m), 3.21 (1H, dt, J = 4.8, 4.8, 4.4, 4.4 Hz), 3.13-3.04 (3H, s) m), 2.96-2.86 (2H, m), 2.72 (1H, d, J = 12.8 Hz), 2.63-2.59 (2H, m), 2.27-2.21 (3H, m), 2.14-2.07 (1H, m) , 1.89-1.85 (4H, m), 1.79-1.56 (10H, m), 1.48-1.41 (4H, m), 0.57 (2H, s); 13 1 C NMR (100 MHz, CD 3 OD) δ: 174.9, 174.1, 173.4, 170.9, 169.2, 164.0, 69.3, 69.2, 68.6, 68.4, 61.3, 59.6, 55.0, 54.1, 52.2, 49.7, 49.5, 41.5, 39.1, 38.4, 38.3, 34.8, 32.4, 31.0, 30.5, 30.4, 30.3, 27.7, 27.5, 25.4, 24.9, 24.0, 23.7, 23.2.
9-BBN protected GS-ESF biotin (5.2 mg, 5.82 μmol) をジオキサン:水 (1:2.7) 混合物に溶解し、そこに12 N 塩酸水溶液 (24.0 μL, 0.291 mmol) を添加した。得られた溶液を40℃で2日間攪拌した。反応混合物を濃縮し、分取RP-HPLC クロマトグラフィー [Hydosphere C18, 250 × 20 mm, 5 μm, UV 220 nm, 10.0 mL/min, 水 (1‰トリフルオロ酢酸)/アセトニトリル (1‰トリフルオロ酢酸)] で精製して、目的化合物を白色固体として得た (0.6 mg, 12%) 。1H NMR (400 MHz, D2O) δ: 4.62 (2H, dd, J= 8.4, 8.2 Hz), 4.43 (1H, dd, J = 8.4, 8.2 Hz), 4.07―4.02 (2H, m), 3.94 (2H, s), 3.84 (1H, dd, J = 6.8, 6.4 Hz), 3.69 (4H, s), 3.64 (4H, dd, J = 5.2, 4.8 Hz), 3.45―3.39 (4H, m), 3.37―3.32 (1H, m), 3.17―3.12 (3H, m), 3.03―2.94 (2H, m), 2.79 (1H, d, J = 13.2 Hz), 2.55 (2H, q, J = 7.6 Hz), 2.29 (2H, t, J = 7.6 Hz), 2.18 (2H, dd, J = 7.6, 6.8 Hz), 1.75―1.60 (4H, m), 1.46―1.41 (2H, m); 13C NMR (100 MHz, D2O) δ: 177.0, 174.4, 172.5, 171.6, 171.0, 165.4, 69.5, 68.9, 68.8, 62.1, 60.3, 55.4, 53.1, 52.3, 50.4, 50.3, 42.6, 39.7, 39.0, 38.9, 35.5, 32.8, 30.9, 27.9, 27.7, 25.5, 25.2, 24.6. 。 9-BBN protected GS-ESF biotin (5.2 mg, 5.82 μmol) was dissolved in a mixture of dioxane: water (1: 2.7), to which a 12 N aqueous hydrochloric acid solution (24.0 μL, 0.291 mmol) was added. The resulting solution was stirred at 40 ° C. for 2 days. Concentrate the reaction mixture and perform preparative RP-HPLC chromatography [Hydosphere C18, 250 x 20 mm, 5 μm, UV 220 nm, 10.0 mL / min, water (1 ‰ trifluoroacetic acid) / acetonitrile (1 ‰ trifluoroacetic acid) )] Purified to obtain the target compound as a white solid (0.6 mg, 12%). 1 H NMR (400 MHz, D 2 O) δ: 4.62 (2H, dd, J = 8.4, 8.2 Hz), 4.43 (1H, dd, J = 8.4, 8.2 Hz), 4.07-4.02 (2H, m), 3.94 (2H, s), 3.84 (1H, dd, J = 6.8, 6.4 Hz), 3.69 (4H, s), 3.64 (4H, dd, J = 5.2, 4.8 Hz), 3.45-3.39 (4H, m) , 3.37―3.32 (1H, m), 3.17―3.12 (3H, m), 3.03-2.94 (2H, m), 2.79 (1H, d, J = 13.2 Hz), 2.55 (2H, q, J = 7.6 Hz) ), 2.29 (2H, t, J = 7.6 Hz), 2.18 (2H, dd, J = 7.6, 6.8 Hz), 1.75-1.60 (4H, m), 1.46-1.41 (2H, m); 13 C NMR ( 100 MHz, D 2 O) δ: 177.0, 174.4, 172.5, 171.6, 171.0, 165.4, 69.5, 68.9, 68.8, 62.1, 60.3, 55.4, 53.1, 52.3, 50.4, 50.3, 42.6, 39.7, 39.0, 38.9, 35.5 , 32.8, 30.9, 27.9, 27.7, 25.5, 25.2, 24.6.
実施例5：GS-ESF BODIPYの合成Example 5: Synthesis of GS-ESF BODIPY
Amine-terminated BODIPY (8.5 mg, 0.0340 mmol) をN,N-ジメチルホルムアミド (0.68 mL) に溶解した。得られた溶液に、GS-ESF (14.2 mg, 0.0340 mmol), 1-ヒドロキシベンゾトリアゾール (4.6 mg, 0.0340 mmol), 1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩 (6.5 mg, 0.0.340 mmol) 及びトリメチルアミン (9.7 μL, 0.0680 mmol) を0℃で添加し、室温下で1時間攪拌した。1時間後、反応混合物に50 mM TEAA 緩衝液 (5% アセトニトリル) を添加して、得られた反応混合物を0.45 μm 親水性 PTFE膜フィルターでろ過した。ろ液を分取RP-HPLC クロマトグラフィー [Hydosphere C18, 250 ×20 mm, 5 μm, UV 400 nm, 10.0 mL/min, 50 mM TEAA 緩衝液 (5% アセトニトリル)/アセトニトリル] で精製して、目的化合物を白色固体として得た (0.5 mg, 2% yield) 。 1H NMR (400 MHz, CD3OD) δ: 7.58 (1H, bs), 7.41―7.32 (3H, m), 6.56 (1H, bs), 6.40 (1H, bs), 4.60 (2H, bs), 4.50 (1H, dd, J = 5.6, 5.2 Hz), 3.95―3.89 (5H, m), 3.73―3.70 (2H, m), 3.64―3.59 (2H, m), 3.101―3.00 (3H, m), 2.86 (1H, dd, J = 8.8 Hz), 2.52―2.48 (2H, m), 2.19―2.12 (1H, m), 2.06―2.03 (1H, m),; 13C NMR (100 MHz, CD3OD) δ: 172.6, 171.1, 170.8, 169.6, 154.0, 130.0, 129.5, 123.8, 123.1, 122.3, 119.6, 112.2, 112.1. 52.3, 51.5, 49.7, 49.5, 47.3, 41.7, 37.7, 32.4, 30.1, 25.0, 24.1。 Amine-terminated BODIPY (8.5 mg, 0.0340 mmol) was dissolved in N, N-dimethylformamide (0.68 mL). In the resulting solution, GS-ESF (14.2 mg, 0.0340 mmol), 1-hydroxybenzotriazole (4.6 mg, 0.0340 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (6.5 mg, 0.0.340 mmol) and trimethylamine (9.7 μL, 0.0680 mmol) were added at 0 ° C., and the mixture was stirred at room temperature for 1 hour. After 1 hour, 50 mM TEAA buffer (5% acetonitrile) was added to the reaction mixture and the resulting reaction mixture was filtered through a 0.45 μm hydrophilic PTFE membrane filter. The filtrate is purified by preparative RP-HPLC chromatography [Hydosphere C18, 250 × 20 mm, 5 μm, UV 400 nm, 10.0 mL / min, 50 mM TEAA buffer (5% acetonitrile) / acetonitrile] for the purpose. The compound was obtained as a white solid (0.5 mg, 2% yield). 1 H NMR (400 MHz, CD 3 OD) δ: 7.58 (1H, bs), 7.41-7.32 (3H, m), 6.56 (1H, bs), 6.40 (1H, bs), 4.60 (2H, bs), 4.50 (1H, dd, J = 5.6, 5.2 Hz), 3.95-3.89 (5H, m), 3.73-3.70 (2H, m), 3.64-3.59 (2H, m), 3.101-3.00 (3H, m), 2.86 (1H, dd, J = 8.8 Hz), 2.52-2.48 (2H, m), 2.19-2.12 (1H, m), 2.06-2.03 (1H, m) ,; 13 C NMR (100 MHz, CD 3 OD) ) δ: 172.6, 171.1, 170.8, 169.6, 154.0, 130.0, 129.5, 123.8, 123.1, 122.3, 119.6, 112.2, 112.1. 52.3, 51.5, 49.7, 49.5, 47.3, 41.7, 37.7, 32.4, 30.1, 25.0, 24.1 ..
実施例6：GST抑制効果解析試験1
図１の下段に示すスキームに従って、次のように試験を行った。各種濃度のGST誘導体, GSH (0.5 mM), 及びGST (4 μg/mL) を含有する1×PBS緩衝溶液 (total 500 μL) を、1分間又は30分間、プレインキュベートした。その後、2,4-ジニトロクロロベンゼン（本明細書において、CDNBと示すこともある。）(0.2 mM) を添加し、添加後の各時間においてUV波長の吸光度を測定した (光路長: 5 mm, 反応時間: 90秒, 37℃, 検出波長: 340 nm)。吸光度に基づいて以下に示す式に従って酵素反応の初速度（nmol/min）を算出した。
Example 6: GST suppression effect analysis test 1
The test was conducted as follows according to the scheme shown in the lower part of FIG. A 1 × PBS buffer solution (total 500 μL) containing various concentrations of GST derivative, GSH (0.5 mM), and GST (4 μg / mL) was pre-incubated for 1 or 30 minutes. Then, 2,4-dinitrochlorobenzene (sometimes referred to as CDNB in the present specification) (0.2 mM) was added, and the absorbance at the UV wavelength was measured at each time after the addition (optical path length: 5 mm, Reaction time: 90 seconds, 37 ° C, detection wavelength: 340 nm). The initial rate (nmol / min) of the enzymatic reaction was calculated according to the formula shown below based on the absorbance.
GST誘導体としてGS-ESFを用いた場合の結果を図１に示す。図１に示されるように、GS-ESF濃度依存的に、さらにはプレインキュベート時間依存的に、GST酵素反応が抑制された。 The results when GS-ESF is used as the GST derivative are shown in FIG. As shown in FIG. 1, the GST enzymatic reaction was suppressed in a GS-ESF concentration-dependent and further pre-incubation time-dependent manner.
実施例7：GST抑制効果解析試験2
実施例6の試験においては、GS-ESF濃度がGSH濃度よりも非常に高い。よって、実施例6の結果は、GSH（求核試薬）がGS-ESF（求電子試薬）と反応し、その結果として酵素反応が起こらなくなったことに起因する可能性がある。この点を確認すべく、GS-ESFとGSHとの反応速度を評価した。具体的には、図２の下段に示すスキームに従って、以下のように行った。
Example 7: GST suppression effect analysis test 2
In the test of Example 6, the GS-ESF concentration is much higher than the GSH concentration. Therefore, the result of Example 6 may be due to the fact that GSH (nucleophile) reacts with GS-ESF (electrophile), and as a result, the enzymatic reaction does not occur. To confirm this point, the reaction rate between GS-ESF and GSH was evaluated. Specifically, it was carried out as follows according to the scheme shown in the lower part of FIG.
各種濃度のGS-ESFを含有する1×PBS緩衝溶液 (total 500 μL)（反応溶液1）, 並びに各種濃度のGS-ESF及びGSH (0.5 mM) を含有する1×PBS緩衝溶液 (total 500 μL)（反応溶液2） を、30分間プレインキュベートした。その後、反応溶液1にはGSH (0.5 mM), CDNB (0.2 mM), 及びGSTπ (4 μg/mL) を添加し、反応溶液2にはCDNB (0.2 mM), 及びGSTπ (4 μg/mL) を添加し、添加後の各時間においてUV波長の吸光度を測定した (光路長: 5 mm, 反応時間: 90秒, 37℃, 検出波長: 340 nm)。吸光度に基づいて定法に従って酵素反応の初速度（nmol/min）を算出した。 1 × PBS buffer solution (total 500 μL) containing various concentrations of GS-ESF (reaction solution 1), and 1 × PBS buffer solution (total 500 μL) containing various concentrations of GS-ESF and GSH (0.5 mM). ) (Reaction solution 2) was pre-incubated for 30 minutes. After that, GSH (0.5 mM), CDNB (0.2 mM), and GSTπ (4 μg / mL) were added to the reaction solution 1, and CDNB (0.2 mM), and GSTπ (4 μg / mL) were added to the reaction solution 2. Was added, and the absorbance of the UV wavelength was measured at each time after the addition (optical path length: 5 mm, reaction time: 90 seconds, 37 ° C, detection wavelength: 340 nm). The initial rate (nmol / min) of the enzyme reaction was calculated according to a conventional method based on the absorbance.
結果を図２に示す。図２に示されるように、GS-ESFとGSHとのプレインキュベートは酵素反応の初速度に対して影響が無かった。このことから、GS-ESFはGSHとほとんど反応しないことが示唆された。 The results are shown in FIG. As shown in FIG. 2, preincubation of GS-ESF and GSH had no effect on the initial rate of enzymatic reaction. This suggests that GS-ESF hardly reacts with GSH.
実施例8：GST抑制効果解析試験3
図３の下段に示すスキームに従って、次のように試験を行った。GS-ESF (0.1 mM), GSH (1.0 mM), 及びGSTπ (4 μg/mL) を含有する1×PBS緩衝溶液 (total 500 μL) を各時間（〜120分間）プレインキュベートした。その後、CDNB (0.2 mM) を添加し、添加後の各時間においてUV波長の吸光度を測定した (光路長: 5 mm, 反応時間: 90秒, 37℃, 検出波長: 340 nm)。吸光度に基づいて定法に従って酵素反応の初速度（nmol/min）を算出した。試験は計3回行った。
Example 8: GST suppression effect analysis test 3
The test was conducted as follows according to the scheme shown in the lower part of FIG. A 1 × PBS buffer solution (total 500 μL) containing GS-ESF (0.1 mM), GSH (1.0 mM), and GSTπ (4 μg / mL) was preincubated for each time (~ 120 min). After that, CDNB (0.2 mM) was added, and the absorbance of the UV wavelength was measured at each time after the addition (optical path length: 5 mm, reaction time: 90 seconds, 37 ° C, detection wavelength: 340 nm). The initial rate (nmol / min) of the enzyme reaction was calculated according to a conventional method based on the absorbance. The test was conducted a total of 3 times.
結果を図３に示す。図３に示されるように、プレインキュベート時間依存的に、GST酵素反応が抑制された。 The results are shown in FIG. As shown in FIG. 3, the GST enzymatic reaction was suppressed in a preincubation time-dependent manner.
実施例9：GST抑制効果解析試験4
実施例8の結果は、GS-ESFがGSHと反応していることに起因する可能性がある。この可能性を調べるために、GSH非存在下においてGSTとGS-ESFとをプレインキュベートした後に、酵素反応の初速度を測定した。具体的には、図４の下段に示すスキームに従って、以下のように行った。
Example 9: GST suppression effect analysis test 4
The results of Example 8 may be due to the reaction of GS-ESF with GSH. To investigate this possibility, the initial rate of enzymatic reaction was measured after pre-incubating GST and GS-ESF in the absence of GSH. Specifically, it was carried out as follows according to the scheme shown in the lower part of FIG.
GSTπ (4 μg/mL) を含有する1×PBS緩衝溶液 (total 500 μL)（反応溶液3）, 並びにGSTπ (4 μg/mL) 及びGS-ESF (0.1 mM) を含有する1×PBS緩衝溶液 (total 500 μL)（反応溶液4） を、各時間（〜120分間）プレインキュベートした。その後、GSH (1.0 mM)及びCDNB (0.2 mM) を添加し、添加後の各時間においてUV波長の吸光度を測定した (光路長: 5 mm, 反応時間: 90秒, 37℃, 検出波長: 340 nm)。吸光度に基づいて定法に従って酵素反応の初速度（nmol/min）を算出した。試験は計3回行った。 1 × PBS buffer solution (total 500 μL) (reaction solution 3) containing GSTπ (4 μg / mL), and 1 × PBS buffer solution containing GSTπ (4 μg / mL) and GS-ESF (0.1 mM). (Total 500 μL) (reaction solution 4) was preincubated for each time (~ 120 minutes). After that, GSH (1.0 mM) and CDNB (0.2 mM) were added, and the absorbance of the UV wavelength was measured at each time after the addition (optical path length: 5 mm, reaction time: 90 seconds, 37 ° C, detection wavelength: 340). nm). The initial rate (nmol / min) of the enzyme reaction was calculated according to a conventional method based on the absorbance. The test was conducted a total of 3 times.
結果を図４に示す。図４に示されるように、実施例8に示される酵素反応の抑制は、GS-ESFがGSHと反応することによるものではなく、GS-ESFがGSTに作用することによるものであることが示唆された。 The results are shown in FIG. As shown in FIG. 4, it is suggested that the suppression of the enzymatic reaction shown in Example 8 is not due to the reaction of GS-ESF with GSH, but due to the action of GS-ESF on GST. Was done.
実施例10：GST抑制効果解析試験5
GS-ESFとGSHとの非酵素的反応の影響を確認するために、GST非存在下においてGS-ESFとGSHとを反応させた後に、酵素反応の初速度を測定した。もし、GS-ESFとGSHとが、GST非存在下で反応するのであれば、酵素反応の開始速度は、GS-ESFとGSHとの反応時間に応じて徐々に減少することが予想される。具体的には、図５の下段に示すスキームに従って、以下のように行った。
Example 10: GST suppression effect analysis test 5
In order to confirm the effect of the non-enzymatic reaction between GS-ESF and GSH, the initial rate of the enzymatic reaction was measured after reacting GS-ESF and GSH in the absence of GST. If GS-ESF and GSH react in the absence of GST, the rate of initiation of the enzymatic reaction is expected to gradually decrease with the reaction time of GS-ESF and GSH. Specifically, it was carried out as follows according to the scheme shown in the lower part of FIG.
GSH (1.0 mM) を含有する1×PBS緩衝溶液 (total 500 μL)（反応溶液5）, 並びにGSH (1.0 mM) 及びGS-ESF (0.1 mM) を含有する1×PBS緩衝溶液 (total 500 μL)（反応溶液6）を、30分間インキュベートした。その後、CDNB (0.2 mM) 及びGSTπ (4 μg/mL) を添加し、添加後の各時間においてUV波長の吸光度を測定した (光路長: 5 mm, 反応時間: 90秒, 37℃, 検出波長: 340 nm)。吸光度に基づいて定法に従って酵素反応の初速度（nmol/min）を算出した。試験は計3回行った。 1 x PBS buffer solution (total 500 μL) containing GSH (1.0 mM) (reaction solution 5), and 1 x PBS buffer solution (total 500 μL) containing GSH (1.0 mM) and GS-ESF (0.1 mM). ) (Reaction solution 6) was incubated for 30 minutes. After that, CDNB (0.2 mM) and GSTπ (4 μg / mL) were added, and the absorbance of the UV wavelength was measured at each time after the addition (optical path length: 5 mm, reaction time: 90 seconds, 37 ° C, detection wavelength). : 340 nm). The initial rate (nmol / min) of the enzyme reaction was calculated according to a conventional method based on the absorbance. The test was conducted a total of 3 times.
結果を図５に示す。図５に示されるように、酵素反応の開始速度は、GS-ESFとGSHとの反応時間の長さに関わらず、ほぼ一定であった。このことから、GS-ESFはGSHとほとんど反応しないことが示唆された。 The results are shown in FIG. As shown in FIG. 5, the initiation rate of the enzymatic reaction was substantially constant regardless of the length of the reaction time between GS-ESF and GSH. This suggests that GS-ESF hardly reacts with GSH.
実施例11：GST抑制効果解析試験6
GS-ESFによるGST活性の抑制が、タンパク質の変性に起因するのか否かを調べた。具体的には、図６の下段に示すスキームに従って、以下のように行った。
Example 11: GST suppression effect analysis test 6
We investigated whether the suppression of GST activity by GS-ESF was due to protein denaturation. Specifically, it was carried out as follows according to the scheme shown in the lower part of FIG.
GSTπ (4 μg/mL) を含有する1×PBS緩衝溶液 (total 500 μL)（反応溶液7）、GSTπ (4 μg/mL) 及びNaOH（3％）を含有する1×PBS緩衝溶液 (total 500 μL)（反応溶液8）、GSTπ (4 μg/mL) 及びGSH (1.0 mM) を含有する1×PBS緩衝溶液 (total 500 μL)（反応溶液9）、並びにGSTπ (4 μg/mL) 及びGS-ESF (0.1 mM) を含有する1×PBS緩衝溶液 (total 500 μL)（反応溶液10）を30分間インキュベートした。その後、CDスペクトルを測定した。 1 x PBS buffer (total 500 μL) (reaction solution 7) containing GSTπ (4 μg / mL), 1 × PBS buffer (total 500) containing GSTπ (4 μg / mL) and NaOH (3%) 1 × PBS buffer solution (total 500 μL) (reaction solution 9) containing μL) (reaction solution 8), GSTπ (4 μg / mL) and GSH (1.0 mM), and GSTπ (4 μg / mL) and GS -1 x PBS buffer (total 500 μL) (reaction solution 10) containing ESF (0.1 mM) was incubated for 30 minutes. Then, the CD spectrum was measured.
結果を図６に示す。図６に示されるように、水酸化ナトリウムを含む反応溶液8の場合はタンパク変性を示すCDスペクトルとなったが、GS-ESFを含む反応溶液10の場合はこのようなCDスペクトルとはならなかった。このことから、GS-ESFによるGST活性の抑制はタンパク質の変性に起因するのではないことが示唆された。 The results are shown in FIG. As shown in FIG. 6, in the case of the reaction solution 8 containing sodium hydroxide, the CD spectrum showing protein denaturation was obtained, but in the case of the reaction solution 10 containing GS-ESF, such a CD spectrum was not obtained. It was. This suggests that the suppression of GST activity by GS-ESF is not due to protein denaturation.
実施例11：GST抑制効果解析試験7
GS-ESFとGSHが反応しているか否かを調べるために、下記スキームで示される反応の速度を評価した。
Example 11: GST suppression effect analysis test 7
In order to investigate whether GS-ESF and GSH are reacting, the reaction rate shown in the following scheme was evaluated.
反応物GS-ESFの損失速度は、次式：-d[GS-ESF]/dt=k[GS-ESF][GSH]である。もし、GSH濃度がGS-ESF濃度よりも大きく高ければ、反応（擬似一次反応）中、GSH濃度は定数（=[GSH]0）であるとみなすことができる。よって、新たな見かけ上の速度定数kappは、kapp=k[GSH]0と公式化することができる。よって、-d[GS-ESF]/dt=k[GS-ESF][GSH]0=kapp[GS-ESF]→-∫1/[GS-ESF] d[GS-ESF]= kapp∫dtとなり、それ故、-In([GS-ESF]/[GS-ESF]0)= kapptとなる。{-In([GS-ESF]/[GS-ESF]0)}を時間に対してプロットすると、kappと等しい傾きの直線が得られる。 The loss rate of the reaction product GS-ESF is given by the following equation: -d [GS-ESF] / dt = k [GS-ESF] [GSH]. If the GSH concentration is larger than the GS-ESF concentration, the GSH concentration can be regarded as a constant (= [GSH] 0 ) during the reaction (pseudo-primary reaction). Therefore, the new apparent rate constant k app can be formulated as k app = k [GSH] 0 . Therefore, -d [GS-ESF] / dt = k [GS-ESF] [GSH] 0 = k app [GS-ESF] →-∫1 / [GS-ESF] d [GS-ESF] = k app ∫ dt and therefore -In ([GS-ESF] / [GS-ESF] 0 ) = k app t. Plotting {-In ([GS-ESF] / [GS-ESF] 0 )} over time gives a straight line with a slope equal to k app .
以上を踏まえて、まずは各種濃度のGS-ESF溶液をHPLC分析し、GS-ESFピークの面積とGS-ESF濃度との検量線を作成した。該検量線を図７に示す。 Based on the above, first, GS-ESF solutions of various concentrations were analyzed by HPLC, and a calibration curve between the area of the GS-ESF peak and the GS-ESF concentration was prepared. The calibration curve is shown in FIG.
次に、以下の試験よって、見かけ上の速度定数kappを決定した。具体的には次のように行った。GS-ESF (50 mM) 及び各種濃度（0.4 M, 1 M, 1.4 M）のGSHを含有する1×PBS緩衝溶液 (total 24 μL) を各時間（〜120分間）インキュベートした。0.1％トリフルオロ酢酸を30μL添加し、得られた溶液の内の20μLをHPLC分析に供した。得られた結果から、-In([GS-ESF]/[GS-ESF]0)及びkappを求めた。結果を図８及び９に示す。 Next, the apparent rate constant k app was determined by the following tests. Specifically, it was done as follows. A 1 × PBS buffer (total 24 μL) containing GS-ESF (50 mM) and various concentrations (0.4 M, 1 M, 1.4 M) of GSH was incubated for each time (~ 120 minutes). 30 μL of 0.1% trifluoroacetic acid was added, and 20 μL of the obtained solution was subjected to HPLC analysis. From the obtained results, -In ([GS-ESF] / [GS-ESF] 0 ) and k app were calculated . The results are shown in FIGS. 8 and 9.
kapp = k[GSH]0の関係式が確立されているので、二次速度定数kは、0.8×10-3 (M-1 min-1)であることが導かれた。この結果から、非酵素反応（上記スキーム）による生成物量を計算した結果、120分間の反応で0.0096％であった（図１０）。以上より、GS-ESFはGSHとほとんど反応しないことが示唆された。 Since the relational expression of k app = k [GSH] 0 has been established, it is derived that the secondary rate constant k is 0.8 × 10 -3 (M -1 min -1 ). From this result, the amount of product produced by the non-enzymatic reaction (the above scheme) was calculated and found to be 0.0096% in the reaction for 120 minutes (Fig. 10). From the above, it was suggested that GS-ESF hardly reacts with GSH.
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