JP2014133704A - Fluorescent probe for measuring hydrogen sulfide - Google Patents

Fluorescent probe for measuring hydrogen sulfide Download PDF

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JP2014133704A
JP2014133704A JP2011095598A JP2011095598A JP2014133704A JP 2014133704 A JP2014133704 A JP 2014133704A JP 2011095598 A JP2011095598 A JP 2011095598A JP 2011095598 A JP2011095598 A JP 2011095598A JP 2014133704 A JP2014133704 A JP 2014133704A
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hydrogen atom
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Tetsuo Nagano
哲雄 長野
Kenjiro Hanaoka
健二郎 花岡
Kiyoshi Shinokura
潔 篠倉
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University of Tokyo NUC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescent probe which can measure hydrogen sulfide with high sensitivity and specifically.SOLUTION: This invention relates to a compound represented by formula (I) or salt thereof. In the formula, Ris a group represented by formula (A); Ris a hydrogen atom or a univalent substituent; Rand Rare a hydrogen atom or a halogen atom; Rand Rare a hydrogen atom, an alkyl carbonyl group or the like; Ris a hydrogen atom, an alkyl group or the like; Rand Rare a hydrogen atom, a halogen atom, or -(CH)-N(R)(R), where x is an integer of 1-4.

Description

本発明は硫化水素測定用の蛍光プローブに関する。   The present invention relates to a fluorescent probe for measuring hydrogen sulfide.

硫化水素(H2S)の毒性については300年前から知られており、今までに数多くの毒性に関する報告がなされてきた。一方、近年、H2Sが生理的シグナル伝達物質として機能していることが明らかとなってきており、一酸化窒素(NO)や一酸化炭素(CO)に次ぐ第三のガス性シグナル伝達物質として注目されている。 The toxicity of hydrogen sulfide (H 2 S) has been known for 300 years, and many reports on toxicity have been made so far. On the other hand, in recent years, it has become clear that H 2 S functions as a physiological signaling substance, and is the third gaseous signaling substance after nitrogen monoxide (NO) and carbon monoxide (CO). It is attracting attention as.

H2Sが果たす役割はH2SドナーであるNaHSやNa2Sを用いて種々検討されているが、最もよく研究されている役割は平滑筋の弛緩に関する作用である。H2Sは血管平滑筋及び腸管平滑筋などの弛緩をもたらし、個体レベルでは頭痛や血圧低下などの症状を引き起こす。一方、神経細胞においてはH2Sが記憶形成に関与することが報告されており、さらに頸動脈小体におけるO2 sensingやインスリンの分泌制御にH2Sが関与しているという報告もある。 Role H 2 S play is being studied using NaHS and Na 2 S is H 2 S donor, the roles that are best studied is an operation related to relaxation of smooth muscle. H 2 S causes relaxation of vascular smooth muscles and intestinal smooth muscles, and causes symptoms such as headache and decreased blood pressure at the individual level. On the other hand, it has been reported that H 2 S is involved in memory formation in nerve cells, and there is also a report that H 2 S is involved in O 2 sensing and insulin secretion control in the carotid body.

このように、H2Sが細胞内シグナル伝達に寄与していることが示唆されているが、生理的なH2Sが本当に存在しているかについてもまだ疑いの余地がある。また、H2Sのターゲットとなる分子の同定も進んでおらず、具体的な細胞内のシグナル伝達機構には未解決の点が多い。このような背景から、生体内のH2Sを可視化する手段の開発が切望されている。 Thus, it has been suggested that H 2 S contributes to intracellular signal transduction, but there is still doubt about whether physiological H 2 S really exists. In addition, identification of molecules that are targets of H 2 S has not progressed, and there are many unresolved points in specific intracellular signal transduction mechanisms. From such a background, development of a means for visualizing H 2 S in a living body is eagerly desired.

蛍光プローブを用いて生体内のH2Sを特異的に可視化して高感度な測定を行うためには、水中でH2Sを測定することができること、他のアニオン種との間に選択性を有すること、及び還元型グルタチオン(GSH)との間で選択性を有することなどの条件が必須となる。従来、H2Sを可視化して測定するための蛍光プローブとしてp-位にアミノ基を有する吸収変化型プローブである2,4,6-トリアリールピリリウムカチオン(Journal of the American Chemical Society, 125, 9000, 2003)が提案されているが、GSHとの反応の競合を避けられないという問題がある。また、蛍光増大型プローブとして2,4-ジニトロベンゼンスルホニルフルオレセインを用いる方法も提案されているが(Analytica Chimica Acta, 631, 91, 2009)、経時的にスルホン酸エステルが加水分解して蛍光強度が変化するという問題を有している。 In order to specifically visualize H 2 S in a living body using a fluorescent probe and perform highly sensitive measurement, H 2 S can be measured in water and selectivity with other anion species And other conditions such as selectivity with reduced glutathione (GSH) are essential. Conventionally, 2,4,6-triarylpyrylium cation (Journal of the American Chemical Society, 125), which is an absorption-change probe having an amino group at the p-position as a fluorescent probe for visualizing and measuring H 2 S. , 9000, 2003) has been proposed, but there is a problem that competition of reaction with GSH cannot be avoided. In addition, a method using 2,4-dinitrobenzenesulfonylfluorescein as a fluorescence-enhanced probe has been proposed (Analytica Chimica Acta, 631, 91, 2009). Have the problem of changing.

また、別の蛍光増大型プローブとして、二価銅イオン(Cu2+)を配位させた2,2'-ジピコリルアミン部分を有するアミノフルオレセイン化合物(DPA-4-AF)が提案されている(Chem. Commun., pp.7390-7392, 2009)。この化合物では、Cu2+の配位によって蛍光が消光されているが、スルフィドイオン(HS-)が作用するとCu2+がCuSとなって不溶化し、消光が解除されて強蛍光を発する。この蛍光プローブは水中においてスルフィドイオンを検出することができ、種々のアニオン(ハロゲンイオン、硫酸イオン、酢酸イオン、硝酸イオン、次亜塩素酸イオンなど)の存在下においても特異的かつ高感度にH2Sを測定できるという特徴がある。しかしながら、本発明者らの追試によれば、この化合物は還元型グルタチオンとの間で選択性が低く、生体内に大量に存在するグルタチオンによりイン・ビボにおいて細胞内などに存在するH2Sを特異的かつ高感度に測定することができないという問題がある。なお、環状ポリアミンを亜鉛イオンの捕捉基として用いた化合物が知られているが(ダンシルアミノエチルサイクレン: J. Am. Chem. Soc., 118, 12696, 1996)、この捕捉基に銅イオンを配位させた化合物は知られておらず、H2Sとの反応性についても全く報告がない。また、環状ポリアミンをカドミウムイオンの捕捉基として用いた化合物も知られているが、この化合物とH2Sとの反応性についても知られていない(J. Am. Chem. Soc., 124, pp.3920-3925, 2002)。 As another fluorescence-enhancing probe, an aminofluorescein compound (DPA-4-AF) having a 2,2′-dipicolylamine moiety coordinated with a divalent copper ion (Cu 2+ ) has been proposed. (Chem. Commun., Pp.7390-7392, 2009). In this compound, the fluorescence is quenched by the coordination of Cu 2+ , but when the sulfide ion (HS − ) acts, Cu 2+ becomes CuS insolubilized, the quenching is released, and strong fluorescence is emitted. This fluorescent probe is capable of detecting sulfide ions in water and is highly specific and sensitive even in the presence of various anions (halogen ions, sulfate ions, acetate ions, nitrate ions, hypochlorite ions, etc.). It has the feature that 2 S can be measured. However, according to a follow-up test by the present inventors, this compound has low selectivity with reduced glutathione, and H 2 S present in cells and the like is present in vivo by glutathione present in large quantities in the living body. There is a problem that it cannot be measured specifically and with high sensitivity. A compound using a cyclic polyamine as a zinc ion capturing group is known (dansylaminoethylcyclene: J. Am. Chem. Soc., 118, 12696, 1996). The coordinated compound is not known, and there is no report on the reactivity with H 2 S. In addition, a compound using a cyclic polyamine as a cadmium ion capturing group is also known, but the reactivity of this compound with H 2 S is not known (J. Am. Chem. Soc., 124, pp .3920-3925, 2002).

Journal of the American Chemical Society, 125, 9000, 2003Journal of the American Chemical Society, 125, 9000, 2003 Analytica Chimica Acta, 631, 91, 2009Analytica Chimica Acta, 631, 91, 2009 Chem. Commun., pp.7390-7392, 2009Chem. Commun., Pp.7390-7392, 2009 J. Am. Chem. Soc., 118, 12696, 1996J. Am. Chem. Soc., 118, 12696, 1996 J. Am. Chem. Soc., 124, pp.3920-3925, 2002J. Am. Chem. Soc., 124, pp. 3920-3925, 2002

本発明の課題は、生体内の硫化水素を特異的に可視化して高感度な測定を行うことができる蛍光プローブを提供することにある。特に、生体内に大量に存在する還元型グルタチオンの影響を受けることなく高感度かつ特異的に硫化水素を測定することができる蛍光プローブを提供することが本発明の課題である。   An object of the present invention is to provide a fluorescent probe capable of specifically visualizing hydrogen sulfide in a living body and performing highly sensitive measurement. In particular, it is an object of the present invention to provide a fluorescent probe capable of measuring hydrogen sulfide with high sensitivity and specificity without being affected by reduced glutathione present in large quantities in a living body.

本発明者らは上記の課題を解決すべく鋭意研究を行った結果、下記の一般式(I)で表される化合物を硫化水素測定用の蛍光プローブとして用いると、還元型グルタチオンの存在下においても極めて高感度かつ特異的に硫化水素を測定することができることを見出した。本発明は上記の知見を基にして完成されたものである。   As a result of intensive studies to solve the above problems, the present inventors have used the compound represented by the following general formula (I) as a fluorescent probe for measuring hydrogen sulfide in the presence of reduced glutathione. It was also found that hydrogen sulfide can be measured with extremely high sensitivity and specificity. The present invention has been completed based on the above findings.

すなわち、本発明により、下記の一般式(IA)又は(IB)：
〔式中、R1は下記式(A)：
（式中、p、q、r、及びsはそれぞれ独立に2又は3の整数を示し、tは0又は1を示し、R11、R12、及びR13はそれぞれ独立に水素原子又は炭素原子数1〜6のアルキル基を示す）で表される基を示し；R2は水素原子又はベンゼン環上に置換する1個ないし3個の一価の置換基を示し；R3及びR4はそれぞれ独立に水素原子又はハロゲン原子を示し；R5及びR6はそれぞれ独立に水素原子、アルキルカルボニル基、又はアルキルカルボニルオキシアルキル基を示し；R7は水素原子、アルキル基、又はアルコキシアルキル基を示し；R8及びR9はそれぞれ独立に水素原子、ハロゲン原子、又は-(CH2)x-N(R14)(R15)(式中、xは1ないし4の整数を示し、R14及びR15はそれぞれ独立に-(CH2)y-COOR16（式中、yは1ないし4の整数を示し、R16は水素原子、アルキルカルボニル基、又はアルキルカルボニルオキシアルキル基を示す）で表される基を示す)で表される基を示す〕で表される化合物又はその塩が提供される。 That is, according to the present invention, the following general formula (IA) or (IB):
[Wherein R 1 represents the following formula (A):
(In the formula, p, q, r, and s each independently represent an integer of 2 or 3, t represents 0 or 1, and R 11 , R 12 , and R 13 each independently represent a hydrogen atom or a carbon atom. R 2 represents a hydrogen atom or 1 to 3 monovalent substituents substituted on a benzene ring; R 3 and R 4 represent R 5 and R 6 each independently represent a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxyalkyl group; R 7 represents a hydrogen atom, an alkyl group, or an alkoxyalkyl group. shown; R 8 and R 9 are each independently a hydrogen atom, a halogen atom, or a - (CH 2) x -N ( R 14) (R 15) ( wherein, x is an integer of 1 to 4, R 14 and R 15 are each independently - (CH 2) y -COOR 16 ( wherein, y is an integer of 1 to 4, R 16 is a hydrogen atom, an alkylcarbonyl group, A compound represented by a group represented by a group represented by showing the alkylcarbonyloxy group))] or a salt thereof is provided.

上記発明の好ましい態様によれば、p、q、r、及びsが2であり、tが1であり、かつR11、R12、及びR13が水素原子である上記一般式(IA)又は(IB)で表される化合物又はその塩；p、q、r、及びsが2であり、tが1であり、R11、R12、及びR13が水素原子であり、かつR2が水素原子である上記一般式(IA)又は(IB)で表される化合物又はその塩；並びに、ベンゼン環上に存在するR1がキサンテン環の結合部位に対してパラ位に結合しており、p、q、r、及びsが2であり、tが1であり、R11、R12、及びR13が水素原子であり、かつR2が水素原子である上記一般式(IA)又は(IB)で表される化合物又はその塩が提供される。 According to a preferred embodiment of the present invention, p, q, r, and s are 2, t is 1, and R 11 , R 12 , and R 13 are hydrogen atoms, A compound represented by (IB) or a salt thereof; p, q, r, and s are 2, t is 1, R 11 , R 12 , and R 13 are hydrogen atoms, and R 2 is A compound represented by the above general formula (IA) or (IB) which is a hydrogen atom or a salt thereof; and R 1 present on the benzene ring is bonded to the para-position relative to the binding site of the xanthene ring, p, q, r, and s are 2, t is 1, R 11 , R 12 , and R 13 are hydrogen atoms, and R 2 is a hydrogen atom, or the above general formula (IA) or ( A compound represented by IB) or a salt thereof is provided.

さらに好ましい態様によれば、R3及びR4が水素原子である上記一般式(IA)又は(IB)で表される化合物又はその塩；R5及びR6がそれぞれ独立に水素原子、アセチル基、又はアセチルオキシメチル基である上記一般式(IA)又は(IB)で表される化合物又はその塩；R7が水素原子、アルキル基、又はメトキシメチル基である上記一般式(IA)又は(IB)で表される化合物又はその塩；並びにR8及びR9がともに水素原子である上記一般式(IA)又は(IB)で表される化合物又はその塩が提供される。 According to a more preferred embodiment, the compound represented by the above general formula (IA) or (IB) or a salt thereof, wherein R 3 and R 4 are hydrogen atoms; R 5 and R 6 are each independently a hydrogen atom, an acetyl group; Or a compound represented by the above general formula (IA) or (IB) which is an acetyloxymethyl group or a salt thereof; R 7 is a hydrogen atom, an alkyl group or a methoxymethyl group; And a compound represented by the above general formula (IA) or (IB) or a salt thereof, wherein R 8 and R 9 are both hydrogen atoms.

また、本発明により、下記の一般式(IIA)又は(IIB)：
〔式中、R21は下記式(B)：
（式中、d、e、f、及びgはそれぞれ独立に2又は3の整数を示し、hは0又は1を示し、R31、R32、及びR33はそれぞれ独立に水素原子又は炭素原子数1〜6のアルキル基を示す）で表される基を示し；R22は水素原子又はベンゼン環上に置換する1個ないし3個の一価の置換基を示し；R23及びR24はそれぞれ独立に水素原子又はハロゲン原子を示し；R25及びR26はそれぞれ独立に水素原子、アルキルカルボニル基、又はアルキルカルボニルオキシアルキル基を示し；R27は水素原子、アルキル基、又はアルコキシアルキル基を示し；R28及びR29はそれぞれ独立に水素原子、ハロゲン原子、又は-(CH2)m-N(R34)(R35)(式中、mは1ないし4の整数を示し、R34及びR35はそれぞれ独立に-(CH2)n-COOR36（式中、nは1ないし4の整数を示し、R36は水素原子、アルキルカルボニル基、又はアルキルカルボニルオキシアルキル基を示す）で表される基を示す)で表される基を示す〕で表される化合物又はその塩が提供される。 Further, according to the present invention, the following general formula (IIA) or (IIB):
[Wherein R 21 represents the following formula (B):
(Wherein, d, e, f and g each independently represents an integer of 2 or 3, h represents 0 or 1, and R 31 , R 32 and R 33 each independently represent a hydrogen atom or a carbon atom. R 22 represents a hydrogen atom or 1 to 3 monovalent substituents substituted on a benzene ring; R 23 and R 24 represent R 25 and R 26 each independently represent a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxyalkyl group; R 27 represents a hydrogen atom, an alkyl group, or an alkoxyalkyl group. shown; each R 28 and R 29 independently represent a hydrogen atom, a halogen atom, or a - (CH 2) m -N ( R 34) (R 35) ( wherein, m is an integer of 1 to 4, R 34 and R 35 are each independently - (CH 2) n -COOR 36 ( wherein, n represents an integer of from 1 4, R 36 is a hydrogen atom, an alkyl carbonylation Group, or an alkylcarbonyloxy group represents a group represented by the illustrated)) compound represented by a group] that represented by or a salt thereof is provided.

上記発明の好ましい態様によれば、d、e、f、及びgが2であり、hが1であり、かつR31、R32、及びR33が水素原子である上記一般式(IIA)又は(IIB)で表される化合物又はその塩；d、e、f、及びgが2であり、hが1であり、R31、R32、及びR33が水素原子であり、かつR22が水素原子である上記一般式(IIA)又は(IIB)で表される化合物又はその塩；並びに、ベンゼン環上に存在するR21がキサンテン環の結合部位に対してパラ位に結合しており、d、e、f、及びgが2であり、hが1であり、R31、R32、及びR33が水素原子であり、かつR22が水素原子である上記一般式(IIA)又は(IIB)で表される化合物又はその塩が提供される。 According to a preferred embodiment of the present invention, d, e, f, and g are 2, h is 1, and R 31 , R 32 , and R 33 are hydrogen atoms, the general formula (IIA) or A compound represented by (IIB) or a salt thereof; d, e, f, and g are 2, h is 1, R 31 , R 32 , and R 33 are hydrogen atoms, and R 22 is A compound represented by the above general formula (IIA) or (IIB) which is a hydrogen atom or a salt thereof; and R 21 present on the benzene ring is bonded to the binding position of the xanthene ring at the para position, d, e, f, and g are 2, h is 1, R 31 , R 32 , and R 33 are hydrogen atoms, and R 22 is a hydrogen atom, or the above general formula (IIA) or ( A compound represented by IIB) or a salt thereof is provided.

さらに好ましい態様によれば、R23及びR24が水素原子である上記一般式(IIA)又は(IIB)で表される化合物又はその塩；R25及びR26がそれぞれ独立に水素原子、アセチル基、又はアセチルオキシメチル基である上記一般式(IIA)又は(IIB)で表される化合物又はその塩；R27が水素原子、アルキル基、又はメトキシメチル基である上記一般式(IIA)又は(IIB)で表される化合物又はその塩；並びにR28及びR29がともに水素原子である上記一般式(IIA)又は(IIB)で表される化合物又はその塩が提供される。 According to a further preferred embodiment, the compound represented by the above general formula (IIA) or (IIB) or a salt thereof, wherein R 23 and R 24 are hydrogen atoms; R 25 and R 26 are each independently a hydrogen atom, an acetyl group; Or a compound represented by the above general formula (IIA) or (IIB) which is an acetyloxymethyl group or a salt thereof; and the above general formula (IIA) or (II) wherein R 27 is a hydrogen atom, an alkyl group or a methoxymethyl group And a compound represented by the above general formula (IIA) or (IIB) or a salt thereof, wherein both R 28 and R 29 are hydrogen atoms.

別の観点からは、本発明により、上記一般式(IA)又は(IB)で表される化合物又はその塩を含む硫化水素測定用蛍光プローブ、及び上記一般式(IA)又は(IB)で表される化合物又はその塩を含む硫化水素測定用試薬が提供される。また、硫化水素測定用蛍光プローブ又は硫化水素測定用試薬の製造のための上記上記一般式(IA)又は(IB)で表される化合物又はその塩の使用も本発明により提供される。   From another point of view, according to the present invention, a fluorescent probe for measuring hydrogen sulfide containing the compound represented by the above general formula (IA) or (IB) or a salt thereof, and the above general formula (IA) or (IB) are used. Provided is a reagent for measuring hydrogen sulfide containing the compound or a salt thereof. The present invention also provides use of the compound represented by the above general formula (IA) or (IB) or a salt thereof for the production of a fluorescent probe for measuring hydrogen sulfide or a reagent for measuring hydrogen sulfide.

また、本発明により、上記一般式(IIA)又は(IIB)で表される化合物又はその塩を含む硫化水素測定用蛍光プローブ、及び上記一般式(IIA)又は(IIB)で表される化合物又はその塩を含む硫化水素測定用試薬が提供される。また、硫化水素測定用蛍光プローブ又は硫化水素測定用試薬の製造のための上記上記一般式(IIA)又は(IIB)で表される化合物又はその塩の使用も本発明により提供される。この蛍光プローブ及び試薬は、測定系内において二価銅イオンと反応させて上記一般式(IA)又は(IB)で表される化合物又はその塩を測定系内において生成させるin situ型の硫化水素測定用蛍光プローブ又は試薬として有用である。   Further, according to the present invention, a fluorescent probe for measuring hydrogen sulfide containing a compound represented by the above general formula (IIA) or (IIB) or a salt thereof, and a compound represented by the above general formula (IIA) or (IIB) or A reagent for measuring hydrogen sulfide containing the salt is provided. The present invention also provides use of the compound represented by the above general formula (IIA) or (IIB) or a salt thereof for the production of a fluorescent probe for measuring hydrogen sulfide or a reagent for measuring hydrogen sulfide. This fluorescent probe and reagent are in situ hydrogen sulfide that reacts with divalent copper ions in the measurement system to produce the compound represented by the above general formula (IA) or (IB) or a salt thereof in the measurement system. It is useful as a fluorescent probe or reagent for measurement.

さらに別の観点からは、本発明により、硫化水素の測定方法であって、下記の工程：(a)上記一般式(IA)又は(IB)で表される化合物又はその塩を硫化水素と接触させる工程；及び(b)上記工程(a)で生成した上記一般式(IIA)又は(IIB)で表される化合物又はその塩の蛍光を測定する工程を含む方法が提供される。   From another aspect, according to the present invention, there is provided a method for measuring hydrogen sulfide, comprising the following steps: (a) contacting a compound represented by the above general formula (IA) or (IB) or a salt thereof with hydrogen sulfide And (b) measuring the fluorescence of the compound represented by the general formula (IIA) or (IIB) produced in the step (a) or a salt thereof.

また、本発明により、硫化水素の測定方法であって、下記の工程：(a)上記一般式(IIA)又は(IIB)で表される化合物又はその塩と二価銅イオンとを反応させて上記一般式(IA)又は(IB)で表される化合物又はその塩を生成させる工程；(b)上記工程(a)で生成した上記一般式(IA)又は(IB)で表される化合物又はその塩と硫化水素とを接触させる工程；及び(c)上記工程(b)で生成した上記一般式(IIA)又は(IIB)で表される化合物又はその塩の蛍光を測定する工程を含む方法が提供される。   According to the present invention, there is also provided a method for measuring hydrogen sulfide, comprising the steps of: (a) reacting a compound represented by the general formula (IIA) or (IIB) or a salt thereof with a divalent copper ion. A step of producing a compound represented by the general formula (IA) or (IB) or a salt thereof; (b) a compound represented by the general formula (IA) or (IB) produced in the step (a); A step of contacting the salt with hydrogen sulfide; and (c) a step of measuring the fluorescence of the compound represented by the general formula (IIA) or (IIB) produced in the step (b) or the salt thereof. Is provided.

本発明により提供される一般式(IA)又は(IB)で表される化合物又はその塩は硫化水素に対して高い反応性を有しており、硫化水素の存在下において強蛍光性の一般式(IIA)又は(IIB)で表される化合物又はその塩を生成する性質を有している。また、一般式(IA)又は(IB)で表される化合物又はその塩は還元型グルタチオンに対しては実質的に反応性を有しないことから、還元型グルタチオンの存在下においても高感度に硫化水素を測定することができる。さらに、一般式(IA)又は(IB)で表される化合物又はその塩は種々のアニオン(ハロゲンイオン、硫酸イオン、酢酸イオン、硝酸イオン、次亜塩素酸イオンなど)の存在下においても特異的かつ高感度に硫化水素と反応することができる。従って、一般式(IA)又は(IB)で表される化合物又はその塩を含む硫化水素測定用蛍光プローブは、生体内に存在する微量の硫化水素を高感度かつ高精度に測定するための蛍光プローブとして有用であり、イン・ビボ環境下において細胞内や組織内における硫化水素をイメージングするための試薬として極めて有用である。   The compound represented by the general formula (IA) or (IB) provided by the present invention or a salt thereof has high reactivity with hydrogen sulfide, and is a general formula having strong fluorescence in the presence of hydrogen sulfide. It has the property of producing a compound represented by (IIA) or (IIB) or a salt thereof. In addition, since the compound represented by the general formula (IA) or (IB) or a salt thereof has substantially no reactivity with reduced glutathione, it is highly sensitive even in the presence of reduced glutathione. Hydrogen can be measured. Furthermore, the compound represented by the general formula (IA) or (IB) or a salt thereof is specific even in the presence of various anions (halogen ion, sulfate ion, acetate ion, nitrate ion, hypochlorite ion, etc.). It can react with hydrogen sulfide with high sensitivity. Accordingly, the hydrogen sulfide measurement fluorescent probe containing the compound represented by the general formula (IA) or (IB) or a salt thereof is a fluorescent probe for measuring a small amount of hydrogen sulfide existing in a living body with high sensitivity and high accuracy. It is useful as a probe and extremely useful as a reagent for imaging hydrogen sulfide in cells and tissues in an in vivo environment.

Cyclne-4-AF-Cu(左側)及びCyclen-4-AF(右側)の吸光及び蛍光スペクトルを測定した結果を示した図である。It is the figure which showed the result of having measured the absorption and fluorescence spectrum of Cyclne-4-AF-Cu (left side) and Cyclen-4-AF (right side). TACN-4-AF(左側)及びCyclam-4-AF(右側)の吸光及び蛍光スペクトルを測定した結果を示した図である。It is the figure which showed the result of having measured the absorption and fluorescence spectrum of TACN-4-AF (left side) and Cyclam-4-AF (right side). TMCyclen-4-AF(左側)及びTMCyclen-4-AF-Cu(右側)の吸光及び蛍光スペクトルを測定した結果を示した図である。It is the figure which showed the result of having measured the absorption and fluorescence spectrum of TMCyclen-4-AF (left side) and TMCyclen-4-AF-Cu (right side). Cyclen-4-AF-Cuの反応性の評価を行った結果を示した図である。10 μM Na2S (赤)、10 μM Na2Sと10 mM GSH (緑)、又は 10 mM GSH (黄)を添加した結果、及び無添加のネガティブコントロール(青)の結果を示した。It is the figure which showed the result of having evaluated the reactivity of Cyclen-4-AF-Cu. The result of adding 10 μM Na 2 S (red), 10 μM Na 2 S and 10 mM GSH (green), or 10 mM GSH (yellow) and the result of adding no negative control (blue) are shown. Cyclen-4-AF-CuをHeLa細胞内にマイクロインジェクションにより導入して硫化水素を測定した結果を示した図である。上段はNa2S添加前(0 sec)、中段はNa2S添加後(360 sec)、及び下段は蛍光強度の経時変化を示し、領域5及び6はマイクロインジェクションをしていない領域を示す。It is the figure which showed the result of having introduced Cyclen-4-AF-Cu into the HeLa cell by microinjection and measuring hydrogen sulfide. The upper row shows before Na 2 S addition (0 sec), the middle row shows after Na 2 S addition (360 sec), and the lower row shows changes in fluorescence intensity over time, and regions 5 and 6 show regions that are not microinjected. Cyclen-4-AF-Cu diacetateを用いてHeLa細胞への取り込みによる硫化水素の測定を行った結果を示した図である。上段はNa2S添加前(0 sec)、中段はNa2S添加後(240 sec)、及び下段は蛍光強度の経時変化を示す。It is the figure which showed the result of having measured the hydrogen sulfide by the uptake | capture to a HeLa cell using Cyclen-4-AF-Cu diacetate. The upper part shows before Na 2 S addition (0 sec), the middle part after Na 2 S addition (240 sec), and the lower part shows the change in fluorescence intensity over time. TACN-4-AF単独、及び2当量のCu2+を加えたTACN-4-AFの吸収スペクトル(上段左)と蛍光スペクトル(上段右)を測定した結果、及びTACN-4-AFにCuSO4を加えて100 μM Na2S (赤)、10 μM Na2S (緑)、又は10 mM GSH (黄)を添加し、無添加の実験をネガティブコントロール (青) として反応性を調べた結果(下段)を示した図である。TACN-4-AF alone and 2 CuSO 4 to equivalents of Cu 2+ absorption spectra of TACN-4-AF was added (upper left) and fluorescence spectrum results obtained by measuring the (upper right), and TACN-4-AF And added 100 μM Na 2 S (red), 10 μM Na 2 S (green), or 10 mM GSH (yellow), and the results of examining the reactivity using the additive-free experiment as a negative control (blue) ( FIG. Cyclam-4-AF単独、及び2当量のCu2+を加えたCyclam-4-AFの吸収スペクトル(左)と蛍光スペクトル(右)を測定した結果を示した図である。It is the figure which showed the result of having measured the absorption spectrum (left) and the fluorescence spectrum (right) of Cyclam-4-AF which added Cyclam-4-AF independent and 2 equivalent Cu2 + . Cyclam-4-AF(上段)にCuSO4を加え、又はTMCyclen-4-AF-Cu(下段)に 100 μM Na2S (赤)、10 μM Na2S (緑)、又は10 mM GSH (黄)を添加し、無添加の実験をネガティブコントロール (青) として反応性を調べた結果を示した図である。The Cyclam-4-AF (top) and CuSO 4 was added, or TMCyclen-4-AF-Cu (bottom) to 100 μM Na 2 S (red), 10 μM Na 2 S (green), or 10 mM GSH (Yellow ) Was added, and the experiment without addition was used as a negative control (blue) to show the results of examining the reactivity.

一般式(IA)又は(IB)においてR1は式(A)で表される基を示す。式(A)で表される基において、p、q、r、及びsはそれぞれ独立に2又は3の整数を示すが、好ましくはp、q、r、及びsがいずれも2の場合である。tは0又は1を示すが、1であることが好ましい。R11、R12、及びR13はそれぞれ独立に水素原子又は炭素原子数1〜6のアルキル基を示すが、R11、R12、及びR13のうちいずれか又は全部が水素原子であることが好ましく、R11、R12、及びR13が全て水素原子であることが好ましい。式(A)で表される基はベンゼン環上の任意の位置に結合することができるが、好ましくはR1とキサンテン環が結合するベンゼン環において、R1とキサンテン環との結合位置がパラ位になることが好ましい。 In the general formula (IA) or (IB), R 1 represents a group represented by the formula (A). In the group represented by the formula (A), p, q, r, and s each independently represent an integer of 2 or 3, preferably p, q, r, and s are all 2. . t represents 0 or 1, but is preferably 1. R 11 , R 12 , and R 13 each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and any or all of R 11 , R 12 , and R 13 are hydrogen atoms. It is preferable that R 11 , R 12 , and R 13 are all hydrogen atoms. The group represented by the formula (A) can be bonded to any position on the benzene ring. Preferably, in the benzene ring where R 1 and the xanthene ring are bonded, the bonding position of R 1 and the xanthene ring is It is preferable to be in a position.

本明細書において、アルキル基の用語は直鎖状、分枝鎖状、環状、及びそれらの組み合わせからなるアルキル基を包含する。アルキル部分を有する他の置換基(例えばアルキルカルボニル基やアルコキシアルキル基)のアルキル部分についても同様である。   In the present specification, the term alkyl group includes alkyl groups composed of linear, branched, cyclic, and combinations thereof. The same applies to the alkyl moiety of other substituents having an alkyl moiety (for example, an alkylcarbonyl group or an alkoxyalkyl group).

R2は水素原子又はベンゼン環上に置換する1個ないし3個の一価の置換基を示し、好ましくは水素原子又はベンゼン環上に置換する1個又は2個の一価の置換基を示し、さらに好ましくは水素原子又はベンゼン環上に置換する1個の一価の置換基を示す。R2が一価の置換基である場合、置換基としては、例えば、ハロゲン原子(本明細書においてハロゲン原子の用語はフッ素原子、塩素原子、臭素原子、又はヨウ素原子を包含する)、水酸基、オキソ基、カルボキシル基、アルコキシカルボニル基、アシル基、アミノ基、アルキル基、アルケニル基、アルキニル基、アルコキシ基、アリール基、又はアラルキル基などが挙げられるが、これらに限定されることはない。これらの置換基はさらに別の置換基で置換されていてもよい。このような例として、例えば、フルオロアルキル基、フルオロアセチル基、メトキシベンジル基などを挙げることができるが、これらに限定されることはない。R2は水素原子であることが特に好ましい。 R 2 represents a hydrogen atom or 1 to 3 monovalent substituents substituted on the benzene ring, preferably 1 or 2 monovalent substituents substituted on the hydrogen atom or benzene ring. More preferably, it represents one monovalent substituent substituted on a hydrogen atom or a benzene ring. When R 2 is a monovalent substituent, examples of the substituent include a halogen atom (in the present specification, the term halogen atom includes a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), a hydroxyl group, Examples thereof include, but are not limited to, an oxo group, a carboxyl group, an alkoxycarbonyl group, an acyl group, an amino group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, and an aralkyl group. These substituents may be further substituted with another substituent. Examples of such include, but are not limited to, a fluoroalkyl group, a fluoroacetyl group, a methoxybenzyl group, and the like. R 2 is particularly preferably a hydrogen atom.

R3及びR4はそれぞれ独立に水素原子又はハロゲン原子を示すが、R3及びR4がともに水素原子であることが好ましい。
R5及びR6はそれぞれ独立に水素原子、アルキルカルボニル基、又はアルキルカルボニルオキシアルキル基を示す。アルキルカルボニル基としては、炭素数2〜7個程度のアルキルカルボニル基が好ましく、例えば、アセチル基などを好ましく用いることができる。アルキルカルボニルオキシアルキル基としては、例えば、炭素数2〜7個程度のアルキルカルボニルオキシ基が炭素数1〜6個程度のアルキル基に置換したアルキルカルボニルオキシアルキル基が好ましいが、例えば、アセトキシメチル基などを好ましく用いることができる。 R 3 and R 4 each independently represent a hydrogen atom or a halogen atom, but it is preferable that both R 3 and R 4 are hydrogen atoms.
R 5 and R 6 each independently represent a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxyalkyl group. As the alkylcarbonyl group, an alkylcarbonyl group having about 2 to 7 carbon atoms is preferable. For example, an acetyl group can be preferably used. As the alkylcarbonyloxyalkyl group, for example, an alkylcarbonyloxyalkyl group in which an alkylcarbonyloxy group having about 2 to 7 carbon atoms is substituted with an alkyl group having about 1 to 6 carbon atoms is preferable, for example, an acetoxymethyl group Etc. can be preferably used.

R7は水素原子、アルキル基、又はアルコキシアルキル基を示す。アルコキシアルキル基としては、炭素数1〜6個程度のアルコキシ基が炭素数1〜6程度のアルキル基に置換したアルコキシアルキル基が好ましいが、例えば、メトキシメチル基やエトキシメチル基などを好ましく用いることができる。 R 7 represents a hydrogen atom, an alkyl group, or an alkoxyalkyl group. As the alkoxyalkyl group, an alkoxyalkyl group in which an alkoxy group having about 1 to 6 carbon atoms is substituted with an alkyl group having about 1 to 6 carbon atoms is preferable. For example, a methoxymethyl group or an ethoxymethyl group is preferably used. Can do.

R8及びR9はそれぞれ独立に水素原子、ハロゲン原子、又は-(CH2)x-N(R14)(R15)で表される基を示す。xは1ないし4の整数を示すが、xは1又は2であることが好ましく、xが1であることが特に好ましい。R14及びR15はそれぞれ独立に-(CH2)y-COOR16で表される基を示す。yは1ないし4の整数を示すが、yは1又は2であることが好ましく、yが1であることが特に好ましい。R16は水素原子、アルキルカルボニル基、又はアルキルカルボニルオキシアルキル基を示すが、アルキルカルボニル基及びアルキルカルボニルオキシアルキル基は上記R5について説明したものと同様である。好ましくはR16として水素原子又はアセトキシメチル基などを用いることができる。 R 8 and R 9 each independently represent a hydrogen atom, a halogen atom, or a group represented by — (CH 2 ) x —N (R 14 ) (R 15 ). x represents an integer of 1 to 4, but x is preferably 1 or 2, and x is particularly preferably 1. R 14 and R 15 each independently represent a group represented by — (CH 2 ) y —COOR 16 . y represents an integer of 1 to 4, but y is preferably 1 or 2, and y is particularly preferably 1. R 16 represents a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxyalkyl group, and the alkylcarbonyl group and the alkylcarbonyloxyalkyl group are the same as those described for R 5 above. Preferably, a hydrogen atom or an acetoxymethyl group can be used as R 16 .

一般式(IIA)又は(IIB)における式(B)で表される基、d、e、f、g、h、R31、R32、R33、R22、R23、R24、R25、R26、R27、R28、R29、m、R34、R35、n、R36は、それぞれ、一般式(IA)又は(IB)において対応する式(A)で表される基、p、q、r、s、t、R11、R12、R13、R2、R3、R4、R5、R6、R7、R8、R9、x、R14、R15、n、及びR16と同様である。 Group represented by formula (B) in general formula (IIA) or (IIB), d, e, f, g, h, R 31 , R 32 , R 33 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , m, R 34 , R 35 , n, and R 36 are groups represented by the corresponding formula (A) in the general formula (IA) or (IB), respectively. , P, q, r, s, t, R 11 , R 12 , R 13 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , x, R 14 , R Same as 15 , n, and R 16 .

一般式(IA)又は(IB)、あるいは一般式(IIA)又は(IIB)で表される化合物は酸付加塩又は塩基付加塩を形成する場合がある。酸付加塩としては、例えば、塩酸塩、硫酸塩、硝酸塩などの鉱酸塩、p-トルエンスルホン酸塩、シュウ酸塩、リンゴ酸塩などの有機酸塩などを用いることができるが、これらに限定されることはない。塩基付加塩としては、例えば、ナトリウム塩、カリウム塩、マグネシウム塩、若しくはカルシウム塩などの金属塩、アンモニウム塩、又はトリエチルアミン塩若しくはエタノールアミン塩などの有機アミン塩などを挙げることができるが、これらに限定されることはない。これらの塩のうち、生理学的に許容される塩は本発明の化合物を生体内や細胞又は組織内の硫化水素測定用試薬として用いる場合に好ましい。   The compound represented by general formula (IA) or (IB), or general formula (IIA) or (IIB) may form an acid addition salt or a base addition salt. Examples of acid addition salts include mineral acid salts such as hydrochloride, sulfate, and nitrate, and organic acid salts such as p-toluenesulfonate, oxalate, and malate. There is no limit. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, magnesium salt, or calcium salt, ammonium salts, and organic amine salts such as triethylamine salt or ethanolamine salt. There is no limit. Among these salts, physiologically acceptable salts are preferable when the compound of the present invention is used as a reagent for measuring hydrogen sulfide in a living body, cell or tissue.

また、一般式(IA)又は(IB)、あるいは一般式(IIA)又は(IIB)で表される化合物は、置換基の種類に応じて1個又は2個以上の不斉炭素を有する場合があるが、これらの不斉炭素に基づく任意の光学異性体、光学異性体の任意の混合物、ラセミ体、2個以上の不斉炭素に基づくジアステレオ異性体、ジアステレオ異性体の任意の混合物などは、いずれも本発明の範囲に包含される。遊離化合物又は塩の形態の化合物の任意の水和物又は溶媒和物も本発明の範囲に包含される。また、R7が水素原子の場合にはカルボキシ基がラクトンを形成する場合もあるが、本発明の範囲にはこのような構造異性体も包含される。一般式(IA)においてR5が水素原子である化合物と一般式(IB)においてR7が水素原子である化合物は互変異性体に相当しているが、このような互変異性体の存在は当業者に容易に理解されることであり、いずれの互変異性体も本発明の範囲に包含される。 Further, the compound represented by the general formula (IA) or (IB), or the general formula (IIA) or (IIB) may have one or more asymmetric carbons depending on the type of the substituent. Any optical isomers based on these asymmetric carbons, any mixture of optical isomers, racemates, diastereoisomers based on two or more asymmetric carbons, any mixture of diastereoisomers, etc. Are included in the scope of the present invention. Any hydrate or solvate of the free compound or salt form of the compound is also encompassed within the scope of the present invention. In addition, when R 7 is a hydrogen atom, the carboxy group may form a lactone, but such structural isomers are also included in the scope of the present invention. A compound in which R 5 is a hydrogen atom in general formula (IA) and a compound in which R 7 is a hydrogen atom in general formula (IB) correspond to tautomers, but such tautomers exist. Is easily understood by those skilled in the art, and any tautomer is included in the scope of the present invention.

本発明の化合物の代表的化合物の製造方法を本明細書の実施例に詳細かつ具体的に示した。当業者は、本実施例の説明を基にして反応原料、反応条件、及び反応試薬などを適宜選択し、必要に応じてこれらの方法に修飾や改変を加えることによって、上記一般式で表される本発明の化合物をいずれも製造することができる。なお、原料化合物として用いることができる4-アミノフルオレセインなどのフルオレセイン誘導体は、例えば、亀谷哲治著、有機合成化学IX、南江堂、215頁(1977年)等に記載の方法に準じて製造できる。また、環状ポリアミン部分構造に関してはJ. Am. Chem. Soc., 118, 12696, 1996に開示された亜鉛プローブの部分構造の合成を参照することができる。   The production methods of representative compounds of the compounds of the present invention are shown in detail and specifically in the examples of the present specification. Those skilled in the art can appropriately select the reaction raw materials, reaction conditions, reaction reagents, and the like based on the description of the present example, and modify or modify these methods as necessary. Any of the compounds of the present invention can be prepared. In addition, a fluorescein derivative such as 4-aminofluorescein that can be used as a raw material compound can be produced according to the method described in, for example, Tetsuji Kameya, Synthetic Organic Chemistry IX, Nanedo, page 215 (1977). Regarding the cyclic polyamine partial structure, the synthesis of the partial structure of the zinc probe disclosed in J. Am. Chem. Soc., 118, 12696, 1996 can be referred to.

本明細書において用いられる「測定」という用語は、定量、定性、又は診断などの目的で行われる測定、検査、検出などを含めて、最も広義に解釈しなければならない。本発明による硫化水素の測定方法は、一般的には、(a)一般式(IA)又は(IB)で表される化合物又はその塩と硫化水素とを反応させる工程、及び(b)上記工程(a)で生成した一般式(IIA)又は(IIB)で表される化合物又はその塩に由来する蛍光を測定する工程を含んでいる。上記一般式(IA)又は(IB)で表される化合物は二価銅イオンの配位により消光されており、それ自体は無蛍光性又は弱蛍光性であるが、硫化水素と接触すると二価銅イオンが硫化水素と反応して不溶性のCuSとなり環状ポリアミン部分から脱落し、強蛍光性の一般式(IIA)又は(IIB)で表される化合物又はその塩を生成する。   As used herein, the term “measurement” should be interpreted in the broadest sense, including measurements, tests, detections, etc. performed for purposes such as quantification, qualitative or diagnostic. The method for measuring hydrogen sulfide according to the present invention generally comprises (a) a step of reacting a compound represented by the general formula (IA) or (IB) or a salt thereof with hydrogen sulfide, and (b) the above step. and a step of measuring fluorescence derived from the compound represented by the general formula (IIA) or (IIB) generated in (a) or a salt thereof. The compound represented by the above general formula (IA) or (IB) is quenched by coordination of divalent copper ions, and is itself non-fluorescent or weakly fluorescent. Copper ions react with hydrogen sulfide to form insoluble CuS, which is removed from the cyclic polyamine moiety to produce a highly fluorescent compound represented by the general formula (IIA) or (IIB) or a salt thereof.

硫化水素測定用の試薬としては、二価銅イオンが配位した状態の一般式(IA)又は(IB)で表される化合物又はその塩を水などの適宜の水性媒体に溶解して使用することができるが、測定系内で一般式(IIA)又は(IIB)で表される化合物又はその塩と二価銅イオンとを反応させてin situに一般式(IA)又は(IB)で表される化合物又はその塩を測定系内で生成させ、その一般式(IA)又は(IB)で表される化合物又はその塩と硫化水素とを反応させてもよい。このような測定方法も本発明の範囲に包含されることは言うまでもない。   As a reagent for measuring hydrogen sulfide, a compound represented by the general formula (IA) or (IB) in which a divalent copper ion is coordinated or a salt thereof is dissolved in an appropriate aqueous medium such as water. However, in the measurement system, the compound represented by the general formula (IIA) or (IIB) or a salt thereof and a divalent copper ion are reacted with each other in situ to represent the general formula (IA) or (IB). Or a salt thereof may be produced in a measurement system and the compound represented by the general formula (IA) or (IB) or a salt thereof may be reacted with hydrogen sulfide. It goes without saying that such a measuring method is also included in the scope of the present invention.

本発明の硫化水素測定用試薬を用いた蛍光測定手段は特に限定されないが、イン・ビトロで蛍光スペクトルを測定する方法や、バイオイメージングの手法を用いてイン・ビボで蛍光スペクトルを測定する方法などを採用することができる。例えば、定量を行う場合には、常法に従って予め検量線を作成しておくことが望ましい。本発明の試薬をマイクロインジェクション法等により細胞内に取り込ませれば、個々の細胞内に局在する硫化水素をバイオイメージング手法により高感度にリアルタイムで測定することができるほか、細胞培養液又は組織切片等の培養液又は灌流液中に用いることで細胞や生体組織が放出する硫化水素を測定できる。従って、本発明の硫化水素測定用試薬を用いることにより、細胞又は生体組織での硫化水素の挙動をリアルタイムに測定することが可能であり、硫化水素によるシグナル伝達のメカニズムの解明のほか、疾患病態の原因究明や治療薬の開発などに好適に利用することができる。   The fluorescence measuring means using the hydrogen sulfide measuring reagent of the present invention is not particularly limited, but a method of measuring a fluorescence spectrum in vitro, a method of measuring a fluorescence spectrum in vivo using a bioimaging technique, etc. Can be adopted. For example, when quantification is performed, it is desirable to prepare a calibration curve in advance according to a conventional method. If the reagent of the present invention is incorporated into cells by a microinjection method or the like, hydrogen sulfide localized in individual cells can be measured in real time with high sensitivity by a bioimaging technique, and a cell culture solution or tissue slice The hydrogen sulfide released from cells and living tissues can be measured by using it in a culture solution or perfusate. Therefore, by using the hydrogen sulfide measurement reagent of the present invention, it is possible to measure the behavior of hydrogen sulfide in cells or living tissues in real time. In addition to elucidating the mechanism of signal transmission by hydrogen sulfide, It can be suitably used for investigating the cause of the disease and developing therapeutic agents.

また、例えば、R5、R6、R7、R8、又はR9のいずれか又は2箇所以上にエステラーゼなどにより加水分解可能なエステル基を導入しておくと、分子全体が脂溶性になって細胞膜を容易に透過して細胞質内に到達するが、細胞質内においてエステラーゼにより加水分解を受けて親水性のカルボキシル基が生成すると細胞膜を容易に透過できなくなる。従って、このようなエステル導入型の化合物(例えばエステルとしてアセトキシメチルエステルやメトキシメチルエステルなどを導入した化合物)は、細胞質内における硫化水素の濃度を測定するための試薬として極めて有用である。 Further, for example, if an ester group that can be hydrolyzed by esterase or the like is introduced into any one or two or more of R 5 , R 6 , R 7 , R 8 , or R 9 , the entire molecule becomes lipophilic. Thus, it easily penetrates the cell membrane and reaches the cytoplasm. However, when a hydrophilic carboxyl group is generated by esterase hydrolysis in the cytoplasm, the cell membrane cannot be easily penetrated. Accordingly, such ester-introduced compounds (for example, compounds into which acetoxymethyl ester or methoxymethyl ester is introduced as an ester) are extremely useful as reagents for measuring the concentration of hydrogen sulfide in the cytoplasm.

本発明の試薬は、必要に応じて試薬の調製に通常用いられる添加剤を配合して組成物として用いてもよい。例えば、生理的環境で試薬を用いるための添加剤として、溶解補助剤、pH調節剤、緩衝剤、等張化剤などの添加剤を用いることができ、これらの配合量は当業者に適宜選択可能である。これらの組成物は、粉末形態の混合物、凍結乾燥物、顆粒剤、錠剤、液剤など適宜の形態の組成物として提供される。   The reagent of this invention may mix | blend the additive normally used for preparation of a reagent as needed, and may use it as a composition. For example, additives such as solubilizers, pH adjusters, buffers, and isotonic agents can be used as additives for using the reagent in a physiological environment, and the amount of these additives is appropriately selected by those skilled in the art. Is possible. These compositions are provided as a composition in an appropriate form such as a mixture in a powder form, a lyophilized product, a granule, a tablet, or a liquid.

以下、実施例により本発明をさらに具体的に説明するが、本発明の範囲は下記の実施例に限定されることはない。
例1
下記合成スキームによりCyclen-4-AF及びCyclen-4-AF-Cuを合成した。
EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.
Example 1
Cyclen-4-AF and Cyclen-4-AF-Cu were synthesized according to the following synthesis scheme.

(a)クロロアセチルアミド-4-AF
4-アミノフルオレセイン (948.5 mg, 2.73 mmol) を脱水テトラヒドロフラン(THF) 30 mLに溶解し、NaHCO3 (761.5 mg, 9.06 mmol) を加えた。脱水THF 40 mLに溶解したClCH2COCl (436.8 mg, 3.87 mmol) を攪拌しながら10分以上かけて滴下した。その後、室温にてアルゴン下で一晩攪拌し、濾過後に溶媒を除去し、残渣をカラムクロマトグラフィー (silica gel、酢酸エチル/ヘキサン = 6/4) で精製して目的物 (756.9 mg, 1.79 mmol, y. 65%) を得た。
1H NMR (300 MHz, CD3OD): δ 8.34 (d, 1H, J = 1.5 Hz), 7.86 (dd, 1H, J = 8.1, 1.5 Hz), 7.16 (d, 1H, J = 8.1 Hz), 6.67 (d, 2H, J = 2.9 Hz), 6.61 (d, 2H, J = 8.8 Hz), 6.52 (dd, 2H, J = 8.8, 2.9 Hz), 4.24 (s, 2H)
13C NMR (75 MHz, CD3OD): δ 171.1, 167.8, 161.4, 154.1, 149.5, 141.2, 130.2, 129.1, 128.3, 125.9, 116.4, 113.6, 111.3, 103.5, 44.0
HRMS (ESI+): Calcd for [M+H]+, 424.0588, Found, 424.0578 (-1.0 mmu) (a) Chloroacetylamide-4-AF
4-Aminofluorescein (948.5 mg, 2.73 mmol) was dissolved in 30 mL of dehydrated tetrahydrofuran (THF), and NaHCO 3 (761.5 mg, 9.06 mmol) was added. ClCH 2 COCl (436.8 mg, 3.87 mmol) dissolved in 40 mL of dehydrated THF was added dropwise over 10 minutes with stirring. After stirring overnight at room temperature under argon, the solvent was removed after filtration, and the residue was purified by column chromatography (silica gel, ethyl acetate / hexane = 6/4) to give the desired product (756.9 mg, 1.79 mmol , y. 65%).
1 H NMR (300 MHz, CD 3 OD): δ 8.34 (d, 1H, J = 1.5 Hz), 7.86 (dd, 1H, J = 8.1, 1.5 Hz), 7.16 (d, 1H, J = 8.1 Hz) , 6.67 (d, 2H, J = 2.9 Hz), 6.61 (d, 2H, J = 8.8 Hz), 6.52 (dd, 2H, J = 8.8, 2.9 Hz), 4.24 (s, 2H)
13 C NMR (75 MHz, CD 3 OD): δ 171.1, 167.8, 161.4, 154.1, 149.5, 141.2, 130.2, 129.1, 128.3, 125.9, 116.4, 113.6, 111.3, 103.5, 44.0
HRMS (ESI + ): Calcd for [M + H] + , 424.0588, Found, 424.0578 (-1.0 mmu)

(b)Cyclen-4-AF
クロロアセチルアミド-4-アミノフルオレセイン (106.6 mg, 251.5 μmol) を脱水アセトニトリル 30 mLに溶解し、cyclen (368.0 mg, 2.14 mmol) とジイソプロピルエチルアミン(DIEA, 300 μL, 1.72 mmol) を加え、アルゴン下において70℃で8時間攪拌した。溶媒を除去した後、HPLCにて精製してCyclen-4-AF (TFA塩) (103.3 mg, 109.0 μmol, y. 43%) を得た。
1H NMR (300 MHz, D2O): δ 8.34 (d, 1H, J = 2.2 Hz), 7.83 (dd, 1H, J = 8.1, 2.2 Hz), 7.28 (d, 2H, J = 8.8 Hza), 7.23 (d, 1H, J = 8.1 Hz), 6.92 (dd, 2H, J = 8.8, 2.2 H), 6.86 (d, 2H, J = 2.2 Hz), 3.69 (s, 2H), 3.05-3.16 (m, 16H)
13C NMR (100 MHz, D2O): δ 170.9, 167.5, 165.9, 155.8, 137.8, 131.7, 130.5, 129.2, 128.1, 124.1, 120.2, 116.8, 116.3, 113.7, 113.4, 100.9, 54.5, 48.3, 42.9, 41.2, 40.7
HRMS (ESI+): Calcd for [M+H]+, 560.2509, Found, 560.2506 (-0.3 mmu) (b) Cyclen-4-AF
Dissolve chloroacetylamido-4-aminofluorescein (106.6 mg, 251.5 μmol) in 30 mL of dehydrated acetonitrile, add cyclen (368.0 mg, 2.14 mmol) and diisopropylethylamine (DIEA, 300 μL, 1.72 mmol), and under argon. Stir at 70 ° C. for 8 hours. After removing the solvent, the residue was purified by HPLC to obtain Cyclen-4-AF (TFA salt) (103.3 mg, 109.0 μmol, y. 43%).
1 H NMR (300 MHz, D 2 O): δ 8.34 (d, 1H, J = 2.2 Hz), 7.83 (dd, 1H, J = 8.1, 2.2 Hz), 7.28 (d, 2H, J = 8.8 Hza) , 7.23 (d, 1H, J = 8.1 Hz), 6.92 (dd, 2H, J = 8.8, 2.2 H), 6.86 (d, 2H, J = 2.2 Hz), 3.69 (s, 2H), 3.05-3.16 ( m, 16H)
13 C NMR (100 MHz, D 2 O): δ 170.9, 167.5, 165.9, 155.8, 137.8, 131.7, 130.5, 129.2, 128.1, 124.1, 120.2, 116.8, 116.3, 113.7, 113.4, 100.9, 54.5, 48.3, 42.9 , 41.2, 40.7
HRMS (ESI + ): Calcd for [M + H] + , 560.2509, Found, 560.2506 (-0.3 mmu)

(c)Cyclen-4-AF-Cu
CuSO4五水和物を30 mM HEPES緩衝液 (pH 7.4) に溶解し、1M CuSO4水溶液を調製した。Cyclen-4-AF (73.8 mg, 77.9 μmol) をCuSO4水溶液 (7792 μL, 7.79 mmol) に溶解し、室温にて一晩攪拌し、HPLCで精製してCyclen-4-AF-Cu (65.2 mg, quant yield) を得た。
HRMS (ESI+): Calcd for [M-H]+, 621.1649, Found, 621.1690 (4.1 mmu) (c) Cyclen-4-AF-Cu
CuSO 4 pentahydrate was dissolved in 30 mM HEPES buffer (pH 7.4) to prepare a 1M CuSO 4 aqueous solution. Cyclen-4-AF (73.8 mg, 77.9 μmol) was dissolved in CuSO 4 aqueous solution (7792 μL, 7.79 mmol), stirred at room temperature overnight, purified by HPLC, and Cyclen-4-AF-Cu (65.2 mg , quant yield).
HRMS (ESI + ): Calcd for [MH] + , 621.1649, Found, 621.1690 (4.1 mmu)

(d)Cyclen-4-AF-Cu diacetate
Cyclen-4-AF-Cu (5.60 μmol) を脱水アセトニトリル 4 mLに溶解し、ピリジン (4.14 mmol)と無水酢酸 (41.9 mmol)を加え、アルゴン下、60℃で6時間攪拌した。溶媒を除去後、HPLCで精製してCyclen-4-AF-Cu diacetate (3.6 mg, 5.1 μmol, y. 91%) を得た。
HRMS (ESI+): Calcd for [M-H]+, 705.1860, Found, 705.1843 (-1.7 mmu) (d) Cyclen-4-AF-Cu diacetate
Cyclen-4-AF-Cu (5.60 μmol) was dissolved in 4 mL of dehydrated acetonitrile, pyridine (4.14 mmol) and acetic anhydride (41.9 mmol) were added, and the mixture was stirred at 60 ° C. for 6 hours under argon. After removing the solvent, the residue was purified by HPLC to obtain Cyclen-4-AF-Cu diacetate (3.6 mg, 5.1 μmol, y. 91%).
HRMS (ESI + ): Calcd for [MH] + , 705.1860, Found, 705.1843 (-1.7 mmu)

例2
TACN-4-AFを以下のスキームで合成した。
Example 2
TACN-4-AF was synthesized by the following scheme.

クロロアセチルアミド-4-アミノフルオレセイン (53.8 mg, 126.9 μmol) を脱水アセトニトリル 8 mLに溶解し、1,4,7-トリアザシクロノナン (TACN, 101.4 mg, 784.8 μmol)、ヨウ化カリウム (6.8 mg, 41.0 μmol) 及び、K2CO3 (111.0 mg, 803.1 μmol) を加え、アルゴン下において室温で一晩攪拌した。溶媒を除去後、HPLCで精製し、TACN-4-AF (TFA塩) (47.4 mg, 55.2 μmol, y. 44%) を得た。
1H NMR (300 MHz, D2O): δ 8.35 (d, 1H, J = 2.2 Hz), 7.84 (dd, 1H, J = 8.4, 2.2 Hz), 7.27-7.30 (m, 3H), 6.98 (d, 2H, J = 1.5 Hz), 6.93 (dd, 2H, J = 9.2, 1.5 Hz), 3.80 (s, 2H), 3.71 (s, 4H), 3.36 (t, 4H, J = 5.5 Hz), 3.15 (t, 4H, J = 5.5 Hz)
13C NMR (100 MHz, D2O): δ 171.4, 168.1, 165.4, 155.6, 137.5, 132.6, 130.2, 129.6, 127.8, 123.9, 119.6, 116.3, 116.2, 113.3, 113.3, 100.9, 55.1, 47.6, 42.8, 41.5
HRMS (ESI+): Calcd for [M+H]+, 517.2087, Found, 517.2106 (1.9 mmu) Chloroacetylamido-4-aminofluorescein (53.8 mg, 126.9 μmol) was dissolved in 8 mL of dehydrated acetonitrile, and 1,4,7-triazacyclononane (TACN, 101.4 mg, 784.8 μmol), potassium iodide (6.8 mg , 41.0 μmol) and K 2 CO 3 (111.0 mg, 803.1 μmol) were added, and the mixture was stirred overnight at room temperature under argon. After removing the solvent, the residue was purified by HPLC to obtain TACN-4-AF (TFA salt) (47.4 mg, 55.2 μmol, y. 44%).
1 H NMR (300 MHz, D 2 O): δ 8.35 (d, 1H, J = 2.2 Hz), 7.84 (dd, 1H, J = 8.4, 2.2 Hz), 7.27-7.30 (m, 3H), 6.98 ( d, 2H, J = 1.5 Hz), 6.93 (dd, 2H, J = 9.2, 1.5 Hz), 3.80 (s, 2H), 3.71 (s, 4H), 3.36 (t, 4H, J = 5.5 Hz), 3.15 (t, 4H, J = 5.5 Hz)
13 C NMR (100 MHz, D 2 O): δ 171.4, 168.1, 165.4, 155.6, 137.5, 132.6, 130.2, 129.6, 127.8, 123.9, 119.6, 116.3, 116.2, 113.3, 113.3, 100.9, 55.1, 47.6, 42.8 , 41.5
HRMS (ESI + ): Calcd for [M + H] + , 517.2087, Found, 517.2106 (1.9 mmu)

例3
Cyclam-4-AFを以下のスキームで合成した。
Example 3
Cyclam-4-AF was synthesized according to the following scheme.

クロロアセチルアミド-4-アミノフルオレセイン (51.5 mg, 121.5 μmol) を脱水アセトニトリル 8 mLに溶解し、1,4,8,11-テトラアザシクロテトラデカン (cyclam, 191.2 mg, 954.5 μmol)、ヨウ化カリウム (8.7 mg, 52.4 μmol) 及び、K2CO3 (136.7 mg, 989.1 μmol) を加え、アルゴン下において室温で一晩攪拌した。溶媒を除去後、HPLCで精製し、Cyclam-4-AF (TFA塩) (22.5 mg, 21.6 μmol, y. 18%) を得た。
1H NMR (300 MHz, D2O): δ 8.53 (d, 1H, J = 2.2 Hz), 7.89 (dd, 1H, J = 8.8, 2.2 Hz), 7.49 (d, 2H, J = 8.4 Hz), 7.37 (d, 1H, J = 8.8 Hz), 7.18 (d, 2H, J = 2.2 Hz), 7.09 (dd, 2H, J = 8.4, 2.2 Hz), 3.56 (s, 2H), 3.13-3.25 (m, 12H), 2.86-2.91 (m, 4H), 1.94-1.98 (m, 4H)
13C NMR (100 MHz, D2O): δ171.1, 167.2, 166.7, 156.4, 138.1, 130.7, 130.5, 128.6, 123.6, 120.4, 117.3, 116.3, 114.3, 113.4, 100.9, 54.4, 53.6, 52.7, 46.5, 46.0, 45.7, 44.8, 43.6, 42.0, 22.7, 21.4
HRMS (ESI+): Calcd for [M+M]+, 588.2822, Found, 588.2794 (-2.8 mmu). Chloroacetylamido-4-aminofluorescein (51.5 mg, 121.5 μmol) is dissolved in 8 mL of dehydrated acetonitrile, and 1,4,8,11-tetraazacyclotetradecane (cyclam, 191.2 mg, 954.5 μmol), potassium iodide ( 8.7 mg, 52.4 μmol) and K 2 CO 3 (136.7 mg, 989.1 μmol) were added and stirred overnight at room temperature under argon. After removing the solvent, the residue was purified by HPLC to obtain Cyclam-4-AF (TFA salt) (22.5 mg, 21.6 μmol, y. 18%).
1 H NMR (300 MHz, D 2 O): δ 8.53 (d, 1H, J = 2.2 Hz), 7.89 (dd, 1H, J = 8.8, 2.2 Hz), 7.49 (d, 2H, J = 8.4 Hz) , 7.37 (d, 1H, J = 8.8 Hz), 7.18 (d, 2H, J = 2.2 Hz), 7.09 (dd, 2H, J = 8.4, 2.2 Hz), 3.56 (s, 2H), 3.13-3.25 ( m, 12H), 2.86-2.91 (m, 4H), 1.94-1.98 (m, 4H)
13 C NMR (100 MHz, D 2 O): δ171.1, 167.2, 166.7, 156.4, 138.1, 130.7, 130.5, 128.6, 123.6, 120.4, 117.3, 116.3, 114.3, 113.4, 100.9, 54.4, 53.6, 52.7, 46.5, 46.0, 45.7, 44.8, 43.6, 42.0, 22.7, 21.4
HRMS (ESI + ): Calcd for [M + M] + , 588.2822, Found, 588.2794 (-2.8 mmu).

例4
TMCyclen-4-AF及びTMCyclen-4-AF-Cuを以下のスキームで合成した。
Example 4
TMCyclen-4-AF and TMCyclen-4-AF-Cu were synthesized by the following scheme.

(a)1,4,7,10-テトラアザシクロドデカン (cyclen, 614.6 mg, 3.57 mmol) を脱水ジクロロメタン 15 mLに溶解し、ジイソプロピルエチルアミン (1.86 g, 14.35 mmol) を加え、0℃にて攪拌した。そして、脱水ジクロロメタン 10 mLに溶解したp-トルエンスルホン酸クロリド (699.2 mg, 3.67 mmol) を10分以上かけて滴下した。その後室温にて、アルゴン下で16時間攪拌し、2 N NaOH水溶液で液性を塩基性にし、ジクロロメタンで抽出後に脱水し、溶媒を除去した。99%ギ酸 (1.83 g) に溶解し、37% ホルムアルデヒド水溶液 (3.10 mL, 41.6 mmol) を加え、110℃において8.5時間攪拌した。塩酸を加え、50℃で3.5時間攪拌した後、液性を塩基性にし、ジクロロメタンで抽出後脱水し、溶媒を除去した。さらに、HPLCで精製して、液性を塩基性にした後、ジクロロメタンで抽出後に脱水し、溶媒を除去して目的物 (669.7 mg, 1.82 mmol, y. 51%) を得た。
1H NMR (300 MHz, CDCl3) δ: 7.53 (d, 2H, J = 8.1 Hz), 7.14 (d, 2H, J = 8.1 Hz), 3.10 (t, 4H, J = 6.2 Hz), 2.65 (t, 4H, J = 6.2 Hz), 2.32-2.36 (m, 8H), 2.26 (s, 3H), 2.11 (s, 6H), 2.07 (s, 3H)
13C NMR (75 MHz, CDCl3) δ: 142.8, 135.8, 129.4, 126.9, 56.0, 55.3, 55.1, 47.5, 43.5, 43.0, 21.2
HRMS (ESI+): Calcd for [M+H]+, 369.2324, Found, 369.2298 (-2.6 mmu) (a) 1,4,7,10-Tetraazacyclododecane (cyclen, 614.6 mg, 3.57 mmol) was dissolved in 15 mL of dehydrated dichloromethane, diisopropylethylamine (1.86 g, 14.35 mmol) was added, and the mixture was stirred at 0 ° C. did. Then, p-toluenesulfonic acid chloride (699.2 mg, 3.67 mmol) dissolved in 10 mL of dehydrated dichloromethane was added dropwise over 10 minutes. Thereafter, the mixture was stirred at room temperature under argon for 16 hours, basified with 2N NaOH aqueous solution, extracted with dichloromethane and dehydrated to remove the solvent. It melt | dissolved in 99% formic acid (1.83 g), 37% formaldehyde aqueous solution (3.10 mL, 41.6 mmol) was added, and it stirred at 110 degreeC for 8.5 hours. Hydrochloric acid was added, and the mixture was stirred at 50 ° C. for 3.5 hours. The solution was basified, extracted with dichloromethane, dehydrated, and the solvent was removed. Furthermore, after refine | purifying with HPLC and making liquid state basic, it dehydrated after extracting with dichloromethane, the solvent was removed, and the target object (669.7 mg, 1.82 mmol, y. 51%) was obtained.
1 H NMR (300 MHz, CDCl 3 ) δ: 7.53 (d, 2H, J = 8.1 Hz), 7.14 (d, 2H, J = 8.1 Hz), 3.10 (t, 4H, J = 6.2 Hz), 2.65 ( t, 4H, J = 6.2 Hz), 2.32-2.36 (m, 8H), 2.26 (s, 3H), 2.11 (s, 6H), 2.07 (s, 3H)
13 C NMR (75 MHz, CDCl 3 ) δ: 142.8, 135.8, 129.4, 126.9, 56.0, 55.3, 55.1, 47.5, 43.5, 43.0, 21.2
HRMS (ESI + ): Calcd for [M + H] + , 369.2324, Found, 369.2298 (-2.6 mmu)

(b)上記工程(a)で得られた化合物 (669.7 mg, 1.82 mmol) を濃硫酸 5 mLに溶解し、アルゴン下において110℃で11時間攪拌した。水で希釈後、2 N NaOH溶液で液性を塩基性にしてジクロロメタンで抽出し、脱水後、溶媒を除去して目的物 (348.7 mg, 1.63 mmol, y. 89%) を得た。
1H NMR (300 MHz, CDCl3) δ: 2.80 (t, 4H, J = 5.0 Hz), 2.61 (t, 4H, J = 5.0 Hz), 2.45-2.48 (m, 8H), 2.36 (s, 6H), 2.20 (s, 3H)
13C NMR (75 MHz, CDCl3) δ: 55.6, 53.6, 53.3, 47.0, 45.2, 21.4
LRMS (ESI+): [M+H]+, 215 (b) The compound (669.7 mg, 1.82 mmol) obtained in the above step (a) was dissolved in 5 mL of concentrated sulfuric acid and stirred at 110 ° C. for 11 hours under argon. After diluting with water, the solution was made basic with 2 N NaOH solution and extracted with dichloromethane. After dehydration, the solvent was removed to obtain the desired product (348.7 mg, 1.63 mmol, y. 89%).
1 H NMR (300 MHz, CDCl 3 ) δ: 2.80 (t, 4H, J = 5.0 Hz), 2.61 (t, 4H, J = 5.0 Hz), 2.45-2.48 (m, 8H), 2.36 (s, 6H ), 2.20 (s, 3H)
13 C NMR (75 MHz, CDCl 3 ) δ: 55.6, 53.6, 53.3, 47.0, 45.2, 21.4
LRMS (ESI + ): [M + H] + , 215

(c)TMCyclen-4-AF
クロロアセチルアミド-4-アミノフルオレセイン (73.2 mg, 172.7 μmol) を脱水ジメチルホルムアミド 40 mLに溶解し、工程(b)で得られた化合物 (117.4 mg, 547.7 μmol) とジイソプロピルエチルアミン (371 mg, 2.87 mmol) を加え、アルゴン下で加熱還流下に1晩攪拌した。溶媒を除去後、HPLCで精製してTMCyclen-4-AF (TFA塩) (42.2 mg, 42.6 μmol, y. 25%) を得た。
1H NMR (300MHz, D2O) δ: 8.41 (d, 1H, J = 2.2 Hz), 7.85 (dd, 1H, J = 8.8, 2.2 Hz), 7.27 (d, 2H, J = 8.4 Hz), 7.19 (d, 1H, J = 8.8 Hz), 6.90 (dd, 2H, J = 8.4, 2.2 Hz), 6.85 (d, 2H, J = 2.2 Hz), 3.75 (s, 2H), 2.90 (s, 6H), 2.62-3.67 (m, 16H), 2.41 (s, 3H)
13C NMR (75 MHz, D2O) δ: 171.8, 167.4, 166.4, 156.4, 137.6, 131.4, 130.7, 129.4, 128.6, 124.1, 120.6, 117.1, 116.8, 114.1, 112.9, 101.0, 55.6, 52.3, 52.0, 49.0, 48.2, 40.5, 40.4
HRMS (ESI+): Calcd for [M+H]+, 602.2979, Found, 602.2939 (-4.0 mmu) (c) TMCyclen-4-AF
Chloroacetylamido-4-aminofluorescein (73.2 mg, 172.7 μmol) was dissolved in 40 mL of dehydrated dimethylformamide and the compound (117.4 mg, 547.7 μmol) obtained in step (b) and diisopropylethylamine (371 mg, 2.87 mmol) ) Was added and stirred overnight under reflux with heating under argon. After removing the solvent, the residue was purified by HPLC to obtain TMCyclen-4-AF (TFA salt) (42.2 mg, 42.6 μmol, y. 25%).
1 H NMR (300MHz, D 2 O) δ: 8.41 (d, 1H, J = 2.2 Hz), 7.85 (dd, 1H, J = 8.8, 2.2 Hz), 7.27 (d, 2H, J = 8.4 Hz), 7.19 (d, 1H, J = 8.8 Hz), 6.90 (dd, 2H, J = 8.4, 2.2 Hz), 6.85 (d, 2H, J = 2.2 Hz), 3.75 (s, 2H), 2.90 (s, 6H ), 2.62-3.67 (m, 16H), 2.41 (s, 3H)
13 C NMR (75 MHz, D 2 O) δ: 171.8, 167.4, 166.4, 156.4, 137.6, 131.4, 130.7, 129.4, 128.6, 124.1, 120.6, 117.1, 116.8, 114.1, 112.9, 101.0, 55.6, 52.3, 52.0 , 49.0, 48.2, 40.5, 40.4
HRMS (ESI + ): Calcd for [M + H] + , 602.2979, Found, 602.2939 (-4.0 mmu)

(d)TMCyclen-4-AF-Cu
CuSO4五水和物を30 mM HEPES緩衝液 (pH 7.4) に溶解し、1 M CuSO4水溶液を調製した。TMCyclen-4-AF (12.1 mg, 12.2 μmol) をCuSO4水溶液 (1220 μL, 1.22 mmol) に溶解し、室温にて4時間攪拌し、HPLCにて精製してTMCyclen-4-AF-Cu (9.52 mg, y. quant) を得た。
HRMS (ESI+): Calcd for [M-H]+, 663.2118, Found, 663.2106 (-1.2 mmu) (d) TMCyclen-4-AF-Cu
CuSO 4 pentahydrate was dissolved in 30 mM HEPES buffer (pH 7.4) to prepare a 1 M CuSO 4 aqueous solution. TMCyclen-4-AF (12.1 mg, 12.2 μmol) was dissolved in CuSO 4 aqueous solution (1220 μL, 1.22 mmol), stirred at room temperature for 4 hours, purified by HPLC and TMCyclen-4-AF-Cu (9.52 mg, y. quant).
HRMS (ESI + ): Calcd for [MH] + , 663.2118, Found, 663.2106 (-1.2 mmu)

例5
Cyclne-4-AF-Cuの吸光及び蛍光スペクトルを測定した。吸収及び蛍光スペクトルは30 mM HEPES緩衝液 (pH 7.4) 中で測定した。蛍光スペクトルの測定ではCyclen-4-AF-Cuを491 nmで励起した。蛍光量子収率測定では0.1N NaOH溶液中でフルオレセイン(Φfl = 0.85) を蛍光標準として用いた。結果を図1(左側)及び表1に示す。Cyclen-4-AF-Cuの蛍光量子収率は低下していることが示された。同様にしてCyclen-4-AF(図1、右側)、TACN-4-AF(図2左側)、Cyclam-4-AF(図2、右側)、TMCyclen-4-AF(図3、左側)、及びTMCyclen-4-AF-Cu(図3、右側)の吸光及び蛍光スペクトルを測定した。 Example 5
The absorption and fluorescence spectra of Cyclne-4-AF-Cu were measured. Absorption and fluorescence spectra were measured in 30 mM HEPES buffer (pH 7.4). For measurement of the fluorescence spectrum, Cyclen-4-AF-Cu was excited at 491 nm. In the fluorescence quantum yield measurement, fluorescein (Φ fl = 0.85) was used as a fluorescence standard in 0.1N NaOH solution. The results are shown in FIG. 1 (left side) and Table 1. It was shown that the fluorescence quantum yield of Cyclen-4-AF-Cu was lowered. Similarly, Cyclen-4-AF (Fig. 1, right side), TACN-4-AF (Fig. 2, left side), Cyclam-4-AF (Fig. 2, right side), TMCyclen-4-AF (Fig. 3, left side), And the absorption and fluorescence spectra of TMCyclen-4-AF-Cu (FIG. 3, right side) were measured.

例6
Cyclen-4-AF-Cuの反応性の評価を行った。1 μM Cyclen-4-AF-Cuに、300秒後に10 μM Na2S (赤), 10 μM Na2Sと10 mM GSH (緑), 10 mM GSH (黄)を添加し、また、無添加の実験をネガティブコントロール (青) として反応性を調べた。30 mM HEPES緩衝液 (pH 7.4) 中において491 nmで励起し、516 nmの蛍光強度を測定した。結果を図4に示す。Cyclen-4-AF-CuはNa2Sと反応後に蛍光上昇を示し、GSHと比較して選択性を有することが示された。また、Cyclen-4-AF-Cuは10 mM GSH共存下中においても10 μM Na2S添加によって蛍光強度が上昇することが明らかとなった。 Example 6
The reactivity of Cyclen-4-AF-Cu was evaluated. To 1 μM Cyclen-4-AF-Cu, add 10 μM Na 2 S (red), 10 μM Na 2 S, 10 mM GSH (green), and 10 mM GSH (yellow) after 300 seconds. As a negative control (blue), the reactivity was examined. Excitation was performed at 491 nm in 30 mM HEPES buffer (pH 7.4), and the fluorescence intensity at 516 nm was measured. The results are shown in FIG. Cyclen-4-AF-Cu showed an increase in fluorescence after reaction with Na 2 S, indicating that it had selectivity compared to GSH. In addition, Cyclen-4-AF-Cu was found to increase in fluorescence intensity when 10 μM Na 2 S was added even in the presence of 10 mM GSH.

例7
Cyclen-4-AF-Cuを用いて生体内の硫化水素の測定が可能であるか否かを評価した。HeLa細胞を37℃、5% CO2混合空気中にてダルペッコ改変イーグル培地(DMEM)に10% ウシ胎児血清(FBS)、1% ペニシリン、及び1% ストレプトマイシンを添加した培地中で培養した。ハンクス平衡塩溶液(HBSS)にて2回洗浄した。HBSS中のHeLa細胞に対して100 μM Cyclen-4-AF-Cuを含むHBSS (0.1% DMSO) をマイクロインジェクション(領域1-4)した。蛍光顕微鏡にて470-490 nmで励起し、515-550 nmの蛍光強度を測定した。測定開始5分後に細胞外液に10 mM Na2Sを添加した。この結果、マイクロインジェクションしたHeLa細胞内の領域1-4で蛍光強度の増大が観察された。図5の上段はNa2S添加前(0 sec)、中段はNa2S添加後(360 sec)、及び下段は蛍光強度の経時変化を示し、領域5及び6はマイクロインジェクションをしていない領域を示す。 Example 7
It was evaluated whether or not hydrogen sulfide in vivo can be measured using Cyclen-4-AF-Cu. HeLa cells were cultured in a medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin in Dulbecco's modified Eagle medium (DMEM) at 37 ° C in 5% CO 2 mixed air. Washed twice with Hank's balanced salt solution (HBSS). HBSS (0.1% DMSO) containing 100 μM Cyclen-4-AF-Cu was microinjected (region 1-4) into HeLa cells in HBSS. Excitation was performed at 470-490 nm with a fluorescence microscope, and the fluorescence intensity at 515-550 nm was measured. 5 mM after the start of measurement, 10 mM Na 2 S was added to the extracellular fluid. As a result, an increase in fluorescence intensity was observed in the region 1-4 in the microinjected HeLa cells. The upper part of FIG. 5 is before addition of Na 2 S (0 sec), the middle part is after addition of Na 2 S (360 sec), and the lower part is a change in fluorescence intensity with time, and regions 5 and 6 are regions without microinjection. Indicates.

同様に、Cyclen-4-AF-Cu diacetateを用いてHeLa細胞への取り込みによる硫化水素の測定を行った結果を図6に示す。上記と同様にして培養したHela細胞を100 μM Cyclen-4-AF-Cu diacetateを含むHBSSで37℃、2時間インキュベーションした。蛍光顕微鏡にて470-490 nmで励起し、515-550 nmの蛍光強度を測定した。測定開始210秒後に細胞外液に10 mM Na2Sを添加した。HeLa細胞内外で蛍光強度の増大が観察された。図6の上段はNa2S添加前(0 sec)、中段はNa2S添加後(240 sec)、及び下段は蛍光強度の経時変化を示す。 Similarly, FIG. 6 shows the results of measurement of hydrogen sulfide by incorporation into HeLa cells using Cyclen-4-AF-Cu diacetate. Hela cells cultured as described above were incubated at 37 ° C. for 2 hours in HBSS containing 100 μM Cyclen-4-AF-Cu diacetate. Excitation was performed at 470-490 nm with a fluorescence microscope, and the fluorescence intensity at 515-550 nm was measured. 210 mM after the start of measurement, 10 mM Na 2 S was added to the extracellular fluid. An increase in fluorescence intensity was observed inside and outside HeLa cells. The upper part of FIG. 6 shows before Na 2 S addition (0 sec), the middle part after Na 2 S addition (240 sec), and the lower part shows the change in fluorescence intensity over time.

例8
TACN-4-AF単独、及び2当量のCu2+を加えたTACN-4-AFの吸収スペクトルと蛍光スペクトルを測定した。測定は30 mM HEPESバッファー (pH 7.4) 中で行い、蛍光スペクトルの測定では491 nmで励起した。Cu2+の添加により蛍光強度は著しく減少した(図7、上段左側：吸収スペクトル、上段右側：蛍光スペクトル)。また、1 μM TACN-4-AFに2 μM CuSO4を加え、300秒後に100 μM Na2S (赤)、10 μM Na2S (緑)、又は10 mM GSH (黄)を添加し、無添加の実験をネガティブコントロール (青) として反応性を調べた。30 mM HEPES緩衝液 (pH 7.4) 中において491 nmで励起し、516 nmの蛍光強度を測定した。この結果、TACN-4-AF+Cu2+はNa2Sと反応後、大きな蛍光上昇を示し、GSHと比較して硫化水素に対して高い選択性を有することが示された(図7、下段)。 Example 8
The absorption spectrum and fluorescence spectrum of TACN-4-AF alone and TACN-4-AF added with 2 equivalents of Cu 2+ were measured. Measurement was performed in 30 mM HEPES buffer (pH 7.4), and excitation was performed at 491 nm for measurement of the fluorescence spectrum. Addition of Cu 2+ significantly decreased the fluorescence intensity (FIG. 7, upper left: absorption spectrum, upper right: fluorescence spectrum). Add 2 μM CuSO 4 to 1 μM TACN-4-AF, add 100 μM Na 2 S (red), 10 μM Na 2 S (green), or 10 mM GSH (yellow) after 300 seconds. The reactivity was examined using the addition experiment as a negative control (blue). Excitation was performed at 491 nm in 30 mM HEPES buffer (pH 7.4), and the fluorescence intensity at 516 nm was measured. As a result, TACN-4-AF + Cu 2+ showed a large increase in fluorescence after reacting with Na 2 S, indicating that it has a high selectivity for hydrogen sulfide compared to GSH (FIG. 7, Lower row).

例9
Cyclam-4-AF単独、及び2当量のCu2+を加えたCyclam-4-AFの吸収スペクトルと蛍光スペクトルを測定した。測定は30 mM HEPESバッファー (pH 7.4) 中で行い、蛍光スペクトルの測定では491 nmで励起した。Cu2+の添加により蛍光強度は著しく減少した(図8)。 Example 9
The absorption spectrum and fluorescence spectrum of Cyclam-4-AF alone and Cyclam-4-AF added with 2 equivalents of Cu 2+ were measured. Measurement was performed in 30 mM HEPES buffer (pH 7.4), and excitation was performed at 491 nm for measurement of the fluorescence spectrum. Addition of Cu 2+ significantly decreased the fluorescence intensity (Fig. 8).

例10
1 μM Cyclam-4-AFに2 μM CuSO4を加え、300秒後に100 μM Na2S (赤)、10 μM Na2S (緑)、又は10 mM GSH (黄)を添加し、無添加の実験をネガティブコントロール (青) として反応性を調べた。30 mM HEPES緩衝液 (pH 7.4) 中において491 nmで励起し、516 nmの蛍光強度を測定した。この結果、Cyclam-4-AF+Cu2+はNa2Sと反応後、蛍光上昇を示し、GSHと比較して選択性を有することが示された(図9上段)。同様にして、1 μM TMCyclen-4-AF-Cuに対して、300秒後に100 μM Na2S (赤)、10 μM Na2S (緑)、又は10 mM GSH (黄)を添加し、無添加の実験をネガティブコントロール (青) として反応性を調べたところ、TMCyclen-4-AF-CuもNa2Sと反応後に蛍光上昇を示し、GSHと比較して選択性を有することが示された(図9下段)。 Example 10
The 2 [mu] M CuSO 4 was added to 1 μM Cyclam-4-AF, after 300 seconds 100 μM Na 2 S (red), was added 10 μM Na 2 S (green), or 10 mM GSH (yellow), additive-free The reactivity was examined using the experiment as a negative control (blue). Excitation was performed at 491 nm in 30 mM HEPES buffer (pH 7.4), and the fluorescence intensity at 516 nm was measured. As a result, Cyclam-4-AF + Cu 2+ showed an increase in fluorescence after reacting with Na 2 S, indicating that it has selectivity compared to GSH (upper part of FIG. 9). Similarly, 100 μM Na 2 S (red), 10 μM Na 2 S (green), or 10 mM GSH (yellow) is added to 1 μM TMCyclen-4-AF-Cu after 300 seconds. When the reactivity was examined using the addition experiment as a negative control (blue), TMCyclen-4-AF-Cu also showed an increase in fluorescence after reaction with Na 2 S, indicating that it had selectivity compared to GSH. (Figure 9, bottom).

Claims (10)

下記の一般式(IA)又は(IB)：
〔式中、R1は下記式(A)：
（式中、p、q、r、及びsはそれぞれ独立に2又は3の整数を示し、tは0又は1を示し、R11、R12、及びR13はそれぞれ独立に水素原子又は炭素原子数1〜6のアルキル基を示す）で表される基を示し；R2は水素原子又はベンゼン環上に置換する1個ないし3個の一価の置換基を示し；R3及びR4はそれぞれ独立に水素原子又はハロゲン原子を示し；R5及びR6はそれぞれ独立に水素原子、アルキルカルボニル基、又はアルキルカルボニルオキシアルキル基を示し；R7は水素原子、アルキル基、又はアルコキシアルキル基を示し；R8及びR9はそれぞれ独立に水素原子、ハロゲン原子、又は-(CH2)x-N(R14)(R15)(式中、xは1ないし4の整数を示し、R14及びR15はそれぞれ独立に-(CH2)y-COOR16（式中、yは1ないし4の整数を示し、R16は水素原子、アルキルカルボニル基、又はアルキルカルボニルオキシアルキル基を示す）で表される基を示す)で表される基を示す〕で表される化合物又はその塩。 The following general formula (IA) or (IB):
[Wherein R 1 represents the following formula (A):
(In the formula, p, q, r, and s each independently represent an integer of 2 or 3, t represents 0 or 1, and R 11 , R 12 , and R 13 each independently represent a hydrogen atom or a carbon atom. R 2 represents a hydrogen atom or 1 to 3 monovalent substituents substituted on a benzene ring; R 3 and R 4 represent R 5 and R 6 each independently represent a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxyalkyl group; R 7 represents a hydrogen atom, an alkyl group, or an alkoxyalkyl group. shown; R 8 and R 9 are each independently a hydrogen atom, a halogen atom, or a - (CH 2) x -N ( R 14) (R 15) ( wherein, x is an integer of 1 to 4, R 14 and R 15 are each independently - (CH 2) y -COOR 16 ( wherein, y is an integer of 1 to 4, R 16 is a hydrogen atom, an alkylcarbonyl group, The compound or a salt thereof is represented by a group represented by a group represented by showing the alkylcarbonyloxy group))]. ベンゼン環上に存在するR1がキサンテン環の結合部位に対してパラ位に結合しており、p、q、r、及びsが2であり、tが1であり、R11、R12、及びR13が水素原子であり、かつR2が水素原子である請求項１に記載の一般式(IA)又は(IB)で表される化合物又はその塩。 R 1 present on the benzene ring is bonded at the para position with respect to the binding site of the xanthene ring, p, q, r, and s are 2, t is 1, R 11 , R 12 , And R 13 is a hydrogen atom, and R 2 is a hydrogen atom, or a compound represented by the general formula (IA) or (IB) or a salt thereof according to claim 1. R3及びR4が水素原子であり、R5及びR6がそれぞれ独立に水素原子、アセチル基、又はアセチルオキシメチル基であり、R7が水素原子、アルキル基、又はメトキシメチル基であり、R8及びR9がともに水素原子である請求項１又は２に記載の一般式(IA)又は(IB)で表される化合物又はその塩。 R 3 and R 4 are a hydrogen atom, R 5 and R 6 are each independently a hydrogen atom, an acetyl group, or an acetyloxymethyl group, R 7 is a hydrogen atom, an alkyl group, or a methoxymethyl group, The compound represented by the general formula (IA) or (IB) or a salt thereof according to claim 1 or 2, wherein R 8 and R 9 are both hydrogen atoms. 下記の一般式(IIA)又は(IIB)：
〔式中、R21は下記式(B)：
（式中、d、e、f、及びgはそれぞれ独立に2又は3の整数を示し、hは0又は1を示し、R31、R32、及びR33はそれぞれ独立に水素原子又は炭素原子数1〜6のアルキル基を示す）で表される基を示し；R22は水素原子又はベンゼン環上に置換する1個ないし3個の一価の置換基を示し；R23及びR24はそれぞれ独立に水素原子又はハロゲン原子を示し；R25及びR26はそれぞれ独立に水素原子、アルキルカルボニル基、又はアルキルカルボニルオキシアルキル基を示し；R27は水素原子、アルキル基、又はアルコキシアルキル基を示し；R28及びR29はそれぞれ独立に水素原子、ハロゲン原子、又は-(CH2)m-N(R34)(R35)(式中、mは1ないし4の整数を示し、R34及びR35はそれぞれ独立に-(CH2)n-COOR36（式中、nは1ないし4の整数を示し、R36は水素原子、アルキルカルボニル基、又はアルキルカルボニルオキシアルキル基を示す）で表される基を示す)で表される基を示す〕で表される化合物又はその塩。 The following general formula (IIA) or (IIB):
[Wherein R 21 represents the following formula (B):
(Wherein, d, e, f and g each independently represents an integer of 2 or 3, h represents 0 or 1, and R 31 , R 32 and R 33 each independently represent a hydrogen atom or a carbon atom. R 22 represents a hydrogen atom or 1 to 3 monovalent substituents substituted on a benzene ring; R 23 and R 24 represent R 25 and R 26 each independently represent a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxyalkyl group; R 27 represents a hydrogen atom, an alkyl group, or an alkoxyalkyl group. shown; each R 28 and R 29 independently represent a hydrogen atom, a halogen atom, or a - (CH 2) m -N ( R 34) (R 35) ( wherein, m is an integer of 1 to 4, R 34 and R 35 are each independently - (CH 2) n -COOR 36 ( wherein, n represents an integer of from 1 4, R 36 is a hydrogen atom, an alkyl carbonylation Group or alkylcarbonyloxy group represents a group represented by the illustrated)) or a salt thereof shows a group] represented by. ベンゼン環上に存在するR21がキサンテン環の結合部位に対してパラ位に結合しており、d、e、f、及びgが2であり、hが1であり、R31、R32、及びR33が水素原子であり、かつR22が水素原子である請求項４に記載の一般式(IIA)又は(IIB)で表される化合物又はその塩。 R 21 present on the benzene ring is bonded at the para position with respect to the binding site of the xanthene ring, d, e, f, and g are 2, h is 1, R 31 , R 32 , And R 33 is a hydrogen atom, and R 22 is a hydrogen atom, or a compound represented by the general formula (IIA) or (IIB) or a salt thereof according to claim 4. R23及びR24が水素原子であり、R25及びR26がそれぞれ独立に水素原子、アセチル基、又はアセチルオキシメチル基であり、R27が水素原子、アルキル基、又はメトキシメチル基であり、R28及びR29がともに水素原子である請求項４又は５に記載の一般式(IIA)又は(IIB)で表される化合物又はその塩。 R 23 and R 24 are a hydrogen atom, R 25 and R 26 are each independently a hydrogen atom, an acetyl group, or an acetyloxymethyl group, R 27 is a hydrogen atom, an alkyl group, or a methoxymethyl group, 6. The compound represented by the general formula (IIA) or (IIB) or a salt thereof according to claim 4 or 5, wherein R 28 and R 29 are both hydrogen atoms. 請求項１ないし３のいずれか１項に記載の一般式(IA)又は(IB)で表される化合物又はその塩を含む硫化水素測定用蛍光プローブ。 A fluorescent probe for measuring hydrogen sulfide comprising the compound represented by the general formula (IA) or (IB) or a salt thereof according to any one of claims 1 to 3. 請求項４ないし６のいずれか１項に記載の一般式(IIA)又は(IIB)で表される化合物又はその塩を含む硫化水素測定用蛍光プローブ。 A fluorescent probe for measuring hydrogen sulfide, comprising the compound represented by the general formula (IIA) or (IIB) according to any one of claims 4 to 6 or a salt thereof. 硫化水素の測定方法であって、下記の工程：
(a)請求項１ないし３のいずれか１項に記載の一般式(IA)又は(IB)で表される化合物又はその塩を硫化水素と接触させる工程；及び
(b)上記工程(a)で生成した上記一般式(IIA)又は(IIB)で表される化合物又はその塩の蛍光を測定する工程
を含む方法。 A method for measuring hydrogen sulfide, comprising the following steps:
(a) contacting the compound represented by the general formula (IA) or (IB) according to any one of claims 1 to 3 or a salt thereof with hydrogen sulfide; and
(b) A method comprising a step of measuring the fluorescence of the compound represented by the general formula (IIA) or (IIB) produced in the step (a) or a salt thereof. 硫化水素の測定方法であって、下記の工程：
(a)請求項４ないし６のいずれか１項に記載の一般式(IIA)又は(IIB)で表される化合物又はその塩と二価銅イオンとを反応させて上記一般式(IA)又は(IB)で表される化合物又はその塩を生成させる工程；
(b)上記工程(a)で生成した上記一般式(IA)又は(IB)で表される化合物又はその塩と硫化水素とを接触させる工程；及び
(c)上記工程(b)で生成した上記一般式(IIA)又は(IIB)で表される化合物又はその塩の蛍光を測定する工程
を含む方法。 A method for measuring hydrogen sulfide, comprising the following steps:
(a) A compound represented by the general formula (IIA) or (IIB) according to any one of claims 4 to 6 or a salt thereof and a divalent copper ion are reacted to form the above general formula (IA) or A step of producing a compound represented by (IB) or a salt thereof;
(b) contacting the compound represented by the general formula (IA) or (IB) produced in the step (a) or a salt thereof with hydrogen sulfide; and
(c) A method comprising a step of measuring the fluorescence of the compound represented by the general formula (IIA) or (IIB) produced in the step (b) or a salt thereof.
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