JP3731618B2 - Epoxy derivatives - Google Patents

Description

〔式中、Ｘは−Ｎ（Ｒ³ ）−、メチレン基（−ＣＨ₂ −）、酸素原子（−Ｏ−）、又は硫黄原子（−Ｓ−）であり、Ｒ¹ 及びＲ² は、同じか又は異なり、それぞれ水素原子、炭素数１〜４のアルキル基、非置換アミノ基、置換されたアミノ基、カルボキシル基、アルコキシ基、シアノ基、ニトロ基又はハロゲン原子であり、Ｒ³ は炭素数１〜４のアルキル基、非置換フェニル基、置換されたフェニル基、ベンジル基、アルコキシカルボニルメチル基、又はアリールオキシカルボニルメチル基である〕で表される化合物又はその塩に関する。
【００１１】
【発明の実施の形態】
以下、本発明を詳細に説明する。
本明細書において「炭素数１〜４のアルキル基」は、直鎖状若しくは分枝状のアルキル基であり、例えば、メチル基、エチル基、ｎ−プロピル基、ｉ−プロピル基、ｎ−ブチル基、ｉ−ブチル基、ｓ−ブチル基、又はｔ−ブチル基である。本明細書において「非置換アミノ基」は、−ＮＨ₂ 基である。「置換されたアミノ基」は、例えば、低級アルキル基、特には炭素数１〜４のアルキル基でモノ−若しくはジ−置換されたアミノ基であり、例えば、モノ若しくはジメチルアミノ基、モノ若しくはジエチルアミノ基、メチルエチルアミノ基、又はモノ若しくはジベンジルアミノ基を挙げることができる。
「アルコキシ基」は、例えば、炭素数１〜４のアルコシキ基、例えば、メトキシ基、エトキシ基、ｎ−プロポキシ基、ｉ−プロポキシ基、ｎ−ブトキシ基、ｉ−ブトキシ基、ｓ−ブトキシ基、又はｔ−ブトキシ基である。「ハロゲン原子」は、例えば、塩素原子、臭素原子、ヨウ素原子、又はフッ素原子である。
【００１２】
「置換フェニル基」は特に限定されないが、炭素数１〜４のアルキル基、非置換アミノ基、置換されたアミノ基、カルボキシル基、アルコキシ基、シアノ基、ニトロ基、ハロゲン原子及びスクシンイミノ基からなる群から選んだ同一若しくは異なる置換基１〜５個、好ましくは１〜３個を担持していることができる。従って、置換フェニル基は、例えば、一般式（II）：
【化５】

（式中、Ｒ⁴ 、Ｒ⁵ 及びＲ⁶ は、同じか又は異なり、それぞれ水素原子、炭素数１〜４のアルキル基、非置換アミノ基、置換されたアミノ基、カルボキシル基、アルコキシ基、シアノ基、ニトロ基、ハロゲン原子又はスクシンイミノ基である）で表される基であることができる。
【００１３】
「アルコキシカルボニルメチル基」は、例えば、炭素数１〜４のアルコキシ部分を有するカルボニルメチル基であり、例えば、メトキシカルボニルメチル基、又はエトキシカルボニルメチル基を挙げることができる。また、「アリールオキシカルボニルメチル基」は、例えば、場合により１又はそれ以上の置換基を担持することのある炭素数６〜１８のアリール部分を有するアリールオキシカルボニルメチル基である。好ましいアリールオキシカルボニルメチル基は、例えば、一般式（III)：
【化６】

（式中、Ｒ⁷ 、Ｒ⁸ 及びＲ⁹ は、同じか又は異なり、それぞれ水素原子、炭素数１〜４のアルキル基、非置換アミノ基、置換されたアミノ基、カルボキシル基、アルコキシ基、シアノ基、ニトロ基、ハロゲン原子又はスクシンイミノ基である）で表される基である。
【００１４】
前記一般式（Ｉ）で表される新規エポキシ誘導体において、Ｘが−Ｎ（Ｒ³ ）−、メチレン基、酸素原子、又は硫黄原子であり、Ｒ¹ 及びＲ² が、同じか又は異なり、それぞれ水素原子又は炭素数１〜４のアルキル基であり、Ｒ³ が炭素数１〜４のアルキル基、非置換フェニル基、置換されたフェニル基、ベンジル基、アルコキシ部分の炭素数が１〜４のアルコキシカルボニルメチル基、又はフェニルオキシカルボニルメチル基である前記一般式（Ｉ）で表されるエポキシ誘導体又はその塩が好ましい。
【００１５】
前記一般式（Ｉ）で表される新規エポキシ誘導体において、Ｘが−Ｎ（Ｒ³ ）−、メチレン基、酸素原子、又は硫黄原子であり、Ｒ¹ 及びＲ² が水素原子であり、Ｒ³ がメチル基、非置換フェニル基、ベンジル基、又はエトキシカルボニルメチル基である前記一般式（Ｉ）で表されるエポキシ誘導体又はその塩が特に好ましい。
【００１６】
前記一般式（Ｉ）で表わされるエポキシ誘導体がカルボキシル基又はアミノ基を有する場合には、前記エポキシ誘導体は塩を形成することができ、それらの塩も本発明の化合物に含まれる。カルボン酸塩としては、例えば、アルカリ金属（例えば、ナトリウム又はカリウム）の塩を挙げることができ、アミノ基の塩としては、例えば、酸付加塩、例えば、塩酸塩、硫酸塩、各種スルホン酸塩、シュウ酸塩、又は過塩素酸塩を挙げることができる
【００１７】
本発明による前記一般式（Ｉ）で表わされるエポキシ誘導体は、それ自体公知の方法によって調製することができる。
調製方法の１例を、工程図によって図１に示す。すなわち、ケトン類（１）とアダマンタノンとを、例えば、無水三塩化チタンアルミニウム還元型と水素化アルミニウムリチウムによる還元的縮合反応によりオレフィン体（２）へ誘導する。続いて、得られたオレフィン体（２）を、例えば、塩化メチレン中でｍ−クロロ過安息香酸を用い、常温で短時間反応させると、目的のエポキシ誘導体（Ｉ）を得ることができる。
【００１８】
前記の工程において原料として用いるケトン類（１）は、公知化合物であり、それ自体公知の方法により調製することができる。例えば、Ｒ¹ 及びＲ² が水素原子であり、Ｘが−Ｎ−ＣＨ₃ であるケトン化合物（１ａ）〔すなわち、１０−メチル−９（１０Ｈ）−アクリドン〕、並びにＲ¹ 及びＲ² が水素原子であり、Ｘが−Ｎ−ＣＨ₂ Ｃ₆ Ｈ₅ であるケトン化合物（１ｂ）〔すなわち、１０−フェニルメチル−９（１０Ｈ）−アクリドン〕は、西らの文献（Ｂｕｌｌ．Ｃｈｅｍ．Ｓｏｃ．Ｊｐｎ．，５４，１８９８，１９８１年）とＪ．Ｊ．Ｃｈａｒｂｉｔらの文献（Ｊ．Ｃｈｅｍ．Ｅｎｇ．Ｄａｔａ，３４，１３６，１９８９年）に従って合成することができる。また、Ｒ¹ 及びＲ² が水素原子であり、Ｘが−Ｎ−ＣＨ₂ ＣＯ₂ Ｃ₂ Ｈ₅ であるケトン化合物（１ｃ）〔すなわち、９−オキン−１０（９Ｈ）−アクリジン酢酸エチル〕は、Ｎ．Ｍｉｏｓｇａらの文献（Ｐｈａｍａｚｉｅ，４０，８００，１９８５年）と、Ｉ．Ｄ．Ｐｏｓｔｅｓｃｕらの文献（Ｊ．Ｐｒａｋｔ．Ｃｈｅｍｉｅ．，３１９，３４７，１９７７年）に従って合成することができる。更に、Ｒ¹ 及びＲ² が水素原子であり、Ｘが−Ｎ−ＣＨ₂ Ｃ₆ Ｈ₅ であるケトン化合物（１ｄ）〔すなわち、１０−フェニル−９（１０Ｈ）−アクリドン〕は、Ｍｉｔｔｅｉｌｕｎｇらの文献（Ｂｅｒ，３９，１６９１，１９０６年）に従って合成することができる。
【００１９】
前記一般式（Ｉ）で表される本発明のエポキシ誘導体は、発光性物質であり、しかも従来のアクリジン誘導体等と比較して、中性付近の水溶液中での保存安定性が格段に優れている。例えば、従来技術の代表的発光アクリジン誘導体である９−フェノキシカルボニル−１０−メチルアクリジニウムフルオロスルホネート（ＰＭＡ）の中性水溶液中での保存安定性は、１ヶ月で約８０％発光量が低下するのに対し、本発明のエポキシ誘導体、例えば、１０−メチルジスピロ〔アクリジン−９（１０Ｈ），２’−オキシラン−３’，２”−トリシクロ（３．３．１．１３，７）デカン〕（Ｉａ）は、中性水溶液中に１ヶ月保存しても、ほとんど発光量の低下はみられなかった。
【００２０】
本発明のエポキシ誘導体は、検体中の特定物質を検出する従来公知のあらゆる分析手段において、発光標識物質として用いることができる。特に、生体試料中に含まれる微量の生理活性物質を免疫学的に、あるいは核酸の親和性を利用し、又はクロマトグラフィーを利用して測定する方法、酵素免疫学的、分子生物学的に測定する方法や装置、あるいはキット等に利用するのが好ましい。例えば、サンドイッチ法と呼ばれる免疫学的測定に利用する場合には、まず、ポリエチレンなどの不溶性担体に固定化した特定物質に対する抗体に、試料を接触させることで試料中に含まれる特定物質は、抗体との親和力により上記不溶性担体上に結合する。次に、特定物質に対する抗体（モノクローナル抗体又はポリクローナル抗体）に本発明新規発光物質を従来公知の手段（例えば、物理吸着法あるいは化学的結合）で担持した標識抗体を、前記の不溶性担体に接触させる。この操作により標識抗体は、不溶性担体上に結合している特定物質の量に相関した分だけ不溶性担体上に結合する。次に、不溶性担体上に結合していない過剰の標識抗体を洗浄操作等により分離してから化学発光測定を行う。
【００２１】
本発明のエポキシ誘導体は、アルカリ性過酸化水素の添加により強く発光することができる。従って、化学発光を誘発する試薬としては、通常、水酸化ナトリウムや水酸化カリウム等のアルカリ水溶液に、過酸化水素等の過酸化物を混合した物を使用することができる。このような化学発光を誘発する試薬を、先の不溶性担体上に結合している標識抗体に接触させ、本発明の新規発光物質を化学反応により発光させることができる。この時の発光強度は、試料中の特定物質濃度に相関しているので、発光強度をフォトンカウンター等により測定することで、試料中の特定物質濃度を求めることができる。
更に本発明によれば、前記抗体を用いた免疫学的検出法だけではなく、核酸に対しても、その核酸と相補的な核酸との親和性を利用し、目的の核酸を検出することができる。
【００２２】
また、本発明者が見出したところによれば、本発明のエポキシ誘導体の発光強度を増加することのできる特定の化合物が存在し、反応の要素であるアルカリ性過酸化水素に、トリフルオロ酢酸やトリエチルアミン等を加えると発光量が増加する現象がみられる。
【００２３】
特定物質が含まれる被検試料としては、血液、血清、血漿、尿、唾液、髄液等の生体液や、細胞及び組織抽出物を挙げることができ、特定物質としては、各種タンパク質、多糖類、脂質、核酸等が挙げられ、より具体的には、フィブリノーゲン、アルブミン、ＣＲＰ、ＩｇＧ、ＩｇＡ、ＩｇＭ等の蛋白、ＡＦＰ、ＣＥＡ等の腫瘍関連蛋白、Ｂ型肝炎ウイルス関連蛋白やその抗体、Ｃ型肝炎ウイルス関連蛋白やその抗体、ＨＩＶ抗原や抗体、ＨＴＬＶ抗体等の感染症関連マーカー、Ｄダイマー、ＰＰＩ、ＴＡＴ等の凝固関連マーカー、インシュリン、ＨＣＧ、サイロキシン等のホルモンや、抗てんかん薬及びジゴキシン等の薬剤を挙げることができる。
また、特定物質が核酸であれば特定の塩基配列を有するＤＮＡ断片などが挙げられる。
【００２４】
【実施例】
以下、実施例によって本発明を具体的に説明するが、これらは本発明の範囲を限定するものではない。また、以下の実施例において、融点は、柳本微量融点測定装置を用いて測定し、全て未補正である。赤外線吸収（ＩＲ）スペクトルは、日本分光ＦＴ／ＩＲ−２００型赤外線光度系を用いて測定した。また、 ¹Ｈ−ＮＭＲスペクトルは、バリアンＶＸＲ−２００型（２００ＭＨｚ）を用い、内部標準にテトラメチルシランを用いて測定した。シリカゲルクロマトグラフィーは、Ｅ．Ｍｅｒｃ Ｋｉｅｓｅｌ ６０（７０〜２３０メッシュ）を用いた。
【００２５】
更に、以下の実施例及び参考例などにおいて、出発材料であるケトン化合物〔図１の化合物（１）〕、中間体であるオレフィン誘導体〔図１の化合物（２）〕及び最終生成物〔図１の化合物（Ｉ）〕における置換基Ｒ¹ 、Ｒ² 、及びＸが共通の化合物群を、説明の便宜のために以下の（ａ）〜（ｇ）に分けて表記する。従って、例えば、以下の（ａ）に属する出発材料（１）はケトン化合物（１ａ）、中間体（２）はオレフィン誘導体（２ａ）、そして最終生成物は、エポキシ誘導体（Ｉａ）となる。
（ａ）：Ｒ¹ ＝Ｒ² ＝Ｈ，Ｘ＝Ｎ−ＣＨ₃
（ｂ）：Ｒ¹ ＝Ｒ² ＝Ｈ，Ｘ＝Ｎ−ＣＨ₂ Ｃ₆ Ｈ₅
（ｃ）：Ｒ¹ ＝Ｒ² ＝Ｈ，Ｘ＝Ｎ−ＣＨ₂ ＣＯ₂ Ｃ₂ Ｈ₅
（ｄ）：Ｒ¹ ＝Ｒ² ＝Ｈ，Ｘ＝Ｎ−Ｃ₆ Ｈ₅
（ｅ）：Ｒ¹ ＝Ｒ² ＝Ｈ，Ｘ＝ＣＨ₂
（ｆ）：Ｒ¹ ＝Ｒ² ＝Ｈ，Ｘ＝Ｓ
（ｇ）：Ｒ¹ ＝Ｒ² ＝Ｈ，Ｘ＝Ｏ
【００２６】
参考例１：出発材料（２ａ）〜（２ｇ）の調製
〔１〕９，１０−ジヒドロ−１０−メチル−９−（トリシクロ〔３．３．１．１^3,7 〕デシリデン）アクリジン（２ａ）の合成
無水三塩化チタンアルミニウム還元型３ｇ（１５ｍｍｏｌ）をテトラヒドロフラン（ＴＨＦ）１２０ｍｌ中に懸濁し、氷冷下で水素化アルミニウムリチウム０．２０ｇ（７．６ｍｍｏｌ）を加えて１０分間撹拌した後、トリエチルアミン１．１ｍｌ（７．６ｍｍｏｌ）を加えた。この反応液を９０分間加熱還流した後、Ｎ−メチル体（１ａ）０．５ｇ（２．４ｍｍｏｌ）と２−アダマンタノン０．３６ｇ（２．４ｍｍｏｌ）とを含む３０ｍｌの無水ＴＨＦ溶液を滴下し、更に還流を３９時間行った。反応混合物を室温にもどし、氷冷下で、氷水６０ｍｌを少量ずつ加えた後、酢酸エチルにより抽出を３回行った。有機層を水及び飽和食塩水で洗浄した後、無水硫酸ナトリウムで乾燥した。溶媒を留去した後、酢酸エチル／へキサン〔１：１０（ｖ／ｖ）〕の混合溶媒を用いたシリカゲルクロマトグラフィーで残渣を精製し、オレフィン体（２ａ）０．４９ｇ（１．５ｍｍｏｌ、収率６３％）を無色結晶として得た。物性値は以下のとおりであった。
融点：２５６〜２５７℃（酢酸エチル）
ＩＲ γ（ＫＢｒ）：２９０８，２８４７，２３５８，１５８９，１４６１，１３３９，１２６９，１１２９，１０９８，１０４２ｃｍ^-1
ＵＶ λ_max ^CH ₃ ^CNｎｍ（ｌｏｇ ε）：３４０（３．７８），２８７（４．０１），２６１（４．１６），２２６（４．４６）
¹Ｈ−ＮＭＲ（ＣＤＣｌ₃ ）δ：１．５３−２．２２（１２Ｈ，ｍ），３．４１（３Ｈ，ｓ），６．９８（４Ｈ，ｔｄ，Ｊ＝８，２Ｈｚ），７．１５−７．２６（４Ｈ，ｍ）
元素分析：Ｃ₂₄Ｈ₂₅Ｎ（分子量＝３２７．４７）に対して：
計算値：Ｃ＝８８．０３；Ｈ＝７．６９；Ｎ＝４．２８
実測値：Ｃ＝８７．９３；Ｈ＝７．６５；Ｎ＝４．２８
【００２７】
〔２〕９，１０−ジヒドロ−１０−ペンジル−９−（トリシクロ〔３．３．１．１^3,7 〕デシリデン）アクリジン（２ｂ）の合成
上記〔１〕で得た生成物の１０−メチル基の代わりに、１０位にベンジル基を有する９，１０−ジヒドロ−１０−ベンジル−９−（トリシクロ〔３．３．１．１^3,7 〕デシリデン）アクリジン（２ｂ）は、対応するケトン体（１ｂ）を出発材料として用い、上記〔１〕と同様な操作を実施し、以下の物性値をもつ無色結晶として収率３５％で得ることができた。
融点：２１８〜２１９．５℃（酢酸エチル）
ＩＲ γ（ＫＢｒ）：３０２６，２９０５，２８４６，１７１８，１５８９，１４９１，１４５８，１３７１，１２６６，１２２５，１０９８，１０６３ｃｍ^-1ＵＶ λ_max ^CH ₃ ^CNｎｍ（ｌｏｇ ε）：３４１（３．９３），２９３（４．０１），２５８（４．２３），２２６（４．６１）
¹Ｈ−ＮＭＲ（ＣＤＣｌ₃ ）δ：１．５３−２．２２（１２Ｈ，ｍ），３．４３−３．４９（２Ｈ，ｍ），５．２３（２Ｈ，ｍ），６．８３（２Ｈ，ｄｄ，Ｊ＝８，２Ｈｚ），７．０３（２Ｈ，ｄｄ，Ｊ＝８，２Ｈｚ），７．１５−７．２７（９Ｈ，ｍ）
元素分析：Ｃ₃₀Ｈ₂₉Ｎ（分子量＝４０３．５７）に対して：
計算値：Ｃ＝８９．２９；Ｈ＝７．２４；Ｎ＝３．４７
実測値：Ｃ＝８９．３１；Ｈ＝７．３２；Ｎ＝３．４６
【００２８】
〔３〕９，１０−ジヒドロ−９−（トリシクロ〔３．３．１．１^3,7 〕デシリデン）−１０−アクリジン酢酸エチル（２ｃ）の合成
同様に、９，１０−ジヒドロ−９−（トリシクロ〔３．３．１．１^3,7 〕デシリデン）−１０−アクリジン酢酸エチル（２ｃ）も、対応するケトン体（１ｃ）を原料として用い、上記〔１〕と同様な操作を実施し、以下の物性値をもつ無色結晶として６３％の収率で得ることができた。
融点：２１６〜２１７℃（酢酸エチル）
ＩＲ γ（ＫＢｒ）：２９０７，２８４７，１７５１，１５９１，１４５８，１３６８，１１８８，１０９８，１０２４ｃｍ^-1
ＵＶ λ_max ^CH ₃ ^CNｎｍ（ｌｏｇ ε）：３３４（３．９０），２８６（３．９９），２５８（４．２０），２２５（４．５５）
¹Ｈ−ＮＭＲ（ＣＤＣｌ₃ ）δ：１．２７−２．２２（３Ｈ，ｔ，Ｊ＝７Ｈｚ），１．５５−２．２１（１２Ｈ，ｍ），３．４１−３．４４（２Ｈ，ｍ），４．６４（２Ｈ，ｓ），６．７７（２Ｈ，ｔｄ，Ｊ＝８，１Ｈｚ），６．９８（２Ｈ，ｔｄ，Ｊ＝１０，１Ｈｚ），７．１１−７．２７（４Ｈ，ｍ），
元素分析：Ｃ₂₅Ｈ₂₉ＮＯ₂ （分子量＝３９９．５３）に対して：
計算値：Ｃ＝８１．１７；Ｈ＝７．３２；Ｎ＝３．５１
実測値：Ｃ＝８０．８２；Ｈ＝７．３６；Ｎ＝３．４７
【００２９】
〔４〕９，１０−ジヒドロ−９−フェニル−９−（トリシクロ〔３．３．１．１^3,7 〕デシリデン）アクリジン（２ｄ）の合成
同様に、１０位にフェニル基を有する９，１０−ジヒドロ−９−フェニル−９−（トリシクロ〔３．３．１．１^3,7 〕デシリデン）アクリジン（２ｄ）も、対応するケトン体（１ｄ）を原料として用い、上記〔１〕と同様な操作を実施し、以下の物性値をもつ無色結晶として５１％の収率で得ることができた。
融点：２４５〜２５５．５℃（酢酸エチル）
ＩＲ γ（ＫＢｒ）：２９０６，２８４５，１５８８，１５５９，１５４０，１５０７，１４９０，１４６６，１４５１，１２７８，１０９８ｃｍ^-1
ＵＶ λ_max ^CH ₃ ^CNｎｍ（ｌｏｇ ε）：３４０（４．００），２８６（４．１８），２５６（４．３２），２２８（４．６７）
¹Ｈ−ＮＭＲ（ＣＤＣｌ₃ ）δ：１．８３−２．０７（１２Ｈ，ｍ），３．４８−３．５１（２Ｈ，ｍ），６．４５（２Ｈ，ｄｄ，Ｊ＝８，２Ｈｚ），６．９７（４Ｈ，ｍ），７．２６（２Ｈ，ｍ），７．３８（２Ｈ，ｄｄ，Ｊ＝７，２Ｈｚ），７．４９−７．６２（３Ｈ，ｍ）
元素分折：Ｃ₂₉Ｈ₂₇Ｎ（分子量＝３８９．５４）に対して：
計算値：Ｃ＝８９．４２；Ｈ＝６．９９；Ｎ＝３．６０
実測値：Ｃ＝８９．３１；Ｈ＝７．２４；Ｎ＝３．６２
【００３０】
〔５〕９−トリシクロ〔３．３．１．１^3,7 〕デシリデン−９，１０−ジヒドロアントラセン（２ｅ）の合成
また、基本骨格の異なる９−トリシクロ〔３．３．１．１^3,7 〕デシリデン−９，１０−ジヒドロアントラセン（２ｅ）についても、対応するケトン体（１ｅ）から上記〔１〕と同様な操作を実施し、４４％の収率で無色結晶として得ることができた。その物性値を以下に示す。
融点：２３２〜２３３．５℃（酢酸エチル）
ＩＲ γ（ＫＢｒ）：３０６０，３０１２，２９７６，２８４５，１６３２，１４６６，１４５１，１４１９，１２２４，１１０７，１０３５ｃｍ^-1
1Ｈ−ＮＭＲ（ＣＤＣｌ₃ ）δ：１．５７−２．２３（１２Ｈ，ｍ），３．４３（２Ｈ，ｓ），３．７６（２Ｈ，ｅａｃｈ，Ｊ＝１８Ｈｚ），７．０９−７．１９（４Ｈ，ｍ），７．２６（４Ｈ，ｍ）
元素分析：Ｃ₂₄Ｈ₂₄（分子量＝３１２．４５）に対して：
計算値：Ｃ＝９２．２６；Ｈ＝７．７４
実測値：Ｃ＝９２．２２；Ｈ＝７．７８
【００３１】
〔６〕９−トリシクロ〔３．３．１．１^3,7 〕デシリデン−９Ｈ−チオキサンテン（２ｆ）の合成
更に、９−トリシクロ〔３．３．１．１^3,7 〕デシリデン−９Ｈ−チオキサンテン（２ｆ）についても、対応するケトン体（１ｆ）から上記〔１〕と同様な操作を実施し、収率３２％で無色結晶として得ることができた。その物性値を以下に示す。
融点：２２０〜２２２．５℃（酢酸エチル）
ＩＲ γ（ＫＢｒ）：３０５６，２９７０，２９１１，２８４５，１６３６，１４５５，１４３６，１３２６，１０６１，１０２９ｃｍ^-1
1Ｈ−ＮＭＲ（ＣＤＣｌ₃ ）δ：１．５０−２．２０（１２Ｈ，ｍ），３．４３（２Ｈ，ｓ），３．２０−３．３６（２Ｈ，ｍ），７．４７（２Ｈ，ｄｄ，Ｊ＝７，ＩＨｚ）
〔７〕９−トリシクロ〔３．３．１．１^3,7 〕デシリデン−９Ｈ−キサンテン（２ｇ）の合成
また、９−トリシクロ〔３．３．１．１^3,7 〕デシリデン−９Ｈ−キサンテン（２ｇ）についても、対応するケトン体（１ｇ）から同様な操作を実施し、収率７８％で無色結晶として得ることができた。その物性値を以下に示す。
融点：２２０〜２２１．５℃（酢酸エチル）
ＩＲ γ（ＫＢｒ）：２９８１，２９１１，２８４４，１５８０，１５６６，１４７１，１４４５，１３４２，１３０３，１２５２，１２０２，１１５０ｃｍ^{-1 1}Ｈ−ＮＭＲ（ＣＤＣｌ₃ ）δ：１．８８−２．０５（１２Ｈ，ｍ），３．４７−３．５３（２Ｈ，ｍ），７．０５−７．２９（２Ｈ，ｍ）
元素分析：Ｃ₂₃Ｈ₂₂Ｏ（分子量＝３１４．４３）に対して：
計算値：Ｃ＝８７．８６；Ｈ＝７．０５
実測値：Ｃ＝８７．７６；Ｈ＝７．０９
【００３２】
実施例１：１０−メチルジスピロ〔アクリジン−９（１０Ｈ），２’−オキシラン−３’，２”−トリシクロ〔３．３．１．１ ^3,7 〕デカン〕（Ｉａ）の合成
前記参考例１〔１〕で得たオレフィン体（２ａ）（５００ｍｇ，１．５ｍｏｌ）の塩化メチレン（２．０ｍｏｌ）溶液に、３−クロロ過安息香酸（６５０ｍｇ，１６８ｍｍｏｌ）を室温で加え、約１０分間撹拌した。薄層クロマトグラフィーにより原料の消失を確認した後、飽和重曹水を加え、過剰の３−クロロ安息香酸を分解した。続いて、ジクロロメタンにより３回抽出を行った。有機層を水及び飽和食塩水で洗浄し、無水硫酸ナトリウムで乾燥した。
溶媒を蒸留した後、残渣をシリカゲルクロマトグラフィー（酢酸エチル：へキサン＝１：１０）で精製し、エポキシ体（Ｉａ）（４７０ｍｇ，１．４ｍｍｏｌ，８９％）を無色結晶として得た。物性値は以下のとおりであった。
融点：２４１〜２４２℃（酢酸エチル）
ＩＲ γ（ＫＢｒ）：２９０５，２８５０，１５９５，１４７０，１２６７ｃｍ^-1
1Ｈ−ＮＭＲ（ＣＤＣｌ₃ ）δ：１．０２−２．１３（１４Ｈ，ｍ），３．４６（３Ｈ，ｓ），６．９５−７．０５（４Ｈ，ｍ），７．２３−７．３６（４Ｈ，ｍ）
元素分析：Ｃ₂₄Ｈ₂₄ＮＯ（分子量＝３４３．４７）に対して：
計算値：Ｃ＝８３．９３；Ｈ＝７．３４；Ｎ＝４．０８
実測値：Ｃ：８３．９８；Ｈ＝７．３１；Ｎ＝４．０３
【００３３】
実施例２：１０−ベンジルジスピロ〔アクリジン−９（１０Ｈ），２’−オキシラン−３’，２”−トリシクロ〔３．３．１．１ ^3,7 〕デカン〕（Ｉｂ）の合成
出発材料として前記参考例１〔２〕で得たオレフィン体（２ｂ）を用いること以外は実施例１と同様の方法で、標記化合物を無色柱状結晶として、９７％の収率で得た。物性値は以下のとおりであった。
融点：２２７〜２２９℃（酢酸エチル）
ＩＲ γ（ＫＢｒ）：２９１３，２８４９，１５９６，１４６４，１３７１，１２６７ｃｍ^-1
1Ｈ−ＮＭＲ（ＣＤＣｌ₃ ）δ：１．０５−２．１７（１４Ｈ，ｍ），５．２７（２Ｈ，ｓ），６．８５（２Ｈ，ｄ，Ｊ＝８Ｈｚ），６．９７（２Ｈ，ｔ，Ｊ＝７Ｈｚ），７．０９−７．４２（９Ｈ，ｍ）
元素分析：Ｃ₃₁Ｈ₂₉ＮＯ（分子量＝４１９．５７）に対して：
計算値：Ｃ＝８５．８８；Ｈ＝６．９７；Ｎ＝３．３４
実測値：Ｃ＝８５．８５；Ｈ＝６．９８；Ｎ＝３．３０
【００３４】
実施例３：ジスピロ〔アクリジン−９（１０Ｈ），２’−オキシラン−３’，２”−トリシクロ〔３．３．１．１ ^3,7 〕デカン〕−１０−酢酸エチル（Ｉｃ）の合成
出発材料として前記参考例１〔３〕で得たオレフィン体（２ｃ）を用いること以外は実施例１と同様の方法で、標記化合物を無色柱状結晶として、８５％の収率で得た。物性値は以下のとおりであった。
融点：１７７〜１７８℃（酢酸エチル）
ＩＲ γ（ＫＢｒ）：２９１２，２８４９，１７５３，１５９８，１４９９，１４６４，１３６６ｃｍ^-1
1Ｈ−ＮＭＲ（ＣＤＣｌ₃ ）δ：１．０５−２．２０（１４Ｈ，ｍ），１．２６（３Ｈ，ｔ，Ｊ＝７Ｈｚ），４．２６（２Ｈ，ｑ，Ｊ＝７Ｈｚ），４．６７（２Ｈ，ｓ），６．７２（２Ｈ，ｄ，Ｊ＝８Ｈｚ），７．０２（２Ｈ，ｔｄ，Ｊ＝１，８Ｈｚ），７．１９−７．３４（２Ｈ，ｍ），７．３７（２Ｈ，ｄ，Ｊ＝８Ｈｚ），７．０９−７．４２（９Ｈ，ｍ）
元素分析：Ｃ₃₁Ｈ₂₉ＮＯ（分子量＝４１９．５７）に対して：
計算値：Ｃ＝７８．０４；Ｈ：７．０３；Ｎ＝３．３７
実測値：Ｃ＝７７．７９；Ｈ＝７．０３；Ｎ＝３．３７
【００３５】
実施例４：１０−フェニルジスピロ〔アクリジン−９（１０Ｈ），２’−オキシラン−３’，２”−トリシクロ〔３．３．１．１ ^3,7 〕デカン〕（Ｉｄ）の合成
出発材料として前記参考例１〔４〕で得たオレフィン体（２ｄ）を用いること以外は実施例１と同様の方法で、標記化合物を無色柱状結晶として、８８％の収率で得た。物性値は以下のとおりであった。
ＩＲ γ（ＫＢｒ）：２９１７，２８５１，１５９７，１４９７，１４５７，１３１９ｃｍ^-1
1Ｈ−ＮＭＲ（ＣＤＣｌ₃ ）δ：１．１６−２．１５（１４Ｈ，ｍ），６．４４（２Ｈ，ｄ，Ｊ＝８Ｈｚ），６．９３−７．１２（３Ｈ，ｍ），７．３７（４Ｈ，ｄ，Ｊ＝８Ｈｚ），７．５０−７．６８（４Ｈ，ｍ）
【００３６】
実施例５：９−１０−ジヒドロジスピロ〔アントラセン−９，２’−オキシラン−３’，２”−トリシクロ〔３．３．１．１ ^3,7 〕デカン〕（Ｉｅ）の合成
出発材料として前記参考例１〔５〕で得たオレフィン体（２ｅ）を用いること以外は実施例１と同様の方法で、標記化合物を無色柱状結晶として、１００％の収率で得た。物性値は以下のとおりであった。
ＩＲ γ（ＫＢｒ）；３０２０，２９６４，２９５０，２８４９，１４７０，１４５５，１４１８，１３５０ｃｍ^-1
1Ｈ−ＮＭＲ（ＣＤＣｌ₃ ）δ：１．１０−２．２０（１４Ｈ，ｍ），３．９９（２Ｈ，ｅａｃｈ ｄ，Ｊ＝１８Ｈｚ），７．１８−７．３７（６Ｈ，ｍ），７．４０（２Ｈ，ｄｄ，Ｊ＝３，６Ｈｚ）
元素分折：Ｃ₂₄Ｈ₂₄Ｏ（分子量＝３２８．４５）に対して：
計算値：Ｃ＝８７．７６；Ｈ＝７．３６
実測値：Ｃ＝８７．４８；Ｈ＝７．３５
【００３７】
実施例６：ジスピロ〔９Ｈ−チオキサンテン−９，２’−オキシラン−３’，２”−トリシクロ〔３．３．１．１ ^3,7 〕デカン〕−１０−オキサイド（Ｉｆ）の合成
出発材料として前記参考例１〔６〕で得たオレフィン体（２ｆ）を用いること以外は実施例１と同様の方法で、標記化合物を無色柱状結晶として、５８％の収率で得た。物性値は以下のとおりであった。
融点：２６７〜２６８．５℃（酢酸エチル）
ＩＲ γ（ＫＢｒ）：３０７６，２９１２，２８５４，２５９４，１５８０，１４５０，１４１３，１３５３，１３０２，１２２７，１１７１，１１３８，１１２７ｃｍ^-1
1Ｈ−ＮＭＲ（ＣＤＣｌ₃ ）δ：１．４０−２．２５（１４Ｈ，ｍ），７．４７−７．６３（６Ｈ，ｍ），８．１０（２Ｈ，ｄｄ，Ｊ＝１，６Ｈｚ）
【００３８】
実施例７：ジスピロ〔トリシクロ〔３．３．１．１ ^3,7 〕デカン２，２’−オキシラン−３’，９”−９Ｈ−キサンテン〕（Ｉｇ）の合成
出発材料として前記参考例１〔７〕で得たオレフィン体（２ｇ）を用いること以外は実施例１と同様の方法で、標記化合物を無色柱状結晶として、９８％の収率で得た。物性値は以下のとおりであった。
融点：１３１〜１３２．５℃（酢酸エチル）
ＩＲ γ（ＫＢｒ）：２９１４，２８５０，１６０３，１５７３，１４５０，１３１７，１２００，１１４７，１１００，１０７５，１０３２ｃｍ^-1
1Ｈ−ＮＭＲ（ＣＤＣｌ₃ ）δ：１．０９−２．２０（１４Ｈ，ｍ），７．０７−７．４５（８Ｈ，ｍ）
元素分折：Ｃ₂₃Ｈ₂₂Ｏ₂ （分子量＝３３０．４３）に対して：
計算値：Ｃ＝８３．６０；Ｈ＝６．７１
実測値：Ｃ＝８３．５６；Ｈ＝６．７４
【００３９】
実施例８：発光量の測定−１
以下の発光量測定にはベルトールド社のＬｕｍａｔ ＬＢ９５０１を用いた。実施例１〜４で得た１０位置換ジスピロ〔アクリジン−９（１０Ｈ），２’−オキシラン−３’，２”−トリシクロ〔３．３．１．１^3,7 〕デカン〕（Ｉａ〜Ｉｄ）の１×１０^-6Ｍ溶液をジメチルスルホキシド（ＤＭＳＯ）、あるいはＤＭＳＯと蒸留水の等量混合液で調整し、得られた溶液それぞれ１００μｌをガラスチューブに採り、測定装置のホルダーにセットし、発光開始液として水酸化ナトリウムと過酸化水素水を各０．１Ｎ含む溶液を１００μｌ注入し、１２０秒間の発光カウントを積算して発光量とし、ＲＬＵ（Ｒｅｌａｔｉｖｅ Ｌｉｇｈｔ Ｕｎｉｔ：相対発光単位）で示した。
【００４０】
【表１】
（Ａ）ｌ００％ＤＭＳＯ溶液中

（Ｂ）５０％ＤＭＳＯ＋５０％蒸留水溶液中

【００４１】
実施例９：発光量の測定−２
実施例２で得た１０−ベンジルジスピロ〔アクリジン−９（１０Ｈ），２’−オキシラン−３’，２”−トリシクロ〔３．３．１．１^3,7 〕デカン〕（Ｉｂ）を、ジクロロメタン（ＣＨ₂ Ｃｌ₂ ）、ＤＭＳＯあるいはＤＭＳＯ−Ｈ₂ Ｏ（蒸留水）（１：１）に溶かし、１×１０^-6Ｍに調整し、発光強度を増すのに効果があったトリフルオロ酢酸、メタクロロ過安息香酸、又はトリエチルアミンをそれぞれ添加剤として加えてからの１２０秒間のカウントを積算して発光量とした。結果を表２に示す。なお、表２において、ｍＣＰＢＡはメタクロロ過安息香酸、ＴＥＡはトリエチルアミンであり、ＴＦＡはトリフルオロ酢酸である。また、２５秒後とは、水酸化ナトリウムと過酸化水素を加えてから２５秒後、更にその下に記載の試薬を加えたときの発光量を示す。
【００４２】
【表２】

表１と表２の結果から明らかなように、本発明によるエポキシ誘導体（Ｉａ）、（Ｉｂ）、（Ｉｃ）、及び（Ｉｄ）は、シャープらの文献（Ｔｅｔｒａｈｅｄｒｏｎ Ｌｅｔｔｅｒｓ Ｖｏｌ．２８，Ｎｏ．１１，ｐ１１５５，１９８７）のアダマンタン誘導体と比較しても、より高い発光力を示した。また、表２の結果から、本発明によるエポキシ誘導体は、添加物によって発光強度が変化し、トリエチルアミン（ＴＥＡ）やトリフルオロ酢酸（ＴＦＡ）を添加したときに発光強度が増すことがわかった。
【００４３】
【発明の効果】
本発明によるエポキシ誘導体は、従来の発光性アクリジン誘導体などと比較して、特に中性付近での保存安定性が格別に優れており、血液などの生物学的検体中の特定物質を検出する際のラベルとして有効に利用することができる。
【図面の簡単な説明】
【図１】本発明によるエポキシ誘導体の合成工程を示す説明図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel epoxy derivative. Since the epoxy derivative according to the present invention is a chemiluminescent substance, for example, a test substance can be detected using the epoxy derivative as a labeling substance or a luminescent substrate.
[0002]
[Prior art]
In recent years, bioluminescence of fireflies and sea fireflies has been studied, and the enzyme luciferase catalyzes the reaction between luciferin and ATP (adenosine triphosphate), and the substance aequorin specifically binds calcium ions. It was clarified that it emits light. It is also well known that peroxidase catalyzes the luminescence reaction of luminol hydrogen peroxide. Recently, a substrate in which a phosphate group is introduced into stabilized dioxetane is produced by synthesis, and a luminescence reaction occurs due to the action of alkaline phosphatase. Has been reported.
[0003]
On the other hand, a reaction in which a chemical substance is excited by an activator to emit light regardless of the action of living organisms or enzymes has been studied and is generally called chemiluminescence. Among these, light emission by alkaline hydrogen peroxide of an acridinium ester and a reaction in which a fluorescent material is excited by peroxalic acid to emit light are known.
[0004]
Luminescent reaction can be detected even in a very small amount of reaction compared to absorbance measurement and fluorescence measurement. Enzymes and luminescent substances themselves are used as label substances instead of radioisotopes, and the measurement target contained in the sample. Attempts have heretofore been made to measure substances (for example, physiologically active substances such as antigens, antibodies, DNA, etc.) with high sensitivity.
[0005]
[Problems to be solved by the invention]
When measuring a substance to be measured with high sensitivity and speed, the luminescent substance itself is used rather than the method of detecting the luminescence of the luminescent substrate using an enzyme that requires a certain amount of time for turnover as a label. A method that uses it as a label and detects it by emitting light with an excitation agent seems to be more advantageous.
As a substance that causes chemiluminescence, for example, stabilized dioxetane is known. This substance contains oxygen molecules, and can be excited by using an unstable and high-energy dioxetane having a four-membered ring structure as excitation energy and exciting the fluorescent substance contained in the same compound. Although dioxetane was considered very unstable and could not be used as a reagent, stabilized dioxetane, a complex with adamantane, ie adamantylidene adamantane-1,2-dioxetane, has a half-life of 20 at 25 ° C. Since it became clear that it was a stable substance for more than a year, methods for using this substance as a labeling substance have been studied. However, in order to cause this substance to emit light, heating at 150 to 250 ° C. is necessary. Further, since the adamantane portion of this substance is a substance having a very weak fluorescence intensity, the substance to be measured in the aqueous solvent is detected. It was inappropriate as a label.
[0006]
As an improvement of the above, Sharp et al. Synthesized 4- (hydroxy-2-naphthyl) -4-methoxyspiro (1,2-dioxetane-3,2′-adamantanone), and further added xanthene and adamantanone and dioxetane. A compound to which fluorin or the like was bonded was also synthesized and examined for stability and emission intensity (Japanese Patent Publication No. 3-505739). With these compounds, stability was maintained and a luminescent reaction triggered by enzyme dephosphorylation was observed, but because the luminescence intensity was weak, only the method used as a substrate in a measurement system with an enzyme label was used. Applications were limited.
[0007]
Since a stable dioxetane compound requires a large amount of energy to excite and emit light, it is unsuitable for normal use such as measurement at room temperature, slight heating or cooling. On the other hand, compounds that emit light with small energy lack stability.
As one solution to these problems, Woodhead et al. Show that when alkaline hydrogen peroxide is added to the phenyl ester of N-methylacridinium-9-carboxylate, a luminescent reaction occurs via dioxetane, Furthermore, it was shown that a protein-binding cross-linking agent can be incorporated into the phenyl ester moiety and used as a label (GB212779). However, this compound also had a light emission yield of about 1%. Therefore, as a labeling substance, a substance that is more stable and exhibits strong light emission during light emission has been desired.
[0008]
On the other hand, when detecting a DNA, a Mac coupler or the like uses a dye such as pyrene as a labeling substance, and uses singlet oxygen generated from dissolved oxygen by irradiating the substance with ultraviolet rays, thereby producing 9- (adamantylidene)- A method has been developed in which dioxetane is produced in N-methylacridan and the luminescence emitted during its thermal decomposition is detected (EP Publication 0345776). This method is suitable for use as a starting material for a luminescent substance because the methyl acridan moiety of 9- (adamantylidene) -N-methylacridane has a high fluorescence intensity, but actually 9- (adamandene). When a dioxetane moiety was introduced into (tylidene) -N-methylacridane, it was not stable and could not be taken out as an isolate. Therefore, it was produced as an intermediate in a reaction solution and used in an unstable manner. is there.
[0009]
In this way, dioxetane is stabilized with a bulky substance such as adamantane and used as an energy source, the ester bond portion is excited with alkaline hydrogen peroxide and a luminescence reaction is caused by this energy, and an excited state is created with ultraviolet light. Although a method for causing a luminescence reaction with the energy has been reported, a system that is usually stable and uses all energy for the luminescence reaction during the luminescence reaction and generates strong light is desired.
Accordingly, an object of the present invention is to provide a novel luminescent substance that has excellent stability and can easily cause a luminescent reaction.
[0010]
[Means for Solving the Problems]
  The above objective can be achieved by the new parent epoxy derivative according to the present invention. That is, the present invention relates to the general formula (I):
[Formula 4]

[Where XIs-N (R^Three )-, Methylene group (-CH₂ -), An oxygen atom (-O-), or a sulfur atom (-S-), and R¹ And R² Are the same or different and each represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an unsubstituted amino group, a substituted amino group, a carboxyl group, an alkoxy group, a cyano group, a nitro group, or a halogen atom, and R^Three Is an alkyl group having 1 to 4 carbon atoms, an unsubstituted phenyl group, a substituted phenyl group, a benzyl group, an alkoxycarbonylmethyl group, or an aryloxycarbonylmethyl group] or a salt thereof.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be described in detail below.
In the present specification, the “alkyl group having 1 to 4 carbon atoms” is a linear or branched alkyl group, for example, methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl. Group, i-butyl group, s-butyl group, or t-butyl group. As used herein, “unsubstituted amino group” refers to —NH₂ It is a group. The “substituted amino group” is, for example, a lower alkyl group, particularly an amino group mono- or di-substituted with an alkyl group having 1 to 4 carbon atoms, such as a mono- or dimethylamino group, mono- or diethylamino group. Mention may be made of a group, a methylethylamino group, or a mono- or dibenzylamino group.
The “alkoxy group” is, for example, an alkoxy group having 1 to 4 carbon atoms, such as a methoxy group, an ethoxy group, an n-propoxy group, an i-propoxy group, an n-butoxy group, an i-butoxy group, an s-butoxy group, Or a t-butoxy group. The “halogen atom” is, for example, a chlorine atom, a bromine atom, an iodine atom, or a fluorine atom.
[0012]
“Substituted phenyl group” is not particularly limited, and consists of an alkyl group having 1 to 4 carbon atoms, an unsubstituted amino group, a substituted amino group, a carboxyl group, an alkoxy group, a cyano group, a nitro group, a halogen atom, and a succinimino group. It can carry 1 to 5, preferably 1 to 3, the same or different substituents selected from the group. Accordingly, the substituted phenyl group is, for example, represented by the general formula (II):
[Chemical formula 5]

(Wherein R^Four , R^Five And R⁶ Are the same or different and each is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an unsubstituted amino group, a substituted amino group, a carboxyl group, an alkoxy group, a cyano group, a nitro group, a halogen atom, or a succinimino group. ).
[0013]
The “alkoxycarbonylmethyl group” is, for example, a carbonylmethyl group having an alkoxy moiety having 1 to 4 carbon atoms, and examples thereof include a methoxycarbonylmethyl group or an ethoxycarbonylmethyl group. Moreover, the “aryloxycarbonylmethyl group” is, for example, an aryloxycarbonylmethyl group having an aryl moiety having 6 to 18 carbon atoms which may optionally carry one or more substituents. Preferred aryloxycarbonylmethyl groups are, for example, general formula (III):
[Chemical 6]

(Wherein R⁷ , R⁸ And R⁹ Are the same or different and each is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an unsubstituted amino group, a substituted amino group, a carboxyl group, an alkoxy group, a cyano group, a nitro group, a halogen atom, or a succinimino group. ).
[0014]
In the novel epoxy derivative represented by the general formula (I), X is —N (R^Three )-, A methylene group, an oxygen atom, or a sulfur atom, and R¹ And R² Are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms;^Three Wherein the general formula is an alkyl group having 1 to 4 carbon atoms, an unsubstituted phenyl group, a substituted phenyl group, a benzyl group, an alkoxycarbonylmethyl group having 1 to 4 carbon atoms in the alkoxy moiety, or a phenyloxycarbonylmethyl group The epoxy derivative represented by (I) or a salt thereof is preferable.
[0015]
In the novel epoxy derivative represented by the general formula (I), X is —N (R^Three )-, A methylene group, an oxygen atom, or a sulfur atom, and R¹ And R² Is a hydrogen atom and R^Three An epoxy derivative represented by the above general formula (I) or a salt thereof is particularly preferable in which is a methyl group, an unsubstituted phenyl group, a benzyl group, or an ethoxycarbonylmethyl group.
[0016]
When the epoxy derivative represented by the general formula (I) has a carboxyl group or an amino group, the epoxy derivative can form a salt, and those salts are also included in the compound of the present invention. Examples of the carboxylate include salts of alkali metals (for example, sodium or potassium). Examples of amino group salts include acid addition salts such as hydrochloride, sulfate, and various sulfonates. , Oxalate, or perchlorate
[0017]
The epoxy derivative represented by the general formula (I) according to the present invention can be prepared by a method known per se.
An example of the preparation method is shown in FIG. That is, the ketones (1) and adamantanone are derived into the olefin body (2) by, for example, a reductive condensation reaction between anhydrous titanium aluminum trichloride reduced type and lithium aluminum hydride. Subsequently, when the obtained olefin (2) is reacted at room temperature for a short time using m-chloroperbenzoic acid in methylene chloride, for example, the desired epoxy derivative (I) can be obtained.
[0018]
The ketones (1) used as a raw material in the above step are known compounds and can be prepared by a method known per se. For example, R¹ And R² Is a hydrogen atom and X is -N-CH_Three A ketone compound (1a) [i.e., 10-methyl-9 (10H) -acridone], and R¹ And R² Is a hydrogen atom and X is -N-CH₂ C₆ H_Five The ketone compound (1b) [i.e., 10-phenylmethyl-9 (10H) -acridone] is described in Nishi et al. (Bull. Chem. Soc. Jpn., 54, 1898, 1981) and J. Am. J. et al. Can be synthesized according to Charbit et al. (J. Chem. Eng. Data, 34, 136, 1989). R¹ And R² Is a hydrogen atom and X is -N-CH₂ CO₂ C₂ H_Five The ketone compound (1c) [that is, 9-okine-10 (9H) -acridine ethyl acetate] is N.I. Miosga et al. (Phamazie, 40, 800, 1985); D. It can be synthesized according to the document of Postescu et al. (J. Prakt. Chemie., 319, 347, 1977). In addition, R¹ And R² Is a hydrogen atom and X is -N-CH₂ C₆ H_Five A ketone compound (1d) [i.e., 10-phenyl-9 (10H) -acridone] can be synthesized according to Mitteilung et al. (Ber, 39, 1691, 1906).
[0019]
The epoxy derivative of the present invention represented by the above general formula (I) is a luminescent substance, and has excellent storage stability in an aqueous solution near neutrality compared to conventional acridine derivatives and the like. Yes. For example, 9-fluoro, which is a typical luminescent acridine derivative of the prior art.YeThe storage stability of noxycarbonyl-10-methylacridinium fluorosulfonate (PMA) in a neutral aqueous solution is reduced by about 80% in one month, whereas the epoxy derivative of the present invention, for example, 10-methyl dispiro [acridine-9 (10H), 2′-oxirane-3 ′, 2 ″ -tricyclo (3.3.1.13,7) decane] (Ia) is stored in neutral aqueous solution for 1 month. However, almost no decrease in light emission was observed.
[0020]
The epoxy derivative of the present invention can be used as a luminescent labeling substance in any conventionally known analysis means for detecting a specific substance in a specimen. In particular, a method of measuring a trace amount of physiologically active substance contained in a biological sample immunologically, utilizing the affinity of a nucleic acid, or utilizing chromatography, enzyme immunological, molecular biological measurement It is preferably used for a method, an apparatus, a kit, or the like. For example, when used for immunoassay called the sandwich method, first, the specific substance contained in the sample is contacted with the antibody against the specific substance immobilized on an insoluble carrier such as polyethylene. It binds on the insoluble carrier due to its affinity. Next, a labeled antibody in which the novel luminescent substance of the present invention is supported on an antibody against a specific substance (monoclonal antibody or polyclonal antibody) by a conventionally known means (for example, physical adsorption method or chemical binding) is brought into contact with the insoluble carrier. . By this operation, the labeled antibody is bound on the insoluble carrier by an amount correlated with the amount of the specific substance bound on the insoluble carrier. Next, after the excess labeled antibody not bound on the insoluble carrier is separated by a washing operation or the like, chemiluminescence measurement is performed.
[0021]
The epoxy derivatives of the present inventionReLight can be emitted strongly by adding hydrogen peroxide. Therefore, as a reagent for inducing chemiluminescence, a mixture of a peroxide such as hydrogen peroxide in an alkaline aqueous solution such as sodium hydroxide or potassium hydroxide can be used. Such a reagent that induces chemiluminescence can be brought into contact with the labeled antibody bound on the insoluble carrier, and the novel luminescent substance of the present invention can emit light by a chemical reaction. Since the emission intensity at this time correlates with the specific substance concentration in the sample, the specific substance concentration in the sample can be obtained by measuring the emission intensity with a photon counter or the like.
Furthermore, according to the present invention, not only the immunological detection method using the antibody, but also the nucleic acid is utilized for the target nucleus by utilizing the affinity between the nucleic acid and the complementary nucleic acid.acidCan be detected.
[0022]
Further, according to the finding of the present inventor, there is a specific compound capable of increasing the emission intensity of the epoxy derivative of the present invention, and alkaline hydrogen peroxide which is a reaction element includes trifluoroacetic acid and triethylamine. There is a phenomenon that the amount of light emission increases when etc. are added.
[0023]
Test samples containing specific substances can include biological fluids such as blood, serum, plasma, urine, saliva, spinal fluid, cell and tissue extracts, and specific substances include various proteins and polysaccharides. , Lipids, nucleic acids, etc., more specifically, proteins such as fibrinogen, albumin, CRP, IgG, IgA, IgM, AFP, CEA, etc.tumorRelated proteins, hepatitis B virus-related proteins and antibodies, hepatitis C virus-related proteins and antibodies, HIV antigens and antibodies, HTLV antibodies and other infection-related markers, D-dimer, PPI, TATetcCoagulation-related markers, insulin, HCG, thyroxine and other hormones, and antiepileptic drugs and digoxin and other drugs.
Moreover, if the specific substance is a nucleic acid, a DNA fragment having a specific base sequence may be used.
[0024]
【Example】
EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention. In the following examples, the melting points were measured using a Yanagimoto trace melting point measuring apparatus and all are uncorrected. The infrared absorption (IR) spectrum was measured using JASCO FT / IR-200 type infrared photometric system. Also, ¹The H-NMR spectrum was measured using a Varian VXR-200 type (200 MHz) and tetramethylsilane as an internal standard. Silica gel chromatography was performed using E.I. Merc Kiesel 60 (70-230 mesh) was used.
[0025]
Furthermore, in the following examples and reference examples, the ketone compound as a starting material [compound (1) in FIG. 1], the olefin derivative as an intermediate [compound (2) in FIG. 1], and the final product [FIG. Substituent R in compound (I)]¹ , R² , And X are divided into the following (a) to (g) for convenience of explanation. Thus, for example, the starting material (1) belonging to the following (a) is the ketone compound (1a), the intermediate (2) is the olefin derivative (2a), and the final product is the epoxy derivative (Ia).
(A): R¹ = R² = H, X = N-CH_Three
(B): R¹ = R² = H, X = N-CH₂ C₆ H_Five
(C): R¹ = R² = H, X = N-CH₂ CO₂ C₂ H_Five
(D): R¹ = R² = H, X = N-C₆ H_Five
(E): R¹ = R² = H, X = CH₂
(F): R¹ = R² = H, X = S
(G): R¹ = R² = H, X = O
[0026]
Reference Example 1: Preparation of starting materials (2a) to (2g)
[1] 9,10-Dihydro-10-methyl-9- (tricyclo [3.3.1.1^3,7 Synthesis of decylidene) acridine (2a)
3 g (15 mmol) of anhydrous titanium aluminum trichloride reduced form was suspended in 120 ml of tetrahydrofuran (THF), 0.20 g (7.6 mmol) of lithium aluminum hydride was added under ice cooling, and the mixture was stirred for 10 minutes. 1 ml (7.6 mmol) was added. The reaction solution was heated to reflux for 90 minutes, and then 30 ml of anhydrous THF solution containing 0.5 g (2.4 mmol) of N-methyl compound (1a) and 0.36 g (2.4 mmol) of 2-adamantanone was added dropwise. Further reflux was performed for 39 hours. The reaction mixture was returned to room temperature, and 60 ml of ice water was added little by little under ice cooling, followed by extraction with ethyl acetate three times. The organic layer was washed with water and saturated brine, and then dried over anhydrous sodium sulfate. After the solvent was distilled off, the residue was purified by silica gel chromatography using a mixed solvent of ethyl acetate / hexane [1:10 (v / v)] to obtain 0.49 g (1.5 mmol, 1.5 mmol, Yield 63%) was obtained as colorless crystals. The physical properties were as follows.
Melting point: 256-257 ° C. (ethyl acetate)
IR γ (KBr): 2908, 2847, 2358, 1589, 1461, 1339, 1269, 1129, 1098, 1042 cm^-1
UV λ_max ^CH _Three ^CNnm (log ε): 340 (3.78), 287 (4.01), 261 (4.16), 226 (4.46)
 ¹H-NMR (CDCl_Three ) Δ: 1.53-2.22 (12H, m), 3.41 (3H, s), 6.98 (4H, td, J = 8, 2 Hz), 7.15-7.26 (4H, m)
Elemental analysis: C_{twenty four}H_{twenty five}For N (molecular weight = 327.47):
Calculated value: C = 88.03; H = 7.69; N = 4.28
Found: C = 87.93; H = 7.65; N = 4.28
[0027]
[2] 9,10-Dihydro-10-pentyl-9- (tricyclo [3.3.1.1^3,7 Synthesis of decylidene) acridine (2b)
In place of the 10-methyl group of the product obtained in [1] above, 9,10-dihydro-10-benzyl-9- (tricyclo [3.3.1.1] having a benzyl group at the 10-position.^3,7 Decylidene) acridine (2b) is obtained as a colorless crystal having the following physical property values in a yield of 35% using the corresponding ketone body (1b) as a starting material and the same operation as in [1] above. I was able to.
Melting point: 218-219.5 ° C. (ethyl acetate)
IR γ (KBr): 3026, 2905, 2846, 1718, 1589, 1491, 1458, 1371, 1266, 1225, 1098, 1063 cm^-1UV λ_max ^CH _Three ^CNnm (log ε): 341 (3.93), 293 (4.01), 258 (4.23), 226 (4.61)
 ¹H-NMR (CDCl_Three ): 1.53-2.22 (12H, m), 3.43-3.49 (2H, m), 5.23 (2H, m), 6.83 (2H, dd, J = 8, 2Hz), 7.03 (2H, dd, J = 8, 2Hz), 7.15-7.27 (9H, m)
Elemental analysis: C₃₀H₂₉For N (molecular weight = 403.57):
Calculated value: C = 89.29; H = 7.24; N = 3.47
Found: C = 89.31; H = 7.32; N = 3.46
[0028]
[3] 9,10-dihydro-9- (tricyclo [3.3.1.1^3,7 Synthesis of decylidene) -10-acridine ethyl acetate (2c)
Similarly, 9,10-dihydro-9- (tricyclo [3.3.1.1^3,7 ] Desilidene) -10-acridine ethyl acetate (2c) was prepared by using the corresponding ketone body (1c) as a raw material and carrying out the same operation as in the above [1], and producing 63% of colorless crystals having the following physical properties. The yield could be obtained.
Melting point: 216-217 ° C. (ethyl acetate)
IR γ (KBr): 2907, 2847, 1751, 1591, 1458, 1368, 1188, 1098, 1024 cm^-1
UV λ_max ^CH _Three ^CNnm (log ε): 334 (3.90), 286 (3.99), 258 (4.20), 225 (4.55)
 ¹H-NMR (CDCl_Three ) Δ: 1.27-2.22 (3H, t, J = 7 Hz), 1.55-2.21 (12H, m), 3.41-3.44 (2H, m), 4.64 ( 2H, s), 6.77 (2H, td, J = 8, 1 Hz), 6.98 (2H, td, J = 10, 1 Hz), 7.11-7.27 (4H, m),
Elemental analysis: C_{twenty five}H₂₉NO₂ For (Molecular weight = 399.53):
Calculated values: C = 81.17; H = 7.32; N = 3.51
Found: C = 80.82; H = 7.36; N = 3.47
[0029]
[4] 9,10-Dihydro-9-phenyl-9- (tricyclo [3.3.1.1^3,7 Synthesis of decylidene) acridine (2d)
Similarly, 9,10-dihydro-9-phenyl-9- (tricyclo [3.3.1.1] having a phenyl group at the 10-position.^3,7 ] Desilidene) acridine (2d) is also obtained in the yield of 51% as colorless crystals having the following physical properties by carrying out the same operation as in [1] above using the corresponding ketone body (1d) as a raw material. I was able to.
Melting point: 245-255.5 ° C. (ethyl acetate)
IR γ (KBr): 2906, 2845, 1588, 1559, 1540, 1507, 1490, 1466, 1451, 1278, 1098 cm^-1
UV λ_max ^CH _Three ^CNnm (log ε): 340 (4.00), 286 (4.18), 256 (4.32), 228 (4.67)
 ¹H-NMR (CDCl_Three ) Δ: 1.83 to 2.07 (12H, m), 3.48 to 3.51 (2H, m), 6.45 (2H, dd, J = 8, 2 Hz), 6.97 (4H, m), 7.26 (2H, m), 7.38 (2H, dd, J = 7, 2 Hz), 7.49-7.62 (3H, m)
Elemental analysis: C₂₉H₂₇For N (molecular weight = 389.54):
Calculated: C = 89.42; H = 6.99; N = 3.60
Found: C = 89.31; H = 7.24; N = 3.62
[0030]
[5] 9-Tricyclo [3.3.1.1^3,7 Synthesis of decylidene-9,10-dihydroanthracene (2e)
Further, 9-tricyclo [3.3.1.1] having a different basic skeleton is used.^3,7 For the decylidene-9,10-dihydroanthracene (2e), the same operation as in the above [1] was carried out from the corresponding ketone body (1e), and it was possible to obtain 44% yield as colorless crystals. The physical property values are shown below.
Melting point: 232-233.5 ° C. (ethyl acetate)
IR γ (KBr): 3060, 3012, 2976, 2845, 1632, 1466, 1451, 1419, 1224, 1107, 1035 cm^-1
1H-NMR (CDCl_Three ): 1.57-2.23 (12H, m), 3.43 (2H, s), 3.76 (2H, each, J = 18 Hz), 7.09-7.19 (4H, m) , 7.26 (4H, m)
Elemental analysis: C_{twenty four}H_{twenty four}For (Molecular weight = 312.45):
Calculated value: C = 92.26; H = 7.74
Found: C = 92.22; H = 7.78
[0031]
[6] 9-Tricyclo [3.3.1.1^3,7 Synthesis of decylidene-9H-thioxanthene (2f)
Furthermore, 9-tricyclo [3.3.1.1^3,7 For desilidene-9H-thioxanthene (2f), the same operation as in the above [1] was carried out from the corresponding ketone body (1f), and colorless crystals were obtained in a yield of 32%. The physical property values are shown below.
Melting point: 220-222.5 ° C. (ethyl acetate)
IR γ (KBr): 3056, 2970, 2911, 2845, 1636, 1455, 1436, 1326, 1061, 1029 cm^-1
1H-NMR (CDCl_Three ): 1.50-2.20 (12H, m), 3.43 (2H, s), 3.20-3.36 (2H, m), 7.47 (2H, dd, J = 7, IHz)
[7] 9-Tricyclo [3.3.1.1^3,7 Synthesis of decylidene-9H-xanthene (2 g)
In addition, 9-tricyclo [3.3.1.1^3,7 With respect to decylidene-9H-xanthene (2 g), the same operation was carried out from the corresponding ketone body (1 g), and it was possible to obtain colorless crystals in a yield of 78%. The physical property values are shown below.
Melting point: 220-221.5 ° C. (ethyl acetate)
IR γ (KBr): 2981, 291, 2844, 1580, 1566, 1471, 1445, 1342, 1303, 1252, 1202, 1150 cm^-1 1H-NMR (CDCl_Three ) Δ: 1.88-2.05 (12H, m), 3.47-3.53 (2H, m), 7.05-7.29 (2H, m)
Elemental analysis: C_{twenty three}H_{twenty two}For O (molecular weight = 314.43):
Calculated value: C = 87.86; H = 7.05
Found: C = 87.76; H = 7.09
[0032]
Example 1: 10-methyl dispiro [acridine-9 (10H), 2'-oxirane-3 ', 2 "-tricyclo [3.3.1.1 ^3,7 Synthesis of decane] (Ia)
To a solution of the olefin compound (2a) (500 mg, 1.5 mol) obtained in Reference Example 1 [1] in methylene chloride (2.0 mol), 3-chloroperbenzoic acid (650 mg, 168 mmol) was added at room temperature. Stir for 10 minutes. After confirming the disappearance of the raw material by thin layer chromatography, saturated aqueous sodium bicarbonate was added to decompose excess 3-chlorobenzoic acid. Subsequently, extraction with dichloromethane was performed three times. The organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate.
After the solvent was distilled, the residue was purified by silica gel chromatography (ethyl acetate: hexane = 1: 10) to obtain the epoxy compound (Ia) (470 mg, 1.4 mmol, 89%) as colorless crystals. The physical properties were as follows.
Melting point: 241-242 ° C. (ethyl acetate)
IR γ (KBr): 2905, 2850, 1595, 1470, 1267 cm^-1
1H-NMR (CDCl_Three ) Δ: 1.02-2.13 (14H, m), 3.46 (3H, s), 6.95-7.05 (4H, m), 7.23-7.36 (4H, m)
Elemental analysis: C_{twenty four}H_{twenty four}For NO (molecular weight = 343.47):
Calculated value: C = 83.93; H = 7.34; N = 4.08
Found: C: 83.98; H = 7.31; N = 4.03
[0033]
Example 2: 10-benzyl dispiro [acridine-9 (10H), 2'-oxirane-3 ', 2 "-tricyclo [3.3.1.1 ^3,7 Synthesis of decane] (Ib)
The title compound was obtained in 97% yield as colorless columnar crystals in the same manner as in Example 1 except that the olefin compound (2b) obtained in Reference Example 1 [2] was used as a starting material. The physical properties were as follows.
Melting point: 227-229 ° C. (ethyl acetate)
IR γ (KBr): 2913, 2849, 1596, 1464, 1371, 1267 cm^-1
1H-NMR (CDCl_Three ) Δ: 1.05-2.17 (14H, m), 5.27 (2H, s), 6.85 (2H, d, J = 8 Hz), 6.97 (2H, t, J = 7 Hz) 7.09-7.42 (9H, m)
Elemental analysis: C₃₁H₂₉For NO (molecular weight = 419.57):
Calculated: C = 85.88; H = 6.97; N = 3.34
Found: C = 85.85; H = 6.98; N = 3.30
[0034]
Example 3: Dispiro [acridine-9 (10H), 2'-oxirane-3 ', 2 "-tricyclo [3.3.1.1 ^3,7 Synthesis of decane] -10-ethyl acetate (Ic)
The title compound was obtained as colorless columnar crystals in a yield of 85% by the same method as in Example 1 except that the olefin (2c) obtained in Reference Example 1 [3] was used as a starting material. The physical properties were as follows.
Melting point: 177-178 ° C (ethyl acetate)
IR γ (KBr): 2912, 2849, 1753, 1598, 1499, 1464, 1366 cm^-1
1H-NMR (CDCl_Three ) Δ: 1.05-2.20 (14H, m), 1.26 (3H, t, J = 7 Hz), 4.26 (2H, q, J = 7 Hz), 4.67 (2H, s) 6.72 (2H, d, J = 8 Hz), 7.02 (2H, td, J = 1, 8 Hz), 7.19-7.34 (2H, m), 7.37 (2H, d, J = 8 Hz), 7.09-7.42 (9H, m)
Elemental analysis: C₃₁H₂₉For NO (molecular weight = 419.57):
Calculated value: C = 78.04; H: 7.03; N = 3.37
Found: C = 77.79; H = 7.03; N = 3.37
[0035]
Example 4: 10-phenyl dispiro [acridine-9 (10H), 2'-oxirane-3 ', 2 "-tricyclo [3.3.1.1 ^3,7 Synthesis of Decane] (Id)
The title compound was obtained as colorless columnar crystals in a yield of 88% in the same manner as in Example 1 except that the olefin compound (2d) obtained in Reference Example 1 [4] was used as a starting material. The physical properties were as follows.
IR γ (KBr): 2917, 2851, 1597, 1497, 1457, 1319 cm^-1
1H-NMR (CDCl_Three ) Δ: 1.16-2.15 (14H, m), 6.44 (2H, d, J = 8 Hz), 6.93-7.12 (3H, m), 7.37 (4H, d, J = 8Hz), 7.50-7.68 (4H, m)
[0036]
Example 5: 9-10-dihydrodispiro [anthracene-9,2′-oxirane-3 ′, 2 ″ -tricyclo [3.3.1.1 ^3,7 Synthesis of Decane] (Ie)
The title compound was obtained as a colorless columnar crystal in a yield of 100% in the same manner as in Example 1 except that the olefin compound (2e) obtained in Reference Example 1 [5] was used as a starting material. The physical properties were as follows.
IR γ (KBr); 3020, 2964, 2950, 2849, 1470, 1455, 1418, 1350 cm^-1
1H-NMR (CDCl_Three ) Δ: 1.10-2.20 (14H, m), 3.99 (2H, each d, J = 18 Hz), 7.18-7.37 (6H, m), 7.40 (2H, dd) , J = 3, 6Hz)
Elemental analysis: C_{twenty four}H_{twenty four}For O (molecular weight = 328.45):
Calculated value: C = 87.76; H = 7.36
Found: C = 87.48; H = 7.35
[0037]
Example 6: Dispiro [9H-thioxanthene-9,2'-oxirane-3 ', 2 "-tricyclo [3.3.1.1 ^3,7 Synthesis of decane] -10-oxide (If)
The title compound was obtained as colorless columnar crystals in a yield of 58% in the same manner as in Example 1, except that the olefin compound (2f) obtained in Reference Example 1 [6] was used as a starting material. The physical properties were as follows.
Melting point: 267-268.5 ° C. (ethyl acetate)
IR γ (KBr): 3076, 2912, 2854, 2594, 1580, 1450, 1413, 1353, 1302, 1227, 1171, 1138, 1127 cm^-1
1H-NMR (CDCl_Three ) Δ: 1.40-2.25 (14H, m), 7.47-7.63 (6H, m), 8.10 (2H, dd, J = 1, 6 Hz)
[0038]
Example 7: Dispiro [tricyclo [3.3.1.1] ^3,7 Synthesis of decane 2,2′-oxirane-3 ′, 9 ″ -9H-xanthene] (Ig)
The title compound was obtained as colorless columnar crystals in a yield of 98% in the same manner as in Example 1 except that the olefin product (2 g) obtained in Reference Example 1 [7] was used as a starting material. The physical properties were as follows.
Melting point: 131-132.5 ° C. (ethyl acetate)
IR γ (KBr): 2914, 2850, 1603, 1573, 1450, 1317, 1200, 1147, 1100, 1075, 1032 cm^-1
1H-NMR (CDCl_Three ) Δ: 1.09-2.20 (14H, m), 7.07-7.45 (8H, m)
Elemental analysis: C_{twenty three}H_{twenty two}O₂ For (Molecular weight = 330.43):
Calculated value: C = 83.60; H = 6.71
Found: C = 83.56; H = 6.74
[0039]
Example 8: Measurement of luminescence amount-1
Lumat LB9501 manufactured by Bertoled was used for the following luminescence measurement. 10-substituted dispiro [acridine-9 (10H), 2'-oxirane-3 ', 2 "-tricyclo [3.3.1.1] obtained in Examples 1 to 4^3,7 ] Decane] (Ia-Id) 1 × 10^-6M solution is adjusted with dimethyl sulfoxide (DMSO) or a mixed solution of equal volume of DMSO and distilled water. 100 μl of each solution is taken in a glass tube and set in a holder of a measuring device. Sodium hydroxide is used as a luminescence starting solution. And 100 N of a solution containing 0.1N each of hydrogen peroxide solution was injected, and the luminescence counts for 120 seconds were integrated to obtain the luminescence amount, which was expressed in RLU (relative light unit).
[0040]
[Table 1]
(A) In 100% DMSO solution

(B) In 50% DMSO + 50% distilled aqueous solution

[0041]
Example 9: Measurement of luminescence amount-2
10-benzyl dispiro [acridine-9 (10H), 2'-oxirane-3 ', 2 "-tricyclo [3.3.1.1] obtained in Example 2^3,7 Decane] (Ib) was converted to dichloromethane (CH₂ Cl₂ ), DMSO or DMSO-H₂ Dissolved in O (distilled water) (1: 1), 1 × 10^-6Adjusted to M, the luminescence amount was obtained by integrating the counts for 120 seconds after adding trifluoroacetic acid, metachloroperbenzoic acid, or triethylamine, which were effective in increasing the luminescence intensity, as additives. The results are shown in Table 2. In Table 2, mCPBA is metachloroperbenzoic acid, TEA is triethylamine, and TFA is trifluoroacetic acid. The term “after 25 seconds” refers to the amount of light emitted when 25 hours after adding sodium hydroxide and hydrogen peroxide and when the reagent described below is further added.
[0042]
[Table 2]

As is apparent from the results in Tables 1 and 2, the epoxy derivatives (Ia), (Ib), (Ic), and (Id) according to the present invention are obtained from Sharp et al. (Tetrahedron Letters Vol. 28, No. 11). , P 1155, 1987), it showed higher luminous power than the adamantane derivative. Further, from the results shown in Table 2, it was found that the emission intensity of the epoxy derivative according to the present invention changes depending on the additive, and the emission intensity increases when triethylamine (TEA) or trifluoroacetic acid (TFA) is added.
[0043]
【The invention's effect】
The epoxy derivative according to the present invention is particularly superior in storage stability near neutrality compared to conventional luminescent acridine derivatives and the like, and is suitable for detecting a specific substance in a biological specimen such as blood. Can be used effectively as a label.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is an explanatory diagram showing a synthesis process of an epoxy derivative according to the present invention.

Claims (4)

Formula (I):

[Wherein, X is —N (R ³ ) —, a methylene group, an oxygen atom, or a sulfur atom, and R ¹ and R ² are the same or different, and each represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. , An unsubstituted amino group, a substituted amino group, a carboxyl group, an alkoxy group, a cyano group, a nitro group, or a halogen atom, and R ³ is an alkyl group having 1 to 4 carbon atoms, an unsubstituted phenyl group, or a substituted phenyl group Group, benzyl group, alkoxycarbonylmethyl group, or aryloxycarbonylmethyl group] or a salt thereof. The substituted phenyl group has the general formula (II):

(In the formula, R ⁴ , R ⁵ and R ⁶ are the same or different and each is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an unsubstituted amino group, a substituted amino group, a carboxyl group, an alkoxy group, cyano. The compound according to claim 1, which is a group represented by a group, a nitro group, a halogen atom or a succinimino group. The aryloxycarbonylmethyl group has the general formula (III):

(Wherein R ⁷ , R ⁸ and R ⁹ are the same or different and each represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an unsubstituted amino group, a substituted amino group, a carboxyl group, an alkoxy group, cyano, The compound according to claim 1, which is a group represented by a group, a nitro group, a halogen atom or a succinimino group. A method for detecting a substance to be examined in a test sample, wherein the compound represented by the general formula (I) according to claim 1 or a salt thereof is used as a labeling substance.

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