JP2004510703A - Enzyme-degradable prodrug compounds - Google Patents

Abstract

A prodrug of the invention is a modified form of a therapeutic agent and comprises a therapeutic agent, an oligopeptide, a stabilizing group, and optionally a linker group. Prodrugs can be degraded by the enzyme thimet oligopeptide or TOP. Also disclosed are methods of designing prodrugs by using a TOP-degradable sequence in a conjugate, and methods of treating a patient with a prodrug of the present invention.

Description

従って、癌細胞トロウアーゼ及びＴＯＰは、ほぼ同一の基質特異性を有する。
【０１５９】
例７．精製された ＣＤ１０ による加水分解
等量の精製されたブタ腎臓ＣＤ１０（Ｅｌａｓｔｉｎ Ｐｒｏｄｕｃｔｓ Ｃｏｍｐａｎｙ）を、１２．５μｇ／ｍｌの種々のペプチジルドキソルビシン化合物と共に、ｐＨ７．４の５０ｍＭのトリス−ＨＣｌ、１５０ｍＭのＮａＣｌ、０．１％のＴｒｉｔｏｎ Ｘ−１００において３７℃で１０時間までの間インキュベートした。反応生成物を、蛍光検出を用いて、ＨＰＬＣにより分析した。加水分解率は、インキュベーション期間で実質的に直線的であった。観察される生成物は、Ｌｅｕ−ドキソルビシンであった。表２は、１０時間にわたって加水分解された個々の試験化合物の％を提供する。さらに、それらの結果は、標準の試験化合物、Ｓｕｃ−Ａｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘに対して表される。
【０１６０】
【表２】

【０１６１】
例８．Ｔｏｐ 及び ＨｅＬａ 細胞トロウアーゼの阻害及び不活性化
金属酵素について予測されるように、ＴＯＰは金属キレート化剤、例えばＥＤＴＡ及び１，１０−フェナントロリンへの暴露により不活性化される（Ｂａｒｒｅｎｔｔなど．， “Ｔｈｉｍｅｔ ｏｌｉｇｏｐｅｐｔｉｄａｓｅ ａｎｄ ｏｌｉｇｏｐｅｐｔｉｄａｓｅ Ｍｏｒ ｎｅｕｒｏｌｙｓｉｎ”， Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏｌ． ２４８： ５２９−５５６ （１９９５））。ペプチド化合物Ｎ−［１−（ＲＳ）−カルボキシプロピル−Ａｌａ−Ａｌａ−Ｐｈｅ−ｐ−アミノベンゾエート（Ｃｐｐ−ＡＡＦ−ｐＡＢ）］は、より選択性で且つ感受性のインヒビターである（Ｋｎｉｇｈｔ ａｎｄ Ｂａｒｒｅｔｔ， “Ｓｔｒｕｃｔｕｒｅ／ｆｕｎｃｔｉｏｎ ｒｅｌａｔｉｏｎｓｈｉｐｓ ｉｎ ｔｈｅ ｉｎｈｉｂｉｔｉｏｎ ｏｆ ｔｈｉｍｅｔ ｌｉｇｏｐｅｐｔｉｄａｓｅ ｂｙ ｃａｒｂｏｘｙｐｈｅｎｙｌｐｒｏｐｙｌ−ｐｅｐｔｉｄｅｓ”， ＦＥＢＳ Ｌｅｔｔ ２９４： １８３−１８６ （１９９１））。
【０１６２】
Ｃｐｐ−ＡＡＦ−ｐＡＢはまた、密接に関連する金属ペプチダーゼ神経溶解素を阻害するが、単なる神経溶解素活性は５ｍＭのジペプチドＰｒｏ−Ｉｌｅにより阻害される（Ｓｅｒｉｚａｗａなど．， “Ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ａ ｍｉｔｏｃｈｅｏｎｄｒｉａｌ ｍｅｔａｌｌｏｐｅｐｔｉｄａｓｅ ｒｅｖｅａｌｓ ｎｅｕｒｏｌｙｓｉｎ ａｓ ａ ｈｏｍｏｌｏｇｕｅ ｏｆ ｔｈｉｍｅｔ ｏｌｉｇｏｐｅｐｔｉｄａｓｅ”， Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２７０： ２０９２−２０９８ （１９９５））。ＭＣＦ−７／６細胞ホモジネートによる研究においては、トロウアーゼ活性は、１ｍＭのＥＤＴＡ及び２ｍＭの１，１０−フェナントロリンにより９倍、阻害されたが、ところが非金属ペプチダーゼのインヒビターは効果的でなかった。
【０１６３】
従って、５０μＭのアミノエチルベンゼンスルホニルフルオリド、４μｇ／ｍｌアプロチニン（両者ともセリンペプチダーゼを阻害する）、２０μＭのＥ−６４（システインペプチダーゼを阻害する）、１．５μＭのペプスタチン（アスパラギン酸ペプチダーゼを阻害する）、２０μＭのロイペプチン（セリン及びシステインペプチダーゼを阻害する）、又は１μＭのＣＡ−０７４（カテプシンＢを阻害する）により阻害されなかった。ＨｅＬａ細胞画分についての試験においては、１．３μＭのＣｐｐ−ＡＡＦ−ｐＡＢがＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ加水分解を完全に阻害したが、しかし５ｍＭのＰｒｏ−Ｉｌｅは効果を有さなかった。
【０１６４】
Ｂａｒｒｅｔｔ ａｎｄ Ｂｒｏｗｎ （“Ｃｈｉｃｋｅｎ ｌｉｖｅｒ Ｐｚ−ｐｅｐｔｉｄａｓｅ， ａ ｔｈｉｏｌ−ｄｅｐｅｎｄｅｎｔ ｍｅｔａｌｌｏ−ｅｎｄｏｐｅｐｔｉｄａｓｅ”， Ｂｉｏｃｈｅｍ． Ｊ． ２７１： ７０１−７０６ （１９９０）） は、ＥＤＴＡに対する透析の後、再活性化を測定するために、精製された鶏ＴＯＰを使用した。５０μＭの濃度で、Ｚｎ^２＋は活性を完全に回復した。同じ濃度での他の２価のカチオンは、次の有効性の順序で活性を一部、回復した：Ｍｎ^２＋＞Ｃａ^２＋＞Ｃｏ^２＋＞Ｃｄ^２＋。他の２価のカチオン、例えばＣｕ^２＋は効果を有さなかった。過剰のＺｎ^２＋（１００μＭ以上）は阻害性であった。ＥＤＴＡにより処理されたＭＣＦ−７／６細胞ホモジネートによる再構成実験においては、活性は、５０μＭのＣｏ^２＋又はＭｎ^２＋により完全に回復されたが、しかしＺｎ^２＋又はＣｕ^２＋によって回復されなかった。
【０１６５】
例９．ゲル濾過及び等電点電気泳動
ＭＣＦ−７／６細胞ホモジネートは、保持用量の活性ゲル濾過クロマトグラフィー画分に基づいて、６８ＫＤのおおよその分子量を有した。それらの測定値に関しては、ＭＣＦ−７／６細胞ホモジネート及びタンパク質分子量標準を、Ｓｕｐｅｒｏｓｅ Ｓ１２， １０×３００カラム（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）上で分別した。ＨｅＬａ細胞からの精製されたトロウアーゼを、例１１に記載される方法を用いて、７４及び６３ＫＤに対応する２種のタンパク質バンドに、ＳＤＳポリアクリルアミドゲル電気泳動（ＰＡＧＥ）により分離した。
【０１６６】
約７４及び６３ＫＤのバンドが、抗−チミットオリゴペプチダーゼ抗体により染色されたＨｅＬａ細胞Ｆ１のＳＤＳ ＰＡＧＥウェスターンイムノブロットにおいて観察された。７４ＫＤのバンドがまた、ＭＣＦ−７／６、ＭＤＳ−ＭＤ−２３１及びＥＡ ｈｙ９２６細胞の粗ホモジネートのＳＤＳ ＰＡＧＥウェスターンブロットにおいて観察された。それらの結果は、７８ＫＤとしてＤＮＡ配列から推定され、そして種々のＳＤＳ ＰＡＧＥ決定において７４〜８０ＫＤとして報告されている、ヒト、ラット及びブタＴＯＰの分子量と一致する（Ｂａｒｒｅｔｔなど．， “Ｔｈｉｍｅｔ ｏｌｉｇｏｐｅｐｔｉｄａｓｅ ａｎｄ ｏｌｉｇｏｐｅｐｔｉｄａｓｅ Ｍｏｒｎｅｕｒｏｌｙｓｉｎ”， Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏｌ． ２４８： ５２９−５５６ （１９９５））。
【０１６７】
５．２の等電点が、Ｍｏｎｏ Ｐ ＨＲ５／５， ５×４０ｍｍカラム（Ａｍｅｒｓｈａｍ−Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ） 上でのクロマトフォーカシングによりＭＣＦ−７／６細胞ホモジネートトロウアーゼについて決定された。この結果は、種々の源からのＴＯＰについて一般的に報告される５．０±０．２７ｐＩ値と一致する（Ｔｉｓｌｊａｒ， “Ｔｈｉｍｅｔ ｏｌｉｇｏｐｅｐｔｉｄａｓｅ−ａ ｒｅｖｉｅｗ ｏｆ ａ ｔｈｉｏｌ ｄｅｐｅｎｄｅｎｔ ｍｏｔａｌｌｏ−ｅｎｄｏｐｅｐｔｉｄａｓｅ ａｌｓｏ ｋｎｏｗｎ ａｓ Ｐｚ−ｐｅｐｔｉｄａｓｅ ｅｎｄｏｐｅｐｔｉｄａｓｅ ２４．１５ ａｎｄ ｅｎｄｏ−ｏｌｉｇｏｐｅｐｔｉｄａｓｅ”， Ｂｉｏｌ． Ｃｈｅｍ． Ｈｏｐｐｅ． Ｓｅｙｌｅｒ． ３７４： ９１−１００ （１９９３））。
【０１６８】
例 １０．チオール活性化
ＭＣＦ−７／６ならし培地を、示される濃度でのジチオトレイトール（ＤＴＴ）と共に室温で３０分間、プレーインキュベートした。次に、１０μｇ／ｍｌのＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘを添加し、そして３７℃でインキュベートした。加水分解生成物を抽出し、そして上記のようにして、Ｌｕｎａ　Ｃ１８−３μ、４．６×１００ｍｍカラム（Ｐｈｅｎｏｍｅｎｅｘ）上で分析した。残留Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ基質を、内部標準として、硼酸よりもむしろｐＨ３．０のクエン酸緩衝液及びＮ−スクシニルドキソルビシンを用いて抽出し、そして、上記のようなＬｕｎａ Ｃ１８−３μカラム上で分析した。
【０１６９】
ほとんどの金属ペプチダーゼとは異なって、ＴＯＰは、低レベルのチオール還元剤、例えば５０μＭのジチオトレイトール（ＤＴＴ）又は１ｍＭのメルカプトエタノールにより活性化されるが、しかし高濃度、例えば５ｍＭのＤＴＴで阻害される（Ｏｒｌｏｗｓｋｉ， など． “Ｅｎｄｏｐｅｐｔｉｄａｓｅ ２４．１５ ｆｒｏｍ ｒａｔ ｔｅｓｔｅｓ． Ｉｓｏｌａｔｉｏｎ ｏｆ ｔｈｅ ｅｎｚｙｍｅ ａｎｄ ｉｔｓ ｓｐｅｃｉｆｉｘｉｔｙ ｔｏｗａｒｄ ｓｙｎｔｈｅｔｉｃ ａｎｄ ｎａｔｕｒａｌ ｐｅｐｔｉｄｅｓ， ｉｎｃｌｕｄｉｎｇ ｅｎｋｅｐｈａｌｉｎ−ｃｏｎｔａｉｎｉｎｇ ｐｅｐｔｉｄｅｓ，” Ｂｉｏｃｈｅｍ Ｊ ２６１： ９５１−９５８ （１９８９）； Ｔｉｓｌｊａｒ ａｎｄ Ｂａｒｒｅｔｔ “Ｔｈｉｏｌ−ｄｅｐｅｎｄｅｎｔ ｍｅｔａｌｌｏ−ｅｎｄｏｐｅｐｔｉｄａｓｅ ｃｈａｒａｃｔｅｒｉｓｔｉｃｓ ｏｆ Ｐｚ−ｐｅｐｔｉｄａｓｅ ｉｎ ｒａｔ ａｎｄ ｒａｂｂｉｔ，” Ｂｉｏｃｈｅｍ Ｊ２６７： ５３１−５３３ （１９９０）； Ｌｅｗ， など． “Ｓｕｂｓｔｒａｔｅ ｓｐｅｃｉｆｉｃｉｔｙ ｄｉｆｆｅｒｅｎｃｅｓ ｂｅｔｗｅｅｎ ｒｅｃｏｍｂｉｎａｎｔ ｒａｔ ｔｅｓｔｅｓ ｅｎｄｏｐｅｐｔｉｄａｓｅ ＥＣ ３． ４． ２４． １５ ａｎｄ ｔｈｅ ｎａｔｉｖｅ ｂｒａｉｎ ｅｎｚｙｍｅ， “Ｂｉｏｃｈｅｍ Ｂｉｏｐｈｙｓ Ｒｅｓ Ｃｏｍｍｕｎ ２０９： ７８８−７９５ （１９９５））。
【０１７０】
Ｓｈｒｉｍｐｔｏｎなど．， “Ｔｈｉｏｌ ａｃｔｉｖａｔｉｏｎ ｏｆ ｅｎｄｏｐｅｐｔｉｄａｓｅ ＥＣ ３． ４． ２４． １５。Ａ ｎｏｖｅｌ ｍｅｃｈａｎｉｓｍ ｆｏｒ ｔｈｅ ｒｅｇｕｌａｔｉｏｎ ｏｆ ｃａｔａｌｙｔｉｃ ａｃｔｉｖｉｔｙ”， Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２７２： １７３９５−１７３９９ （１９９７） は、還元剤がＴＯＰ酵素タンパク質の不活性ジスルフィド結合のダイマーの形成を逆転することによって活性化することを示した。同様に、ＭＣＦ−７／６細胞ならし培地によるＤＴＴ前処理実験は、高レベルでの阻害性を伴なって、１ｍＭのＤＴＴでＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ加水分解の１０倍の活性化を示した（図１３）。従って、ＴＯＰ及び癌細胞トロウアーゼの両者は、低レベルのチオール還元剤により活性化される。
【０１７１】
例 １１．ｐＨ 最適性
上記例１において調製されるようなＭＦＣ−７／６細胞ホモジネートを、種々のｐＨレベルで緩衝された１００ｍＭのトリエタノールアミンにおいて、βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｃｏｎと共に３７℃でインキュベートした。Ｌｅｕ−Ｃｏｕの量を、ロイシンアミノペプチダーゼによる反応生成物の処理、及び得られるアミノメチルクマリン濃度の分光蛍光計による測定により決定した。ＴＯＰは、消光された蛍光基質による１０分間のアッセイに関して、７．８のｐＨ最適性を有する（Ｂａｒｒｅｔｔ、１９９５）。
【０１７２】
消光された蛍光基質Ｍｃｃ−Ｐｒｏ−Ｌｅｕ−Ｇｌｙ−Ｐｒｏ−Ｄ−Ｌｙｓ（ＤＮＰ）によるアッセイは、Ｂａｒｒｅｔｔなど．， （“Ｔｈｉｍｅｔ ｏｌｉｇｏｐｅｐｔｉｄａｓｅ ａｎｄ ｏｌｉｇｏｐｅｐｔｉｄａｓｅ Ｍ ｏｒ ｎｅｕｒｏｌｙｓｉｎ”， Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏｌ． ２４８： ５２９−５５６ （１９９５）） に記載される通りであった。ＭＣＦ−７／６細胞ホモジネートトロウアーゼ活性ｐＨ最適性は、βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｃｏｕアッセイを用いてのＭＣＦ−７／６細胞ホモジネート活性について７．２〜７．７であった。従って、ＴＯＰ及びトロウアーゼ活性は、類似するｐＨで最適化される。
【０１７３】
例 １２．精製された ＨｅＬａ 細胞トロウアーゼトリプシン消化物の質量分光学
精製されたＨｅＬａトロウアーゼを一晩、凍結乾燥し、３０μｌのサンプル充填緩衝液に９０℃で１分間、溶解し、遠心分離し、不溶性材料を除去し、そして小形式ゲルに基づいてのＳＤＳポリアクリルアミドゲル電気泳動により分離した。操作条件は、４５分間、３０ｍＡ， ３００Ｖであった。クーマシーブルーＲ２５０による染色の後、７４及び６３ＫＤの分子量に対応する２つのバンドが明らかであった。
【０１７４】
個々のバンドの２×２ｍｍ断片を、小管に移し、そして次の個々の溶液と共に撹拌しながら、１５分の段階で洗浄した：２５ｍＭの炭酸水素（アンモニウム）、水中、５０％のアセトニトリル、２５ｍＭの炭酸水素（アンモニウム）。次に、サンプルを、遠心分離濃縮機（Ｓｐｅｅｄｖａｃ）において乾燥し、そして０．５μｇのトリプシンを含む、２５ｍＭの炭酸水素アンモニウム溶液１０μｌと共に３７℃で３時間インキュベートした。Ｎａｎｏｓｐｒａｙ−ＭＳのために、トリプシン消化物の一部を、アセトニトリルにより抽出し、遠心分離真空濃縮機（Ｓｐｅｅｄｖａｃ）において乾燥し、そして分析の前、ＺｉｐＴｉｐ^ＴＭ Ｃ１８ 微抽出装置により精製した。
【０１７５】
７４ＫＤのバンドからのトリプシン消化されたタンパク質を、ＭＡＬＤＩ−ｔｏｆ（マトリックス助力のレーザー脱着イオン化−飛行の時間）質量分光計により分析した。トリプシン消化物の質量スペクトルを、レフレクターモードで作動する遅延された抽出性を備えたＢｉｆｌｅｘ（Ｂｒｕｋｅｒ）ＭＡＬＤＩ−ｔｏｆ質量分光計上で獲得した。０．４μｌの個々の消化物（２５ｍＭの炭酸水素アンモニウムにおける）を、ニトロセルロースと共に混合された乾燥薄層α−シアノ−４−ヒドロキシ−桂皮酸（ＣＣＡ）マトリックス（イソプロパノール：アセトン（１：１）中、飽和ＣＣＡ：５ｎｇ／ｍｌのニトロセルロース（４：３；ｖ／ｖ））において、サンプルプローブ上に直接的に折出した。サンプルを、分析の前、０．１％のＴＦＡにより洗浄した。
【０１７６】
個々の消化物に関して得られるペプチド質量フィンガープリントを、ＭＳ−ＦＩＴ（ｈｔｔｐ：／／ｐｒｏｓｐｅｃｔｏｒ．ｕｃｓｆ．ｅｄｕ／ｕｃｓｆｈｔｍ１３．２／ｍｓｆｉｔ．ｈｔｍ）を用いて既知のタンパク質配列からの予測される消化物パターンに適合せしめた。表３に示されるように、予測されるヒトＴＯＰトリプシン断片化パターンへの観察される分子イオンの比較は、予測される質量を有する２０のバンドをもたらした。それらの適合性は、既知のヒトＴＯＰ配列の３３％を占めた（Ｔｈｏｍｐｓｏｎなど．， “Ｃｌｏｎｉｎｇ ａｎｄ ｆｕｎｃｔｉｏｎａｌ ｅｘｐｒｅｓｓｉｏｎ ｏｆ ａ ｍｅｔａｌｌｏｅｎｄｏｐｅｐｔｉｄａｓｅ ｆｒｏｍ ｈｕｍａｎ ｂｒａｉｎ ｗｉｔｈ ｔｈｅ ａｂｉｌｉｔｙ ｔｏ ｃｌｅａｖｅ ａ ｂｅｔａ−ＡＰＰ ｓｕｂｓｔｒａｔｅ”， Ｂｉｏｃｈｅｍ． Ｂｉｏｐｈｙｓ． Ｒｅｓ． Ｃａｍｍｕｎ． ２１３： ６６−７３ （１９９５））。トリプシン消化の後、６３ｋＤのバンドのＭＡＬＤＩ−ｔｏｆ分析は、それが、カルボキシ末端の小さな部分の不在を除いて、同じ配列を共有したことを示した。
【０１７７】
【表３】

【０１７８】
エレクトロスプレーイオン化（ＥＳＩ）四極子飛行時間（Ｑ−ｔｏｆ）タンデム質量分光測定を、ナノスプレーモードで作用するＺ−スプレーイオン源を有するＱ−ｔｏｆ装置（Ｍｉｃｒｏｍａｓｓ）を用いて行った。約３〜５μｌの精製されたサンプルを、サンプル針（ＰＲＯＴＡＮＡ Ｉｎｃ．， Ｏｄｅｎｓｅ， ＤＫ）中に導入し、ＭＳ及びＭＳ／ＭＳ実験を行った。平均細管電位は１０００Ｖであり、そしてサンプルコーンを５０Ｖに設定した。ヒト［Ｇｌｎ^１］−フィブリノペプチドＢを用いて、ＭＳ／ＭＳモードで装置を検量した。ＭＳ／ＭＳスペクトルを、ＭａｘＥｎｔ３を用いて変換し、そして配列を、ＰｅｐＳｅｑ（Ｍｉｃｒｏｍａｓｓ Ｂｉｏｌｙｎｘ）を用いて決定した。
【０１７９】
構造同一性のさらなる確認のために、２種のＨｅＬａ細胞トリプシン処理された７４ＫＤのフラグメントを、エレクトロスプレーイオン化（ＥＳＩ）四極子飛行時間（Ｑ−ｔｏｆ）タンデム質量分光測定により配列決定した。表４に示されるように、両フラグメントは、ヒトＴＯＰの既知配列と完全に一致した（Ｔｈｏｍｐｓｏｎなど．， “Ｃｌｏｎｉｎｇ ａｎｄ ｆｕｎｃｔｉｏｎａｌ ｅｘｐｒｅｓｓｉｏｎ ｏｆ ａ ｍｅｔａｌｌｏｅｎｄｏｐｅｐｔｉｄａｓｅ ｆｒｏｍ ｈｕｍａｎ ｂｒａｉｎ ｗｉｔｈ ｔｈｅ ａｂｉｌｉｔｙ ｔｏ ｃｌｅａｒｅ ａ ｂｅｔａ−ＡＰＰ ｓｕｂｅｔｒａｔｅ ｐｅｐｔｉｄｅ”， Ｂｉｏｃｈｅｍ． Ｂｉｏｐｈｙｓ Ｒｅｓ． Ｃｏｍｍｕｎ． ２１３： ６６−７３ （１９９５））。比較によれば、前記配列は、ラット及びブタＴＯＰの配列と接近するが、しかし同一ではない。
【０１８０】
【表４】

【０１８１】
例 １３．免疫沈殿
免疫沈殿を２段階で行った。第１段階においては、５μｌの試験酵素を、１０ｍＭのヒドロキシエチルピペラジン（ＨＥＰＥＳ）（ｐＨ７．２）、１５０ｍＭのＮａＣｌ溶液において、１：２５０に希釈された抗−ＴＯＰ又は不適切なウサギＩｇＧ（１０μｌ）と共に４℃で１時間インキュベートした。第２段階においては、この混合物を、同じ緩衝液において平衡化された１５μｌのプロテインＡセファロース（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ） に添加し、そして４℃で１時間インキュベートした。微小遠心分離の後、残留酵素活性を、上清液において決定した。
【０１８２】
免疫沈殿は、構造同一性のさらなる表示を提供する。部分的に精製されたＨｅＬａトロウアーゼ（Ｆ１）活性を、ウサギ抗−ＴＯＰ、続くＳｅｐｈａｒｏｓｅ（商標）ビース固定のプロテインＡとのインキュベーションの後、溶液から完全に除去した。同様に、ＭＣＦ−７／６細胞ホモジネートトロウアーゼ活性を、ウサギ抗−ＴＯＰ抗体による免疫沈殿により８０％に低めた。対照実験は、同じ条件下で、ｒＲ−ＴＯＰが完全に免疫沈殿されたが、しかし不適切なウサギ抗体とのインキュベーションはｒＲ−ＴＯＰ、ＨｅＬａ細胞トロウアーゼ活性、又はＭＣＦ７／６トロウアーゼ活性のいずれも免疫沈殿しなかったことを示した。
【０１８３】
例 １４．トロウアーゼについての特異性は、位置 Ｐ２ での非遺伝的にコードされたアミノ酸により提供される
特異性は、位置Ｐ２での、遺伝的にコードされたアミノ酸によりむしろ非遺伝的にコードされたアミノ酸の組み込みにより付与される。特に、位置Ｐ２での非遺伝的にコードされたアミノ酸β−アラニンを含む、Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｎｒを、例１〜３に記載される３種の調製物の個々と共に、例５に記載のようにしてインキュベートした。次に、分解の程度を、その得られる混合物のＨＰＬＣ分析により評価した。
【０１８４】
それらの結果を、Ｐ２位置での遺伝的にコードされたアミノ酸Ｌ−アラニンの位置を除いて同じ化合物により行われる同じインキュベーションについて得られる結果と比較し、例えばＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｎｒ対Ｓｕｃ−Ａｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｎｒを比較した。細胞ホモジネートによる分解の程度（割合）は、Ｐ２のＬ−アラニン化合物対Ｐ２のβ−アラニン化合物について１．３倍高かった。しかしながら、部分的に精製されたトロウアーゼ調製物に関して、Ｐ２のＬ−アラニン化合物の分解の程度は、Ｐ２のβ−アラニン化合物のその程度のわずか０．６倍であった。それらの結果は、Ｐ２でのＬ−アラニンの存在が酵素のより粗製の混合物のために第２の分解部位を提供したことを示唆し；従って、インビボでの活性薬剤の開放が腫瘍組織に局在化される傾向を減じる。
【０１８５】
例 １５．プロドラッグは分解の前、不良な細胞摂取を有する
前骨髄球性白血病細胞、すなわちＨＬ−６０を、１０％熱不活性化されたウシ胎児血清（ＦＣＳ）を含むＲＰＭＩ培地において培養した。研究の日、細胞を集め、洗浄し、そして１０％のＦＣＳを含むＲＰＭＩに、０．５×１０^６個の細胞／ｍｌの濃度で再建濁した。１００μｌ／ウェルの細胞懸濁液を、９６ウェルプレートに添加した。ドキソルビシン又は試験化合物の一連の希釈溶液（３倍の増大）を製造し、そして１００μｌの化合物をウェルに添加した。最終的に、１０μｌの１００μＣｉ／ｍｌの^３Ｈ−チミジンをウェル当たり添加し、そしてプレートを２４時間インキュベートした。プレートを、９６ウェル収穫機（Ｐａｃｋａｒｄ Ｉｎｓｔｒｕｍｅｎｔｓ） を用いて収穫し、そしてＰａｃｋａｒｄ Ｔｏｐ Ｃｏｕｎｔカウンター上で計数した。４パラメーターロジスティック曲線を、Ｐｒｉｓｍソフトフェアを用いて、薬剤モル濃度の関数として、^３Ｈ−チミジン組み込みに適合せしめ、ＩＣ_５０値を決定した。
【０１８６】
【表５】

【０１８７】
ドキソルビシンは、ＨＬ６０細胞における０．０７５μＭのＩＣ_５０を伴なって、可能性ある細胞毒性活性を示す。対照的に、プロドラッグＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘは、ＨＬ−６０増殖アッセイにおいては、不良な細胞摂取及び５０μＭ以上のＩＣ_５０を有する。インビボ分解研究は、ロイシル−ドキソルビシンが、プロドラッグＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘのタンパク質加水分解の後に形成される中間体であることを示す。従って、ロイシル−ドキソルビシンを試験し、そして０．２２２μＭのＩＣ_５０を有することを示した。それらのデータは、ロイシル−ドキソルビシンが、それが活性ドキソルビシンを開放するために分解される細胞により摂取される概念を支持する。
【０１８８】
例 １６．マウスにおける比較代謝
２つのグループのＩＣＲ正常雌マウスに、約１００μモル／ｋｇのＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ又は１０μモル／ｋｇのドキソルビシン（Ｄｏｘ）の１回のＩＶボーラス用量を投与した。血漿を、５分、１，２，４又は６時間で、個々のグループにおける３匹の個々の動物から得た。親、ジペプチジル−ドキソルビシン（ＡＬ−Ｄｏｘ）、α−アミノ−ドキソルビシン（Ｌ−Ｄｏｘ）及びドキソルビシン濃度を、蛍光検出（λｅｘ＝４８０ｎｍ，λｅｍ＝５６０）を用いて、逆相グラジエントＨＰＬＣにより、血漿サンプルの抽出物において分析した。量を、マウスの血漿における１０〜２０００ｎｇ／ｍｌのドキソルビシン溶液の測定値に対する線状標準曲線適合性を用いて決定した。
【０１８９】
６時間までの時間の経路に基づけば、Ｌ−Ｄｏｘが最初の２時間、主要代謝物であり、そしてジペプチジル−接合体ＡＬ−Ｄｏｘが、Ｌ−Ｄｏｘとほぼ同じ時間で形成されるよりマイナーな生成物であった。ドキソルビシンは、後で出現し、そしてその血漿濃度は、現在及び前に測定されたドキソルビシン薬物動力学プロフィールから（Ｖａｎ ｄｅｒ ｖｉｊｇｈなど．， “Ｃｏｍｐａｒａｔｉｖｅ ｍｅｔａｂｏｌｉｓｍ ａｎｄ ｐｈａｒｍａｃｏｋｉｎｅｔｉｃｓ ｏｆ ｄｏｘｏｒｕｂｉｃｉｎ ａｎｄ ４’−ｅｐｉｄｏｘｏｒｕｂｉｃｉｎ ｉｎ ｐｌａｓｍａ， ｈｅａｒｔ ａｎｄ ｔｕｍｏｒ ｏｆ ｔｕｍｏｒ−ｂｅａｒｉｎｇ ｍｉｃｅ”， Ｃａｎｃｅｒ Ｃｈｅｍｏｔｈｅｒ Ｐｈａｒｍａｃｏｌ． ２６ （１）： ９−１２ （１９９０）； 及びＴａｂｒｉｚｉ−Ｆａｒｄなど．， “Ｅｖａｌｕａｔｕｉｎ ｏｆ ｔｈｅ Ｐｈａｒｍａｃｏｋｉｎｅｔｉｃ Ｐｒｏｐｅｒｔｉｅｓ ｏｆ ａ Ｄｏｘｏｒｕｂｉｃｉｎ Ｐｒｏｄｒｕｇ ｉｎ Ｆｅｍａｌｅ ＩＣＲ （ＣＤ１（商標）） Ｍｉｃｅ Ｆｏｌｌｏｗｉｎｇ Ｉｎｔｒａｖｅｎｏｕｓ Ａｄｍｉｎｉｓｔｒａｔｉｏｎ”， Ｐｒｏｃ． Ａｍｅｒ． Ａｓｓｏｃ． Ｃａｎｃｅｒ Ｒｅｓ． ４２： ２３４ （２００１））及びドキソルビシン対照グループにより予測されるような他の代謝物よりも、時間にわたってよりゆっくり低下する。
【０１９０】
これは、続くエキソペプチダーゼ活性による連続的な分解と一致する。ドキソルビシンについての血漿濃度時間曲線下の領域（ＡＵＣ）（表６）は、Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘが、ドキソルビシンのみへの同等のドキソルビシン暴露を生成したことを示す。これらの化合物の投与の後、ドキソルビシン暴露は、マウス安全性研究において最大の許容される用量として表される相対的安全性に類似することが注目されるべきである。
【０１９１】
【表６】

【０１９２】
その結果、１０倍の用量のＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘが、ドキソルビシンに対する類似する暴露を生成した。この研究においては、Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘは、その単一回の用量（ＳＤ）及び反復用量（ＲＤ）ＭＴＤの約２倍で投与され、そしてドキソルビシンはそのＳＤ ＭＴＤの約２５％で投与されるが、しかしＲＤ ＭＴＤに等しい用量で投与された。血漿からの分解されていないＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘの急速なクリアランスは明らかに、ドキソルビシンに比較して、Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ化合物の高い相対的許容性をもたらす。ドキソルビシンは比較的より遅くクリアランスされる。従って、プロドラッグは、ＭＴＤにおける相対的差異により示されるように、過剰の薬物を安全にクリアランスしながら、効果的レベルのドキソルビシン暴露への投与を可能にするので、反復−用量処理、例えば化学療法に使用されるそれらの処理において好都合である。
【０１９３】
例 １７．プロドラッグは、腫瘍異種移植モデルにおいて効果的であり、且つ十分に許容できる
Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘは、エストロゲン−依存性ＭＣＦ−７／６乳癌及びアトリアマイシン体制結腸直腸癌ＣＸＦ２８０／１０及びＬＳ−１７４Ｔを包含する、いくつかのヌードマウス異種移植モデルにおけるヒト腫瘍の増殖を阻害することにおいて効果的であることがわかっている。
【０１９４】
例えば、皮下移植されたＬＳ１７４Ｔ腫瘍を有する１０匹のマウスのグループがＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ治療剤の週５回静脈内用量により処理される場合、生存の平均日（ＭＳＤ）における有意な用量依存性延長が観察され、そして５７（グループ２）、６４（グループ３）及び７１（グループ４）ｍｇ／ｋｇのＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘの用量で、ビークル処理された対照（グループ１）に比較して、腫瘍のサイズ（腫瘍体積）の低下が観察され、そして最高の用量は４０ｍｇ／ｋｇのドキソルビシンに等しかった（図１１を参照のこと）。薬剤は、抗腫瘍効能を示す用量の反復用量レベル及び頻度下で、安全且つ十分に許容できた。いくらかの用量依存性体重の低下が観察された。研究の支持においては、腎臓毒性及び骨髄抑制は、１０６．８ｍｇ／ｋｇまでの用量のＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘで観察されなかった。
【０１９５】
例 １８．Ｓｕｃ −β Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ はドキソルビシンよりもインビボでより許容される
本発明の典型的なテトラペプチドプロドラッグＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘは、マウスにおいて良好に許容される。第２の１回の用量の最大許容用量（ＳＤ−ＭＴＤ）研究においては、５匹の正常ＩＣＲマウスのグループが、ボーラス用量のＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘを静脈内投与された。マウスを毎日、４９日間、観察し、そして体重を週２度、測定した。試験される用量レベルは、それぞれ、０， ２９， ４２又は５６ｍｇ／ｋｇのドキソルビシンに等しい、０， ５０， ７５又は１００ｍｇ／ｋｇであった。いずれの用量レベルでも、２４時間以内に急性毒素は存在しなかった。
【０１９６】
毒性の用量及び時間依存性徴候が、この研究の間、観察された。毒性、例えば部分的な後脚の麻痺及び有意な体重の低下（それらの初期体重の２０％以上）が、７５及び１００ｍｇ／ｋｇの用量グループにおいて観察された。３５日までに、死亡率は、７５ｍｇ／ｋｇの用量グループの４０％に観察された。４９日での生存率及び毒性の徴候の欠失に基づいて、Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−ＤｏｘについてのＳＤ−ＭＴＤは、５０ｍｇ／ｋｇ（２８ｍｇ／ｋｇのドキソルビシンに相当する）であることが決定された。
【０１９７】
この用量は非常に許容され、そして悪影響は観察されなかった。従って、ＳＤ−ＭＴＤは、ドキソルビシンのみについてのＳＤ−ＭＴＤ（１６ｍｇ／ｋｇ）よりも、モル濃度に基づいて約１．８倍、高かった。表７を参照のこと。これは、試験される範囲にわたって１４ｍｇ／ｋｇのドキソルビシン等量の増分での用量の範囲に基づいてのおおよそのＳＤ−ＭＴＤ決定である。
【０１９８】
プロドラッグの毒性プロフィールに関する予備観察は、それが一般的に、ドキソルビシンのその観察、例えば後での麻痺及びるいそうと一致することを示した。しかしながら、ドキソルビシンの特徴的なＧＩ−毒性、心臓毒性及び骨髄抑制の形跡の欠失を包含する、毒性プロフィールにおける好ましいシフトを示すいくつかの差異が見られた。腎臓に対する著しい効果は観察されなかった。
【０１９９】
【表７】

【０２００】
例 １９．プロドラッグは比較体よりもより安全で且つより効果的である
有意に高い用量のＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘが投与され、それは、ドキソルビシンと比較して、ＬＳ−１７４Ｔヒト結腸直腸癌異種移植モデルにおける有意な毒性を有さない効力を達成することができる。４９（グループ３）、５７（グループ４）及び６５ｍｇ／ｋｇ（グループ５）の十分に許容される用量でのＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘは、急速に増殖するアドリアマイシン耐性ＬＳ−１７４Ｔ腫瘍の阻害（図５）及び腫瘍担持のマウスの生存性の延長（図１２）において、３．０ｍｇ／ｋｇ （グループ２）及び塩溶液（グループ１）でのドキソルビシンに比較して、卓越した効力を示した。用量制限毒性（心臓毒性及び骨髄抑制）が、ヒトにけるドキソルビシンの反復投与により観察された。従って、本発明者のドキソルビシンよりも高い用量のＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘが投与され、それが全身性毒性よりも腫瘍阻害を好むことを示した。
【０２０１】
例 ２０．プロドラッグは適度なドキソルビシン感受性腫瘍に対して有用である
適度なドキソルビシン感受性のヒト乳癌異種移植体であるＭＸ−１腫瘍を、皮下（Ｓ．Ｃ．）移植し、そして投与の開始（日０）の前、少なくとも週１度、次に研究の間、週２度、マウスを計量し、そして腫瘍を測定した（カリパスによる）。投与の開始の直前（研究日２〜日０）、マウスを、腫瘍の重量に基づいて、種々のグループにランダマイズした。腫瘍が１．５ｇのカットオフ重量（エンドポイント）に達した後、マウスを安楽死せしめた。研究は、日６０で終結された。
【０２０２】
【表８】

【０２０３】
対照グループ腫瘍は急速に増殖し、そしてすべての動物を、日２４までに、癌エンドポイントで終結した。腫瘍は比較的均等な増殖特性を示し、日１５〜２４の範囲の癌エンドポンとに達した。ドキソルビシンは、腫瘍担持のマウスの存在性を有意に延長することにおいて効果的であった（表８）。用量応答性を、ＭＸ−１腫瘍増殖の阻害及びマウス生存性の延長の両者においてＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘについて確立した（図１８及び表８）。７１ｍｇ／ｋｇでのＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘは、腫瘍増殖の阻害及びマウス生存性の延長において、ドキソルビシンよりも有意に良好であった（表８）。Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ及びドキソルビシンのすべての３種の用量レジメは、非常に良好に許容された。Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ７１ｍｇ／ｋｇグループにおける１０匹のマウスのうちわずか１匹が、研究の最後の方で、２０％までの重量の低下を有した（日４６の後）。
【０２０４】
例 ２１．プロドラッグは、多薬剤耐性機構の回避において有用である
Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘは、異種移植モデルにおいてマウスに移植されるＭＤＲヒト細胞系に対して、遊離ドキソルビシンよりも活性的であることが示されている。変性された形、特にＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘとしてのプロドラッグ形で腫瘍に供給されるドキソルビシンは、その用量グループにおいて生存の有意な延長をもたらす腫瘍増殖の遅延において活性を示す。
【０２０５】
表９及び図１５は、Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘに関して、ＭＤＲヒト結腸直腸癌ＬＳ１７４−Ｔにおける生存性の用量依存性上昇が存在することを示す。ＬＳ１７４Ｔは、壊死中心を有する異種細胞形態学を示す、非常に攻撃的で且つ急速に増殖する腫瘍である。それは従来の化学療法に対して非常に耐性であり、そして数日以内に十分に確立されたようになる腫瘍がいくつかの動物において常に存在するので、それらの動物は腫瘍の増殖を阻害する試みを急速に促進し、従って、動物は、処理にもかかわらず、腫瘍エンドポイントに達する。ドキソルビシンは単独で、このモデルにおいて完全に不活性であり、腫瘍の増殖又は生存性に対して効果をもたらさない。
【０２０６】
【表９】

【０２０７】
特に、表９は、ヌードマウス（Ｑ７Ｄｘ５）におけるＬＳ１７４Ｔ結腸直腸癌異種移植において、ドキソルビシンに比較して、３用量レベルでのＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘの効果の要約を提供する。測定されるパラメーターは、１５００ｍｇ（腫瘍死）の予定されたカットオフサイズに達する腫瘍のために、終結により決定される、計算された平均生存日（ＭＤＳ）、長期生存体（ＬＴＳ）の数、及び毒性死（体重の２０％以上の損失）を示すマウスの数による用量レジメの許容性を包含する。日６０でのＬＴＳの数は、すべてのグループにおいてゼロであった。毒性死は、いずれのグループにおいても観察されなかった。ビークル対象グループからの統計学的有意性：^＊：ｐ＜０．０５；^＊＊：ｐ＜（不対ｔ−検定）。
【０２０８】
対照グループ腫瘍は急速に増殖し、そしてすべての動物を、日３３までに、癌エンドポイントで終結した。腫瘍は異質の増殖特性を示し、日１５〜３３の癌エンドポイントに達した。ドキソルビシン（３ｍｇ／ｋｇ）は、ＬＳ１７４Ｔ腫瘍増殖の阻害又は生存性の延長において無効果であった。用量応答性を、マウス生存性の延長においてＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘについて確立した（表９）。６４ｍｇ／ｋｇでのＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘは、マウス生存性の延長において、ドキソルビシンよりも有意に良好であった（表９）。５７ｍｇ／ｋｇでのＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘは、ＬＳ１７４Ｔ腫瘍増殖を有意に阻害した（図１５及び表９）。Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ及びドキソルビシンのすべての３種の用量レジメは、非常に良好に許容され、そして毒性エンドポイントのために終結は存在しなかった。
【０２０９】
さらに、３種の良好に許容される用量レベル（Ｑ７ｄｘ５）で投与されるＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ（但し、ドキソルビシンではない）は、メジアン腫瘍重量に対して用量依存性阻害効果を示した（図１５）。
図１５は、ヌードマウス及びビークル対照におけるＬＳ１７４Ｔ腫瘍結腸直腸癌異種移植の腫瘍増殖に対する、ドキソルビシンに比較してのＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘの効果を示す。グループＤは、日１９でビークル対照グループとは統計学的に有意に異なった（ｐ＜０．０５）。
それらの結果は、腫瘍における高濃度での活性細胞毒性の局部生成がＭＤＲ経路を克服する治療剤のプロドラッグ形の秘訣であり得ることを示唆する。
【０２１０】
例 ２２．β Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ 凝集
不十分な溶解性のアントラサイクリン薬剤は、水性緩衝液において調製される場合、凝集体を形成することが示されている（Ｍｅｎｏｚｚｉなど．， “Ｓｅｌｆ−ａｓｓｏｃｉａｔｉｏｎ ｏｆ ｄｏｘｏｒｕｂｉｃｉｎ ａｎｄ ｒｅｌａｔｅｄ ｃｏｍｐｏｕｎｄｓ ｉｎ ａｑｕｅｏｕｓ ｓｏｌｕｔｉｏｎｓ”， Ｊ． Ｐｈａｒｍａｃｅｕｔ． Ｓｃｉ．， ７３： ７６６−７７０ （１９８４）； Ｃａｎｆａｌｏｎｉｅｒｉなど．， “Ｔｈｅ ｕｓｅ ｏｆ ｎｅｗ ｌｅｓｅｒ ｐａｒｔｉｃｌｅ ｓｉｚｅｒ ａｎｄ ｓｈａｐｅ ａｎａｌｙｓｅｒ ｔｏ ｄｅｔｅｃｔ ａｎｄ ｅｖａｌｕａｔｅ ｇｅｌａｔｉｎｏｕｓ ｍｉｃｒｏｐａｒｔｉｃｌｅｓ ｓｕｓｐｅｎｄｅｄ ｉｎ ｒｅｃｏｎｓｔｉｔｕｔｅｄ ａｎｔｈｒａｃｙｃｌｉｎｅ ｉｎｆｕｓｉｏｎ ｓｏｌｕｔｉｏｎｓ”， Ｊ． Ｐｈａｒｍａｃｅｕｔ． Ｂｉｏｍｅｄ． Ａｎａｌ． ９： １−８ （１９９１））。
【０２１１】
１７．４μＭ／ｍｌの水溶液におけるβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ凝集体サイズの評価を、それらの溶液をＡｍｉｃｏｎ Ｃｅｎｔｒｉｃｏｎ^ＴＭ フィルター単位を通して濾過する試みにより行った。ドキソルビシン（１７．４μＭ／ｍｌ）及びβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ（１７．４μＭ／ｍｌ）をそれぞれ蒸留水に溶解し、そして３，０００， １０，０００， ３０，０００及び５０，０００分子量カットオフ（ＭＷＣＯ）を有するＣｅｎｔｒｉｃｏｎフィルター中に配置した。個々のフィルター単位を、１５００ｇで２時間、遠心分離した。フィルターを通して保持され、そして通過する薬剤の量を、λ４７５ｎｍで定量化し、そして％に転換した。
【０２１２】
表１０は、ドキソルビシンの８１％が３，０００ＭＷＣＯフィルターを通して通過し、そして接合体、すなわちβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘのわずか５％が３，０００ＭＷＣＯフィルターを通過したことを示す。データは、５０，０００ＭＷＣＯがβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘの４０％以上を保持することを示す。それらのデータは、有意な％のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ凝集体が５０ＫＤよりも大きい（＞５０分子／凝集体）ことを示す。従って、βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘはいくつかの条件下で凝集することができる。
【０２１３】
【表１０】

【０２１４】
例 ２３．マウスにおけるβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ の静脈内注入
急性毒性はたぶん、正に荷電されたポリマー、例えばプロタミン、ポリリシン又はそれらの凝集体、及び血管の管腔表面の相互作用を通して生じることは知られている（Ｄｅｌｕｃｉａなど．， “Ｅｆｆｉｃａｃｙ ａｎｄ ｔｏｘｉｃｉｔｙ ｏｆ ｄｉｆｆｅｒｅｎｔｌｙ ｃｈａｒｇｅｄ ｐｏｌｙｃａｔｉｏｎｉｃ ｐｒｏｔａｍｉｎｅ−ｌｉｋｅ ｐｅｐｔｉｄｅｓ ｆｏｒ ｈｅｐａｒｉｎ ａｎｔｉｃｏａｇｕｌａｔｉｏｎ ｒｅｖｅｒｓａｌ”， Ｊ． Ｖａｓｅ． Ｓｕｒｇ． １８： ４９−６０ （１９９３）； Ｅｋｒａｍｉなど．， “Ｃａｒｂａｍｙｌａｔｉｏｎ ｄｅｃｒｅａｓｅ ｔｈｅ ｃｙｔｏｔｏｘｉｃｉｔｙ ｂｕｔ ｎｏｔ ｔｈｅ ｄｒｕｇ−ｃａｒｒｉｅｒ ｐｒｏｐｅｒｔｉｅｓ ｆｏ ｐｏｌｙｌｙｓｉｎｅｓ”， Ｊ． Ｄｒｕｇ Ｔａｒｇ． ２： ４６９−４７５ （１９９４））。
【０２１５】
ヘパリンはウサギ心筋に対する硫酸プロタミンの毒性効果を減じることはさらに示されている（Ｗａｋｅｆｉｌｄなど．， “Ｈｅｐａｒｉｎ−ｍｅｄｉａｔｅｄ ｒｅｄｕｃｔｉｏｎｓ ｏｆ ｔｈｅ ｔｏｘｉｃ ｅｆｆｅｃｔｓ ｏｆ ｐｒｏｔａｍｉｎｅ ｓｕｌｆａｔｅ ｏｎ ｒａｂｂｉｔ ａｙｏｃａｒｄｉｕｍ”， Ｊ． Ｖａｓｃ． Ｓｕｒｇ． １６： ４７−５３ （１９９２））。ここで見られる急性毒性が正に荷電されたプロドラッグ凝集体によるものであった仮説を試験するために、βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ（１７４μＭ／ｍｌ）を、対照に比較されるように、４．０００Ｉ．Ｕ．のヘパリンによる１時間の静脈内前処理に続いて、マウスに与えた。表１２は、ヘパリンに続いて、これまで急性致死容量のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘが有意に低い毒性であったことを示す。
【０２１６】
それらのデータは、急性毒性が、プロタミン又はポリリシンについて見られる効果に類似する効果を引き起こす正に荷電された凝集体によるものである仮説を指示する。本発明の負及び中性に荷電されたプロドラッグは、この所望しない副作用を克服する。
【０２１７】
【表１１】

【０２１８】
前述の仮説に従って、負に荷電された成分による、βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘの末端アミノ基のキャッピングが、２５０ｍｇ／ｋｇ（Ｄｏｘ−ＨＣｌ）ほどの高い用量レベルでの急性毒素効果の完全な消出をもたらした。
この証拠として、関連する実験において、すべての動物は、グループ当たり３〜５匹のマウスが２５０ｍｇ／ｋｇ（Ｄｏｘ−ＨＣｌ）のＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ又はＧ１−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘの静脈内ボーラスにより処理された場合、８日まで生存した。
【０２１９】
残る例についての分析方法：
固相又は溶液相アプローチを用いて合成されるペプチド配列は、分析用ＨＰＬＣ（方法Ａ， Ｂ ＆ Ｄ）が８０％以上の純度である粗生成物を示す場合、さらに精製しないで使用された。そうでなければ、材料は分離用ＨＰＬＣ方法Ｃを用いて精製された。
【０２２０】
ＨＰＬＣ方法Ａ：
分析用ＨＰＬＣ分析は、溶媒Ａ（０．１％ＴＦＡ／Ｈ_２Ｏ）及び溶媒Ｂ（０．１％ＴＦＡ／ＡＣＮ）のグラジエントにより溶離するＣ−１８カラム（４μｍ， ３．９×１５０ｍｍのＩＤ、１ｍｌ／分の流速）を用いてＷａｔｅｒｓ ２６９０上で行われ、そしてデータはＷａｔｅｒ Ｍｉｌｌｅｎｎｉｕｍシステムを用いてλ２５４ｎｍで処理された。分析用ＨＰＬＣグラジエントは、９０％の溶媒Ａで開始し、そして１４分間にわたって（線状）の１００％の溶媒Ｂで終結した。この方法及び次の方法についての化合物の純度は、ピークの曲線下での相対的％領域として評価された。
【０２２１】
ＨＰＬＣ方法Ｂ：
分析用ＨＰＬＣ分析は、溶媒Ａ（８０％の２０ｍＭの蟻酸アンモニウム及び２０％のアセトニトリル）及び溶媒Ｂ（２０％の２０ｍＭの蟻酸アンモニウム及び８０％のアセトニトリル）のグラジエントにより溶離するＣ−１８カラム（３．５μｍ， ４．６×１５０ｍｍのＩＤ、１ｍｌ／分の流速）を用いてＷａｔｅｒｓ ２６９０上で行われ、そしてデータはＷａｔｅｒ Ｍｉｌｌｅｎｎｉｕｍシステムを用いてλ２５４ｎｍで処理された。分析用ＨＰＬＣグラジエントは、１００％の溶媒Ａで開始し、そして１４分間にわたって（線状）の１００％の溶媒Ｂで終結した。
【０２２２】
ＨＰＬＣ方法Ｃ：
粗生成物の分離用精製は、溶媒Ａ（Ｈ_２Ｏ）及び溶媒Ｂ（ＭｅＯＨ）のグラジエントにより溶離するＣ−４カラム（１５μｍ， ４０×１００ｍｍのＩＤ、３０ｍｌ／分の流速）を用いて、Ｗａｔｅｒｓ Ｄｅｌｔａ Ｐｒｅｐ ４０００システムにより達成された。分離用ＨＰＬＣグラジエントは、８０％の溶媒により開始し、そして７０分間にわたって１００％の溶媒Ｂに進行する（線状）。データは、Ｗａｔｅｒｓ Ｍｉｌｌｅｎｎｉｕｍ Ｓｙｓｔｅｍを用いてλ２５４ｎｍで処理された。
【０２２３】
ＨＰＬＣ方法Ｄ：
分析用ＨＰＬＣは、ＴＳＫ ｓｕｐｅｒＯＤＳカラム（ＴｏｓｏＨａａｓ）を用いて、Ｈｅｗｌｅｔｔ Ｐａｃｋａｒｄ装置上で達成された；溶媒Ａ（水中、０．１％のＴＦＡ）；溶媒Ｂ（アセトニトリル中、０．１％のＴＦＡ）；グラジエント：２分での３０〜３６％のＢ、１０分での３６〜４１％のＢ、３分での４１〜９０％のＢ、５分での９０％のＢ、検出波長λ２５４ｎｍ。
【０２２４】
ＮＭＲ及びＭＳ：
追加の構造決定は、ＮＭＲ及びＭＳ技法により行われ、そして結果は、本発明の化合物を支持した。
ＴＬＣ方法：
ＴＬＣ分析は、溶出のためのＤＣＭ／ＭｅＯＨ／Ｈ_２Ｏ／８８％の蟻酸（８５／１５／１／２）を用いて、シリカゲル６０Ｆ−２５４ｎｍ−０．２５ｍｍプレート（Ｍｅｒｃｋ）上で行われた。
【０２２５】
ニンヒドリン試験：
数ミリグラムの生成物を試験に導入し、そして２滴の溶液Ａ（エタノール中、５０ｍｇ／ｍｌのニンヒドリン）、２滴の溶液Ｂ（エタノール中、４ｍｇ／ｍｌのフェノール）、次に、２滴の溶液Ｃ（１００ｍｌのピリジン中、２ｍｌの０．０１ＭのＫＳＣＮ）を添加した。その混合物を、煮沸水浴に５分間放置した。遊離アミンの存在下で、溶液は紫色になる。
【０２２６】
特異的オリゴペプチド合成例：
市販の試薬源：
ドキソルビシン及びダウノルビシンはＭｅｉｊｉ（Ｊａｐａｎ）により、Ｐｄ （ＰＰｈ_３）_４はＳｔｒｅｍ Ｃｈｅｍ （Ｎｅｗｂｕｒｙｐｏｒｔ， ＭＡ） により、ＰＥＧはＳｈｅａｒｗａｔｅｒ（Ｈｕｎｔｓｖｉｌｌｅ， ａｌａｂａｍａ）により、溶媒ＨＡＴＵはＡｌｄｒｉｃｈ （Ｍｉｌｗａｕｋｅｅ， ＷＩ） により供給され；すべての樹脂及びアミノ酸は、ＡＢＩ （Ｆｏｓｔｅｒ Ｃｉｔｙ， ＣＡ）， Ｎｏｖａｂｉｏｃｈｅｍ （Ｓａｎ Ｄｉｅｇｏ， ＣＡ）， Ａｄｖａｎｃｅｄ ＣｈｅｒｎＴｅｃｈ （Ｌｏｕｉｓｖｉｌｌｅ， ＫＹ）， Ｐｅｐｔｉｄｅ Ｉｎｔｅｒｎａｔｉｏｎａｌ （Ｌｏｕｉｓｖｉｌｌ， ＫＹ） 又はＳｙｎＰｅｐ（Ｄｕｂｌｉｎ， ＣＡ）により供給された。
【０２２７】
例 ２４．Ｆｍｏｃ 形のβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ ベンジルエステル
Ｆｍｏｃ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ（２４．３４ｇ， ０．０４モル）を、ＤＭＦ（３５０ｍｌ）及び磁気撹拌機と共に丸底フラスコ中に添加した。テトラペプチドを溶解した後、臭化ベンジル（４．７６ｍｌ、０．０４モル）、続いて炭酸セシウム（１３．０４ｇ、０．０４モル）を、前記溶液に撹拌しながら添加した。その反応混合物を室温で１．５時間、撹拌した。次に、反応混合物を、４５０ｍｌの氷冷却された水を含むフラスコ中にゆっくりと注いだ。多量の白色固形物が沈殿し、これを吸引濾過により集めた。生成物を水（２×２００ｍｌ）により洗浄し、そして真空デシケーターに配置した。生成物（２４．２ｇ， ８７％）をＨＰＬＣにより同定した（純度：９５％）。Ｃ_４０Ｈ_５０Ｎ_４Ｏ_７について計算されたＭＳ ｍ／ｚ： ６９．４； 実測値：６９９．５。
【０２２８】
例 ２５．β Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ ベンジルエステル
丸底フラスコ（２５ｍｌ）において、Ｆｍｏｃ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕベンジルエステル（０．７ｇ、１．０ｍモル）を、無水ＤＭＦ５ｍｌに溶解した。ピペリジン（１．２ｍｌ、１２．１ｍモル）を前記溶液に添加し、そしてその混合物を室温で２４分間、撹拌した。反応を水（６ｍｌ）により停止し、そして酢酸エチル（２×１０ｍｌ）により抽出した。組合された有機層をさらに、水（２×５ｍｌ）、ブライン（５ｍｌ）により洗浄し、そして硫酸ナトリウム上で乾燥した。白色固形物（０．８ｇ）を、溶媒の除去の後に得た。生成物の純度はわずか６７％であった。Ｃ_２５Ｈ_４０Ｎ_４Ｏ_５について計算されるＭＳｍ／ｚ：４７６．３；実測値：４７７．２。
【０２２９】
例 ２６．メチルスクシニル− Ｎ −キャップ形のβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ ベンジルエステル
丸底フラスコ（２５０ｍｌ）において、メチルヘミスクシネート（３．１９８， ２４．２ｍモル）を、無水ＤＭＦ （５０ｍｌ） に溶解した。ＤＩＥＡ（４．２２ｍｌ、２４．２ｍモル）、続いてＨＢＴＵ（９．１７ｇ、２４．２ｍモル）を前記溶液に添加した。その混合物を室温で４５分間、撹拌した。この混合物に、無水ＤＭＦ（１５０ｍｌ）中、βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕベンジルエステル（粗、１０．１４ｇ、２１．３ｍモル）の溶液を添加した。その混合物を、室温で２．５時間、連続して撹拌した。
【０２３０】
次に、その反応混合物を、２００ｍｌの氷冷却された水を有するフラスコ中に、撹拌しながらゆっくりと注いだ。多量の白色固形物が沈殿し、これを酢酸エチル（３×２００ｍｌ）により抽出した。組合された有機層をさらに、水（２×２００ｍｌ）、ブライン（２００ｍｌ）により洗浄し、そして硫酸ナトリウム上で乾燥した。白色固形物を、溶媒の除去の後に得た。酢酸エチルにおけるこの粗生成物の再結晶化により、８０％の純度の生成物７．５３ｇ（６０％）を得た。Ｃ_３０Ｈ_４６Ｎ_４Ｏ_８について計算されるＭＳ ｍ／ｚ： ５９１．４； 実測値：５９０．３３．
【０２３１】
例 ２７．メチルスクシニル− Ｎ −キャップ形のβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ
メチルスクシニル−Ｎ−キャップ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕベンジルエステル（１．０ｇ、８６％の純度、１．４６ｍモル）を、メタノール１００ｍｌを含む三角フラスコ中に添加した。その溶液は、数分間の撹拌の後、曇った。メタノール５０ｍｌを添加したが、しかし溶液はまだ透明ではなかった。その溶液を水素化反応容器中に移した。この容器に、Ｐｄ−Ｃ（９０ｍｇ、１０％の湿潤、５０％の水、０．０４２ｍモル）を添加した。室温で２時間の水素化の後、反応を停止し、そして触媒を濾過した。白色固形物（０．７７ｇ、７８％）を、溶媒の除去の後に得た。Ｃ_２３Ｈ_４０Ｎ_４Ｏ_８について計算されたＭＳｍ／ｚ：５０１．２；実測値：５００．３。
【０２３２】
例 ２８．Ｎ −キャップアリル−ヘミスクシネートの合成
この分子を、Ｃａｓｉｍｉｒ， Ｊ．Ｒなど．， Ｔｅｔ． Ｌｅｔｔ．３６ （１９）： ３４０９， （１９９５） の方法に従って調製した。１０．０７ｇ（０．１モル）の無水琥珀酸及び５．８０８ｇ（０．１モル）のアリル−アルコールを、１００ｍｌのトルエンにおいて６時間、環流した。その反応混合物を減圧下で濃縮した（１５．５ｇ；９８％）。得られる材料は、続く反応への使用のために十分な純度であった。半固形生成物の純度及び同一性を、^１Ｈ ＮＭＲ及び^１３Ｃ ＮＭＲ， 並びにＬＣ／ＭＳにより確かめた。
【０２３３】
例 ２９．アリル−スクシニル−β Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ の合成
丸底フラスコ（５０ｍｌ）において、Ｎ−キャップ−アリールヘミスクシニル形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ（１ｇ， １．９ｍモル）及びドキソルビシン（１．１ｇ、１．９ｍモル）を、無水ＤＭＦ（５０ｍｌ）に溶解した。その混合物を５分間撹拌した後、ＤＩＥＡ（０．６６ｍｌ、３．８ｍモル）、続いてＨＡＴＵ（０．７６ｇ、１．９ｍモル）を前記溶液に添加し、その混合物を室温で２時間、撹拌した。ＤＭＦを回転蒸発器により除去し、そして残留物を１：１のＤＣＭ：ＭｅＯＨ（４．０ｍｌ）に採取した。この溶液に、１００ｍｌのエーテルを、撹拌しながら、ゆっくりと添加した。赤色の沈殿物が形成され、そして吸引濾過によりそれを集めた。固形物をエーテル（２×２ｍｌ）におより洗浄し、そして真空デシケーターにおいて乾燥し、アリル−スクシニル−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ治療剤を得、これは方法Ｂによれば、９０％ＨＰＬＣ純度を有した。
【０２３４】
例 ３０．アリル−スクシニル−β Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ ドキソルビシンからの Ｓｕｃ −β Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ の調製
ＴＨＦ２ｍｌ中、０．１ｇ（０．０９５ｍモル）のアリル−スクシニル−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕドキソルビシンの撹拌溶液に、窒素雰囲気下で、０．０５ｇ（０．０９５ｍモル）のテトラキス（トリフェニルホスフィン）パラジウムを固形物として添加した。１０分後、反応の間に形成される沈殿物を濾過し、ＴＨＦにより洗浄した。乾量：０．１ｇ。その固形物を、スクシニル−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘであることが、ＨＰＬＣ、^１Ｈ ＮＭＲ， ＬＣ／ＭＳにより同定された。
【０２３５】
例 ３１．Ｆｍｏｃ 形のβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ の合成
Ｆｍｏｃ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕを、標準のＦｏｍｃ化学と共に固相アプローチを用いて合成した。典型的な合成は、Ｗａｎｇのアルコキシ樹脂（０．６０ｍモル／ｇ負荷量）を使用した。Ｆｏｍｃ−保護されたアミノ酸を、固相ペプチド合成のために使用した。樹脂上での１ｍＭのペプチドの規模に関しては、３当量のアミノ酸を、２当量のＤＩＥＡと共に樹脂に添加される５分前、活性剤としてのＨＢＴＵにより予備活性化した。カップリング反応を２時間、行い、そして次に、ＤＭＦ（２５ｍｌ×３）及びＤＣＭ（２５ｍｌ×３）により洗浄した。カップリング反応を、類似する条件下で２当量のアミノ酸を用いて反復した。
【０２３６】
反応の進行を、ニンヒドリン試験を用いてモニターし、そしてニンヒドリン試験が２時間後、不完全な反応を示す場合、カップリング段階を３度、反復した。保護解除を、ＤＭＦ中、２０％ピペリシンを用いて、１５〜２０分間、行った。カップリング段階を、所望するペプチドが樹脂上にアセンブリーされるまで、次のアミノ酸により反復した。樹脂からのペプチドの最終分解を、９５％のＴＦＡ及び５％の水の溶液により樹脂を処理することによって達成した。反応混合物を室温で２時間、撹拌した後、樹脂を減圧下で濾過し、そしてＴＦＡにより２度、洗浄した。濾液を組合し、そしてペプチドを、４００ｍｌの冷エーテルを添加することによって沈殿した。ペプチドを減圧下で濾過し、そして乾燥し、Ｆｍｏｃ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ（方法Ａによる９４％のＨＰＬＣ純度）を得た。粗ペプチドを、さらに精製しないで、次の段階のために使用した。
【０２３７】
例 ３２．Ｆｍｏｃ 形の Ｔｈｉ − Ｔｙｒ − Ｇｌｙ − Ｌｅｎ の合成
Ｆｍｏｃ形のＴｈｉ−Ｔｙｒ−Ｇｌｙ−Ｌｅｎを、標準のＦｏｍｃ化学及びＷａｎｇのアルコキシ樹脂（０．６０ｍモル／ｇ負荷量）と共に固相アプローチを用いて合成した。Ｆｏｍｃ−保護されたアミノ酸及びＦｏｍｃ−Ｔｈｉ−ＯＨを、固相ペプチド合成のために使用した。樹脂上での１ｍＭのペプチドの規模に関しては、３当量のアミノ酸を、２当量のＤＩＥＡと共に樹脂に添加される５分前、活性剤としてのＨＢＴＵにより予備活性化した。
【０２３８】
カップリング反応を２時間、行い、そして次に、ＤＭＦ（２５ｍｌ×３）及びＤＣＭ（２５ｍｌ×３）により洗浄した。カップリング反応を、類似する条件下で２当量のアミノ酸を用いて反復した。反応の進行を、ニンヒドリン試験を用いてモニターし、そしてニンヒドリン試験が２時間後、不完全な反応を示す場合、カップリング段階を３度、反復した。保護解除を、ＤＭＦ中、２０％ピペリシンを用いて、１５〜２０分間、行った。カップリング段階を、所望するペプチドが樹脂上にアセンブリーされるまで、次のアミノ酸により反復した。
【０２３９】
樹脂からのペプチドの最終分解を、９５％のＴＦＡ及び５％の水の溶液により樹脂を処理することによって達成した。反応混合物を室温で２時間、撹拌した後、樹脂を減圧下で濾過し、そしてＴＦＡにより２度、洗浄した。濾液を組合し、そしてペプチドを、４００ｍｌの冷エーテルを添加することによって沈殿した。ペプチドを減圧下で濾過し、そして乾燥し、Ｆｍｏｃ形のＴｈｉ−Ｔｙｒ−Ｇｌｙ−Ｌｅｎ（方法Ａによる８８％のＨＰＬＣ純度）を得た。粗Ｆｍｏｃ形のＴｈｉ−Ｔｙｒ−Ｇｌｙ−Ｌｅｎを、さらに精製しないで、次の段階のために使用した。
【０２４０】
例 ３３．Ｆｍｏｃ 形のβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｎｒ 治療剤の合成
ダウノルビシン・ＨＣｌ（１８５ｍｇ， ０．３２９ｍモル）及びＦｍｏｃ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ（２００ｍｇ， ０．３２ｍモル）を、室温で無水ＤＭＦ（１５ｍｌ）に溶解した。この急速に撹拌された溶液に、ＤＩＥＡ（０．１１５ｍｌ， ０．６５８ｍモル）を、１回で添加し、そしてその反応混合物を室温で１５分間、撹拌した。その反応混合物を、氷浴を用いて０℃に冷却し、そして１３８ｍｇ（０．３６２ｍモル）のＨＡＴＵを、１０分間にわたってゆっくりと添加した。
【０２４１】
反応混合物を、室温でさらに９０分間、撹拌した。氷冷却された水（２００ｍｌ）を、反応混合物に添加し、赤色の沈殿物の形成をもたらした。沈殿物を、粗フリット上に集め、３×５０ｍｌの水及び３×５０ｍｌのジエチルエーテルにより洗浄し、そして減圧下で乾燥し、Ｆｍｏｃ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｎｒ治療剤を得た（９４％の収率、方法Ａによる９５％のＨＰＬＣ純度）。この生成物を、さらに精製しないで、次の段階のために使用した。
【０２４２】
例 ３４．Ｆｍｏｃ 形の Ｔｈｉ − Ｔｙｒ − Ｇｌｙ − Ｌｅｕ − Ｄｎｒ 治療剤の合成
ダウノルビシン・ＨＣｌ（９０ｍｇ， ０．１６ｍモル）及びＦｍｏｃ形のＴｈｉ−Ｔｙｒ−Ｇｌｙ−Ｌｅｕ−Ｄｎｒ（１２０ｍｇ， ０．１６ｍモル）を、室温で無水ＤＭＦ（１５ｍｌ）に溶解した。この急速に撹拌された溶液に、ＤＩＥＡ（０．５６ｍｌ， ０．１６ｍモル）を、１回で添加し、そしてその反応混合物を室温で１５分間、撹拌した。その反応混合物を、氷浴を用いて０℃に冷却し、そして６１ｍｇ（０．１６ｍモル）のＨＡＴＵを、１０分間にわたってゆっくりと添加した。
【０２４３】
反応混合物を、室温でさらに９０分間、撹拌した。氷冷却された水（１５０ｍｌ）を、反応混合物に添加し、赤色の沈殿物の形成をもたらした。沈殿物を、粗フリット上に集め、３×５０ｍｌの水及び３×５０ｍｌのジエチルエーテルにより洗浄し、そして減圧下で乾燥し、Ｆｍｏｃ形のＴｈｉ−Ｔｙｒ−Ｇｌｙ−Ｌｅｕ−Ｄｎｒ治療剤を得た（９４％の収率、方法Ａによる９１％のＨＰＬＣ純度）。この生成物を、さらに精製しないで、次の段階のために使用した。
【０２４４】
例 ３５．Ｆｍｏｃ −β Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ −ドキソルビシンの調製
ダウノルビシン・ＨＣｌ（３．０ｇ， ５．１７ｍモル）及びＦｍｏｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ（３．１５ｇ， ５．１７ｍモル）を、室温で窒素雰囲気下で無水ＤＭＦ（２３０ｍｌ）に溶解した。この急速に撹拌された溶液に、ＤＩＥＡ（１．７９８ｍｌ， １０．３４ｍモル）を、１回で添加し、そしてその反応混合物を室温で１５分間、撹拌した。その反応混合物を、氷／ブライン浴において約−２℃に冷却し、そして５８ｍｌのＤＭＦ中、２．５６ｇ（６．７３ｍモル）のＨＡＴＵを、撹拌しながら１２分間にわたって滴化した。
【０２４５】
反応混合物を、−２℃でさらに３０分間、撹拌し、次にＤＩＥＡ（０．２８５ｍｌ， １．６４ｍモル）１回で添加した。０℃での水（５８０ｍｌ）を、反応混合物に添加し、柔毛状の赤色の沈殿物の形成をもたらした。沈殿物を、粗ガラスフリット上に集め、３×５０ｍｌの水及び３×５０ｍｌの水性ジエチルエーテルにより洗浄し、そして１６時間、空気乾燥し、Ｆｍｏｃ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ５．１２ｇを得た（８９．７％の収率、方法Ｂによる９０．２３％のＨＰＬＣ純度）。
【０２４６】
例 ３６．Ｆｍｏｃ −β Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ からのスクシニル−β Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ の調製
無水ＤＭＦ２３０ｍｌ中、５．０ｇ（４．４１ｍモル）のＦｍｏｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕの溶液に、室温で窒素下で、２１．８ｍｌ （２２０ｍモル）のピペリジンを一度に添加し、赤色から紫色への色彩変化をもたらした。反応混合物を室温で５分間、撹拌し、次にドライアイス／アセトン浴において約−２０℃に冷却した。２２．５ｇ（０．２２５モル）の無水琥珀酸を一度に添加し、そして反応温度を−５℃以下に維持した。−１０℃〜−５℃で約２分間の撹拌の後、色彩は、紫色から赤／オレンジ色に変化した。冷却浴を除き、そしてその反応混合物を１０分間、撹拌した。次に、反応混合物の体積を、回転蒸発により約１００ｍｌに減じ、そして次に、１２５ｍｌのクロロホルムにより希釈した。
【０２４７】
この溶液に、１４００ｍｌのジエチルエーテルをすばやく添加し、赤色の沈殿物の形成をもたらした。この沈殿物を、中位のガラスフリット上に単離し、そして５×２００ｍｌのジエチルエーテルと共に粉砕し、８９．１３％のＨＰＬＣ純度の材料を得た。沈殿物を１×２０ｍｌのジエチルエーテルにより再び洗浄し、そして空気乾燥し、３．６２ｇのＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘを得た（８１％の物理的収率、８８．２％のＨＰＬＣ純度）。この材料を０℃で３０ｍｌの水において撹拌し、そして３３．９８ｍｌ（０．９５当量）の０．１Ｍの水性ＮａＨＣＯ_３を添加し、そして得られる懸濁液を、すべての固形物が溶解するまで、撹拌した。この溶液を凍結乾燥し、３．７７ｇのＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘを得た（９９％の物理的収率、方法Ｂによる８９．０６％のＨＰＬＣ純度）。
【０２４８】
例 ３７．Ｎ −キャップスクシニル形のβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｎｒ −治療剤の合成
ピペリジン（０．４４２ｍｌ、４．４８ｍモル）を、５ｍｌの無水ＤＭＦ中、Ｆｍｏｃ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｎｒ（１００ｍｇ、０．０８９ｍモル）の溶液に添加した。その反応混合物を室温で５分間、撹拌し、そして次に、ドライアイス／アセトン浴を用いて、−２０℃に冷却した。無水琥珀酸（４５８ｍｇ、４．５４ｍモル）を、前記冷却された反応応答物に一度に添加した。
【０２４９】
反応を、−５℃で５分間、次に室温でさらに９０分間、急速に撹拌した。２５０ｍｌの無水ジエチルエーテルを、反応混合物に添加し、そして得られる赤色の沈殿物を、中位のガラスフリット上で単離した。フィルターケークを、２回の連続的な５０ｍｌのジエチルエーテルにより洗浄し、そして減圧下で乾燥し、Ｎ−キャップスクシニル形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｎｒ−治療剤を得た（８０％の収率、方法Ｂによる８８％のＨＰＬＣ純度）。ＬＣ／ＭＳは、９９５（予測される分子量９９６）の分子量を付与した。
【０２５０】
例 ３８．Ｎ −キャップスクシニル形の Ｔｈｉ − Ｔｙｒ − Ｇｌｙ − Ｌｅｕ − Ｄｎｒ 治療剤の合成
５ｍｌの無水ＤＭＦ中、Ｆｍｏｃ形のＴｈｉ−Ｔｙｒ−Ｇｌｙ−Ｌｅｕ−Ｄｎｒ（１００ｍｇ、０．０７９ｍモル）の溶液に、ピペリジン（０．３９１ｍｌ、３．９５ｍモル）を一度に添加し、赤色から紫色への色彩変化をもたらした。その反応混合物を、室温で５分間、撹拌し、そして次に、ドライアイス／アセトン浴を用いて−２０℃に冷却した。次に４０７ｍｇ（４．０２ｍモル）の無水琥珀酸を、前記冷却された混合物に一度に添加した。反応を−５℃で５分間、次に室温でさらに９０分間、急速に撹拌した。２００ｍｌの無水ジエチルエーテルを反応混合物に添加し、赤色の沈殿物をもたらした。この沈殿物を中位のガラスフリット上で単離し、３×５０ｍｌのジエチルエーテルにより洗浄し、そして減圧下で乾燥し、Ｎ−キャップスクシニル形のＴｈｉ−Ｔｙｒ−Ｇｌｙ−Ｌｅｕ−Ｄｎｒ治療剤を得た（８０％の収率、方法Ａによる８１％のＨＰＬＣ純度）。ＬＣ／ＭＳは、１１４１の分子量（１１４２の予測される分子量）を与えた。
【０２５１】
例 ３９．Ｎ −キャップグルタリル形のβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ 治療剤のナトリウム塩の合成
ピペリジン（４３６μｌ、４．４１３ｍモル）を、ＤＭＦ（４．５ｍｌ）中、Ｆｍｏｃ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ（１００ｍｇ、０．０８８ｍモル）の溶液に添加した。室温で５分間、撹拌した後、反応混合物を−５℃に冷却し、そして無水グルタミン酸（６２４ｍｇ、５．４７２ｍモル）をすばやく添加した。冷却浴を、色彩が変化するとできるだけ早く除去し、そしてその混合物を室温でさらに１０分間、撹拌した。
【０２５２】
ＤＭＦを回転蒸発により除去し、そして残留物をクロロホルム（２．５ｍｌ）に溶解した。ジエチルエーテル（１４ｍｌ）を添加し、そして得られる沈殿物を濾過した。フィルターケークをジエチルエーテルにより洗浄し、空気乾燥し、次に、水（１４ｍｌ）に再懸濁した。ナトリウム塩を、固体の完全な溶解まで、０．０２５ＭのＮａＯＨ（４ｍｌ、０．１０ｍモル）の懸濁液への滴下により形成した。次に、この溶液を凍結乾燥し、Ｇ１−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘのナトリウム塩を得た（９７％の収率、方法Ｄによる８７％のＨＰＬＣ純度）。
【０２５３】
例 ４０．
接合体、すなわち酵素経路のための前駆体を調製するための“ウレア方法”
メチルスクシニル− Ｎ −キャップ形のβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ 及びドキソルビシンのカップリング
無水窒素雰囲気下で、２６．０４ｇ（５２．０ｍモル）のメチルスクシニル−Ｎ−キャップ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ、２３．２６ｇ （４０．２ｍモル） のドキソルビシン塩酸塩を、８００ｍｌの無水ウレア−飽和（約３０％ｗ／ｖ）ＤＭＦ及び１９．９４８ｍｌ（１１４．１６ｍモル）のＤＩＥＡに懸濁し／溶解した。この混合物を、約２５分間にわたって、０〜３℃に冷却した。
【０２５４】
この点で、２１．２ｇ（５６．０ｍモル）のＨＡＴＵを、約１００ｍｌのウレア飽和ＤＭＦにおける溶液として、１０分間にわたって添加した（この溶液の体積は最少に維持されるべきである）。反応混合物を−２〜２℃で１０分間、撹拌し、そして２％（ｖ／ｖ）の酢酸を含む氷冷却されたブライン４０００ｍｌ中に、激しく撹拌しながら、約５分間にわたって注いだ、生成物を、中位の多孔性ガラス濾過器上で濾過し、水により十分に洗浄し、そして減圧下で乾燥した。４３ｇの物理的収率：１０４．４７％、方法Ｂによる９３．４５％のＨＰＬＣ純度。
【０２５５】
例 ４１．メチルスクシニル− Ｎ −キャップ形のβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ 治療剤の合成
丸底フラスコ（５０ｍｌ）において、Ｎ−キャップ−メチルヘミスクシニル形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ（０．２５ｇ， ０．５ｍモル）及びドキソルビシン（０．２９ｇ、０．５ｍモル）を、無水ＤＭＦ（２０ｍｌ）に溶解した。その混合物を５分間撹拌した後、ＤＩＥＡ（０．１７ｍｌ、１．０ｍモル）、続いてＨＡＴＵ（０．１９ｇ、０．５ｍモル）を前記溶液に添加した。その混合物を室温で４時間、撹拌した。ＤＭＦを回転蒸発器により除去し、そして残留物を１：１の塩化メチレン：メタノール（４．０ｍｌ）に採取した。この溶液に、４０ｍｌのエーテルを、撹拌しながら、ゆっくりと添加した。赤色の沈殿物が形成され、そして吸引濾過によりそれを集めた。固形物をエーテル（２×１０ｍｌ）により洗浄し、そして真空デシケーターにおいて乾燥た。０．５０ｇの生成物（９８％）を得た。
【０２５６】
例 ４２．ＭｅＯＳｕｃ −β Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ からの遊離ドキソルビシンの除去
ＭｅＯＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ（２００ｍｇ、０．１９４ｍモル）、ＤＩＥＡ（０．０６８ｍｌ、０．３８８ｍモル）及び無水ＤＭＦ（１０ｍｌ）を、磁気撹拌棒を備えた５０ｍｌのフラスコに配置した。ＭｅＯＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘが完全に溶解した場合、イソシアネート樹脂（３４０ｍｇ、０．５８２ｍモル；５ｍｌのジクロロメタンにおいて５分間、予備膨潤された）を添加し、そして得られる溶液を室温で２時間、定期的なＨＰＬＣモニターを伴なって、撹拌した。ＨＰＬＣクロマトグラフィーは、Ｄｏｘが４５分以内の樹脂処理で完全に除去されたことを示した。
【０２５７】
次に、反応混合物をフリットを通して濾過し、樹脂を除去した。樹脂を１０ｍｌのＤＭＦにより洗浄し、そしてそのＤＭＦ洗浄物を、前記濾過された反応混合物と組合した。次に、濾過された反応混合物を、高い真空ポンプ及び３０℃の水浴を備えた回転蒸発機上で赤色のガム上に濃縮した。その赤色のガム上残留物を、５ｍｌのＤＭＦに懸濁し、そして次に、その溶液を、急速に撹拌された無水ジエチルエーテル溶液中にゆっくりと添加した。赤色の生成物が形成され、次にこれを、フリット上で濾過し、そしてジエチルエーテルにより洗浄し、そして減圧下で乾燥し、ＭｅＯＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ（１７６ｍｇ、８６％の収率）を得た。
【０２５８】
例 ４３．架橋された酵素の使用によるメチルスクシニル− Ｎ −キャップ形のβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ 治療剤の加水分解
メチルスクシニル−Ｎ−キャップ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ治療剤（１．０ｇ、０．９７５ｍモル）及び１００ｍｌのＤＭＦを、５００ｍｌのフラスコに配置した。その懸濁液を磁気撹拌機により激しく撹拌した。メチルスクシニル−Ｎ−キャップ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ治療剤が完全に溶解した場合、４００ｍｌの脱イオン水を添加し、そしえ得られる溶液を３５℃で撹拌した。１ｇの洗浄されたＣＬＥＣ−ＰＣ（Ａｌｔｕｓ Ｂｉｏｌｏｇｉｃｓ）固定された酵素のスラリーを、３アリコートの脱イオン水によりすすぎ、次に使用の前、２０％の水性ＤＭＦ１０ｍｌに再懸濁した。得られる懸濁液を、定期的なＨＰＬＣモニターを伴なって、３５℃で撹拌した。
【０２５９】
すべてのメチルスクシニル−Ｎ−キャップ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ治療剤が消費された場合（約１８時間）、その反応混合物を、０．４５μＭのナイロン膜フィルターを通して濾過し、ＣＬＥＣ−ＰＣ酵素を除去した。ＣＬＥＣ−ＰＣケークを、３×１０ｍｌのメタノールにより洗浄し、そしてメタノール洗浄物を、前記濾過された反応混合物と組合した。次に、濾過された反応混合物及びメタノール洗浄物を、高い真空ポンプ及び３０℃での水浴を備えた回転蒸発器上で、赤色のガム状物に濃縮した。次に、その赤色のガム状物を、室温で５０ｍｌの脱イオン水に懸濁し、そして機械的撹拌機を通して激しく撹拌した。
【０２６０】
この懸濁液に、１００ｍｌの脱イオン水中、７７．８ｍｇ（０．９２６ｍモル、０．９５当量）の炭酸水素ナトリウムの溶液を、２分間にわたって添加した。その懸濁液を室温で２０分間、撹拌した。反応混合物を、０．４５μＭのナイロン膜フィルターを通して濾過し、そして連結乾燥した。スクシニル−Ｎ−キャップ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ治療剤のナトリウム塩０．９３６ｇを単離した（約１００％の収率；方法Ｂによる８４％のＨＰＬＣ純度）。^１Ｈ及び^１３Ｃ　ＮＭＲスペクトルを、それぞれ６００及び１５０ＭＨｚの分光計、及びエレクトロスプレーＭＳ上で記録し、それは、所望する構造と一致した。
【０２６１】
例 ４４．可溶性酵素の使用によるメチルスクシニル− Ｎ −キャップ形のβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ 治療剤の加水分解
１１．０ｇ（１０．７２ｍモル）のメチルスクシニル−Ｎ−キャップ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ治療剤を、ＨＰＬＣ品種の水に懸濁し、そしてＵｌｔｒａｔｕｒｒａｘ Ｔ８ホモジナイザーにより６０分間、均質化し、細かく分割された懸濁液を生成した。この懸濁液を３５℃で撹拌し（５００ｒｐｍ）、そして７６ｍＭの水性炭酸水素ナトリウムによりｐＨ＝６．０５に調節した。次に、１．０ｇのＣ．アンタルクチカ“Ｂ”リパーゼ（Ａｌｔｕｓ Ｂｉｏｌｏｇｉｃｓ）を添加し、そして反応混合物を３５℃で４８時間、撹拌した。４８時間の反応時間の間、ｐＨを、７６ｍＭの炭酸水素ナトリウムの定期的な添加による５．３〜６．２の間に維持し、そして反応をＨＰＬＣにより定期的にモニターした。４８時間後、反応はＨＰＬＣによれば、約９８％の完結であった。
【０２６２】
次に、反応混合物を７６ｍＭの水性炭酸水素ナトリウムによりｐＨ＝７に調節し、そしてセライト５２１のパッドを通して濾過した。次に、透明にされた反応混合物を、５ｍｌの氷酢酸により約３のｐＨに酸性化し、ゴム状の赤色沈殿物の形成をもたらした。沈殿物をセライト５２１濾過、セライトパッドのメタノールによる続くすすぎ、１０〜２０μＭのガラス濾過器を通してのメタノール溶液の濾過及び濾過された溶液の回転蒸発により単離した。７．３１ｇのゴム状の赤色生成物を得た。この生成物を、７０ｍｌの７６ｍＭの炭酸水素ナトリウム（０．９５当量）における溶解によりナトリウム塩に転換し、そして凍結乾燥し、７．３０ｇ（６６．１％の物理的収率）のスクシニル−Ｎ−キャップ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ治療剤のナトリウム塩を得た（ＨＰＬＣによる８４．５％の純度）。
生成物は、例４５のものと同一であった。
【０２６３】
例 ４５．メチルスクシニル− Ｎ −キャップ形のβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ 治療剤の固定されたカンジダ・アンタルクチカ“ Ｂ ”リパーゼ加水分解
３０．０ｇのカンジダ　アンタルクチカ“Ｂ”リパーゼ（Ａｌｔｕｓ Ｂｉｏｌｏｇｉｃｓ）を、３００ｍｌの水に溶解し、そして３×４Ｌの５０ｍＭの水性炭酸水素ナトリウム（ｐＨ＝６．４）に対して透析した。透析の後、その透析された溶液の体積は３００ｍｌであった。３６０ｍｌのＦｈａｒｍａｃｉａ ＮＨＳ−Ａｃｔｉｖａｔｅｄ Ｓｅｐｈａｒｏｓｅ ４ Ｆａｓｔ Ｆｌｏｗを、粗ガラス濾過器に置き、そして５×４５０ｍｌの氷冷却された１ｍＭの水性ＨＣｌによりすすいだ。そのすすがれたＮＨＳ−Ａｃｔｉｖａｔｅｄ Ｓｅｐｈａｒｏｓｅを、前記透析された酵素溶液と組合した。得られる懸濁液を、周囲温度（約２２℃）で２．０時間、撹拌した。
【０２６４】
セファロース／酵素接合体を、粗ガラス濾過器上で単離し、そして次に、１０００ｍｌの１００ｍＭの水性ＴＲＩＳ（ｐＨ＝７．４５）において１５分間、撹拌した。この懸濁液を濾過し、そしてもう１つの１０００ｍｌの１００ｍＭの水性ＴＲＩＳ緩衝液（ｐＨ＝７．４５）と共に４℃で一晩インキュベートした。朝、固定された酵素を濾過し、そして水による洗浄の後、２０００ｍｌの三ツ首丸底フラスコに配置した。４３ｇのメチルスクシニル−Ｎ−キャップ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘを添加し、そして固形物を８００ｍｌの脱イオン水に懸濁した。
【０２６５】
フラスコに、オーバーヘッド撹拌機、及び注射器ポンプを調節することによって反応混合物のｐＨを５．９〜６．２に維持するよう設定されたｐＨ−安定装置を固定した。注射器ポンプを、０．１Ｍの炭酸水素ナトリウムにより充填した。反応の進行をＨＰＬＣにより追跡した。６日後、固定された酵素を濾過し、そして溶液相を凍結乾燥した。次に、乾燥固形物を約１１ｍｌの無水ＴＨＦに懸濁し、そして濾過した。４２．６６ｇ、９８．３４％の物理的収率、方法Ｂによる９３．４５％（２５４ｎｍ）、９４．４３％（４８０ｎｍ）ＨＰＬＣ純度。
【０２６６】
例 ４６．β Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ 治療剤の乳酸塩［β Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ ラクテート］の合成
ピペリジン（２６ｍｌ、２６４ｍモル）を、ＤＭＦ（２６５ｍｌ）中、Ｆｍｏｃ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ治療剤（６．００ｇ、５．３ｍモル）の溶液に添加した。室温で５分間の撹拌の後、反応混合物を、氷−塩浴に配置し、そして予備冷却（４℃）をされた１０％乳酸緩衝液（ｐＨ３）（６００ｍｌ）をすぐに添加した。その水溶液を、ＤＣＭ（３×５００ｍｌ）により抽出し、そして過剰の塩を、固相抽出により除去した。
【０２６７】
Ｃ１８ ＯＤＳ−Ａシリカゲル（１２０ｇ）を、ガラス濾過器において条件づけし（５００ｍｌのメタノール、２×５００ｍｌの水）、そして粗生成物乳酸塩の水溶液において条件づけし（５００ｍｌのメタノール、２×５００ｍｌの水）、そして粗生成物乳酸塩の水溶液を充填した。水（２×５００ｍｌ）による洗浄及び乾燥の後、フィルターケークをメタノールに溶解した。メタノールを蒸発し、そして残留物を水に溶解した。得られる溶液を、凍結乾燥し、３．５４ｇのβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ治療剤の乳酸塩を得た（６７％の収率、方法Ｂによる８９％ＨＰＬＣ純度）。
【０２６８】
例 ４７．β Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ 治療剤の乳酸塩から出発してのスクシニル− Ｎ −キャップ形のβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ 治療剤の合成
ＤＩＥＡ（４１７μｌ、２．４０ｍモル）を、ＤＭＦ（３５ｍｌ）中、βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ治療剤の乳酸塩（１．２００ｇ、１．２０ｍモル）の溶液に添加した。室温での１５分間の撹拌の後、９７％の無水琥珀酸（０．１４４ｇ、１．４４ｍモル）を添加した。混合物を２時間、撹拌し、そしてＤＭＦを回転蒸発により除去した。
【０２６９】
残留物を、ＣＨＣｌ_３／ＣＨ_３ＯＨ（４／１）（６ｍｌ）の混合物に溶解し、そして１：１のＥｔ_２Ｏ：ヘキサンの混合物２００ｍｌを添加した。その混合物を３０分間、撹拌した後、沈殿物を定量紙（Ｗｈａｔｍａｎ４２）上で濾過し、洗浄し（１：１のＥｔ_２Ｏ：ヘキサン）、そして空気乾燥した。フィルターケークを、水（１５０ｍｌ）に懸濁し、そして１ＭのＮａＯＨ（±１．２当量、１．５ｍｌ）を、完全な溶解（ｐＨ＝７．２）まで、滴下した。その溶液を凍結乾燥し、１．２１８ｇのスクシニル−Ｎ−キャップ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ治療剤（９７％の収率；方法ＢによるＨＰＬＣ純度：８０．２％）。
【０２７０】
例 ４８．Ｆｍｏｃ 形のβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ 治療剤から出発してのスクシニル− Ｎ −キャップ形のβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ 治療剤の合成
ピペリジン（２１８０μｌ、２２．０６ｍモル）を、ＤＭＦ（２１．５ｍｌ）中、Ｆｍｏｃ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ治療剤（０．５０ｇ、０．４４ｍモル）の溶液に添加した。室温での５分間の撹拌の後、反応混合物を、すばやく、−５℃に冷却し、そして無水琥珀酸（２．２５ｇ、２２．５１ｍモル）をすぐに添加した。冷浴を、色彩が変化するとできるだけすぐに除去し、その混合物を室温で１０分間、撹拌した。
【０２７１】
ＤＭＦを回転蒸発により除去し、そして残留物をクロロホルム（１２．５ｍｌ９に溶解した。ジエチルエーテル（３６０ｍｌ）をすばやく添加した。沈殿物がすぐに出現した。沈殿物をＷｈａｔｍａｎ４２紙上で濾過し、Ｅｔ_２Ｏにより洗浄した。固形物を水（１２０ｍｌ、ｐＨ＝４．１）に懸濁し、そして０．０２５ＭのＮａＯＨ（２０ｍｌ、０．５３ｍモル）を、完全な溶解（ｐＨ＝７．４）まで、滴下した。次に、溶液を凍結乾燥し、スクシニル−Ｎ−キャップ形のβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ治療剤を得た（８９％の収率；及び方法Ｄによる９１％のＨＰＬＣ純度）。
【０２７２】
例 ４９．メチルスクシニル− Ｎ −キャップのβ Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ 治療剤の大規模合成
６９．６ｇのドキソルビシン・ＨＣｌ（１２０ｍモル）及び１００ｇのＭｅＯＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ（１９９ｍモル）を、室温下で無水ＤＭＦ（１０Ｌ）に溶解した。７６ｍｌのＤＩＥＡ（４３４ｍモル）を、反応混合物に添加し、そしてその反応混合物を、室温で、窒素下で１０分間、撹拌した。次に、反応混合物を、１０分にわたって０℃に冷却した。別々のフラスコにおいて、ＤＭＦ（５００ｍｌ）中、８６４ｇＨＡＴＵ（２２０ｍモル）の溶液を分離した。そのＨＡＴＵ溶液を、前記反応混合物に、その混合物を０℃で維持しながら、２０分間にわたってゆっくりと添加した。反応混合物を３０分間、０℃で撹拌した。
【０２７３】
水（２５Ｌ）中、ＮａＣｌ（７．５ｋｇ、少なくとも３０％ｗ／ｖ）の溶液を分離し、そして０℃に冷却した。次に、反応混合物を、冷却されたブライン溶液に、撹拌しながら１２０分間にわたってゆっくりと添加した。前記溶液の色は赤色を維持し、青色の溶液は、ｐＨが酢酸の添加により、５．８〜６．０にすぐに調節する必要があることを示した。温度を、約５℃で維持した。赤色の沈殿物を、中位の多孔性ガラス濾過器上で濾過し、水により洗浄し、そしてＰ_２Ｏ_５上で真空下を乾燥し、１１５ｇのＭｅＯＳｕｓ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘを得た。
【０２７４】
例 ５０．微量のドキソルビシンを除去するために Ｐｓ −イソシアネートビーズによる ＭｅＯＳｕｃ −β Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ の処理
１４６．４のＰＳ−イソシアネートビース（２４０ｍモル；Ａｒｇｏｎａｕｔ Ｌａｂ， Ｓａｎ Ｃａｒｌｏｓ， ＣＡにより供給される）を、１．５Ｌの無水ＤＭＦに溶解し、そして室温で５〜１０分間、膨潤した。膨潤されたビーズを、ガラス濾過器を通して濾過し、そして追加の５００ｍｌの無水ＤＭＦにより洗浄した。１１５ｇのＭｅＯＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ（１１２ｍモル）を、１０００ｍｌの無水ＤＭＦに溶解し、そして２．１ｍｌのＤＩＥＡ（１２ｍモル）、続いて膨潤されたＰＳ−イソシアネートビーズを添加した。反応混合物を室温で撹拌し、そしてドキソルビシンピークの量が０．１％以下になるまで、ＨＰＬＣを用いてモニターした。
【０２７５】
バッチのサイズに依存して、２〜１２時間を要した。ＨＰＬＣ分析を、Ｗａｔｅｒ ２６９０カラムを用いて行った：Ｗａｔｅｒ Ｓｙｍｍｅｔｒｙ ｓｈｉｅｌｄ Ｃ_８ ３．５μＭ ４．６×１５０ｍｍ（カタログ番号ＷＡＴ０９４２６９）、溶媒：Ａ−８０％の２０ｍＭの蟻酸アンモニウム（ｐＨ＝４．５）、２０％アセトニトリル、溶媒：Ｂ−２０％蟻酸アンモニウム（ｐＨ＝４．５）、８０％アセトニトリル。カラム温度：調節された室温、サンプル温度；４℃、運転時間：３７．５分、検出器：２５４ｎｍ， 流速：１．０ｍｌ／分、注入量１０μｇ（０．５ｍｇ／ｍｌ×０．０２ｍｌ）、移動相Ａ及びＢ．グラジエント：１００％移動相〜１００％移動相Ｂの３７．５分の洗浄グラジエント（７．５分の平衡化遅延を伴なう）。
【０２７６】
ドキソルビシンピークの量が０．１％以下である６時間で、反応混合物を、粗ガラス濾過を通して濾過し、ビーズを除いた。３．５ｌの水中、１．１ｋｇのＮａＣｌのブライン溶液（少なくとも３０％ｗ／ｖ）を調製し、そして０℃に冷却した。次に、濾過された反応混合物を、冷却されたブライン溶液に撹拌しながら４５分間にわたってゆっくりと添加した。溶液の色彩は赤のままであり、青色の溶液は、ｐＨが酢酸の添加により５．８〜６．０にすぐに調節する必要があることを示した。赤色の沈殿物を中位のガラス濾過器を通して濾過し、水により洗浄し、そしてＰ_２Ｏ_５上で減圧下で乾燥し、いずれの残留ドキソルビシンも有さないＭｅＯＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘを得た。
【０２７７】
ＭｅＯＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘを、ＩＬのＭｅＯＨに溶解し、そして次に、メタノール溶液を、撹拌しながら、６０分間にわたって、１４Ｌの冷却されたエチルエーテルにゆっくりと添加した。赤色の沈殿物を、中位のガラス濾過器を通して濾過し、エーテル（１Ｌ）により洗浄し、そして真空下で乾燥し、１１０ｇのＭｅＯＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘを得た。純度は、例４４に記載のようにして、ＨＰＬＣにより９６．５％であることが決定された。Ｃ_５０Ｈ_６７Ｎ_５Ｏ_１８についてのＭＳｍ／ｚ計算値：１０２５；実測値：１０４８（Ｍ^＋＋Ｎａ）。
【０２７８】
例 ５１．Ｓｕｃ −β Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ を生成するために ＭｅＯＳｕｃ −β Ａｌａ − Ｌｅｕ − Ａｌａ − Ｌｅｕ − Ｄｏｘ の酵素的加水分解
ＣＬＥＣ−ＣＡＢ（Ｃａｎｄｉｄａ Ａｎｔａｒｔｉｃａ “Ｂ” リパーゼ）酵素を、溶液形で購入し（Ａｌｔｕｓ Ｂｉｏｌｏｇｉｃｓ．， Ｂｏｓｔｏｎ， ＭＡから）、ここで酵素の濃度は溶液１ｍｌ当たりの乾燥酵素の重量により定義される。粗酵素懸濁液を数分間、振盪し、均質溶液を得た。５０４ｍｌ（３２９ｍモル）のこの均質溶液をフラスコにアリコートした。２．５Ｌの脱イオン水を添加し、そしてこのスラリーを、磁気撹拌機を用いて１０分間、撹拌した。酵素溶液を粗ガラス濾過器を用いて、酵素を乾燥しないで濾過した。酵素をフラスコに戻した。酵素を水に懸濁し、そしてさらに３度、濾過した。
【０２７９】
酵素ケークを、５５０ｍｌの脱イオン水に再懸濁し、そしてＲＢフラスコに移した。この懸濁液に、１０９ｇのＭｅＯＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ（１０５ｍモル）を添加し、そしてその反応混合物を室温（２５℃）で撹拌した。反応混合物のｐＨを、１ＮのＮａＨＣＯ_３溶液により充填された注射器ポンプを備えたｐＨ−装置により、５．８〜６．１に維持した。反応の進行を、例４４に記載のようにして、定期的なＨＰＬＣモニターにより追跡した。２４時間後、反応は、ＨＰＬＣにより決定される場合、９４％の完結性であると思われる。
【０２８０】
反応を早めるために、追加のＣＬＥＣ酵素を、２４時間後、必要とした。１６８ｍｌのＣＬＥＣ酵素（均質溶液）を、上記のようにカラム型で洗浄した。酵素ケークを、１．１Ｌの脱イオン水に再懸濁し、そして反応金剛物に添加した。反応混合物を、定義的にＨＰＬＣによりモニターしながら、室温で撹拌し、そしてｐＨを、５．８〜６．１に維持した。６０時間後、反応は、ＨＰＬＣによりモニターされる場合、９９．９％の完結性であった。
【０２８１】
ＣＬＥＣ酵素を、０．２μＭのフィルターを通しての濾過により反応混合物から除去し、そして５００ｍｌの脱イオンによりすすいだ。次に、濾過を凍結乾燥し、９５．２ｇのＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ・Ｎａを得た（８７％の物理的収率；Ｃ_４９Ｈ_６５Ｎ_５Ｏ_１８についてのＭＳｍ／ｚ計算値：１０１１；実測値：（Ｍ^＋＋Ｎａ）。
プロドラッグ化合物Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘを、質量スペクトル分析、ＦＴＩＲ、ＮＭＲにより十分に特徴づけた。
Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘの質量スペクトル分析は、Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ（Ｃ_４９Ｈ_６５Ｎ_５Ｏ_１８Ｎａ）についての計算されたｍ／ｚ（１０３３）と適合する、分子イオンピーク（ｍ／ｚ；１０３４）の存在を明確に示す。
【０２８２】
Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘのサンプルをまた、ＦＴＩＲにより分析した。そのスペクトルは、上記材料の対照標準のスペクトルと適合した。主要吸収率についての割り当ては、次の通りである：
ヒドロキシル　　　　３３７９ｃｍ^−１
Ｃ−Ｈ　　　　　　　　３０００−２７００
カルボニル　　　　１６５０−１７２５
【０２８３】
最終的に、サンプルをＮＭＲにより分析した。化学シフト及び割り当ては、表１３に列挙され、そして図１７に示される。Ｓｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘの構造と一致する、２１５．２， １８７．７， １８７．４ｐｐｍでケトン領域に３個の炭素が存在する。後者の２つは、類似する化学シフトを有し、その結果、１８７．７及び１８７．４ｐｐｍで、（２）及び（３）に割り当てられる。従って、残るケトンは（１）である。それらの割り当てからのさらなる証拠は、ＨＭＱＣ及びＨＭＢＣスペクトルから発生し；それらの３個はいずれのＨＭＱＣピークも示さず、その結果、陽子化されず、そして（１）のみが、４．７７ｐｐｍでのプロトンに対して、広範囲のＣ−Ｈカップリング（ＨＭＢＣ）を有し、これはプロトンシングレットであり、従ってこれが（４）である。ＨＭＱＣスペクトルから、６５．７ｐｐｍでの炭素がそれらのプロトンに連結される。
【０２８４】
３．９６ｐｐｍでの^１Ｈ ＮＭＲシグナルは化学シフト及びメトキシ基の領域を有し；それらのプトロンは５７．２ｐｐｍで炭素にカップリングされ、そして構造（５）における唯一のメトキシに割り当てられる。^１３Ｃ化学シフトはまた、メトキシと一致する。プロトンの広範囲Ｃ−Ｈカップリングは、（６）であるべきである、１６２．３ｐｐｍでの炭素に対してである。
【０２８５】
ＨＭＱＣスペクトルは、１２０．５、１２０．３及び１３７．２ｐｐｍで３種の陽子化された芳香族が存在することを示し；前記芳香族プロトンは、いずれの広範囲のＣ−Ｈカップリングも、隣接するプロトン間でのいずれのカップリングも示さなかった。芳香族プロトンシグナルは非常に広く、このことは、いずれかの観察されるカップリングの欠失を説明する、短いＴ_２緩和時間を示す。このカップリングの欠失が存在する場合、それらの３種の部位をユニークに割り当てることは不可能でありそして集合的に、（７）（８）及び（９）に割り当てられる。
【０２８６】
１５７．２及び１５６．１ｐｐｍでの２種の陽子化されていない芳香族炭素は、（１０）及び（１１）と一致する化学シフト、すなわち酸素に結合される芳香族炭素を有する。広範囲のカップリングは観察されない。
１１２．３及び１１２．０ｐｐｍでの^１３Ｃ ＮＭＲシグナルは、芳香族炭素と一致し、そして（２）及び（１３）に割り当てられる。１２１．３での^１３Ｃ ＮＭＲシグナルもまた、この効果を示し、その結果、（１４）に割り当てられる。残る３種の陽子化されていない芳香族炭素は、その領域において、（１５）、（１６）及び（１７）の最後の３種の炭素に割り当てられる。
【０２８７】
いずれかの芳香族炭素へのいずれかの広範囲のＣ−Ｈカップリングの欠失は、予測されず、そしてカップリングが、短いＴ_２緩和時間又は平面形状のために、非常に小さいか又は存在しないことを示す。
１８１〜１７４ｐｐｍで６種のカルボニル炭素が存在し；それらのうち、１８０．６ｐｐｍでの１つが、ナトリウム塩と一致する化学シフトを有する唯一の１つであり、その結果、（１８）に割り当てられる。このピークは、解決されていない、２．４ｐｐｍでのプロトンへの広範囲のＣ−Ｈカップリングを示す。残る５種のカルボニル炭素は、すべて１ｐｐｍ化学シフト範囲内に存在し、そして可能な（１９）、（２０）、（２１）、（２２）、（２３）ではない。
【０２８８】
１０２．３ｐｐｍでの炭素は、２種の別々の酸素に結合される炭素と一致する化学シフトを有し、これは、（２５）に割り当てられる。このプロトンは、３０．６ｐｐｍで炭素にカップリングされる。Ｃ−Ｏ領域（８０〜６０ｐｐｍ）における炭素のうち、１つのみが７７．４ｐｐｍで陽子化され、その結果、（２６）であるべきである。これは、プロトン又は炭素のいずれかへの広範囲のＣ−Ｈカップリングを有さない。
【０２８９】
すべてのメチンが酵素に結合されている、８０〜６０ｐｐｍにおいて、まだ割り当てられていない３個の炭素が存在する。それらはすべて、類似する化学シフト（７１．２、６９．９及び６８．８ｐｐｍ）を有するが、しかし６８．８ｐｐｍでの炭素が、（２８）でのメチルへの広範囲のカップリングを示すことは明白であると思われる。６９．９での炭素はまた、（２８）でのメチルへの広範囲のカップリングを示し、その結果、（２７）に隣接すべきであり、そして（２９）に割り当てられる。従って、残るメチンは（３０）である。
【０２９０】
３．６ｐｐｍ（２９）でのプロトンは、４７．２ｐｐｍで、１つの炭素のみへの広範囲のＣ−Ｈカップリングを示す。唯一の隣接する割り当てられていない炭素は（３１）であるべきである。（３１）のプロトンは、他のプロトンをオーバーラップし、そして広範囲の相互関係は不可能である。
残る４個のメチルは、イソプロピル領域に存在し、そして１つは１．２５／１．３４ｐｐｍで存在し、そして最後の残るメチル（３２）に対応するべきである。このメチルプロトンは、（３３）であるべきである。５１．３ｐｐｍでの１つの炭素のみへの広範囲のカップリングを示す。（３３）のプトロンは、他のプロトンと厳格にオーバーラップし、そしていずれかの広範囲の相互関係のためには使用され得ない。
【０２９１】
残る４個のメチルはすべて、イソプロピルメチル、すなわち集合的にラベルされた（３４）から生じるべきである。（３４）のプロトンは、対合されたメチル間での、及び２５．９／２５．８及び４１．６／４１．７ｐｐｍでの炭素に対して広範囲のカップリングを示し；メチンは、（３５）（３６）に割り当てられ、そしてメチレンは（３７）（３８）に割り当てられる。それらのプロトンのすべては、１．５〜１．８ｐｐｍでオーバーラップするが、しかし５４．７／５３．５ｐｐｍでのメチニンへの広範囲のカップリングを示し、これはアミドに隣接するメチニンであり、そして（３９）（４０）に割り当てられる。
【０２９２】
すべてメチレンである残る５個の炭素は、カルボニルへの広範囲のカップリングを示し、その結果、そのようなカルボニルに隣接すべきであり、そして（４１）、（４２）及び（４３）に割り当てられ；^１Ｈ ＮＭＲ化学シフトはすべて、２．４ｐｐｍでオーバーラップし、そして３７．２、３４．４及び３３．８ｐｐｍで炭素に対応する。３７．２での炭素がほとんどの差異であるので、それはナトリウム塩カルボニル（４１）に割り当てられ、そして他の２つは、アミドカルボニル（４２）、（４３）に割り当てられる。残る２つのメチレンは、特定の割り当て（４４）、（４５）のためには類似すぎる。
割り当てられない１つの部位が存在する。この割り当てられた部位を包含する、構造と一致する、５．５〜１．５ｐｐｍ領域に３０のプロトンが存在する。従って、炭素シグナルは、窒素に隣接するメチレンに関して一致する、約５０ｐｐｍでの溶媒シグナル下に隠されていると思われる。
【０２９３】
【表１２】

【０２９４】
本明細書に言及されるすべての出版物及び特許出願は、個々の出版物又は特許出願が特異的に且つ個々に引用により組み込まれていることを示されているかのように、同じ程度に引用により本明細書に組み込まれる。
本発明は現在、十分に記載されているが、多くの変更及び修飾が本発明の範囲内で行われ得ることは、当業者に明らかであろう。
【図面の簡単な説明】
【図１】
図１Ａ−１Ｄは、略語、名称及び構造の表である。
【図２】
図２は、標的細胞の細胞外付近及び標的細胞内での本発明のプロドラッグの分解の典型的なスキームである。
【図３】
図３は、本発明の典型的な中間体であるＦｍｏｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕの合成を示す。
【図４】
図４は、本発明の典型的な中間体であるメチル−スクシニル−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕの“Ｆｍｏｃ−経路”合成を示す。
【図５】
図５は、本発明の典型的な化合物であるＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘの塩形の“Ｆｍｏｃ−経路”合成を示す。
【図６】
図６は、本発明の典型的な化合物であるＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘの塩形の“エステル−経路”合成を示す。
【図７】
図７は、本発明の典型的な中間体であるアミノ保護されたβＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘの合成を示す。
【図８】
図８は、本発明の典型的な化学であるＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘの塩形の“アリルエステル経路”を示す。
【図９】
図９は、本発明の典型的な化合物であるＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘの“樹脂経路”を示す。
【図１０】
図１０Ａ−１０Ｃは、本発明のプロドラッグにおいて有用なオリゴペプチドの表である。
【図１１】
図１１は、薬剤と共に又は薬剤を伴わないでビークルを与えられる動物についてのマウス異種移植片モデルにおける生存性のグラフである。
【図１２】
図１２は、ドキソルビシンプロドラッグ及びドキソルビシンを比較するマウス異種移植片モデルにおける生存性のグラフである。
【図１３】
図１３は、ＤＴＴの濃度を高めることによるＨｅＬａ細胞トロウアーゼの活性化及び次に阻害のグラフである。
【図１４】
図１４は、スキャベンジング樹脂又はビーズの使用を通しての遊離治療剤の除去を示す。
【図１５】
図１５は、マウス異種移植片モデルにおける腫瘍増殖阻害のグラフである。
【図１６】
図１６は、本発明の典型的な中間体であるＭｅＯＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘの大規模合成を示す。
【図１７】
図１７は、本発明の典型的な化合物であるＭｅＯＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−ＤｏｘについてのＮＭＲ割り当てを示す。
【図１８】
図１８は、雌ヌードマウスにおけるＭＸ−１ヒト乳癌腫瘍の増殖に対してのＳｕｃ−βＡｌａ−Ｌｅｕ−Ａｌａ−Ｌｅｕ−Ｄｏｘ及びドキソルビシン化合物、及びビークルの効果の比較を示す。[0001]
Introduction:
Technical field:
The present invention relates to novel compounds useful as prodrugs. Such prodrugs can be used for the treatment of a disease, especially a tumor, in a patient.
[0002]
background:
Many therapeutic agents, such as anthracyclines and vinca alkaloids, are particularly effective for treating cancer. However, these molecules are often characterized in vivo by acute toxicity, especially bone marrow and mucosal toxicity, and chronic cardiotoxicity in the case of anthracyclines and chronic neurological toxicity in the case of vinca alkaloids. Similarly, methotrexate can be used for the treatment of inflammatory reactions, such as rheumatic patients, but its high toxicity limits its application. The development of more specific and safe anti-tumor agents is desired due to the high efficacy against tumor cells and the reduced number and severity of side effects (toxicity, destruction of non-tumor cells, etc.) of their products Is done. The development of more specifically anti-inflammatory agents is also desirable.
[0003]
To minimize toxicity problems, the therapeutic agent is conveniently provided to the patient in the form of a prodrug. Prodrugs are molecules that can be converted in vivo into drugs (active therapeutic compounds) by chemical or enzymatic modification of their structure. In order to reduce toxicity, this conversion should be restricted to the site of action or target tissue rather than the circulatory system or non-target tissue. However, prodrugs often have low stability in blood and serum because blood and serum contain enzymes that degrade or activate the prodrug before it reaches the desired site in the patient's body. It is characterized by
[0004]
Desirable types of prodrugs that overcome such problems are disclosed in the Patent Cooperation Treaty International Publication No. WO 96/05863 and US Pat. No. 5,962,216, which are incorporated herein by reference. I have. However, more useful prodrug compounds and methods of making such prodrugs are desired.
Prodrugs that exhibit high specificity of action, reduced toxicity and improved stability in blood are particularly desirable for prodrugs of similar structure (especially the closest structure) that exist in the absence of rights. .
[0005]
Summary of the Invention:
The compounds of the invention are prodrug forms of a therapeutic agent conjugated directly or indirectly to an oligopeptide that is linked to a stabilizing group. The compound can be degraded by enzymes bound by the target cells.
[0006]
More generally, the present invention relates to novel prodrug compounds of therapeutic agents, in particular improved therapeutic properties over the products of the prior art, in particular the treatment of cancerous tumors and / or inflammatory reactions, such as rheumatic diseases Can be described as prodrugs comprising anti-tumor therapeutics that exhibit improved therapeutic properties in the treatment of Improved therapeutic properties include reduced toxicity and increased efficacy. Particularly desirable are prodrugs that exhibit high specificity of action in serum and blood, reduced toxicity, improved stability, and that do not migrate into target cells until activated by the target cell binding enzyme. Prodrug chemistry of markers that allow characterization of tumors (diagnosis, tumor progression, assays for factors secreted by tumor cells, etc.) are also designed. Accordingly, the present invention includes diagnostic or assay kits using the compounds of the present invention.
[0007]
The present invention also relates to a pharmaceutical composition comprising a compound of the present invention and, optionally, a pharmaceutically acceptable carrier, adjuvant, vehicle or the like.
In addition, methods are disclosed for reducing toxicity and improving safety index by modifying therapeutic agents to create prodrugs. Other aspects of the invention include methods of designing prodrugs for administration to a patient, and methods of treating a patient by administering a therapeutic dose of a compound.
Several methods for creating the prodrugs of the present invention are also described.
[0008]
Specific statement:
Abbreviations:
ACN = acetonitrile; Aib = aminoisobutyric acid; All = allyl; Aloc = allyloxycarbonyl; Amb = 4- (aminomethyl) benzoic acid; AAP = 3-amino-3-phenylpropionic acid; DCC = N, N′-dicyclohexyl Carbodiimide; Boc = t-butyloxycarbonyl; Cap = aminocaproic acid; Cou = aminomethylcoumarin; DBN = 1,5-diazabicyclo [4. 3. 0] non-5-ene; DBO = 1,4-diazabicyclo [2. 2. 2] octane; DBU = 1,8-diazabicyclo [5. 4. 0] undec-7-ene; DCM = dichloromethane; DIC = N, N'-diisopropylcarbodiimide; DIEA = diisopropylethyleneamine; Dg = diglycolic acid; DMF = dimethylformamide;
[0009]
Dnr = Daunorubicin; Dox = Doxorubicin; Dox-HCL = Doxorubicin hydrochloride; Et₂O = diethyl ether; Fmoc = 9-fluorenylmethyloxycarbonyl; Gl = glutaric acid; OSU = N-hydroxysuccinimide; HATU = O- (7-azabenzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate; HBTU = 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate; HEPES = hydroxyethylpiperidine; HOBt = N -Hydroxybenzotriazole; HPLC = high pressure liquid chromatography; MeOH = methanol; MeOSuc = methylhemisuccinate / methylhemisuccinyl; MDR = multidrug resistance; MTD = maximally tolerated dose; NAA = 3-amino-4,4-diphenyl. Butyric acid;
[0010]
NaI = 2-naphthylalanine; Naph = 1,8-naphthalenedicarboxylic acid; NIe = norleucine; NMP = N-methylpyrrolidine; Nva = norvaline; PAM resin = 4-hydroxymethylphenylacetamidomethyl; Phg = phenylglycine; Pyg = Pyrglutamic acid; Pyr = 3-pyridylalanine; rRTOP = recombinant rat TOP; RT, rt = room temperature; SD-MTD = single dose-maximum allowed dose; SD = single dose; RD-MTD = repeated dose-maximum allowed Dose; RD = repeated dose; Suc = succinic / succinyl; TCE = trichloroethyl; TFA = trifluoroacetic acid; THF = tetrahydrofuran; Thi = 2-thienylalanine; Thz = thiazolidine-4-carboxylic acid; Tic = tetrahydroisoquinoline- 3-Carbon ; TOP = Chimetto (Timet) oligo peptidase.
[0011]
The invention includes compounds that may be described as prodrug forms of a therapeutic agent. Each of these therapeutic agents is modified by direct or indirect attachment to an oligopeptide attached to a stabilizing group. The prodrug and, in particular, the oligopeptide portion of the prodrug can be degraded by enzymes bound by the target cells. The enzyme is preferably a troase, and more preferably a thimeto oligopeptidase.
[0012]
Prodrugs:
A prodrug of the invention is a modified form of a therapeutic agent, which comprises several moieties, for example,
(1) therapeutic agents,
(2) an oligopeptide, and
(3) a stabilizing group, and
(4) optionally comprising a linker group.
Each of the prodrug moieties is discussed in more detail below. Typical orientations of those portions of the prodrug are as follows:
[0013]
(Stabilizing group)-(oligopeptide)-(optional linker group)-(therapeutic agent).
The stabilizing group is directly linked to the oligopeptide at the first binding site of the oligopeptide. The oligopeptide is linked directly or indirectly to the therapeutic agent at the second binding site of the oligopeptide. Where the oligopeptide and the therapeutic agent are indirectly linked, a linker group is present.
[0014]
A direct bond between two parts of a prodrug means a covalent bond that exists between the two parts. Thus, the stabilizing group and the oligopeptide are strongly attached directly through a covalent chemical bond at the first attachment site of the oligopeptide, typically at the N-terminus of the oligopeptide. When the oligopeptide and the therapeutic agent are directly linked, they are covalently linked to one another at the second binding site of the oligopeptide. The second binding site of the oligopeptide is typically at the C-terminus of the oligopeptide, but can be at other positions on the oligopeptide.
[0015]
Indirect association of the two parts of the prodrug means that each of the two parts is covalently linked to a linker group. In other embodiments, the prodrug has indirect binding of the oligopeptide to the therapeutic. Thus, typically, the oligopeptide is covalently linked to a linker group that is covalently linked to the therapeutic agent.
[0016]
A prodrug of the invention can be degraded within its oligopeptide moiety. Prodrugs typically undergo in vivo denaturation, and the active site of the prodrug, the transport-component moiety, enters the target cell. A first degradation within the oligopeptide portion of the prodrug can leave the transport component portion of the prodrug as one of the degradation products. On the other hand, further degradation by one or more peptidases is required to provide some of the prodrug that can enter cells. The active or transport component portion of the prodrug is at least a portion of the prodrug that has a therapeutic agent and can enter the target cell to exert a therapeutic effect either directly or based on further transformation within the target cell. It is. Thus, the compound has an active moiety, and the active moiety can enter the target cell after degradation by the enzyme bound by the target cell rather than before degradation by the enzyme bound by the target cell. .
[0017]
The stabilizing groups and the structure of the oligopeptide are selected to limit the clearance and metabolism of the prodrug by enzymes that can be present in blood or non-target tissues, and further, to limit the transport of the prodrug into cells. You. Stabilizing groups prevent prodrug degradation and can be used in providing the prodrug's favorable charge or other physical properties. The amino acid sequence of the oligopeptide is designed to ensure that the enzyme bound by the target cell, more particularly the troidase enzyme, and even more particularly, the thimet oligopeptidase, is specifically degraded by ("TOP"). You.
[0018]
It is desirable to produce a therapeutic agent, particularly an anti-tumor and / or anti-inflammatory therapeutic agent, that is inactivated by denaturation of the therapeutic agent into a prodrug form. According to the invention, the target cells are usually tumor cells or cells that precipitate in an inflammatory response, especially those cells associated with rheumatic diseases, such as macrophages, neutrophils and monocytes. Denaturation of the therapeutic into the prodrug form somewhat reduces the side effects of the therapeutic. Modification of the therapeutic agent to the prodrug form further allows administration of an increased dose of the therapeutic agent in the prodrug form to the patient relative to the dose of the therapeutic agent in the unconjugated form.
[0019]
In the target cell, the therapeutic agent (optionally attached to one or two amino acids and possibly also a linker group) acts directly on that particular intracellular site of action, or After denaturation under the action of the peptidase, the target cell is killed or its growth is arrested. Since normal cells release little or no TOP in vivo, the compounds of the present invention remain inactive and do not enter normal cells or enter relatively small amounts. Although TOP appears to be widely distributed in the body, it typically exists as an intracellular enzyme. Therefore, it is generally inaccessible to peptide prodrugs in the circulation. In a tumor environment, TOP appears to be released from necrotic tissue.
[0020]
The prodrug is administered to the patient, carried in a stable form through the bloodstream, and is affected by TOP when near the target cells. Because the enzymatic activity is minimally in the extracellular vicinity of normal cells, the prodrug is maintained, and its active portion (including the therapeutic agent) minimally enters normal cells. However, near the tumor or other target cells, the presence of TOP in the local environment causes prodrug degradation. Once the stabilizing group is removed, additional amino acids can be removed by other peptidases near the target cell. The example shown in FIG. 2 shows an N-capped tetrapeptide prodrug that is extracellularly degraded and enters the target cell. Within the target cell, it can be further modified to provide a therapeutic effect, for example, by killing the target cell or inhibiting its growth. The active site of the prodrug penetrates into normal cells to some extent, but its active part is released from the rest of the prodrug mainly near the target cell. Thus, toxicity to normal cells is minimized.
[0021]
This method is particularly useful for, and designed for, target cell destruction, where the target tissue releases enzymes that are not released by normal cells or tissue. As used herein, “normal cells” means non-target cells that are encountered by a prodrug upon administration of the prodrug, in a manner appropriate for its intended use. Normal (i.e., non-specific) cells may have little or no target cell enzyme, e.g., TOP, responsible for breaking the bond that binds the active portion of the prodrug (e.g., therapeutic agent) from the rest of the prodrug in vivo. As it does not form at all, the compounds of the present invention remain inactive and do not enter normal cells.
[0022]
In other embodiments, the orientation of the prodrug is reversed so that the stabilizing group is attached to the C-terminus of the oligopeptide and the therapeutic agent is directly or indirectly attached to the N-terminus of the oligopeptide. Be combined. Thus, in other embodiments, the first binding site of the oligopeptide may be at the C-terminus of the oligopeptide, and the second binding site of the oligopeptide may be at the N-terminus of the oligopeptide. A linker group can optionally be present between the therapeutic agent and the oligopeptide. Another embodiment of the prodrug of the present invention functions in the same manner as the primary embodiment.
[0023]
Target cell binding enzyme:
Prodrugs of the present invention are designed to take advantage of selective activation through interaction with enzymes bound by target cells at or near a targeted site in the patient's body. One such type of enzyme is troase, described in more detail in PCT / US99 / 30393, which is incorporated herein by reference.
[0024]
Troases are a type of enzyme that are thought to activate prodrugs in target tissues. Troases are a class of endopeptidases that exhibit a significant degree of distinction between leucine and isoleucine on the carboxy side of the oligopeptide degradation site. Under the appropriate assay conditions, Troase readily degrades Suc-βAla-Leu-Ala-Leu-Dnr, but it has at least a 20-fold lower activity than Suc-βAla-Ile-Ala-Leu-Dnr. It is characterized by having. TOP is a member of the Troase class of enzymes.
[0025]
The target cells appear to release the troase. Maybe the enzyme is produced by the target cell or by a normal cell bound by the target cell, for example, a stromal cell, neutrophil, macrophage or B cell. The target cell binding enzyme is bound by the extracellular surface or is bound on its surface (at least the active site), secreted, released, or otherwise present near the extracellular area of the target cell. Can be. As a result, the troase may be secreted near the extracellular vicinity of the target cell or may be otherwise present near it. In many cases, the prodrugs of the invention will include a therapeutic for the treatment of cancer, and the target cells will be tumor cells. Thus, the troase can be secreted extracellularly by the tumor cells, or it can be extracellular, for example, because there is a significant amount of cell lysate that is generally bound by the tumor. The cell lysate is also bound by inflammatory tissue, another target cell site.
[0026]
However, troase activity is low in human plasma. TroWase activity has been observed in cancer cell extracts and in conditioned media from cultured cancer cells, red blood cells and various human tissues, especially kidneys. The partial purification scheme of troase from ultracentrifugation (145,000 ×, 30 min) supernatant of HeLa (cervical cancer) cell homogenate consists of four steps as follows:
Anion exchange chromatography using a 15Q column (Pharmacia) dissolved by a 0-0.5 M NaCl linear gradient in 1.20 mM triethylamine chloride, pH 7.2, 0.01% Triton X-100;
[0027]
2. CoCl₂And 10 mM sodium phosphate, 0.5 M NaCl, pH 7.2, 0.01% Triton X-100, 0.02% NaN₃Affinity chromatography using Chelating Sepharose Fast Flow (Pharmacia), in which a linear gradient of 0-200 mM imidazole is dissolved;
3. Separation electrophoresis; and
4. Gel filtration high performance liquid using 7.8 mm x 60 cm TSK Gel G-3000SWXL (TosoHaas) eluted with 50 mM potassium phosphate, 200 mM potassium sulfate (pH 7.0) at 0.3 ml / min. Chromatography.
[0028]
Further degradation of some of the prodrugs that are released after trolytic degradation may occur intracellularly or extracellularly, probably due to amino-exopeptidases. In vitro experiments indicate that a broad specificity of amino-exopeptidase is present in the human blood and cancer cell environment.
[0029]
Evidence indicates that TOP is an example of troase. Troases isolated from HeLa cell extracts and studied in conditioned media or homogenates from MCF-7 / 6 human cancer cells catalyze the early degradation of Suc-βAla-Leu-Ala-Leu-Dox. . Trousers isolated from those sources appear to be TOP. Structural and functional evidence indicates that the troase found in cancer cells is thimet oligopeptidase or "TOP".
[0030]
According to the literature, TOP or EC3. 4. 24. 15 is an ethyl-activated zinc metallopeptidase that catalyzes the internal (endo) degradation of various oligopeptides having 6 to 17 amino acids (Dando, et al., "Human thimegolipodeptidase", Biochem. 294: 451-457 (1993)). It is also referred to as Pz-peptidase, collagenase-like peptidase, kinase A, amyloidin protease, and metalloendopeptidase.
[0031]
The enzyme is a chicken embryo (Morales, et al., “PZ-peptidase from chicken embryos. Purification, properties, and action on collagen peptides”, J. Biol. 25-Ch. 48-Chicken. Barrett, et al., "Cicken river PZ-peptide, a thiol-dependant metallo-endopeptidase", Biochem. J. 271: 701-706 (1990), Rat testis (O.d. tests.Isolation of the Enzyme a nd its specificity toward synthetic and natural peptides, including enkephalin-containing peptides. ", Biochem J. 261:.. 951-958 (1989)), and human erythrocytes (Dando, such as," Human thimet oligopeptidase ", Biochem J. 294 : 451-457 (1993)).
[0032]
The gene for this enzyme has been cloned, and the DNA sequence has been cloned into human brain (Dovey et al., WO 92/07068), rat testis (Pierotti et al., "Endopeptidase-24.15 in rat hypothalamic / pitadialy / gonadarax." . ", Mol Cell Endocrinol 76:.... 95-103 (1991)), and pig liver (Kato such as," Cloning, amino acid sequence and tissue distribution of porcine thimet oligopeptidase A comparison with soluble angiotensin-binding protein ", Eur. J. Bi chem 221:. was obtained from 159-165 (1994)).
[0033]
TOP is described in HeLa (Krause et al., "Characterization and localization of mitochondrial alipopeptidase (Mop)" (EC 3.4.24.elco.com. AT-20 cells (Crack et al., "The association of metalloendopeptidase EC 3.4.24.15 at the extracellular surface of the radiation vector. 35:.. 113-124 (1999); Ferro, such as, "Secretion of metalloendopeptidase 24. 15 (EC 3. 4. 24. 15)", DNA Cell Biol 18: 781-789 (1999);
[0034]
Garrido et al. , "Confocal microscopy reviews thim oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) in the classical digital creator. Biol. 18: 323-331 (1999)); Madin-Darby canine kidney cells (Oliveira et al., "Characterization of thiol-, aspartyl-, and thiol-metal-all-in-one-department of drug-in-derivatives initiative-in-departure-in-department-industry-industry-industry-in-de-certification-in-department-in-de-certification-in-department. Biochem. 76: 478-488 (2000)); and prostate cancer cell lines (such as, "Neurotensin is metabolized by endogenous proteases in prostate cancer cells", Extracts 25, Peptides 25, p. Biologically or sensoryly identified You.
[0035]
Troase purified from HeLa cells, as seen in Example 12, has a mass: charge ratio of the trypsin fragment that fills 33% of all residues distributed over the full length of the known human enzyme sequence. Based on human TOP. Immunoprecipitation using a specific anti-TOP antibody preparation with partially purified HeLa cell fraction (F1) and MCF-7 / 6 cell homogenate was also performed as described in Example 13. Indicates identity. The size of the 74 KD purified HeLa cell troase by SDS polyacrylamide gel electrophoresis is in the range reported for TOP.
[0036]
The minor 63KD band co-purifying from HeLa cells has not been previously reported and may be a proteolytic product of TOP formed during extraction. As seen in Example 9, the gel filtration estimated size of native HeLa cell troWase is 68 KD, rather than the reported TOP size of 78 KD; however, the difference is such native protein sizing. It can be explained by the inherent error of the method. The isoelectric point of the purified cancer cell troWase is 5.2 found in Example 9, Which is in the range reported for TOP.
[0037]
TOP and human cancer cell troase show the same substrate specificity as the nine different experimental compounds, as seen in Example 6. This specificity includes the ability to degrade Suc-βAla-Leu-Ala-Leu-Dox at a rate about 12 times faster than that of Suc-βAla-Ile-Ala-Leu-Dox. Said cancer cell troWase also has substantially the same pH optimum (see Example 11) and inhibitor profile (see Example 8) as TOP.
[0038]
Regarding TOP (Barrett et al., “Cicken river PZ-peptide, a thiol-dependent metall-endopeptidase”, Biochem. J. 271: 701-706 (1997)), cancer cell metabolic inhibitors and cancer trousers, Inhibited by 10-phenanthroline, but not by serine, thiol, or acid proteinase inhibitors such as aminoethylbenzene-sulfonate, E64, pepstatin, leupeptin, aprotinin, CA074 or fumagillin. As reported for TOP, EDTA-treated cancer cell troases were²⁺(50-100 μM) or Mn²⁺(50-100 μM).
[0039]
EDTA-inactivated chickens (Barrett et al., "Cicken river PZ-peptide, a thiol-dependent metallo-endopeptidase", Biochem. J. 271: 701-706 (1990), or rat (1990), or rat. ". Endopeptidase 24. 15 from rat testes Isolation of the enzyme and its specificity toward synthetic and natural peptides, including enkephalin-containing peptides", Biochem J. 261:. 951-958 (1989)) can be re-activate also It is a function.
[0040]
TOP is also Zn²⁺But is reactivated by Zn²⁺Reactivation is not seen for EDTA-treated MCF-7 / 6 cell homogenates. The particular method used for EDTA treatment and removal can influence the outcome with cancer cell troases. Zn at a concentration as low as 100 μM²⁺The fact that is inhibitory for TOP can also be a factor. Zn at 100 μM²⁺Completely inhibits the hydrolysis of Suc-βAla-Leu-Ala-Leu-Dox by Hela cell fraction 1. Cancer cell troase inactivated by EDTA cannot be reactivated by cupric ions. Finally, human cultured cell troWase exhibits the unique thiol sensitivity of TOP metallo endopeptidase, as seen in Example 10.
[0041]
TOP activity is inhibited in enzymatically added solutions (eg, blood) and activated in a light reducing (hypoxic) environment, as indicated by thiol activation of the air-inactivated preparation. (Shripton et al., "Thiolactation of endopeptidase EC 3.4.24.15. A novel mechanism for the regulation of catalytic activity", 19-27. Therefore, it is a useful enzyme for a general approach to designing prodrugs that are activated in a hypoxic environment, such as tumor tissue.
[0042]
CD10 (CALLA, neprilysin, neutral endopeptidase, EC 3.4.24.11) is an oligopeptidase that is bound to the outer cell membrane of many cells, including a limited number of cancer tumor types. It also contributes to the systemic activation of peptidyl prodrugs, as it is present at high concentrations at the brush border of the proximal renal tubules and at low levels in colon tissue and many immune system cells, such as B-lymphocytes. be able to. This conferred systemic activation could lead to enhanced toxin in normal tissues when compared to peptidyl prodrugs that were not CD10 substrates. Su-βAla-Leu-Ala-Leu-Dox is a substrate for CD10, and as shown in Example 17, degradation occurs between Ala and Leu (AA2 and AA1).
[0043]
CD10 is poorly degraded when glycine or alanine is present at the P1 ′ degradation site (Pozgay et al., Biochemistry (1986)) [see CD10 Patent under Substrate Specificity], and therefore Suc- βAla-Leu-Ala-Gly-Dox and βAla-Leu-Ala-Ala-Dox are predicted to be poorly degraded by CD10. On the other hand, as shown below, Suc-βAla-Leu-Ala-Gly-Dox and probably Suc-βAla-Leu-Ala-Ala-Dox were well degraded by TOP. Thus, a preferred embodiment of the present invention is activated by TOP rather than by CD10, as exemplified by Suc-βAla-Leu-Ala-Gly-Dox or Suc-βAla-Leu-Ala-Ala-Dox. Compound.
Ideally, when treating non-CD10 containing tumors, the prodrug cannot be degraded by the CD10 enzyme.
[0044]
Stabilizing group:
An important part of the prodrug is that when the prodrug is administered to a patient, it acts to protect the prodrug compound from degradation in the circulating blood and to reach the prodrug near the relatively intact target cells. Is a stabilizing group that enables Stabilizing groups typically protect the prodrug from degradation by proteinases and peptidases present in blood, serum and normal tissues. In particular, because the stabilizing group caps the N-terminus of the oligopeptide, and thus is sometimes referred to as an N-cap or N-block, it, on the other hand, protects against the peptidases to which the prodrug is sensitive Act to
[0045]
Ideally, the stabilizing group acts to protect the prodrug from denaturation, ie, degradation, when tested by storage of the prodrug compound in human blood at 37 ° C. for 2 hours, and Useful in the prodrugs of the present invention if they provide for the degradation of the prodrug by enzymes present in human blood under the assay conditions to be less than 20%, preferably less than 2%.
[0046]
More particularly, the stabilizing group is
(1) It is other than an amino acid, or
(2) (i) a non-genetically encoded amino acid having 4 or more carbon atoms, or (ii) a β-carboxyl group of aspartic acid or a γ-carboxyl group of glutamic acid, An amino acid that is aspartic or glutamic acid attached to the N-terminus.
[0047]
For example, a dicarboxylic acid (or higher carboxylic acid) or a pharmaceutically acceptable salt thereof can be used as a stabilizing group. Chemical groups with multiple carboxylic acids can still be used as stabilizing groups. End groups with dicarboxylic acids (or higher carboxylic acids) are typical N-caps, as chemical groups with multiple carboxylic acids are also acceptable as part of the prodrug. Thus, the N-cap may be a monoamide derivative of a chemical group having more than one carboxylic acid, where the amide is attached to the amino terminus of the peptide and the remaining carboxylic acid is free and unattached. To this end, the N-cap is preferably succinic, adipic, glutamic or phthalic acid, and succinic acid is most preferred.
[0048]
Other examples of N-caps useful in the prodrug compounds of the invention include diglycolic acid, fumaric acid, naphthalenedicarboxylic acid, pyroglutamic acid, acetic acid, 1- or 2-naphthylcarboxylic acid, 1,8-naphthyldicarboxylic acid , Aconitic acid, carboxycinnamic acid, triazole dicarboxylic acid, gluconic acid, 4-carboxyphenylboronic acid, (PEG)_n-Analogs, such as polyethylene glycolic acid, butanedisulfonic acid, maleic acid, isonipecotic acid and nipecotic acid.
[0049]
Furthermore, intravenous administration of aggregating positively charged prodrugs in mice resulted in acute toxicity. However, such toxicity was not observed when the charge on the prodrug was reversed by derivatization with a negatively charged stabilizing group. This effect is discussed in more detail below.
Thus, where aggregation of the therapeutic agent is involved, the attached stabilizing groups are negatively charged or neutral.
[0050]
Acute toxicity:
Many cytotoxic compounds inherently have low solubility. For example, positively charged anthracyclines form aggregates at high concentrations, and those aggregates, when administered intravenously, induce intravenous coagulation. Troases recognize specific sites in the peptide sequence. If one of their hydrophobic sequences (eg, βAla-Leu-Ala-Leu) is conjugated to a cytotoxic compound (eg, doxorubicin), it will have a large aggregation when injected intravenously as a concentrated bolus. This results in low soluble compounds that can form aggregates.
[0051]
Since most peptides have a positively charged amino terminus exposed at physiological pH, their aggregates can form multipositively charged surfaces in vivo. Those aggregates given intravenously trigger the coagulation cascade and death in mice within minutes (usually 30 minutes or less) of administration. This renders any positively charged prodrug formulated by means of forming aggregates in suspension unsuitable for therapeutic use.
[0052]
Some experiments support the hypothesis that positively charged aggregates are formed by peptide-conjugated doxorubicin. Testing of similarly arranged solutions by laser light scattering and size exclusion ultrafiltration showed that only a small amount of material had a molecular weight of 10 kD or less. The average molecular size of the aggregate was found to be about 70 kD. When animals were concomitantly administered heparin at an IV dose (see Example 22), acute toxicity was greatly reduced or eliminated. No acute toxicity was present when animals received a dilute solution of the same drug (same total dose). The results, when considered in conjunction with the literature reports, support the conclusion that peptide prodrugs of aggregate-forming compounds do not provide optimal therapy because of poor solubility.
[0053]
Solutions of the aggregates in question make those peptide prodrugs more practical. If the peptide prodrugs form aggregates due to insufficient solubility at the desired compounded concentrations, the stabilizing groups on the peptide chain may be negatively charged or neutral. Should end with sensuality. For example, use of succinyl as a stabilizing group on a peptide prodrug alleviates the acute toxicity of the prodrug (see Example 22). This solves a significant problem in using peptide prodrugs as experimental treatments for humans.
[0054]
Oligopeptide:
Oligopeptides are generally defined as polypeptides of short length, typically 12 or fewer amino acids. However, oligopeptides useful in the prodrugs of the invention are at least four amino acids in length. At the upper end, oligopeptides of 12 or less or 12 amino acids are most useful, provided that the oligopeptides have a chain length of more than 12 amino acids and are commonly used in the scientific art. It is within the scope of the present invention, as well as the definition of terms as recognized. Thus, the oligopeptide portion of a prodrug of the invention has four or more amino acids. Typically, the oligopeptide portion of a prodrug of the invention has from 4 to 12 amino acids. Preferably, it has 4 or 5 amino acids.
[0055]
Numbering scheme:
The oligopeptide has the formula or sequence (AA)_n-AA⁴-AA³-AA²-AA¹:
Wherein each AA independently represents an amino acid,
n is an integer of 0 to 16,
AA⁴Represents an amino acid that is not genetically encoded,
AA³Represents any amino acid,
AA²Represents any amino acid, and
AA¹Represents any amino acid].
This corresponds to the position array P (n + 2)... P2-P1-P1'-P2 '. TOP appears to resolve between the P1 and P1 'positions. Oligopeptides are written in a conventional manner, with the carboxyl terminus (or C-terminus) on the right and the amino terminus (or N-terminus) on the left. Therefore, in the above equation, AA¹Is the carboxyl terminus.
[0056]
Preferred amino acids:
Unless otherwise stated, all amino acids are in the L configuration. Provides a blocking function as described in further detail below. AA, a non-genetically encoded amino acid⁴With the exception of, any amino acid can be present in the oligopeptide portion of the prodrug, but certain amino acids are preferred.
[0057]
P2 position, ie AA⁴Wherein one of the following amino acids is most preferably present: β-alanine, thiazolidine-4-carboxylic acid, 2-thienylalanine, 2-naphthylalanine, D-alanine, D-leucine, D-methionine, D-methionine. -Phenylalanine, 3-amino-3-phenylpropionic acid, [gamma] -aminobutyric acid and 3-amino-4,4-diphenylbutyric acid. Tetrahydroisoquinoline-3-carboxylic acid, 4-aminomethylbenzoic acid, nipecotic acid, isonipecotic acid or aminoisobutyric acid are also preferred at the P2 position.
[0058]
P1 or AA³In position, one of the following amino acids is most preferred: leucine, tyrosine, phenylalanine, p-C1-phenylalanine, p-nitrophenylalanine, valine, norleucine, norvaline, phenylglycine, tryptophan, tetrahydroisoquinoline-3-carboxylic acid, 3 -Pyridylalanine, alanine, glycine or thienylalanine. At the P1 position, methionine, valine or proline is also preferred.
[0059]
At the P1 'position, AA²Is most preferably selected from the following amino acids: alanine, leucine, tyrosine, glycine, serine, 3-pyridylalanine, 2-thienylalanine, norleucine, homoserine, homophenylalanine, p-C1-phenylalanine or p-nitrophenylalanine. . In this position, aminoisobutyric acid, threonine and phenylalanine are also preferred.
[0060]
P2 or AA¹In position, one of the following amino acids is most preferably present: leucine, phenylalanine, isoleucine, alanine, glycine, tyrosine, 2-naphthylalanine, serine, p-C1-phenylalanine, p-nitrophenylalanine, 1-naphthylalanine. , Threonine, homoserine, cyclohexylalanine, thienylalanine, homophenylalanine or norleucine. Β-alanine at the P2 ′ position is also preferred.
[0061]
Oligopeptides particularly useful in the prodrugs of the invention include those shown in FIGS. 10A-10D, particularly one of the following: D-AlaThβAlaβAlaLeuAlaLeu (SEQ ID NO: 1), ThiβAlaβAlaLeuAlaLeu (SEQ ID NO: 2), βAlaβAlaLeuAluLeu (SEQ ID NO: 2). SEQ ID NO: 3) βAlaAlaAlaIle (SEQ ID NO: 4), βAlaAlaAlaLeu (SEQ ID NO: 5), βAlaPheTyrLeu (SEQ ID NO: 6), βAlaPheThrPhe (SEQ ID NO: 7), βAlaPheGlyIle (SEQ ID NO: 8), βAlaPhePhePheGayPePheGy 10), βAlaPhePheIle (SEQ ID NO: 11), βAlaPhePheLeu (SEQ ID NO: 12), β laPheAlaIle (SEQ ID NO: 13), βAlaPheAlaLeu (SEQ ID NO: 14), ThiGlyAlaLeu (SEQ ID NO: 15), NalGlyAlaLeu (SEQ ID NO: 16), βAlaLeuTyrLeu (SEQ ID NO: 17), βAlaLeuThuLu, Sequence ID No. (SEQ ID NO: 20),
[0062]
βAlaLeuThrLeu (SEQ ID NO: 21), βAlaLeuSerLeu (SEQ ID NO: 22), βAlaLeuPyrLeu (SEQ ID NO: 23), βAlaLeuLeuLeu (SEQ ID NO: 24), βAlaLeuGlyPhe (SEQ ID NO: 25), βAlaLeuLuGyLuLuLuGuY (Sequence ID No. (SEQ ID NO: 28), AibLeuGlyLeu (SEQ ID NO: 29), βAlaLeuPheIle (SEQ ID NO: 30), βAlaLeuPheLeu (SEQ ID NO: 31), βAlaLeuAibLeu (SEQ ID NO: 32), βAlaLeuAlaAla (SEQ ID NO: 33), βAlaLae SEQ ID NO: 35), βAlaLeuAl Gly (SEQ ID NO: 36), βAlaLeuAlaIle (SEQ ID NO: 37), βAlaLeuAlaLeu (SEQ ID NO: 38), TicLeuAlaLeu (SEQ ID NO: 39), ThzLeuAlaLeu (SEQ ID NO: 40),
[0063]
ThiLeuAlaLeu (SEQ ID NO: 41), NalLeuAlaLeu (SEQ ID NO: 42), NAALeuAlaLeu (SEQ ID NO: 43), D-LeuLeuAlaLeu (SEQ ID NO: 44), D-AlaLeuAlaLeu (SEQ ID NO: 45), D-MetLeuAluP SEQ ID NO: 47), AmbLeuAlaLeu (SEQ ID NO: 48), βAlaLeuAlaNal (SEQ ID NO: 49), βAlaLeuAlaSer (SEQ ID NO: 50), βAlaLeuAlaTyr (SEQ ID NO: 51), βAlaMetTyrPhe (SEQ ID NO: 52), βAlaMetTyL (Sequence No. No. 54), ThiMetGlyLeu (SEQ ID NO. 55), βAlaMetP ephe (SEQ ID NO: 56), βAlaMetPheIle (SEQ ID NO: 57), TicMetAlaLeu (SEQ ID NO: 58), NalMetAlaLeu (SEQ ID NO: 59), NAAMetAlaLeu (SEQ ID NO: 60),
[0064]
βAlaMetAlaLeu (SEQ ID NO: 61), APPMetAlaLeu (SEQ ID NO: 62), βAlaNleTyrIle (SEQ ID NO: 63), βAlaNleTyrLeu (SEQ ID NO: 64), βAlaNleThrIle (SEQ ID NO: 65), βAlaNleThrLeu (SEQ ID NO: 66), AlNPleGy, βAlaNleGy (SEQ ID NO: 68), βAlaNleGlyLeu (SEQ ID NO: 69), βAlaNlePheIle (SEQ ID NO: 70), βAlaNleAlaIle (SEQ ID NO: 71), βAlaNleAlaLeu (SEQ ID NO: 72), βAlaNleAlaPhe (SEQ ID NO: 73), βAlaNvaAleTra (AlaNleAleTra) SEQ ID NO: 75), ThiProGly Leu (SEQ ID NO: 76), ThiProAlaLeu (SEQ ID NO: 77), NalProAlaLeu (SEQ ID NO: 78), βAlaProAlaLeu (SEQ ID NO: 79), βAlaPhe (Cl), AlaLeu (SEQ ID NO: 80),
[0065]
βAlaPhe (NO₂), AlaIle (SEQ ID NO: 81), βAlaPhe (NO₂), AlaLeu (SEQ ID NO: 82), βAlaPhgAlaLeu (SEQ ID NO: 83), βAlaPyrAlaLeu (SEQ ID NO: 84), TicThrGlyLeu (SEQ ID NO: 85), βAlaThiGlyIle (SEQ ID NO: 86), βAlaThiAlaLeu (SEQ ID NO: 87), (SEQ ID NO: 87) , ΒAlaTicAlaLeu (SEQ ID NO: 89), βAlaValAlaLeu (SEQ ID NO: 90), βAlaTrpAlaLeu (SEQ ID NO: 91), βAlaTyrTyrPhe (SEQ ID NO: 92), βAlaTyrTyrIle (SEQ ID NO: 93), βAlaTyrTyrLue, βAlaTyrTyrLuer, and βAlaTyrPheLeu (SEQ ID NO: 96), βAlaTyrGlyIle ( SEQ ID NO: 97), ThiTyrGlyLeu (SEQ ID NO: 98), βAlaTyrGlyLeu (SEQ ID NO: 99), βAlaTyrPheIle (SEQ ID NO: 100), βAlaTyrAlaIle (SEQ ID NO: 101), ThiTyrAlaLeu (SEQ ID NO: 102), and βALaTru (SEQ ID NO: 102).
[0066]
Blocking amino acids:
The oligopeptide of the prodrug is, according to the numbering scheme described above, the AA of the oligopeptide sequence.⁴, Ie, the blocking amino acid at position P2 of the position sequence. The blocking amino acids are those that are not genetically encoded.
[0067]
The function of the blocking amino acid at position P2 maintains selectivity for prodrug degradation by TOP and is most closely linked (directly or indirectly) to the therapeutic moiety of the prodrug compound. To prevent degradation of the oligopeptide by other enzymes in that part of the oligopeptide, or to avoid providing a degradation site for said enzyme. More particularly, by placing a blocking amino acid at position P2, the oligopeptide sequence AA⁴-AA³-AA²-AA¹And unwanted degradation within the peptide chain of the four amino acids of the position sequence P2-P1-P1'-P2 'is reduced. It is believed that the troase degrades between the P1 and P1 'positions of the oligopeptide.
[0068]
Since blood and normal cells are associated with various peptidases, the placement of a blocking amino acid at position P2 acts to protect the oligopeptide portion of the prodrug until the prodrug is in close proximity to the target cell. . In particular, by placing a blocking amino acid at position P2, the oligopeptide is protected from unwanted degradation between P2 and P1. In the absence of the blocking amino acid, the prodrug is susceptible to both exopeptidases and endopeptidases present in blood and normal tissue, where enzymes of the type, on the other hand, have the prodrug in its target Before reaching, the prodrug is degraded. Example 14 below illustrates this important feature of the prodrug.
[0069]
Screening by TOP:
TOP is an important enzyme that can be used to select oligopeptides for further use, and thus another aspect of the invention is that
Formula (AA)_n-AA⁴-AA³-AA²-AA¹:
[Wherein each AA independently represents an amino acid;
n is an integer of 0 to 16,
AA⁴Represents an amino acid that is not genetically encoded,
AA³Represents any amino acid,
AA²Represents any amino acid, and
AA¹Represents any amino acid], and can be degraded by TOP.
The oligopeptide can be conjugated to a therapeutic agent and / or a stabilizing group when tested for degradability by TOP.
[0070]
Therapeutic agents:
Therapeutic agents that are particularly advantageous for converting into prodrugs according to the present invention are those agents that have a narrow therapeutic window. Drugs or therapeutics with a narrow therapeutic window are those whose toxicity is evident by general medical standards, very close to that at which efficacy is evident.
The therapeutic agent conjugated to the stabilizing group and the oligopeptide and, optionally, the linker group to form a prodrug of the invention may be used to treat cancer, inflammatory diseases, or some other medical condition. May be useful for Preferably, the therapeutic agent is selected from the following types of components:
[0071]
Alkylating agents, antiproliferative agents, tubulin binders, vinca alkaloids, enyne, podophyllotoxin or podophyllotoxin derivatives, drugs of the pteridine class, taxanes, anthracyclines, dolastatin, topoisomerase inhibitors, and platinum complex chemotherapeutic agents .
[0072]
In particular, the therapeutic agent is advantageously selected from the following compounds or derivatives or analogs thereof: doxorubicin, daunorubicin, vinblastine, vincristine, calicheamicin, etoposide, etoposide phosphate, CC-1065, zuocarmycin, KW-2189. , Methotrexate, metopterin, aminopterin, dichloromethotrexate, docetaxel, paclitaxel, epithiolone, combretastatin, combretastatin A4 phosphate, dolastatin 10, dolastatin 11, dolastatin 15, topotecan, camphothecin, mitomycin C, porphyromycin , 6-mercaptopurine, fludarabine, tamoxifen, cytosine arabinoside, adenosine arabinoside, colchici , Cisplatin, carboplatin, mitomycin C, bleomycin, melphalan, chloroquine, Shikuroporin A, chloroquine, maytansine or cisplatin.
[0073]
Derivative means a compound resulting from the reaction of a named compound with another chemical moiety and including pharmaceutically acceptable salts, acids, bases or esters of the named compound. By analog is meant a compound having similar structural and functional properties to the named compound, eg, biological activity.
[0074]
Linker group:
A linker group between the oligopeptide and the therapeutic agent may be advantageous, for example, for the following reasons:
1. AA¹As a spacer for steric considerations to facilitate the enzymatic release of amino acids or other enzymatic activation steps;
2. To provide the appropriate coupling chemistry between the therapeutic agent and the oligopeptide;
3. To improve synthetic methods of producing prodrug conjugates (eg, by pre-derivatizing a therapeutic agent or oligopeptide with a linker group prior to conjugation to enhance yield or specificity);
[0075]
4. To improve the physical properties of prodrugs;
5. To provide an additional mechanism for intracellular release of the drug.
The linker structure is dictated by the functionality required. Examples of possible linker chemistries are hydrazides, esters, ethers and sulfhydryls. Aminocaproic acid is an example of a bifunctional linker group. If aminocaproic acid is used for the linker group, it is not counted as an amino acid in the oligopeptide numbering scheme.
[0076]
An optionally present linker group cannot be degraded by TOP, ie it cannot be degraded by TOP under physiological conditions.
Particularly useful embodiments are compounds that can be degraded by troases, but are resistant to degradation by CD10 or other systemic or blood enzymes.
[0077]
Prodrug planning:
Prodrug design methods are another aspect of the invention and, as described above, first require the identification of oligopeptides. The oligopeptide is then attached at a first binding site of the oligopeptide to a stabilizing group that prevents oligopeptide degradation by enzymes present in safe blood, and at a second binding site of the oligopeptide the therapeutic agent. Directly or indirectly. The conjugation of the oligopeptide to the therapeutic agent and the stabilizing group can be performed in any order or simultaneously. The resulting conjugate is tested for degradability by TOP. The resulting conjugate can also be tested for stability in whole blood. A test compound that is stable in whole blood is selected.
[0078]
The first binding site is usually at the N-terminus of the oligopeptide, but can also be at the C-terminus of the oligopeptide, or another portion of the oligopeptide. The second binding site is usually at the C-terminus of the oligopeptide, but can also be at the N-terminus of the oligopeptide or another portion of the oligopeptide. Prodrugs designed by such methods are also part of the present invention.
[0079]
In addition, the present invention includes a method for reducing the toxicity of a therapeutic agent intended for administration to a patient. In particular, a modified prodrug form of the therapeutic agent is formed by directly or indirectly attaching the therapeutic agent to an oligopeptide that can be degraded by troase, or more specifically, degraded by TOP. The oligopeptide is also attached to a stabilizing group. The prodrugs thus formed, when administered to a patient, provide for reduced toxicity of the therapeutic agent. Modification of the therapeutic in this manner also allows for the administration of an elevated dose of the therapeutic to the patient relative to the dose of the therapeutic in an unconjugated form.
[0080]
Pharmaceutical composition:
The invention also encompasses pharmaceutical compositions comprising a compound of the invention, particularly a prodrug compound, and optionally a pharmaceutically acceptable carrier, such as an adjuvant or vehicle, or the like.
The invention also relates to the use of said pharmaceutical composition for the preparation of a pharmaceutical product directed for the treatment of a pharmaceutical condition.
[0081]
For example, the pharmaceutical composition can be administered to the patient parenterally, especially intravenously, intramuscularly or intraperitoneally. Pharmaceutical compositions of the present invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. As pharmaceutically acceptable solvents or beers, propylene glycol, polyethylene glycol, injectable organic esters such as ethyl oleate, or cyclodextrin may be used. The isotonic salt solution can be part of a pharmaceutical composition. These compositions may also contain wetting agents, emulsifying and / or dispersing agents.
[0082]
Disinfection may be effected in several ways, for example using a bacteriological filter, by introducing a disinfectant into the composition, or by irradiation. They can also be prepared in the form of sterile solid compositions which can be dissolved at the time of use in sterile water or any other sterile injectable medium.
Pharmaceutical compositions are also well known in the art and can be used in combination with the compounds of the present invention to improve and extend the treatment of the medical condition to which they are administered, such as adjuvants (eg, , Vitamin C, antioxidants, etc.).
[0083]
Doses for administration of a compound of the present invention to a patient are generally described in Bruce A. et al. Chabner and Jerry M. Collins, Cancer Chemotherapy, Lippincott Ed. , ISBN 0-397-50900-6 (1990), or at least the usual doses of therapeutic agents known in the art, or they are the superior efficacy of prodrug formulations or It can be adjusted within the judgment of the treating physician to adapt to the particular environment of the patient. Thus, the dose administered will vary according to the therapeutic used for the preparation of the compound of the invention.
[0084]
Treatment with prodrug compounds:
Methods for therapeutic treatment of a pharmaceutical condition, including administering a therapeutically effective dose of a pharmaceutical composition to a patient, particularly parenterally or intravenously, are also within the scope of the invention. Thus, methods for treating patients require therapeutically effective amounts of
(1) a therapeutic agent that can be inserted into target cells,
(2) Formula (AA)_n-AA⁴-AA³-AA²-AA¹:
[Wherein each AA independently represents an amino acid;
n is an integer of 0 to 16,
AA⁴Represents an amino acid that is not genetically encoded,
AA³Represents any amino acid,
AA²Represents any amino acid, and
AA¹Represents any amino acid], an oligopeptide represented by the formula:
(3) a stabilizing group, and
(4) optionally comprising a linker group that cannot be degraded by TOP;
[0085]
Wherein the oligopeptide is directly linked to the stabilizing group at a first binding site of the oligopeptide, and the oligopeptide is linked directly to the therapeutic agent or the oligopeptide Indirectly attached to the therapeutic agent through the linker group at the second binding site of
The stabilizing group prevents degradation of the compound by enzymes present in whole blood, and
[0086]
Administering to the patient a compound characterized in that the compound can be degraded by enzymes associated with target cells. The enzyme that is bound by the target cell is preferably Troase and more preferably TOP.
Prodrug compounds are useful for the treatment of many medical conditions, such as cancer, neoplastic diseases, tumors, inflammatory patients, and infectious diseases. Examples of preferred diseases are breast, colorectal, liver, lung, prostate, ovarian, brain and pancreatic cancer. The prodrug compounds of the present invention are also useful in addressing the problem of multidrug resistant target cells.
[0087]
For example, following repeated chemotherapy, many tumor cells develop multidrug resistance (MDR). MDR is evident in vitro and clinically, and especially in the case of doxorubicin and other adriamycin analogs. There are a variety of basic mechanisms of MDR action, including changes in the expression or activity of a wide range of transport-associated cell membrane proteins in general. They include P-glycoprotein pumps that actively transport doxorubicin or other therapeutic agents from cells.
[0088]
In tumors that develop MDR, the dose effective to kill the tumor cells increases until it reaches an overall toxic dose. Thus, on the other hand, very effective chemotherapy is no longer useful as a drug due to unacceptable side effects levels and mortality. However, chemotherapy or other therapeutic agents that are modified to form prodrugs as taught herein are useful in preventing such resistance of target cells to the therapeutic agent.
[0089]
Accordingly, a therapeutically effective amount of
(1) a therapeutic agent that can be inserted into target cells,
(2) Formula (AA)_n-AA⁴-AA³-AA²-AA¹:
[Wherein each AA independently represents an amino acid;
n is an integer of 0 to 16,
AA⁴Represents an amino acid that is not genetically encoded,
AA³Represents any amino acid,
AA²Represents any amino acid, and
AA¹Represents any amino acid], an oligopeptide represented by the formula:
(3) a stabilizing group, and
(4) optionally comprising a linker group that cannot be degraded by TOP;
[0090]
Wherein the oligopeptide is directly linked to the stabilizing group at a first binding site of the oligopeptide, and the oligopeptide is linked directly to the therapeutic agent or the oligopeptide Indirectly attached to the therapeutic agent through the linker group at the second binding site of
The stabilizing group prevents degradation of the compound by enzymes present in whole blood, and
A method for treating resistance to a therapeutic agent in a patient in need thereof, comprising administering a compound characterized in that the compound is degradable by TOP.
[0091]
Prodrug compounds formulated in a pharmaceutically acceptable vehicle (eg, an isotonic salt solution) can be administered to an animal or human at an intravenous dose ranging from 0.05 mg / kg / dose / day to 300 mg / kg / dose / day. Can be administered. It can also be administered via intravenous infusion or other slow infusion methods. Human patients are normal receptors for the prodrugs of the invention, although veterinary use is also contemplated.
[0092]
Diagnosis or assay:
Products, such as kits, for diagnosis or assays are also within the scope of the invention. Such products preferably employ a compound as described above, except that a marker such as coumarin is conjugated to the oligopeptide and stabilizing group instead of the therapeutic agent. Marker refers to any component that can be conjugated to an oligopeptide and that can be readily detected by any method known in the art. At least one reagent useful in the detection of the marker is typically included as part of the kit.
[0093]
Therefore, the product
(1) (a) a marker,
(B) Formula (AA)_n-AA⁴-AA³-AA²-AA¹:
[Wherein each AA independently represents an amino acid;
n is an integer of 0 to 16,
AA⁴Represents an amino acid that is not genetically encoded,
AA³Represents any amino acid,
AA²Represents any amino acid, and
AA¹Represents any amino acid], an oligopeptide represented by the formula:
(C) a stabilizing group, and
(D) optionally comprising a linker group that cannot be degraded by TOP;
[0094]
Wherein the oligopeptide is directly attached to the stabilizing group at a first binding site of the oligopeptide, and the oligopeptide is directly attached to the marker or Indirectly attached to the marker at said second binding site through said linker group;
The stabilizing group prevents degradation of the compound by enzymes present in whole blood, and
A compound characterized in that the compound can be decomposed by TOP, and
(2) Optionally, at least one reagent useful in detecting the marker is included.
The product can be used with patient samples to diagnose tumors or identify patients who are sensitive to treatment with prodrug treatment.
[0095]
General methods of process chemistry:
Oligopeptides: General methods for peptide synthesis:
The peptide or oligopeptide sequences in the prodrug conjugates of the invention can be synthesized by solid phase peptide synthesis (using either Boc or Fmoc chemistry) or by solution phase synthesis. The general Boc and Fmoc methods are widely used and are described in: Merrifield, J .; A. Chem. Soc. , 88: 2148 (1963); Bodanszky and Bodanszky, The Correction of Peptide Synthesis, Springer-Verlay, Berlin, 7-161, 1994, Steph, Desert, Petr.
[0096]
General Fmoc solid phase method:
Using the preferred solid phase synthesis method (automatic or manual), peptides of the desired length and sequence are synthesized through the stepwise addition of amino acids to a growing chain that is bound to a solid resin. Examples of useful Fmoc compatible resins include, but are not limited to, Wang resin, HMPA-PEGA resin, Rink acid resin or hydroxyethyl-photolinker resin. The C-terminus of the peptide chain was covalently linked to the polymer resin, and the protected α-amino acid was added in a stepwise manner along with the coupling reagent. Preferred α-amino protecting groups are Fmoc groups that are stable to coupling conditions and can be easily removed under mild alkaline conditions. The reaction solvent is preferably, but not limited to, DMF, NMP, DCM, MeOH and EtOH.
[0097]
Examples of coupling agents are DCC, DIC, HATU, HBTU. Decomposition of the N-terminal protecting group is achieved at 10 to 100% piperidine in DMF at 0 to 40 ° C, and the temperature is preferably ambient. At the end of the synthesis, the final Fmoc protecting group is removed using the N-terminal cleavage method described above. The remaining peptide on the resin is degraded from the resin along with any acid sensitive side chain protecting groups by treating the resin under acidic conditions. For example, acidic decomposition conditions are a mixture of trifluoroacetic acid (TFA) in dichloromethane. If a hydroxyethyl-photolinker resin is used, a suitable wavelength to induce degradation is UV at λ 365 nm. An illustration of this process is given in FIG.
[0098]
General N-cap method through solid phase synthesis:
Preparation of the N-terminally derivatized peptide is conveniently achieved on a solid phase. When peptide synthesis is complete, the terminal Fmoc is removed and the peptide is still present on the solid support. The selected N-cap is then coupled onto the N-terminus of the peptide using standard peptide coupling conditions. Upon completion of the N-cap coupling, the peptide is degraded from the resin using the method described above.
[0099]
General Boc solid phase method:
For solid phase methods using Boc chemistry, Merrivield resins or PAM resins are useful. Amino acids are coupled to the growing chain on the solid phase by sequential addition of Boc-protected amino acids activated by a coupling agent. Examples of coupling agents are DCC, DIC, HATU, HBTU. The reaction solvent can be DMF, DCM, MeOH and NMP. Decomposition of the Boc protecting group is achieved at 0-40 ° C. in 10-100% TFA in DCM, with regard to temperature, ambient temperature being preferred. Upon completion of the peptide chain assembly, the N-terminal protecting group (usually Boc) is removed as described above. The peptide is removed from the resin using liquid HF or trifluoromethanesulfonic acid in dichloromethane. General method for preparing Fmoc oligopeptides by solution phase synthesis:
[0100]
On the other hand, prodrug peptide intermediates can be made through solution phase synthesis using Boc or Fmoc chemistry. In the illustration of this method (FIG. 4), the C-terminal Leu tetrapeptide is generally used as an example, but it will be understood that similar reactions can be performed with other C-terminal tetrapeptides. There will be. This peptide is amplified by step-wise assembly (N-terminal or C-terminal) similar to the solid phase method, or through coupling of two suitably protected dipeptides or tripeptides with a single amino acid. Can be done.
[0101]
One method of solution phase synthesis is the step-wise increase of a prodrug peptide intermediate using Fmoc chemistry, shown in FIG. The C-terminus should be protected to reduce by-product formation. The C-terminal R group in FIG. 4 is Me, tBu, benzyl or TCE. (Note that if N-cap is methylsuccinyl, the C-terminal R group cannot be methyl.) DMF is provided as a solvent, but other solvents such as DMSO, CH₃CN or NMP (or mixtures thereof) can be substituted for it.
[0102]
Pyridine, Et₃N or other bases can be replaced by piperidine in deprotection of the amino terminus of the growing peptide chain protection. Similarly, HBTU is given as an activator in the above figure, but other activators such as DCC, DIC, DCC + HOBt, Osu, activated ester, azide or triphenylphosphoryl azide may be used. In addition, protected peptide acid chlorides or acid bromides can be used to couple directly to amino acids or peptide fragments. Based on the completion of the oligopeptide assembly, the N-terminally deprotected and C-terminally protected peptides are readily accepted by the desired N-cap.
[0103]
General method for preparing N-capped oligopeptides through solution phase synthesis:
When constructing N-capped oligopeptides by solution phase synthesis, N-caps need to be synthesized by a slightly improved method (FIG. 4). First, the C-terminus of the Fmoc oligopeptide needs to be protected by an acid labile or hydrogenation sensitive protecting group that is compatible with the selective deprotection of the C-terminus on the N-cap. Next, the Fmoc protecting group needs to be removed from the oligopeptide to represent the N-terminus.
[0104]
Oligopeptides having a deprotected N-terminus and a protected C-terminus are reacted with an activated N-cap activated hemiester. The N-cap can be activated using a method for activating an amino acid, such as DCC or HATU, in a base and a suitable solvent. On the other hand, if methyl-hemisuccinate is used, the coupling may also be with an organic or inorganic base such as DIEA, triethylamine or Cs₂CO₃Using an inert solvent in the presence of methylhemisuccinyl chloride (or other acid halide).
[0105]
One example of such a synthesis may be by reacting methyl-hemisuccinate and βAla-Leu-Ala-Leu benzyl ester. The coupling method may be any one of the methods commonly used in the art (eg, Bodanszky, M., The Practice of Peptide Synthesis, Springer Verlag, 185 (1984); Bodanszky, M.D.). , Principles of Peptide Synthesis, Springer Verlag, 159 (1984)). Next, the benzyl group can be removed by catalytic hydrogenation to provide the desired N-cap methyl-succinyl form of βAla-Leu-Ala-Leu. Other examples of suitable, selectively removable C-terminal protecting groups may be, but are not limited to, tBu, alkoxy-methyl and TCE. Other methods for accomplishing this step are described in the literature.
[0106]
Combinations of any of the above methods, such as "fragment degeneracy" of di- or tripeptides, may be considered. Reaction conditions are well known in the art and are described in the references given. The advantage of the above method is the easy purification of the product produced by the solution phase synthesis.
[0107]
Prodrug conjugate:
General methods for the joining and deprotection steps:
The N-capped forms of the oligopeptide-therapeutic agents described herein can be oligo-drugated with daunorubicin, doxorubicin, or any suitable therapeutic agent using any of the standard activating reagents used in peptide synthesis. It can be synthesized by coupling the Fmoc form of the peptide (meaning that Fmoc is attached to the N-terminus of the oligopeptide) (Figure 5). Solvents are toluene, ethyl acetate, DMF, DMSO, CH₃It can be CN, NMP, THF, DCM or any other inert solvent as known in the art, and the reagent can be dissolved therein. Preferred solvents are DMF and NMP.
[0108]
A suitable temperature range is −25 to + 25 ° C., and ambient temperature is preferred. The activator may be selected from one of the following: PyBOP, HBTU, HATU, EDC, DIC, DCC, DCC + HOBT, OSu, activated ester, azide or triphenylphosphoryl azide. HBTU or HATU is the preferred activator. On the other hand, the protected peptide acid chloride or acid bromide can also be used for this coupling reaction. 2-4 equivalents, conveniently 2-2.5 equivalents, of base are required for the coupling reaction. The base is an inorganic base such as CsCO₃, Na₂Or K₂CO₃Or an organic base such as TEA, DIEA, DBU, DBN, DBO, pyridine, substituted pyridine, N-methyl-morpholine, and the like, preferably TEA or DIEA. The reaction may be carried out at a temperature between -15 and 50C, conveniently between -10 and 10C.
[0109]
The reaction time is between 5 and 90 minutes and conveniently between 20 and 40 minutes. The product is isolated by pouring the reaction mixture into water and filtering off the precipitate formed. The crude product can be further purified by recrystallization from DCM, THF, ethyl acetate, or acetonitrile, preferably dichloromethane or acetonitrile. Next, the isolated Fmoc form of the oligopeptide therapeutic agent conjugate is treated with a 10-100 fold excess of base at a temperature of -10 to 50 ° C for 2 to 90 minutes, preferably 3 to 8 minutes. The protection is released. Ideally, 5 to 60 equivalents of base are preferred. Pipetidine is a preferred base for deprotecting the Fmoc group. The deprotected amino terminus of the oligopeptide therapeutic conjugate is acylated with the diacid anhydride as an activated hemi-ester to obtain the final N-cap form of the oligopeptide-therapeutic.
[0110]
On the other hand, final prodrugs can be similarly prepared from protected N-capped forms of the oligopeptide, such as the methyl-hemiester of a succinyl-N-capped oligopeptide, and conjugated to a therapeutic agent. This method is illustrated in FIG.
The protected N-cap-oligopeptide therapeutic is deprotected in a manner compatible with the stability of the therapeutic. For example, anthracyclines can be protected by methyl groups and deprotected by esterases. For other therapeutic agents, a benzyl protecting group and catalytic hydrogenation may be selected for deprotection.
[0111]
Conversion of the negatively charged N-cap oligopeptide therapeutic to a salt form can be performed with a solvent selected from the following group: alcohol (eg, methanol, ethanol or isopropanol), water, acetonitrile, tetrahydrofuran, Diglyme or other polar solvent. The sodium source was 1 molar equivalent of NaHCO₃, NaOH, Na₂CO₃, NaOAc, NaOCH₃(Generally sodium alkoxide) or NaH. Na⁺Ion exchange columns charged with (e.g., strong or weak ion exchangers) are also useful, where appropriate, for this last step in making the salt form of the N-cap oligopeptide therapeutic. Sodium is described merely as an example.
[0112]
Generally, a prodrug can be converted to a pharmaceutically acceptable salt form to improve the solubility of the prodrug. The N-cap-oligopeptide therapeutic is a pharmaceutically acceptable salt, such as NaHCO₃, Na₂CO₃, NaOH, tris (hydroxymethyl) aminomethane, KHCO₃, K₂CO₃, CaCO₃, NH₄OH, CH₃NH₂, (CH₃)₂NH, (CH₃)₃Neutralized with N, acetyltriethylammonium. The preferred salt form of the prodrug is sodium and the preferred neutralizing salt is NaHCO₃It is.
[0113]
It has been described that anthracycline-type molecules, such as doxorubicin and daunorubidine, form gels in organic solvents at very low concentrations (Matzanke et al., Eur. J. Biochem. 207: 747-55 (1992); Chaires). Etc., Biochemistry 21: 3927-32 (1982); Hayagawa et al., Chem. Pharm. Bull. 39: 1282-6 (1991) This is a high yield of contaminants when producing peptide anthracycline conjugates. Gel formation can contribute to the formation of undesired side reactions.
[0114]
One means for minimizing this problem is to use a very dilute solution (1-2%) for the coupling reaction, however, it is not suitable for the process environment (high waste, complex Isolation is not practical. To overcome this problem, urea or other chaotropic agents can be used to break the strong hydrophobic and hydrogen bonding forces that form the gel. Thus, when the coupling reaction is carried out in a urea-containing solvent, conveniently from 20% to a saturated solution of urea in DMF or NMP, the side reaction is less than 2%, even when the concentration of the reaction exceeds 10%. Can be maintained. This makes the bonding step at high concentrations practical and produces good yields.
[0115]
General enzyme methods:
Acid or base catalyzed hydrolysis of a protected N-cap-oligopeptide therapeutic to a sufficient N-cap compound results in the instability of many therapeutics, even under moderately acidic or basic conditions. To derive a plurality of reaction mixtures. Enzymes can promote hydrolysis without destroying the substrate or product. Suitable enzymes for this reaction may be esterases or lipases and, by their nature, exist in water-soluble form or are immobilized on commercially available solid support materials by cross-coupling or conjugation. obtain.
[0116]
Among the soluble enzymes that are evaluated, Candida Antarctica "B" lipase (Altus Biologics) is particularly useful. An example of an enzyme immobilized by cross-coupling is ChiroCLEC-PC^TM (Alus Biologicals). Candida antarctica "B" lipase (Altus Biologics) is an NHS-activated Sepharose^TM 4 Fast Flow (American Pharmacia Biotech). During the hydrolysis, the pH of the reaction mixture was carefully adjusted and the NaHCO₃Through a controlled addition of the solution, a pH of 5.5-7.5, advantageously 5.7-6.5, is maintained with a pH onset. When the reaction is completed, the product is isolated by lyophilization of the filtered reaction mixture. The immobilized enzyme remains on the filter cake and can be reused if desired.
[0117]
General allyl or alkyl ester method:
Prodrugs can also be prepared by coupling the allyl- or alkyl-hemiester form of the N-capped oligopeptide with a therapeutic agent and generating the free acid form from the conjugate. FIG. 8 illustrates this step with succinyl-β-Ala-Leu-Ala-Leu and doxorubicin.
Coupling of allyl-succinyl-β-Ala-Leu-Ala-Leu with doxorubicin can be performed by any one of the oligopeptide conjugation methods.
[0118]
Allyl-succinyl-βAla-Leu-Ala-Leu-doxorubicin is also analogous to βAla-, as described in the method wherein coupling of the protected tetrapeptide precursor to doxorubicin is shown in FIG. It can be synthesized by reacting Leu-Ala-Leu with allyl hemisuccinate prepared through a known method (Casimir et al., Tet. Lett. 36: 3409 (1995)). Suitable inert solvents are THF, dichloromethane, ethyl acetate, toluene, preferably THF in which the product acid form precipitates as the reaction proceeds. The isolated acid is converted to its sodium salt as described above. The reaction time is from 10 to 180 minutes, conveniently from 10 to 60 minutes, at a temperature of from 0 to 60C, preferably from 15 to 30C.
[0119]
Removal of allyl or alkyl groups is well known in the art and, as recorded in the specialist literature, Pd (0), or Ni (0), of the allyl or alkyl group on the receptor molecule, conveniently. Tet. Lett. 50: 497 (1994); Bricot et al., Tet. Lett. 54: 1073 (1998); Genet et al., Synlett. 680 (1993); Waldmann et al., Bioorg. Med. Chem. 7: 749 (1998); Shaphilo et al., Tet. Lett. 35: 5421 (1994)). The amount of the catalyst may be 0.5 to 25 mol% based on the substrate.
[0120]
General trityl or substituted trityl method:
Prodrugs can also be synthesized by the method shown in FIG. This approach utilizes R'-oligopeptides, where R 'is trityl or substituted trityl. Coupling of the R'-oligopeptide by the therapeutic agent may be performed at 0-20 <0> C for 30-120 minutes by any one of the methods described above for conjugation of protected oligopeptide by the therapeutic agent.
[0121]
Removal of the trityl or substituted trityl group can be achieved under acidic conditions to obtain a positively charged prodrug. This positively charged prodrug is shown in FIG. 4 and is N-capped as described above. Trityl deprotection can be achieved with acetic acid, formic acid and dilute hydrochloric acid.
Prodrugs can be converted to succinyl or glutaryl βAla-Leu-Ala-Leu therapeutics by reaction with succinic anhydride. The succinyl or glutaryl βAla-Leu-Ala-Leu therapeutic can be converted to any pharmaceutically acceptable salt. Solvent for coupling step, DMF, DMSO, CH₃CN, NMP, or any other suitable solvent is known in the art.
[0122]
General reverse solid phase joining method:
The prodrug compounds of the present invention can be synthesized by using solid-phase chemistry through a “step-wise” reverse (N-terminal to C-terminal) method.
One means is to use a resin to fix the succinyl-hemiester, for example succinyl-benzyl or -allyl ester. Examples of resins selected are "Wang resin" (Wong, J. Am. Chem. Soc, 95: 1328 (1973); Zhang et al., Tet. Lett. 37: 5457 (1996)), "Rink resin". (Rink, Tet. Lett. 28: 3787 (1987)), “Trityl- or substituted-trityl resin” (Chen et al., J. Am. Chem. Soc. 116: 2611 (1994); Bartos et al. , Peptides Proc.^nd European Peptide Symposium (1992); Schneider and Eberle (Eds.), Escom Leiden. Pp. 281 (1993)).
[0123]
The immobilized ester is then deprotected and reacted, for example, with similarly C-terminally protected β-alanine. The steps are then repeated with leucine, alanine and finally the leucine ester, followed by coupling of doxorubicin to the immobilized succinyl-tetrapeptide. This methodology in which molecules are then generated from the resin by using mild acidic conditions to form a free prodrug, eg, free Suc-βAla-Leu-Ala-Leu-Dox, is shown in the scheme of FIG. Represented above. Another version of phase synthesis uses immobilized succinyl oligopeptides. This is then C-terminally deprotected, followed by a coupling step to doxorubicin or other therapeutic agent, and finally produced from the resin, as depicted on the scheme of FIG. . The acid form of the prodrug molecule can then be ultimately converted to its sodium salt as described above.
[0124]
General large-scale compound synthesis:
Prodrug compounds can be synthesized using the simple and effective three-step process of the present invention. The first step involves coupling of an alkyl-ester protected oligopeptide fragment to a therapeutic agent. A preferred embodiment of the first step is to couple MeOSuc-βAla-Leu-Ala-Leu-OH by doxorubici using HATU as a coupling agent to obtain MeOSuc-βAla-Leu-Ala-Leu-Dox. Include (FIG. 16). The focus of this step is on the purity and yield of the methyl ester, as the hydrolysis step was found to have no effect on purity.
[0125]
The second step is the hydrolysis of the alkyl-ester group by an enzyme (esterase), which directly gives the prodrug compound with a final purity of at least 90% in good yield. For example, the second stage involves the enzyme (CLEC CAB, cross-linked Candida antarctica "B"), which directly provides the sodium salt of Suc-βAla-Leu-Ala-Leu-Dox in high purity and quantitative yield. Lipase) by hydrolysis of the methyl ester group in MeOSuc-βAla-Leu-Ala-Leu-Dox.
[0126]
The final step is to isolate the product after the hydrolysis step. Since most therapeutic agents are toxic, it is preferable to add an extra step to eliminate any free therapeutic agent from the coupled product. The focus of the final step is to isolate the final product, for example Suc-βAla-Leu-Ala-Leu-Dox, after the hydrolysis step. This uses a 0.2 micron filter to filter the reaction mixture from the hydrolysis step and then filter the Suc-βAla-Leu-Ala-Leu-Dox. It is simply achieved by freeze-drying the filtration to obtain Na.
[0127]
Removal of free therapeutic agent:
The unconjugated therapeutic can be present later in the prodrug manufacturing phase. For example, during the coupling step of the (stabilizing group)-(oligopeptide) conjugate with doxorubicin as a therapeutic agent, it was found that in many cases the reaction did not proceed completely. There was about 2-4% residual doxorubicin remaining in the coupled product. Initial attempts to completely remove doxorubicin from the product by acid washing did not result in complete removal. For large-scale synthesis of MeOSuc-βAla-Leu-Ala-Leu-Dox, complete removal of doxorubicin is crucial. Complete removal of free therapeutic agent was provided by the steps outlined in Example 41 and FIG. 14 using scavenging resin or beads.
[0128]
The crude product containing MeOSuc-βAla-Leu-Ala-Leu-Dox and residual doxorubicin was dissolved in DMF and polystyrene methyl isocyanate or polystyrene sulfonyl chloride resin or beads were added. The reaction was stirred for 60 minutes. The free amino groups of doxorubicin react with isocyanate or sulfonyl chloride groups on the beads to form urea or sulfonamide derivatives. Next, those beads with doxorubicin bound to the solid beads were separated from the desired product by filtration. The desired product remains in the DMF solution. This approach appears to be a very gentle and effective method for removing residual therapeutic agents from the product.
[0129]
Therefore, the present invention
(1) Formula (AA)_n-AA⁴-AA³-AA²-AA¹:
[Wherein each AA independently represents an amino acid;
n is an integer of 0 to 16,
AA⁴Represents an amino acid that is not genetically encoded,
AA³Represents any amino acid,
AA²Represents any amino acid, and
AA¹Represents any amino acid], and selects an Fmoc-protected oligopeptide represented by
[0130]
(2) activating the Fmoc-protected oligopeptide with an activating agent in the presence of the therapeutic agent to form an Fmoc-protected oligopeptide-therapeutic agent conjugate, thereby providing the therapeutic agent with the Fmoc-protected oligopeptide. Coupling the protected oligopeptide,
(3) deprotecting the Fmoc-protected oligopeptide-therapeutic agent conjugate by contacting it with a base to form an oligopeptide-therapeutic agent conjugate; and
(4) a method of preparing a compound comprising coupling the oligopeptide-therapeutic agent conjugate to a stabilizing group to form a compound.
[0131]
On the other hand, the process for preparing the compounds comprises the following steps:
(1) Formula (AA)_n-AA⁴-AA³-AA²-AA¹:
[Wherein each AA independently represents an amino acid;
n is an integer of 0 to 16,
AA⁴Represents an amino acid that is not genetically encoded,
AA³Represents any amino acid,
AA²Represents any amino acid, and
AA¹Represents any amino acid], and an oligopeptide represented by
[0132]
(2) coupling the oligopeptide to an alkyl ester-protected stabilizing group to form an alkyl ester-protected stabilizing group-oligopeptide conjugate;
(3) The alkyl ester-protected stabilizing group-oligopeptide-therapeutic agent conjugate is converted to the active agent in the presence of the therapeutic agent to form an alkyl ester-protected stabilizing group-oligopeptide conjugate. Coupling the alkyl ester-protected stabilizing group-oligopeptide conjugate to the therapeutic agent by activation with
(4) deprotecting the alkyl ester-protected stabilizing group-oligopeptide therapeutic conjugate to form a compound.
[0133]
The compounds of the present invention also include the following steps:
(1) Formula (AA)_n-AA⁴-AA³-AA²-AA¹:
[Wherein each AA independently represents an amino acid;
n is an integer of 0 to 16,
AA⁴Represents an amino acid that is not genetically encoded,
AA³Represents any amino acid,
AA²Represents any amino acid, and
AA¹Represents any amino acid], and an oligopeptide represented by
[0134]
(2) coupling the oligopeptide to an allyl ester-protected stabilizing group to form an allyl ester-protected stabilizing group-oligopeptide conjugate;
(3) Allyl ester-protected stabilizing group-oligopeptide conjugate is treated with an active agent in the presence of a therapeutic agent to form an allylic ester-protected stabilizing group-oligopeptide conjugate in order to form a conjugate. Coupling the allyl ester-protected stabilizing group-oligopeptide conjugate to the therapeutic agent by activation with
(4) deprotecting said allyl ester-protected stabilizing group-oligopeptide therapeutic conjugate to form a compound;
Manufactured by stages.
[0135]
Yet another method for preparing the compounds of the present invention comprises the following steps:
(1) Formula (AA)_n-AA⁴-AA³-AA²-AA¹:
[Wherein each AA independently represents an amino acid;
n is an integer of 0 to 16,
AA⁴Represents an amino acid that is not genetically encoded,
AA³Represents any amino acid,
AA²Represents any amino acid, and
AA¹Represents any amino acid], and selects a trityl-protected oligopeptide represented by the formula:
[0136]
(2) activating the trityl-protected oligopeptide with an activating agent in the presence of the therapeutic agent to form a trityl-protected oligopeptide-therapeutic agent conjugate, thereby providing the therapeutic agent with said trityl Coupling the protected oligopeptide,
(3) deprotecting the trityl-protected oligopeptide-therapeutic agent conjugate under acidic conditions to form an oligopeptide-therapeutic agent conjugate;
(4) coupling the oligopeptide-therapeutic agent conjugate to a stabilizing group to form a compound;
Comprising.
[0137]
Another possible step for any of these methods is to remove the uncoupled therapeutic agent by using a scavenging resin or beads. In addition, the compounds can be neutralized, if desired, with pharmaceutically acceptable salts.
[0138]
Therefore, in the method for producing a prodrug, the present invention
(1) coupling the optionally protected stabilizing group-oligopeptide conjugate with a free therapeutic agent;
(2) contacting the polymer resin binding free therapeutic agent remaining after step (1) with the reactant of step (1) to form a therapeutic agent-polymer resin complex;
(3) A method for removing free therapeutic agent, comprising removing the therapeutic agent-polymer resin complex.
The polymer resin may be polystyrene methyl isocyanate or polystyrene sulfonyl chloride.
[0139]
Specific compounds:
The compounds of the present invention include prodrugs, namely, Suc-βAla-Leu-Ala-Leu-Dox, Suc-βAla-Leu-Ala-Leu-Dnr, and G1-βAla-Leu-Ala-Leu-Dox.
In addition, the following key intermediate compounds in the process for preparing the prodrugs of the present invention are part of the present invention:
[0140]
βAla-Leu-Ala-Leu
Trityl-βAla-Leu-Ala-Leu-Dox
Diphenylmethyl-βAla-Leu-Ala-Leu-Dox
Benzyloxycarbonyl-βAla-Leu-Ala-Leu-Dox
Fmoc-βAla-Leu-Ala-Leu-OBn
βAla-Leu-Ala-Leu-OBn
Methyl-succinyl-βAla-Leu-Ala-Leu-OBn
Methyl-succinyl-βAla-Leu-Ala-Leu
Fmoc-βAla-Leu-Ala-Leu
Fmoc-Thi-Tyr-Gly-Leu
Fmoc-βAla-Leu-Ala-Leu-Dnr
Fmoc-Thi-Tyr-Gly-Leu-Dnr
Suc-Thi-Tyr-Gly-Leu-Dnr
G1-βAla-Leu-Ala-Leu-Dox
βAla-Leu-Ala-Leu-Dox lactate
Allyl-succinyl-βAla-Leu-Ala-Leu-Dox
Suc-βAla-Leu-Ala-Leu
Methyl ester of Suc-βAla-Leu-Ala-Leu
Fmoc-βAla-Leu-Ala-Leu-Dox
Methyl-succinyl-βAla-Leu-Ala-Leu-Dox, and
Allyl-hemisuccinate.
[0141]
Example
Example 1.MCF7 / 6 Preparation of Cell Homodnate
MCF7 / 6 cells were treated with DMEM: F12 (1: 1), 50 mg / l bovine serum albumin, ITS-X (10 mg / l insulin, 5.5 mg / l transferrin, 6.7 μg / l selenite) Grow to confluency in serum-free medium containing sodium, 2 mg / l ethanolamine) and lipid concentrate (Gibco # 21900-030). 100 ml of cells were centrifuged at 10,000 xg for 20 minutes at 4 ° C and harvested by decanting the supernatant. The pellet was resuspended in 2 ml of phosphate buffer solution (Gibco) and centrifuged at 18,000 xg for 10 minutes. After decanting the supernatant, the cells (about 300 μl wet) were homogenized by grinding in 10 ml of 10 mM HEPES buffer (pH 7.2) (sodium salt). The homodunate was centrifuged at 18,000 × g for 5 minutes at 4 ° C., and the supernatant was aliquoted and stored at −20 ° C. or lower for subsequent use in compound screening.
[0142]
Example 2.MCF7 / 6 Preparation of conditioned medium
MCF7 / 6 cells were confluent in DMEM / F12 (1: 1) containing 10% fetal calf serum, 0.05% (w / v) L-glutamine, 250 IU / ml penicillin and 100 μg / ml streptomycin. Proliferated to sex. The cells are then washed twice with phosphate buffered saline and MDEM / F12 (1: 1), 0.02% BSA, ITS-X (10 mg / l insulin, 5.5 mg / l Transferrin, 6.7 μg / l sodium selenite, 2 mg / l ethanolamine) with 5% CO 2₂Incubated at 37 ° C for 24 hours. The conditioned medium is then decanted and 10 mM HEPES buffer (pH 7.2) using a stirred cell apparatus equipped with a YM10 (10,000 MW cutoff) ultrafiltration membrane (Millipore). Once, and concentrated 20-fold. This solution was stored in aliquots at −20 ° C. for use in compound screening.
[0143]
Example 3.HeLa Cell anion exchange fraction pool ( PI Preparation of
30x10 commercially produced¹⁰HeLa cells (human cervical cancer, Computer Cell Culture Center, Seneffe, Belgium) were homogenized in a 108 ml aqueous lysine solution with a sonicator and a Dounce homogenizer. The lysine solution was a cocktail of 0.02% (w / v) Triton X-100, 0.04% (w / v) sodium azide, and an inhibitor of protease (2 tablets / 50 ml Complete).^TM, EDTA-free tablets (Roche Molecular Biochemicals). The cell homogenate was centrifuged at 5000 × g for 30 minutes at 4 ° C., and the pellet was homogenized in a second 108 ml lysine solution using a Dounce homogenizer and centrifuged as above. The supernatants were combined and centrifuged at 145,000 × g at 4 ° C. for 90 minutes.
[0144]
A portion of the ultracentrifuged supernatant was aliquoted into 20 mM triethanolamine-HCl (0.01% (w / v) X-100 and 0.02% (w / v) sodium azide). pH 7.2) Diluted 2-fold with buffer (equilibration buffer). 30 ml of the above obtained solution, corresponding to about 180 mg of protein, was added to a 2.6 × 9.4 cm Source^TM 15Q (Amersham Pharmacia Biotech) was loaded on a low pressure anion exchange chromatography column at 4 ° C (1 ml / min). Next, the column was washed with 250 ml of equilibration buffer at a flow rate of 1 ml / min.
[0145]
Protein was eluted at a flow rate of 3 ml / min in a NaCl linear concentration gradient (0-0.5 M in equilibration buffer; total volume of the gradient was 1000 ml). Two minute fractions were collected and used for enzyme activity determination using βAla-Leu-Ala-Leu-Dox as substrate. Its translocation to Ala-Leu-Dox was quantified by reverse-phase high performance chromatography using fluorescence detection of the anthracycline component. Fractions containing the highest activity level were pooled (fractions # 43-46; about 0.13 M NaCl) and protease inhibitors (Complete^TM, EDTA-free tablets, Roche Molecular Biochemicals) and stored as aliquots at -80 ° C.
[0146]
Example 4.HeLa Purification of cell troase
HeLa cell fraction 1 (F1) was treated as described in Example 3 with 50 × 10¹⁰Of HeLa cells, but unlike Example 3, six experiments were performed with a load of about 350 mg of protein and 50 μM CoCl₂Was added to the equilibration and elution buffer. The F1 fraction was concentrated by ultrafiltration (30 KD MWCO) and incubated at 4 ° C. for 2 hours in the presence of 1.25% EDTA. EDTA is removed on an equilibrated desalting column (PD10) and equilibration buffer (20 mM phosphoric acid, 0.01% Triton-X100, 0.02% NaN₃, 0.5M NACl, pH 7.2).
[0147]
Next, about 20 mg of the protein corresponding to the F1 fraction was added to 250 ml of 5% EDTA, 250 ml of water, 250 ml of 0.1 ml of 0.1 M CoCl.₂, Loaded on a 12 x 150 mm Chelating-Sepharose (Amersham Pharmacia Biotech) column, which was previously treated sequentially with 250 ml of water and 250 ml of equilibration buffer. After sample adsorption, the column was washed with 150 ml of equilibration buffer and eluted with a 600 ml 0-0.2 M imidazole gradient. All steps were performed at a flow rate of 0.1 ml / min. The 40 minute fraction was collected. The activity-containing fractions (about 1 ml of protein) were pooled, concentrated by ultrafiltration, and electrophoresis sample buffer (0.12 M Tris-HCl, 5% glycerol, 0.01% bromophenol blue, pH 6.8) (1: 1).
[0148]
This sample was fractionated by preparative polyacrylamide gel electrophoresis. Model 491 PrepCell (BioRad) was run on a 37 × 120 mm 7% T, 2.5% C degradation gel, buffered with 0.37 M Tris-HCl (pH 8.8), and 0.12 M Tris-HCl (pH 6). .8), used with 37 × 5 mm 4% T, 2.6% C condensed gel. The electrode buffer was 25 mM Tris, 192 mM glycine, pH 8.3, and the elution buffer was 100 mM triethanolamine, 0.01% Triton X-100, 50 μM CoCl.₂(PH 7.2). After 30 minutes at 30 mA, separation was performed at 40 mA for about 24 hours. The 12 min fraction was collected with an elution flow rate of 0.4 ml / min.
[0149]
Active fractions (about 150 μg protein) were pooled, concentrated by ultrafiltration, and the sample was run on an equilibrated gel filtration HPLC column (Toso Haas TSK G3000SK)._XL, 7.8 x 600 mM) and 0.2 M K₂SO₄And eluted at 0.3 ml / min with a 50 mM phosphate buffer (pH 7.2). The 0.5 minute fraction was collected. Fractions containing activity were stored at -80 ° C.
[0150]
Example 5.Screening for potential prodrugs with troase and human blood
Based on HPLC analysis of the digestion products, activation of the prodrug to free toxicity occurs through a series of enzyme-catalyzed degradation reactions. For example, the prodrug, Suc-βAla-Leu-Ala-Leu-Dox, is converted to Leu-Dox in two steps catalyzed by at least two enzymes in extracts of cancer cells or conditioned medium of cancer cells. . The initial endopeptidase degradation is AA³(P1) and AA²(P1 ') Generate Ala-Leu-Dox, which occurs between amino acids. Subsequently, exopeptidase removes alanine, yielding the active toxin, leucyl-doxorubicin, which is known to be taken into cells where doxorubicin is released.
[0151]
Good candidates for prodrugs with improved therapeutic index are activated by cancer cells that are relatively stable in whole human blood. Various N-capped peptidyl-toxins were screened using three different cancer preparations. The three preparations were as follows:
(A) MCF7 / 6 (breast cancer) cell homogenate;
(B) MCF7 / 6 (breast cancer) conditioned medium; and
(C) HeLa (cervical cancer) cell extract anion exchange fraction pool.
[0152]
A single amino acid toxin conjugate (ie, AA¹-(Optional linker) -Therapeutic agent) was further tested for stability in whole human blood. Whole blood was collected using a commercially available acid-buffered citrate complete blood collection tube (Decton Dickinson).
The test chemistry was incubated at 37 ° C. for 2 hours with three different preparations of the cancer enzyme and whole blood at a concentration of 12.5 μg / ml. Following incubation, three volumes of acetonitrile were added, the reaction was stopped, and proteins were removed from the mixture. The sample was centrifuged at 18.000 g for 5 minutes and 100 μl of the supernatant were mixed with 300 μl of water before analysis by HPLC.
[0153]
For HPLC analysis, 50 μl of the sample was injected at 40 ° C. onto a 4.6 × 50 mM 2 μ TSK Super-ODS chromatography column and 26 μm in 20 mM aqueous ammonium formate (pH 4.5) buffer. Eluted at 2 ml / min with a 3 minute linear gradient of% -68% acetonitrile. Detection was performed by fluorescence using an excitation wavelength of 235 nm and an emission wavelength of 560 nm.
[0154]
The oligopeptide moieties of the test compounds that are degraded by troase under defined conditions and are stable in human blood are shown in FIGS. 10A-10C. For all oligopeptides shown in FIGS. 10A-10C, the test compound had a succinyl stabilizing group and a daunorubicin therapeutic. In addition, the oligopeptide having SEQ ID NO: 1 was tested using aminomethylbenzoic acid as a stabilizing group and daunorubicin as a therapeutic. The oligopeptide having SEQ ID NO: 35 was tested using diglycolic acid and maleic acid as stabilizing groups and daunorubicin as a therapeutic.
[0155]
The oligopeptide having SEQ ID NO: 38 was also tested with a number of additional stabilizing groups and therapeutic agents. In particular, the test compounds of the oligopeptide of SEQ ID NO: 38 included the following: Adamantenecarbonyl-βAla-Leu-Ala-Leu-Dnr, diphenyl-acetyl βAla-Leu-Ala-Leu-Dnr, maleic acid- βAla-Leu-Ala-Leu-Dox, 4-morpholinecarbonyl-βAla-Leu-Ala-Leu-Dnr, PEG-βAla-Leu-Ala-Leu-Dox, 2-furoyl-βAla-Leu-Ala-Leu-Dnr Acetyl-βAla-Leu-Ala-Leu-Dnr, diglycolic acid-βAla-Leu-Ala-Leu-Dox and Napth-βAla-Leu-Ala-Leu-Dox.
With a few exceptions, the results for cancer enzymatic degradation were the same for partially purified fractions from HeLa cells, MFC7 / 6 cell homogenates or MCF7 / 6 conditioned media.
[0156]
Example 6.Hydrolysis rate
After hydrolysis rates for different prodrugs, or after immunoprecipitation, enzyme test solutions (as prepared in Examples 1-4 above) were treated with 100 μM MnCl for comparison of troase and TOP activity measurements.₂Were incubated with 10 μg / ml substrate for up to 2 hours at 37 ° C. in 10 mM HEPES, pH 7.2. The reaction was stopped by adding 3 volumes of acetonitrile. The precipitated protein was removed by centrifugation, and the supernatant was diluted in 3 volumes of water before HPLC analysis as described in Example 5 above. The fraction of hydrolyzed substrate was calculated by dividing the peak area for product by the total peak area for substrate and product.
[0157]
The substrate specificity of the partially purified HeLa cell troase was determined by the method of Glucksman and Roberts, "Stratagies for characterizing, cloning, and expressing solutions, available from the end of the method of eds. , E .; The specificity of the recombinant rat TOP (rRTOP) produced in E. coli was substantially identical. Nine peptidyl test compounds were found to have similar rates of hydrolysis by HeLa cell F1 and rR-TOP (Table 1). For all peptidyl Dox substrates, the reaction product of Dox binding is AA²-AA¹-Dox. For example, Suc-βAla-Leu-Ala-Leu-Dox was decomposed into Ala-Leu-Dox and possibly Suc-βAla-Leu.
[0158]
[Table 1]

Thus, cancer cell troase and TOP have nearly identical substrate specificities.
[0159]
Example 7.Refined CD10 Hydrolysis
Equal amounts of purified porcine kidney CD10 (Elastin Products Company) were combined with various peptidyl doxorubicin compounds at 12.5 μg / ml with 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Triton X at pH 7.4. Incubate at -100 at 37 ° C for up to 10 hours. The reaction products were analyzed by HPLC using fluorescence detection. The hydrolysis rate was substantially linear over the incubation period. The product observed was Leu-doxorubicin. Table 2 provides the percentage of individual test compound that was hydrolyzed over 10 hours. In addition, the results are expressed against a standard test compound, Suc-Ala-Leu-Ala-Leu-Dox.
[0160]
[Table 2]

[0161]
Example 8.Top as well as HeLa Inhibition and inactivation of cellular troase
As expected for metal enzymes, TOP is inactivated by exposure to metal chelators such as EDTA and 1,10-phenanthroline (Barrentt et al., "Thime oligopeptidase and oligopeptidase Mor neurosin", Methods. 248: 529-556 (1995)). The peptide compound N- [1- (RS) -carboxypropyl-Ala-Ala-Phe-p-aminobenzoate (Cpp-AAF-pAB)] is a more selective and sensitive inhibitor (Knight and Barrett, " Structure / function relations in the inhibition of this lipopeptidase by carboxyphenylpropyl-peptides, FEBS Lett 189: FEBS Lett.
[0162]
Cpp-AAF-pAB also inhibits the closely related metalpeptidase neurolysin, but the mere neurolysin activity is inhibited by the 5 mM dipeptide Pro-Ile (Serizawa et al., “Characterization of a mitochondrial metabolites). neurosin as a homologue of this oligopeptide ", J. Biol. Chem. 270: 2092-2098 (1995)). In studies with MCF-7 / 6 cell homogenate, troase activity was inhibited 9-fold by 1 mM EDTA and 2 mM 1,10-phenanthroline, whereas inhibitors of non-metallic peptidases were not effective.
[0163]
Thus, 50 μM aminoethylbenzenesulfonyl fluoride, 4 μg / ml aprotinin (both inhibit serine peptidase), 20 μM E-64 (inhibit cysteine peptidase), 1.5 μM pepstatin (inhibit aspartate peptidase) , 20 μM leupeptin (which inhibits serine and cysteine peptidase), or 1 μM CA-074 (which inhibits cathepsin B). In tests on HeLa cell fractions, 1.3 μM Cpp-AAF-pAB completely inhibited Suc-βAla-Leu-Ala-Leu-Dox hydrolysis, but 5 mM Pro-Ile had an effect. I didn't.
[0164]
Barrett and Brown ("Cicken river Pz-peptidase, a thiol-dependant metallo-endopeptidase", Biochem. J. 271: 701-706 (1990)). Purified chicken TOP was used. At a concentration of 50 μM, Zn²⁺Completely recovered activity. Other divalent cations at the same concentration partially recovered activity in the following order of effectiveness: Mn²⁺> Ca²⁺> Co²⁺> Cd²⁺. Other divalent cations such as Cu²⁺Had no effect. Excess Zn²⁺(100 μM or more) was inhibitory. In reconstitution experiments with MCF-7 / 6 cell homogenate treated with EDTA, the activity was 50 μM Co²⁺Or Mn²⁺Was completely recovered by²⁺Or Cu²⁺Was not recovered by.
[0165]
Example 9.Gel filtration and isoelectric focusing
The MCF-7 / 6 cell homogenate had an approximate molecular weight of 68 KD based on a retained dose of active gel filtration chromatography fraction. For their measurements, MCF-7 / 6 cell homogenates and protein molecular weight standards were fractionated on a Superose S12, 10 × 300 column (Amersham Pharmacia Biotech). Purified tropase from HeLa cells was separated by SDS polyacrylamide gel electrophoresis (PAGE) into two protein bands corresponding to 74 and 63 KD using the method described in Example 11.
[0166]
Approximately 74 and 63 KD bands were observed in SDS PAGE Western immunoblots of HeLa cells F1 stained with anti-thymite oligopeptidase antibody. A 74 KD band was also observed on SDS PAGE Western blots of crude homogenates of MCF-7 / 6, MDS-MD-231 and EA hy926 cells. The results are consistent with the molecular weights of human, rat and porcine TOP, estimated from the DNA sequence as 78 KD and reported as 74-80 KD in various SDS PAGE determinations (Barrett et al., "Thymet oligopeptidase and oligopeptidase"). Morneurolysin ", Methods Enzymol. 248: 529-556 (1995)).
[0167]
An isoelectric point of 5.2 was determined for MCF-7 / 6 cell homogenate troase by chromatofocusing on a Mono P HR5 / 5, 5 × 40 mm column (Amersham-Pharmacia Biotech). This result is in agreement with the commonly reported 5.0 ± 0.27 pI values for TOP from various sources (Tisljar, “Thiemet oligopeptidase-a review of a thiol dependent metallo-dose catalysis. Endopeptidase 24.15 and endo-oligopeptidase ", Biol. Chem. Hoppe. Seyler. 374: 91-100 (1993)).
[0168]
An example 10.Thiol activation
MCF-7 / 6 conditioned media was pre-incubated with dithiothreitol (DTT) at the indicated concentrations for 30 minutes at room temperature. Next, 10 μg / ml Suc-βAla-Leu-Ala-Leu-Dox was added and incubated at 37 ° C. The hydrolysis products were extracted and analyzed on a Luna @ C18-3μ, 4.6 × 100 mm column (Phenomenex) as described above. Residual Suc-βAla-Leu-Ala-Leu-Dox substrate was extracted using citrate buffer at pH 3.0 and N-succinyl doxorubicin rather than boric acid as an internal standard, and Luna C18 as described above. Analyzed on a -3μ column.
[0169]
Unlike most metal peptidases, TOP is activated by low levels of thiol reducing agents, such as 50 μM dithiothreitol (DTT) or 1 mM mercaptoethanol, but is inhibited at high concentrations, such as 5 mM DTT. . is the (Orlowski, etc. ". Endopeptidase 24.15 from rat testes Isolation of the enzyme and its specifixity toward synthetic and natural peptides, including enkephalin-containing peptides," Biochem J 261: 951-958 (1989); Tisljar and Barrett “Thiol-depend nt metallo-endopeptidase characteristics of Pz-peptidase in rat and rabbit, "Biochem J267:. 531-533 (1990); Lew, such as" Substrate specificity differences between recombinant rat testes endopeptidase EC 3. 4. 24. 15 and the native brain Enzyme, "Biochem Biophys Res Commun 209: 788-795 (1995)".
[0170]
Shrimpton and others. , "Thiol activation of endopeptidase EC 3.4.24.15. A novel mechanism for the regulation of catalytic activity", J. Biol. Biol. Chem. 272: 17395-17399 (1997) showed that reducing agents activate the TOP enzyme protein by reversing the formation of inactive disulfide bond dimers. Similarly, DTT pretreatment experiments with MCF-7 / 6 cell conditioned medium, with a high level of inhibitory activity, were 10 times higher than Suc-βAla-Leu-Ala-Leu-Dox hydrolysis at 1 mM DTT. (Fig. 13). Thus, both TOP and cancer cell troWase are activated by low levels of thiol reducing agents.
[0171]
An example 11.pH Optimality
MFC-7 / 6 cell homogenate as prepared in Example 1 above was incubated at 37 ° C. with βAla-Leu-Ala-Leu-Con in 100 mM triethanolamine buffered at various pH levels. The amount of Leu-Cou was determined by treating the reaction product with leucine aminopeptidase and measuring the resulting aminomethyl coumarin concentration by spectrofluorometer. TOP has a pH optimum of 7.8 for a 10 minute assay with a quenched fluorescent substrate (Barrett, 1995).
[0172]
Assays with the quenched fluorescent substrate Mcc-Pro-Leu-Gly-Pro-D-Lys (DNP) are described in Barrett et al. , ("Thymet oligopeptidase and oligopeptidase Mor neurosin", Methods Enzymol. 248: 529-556 (1995)). The MCF-7 / 6 cell homogenate troase activity pH optimum was 7.2-7.7 for MCF-7 / 6 cell homogenate activity using the βAla-Leu-Ala-Leu-Cou assay. Thus, TOP and troase activities are optimized at similar pH.
[0173]
An example 12.Refined HeLa Mass Spectroscopy of Cell Trouser Trypsin Digest
The purified HeLa troWase is lyophiliZed overnight, dissolved in 30 pl of sample loading buffer at 90 ° C. for 1 min, centrifuged to remove insoluble material, and SDS polyacrylamide based on a small format gel. Separated by gel electrophoresis. Operating conditions were 30 mA, 300 V for 45 minutes. After staining with Coomassie Blue R250, two bands corresponding to molecular weights of 74 and 63 KD were evident.
[0174]
A 2x2 mm piece of each band was transferred to a small tube and washed in 15 min steps with stirring with the following individual solutions: 25 mM bicarbonate (ammonium), 50% acetonitrile in water, 25 mM Bicarbonate (ammonium). The samples were then dried in a centrifugal concentrator (Speedvac) and incubated with 10 μl of a 25 mM ammonium bicarbonate solution containing 0.5 μg of trypsin at 37 ° C. for 3 hours. For Nanospray-MS, a portion of the trypsin digest was extracted with acetonitrile, dried in a centrifuge vacuum concentrator (Speedvac), and analyzed before ZipTip analysis.^TM The product was purified by a C18 fine extraction device.
[0175]
Tryptic digested protein from the 74 KD band was analyzed by MALDI-tof (matrix assisted laser desorption ionization-time of flight) mass spectrometer. Mass spectra of tryptic digests were acquired on a Biflex (Bruker) MALDI-tof mass spectrometer with delayed extractability operating in reflector mode. 0.4 μl of each digest (in 25 mM ammonium bicarbonate) was mixed with a dry thin-layer α-cyano-4-hydroxy-cinnamic acid (CCA) matrix (isopropanol: acetone (1: 1)) mixed with nitrocellulose. Medium, saturated CCA: 5 ng / ml in nitrocellulose (4: 3; v / v)) directly on the sample probe. Samples were washed with 0.1% TFA before analysis.
[0176]
Peptide mass fingerprints obtained for individual digests were obtained using MS-FIT (http://spector.ucsf.edu/ucsfhtm13.2/msfit.htm) to predict the expected digest pattern from the known protein sequence. Adapted to. As shown in Table 3, comparison of the observed molecular ions to the expected human TOP trypsin fragmentation pattern resulted in twenty bands with the expected mass. Their suitability accounted for 33% of the known human TOP sequences (Thompson et al., "Cloning and functional expression of a metallodope peptidase from the biobrain bioavailability of the biobrain bioavailability of the biobrain bioavailability of the biochemistry and the biochemistry of the biochemistry and the biobrain biotherapeutics and the biopsy). 213: 66-73 (1995)). After trypsin digestion, MALDI-tof analysis of the 63 kD band showed that it shared the same sequence except for the absence of a small portion at the carboxy terminus.
[0177]
[Table 3]

[0178]
Electrospray ionization (ESI) quadrupole time-of-flight (Q-tof) tandem mass spectrometry measurements were performed using a Q-tof instrument (Micromass) with a Z-spray ion source operating in nanospray mode. About 3-5 μl of the purified sample was introduced into a sample needle (PROTANA Inc., Odense, DK) and MS and MS / MS experiments were performed. The average capillary potential was 1000V and the sample cone was set at 50V. Human [Gln¹-The instrument was calibrated in fibrinopeptide B in MS / MS mode. MS / MS spectra were converted using MaxEnt3 and sequences were determined using PepSeq (Micromass Biolynx).
[0179]
For further confirmation of structural identity, two HeLa cell trypsinized 74 KD fragments were sequenced by electrospray ionization (ESI) quadrupole time-of-flight (Q-tof) tandem mass spectrometry. As shown in Table 4, both fragments were completely identical to the known sequence of human TOP (Thompson et al., "Cloning and functional expression of a metallo-endopeptidase from the brain after the ceremony of the ceremony of the brain in the ceremony). , Biochem. Biophys Res. Commun. 213: 66-73 (1995)). By comparison, the sequences are close, but not identical, to those of rat and pig TOP.
[0180]
[Table 4]

[0181]
An example Thirteen.Immunoprecipitation
Immunoprecipitation was performed in two steps. In the first step, 5 μl of the test enzyme was added to a 10 mM hydroxyethylpiperazine (HEPES) (pH 7.2), 150 mM NaCl solution, diluted anti-TOP or inappropriate rabbit IgG (10 μl) diluted 1: 250. ) At 4 ° C. for 1 hour. In the second step, the mixture was added to 15 μl of Protein A Sepharose (Amersham Pharmacia Biotech) equilibrated in the same buffer and incubated at 4 ° C. for 1 hour. After microcentrifugation, residual enzyme activity was determined in the supernatant.
[0182]
Immunoprecipitation provides a further indication of structural identity. Partially purified HeLa Troase (F1) activity was completely removed from the solution after incubation with rabbit anti-TOP followed by Sepharose ™ Bees immobilized Protein A. Similarly, MCF-7 / 6 cell homogenate troase activity was reduced to 80% by immunoprecipitation with rabbit anti-TOP antibody. Control experiments showed that under the same conditions, rR-TOP was completely immunoprecipitated, but incubation with an improper rabbit antibody did not immunize with either rR-TOP, HeLa cell troWase activity, or MCF7 / 6 troWase activity. It did not precipitate.
[0183]
An example 14.Specificity for Troases depends on location P2 Provided by non-genetically encoded amino acids in
Specificity is conferred by incorporation of non-genetically encoded amino acids at position P2 rather than genetically encoded amino acids. In particular, Suc-βAla-Leu-Ala-Leu-Dnr, which contains the non-genetically encoded amino acid β-alanine at position P2, along with each of the three preparations described in Examples 1-3 Incubation was as described in Example 5. Next, the extent of degradation was evaluated by HPLC analysis of the resulting mixture.
[0184]
The results are compared with those obtained for the same incubation performed with the same compound except for the position of the genetically encoded amino acid L-alanine at the P2 position, for example Suc-βAla-Leu-Ala-Leu- Dnr versus Suc-Ala-Leu-Ala-Leu-Dnr was compared. The degree of degradation (proportion) by cell homogenate was 1.3 times higher for P2 L-alanine compound versus P2 β-alanine compound. However, for the partially purified tropase preparation, the degree of degradation of the P2 L-alanine compound was only 0.6 times that of the P2 β-alanine compound. These results suggest that the presence of L-alanine at P2 provided a second degradation site for a cruder mixture of enzymes; thus, release of the active agent in vivo was localized to tumor tissue. Reduce the tendency to be localized.
[0185]
An example Fifteen.Prodrugs have poor cell uptake before degradation
Promyelocytic leukemia cells, HL-60, were cultured in RPMI medium containing 10% heat inactivated fetal calf serum (FCS). On the day of the study, cells are collected, washed, and placed in RPMI containing 10% FCS at 0.5 × 10 5⁶Reconstituted at a concentration of cells / ml. 100 μl / well of the cell suspension was added to a 96-well plate. Serial dilutions of doxorubicin or test compound (3-fold increase) were made and 100 μl of compound was added to the wells. Finally, 10 μl of 100 μCi / ml³H-thymidine was added per well and the plates were incubated for 24 hours. Plates were harvested using a 96-well harvester (Packard Instruments) and counted on a Packard Top Count counter. Four-parameter logistic curves were plotted using Prism software as a function of drug molarity.³IC adapted to H-thymidine incorporation₅₀The value was determined.
[0186]
[Table 5]

[0187]
Doxorubicin has an IC of 0.075 μM in HL60 cells₅₀With potential cytotoxic activity. In contrast, the prodrug Suc-βAla-Leu-Ala-Leu-Dox showed poor cell uptake and IC> 50 μM in the HL-60 proliferation assay.₅₀Having. In vivo degradation studies indicate that leucyl-doxorubicin is an intermediate formed after proteolysis of the prodrug Suc-βAla-Leu-Ala-Leu-Dox. Therefore, leucyl-doxorubicin was tested and an IC of 0.222 μM was used.₅₀Has been shown. These data support the concept that leucyl-doxorubicin is taken up by cells where it is degraded to release active doxorubicin.
[0188]
An example 16.Comparative metabolism in mice
Two groups of ICR normal female mice received a single IV bolus dose of approximately 100 μmol / kg Suc-βAla-Leu-Ala-Leu-Dox or 10 μmol / kg doxorubicin (Dox). Plasma was obtained from three individual animals in individual groups at 5 minutes, 1, 2, 4, or 6 hours. The parent, dipeptidyl-doxorubicin (AL-Dox), α-amino-doxorubicin (L-Dox) and doxorubicin concentrations were measured by reverse phase gradient HPLC using fluorescence detection (λex = 480 nm, λem = 560). The extracts were analyzed. Amounts were determined using linear standard curve fit to measurements of 10-2000 ng / ml doxorubicin solution in mouse plasma.
[0189]
Based on a time course of up to 6 hours, L-Dox is the major metabolite for the first 2 hours, and the dipeptidyl-conjugate AL-Dox is formed at about the same time as L-Dox than the minor. It was a product. Doxorubicin appears later, and its plasma concentration can be determined from current and previously measured doxorubicin pharmacokinetic profiles (Van der vijgh et al., "Comparative metabolism and pharmacokinetics of doxorubicin drug and dioxin ubiquitin and dioxorubicin drug and dioxorubicin drug). and Tumor of tumor-bearing mice, "Cancer Chemother Pharmacol. 26 (1): 9-12 (1990); and Tabrizi-Fard et al. CR (CD1 ™) Mice Following Intravenous Administration ”, Proc. Amer. Assoc. Cancer Res. 42: 234 (2001)) and other metabolites as predicted by the doxorubicin control group more slowly over time. descend.
[0190]
This is consistent with the subsequent continuous degradation by exopeptidase activity. The area under the plasma concentration time curve (AUC) for doxorubicin (Table 6) shows that Suc-βAla-Leu-Ala-Leu-Dox produced equivalent doxorubicin exposure to doxorubicin alone. It should be noted that after administration of these compounds, doxorubicin exposure is similar to the relative safety expressed as the maximum tolerated dose in mouse safety studies.
[0191]
[Table 6]

[0192]
As a result, a 10-fold dose of Suc-βAla-Leu-Ala-Leu-Dox produced similar exposure to doxorubicin. In this study, Suc-βAla-Leu-Ala-Leu-Dox was administered at about twice its single dose (SD) and repeated dose (RD) MTD, and doxorubicin was produced at about twice its SD MTD. It was administered at 25% but at a dose equal to the RD MTD. The rapid clearance of undegraded Suc-βAla-Leu-Ala-Leu-Dox from plasma is clearly due to the high relative tolerance of the Suc-βAla-Leu-Ala-Leu-Dox compound compared to doxorubicin Bring. Doxorubicin is cleared relatively slowly. Thus, prodrugs allow for the administration of effective levels of doxorubicin exposure while safely clearing excess drug, as indicated by the relative differences in MTD, so that repeat-dose treatments such as chemotherapy Is advantageous in those treatments used.
[0193]
An example 17.Prodrugs are effective and well tolerated in tumor xenograft models
Suc-βAla-Leu-Ala-Leu-Dox has been demonstrated in several nude mouse xenograft models, including estrogen-dependent MCF-7 / 6 breast cancer and atriamycin regimen colorectal cancer CXF280 / 10 and LS-174T. It has been found to be effective in inhibiting the growth of human tumors.
[0194]
For example, if a group of 10 mice with LS174T tumors implanted subcutaneously are treated with an intravenous dose of Suc-βAla-Leu-Ala-Leu-Dox therapy five times a week, the mean day of survival (MSD) And a vehicle treatment at doses of 57 (Group 2), 64 (Group 3) and 71 (Group 4) mg / kg Suc-βAla-Leu-Ala-Leu-Dox. A decrease in tumor size (tumor volume) was observed, as compared to the control (Group 1) given, and the highest dose was equal to 40 mg / kg doxorubicin (see FIG. 11). The drug was safe and well tolerated under repeated dose levels and frequency of doses showing antitumor efficacy. Some dose-dependent weight loss was observed. In support of the study, no nephrotoxicity and myelosuppression was observed with doses of up to 106.8 mg / kg of Suc-βAla-Leu-Ala-Leu-Dox.
[0195]
An example 18.Suc −β Ala − Leu − Ala − Leu − Dox Is more tolerated in vivo than doxorubicin
The typical tetrapeptide prodrug Suc-βAla-Leu-Ala-Leu-Dox of the present invention is well tolerated in mice. In a second single dose maximum tolerated dose (SD-MTD) study, a group of five normal ICR mice received a bolus dose of Suc-βAla-Leu-Ala-Leu-Dox intravenously. . Mice were observed daily for 49 days and body weight was measured twice weekly. The dose levels tested were 0, 50, 75 or 100 mg / kg, equivalent to 0, 29, 42 or 56 mg / kg doxorubicin, respectively. There was no acute toxin within 24 hours at any dose level.
[0196]
Dose- and time-dependent signs of toxicity were observed during this study. Toxicity, such as partial hind limb paralysis and significant weight loss (more than 20% of their initial body weight) were observed in the 75 and 100 mg / kg dose groups. By day 35, mortality was observed in 40% of the 75 mg / kg dose group. Based on survival at 49 days and lack of signs of toxicity, the SD-MTD for Suc-βAla-Leu-Ala-Leu-Dox is 50 mg / kg (equivalent to 28 mg / kg doxorubicin). It was decided.
[0197]
This dose was very tolerated and no adverse effects were observed. Thus, the SD-MTD was approximately 1.8-fold higher on a molar basis than the SD-MTD for doxorubicin alone (16 mg / kg). See Table 7. This is an approximate SD-MTD determination based on a range of doses in 14 mg / kg doxorubicin equivalent increments over the range tested.
[0198]
Preliminary observations on the toxicity profile of the prodrug have shown that it is generally consistent with that observation of doxorubicin, eg, later paralysis and wasting. However, some differences were seen indicating a favorable shift in the toxicity profile, including the characteristic GI-toxicity of doxorubicin, the lack of evidence of cardiotoxicity and myelosuppression. No significant effect on the kidney was observed.
[0199]
[Table 7]

[0200]
An example 19.Prodrugs are safer and more effective than comparisons
A significantly higher dose of Suc-βAla-Leu-Ala-Leu-Dox is administered, which achieves efficacy without significant toxicity in the LS-174T human colorectal cancer xenograft model compared to doxorubicin be able to. Suc-βAla-Leu-Ala-Leu-Dox at well-tolerated doses of 49 (Group 3), 57 (Group 4) and 65 mg / kg (Group 5) produced a rapidly growing adriamycin-resistant LS-174T Outstanding potency in inhibiting tumors (FIG. 5) and extending the survival of tumor-bearing mice (FIG. 12) compared to doxorubicin at 3.0 mg / kg (Group 2) and salt solution (Group 1) showed that. Dose limiting toxicity (cardiotoxicity and myelosuppression) was observed with repeated administration of doxorubicin in humans. Thus, a higher dose of Suc-βAla-Leu-Ala-Leu-Dox than our doxorubicin was administered, indicating that it favored tumor inhibition over systemic toxicity.
[0201]
An example 20.Prodrugs are useful for moderate doxorubicin-sensitive tumors
MX-1 tumors, moderately doxorubicin-sensitive human breast cancer xenografts, were implanted subcutaneously (SC) and at least once a week before the start of dosing (Day 0) and then during the study. Twice a week, mice were weighed and tumors were measured (by calipers). Immediately before the start of dosing (Study Day 2 to Day 0), mice were randomized into various groups based on tumor weight. Mice were euthanized after the tumor reached a cut-off weight (end point) of 1.5 g. The study was terminated on day 60.
[0202]
[Table 8]

[0203]
Control group tumors grew rapidly and all animals had terminated by day 24 at the cancer endpoint. The tumors showed relatively uniform growth characteristics, reaching cancer end-pons ranging from 15 to 24 days. Doxorubicin was effective in significantly extending the presence of tumor-bearing mice (Table 8). Dose responsiveness was established for Suc-βAla-Leu-Ala-Leu-Dox in both inhibiting MX-1 tumor growth and increasing mouse survival (FIG. 18 and Table 8). Suc-βAla-Leu-Ala-Leu-Dox at 71 mg / kg was significantly better than doxorubicin in inhibiting tumor growth and prolonging mouse survival (Table 8). All three dose regimes of Suc-βAla-Leu-Ala-Leu-Dox and doxorubicin were very well tolerated. Only one of the 10 mice in the Suc-βAla-Leu-Ala-Leu-Dox 71 mg / kg group had a weight loss of up to 20% towards the end of the study (after day 46).
[0204]
An example 21.Prodrugs are useful in evading multidrug resistance mechanisms
Suc-βAla-Leu-Ala-Leu-Dox has been shown to be more active than free doxorubicin on the MDR human cell line implanted in mice in a xenograft model. Doxorubicin supplied to tumors in denatured form, especially as a prodrug as Suc-βAla-Leu-Ala-Leu-Dox, is active in delaying tumor growth resulting in a significant prolongation of survival in that dose group .
[0205]
Table 9 and FIG. 15 show that for Suc-βAla-Leu-Ala-Leu-Dox, there is a dose-dependent increase in survival in MDR human colorectal cancer LS174-T. LS174T is a very aggressive and rapidly growing tumor that displays heterogeneous cell morphology with necrotic centers. Because it is very resistant to conventional chemotherapy and tumors that become well-established within a few days are always present in some animals, those animals try to inhibit tumor growth , And animals reach tumor endpoints despite treatment. Doxorubicin alone is completely inactive in this model and has no effect on tumor growth or viability.
[0206]
[Table 9]

[0207]
In particular, Table 9 provides a summary of the effects of Suc-βAla-Leu-Ala-Leu-Dox at three dose levels compared to doxorubicin in LS174T colorectal cancer xenografts in nude mice (Q7Dx5). The parameters measured are calculated mean survival days (MDS), number of long-term survivors (LTS), determined by termination, for tumors that reach a predetermined cutoff size of 1500 mg (tumor death), And tolerability of the dose regimen by the number of mice that show toxic death (> 20% loss of body weight). The number of LTS at day 60 was zero in all groups. No toxic death was observed in any of the groups. Statistical significance from vehicle control group:^*: P <0.05;^**: P <(unpaired t-test).
[0208]
Control group tumors grew rapidly and all animals had terminated by day 33 at the cancer endpoint. Tumors displayed heterogeneous growth characteristics and reached the cancer endpoint on days 15-33. Doxorubicin (3 mg / kg) was ineffective at inhibiting LS174T tumor growth or prolonging survival. Dose responsiveness was established for Suc-βAla-Leu-Ala-Leu-Dox in prolonging mouse survival (Table 9). Suc-βAla-Leu-Ala-Leu-Dox at 64 mg / kg was significantly better than doxorubicin in prolonging mouse survival (Table 9). Suc-βAla-Leu-Ala-Leu-Dox at 57 mg / kg significantly inhibited LS174T tumor growth (FIG. 15 and Table 9). All three dose regimes of Suc-βAla-Leu-Ala-Leu-Dox and doxorubicin were very well tolerated and there was no termination due to toxicity endpoint.
[0209]
Furthermore, Suc-βAla-Leu-Ala-Leu-Dox (but not doxorubicin) administered at three well-tolerated dose levels (Q7dx5) has a dose-dependent inhibitory effect on median tumor weight. (FIG. 15).
FIG. 15 shows the effect of Suc-βAla-Leu-Ala-Leu-Dox compared to doxorubicin on tumor growth of LS174T tumor colorectal cancer xenografts in nude mice and vehicle controls. Group D was statistically significantly different from the vehicle control group at day 19 (p <0.05).
These results suggest that local generation of active cytotoxicity at high concentrations in tumors may be the key to prodrug forms of therapeutics that overcome the MDR pathway.
[0210]
An example 22.β Ala − Leu − Ala − Leu − Dox Aggregation
Poorly soluble anthracycline drugs have been shown to form aggregates when prepared in aqueous buffers (Menozzi et al., "Self-association of doxorubicin and related compounds in aqueous solutions", . J. Pharmaceut Sci, 73:.. 766-770 (1984); Canfalonieri such as, "The use of new leser particle sizer and shape analyser to detect and evaluate gelatinous microparticles suspended in reconstituted anthracycline infusion solutions ", J. Pharmaceut. Biomed. Anal. 9: 1-8 (1991)).
[0211]
Evaluation of the size of βAla-Leu-Ala-Leu-Dox aggregates in an aqueous solution at 17.4 μM / ml was performed by using Amicon Centricon.^TM An attempt was made to filter through the filter unit. Doxorubicin (17.4 μM / ml) and βAla-Leu-Ala-Leu-Dox (17.4 μM / ml) were dissolved in distilled water, respectively, and 3,000, 10,000, 30,000 and 50,000 molecular weights Placed in a Centricon filter with cut-off (MWCO). Individual filter units were centrifuged at 1500 g for 2 hours. The amount of drug retained and passing through the filter was quantified at λ 475 nm and converted to%.
[0212]
Table 10 shows that 81% of the doxorubicin passed through the 3,000 MWCO filter and only 5% of the conjugate, βAla-Leu-Ala-Leu-Dox, passed the 3,000 MWCO filter. The data shows that 50,000 MWCO retains more than 40% of βAla-Leu-Ala-Leu-Dox. The data show that significant% of βAla-Leu-Ala-Leu-Dox aggregates are greater than 50 KD (> 50 molecules / aggregate). Therefore, βAla-Leu-Ala-Leu-Dox can aggregate under some conditions.
[0213]
[Table 10]

[0214]
An example 23.Β in mice Ala − Leu − Ala − Leu − Dox Intravenous infusion of
It is known that acute toxicity probably occurs through the interaction of positively charged polymers such as protamine, polylysine or their aggregates, and the luminal surface of blood vessels (Delucia et al., “Efficacy and toxicity of differentiation”. charged polycationic protamine-like peptides for heparin anticoagulation reversal ", J. Vase Surg 18:... 49-60 (1993); Ekrami such as," Carbamylation decrease the cytotoxicity but not the drug-carrier properties fo polylysines ", J. DrugTarg. 2: 469-475 (1994)).
[0215]
It has further been shown that heparin reduces the toxic effects of protamine sulfate on rabbit myocardium (Wakefield et al., "Heparin-mediated reductions of the toxic effects of protamine sulphate. -53 (1992)). To test the hypothesis that the acute toxicity seen here was due to positively charged prodrug aggregates, βAla-Leu-Ala-Leu-Dox (174 μM / ml) was compared to controls. At 4.000 I. U. Mice were given following 1 hour intravenous pretreatment with heparin. Table 12 shows that, following heparin, the so far acute lethal volume of βAla-Leu-Ala-Leu-Dox was significantly less toxic.
[0216]
These data support the hypothesis that acute toxicity is due to positively charged aggregates causing effects similar to those seen for protamine or polylysine. The negative and neutrally charged prodrugs of the present invention overcome this unwanted side effect.
[0217]
[Table 11]

[0218]
According to the previous hypothesis, the capping of the terminal amino groups of βAla-Leu-Ala-Leu-Dox by the negatively charged component is sufficient to complete the acute toxic effect at dose levels as high as 250 mg / kg (Dox-HCl). Quenching.
As evidence of this, in the relevant experiments, all animals had 3-5 mice per group with 250 mg / kg (Dox-HCl) of Suc-βAla-Leu-Ala-Leu-Dox or G1-βAla-Leu-Dox. Survived up to 8 days when treated with an intravenous bolus of Ala-Leu-Dox.
[0219]
How to analyze the remaining examples:
Peptide sequences synthesized using the solid or solution phase approach were used without further purification if analytical HPLC (Methods A, B & D) indicated a crude product with a purity of 80% or more. Otherwise, the material was purified using preparative HPLC method C.
[0220]
HPLC Method A:
Analytical HPLC analysis was performed using solvent A (0.1% TFA / H₂O) and solvent B (0.1% TFA / ACN) on a Waters 2690 using a C-18 column (4 μm, 3.9 × 150 mm ID, 1 ml / min flow rate), eluting with a gradient of The data was then processed at λ 254 nm using a Water Millennium system. The analytical HPLC gradient started with 90% solvent A and ended with (linear) 100% solvent B over 14 minutes. The purity of the compounds for this and the next method was evaluated as the relative% area under the peak curve.
[0221]
HPLC method B:
Analytical HPLC analysis was performed on a C-18 column (3) eluting with a gradient of solvent A (80% 20 mM ammonium formate and 20% acetonitrile) and solvent B (20% 20 mM ammonium formate and 80% acetonitrile). 0.5 μm, 4.6 × 150 mm ID, flow rate 1 ml / min) was performed on a Waters 2690, and the data was processed at λ 254 nm using a Water Millennium system. The analytical HPLC gradient started with 100% solvent A and ended with (linear) 100% solvent B over 14 minutes.
[0222]
HPLC Method C:
Purification for separation of the crude product is carried out using solvent A (H₂This was achieved with a Waters Delta Prep 4000 system using a C-4 column (15 μm, 40 × 100 mm ID, 30 ml / min flow rate) eluting with a gradient of O) and solvent B (MeOH). The preparative HPLC gradient starts with 80% solvent and progresses to 100% solvent B over 70 minutes (linear). Data was processed at λ254 nm using a Waters Millennium System.
[0223]
HPLC Method D:
Analytical HPLC was accomplished on a Hewlett Packard instrument using a TSK superODS column (TosoHaas); solvent A (0.1% TFA in water); solvent B (0.1% TFA in acetonitrile). Gradient: 30-36% B at 2 minutes, 36-41% B at 10 minutes, 41-90% B at 3 minutes, 90% B at 5 minutes, detection wavelength λ 254 nm.
[0224]
NMR and MS:
Additional structure determination was performed by NMR and MS techniques, and the results supported the compounds of the present invention.
TLC method:
TLC analysis showed DCM / MeOH / H for elution₂Performed on silica gel 60F-254 nm-0.25 mm plate (Merck) using O / 88% formic acid (85/15/1/2).
[0225]
Ninhydrin test:
A few milligrams of the product were introduced into the test and two drops of solution A (50 mg / ml ninhydrin in ethanol), two drops of solution B (4 mg / ml phenol in ethanol), and then two drops Solution C (2 ml of 0.01 M KSCN in 100 ml of pyridine) was added. The mixture was left in a boiling water bath for 5 minutes. In the presence of free amine, the solution turns purple.
[0226]
Example of specific oligopeptide synthesis:
Commercial reagent sources:
Doxorubicin and daunorubicin were obtained from Meiji (Japan) using Pd (PPh₃)₄Supplied by Strem Chem (Newburyport, Mass.), PEG by Shearwater (Huntsville, Alabama) and solvent HATU by Aldrich (Milwaukee, Wis.); (San Diego, CA), Advanced ChernTech (Louisville, KY), Peptide International (Louisville, KY) or SynPep (Dublin, CA).
[0227]
An example 24.Fmoc Β of shape Ala − Leu − Ala − Leu Benzyl ester
ΒAla-Leu-Ala-Leu in Fmoc form (24.34 g, 0.04 mol) was added to a round bottom flask with DMF (350 ml) and a magnetic stirrer. After dissolving the tetrapeptide, benzyl bromide (4.76 ml, 0.04 mol) was added to the solution with stirring, followed by cesium carbonate (13.04 g, 0.04 mol). The reaction mixture was stirred at room temperature for 1.5 hours. The reaction mixture was then slowly poured into a flask containing 450 ml of ice-cooled water. A large amount of white solid precipitated, which was collected by suction filtration. The product was washed with water (2 × 200 ml) and placed in a vacuum desiccator. The product (24.2 g, 87%) was identified by HPLC (purity: 95%). C₄₀H₅₀N₄O₇MS m / z calculated for: 69.4; Found: 699.5.
[0228]
An example 25.β Ala − Leu − Ala − Leu Benzyl ester
In a round bottom flask (25 ml), βAla-Leu-Ala-Leu benzyl ester in Fmoc form (0.7 g, 1.0 mmol) was dissolved in 5 ml of anhydrous DMF. Piperidine (1.2 ml, 12.1 mmol) was added to the solution and the mixture was stirred at room temperature for 24 minutes. The reaction was quenched with water (6ml) and extracted with ethyl acetate (2x10ml). The combined organic layers were further washed with water (2 × 5 ml), brine (5 ml) and dried over sodium sulfate. A white solid (0.8 g) was obtained after removal of the solvent. The purity of the product was only 67%. C₂₅H₄₀N₄O₅MS m / z calculated for 476.3; found 477.2.
[0229]
An example 26.Methylsuccinyl- N -Cap-shaped β Ala − Leu − Ala − Leu Benzyl ester
In a round bottom flask (250 ml), methyl hemisuccinate (3.198, 24.2 mmol) was dissolved in anhydrous DMF (50 ml). DIEA (4.22 ml, 24.2 mmol) was added to the solution, followed by HBTU (9.17 g, 24.2 mmol). The mixture was stirred at room temperature for 45 minutes. To this mixture was added a solution of βAla-Leu-Ala-Leu benzyl ester (crude, 10.14 g, 21.3 mmol) in anhydrous DMF (150 ml). The mixture was continuously stirred at room temperature for 2.5 hours.
[0230]
The reaction mixture was then slowly poured with stirring into a flask with 200 ml of ice-cold water. A large amount of white solid precipitated, which was extracted with ethyl acetate (3 × 200 ml). The combined organic layers were further washed with water (2 × 200 ml), brine (200 ml) and dried over sodium sulfate. A white solid was obtained after removal of the solvent. Recrystallization of this crude product in ethyl acetate yielded 7.53 g (60%) of the product with 80% purity. C₃₀H₄₆N₄O₈MS m / z calculated for: 591.4; found: 590.33.
[0231]
An example 27.Methylsuccinyl- N -Cap-shaped β Ala − Leu − Ala − Leu
ΒAla-Leu-Ala-Leu benzyl ester in methyl succinyl-N-cap form (1.0 g, 86% purity, 1.46 mmol) was added to an Erlenmeyer flask containing 100 ml of methanol. The solution became cloudy after several minutes of stirring. 50 ml of methanol were added, but the solution was not yet clear. The solution was transferred into a hydrogenation reactor. To this vessel was added Pd-C (90 mg, 10% wet, 50% water, 0.042 mmol). After hydrogenation at room temperature for 2 hours, the reaction was stopped and the catalyst was filtered. A white solid (0.77 g, 78%) was obtained after removal of the solvent. C₂₃H₄₀N₄O₈MS m / z calculated for: 501.2; found: 500.3.
[0232]
An example 28.N Synthesis of cap-allyl-hemisuccinate
This molecule is described in Casimir, J. et al. R etc. , Tet. Lett. 36 (19): Prepared according to the method of 3409, (1995). 10.07 g (0.1 mol) of succinic anhydride and 5.808 g (0.1 mol) of allyl-alcohol were refluxed in 100 ml of toluene for 6 hours. The reaction mixture was concentrated under reduced pressure (15.5 g; 98%). The resulting material was of sufficient purity for use in subsequent reactions. The purity and identity of the semi-solid product¹H NMR and^ThirteenIt was confirmed by C NMR and LC / MS.
[0233]
An example 29.Allyl-succinyl-β Ala − Leu − Ala − Leu − Dox Synthesis of
In a round bottom flask (50 ml), β-Ala-Leu-Ala-Leu in the N-cap-arylhemisuccinyl form (1 g, 1.9 mmol) and doxorubicin (1.1 g, 1.9 mmol) were added to anhydrous DMF (50 ml). ). After stirring the mixture for 5 minutes, DIEA (0.66 ml, 3.8 mmol) was added to the solution followed by HATU (0.76 g, 1.9 mmol) and the mixture was stirred at room temperature for 2 hours. did. DMF was removed by rotary evaporation and the residue was taken up in 1: 1 DCM: MeOH (4.0 ml). To this solution, 100 ml of ether was added slowly with stirring. A red precipitate formed and was collected by suction filtration. The solid was washed with ether (2 × 2 ml) and dried in a vacuum desiccator to give the allyl-succinyl-βAla-Leu-Ala-Leu-Dox therapeutic, which, according to Method B, was 90% It had HPLC purity.
[0234]
An example 30.Allyl-succinyl-β Ala − Leu − Ala − Leu From doxorubicin Suc −β Ala − Leu − Ala − Leu − Dox Preparation of
To a stirred solution of 0.1 g (0.095 mmol) of allyl-succinyl-βAla-Leu-Ala-Leu doxorubicin in 2 ml of THF, under a nitrogen atmosphere, 0.05 g (0.095 mmol) of tetrakis (triphenylphosphine) ) Palladium was added as a solid. After 10 minutes, the precipitate formed during the reaction was filtered and washed with THF. Dry weight: 0.1 g. The solid was determined to be succinyl-βAla-Leu-Ala-Leu-Dox by HPLC,¹1 H NMR, identified by LC / MS.
[0235]
An example 31.Fmoc Β of shape Ala − Leu − Ala − Leu Synthesis of
The Fmoc form of βAla-Leu-Ala-Leu was synthesized using a solid-phase approach with standard Fomc chemistry. A typical synthesis used Wang's alkoxy resin (0.60 mmol / g loading). Fomc-protected amino acids were used for solid phase peptide synthesis. For a 1 mM peptide scale on the resin, 3 equivalents of amino acids were preactivated with HBTU as activator 5 minutes before being added to the resin with 2 equivalents of DIEA. The coupling reaction was performed for 2 hours and then washed with DMF (25 ml × 3) and DCM (25 ml × 3). The coupling reaction was repeated with two equivalents of amino acid under similar conditions.
[0236]
The progress of the reaction was monitored using the ninhydrin test, and if the ninhydrin test showed an incomplete reaction after 2 hours, the coupling step was repeated three times. Deprotection was performed with 20% pipericin in DMF for 15-20 minutes. The coupling step was repeated with the next amino acid until the desired peptide was assembled on the resin. Final degradation of the peptide from the resin was achieved by treating the resin with a solution of 95% TFA and 5% water. After stirring the reaction mixture at room temperature for 2 hours, the resin was filtered under reduced pressure and washed twice with TFA. The filtrates were combined and the peptide was precipitated by adding 400 ml of cold ether. The peptide was filtered under reduced pressure and dried to give βAla-Leu-Ala-Leu in Fmoc form (94% HPLC purity by Method A). The crude peptide was used for the next step without further purification.
[0237]
An example 32.Fmoc Shaped Thi − Tyr − Gly − Len Synthesis of
The Fmoc form of Thi-Tyr-Gly-Len was synthesized using a solid phase approach with standard Fomc chemistry and Wang's alkoxy resin (0.60 mmol / g loading). Fomc-protected amino acids and Fomc-Thi-OH were used for solid phase peptide synthesis. For a 1 mM peptide scale on the resin, 3 equivalents of amino acids were preactivated with HBTU as activator 5 minutes before being added to the resin with 2 equivalents of DIEA.
[0238]
The coupling reaction was performed for 2 hours and then washed with DMF (25 ml × 3) and DCM (25 ml × 3). The coupling reaction was repeated with two equivalents of amino acid under similar conditions. The progress of the reaction was monitored using the ninhydrin test, and if the ninhydrin test showed an incomplete reaction after 2 hours, the coupling step was repeated three times. Deprotection was performed with 20% pipericin in DMF for 15-20 minutes. The coupling step was repeated with the next amino acid until the desired peptide was assembled on the resin.
[0239]
Final degradation of the peptide from the resin was achieved by treating the resin with a solution of 95% TFA and 5% water. After stirring the reaction mixture at room temperature for 2 hours, the resin was filtered under reduced pressure and washed twice with TFA. The filtrates were combined and the peptide was precipitated by adding 400 ml of cold ether. The peptide was filtered under reduced pressure and dried to give Thi-Tyr-Gly-Len in Fmoc form (88% HPLC purity by Method A). The crude Fmoc form of Thi-Tyr-Gly-Len was used for the next step without further purification.
[0240]
An example 33.Fmoc Β of shape Ala − Leu − Ala − Leu − Dnr Synthesis of therapeutic agents
Daunorubicin.HCl (185 mg, 0.329 mmol) and βAla-Leu-Ala-Leu in Fmoc form (200 mg, 0.32 mmol) were dissolved in anhydrous DMF (15 ml) at room temperature. To this rapidly stirred solution DIEA (0.115 ml, 0.658 mmol) was added in one portion and the reaction mixture was stirred at room temperature for 15 minutes. The reaction mixture was cooled to 0 ° C. using an ice bath and 138 mg (0.362 mmol) HATU was added slowly over 10 minutes.
[0241]
The reaction mixture was stirred at room temperature for a further 90 minutes. Ice-cooled water (200 ml) was added to the reaction mixture, resulting in the formation of a red precipitate. The precipitate was collected on a crude frit, washed with 3 × 50 ml of water and 3 × 50 ml of diethyl ether, and dried under reduced pressure to obtain the Fmoc form of βAla-Leu-Ala-Leu-Dnr therapeutic. (94% yield, 95% HPLC purity by Method A). This product was used for the next step without further purification.
[0242]
An example 34.Fmoc Shaped Thi − Tyr − Gly − Leu − Dnr Synthesis of therapeutic agents
Daunorubicin.HCl (90 mg, 0.16 mmol) and Thi-Tyr-Gly-Leu-Dnr in Fmoc form (120 mg, 0.16 mmol) were dissolved in anhydrous DMF (15 ml) at room temperature. To this rapidly stirred solution DIEA (0.56 ml, 0.16 mmol) was added in one portion and the reaction mixture was stirred at room temperature for 15 minutes. The reaction mixture was cooled to 0 ° C. using an ice bath and 61 mg (0.16 mmol) of HATU was added slowly over 10 minutes.
[0243]
The reaction mixture was stirred at room temperature for a further 90 minutes. Ice-cooled water (150 ml) was added to the reaction mixture, resulting in the formation of a red precipitate. The precipitate was collected on a crude frit, washed with 3 × 50 ml of water and 3 × 50 ml of diethyl ether, and dried under reduced pressure to obtain the Fmoc form of the Thi-Tyr-Gly-Leu-Dnr therapeutic. (94% yield, 91% HPLC purity by Method A). This product was used for the next step without further purification.
[0244]
An example 35.Fmoc −β Ala − Leu − Ala − Leu -Preparation of doxorubicin
Daunorubicin.HCl (3.0 g, 5.17 mmol) and Fmoc-βAla-Leu-Ala-Leu (3.15 g, 5.17 mmol) were dissolved in anhydrous DMF (230 ml) at room temperature under a nitrogen atmosphere. To this rapidly stirred solution DIEA (1.798 ml, 10.34 mmol) was added in one portion and the reaction mixture was stirred at room temperature for 15 minutes. The reaction mixture was cooled to about −2 ° C. in an ice / brine bath and 2.56 g (6.73 mmol) of HATU in 58 ml of DMF was added dropwise with stirring over 12 minutes.
[0245]
The reaction mixture was stirred at −2 ° C. for a further 30 minutes, then added in one portion of DIEA (0.285 ml, 1.64 mmol). Water (580 ml) at 0 ° C. was added to the reaction mixture, resulting in the formation of a fluffy red precipitate. The precipitate is collected on a coarse glass frit, washed with 3 × 50 ml of water and 3 × 50 ml of aqueous diethyl ether and air-dried for 16 hours, 5.12 g of βAla-Leu-Ala-Leu-Dox in Fmoc form. (89.7% yield, 90.23% HPLC purity by Method B).
[0246]
An example 36.Fmoc −β Ala − Leu − Ala − Leu − Dox Succinyl-β from Ala − Leu − Ala − Leu − Dox Preparation of
To a solution of 5.0 g (4.41 mmol) of Fmoc-βAla-Leu-Ala-Leu in 230 ml of anhydrous DMF at room temperature under nitrogen was added 21.8 ml (220 mmol) of piperidine all at once. It caused a color change to purple. The reaction mixture was stirred at room temperature for 5 minutes, then cooled to about -20 C in a dry ice / acetone bath. 22.5 g (0.225 mol) of succinic anhydride were added in one portion and the reaction temperature was kept below -5 ° C. After stirring at −10 ° C. to −5 ° C. for about 2 minutes, the color changed from purple to red / orange. The cooling bath was removed and the reaction mixture was stirred for 10 minutes. Next, the volume of the reaction mixture was reduced to about 100 ml by rotary evaporation and then diluted with 125 ml of chloroform.
[0247]
To this solution, 1400 ml of diethyl ether was quickly added, resulting in the formation of a red precipitate. The precipitate was isolated on a medium glass frit and triturated with 5 × 200 ml of diethyl ether to give 89.13% HPLC pure material. The precipitate was washed again with 1 × 20 ml of diethyl ether and air dried to give 3.62 g of Suc-βAla-Leu-Ala-Leu-Dox (81% physical yield, 88.2%). HPLC purity). This material is stirred at 0 ° C. in 30 ml of water and 33.98 ml (0.95 eq.) Of 0.1 M aqueous NaHCO 3₃Was added and the resulting suspension was stirred until all solids were dissolved. The solution was lyophilized to give 3.77 g of Suc-βAla-Leu-Ala-Leu-Dox (99% physical yield, 89.06% HPLC purity by Method B).
[0248]
An example 37.N -Β in capsuccinyl form Ala − Leu − Ala − Leu − Dnr -Synthesis of therapeutic agents
Piperidine (0.442 ml, 4.48 mmol) was added to a solution of βAla-Leu-Ala-Leu-Dnr in Fmoc form (100 mg, 0.089 mmol) in 5 ml of anhydrous DMF. The reaction mixture was stirred at room temperature for 5 minutes and then cooled to -20 ° C using a dry ice / acetone bath. Succinic anhydride (458 mg, 4.54 mmol) was added in one portion to the cooled reaction mass.
[0249]
The reaction was stirred rapidly at -5 ° C for 5 minutes, then at room temperature for a further 90 minutes. 250 ml of anhydrous diethyl ether were added to the reaction mixture and the resulting red precipitate was isolated on a medium glass frit. The filter cake was washed with two successive 50 ml portions of diethyl ether and dried under reduced pressure to give the β-Ala-Leu-Ala-Leu-Dnr-therapeutic in N-cap succinyl form (80%). Yield, 88% HPLC purity according to method B). LC / MS gave a molecular weight of 995 (expected molecular weight 996).
[0250]
An example 38.N -Cap succinyl form Thi − Tyr − Gly − Leu − Dnr Synthesis of therapeutic agents
To a solution of Thi-Tyr-Gly-Leu-Dnr (100 mg, 0.079 mmol) in Fmoc form in 5 ml of anhydrous DMF was added piperidine (0.391 ml, 3.95 mmol) in one portion and red to purple. Brought a change in color. The reaction mixture was stirred at room temperature for 5 minutes and then cooled to -20 <0> C using a dry ice / acetone bath. Then 407 mg (4.02 mmol) of succinic anhydride was added in one portion to the cooled mixture. The reaction was stirred rapidly at -5 ° C for 5 minutes, then at room temperature for a further 90 minutes. 200 ml of anhydrous diethyl ether was added to the reaction mixture, resulting in a red precipitate. The precipitate is isolated on a medium glass frit, washed with 3 × 50 ml of diethyl ether and dried under reduced pressure to give the N-cap succinyl form of the Thi-Tyr-Gly-Leu-Dnr therapeutic. (80% yield, 81% HPLC purity by Method A). LC / MS gave a molecular weight of 1141 (expected molecular weight of 1142).
[0251]
An example 39.N -Β of cap glutaryl form Ala − Leu − Ala − Leu − Dox Synthesis of sodium salt of therapeutic agent
Piperidine (436 μl, 4.413 mmol) was added to a solution of βAla-Leu-Ala-Leu-Dox in Fmoc form (100 mg, 0.088 mmol) in DMF (4.5 ml). After stirring at room temperature for 5 minutes, the reaction mixture was cooled to -5 ° C and glutamic anhydride (624 mg, 5.472 mmol) was added quickly. The cooling bath was removed as soon as the color changed and the mixture was stirred at room temperature for a further 10 minutes.
[0252]
DMF was removed by rotary evaporation, and the residue was dissolved in chloroform (2.5 ml). Diethyl ether (14 ml) was added and the resulting precipitate was filtered. The filter cake was washed with diethyl ether, air dried and then resuspended in water (14 ml). The sodium salt was formed by dropwise addition to a suspension of 0.025 M NaOH (4 ml, 0.10 mmol) until complete dissolution of the solid. The solution was then lyophilized to give the sodium salt of G1-βAla-Leu-Ala-Leu-Dox (97% yield, 87% HPLC purity by Method D).
[0253]
An example 40.
"Urea method" for preparing conjugates, ie precursors for enzymatic pathways
Methylsuccinyl- N -Cap-shaped β Ala − Leu − Ala − Leu And doxorubicin coupling
Under an atmosphere of anhydrous nitrogen, 26.04 g (52.0 mmol) of β-Ala-Leu-Ala-Leu in methyl succinyl-N-cap form, 23.26 g (40.2 mmol) of doxorubicin hydrochloride were added to 800 ml of anhydrous Suspended / dissolved in urea-saturated (about 30% w / v) DMF and 19.948 ml (114.16 mmol) DIEA. The mixture was cooled to 0-3 C over about 25 minutes.
[0254]
At this point, 21.2 g (56.0 mmol) of HATU was added over 10 minutes as a solution in about 100 ml of urea-saturated DMF (the volume of this solution should be kept to a minimum). The reaction mixture was stirred at −2 to 2 ° C. for 10 minutes and poured into 4000 ml of ice-cooled brine containing 2% (v / v) acetic acid for about 5 minutes with vigorous stirring. Was filtered on a medium porous glass filter, washed well with water and dried under reduced pressure. 43 g physical yield: 104.47%, HPLC purity 93.45% according to method B.
[0255]
An example 41.Methylsuccinyl- N -Cap-shaped β Ala − Leu − Ala − Leu − Dox Synthesis of therapeutic agents
In a round bottom flask (50 ml), N-cap-methylhemisuccinyl form of βAla-Leu-Ala-Leu (0.25 g, 0.5 mmol) and doxorubicin (0.29 g, 0.5 mmol) were added to anhydrous DMF. (20 ml). After the mixture was stirred for 5 minutes, DIEA (0.17 ml, 1.0 mmol) was added to the solution followed by HATU (0.19 g, 0.5 mmol). The mixture was stirred at room temperature for 4 hours. DMF was removed by rotary evaporation and the residue was taken up in 1: 1 methylene chloride: methanol (4.0 ml). To this solution, 40 ml of ether was added slowly with stirring. A red precipitate formed and was collected by suction filtration. The solid was washed with ether (2 × 10 ml) and dried in a vacuum desiccator. 0.50 g of the product (98%) was obtained.
[0256]
An example 42.MeOSuc −β Ala − Leu − Ala − Leu − Dox Of free doxorubicin from food
MeOSuc-βAla-Leu-Ala-Leu-Dox (200 mg, 0.194 mmol), DIEA (0.068 ml, 0.388 mmol) and anhydrous DMF (10 ml) were placed in a 50 ml flask equipped with a magnetic stir bar. did. When MeOSuc-βAla-Leu-Ala-Leu-Dox is completely dissolved, isocyanate resin (340 mg, 0.582 mmol; pre-swollen in 5 ml of dichloromethane for 5 minutes) is added and the resulting solution is allowed to stand at room temperature. For 2 hours with periodic HPLC monitoring. HPLC chromatography showed that Dox was completely removed by resin treatment within 45 minutes.
[0257]
Next, the reaction mixture was filtered through a frit to remove the resin. The resin was washed with 10 ml of DMF and the DMF washes were combined with the filtered reaction mixture. The filtered reaction mixture was then concentrated onto a red gum on a rotary evaporator equipped with a high vacuum pump and a 30 ° C. water bath. The red gum residue was suspended in 5 ml of DMF and then the solution was added slowly into a rapidly stirred solution of anhydrous diethyl ether. A red product is formed, which is then filtered on a frit and washed with diethyl ether and dried under reduced pressure to give MeOSuc-βAla-Leu-Ala-Leu-Dox (176 mg, 86% Yield).
[0258]
An example 43.Methyl succinyl- by using a cross-linked enzyme N -Cap-shaped β Ala − Leu − Ala − Leu − Dox Hydrolysis of therapeutic agents
The βAla-Leu-Ala-Leu-Dox therapeutic in methylsuccinyl-N-cap form (1.0 g, 0.975 mmol) and 100 ml DMF were placed in a 500 ml flask. The suspension was stirred vigorously with a magnetic stirrer. When the β-Ala-Leu-Ala-Leu-Dox therapeutic in methyl succinyl-N-cap form was completely dissolved, 400 ml of deionized water was added and the resulting solution was stirred at 35 ° C. A slurry of 1 g of washed CLEC-PC (Altus Biologics) immobilized enzyme was rinsed with 3 aliquots of deionized water and then resuspended in 10 ml of 20% aqueous DMF before use. The resulting suspension was stirred at 35 ° C. with regular HPLC monitoring.
[0259]
When all the methyl succinyl-N-capped form of βAla-Leu-Ala-Leu-Dox therapeutic has been consumed (about 18 hours), the reaction mixture is filtered through a 0.45 μM nylon membrane filter and subjected to CLEC- The PC enzyme was removed. The CLEC-PC cake was washed with 3 × 10 ml of methanol, and the methanol wash was combined with the filtered reaction mixture. The filtered reaction mixture and methanol wash were then concentrated to a red gum on a rotary evaporator equipped with a high vacuum pump and a water bath at 30 ° C. The red gum was then suspended in 50 ml of deionized water at room temperature and stirred vigorously through a mechanical stirrer.
[0260]
To this suspension was added a solution of 77.8 mg (0.926 mmol, 0.95 equiv) of sodium bicarbonate in 100 ml of deionized water over 2 minutes. The suspension was stirred at room temperature for 20 minutes. The reaction mixture was filtered through a 0.45 μM nylon membrane filter and ligated and dried. 0.936 g of the sodium salt of the βAla-Leu-Ala-Leu-Dox therapeutic in succinyl-N-cap form was isolated (approximately 100% yield; 84% HPLC purity by Method B).¹H and^ThirteenC NMR spectra were recorded on a spectrometer at 600 and 150 MHz, respectively, and on electrospray MS, which was consistent with the desired structure.
[0261]
An example 44.Methyl succinyl by using a soluble enzyme N -Cap-shaped β Ala − Leu − Ala − Leu − Dox Hydrolysis of therapeutic agents
11.0 g (10.72 mmol) of the methyl succinyl-N-cap form of βAla-Leu-Ala-Leu-Dox therapeutic is suspended in HPLC grade water and homogenized with an Ultraturrax T8 homogenizer for 60 minutes, A finely divided suspension was produced. The suspension was stirred at 35 ° C. (500 rpm) and adjusted to pH = 6.05 with 76 mM aqueous sodium bicarbonate. Next, 1.0 g of C.I. Antarctica "B" lipase (Altus Biologics) was added and the reaction mixture was stirred at 35 ° C for 48 hours. During the 48 hour reaction time, the pH was maintained between 5.3 and 6.2 by periodic addition of 76 mM sodium bicarbonate, and the reaction was monitored periodically by HPLC. After 48 hours, the reaction was about 98% complete by HPLC.
[0262]
The reaction mixture was then adjusted to pH = 7 with 76 mM aqueous sodium bicarbonate and filtered through a pad of Celite 521. The clarified reaction mixture was then acidified to a pH of about 3 with 5 ml of glacial acetic acid, resulting in the formation of a gummy red precipitate. The precipitate was isolated by filtration through Celite 521, subsequent rinsing of the celite pad with methanol, filtration of the methanol solution through a 10-20 μM glass filter and rotary evaporation of the filtered solution. 7.31 g of a rubbery red product were obtained. The product was converted to the sodium salt by dissolution in 70 ml of 76 mM sodium bicarbonate (0.95 eq) and lyophilized, giving 7.30 g (66.1% physical yield) of succinyl-N -The sodium salt of the beta-Ala-Leu-Ala-Leu-Dox therapeutic in capped form was obtained (84.5% purity by HPLC).
The product was identical to that of Example 45.
[0263]
An example 45.Methylsuccinyl- N -Cap-shaped β Ala − Leu − Ala − Leu − Dox Candida antarctica with fixed therapeutic agent B ”Lipase hydrolysis
30.0 g of Candida antarctica "B" lipase (Altus Biologics) was dissolved in 300 ml of water and dialyzed against 3 x 4 L of 50 mM aqueous sodium bicarbonate (pH = 6.4). After dialysis, the volume of the dialyzed solution was 300 ml. 360 ml of Pharmacia NHS-Activated Sepharose 4 Fast Flow was placed on a coarse glass filter and rinsed with 5 × 450 ml of ice-cold 1 mM aqueous HCl. The rinsed NHS-Activated Sepharose was combined with the dialyzed enzyme solution. The resulting suspension was stirred at ambient temperature (about 22 ° C) for 2.0 hours.
[0264]
The Sepharose / enzyme conjugate was isolated on a coarse glass filter and then stirred in 1000 ml of 100 mM aqueous TRIS (pH = 7.45) for 15 minutes. The suspension was filtered and incubated overnight at 4 ° C. with another 1000 ml of 100 mM aqueous TRIS buffer (pH = 7.45). In the morning, the immobilized enzyme was filtered and, after washing with water, placed in a 2000 ml three-necked round bottom flask. 43 g of β-Ala-Leu-Ala-Leu-Dox in methylsuccinyl-N-cap form was added and the solid was suspended in 800 ml of deionized water.
[0265]
The flask was fitted with an overhead stirrer and a pH-stabilizer set to maintain the pH of the reaction mixture at 5.9-6.2 by adjusting the syringe pump. The syringe pump was filled with 0.1 M sodium bicarbonate. The progress of the reaction was followed by HPLC. After 6 days, the immobilized enzyme was filtered and the solution phase was lyophilized. Next, the dried solid was suspended in about 11 ml of anhydrous THF and filtered. 42.66 g, 98.34% physical yield, 93.45% (254 nm), 94.43% (480 nm) HPLC purity by Method B.
[0266]
An example 46.β Ala − Leu − Ala − Leu − Dox Lactate [β Ala − Leu − Ala − Leu − Dox Of lactate]
Piperidine (26 ml, 264 mmol) was added to a solution of the β-Ala-Leu-Ala-Leu-Dox therapeutic in Fmoc form (6.00 g, 5.3 mmol) in DMF (265 ml). After stirring at room temperature for 5 minutes, the reaction mixture was placed in an ice-salt bath and precooled (4 ° C.) 10% lactate buffer (pH 3) (600 ml) was added immediately. The aqueous solution was extracted with DCM (3 × 500 ml) and excess salts were removed by solid phase extraction.
[0267]
C18 ODS-A silica gel (120 g) was conditioned in a glass frit (500 ml methanol, 2 × 500 ml water) and in an aqueous solution of the crude lactate (500 ml methanol, 2 × 500 ml). Water) and an aqueous solution of the crude lactate. After washing with water (2 × 500 ml) and drying, the filter cake was dissolved in methanol. The methanol was evaporated and the residue was dissolved in water. The resulting solution was lyophilized to give 3.54 g of the lactate salt of the βAla-Leu-Ala-Leu-Dox therapeutic (67% yield, 89% HPLC purity by Method B).
[0268]
An example 47.β Ala − Leu − Ala − Leu − Dox Succinyl- starting from the lactate of the therapeutic agent N -Cap-shaped β Ala − Leu − Ala − Leu − Dox Synthesis of therapeutic agents
DIEA (417 μl, 2.40 mmol) was added to a solution of the lactate salt of the βAla-Leu-Ala-Leu-Dox therapeutic (1.200 g, 1.20 mmol) in DMF (35 ml). After stirring for 15 minutes at room temperature, 97% succinic anhydride (0.144 g, 1.44 mmol) was added. The mixture was stirred for 2 hours and DMF was removed by rotary evaporation.
[0269]
The residue was washed with CHCl₃/ CH₃Dissolved in a mixture of OH (4/1) (6 ml) and 1: 1 Et₂200 ml of a mixture of O: hexane was added. After stirring the mixture for 30 minutes, the precipitate was filtered on a metering paper (Whatman 42) and washed (1: 1 Et.sub.2).₂O: hexane) and air dried. The filter cake was suspended in water (150 ml) and 1 M NaOH (± 1.2 eq, 1.5 ml) was added dropwise until complete dissolution (pH = 7.2). The solution was lyophilized and 1.218 g of succinyl-N-capped βAla-Leu-Ala-Leu-Dox therapeutic (97% yield; HPLC purity by Method B: 80.2%).
[0270]
An example 48.Fmoc Β of shape Ala − Leu − Ala − Leu − Dox Succinyl- starting from a therapeutic agent N -Cap-shaped β Ala − Leu − Ala − Leu − Dox Synthesis of therapeutic agents
Piperidine (2180 μl, 22.06 mmol) was added to a solution of βAla-Leu-Ala-Leu-Dox therapeutic in Fmoc form (0.50 g, 0.44 mmol) in DMF (21.5 ml). After stirring at room temperature for 5 minutes, the reaction mixture was quickly cooled to −5 ° C., and succinic anhydride (2.25 g, 22.51 mmol) was added immediately. The cold bath was removed as soon as the color changed, and the mixture was stirred at room temperature for 10 minutes.
[0271]
DMF was removed by rotary evaporation, and the residue was dissolved in chloroform (12.5 ml. 9. Diethyl ether (360 ml.) Was added quickly. A precipitate appeared immediately. The precipitate was filtered over Whatman 42 paper and Et.₂Washed with O. The solid was suspended in water (120 ml, pH = 4.1) and 0.025 M NaOH (20 ml, 0.53 mmol) was added dropwise until complete dissolution (pH = 7.4). The solution was then lyophilized to give the βAla-Leu-Ala-Leu-Dox therapeutic in succinyl-N-cap form (89% yield; and 91% HPLC purity by Method D).
[0272]
An example 49.Methylsuccinyl- N -Cap β Ala − Leu − Ala − Leu − Dox Large-scale synthesis of therapeutic agents
69.6 g of doxorubicin.HCl (120 mmol) and 100 g of MeOSuc-βAla-Leu-Ala-Leu (199 mmol) were dissolved in anhydrous DMF (10 L) at room temperature. 76 ml of DIEA (434 mmol) was added to the reaction mixture, and the reaction mixture was stirred at room temperature under nitrogen for 10 minutes. Next, the reaction mixture was cooled to 0 ° C. over 10 minutes. In a separate flask, a solution of 864 g HATU (220 mmol) in DMF (500 ml) was separated. The HATU solution was added slowly to the reaction mixture over 20 minutes while maintaining the mixture at 0 ° C. The reaction mixture was stirred at 0 ° C. for 30 minutes.
[0273]
A solution of NaCl (7.5 kg, at least 30% w / v) in water (25 L) was separated and cooled to 0 ° C. The reaction mixture was then added slowly to the cooled brine solution over 120 minutes with stirring. The color of the solution remained red and the blue solution indicated that the pH needed to be adjusted immediately to 5.8-6.0 by addition of acetic acid. The temperature was maintained at about 5 ° C. The red precipitate is filtered on a medium porous glass filter, washed with water and₂O₅The above was dried under vacuum to obtain 115 g of MeOSus-βAla-Leu-Ala-Leu-Dox.
[0274]
An example 50.To remove trace amounts of doxorubicin Ps -By isocyanate beads MeOSuc −β Ala − Leu − Ala − Leu − Dox Processing
146.4 PS-isocyanate bead (240 mmol; supplied by Argonaut Lab, San Carlos, Calif.) Was dissolved in 1.5 L of anhydrous DMF and swelled at room temperature for 5-10 minutes. The swollen beads were filtered through a glass filter and washed with an additional 500 ml of anhydrous DMF. 115 g of MeOSuc-βAla-Leu-Ala-Leu-Dox (112 mmol) was dissolved in 1000 ml of anhydrous DMF and 2.1 ml of DIEA (12 mmol) was added, followed by swollen PS-isocyanate beads. . The reaction mixture was stirred at room temperature and monitored using HPLC until the amount of doxorubicin peak was less than 0.1%.
[0275]
It took between 2 and 12 hours, depending on the size of the batch. HPLC analysis was performed using a Water 2690 column: Water Symmetry shield C₈ 3.5 μM 4.6 × 150 mm (catalog number WAT094269), solvent: A-80% 20 mM ammonium formate (pH = 4.5), 20% acetonitrile, solvent: B-20% ammonium formate (pH = 4. 5), 80% acetonitrile. Column temperature: adjusted room temperature, sample temperature; 4 ° C., operation time: 37.5 minutes, detector: 254 nm, flow rate: 1.0 ml / min, injection amount 10 μg (0.5 mg / ml × 0.02 ml), Mobile phases A and B. Gradient: 37.5 minute wash gradient from 100% mobile phase to 100% mobile phase B (with 7.5 minute equilibration delay).
[0276]
At 6 hours when the amount of doxorubicin peak was less than 0.1%, the reaction mixture was filtered through coarse glass filtration to remove beads. A solution of 1.1 kg of NaCl in brine (at least 30% w / v) in 3.5 l of water was prepared and cooled to 0 ° C. The filtered reaction mixture was then added slowly to the cooled brine solution with stirring over 45 minutes. The color of the solution remained red, and a blue solution indicated that the pH needed to be immediately adjusted to 5.8-6.0 by addition of acetic acid. The red precipitate is filtered through a medium fritted filter, washed with water and₂O₅Drying under reduced pressure yielded MeOSuc-βAla-Leu-Ala-Leu-Dox without any residual doxorubicin.
[0277]
MeOSuc-βAla-Leu-Ala-Leu-Dox was dissolved in IL in MeOH and the methanol solution was then added slowly with stirring to 14 L of cooled ethyl ether over 60 minutes. The red precipitate was filtered through a medium glass funnel, washed with ether (1 L) and dried under vacuum to give 110 g of MeOSuc-βAla-Leu-Ala-Leu-Dox. Purity was determined to be 96.5% by HPLC as described in Example 44. C₅₀H₆₇N₅O₁₈MS / z calculated for: 1025; found: 1048 (M⁺+ Na).
[0278]
An example 51.Suc −β Ala − Leu − Ala − Leu − Dox To generate MeOSuc −β Ala − Leu − Ala − Leu − Dox Enzymatic hydrolysis of
CLEC-CAB (Candida Antartica "B" lipase) enzyme is purchased in solution form (from Altus Biologics., Boston, Mass.), Where the concentration of the enzyme is defined by the weight of dry enzyme per ml of solution. The crude enzyme suspension was shaken for several minutes to obtain a homogeneous solution. 504 ml (329 mmol) of this homogeneous solution was aliquoted into the flask. 2.5 L of deionized water was added and the slurry was stirred using a magnetic stirrer for 10 minutes. The enzyme solution was filtered using a coarse glass filter without drying the enzyme. The enzyme was returned to the flask. The enzyme was suspended in water and filtered three more times.
[0279]
The enzyme cake was resuspended in 550 ml of deionized water and transferred to an RB flask. To this suspension was added 109 g of MeOSuc-βAla-Leu-Ala-Leu-Dox (105 mmol) and the reaction mixture was stirred at room temperature (25 ° C.). The pH of the reaction mixture was adjusted to 1N NaHCO₃The pH was maintained at 5.8-6.1 by means of a pH device equipped with a syringe pump filled with the solution. The progress of the reaction was monitored by a regular HPLC monitor as described in Example 44. After 24 hours, the reaction appears to be 94% complete as determined by HPLC.
[0280]
Additional CLEC enzyme was required after 24 hours to speed up the reaction. 168 ml of CLEC enzyme (homogeneous solution) was washed in column form as described above. The enzyme cake was resuspended in 1.1 L of deionized water and added to the reaction. The reaction mixture was stirred at room temperature, while being monitored by definition by HPLC, and the pH was maintained between 5.8 and 6.1. After 60 hours, the reaction was 99.9% complete as monitored by HPLC.
[0281]
CLEC enzyme was removed from the reaction mixture by filtration through a 0.2 μM filter and rinsed by 500 ml deionization. The filtration was then lyophilized to give 95.2 g of Suc-βAla-Leu-Ala-Leu-Dox.Na (87% physical yield; C₄₉H₆₅N₅O₁₈MS m / z calculated for: 1011; found: (M⁺+ Na).
The prodrug compound Suc-βAla-Leu-Ala-Leu-Dox was well characterized by mass spectrometry, FTIR, NMR.
The mass spectrum analysis of Suc-βAla-Leu-Ala-Leu-Dox was performed using Suc-βAla-Leu-Ala-Leu-Dox (C₄₉H₆₅N₅O₁₈The presence of the molecular ion peak (m / z; 1034) is clearly shown, which is compatible with the calculated m / z for Na) (1033).
[0282]
A sample of Suc-βAla-Leu-Ala-Leu-Dox was also analyzed by FTIR. The spectrum matched that of the control standard for the material. The assignments for the main absorption rates are as follows:
Hydroxyl 3379cm^-1
CH 3000-2700
Carbonyl @ 1650-1725
[0283]
Finally, the samples were analyzed by NMR. Chemical shifts and assignments are listed in Table 13 and shown in FIG. There are three carbons in the ketone region at 215.2, 187.7, 187.4 ppm, consistent with the structure of Suc-βAla-Leu-Ala-Leu-Dox. The latter two have similar chemical shifts and are therefore assigned to (2) and (3) at 187.7 and 187.4 ppm. Therefore, the remaining ketone is (1). Further evidence from their assignment arises from the HMQC and HMBC spectra; three of them do not show any HMQC peaks, so they are not protonated, and only (1) at 4.77 ppm It has a wide range of CH couplings (HMBCs) to protons, which is a proton singlet and therefore this is (4). From the HMQC spectrum, the carbon at 65.7 ppm is linked to their protons.
[0284]
At 3.96 ppm¹The 1 H NMR signal has regions of chemical shifts and methoxy groups; their ptrons are coupled to carbon at 57.2 ppm and assigned to the only methoxy in structure (5).^ThirteenThe C chemical shift is also consistent with methoxy. The broad CH coupling of the proton is to carbon at 162.3 ppm, which should be (6).
[0285]
The HMQC spectrum shows that there are three protonated aromatics at 120.5, 120.3 and 137.2 ppm; Did not show any coupling between the protons. The aromatic proton signal is very broad, which indicates that a short T₂Indicates relaxation time. If a deletion of this coupling is present, it is not possible to assign these three sites uniquely and collectively to (7), (8) and (9).
[0286]
The two unprotonated aromatic carbons at 157.2 and 156.1 ppm have chemical shifts consistent with (10) and (11), ie, the aromatic carbon bonded to oxygen. No extensive coupling is observed.
At 112.3 and 112.0 ppm^ThirteenThe C NMR signal is consistent with the aromatic carbon and is assigned to (2) and (13). In 121.3^ThirteenThe C NMR signal also shows this effect and is consequently assigned to (14). The remaining three unprotonated aromatic carbons are assigned in that region to the last three carbons of (15), (16) and (17).
[0287]
Deletion of any of the widespread CH couplings to any of the aromatic carbons is not expected, and the coupling will be short T₂Indicates very small or non-existent due to relaxation time or planar shape.
There are six carbonyl carbons at 181 to 174 ppm; of them, one at 180.6 ppm is the only one with a chemical shift consistent with the sodium salt, and is therefore assigned to (18) . This peak shows an unresolved broad CH coupling to the proton at 2.4 ppm. The remaining five carbonyl carbons are all within the 1 ppm chemical shift range and are not possible (19), (20), (21), (22), (23).
[0288]
The carbon at 102.3 ppm has a chemical shift consistent with the carbon bound to two separate oxygens, which is assigned to (25). This proton is coupled to carbon at 30.6 ppm. Of the carbon in the CO region (80-60 ppm), only one should be protonated at 77.4 ppm, resulting in (26). It does not have extensive C—H coupling to either protons or carbon.
[0289]
At 80-60 ppm, where all methine is bound to the enzyme, there are three carbons that have not yet been assigned. They all have similar chemical shifts (71.2, 69.9 and 68.8 ppm), but the carbon at 68.8 ppm shows extensive coupling to methyl at (28). Seems obvious. The carbon at 69.9 also shows extensive coupling to methyl at (28), so that it should be adjacent to (27) and assigned to (29). Therefore, the remaining methine is (30).
[0290]
The proton at 3.6 ppm (29) shows a broad CH coupling to only one carbon at 47.2 ppm. The only adjacent unassigned carbon should be (31). The (31) protons overlap other protons, and extensive correlation is not possible.
The remaining four methyls are in the isopropyl region, and one is at 1.25 / 1.34 ppm and should correspond to the last remaining methyl (32). This methyl proton should be (33). Extensive coupling to only one carbon at 51.3 ppm is shown. The (33) putron strictly overlaps with other protons and cannot be used due to any extensive correlation.
[0291]
All four remaining methyls should come from isopropylmethyl, ie, collectively labeled (34). The proton of (34) shows extensive coupling to the carbon between paired methyls and at 25.9 / 25.8 and 41.6 / 41.7 ppm; methine has (35) ) (36) and methylene is assigned to (37) (38). All of those protons overlap at 1.5-1.8 ppm, but show extensive coupling to methinein at 54.7 / 53.5 ppm, which is methineine adjacent to the amide, Then, (39) and (40) are assigned.
[0292]
The remaining five carbons, all methylene, exhibit extensive coupling to the carbonyl, so that they should be adjacent to such a carbonyl and assigned to (41), (42) and (43) ;¹All 1 H NMR chemical shifts overlap at 2.4 ppm and correspond to carbon at 37.2, 34.4 and 33.8 ppm. Since the carbon at 37.2 is the most different, it is assigned to the sodium salt carbonyl (41), and the other two are assigned to the amidocarbonyl (42), (43). The remaining two methylenes are too similar for the specific assignments (44), (45).
There is one site that cannot be assigned. There are 30 protons in the 5.5-1.5 ppm region, consistent with the structure, encompassing this assigned site. Thus, the carbon signal appears to be hidden under the solvent signal at about 50 ppm, consistent with the methylene adjacent to the nitrogen.
[0293]
[Table 12]

[0294]
All publications and patent applications cited herein are cited to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Hereby incorporated by reference.
While the present invention is now fully described, it will be apparent to those skilled in the art that many changes and modifications can be made within the scope of the invention.
[Brief description of the drawings]
FIG.
1A-1D are tables of abbreviations, names and structures.
FIG. 2
FIG. 2 is an exemplary scheme for the degradation of a prodrug of the present invention near and outside of target cells.
FIG. 3
FIG. 3 shows the synthesis of Fmoc-βAla-Leu-Ala-Leu, a typical intermediate of the present invention.
FIG. 4
FIG. 4 shows the “Fmoc-route” synthesis of methyl-succinyl-βAla-Leu-Ala-Leu, a typical intermediate of the present invention.
FIG. 5
FIG. 5 shows the “Fmoc-route” synthesis of the salt form of Suc-βAla-Leu-Ala-Leu-Dox, a typical compound of the invention.
FIG. 6
FIG. 6 shows the “ester-route” synthesis of the salt form of Suc-βAla-Leu-Ala-Leu-Dox, a typical compound of the invention.
FIG. 7
FIG. 7 shows the synthesis of an amino-protected βAla-Leu-Ala-Leu-Dox, a typical intermediate of the present invention.
FIG. 8
FIG. 8 shows the “allyl ester route” of the salt form of Suc-βAla-Leu-Ala-Leu-Dox, a typical chemistry of the invention.
FIG. 9
FIG. 9 shows the “resin pathway” of Suc-βAla-Leu-Ala-Leu-Dox, a typical compound of the present invention.
FIG. 10
FIGS. 10A-10C are a table of oligopeptides useful in the prodrugs of the present invention.
FIG. 11
FIG. 11 is a graph of viability in a mouse xenograft model for animals receiving a vehicle with or without a drug.
FIG.
FIG. 12 is a graph of viability in a mouse xenograft model comparing doxorubicin prodrug and doxorubicin.
FIG. 13
FIG. 13 is a graph of the activation and then inhibition of HeLa cell troase by increasing the concentration of DTT.
FIG. 14
FIG. 14 illustrates the removal of free therapeutic agent through the use of scavenging resin or beads.
FIG.
FIG. 15 is a graph of tumor growth inhibition in a mouse xenograft model.
FIG.
FIG. 16 shows a large scale synthesis of MeOSuc-βAla-Leu-Ala-Leu-Dox, a typical intermediate of the present invention.
FIG.
FIG. 17 shows the NMR assignment for a typical compound of the invention, MeOSuc-βAla-Leu-Ala-Leu-Dox.
FIG.
FIG. 18 shows a comparison of the effects of Suc-βAla-Leu-Ala-Leu-Dox and doxorubicin compounds and vehicle on the growth of MX-1 human breast cancer tumors in female nude mice.

Claims (79)

(1) a therapeutic agent that can be inserted into target cells,
(2) Formula (AA) _n -AA ⁴ -AA ³ -AA ² -AA ¹ :
Wherein each AA independently represents an amino acid,
n is an integer of 0 to 16,
AA ⁴ represents an amino acid that is not genetically encoded;
AA ³ represents any amino acid,
AA ² represents any amino acid, and AA ¹ represents any amino acid.
(3) a stabilizing group, and (4) optionally a linker group that cannot be degraded by TOP;
Wherein the oligopeptide is directly linked to the stabilizing group at a first binding site of the oligopeptide, and the oligopeptide is linked directly to the therapeutic agent. Or indirectly attached to the therapeutic agent at the second binding site of the oligopeptide through the linker group,
The compound wherein the stabilizing group prevents degradation of the compound by enzymes present in whole blood and the compound can be degraded by TOP. The compound according to claim 1, wherein n is an integer of 0 to 8. The compound according to claim 1, wherein the target cell is a tumor or an inflammatory cell. 2. The compound of claim 1, wherein said TOP is in the extracellular vicinity of a target cell for said therapeutic agent. The TOP is a compound of claim 1 wherein decomposing a bond between the AA ³ and AA ² of the oligopeptide. A prodrug having an active moiety, wherein the active moiety of the prodrug is more capable of entering the target cell after degradation by TOP than before degradation by TOP, wherein the active moiety is at least the therapeutic agent. 2. The compound according to claim 1, comprising: 7. The compound of claim 6, wherein the active portion of said prodrug comprises said therapeutic agent. 7. The compound of claim 6, wherein the active portion of the prodrug comprises the therapeutic agent and at least the linker group. Active portion of the prodrug, the therapeutic agent, and a compound of claim 6, further comprising a AA ¹ of the oligopeptide. The pro-active portion of the drag further compound of claim 9, wherein comprising AA ² of the oligopeptide coupled to AA ^1. The oligopeptides are D-AlaThβAlaβAlaLeuAlaLeu (SEQ ID NO: 1), ThiβAlaβAlaLeuAlaLeu (SEQ ID NO: 2), βAlaβAlaLeuAlaLeu (SEQ ID NO: 3), βAlaAlaAlaIle (SEQ ID NO: 4), βAlaAlaLaElaH SEQ ID NO: 7), βAlaPheGlyIle (SEQ ID NO: 8), βAlaPheGlyLeu (SEQ ID NO: 9), βAlaPhePhePhe (SEQ ID NO: 10), βAlaPhePheIle (SEQ ID NO: 11), βAlaPhePheLeu (SEQ ID NO: 12), βAla (SeI) No. 14), ThiGlyA laLeu (SEQ ID NO: 15), NalGlyAlaLeu (SEQ ID NO: 16), βAlaLeuTyrLeu (SEQ ID NO: 17), βAlaLeuThuLeu (SEQ ID NO: 18), βAlaLeuThrPhe (SEQ ID NO: 19), βAlaLeuThrIle (SEQ ID NO: Lu, AlA) (SEQ ID NO: 22), βAlaLeuPyrLeu (SEQ ID NO: 23), βAlaLeuLeuLeu (SEQ ID NO: 24), βAlaLeuGlyPhe (SEQ ID NO: 25), βAlaLeuGlyIle (SEQ ID NO: 26), ThiLeuGlyLeu (SEQ ID NO: 27), AyLuGuL (GluLu) SEQ ID NO: 29), βAlaLeuPheIle (SEQ ID NO: 0), βAlaLeuPheLeu (SEQ ID NO: 31), βAlaLeuAibLeu (SEQ ID NO: 32), βAlaLeuAlaAla (SEQ ID NO: 33), βAlaLeuAlaβAla (SEQ ID NO: 34), βAlaLeuAlaPhe (SEQ ID NO: 35), βAlaLeuAlaGly (SEQ ID NO: 36), βAlaLeuAlaIle (SEQ ID NO: 37 ), ΒAlaLeuAlaLeu (SEQ ID NO: 38), TicLeuAlaLeu (SEQ ID NO: 39), ThzLeuAlaLeu (SEQ ID NO: 40), ThiLeuAlaLeu (SEQ ID NO: 41), NalLeuAlaLeu (SEQ ID NO: 42), NAALeuAlaLeu (Sequence ID No. 44), D-AlaLeuAlaLeu (SEQ ID NO: 45), D-Me LeuAlaLeu (SEQ ID NO: 46), APPLeuAlaLeu (SEQ ID NO: 47), AmbLeuAlaLeu (SEQ ID NO: 48), βAlaLeuAlaNal (SEQ ID NO: 49), βAlaLeuAlaSer (SEQ ID NO: 50), βAlaLeuAlaTyr (SEQ ID NO: 52Met), βAlaLeuAlaTyr (SEQ ID NO: 53), βAlaMetGlyIle (SEQ ID NO: 54), ThiMetGlyLeu (SEQ ID NO: 55), βAlaMetPhePhe (SEQ ID NO: 56), βAlaMetPheIle (SEQ ID NO: 57), TicMetAlaLeu (SEQ ID NO: 58), NalMetAlaLeuM (Sequence No. SEQ ID NO: 60), βAlaMetAlaLeu (sequence SEQ ID NO: 61), APPMeAlaLeu (SEQ ID NO: 62), βAlaNleTyrIle (SEQ ID NO: 63), βAlaNleTyrLeu (SEQ ID NO: 64), βAlaNleThrIle (SEQ ID NO: 65), βAlaNleThrLeu (SEQ ID NO: 66), βAlaNleGlyPhe (Sequence No. 68), βAlaNleGlyLeu (SEQ ID NO: 69), βAlaNlePheIle (SEQ ID NO: 70), βAlaNleAlaIle (SEQ ID NO: 71), βAlaNleAlaLeu (SEQ ID NO: 72), βAlaNleAlaPhe (SEQ ID NO: 73), βAlaNvaAlaLeu, SEQ ID NO: 74 ), ThiProGlyLeu (SEQ ID NO: 76), Thi RoAlaLeu (SEQ ID NO: 77), NalProAlaLeu (SEQ ID NO: 78), βAlaProAlaLeu (SEQ ID NO: 79), βAlaPhe (Cl), AlaLeu ( SEQ ID NO: _{80), βAlaPhe (NO 2)} , AlaIle ( SEQ ID NO: 81), βAlaPhe (NO ₂ ), AlaLeu (SEQ ID NO: 82), βAlaPhgAlaLeu (SEQ ID NO: 83), βAlaPyrAlaLeu (SEQ ID NO: 84), TicThrGlyLeu (SEQ ID NO: 85), βAlaThiGlyIle (SEQ ID NO: 86), βAlaThiAlaLeu (SEQ ID NO: 87), (SEQ ID NO: 87) , ΒAlaTicAlaLeu (SEQ ID NO: 89), βAlaValAlaLeu (SEQ ID NO: 90), βAlaTrpAlaLeu (SEQ ID NO: 90) 91), βAlaTyrTyrPhe (SEQ ID NO: 92), βAlaTyrTyrIle (SEQ ID NO: 93), βAlaTyrTyrLeu (SEQ ID NO: 94), βAlaTyrThrLeu (SEQ ID NO: 95), βAlaTyrPheLeu (SEQ ID NO: 96), βAlaTyrGlyTyGly ), ΒAlaTyrGlyLeu (SEQ ID NO: 99), βAlaTyrPheIle (SEQ ID NO: 100), βAlaTyrAlaIle (SEQ ID NO: 101), ThiTyrAlaLeu (SEQ ID NO: 102), and βAlaTyrAlaLeu (SEQ ID NO: 103). . AA ^{1 of the} oligopeptide is leucine, phenylalanine, isoleucine, alanine, glycine, tyrosine, 2-naphthylalanine, serine, p-Cl-phenylalanine, p-nitrophenylalanine, 1-naphthylalanine, threonine, homoserine, cyclohexylalanine, The compound according to claim 1, wherein the compound is selected from the group consisting of thienylalanine, homophenylalanine, norleucine and β-alanine. Wherein AA ² of the oligopeptide, alanine, leucine, tyrosine, glycine, serine, 3-pyridylalanine, thienylalanine, norleucine, homoserine, homophenylalanine, p-Cl @ - phenylalanine, p- nitro-phenylalanine, aminoisobutyric acid, threonine The compound of claim 1, wherein the compound is selected from the group consisting of: Wherein AA ³ of the oligopeptide, leucine, tyrosine, phenylalanine, p-Cl @ - phenylalanine, p- nitrophenylalanine, valine, norleucine, norvaline, phenylglycine, tryptophan, tetrahydroisoquinoline-3-carboxylic acid, 3-pyridylalanine, alanine 2. The compound of claim 1, wherein the compound is selected from the group consisting of, glycine, thienylalanine, methionine, valine and proline. The AAA ⁴ oligopeptide, beta-alanine, thiazolidine-4-carboxylic acid, 2-thienyl alanine, 2-naphthylalanine, D- alanine, D- leucine, D- Ichionin, D- phenylalanine, 3-amino-3 2. The composition of claim 1, wherein the compound is selected from the group consisting of -phenylpropionic acid, [gamma] -aminobutyric acid, 3-amino-4,4-diphenylbutyric acid, tetrahydroisoquinoline-3-carboxylic acid, 4-aminomethylbenzoic acid and aminoisobutyric acid. Compound. The compound according to claim 1, wherein the stabilizing group is a dicarboxylic acid or a higher carboxylic acid. The stabilizing group is succinic acid, adipic acid, glutaric acid, phthalic acid, diglycolic acid, fumaric acid, naphthalenedicarboxylic acid, pyrocurutamic acid, acetic acid, 1-naphthylcarboxylic acid, 2-naphthylcarboxylic acid, 1,8- The compound of claim 1, wherein the compound is selected from the group consisting of naphthyldicarboxylic acid, aconitic acid, carboxycinnamic acid, triazoledicarboxylic acid, gluconic acid, 4-carboxyphenylboronic acid, polyethylene glycolic acid, butanedisulfonic acid, and maleic acid. 2. The compound of claim 1, wherein said stabilizing group is a non-genetically encoded amino acid having 4 or more carbon atoms. The method according to claim 1, wherein the stabilizing group is one of aspartic acid bound to the oligopeptide at the β-carboxy group of aspartic acid, or glutamic acid bound to the oligopeptide at the γ-carboxy group of glutamic acid. Compound. 2. The compound of claim 1, wherein said stabilizing group is negatively charged or neutral. 2. The compound of claim 1, wherein the stabilizing group is selected to reduce the interaction between the compound and endothelial cells lining a blood vessel when administered intravenously to a patient. The therapeutic agent, an alkylating agent, an antiproliferative agent, a tubulin binder, a vinca alkaloid, an enediyne, a podophyllotoxin or a podophyllotoxin derivative, a pteridine-type drug, a taxane, an anthracycline, dolastatin, a topoisomerase inhibitor, and The compound of claim 1, wherein the compound is selected from the group consisting of platinum complex chemotherapeutic agents. The therapeutic agent is doxorubicin, daunorubicin, vinblastine, vincristine, calicheamicin, etoposide, etoposide phosphate, CC-1065, duocarmycin, KW-2189, methotrexate, methopterin, aminopterin, dichloromethrexate, docetaxel, epiclitaxel, paclitaxel, paclitaxel, paclitaxel Statins, combretastatin A4 phosphate, dolastatin 10, dolastatin 11, dolastatin 15, topotecan, camfothecin, mitomycin C, porphyromycin, 5-fluorouracil, 6-mercaptopurine, fludarabine, tamoxifen, cytosine arabinoside, adenosine arabinoside , Colchicine, cisplatin, carboplatin, mitomycin C, bleomyci , Melphalan, chloroquine, Shikuroporin A, compound of claim 1 wherein is selected from any one of the derivatives and the group consisting of analogs of any of the above. 2. The compound of claim 1, wherein said oligopeptide is directly linked to said therapeutic agent. The oligopeptide sequence is indirectly linked to a therapeutic agent at a second binding site of the oligopeptide through a linker group selected from the group consisting of aminocaproic acid, hydride groups, ester groups, ether groups and sulfhydryl groups. A compound according to claim 1. The compound according to claim 1, wherein the compound is selected from the group consisting of Suc-βAla-Leu-Ala-Leu-Dox, Suc-βAla-Leu-Ala-Leu-Dnr and glutaryl-βAla-Leu-Ala-Leu-Dox. Compound. 2. The compound of claim 1, wherein said compound is resistant to degradation by CD10. Formula ^{_{(AA) n -AA 4 -AA 3}} -AA 2 -AA 1:
Wherein each AA independently represents an amino acid,
n is an integer of 0 to 16,
AA ⁴ represents an amino acid that is not genetically encoded;
AA ³ represents any amino acid,
AA ² represents any amino acid, and AA ¹ represents any amino acid], comprising an oligopeptide degradable by TOP. 29. The conjugate of claim 28, wherein said oligopeptide is attached to a stabilizing group. 29. The conjugate of claim 28, wherein said oligopeptide is conjugated to a therapeutic agent. (1) (a) a therapeutic agent that can be inserted into a target cell,
(B) Formula ^{_{(AA) n -AA 4 -AA 3}} -AA 2 -AA 1:
Wherein each AA independently represents an amino acid,
n is an integer of 0 to 16,
AA ⁴ represents an amino acid that is not genetically encoded;
AA ³ represents any amino acid,
AA ² represents any amino acid, and AA ¹ represents any amino acid.
(C) comprising a stabilizing group, and (d) optionally a linker group that is not degradable by TOP.
Wherein the oligopeptide is directly linked to the stabilizing group at a first binding site of the oligopeptide, and the oligopeptide is linked directly to the therapeutic agent or the oligopeptide Indirectly attached to the therapeutic agent through the linker group at the second binding site of
The stabilizing group comprises a compound characterized by preventing degradation of the compound by enzymes present in whole blood, and wherein the compound is degradable by TOP, and (2) a pharmaceutically acceptable carrier. A pharmaceutical composition comprising: A method of treating a patient, comprising: a therapeutically effective amount of (1) a therapeutic agent that can be inserted into a target cell;
(2) Formula (AA) _n -AA ⁴ -AA ³ -AA ² -AA ¹ :
Wherein each AA independently represents an amino acid,
n is an integer of 0 to 16,
AA ⁴ represents an amino acid that is not genetically encoded;
AA ³ represents any amino acid,
AA ² represents any amino acid, and AA ¹ represents any amino acid.
(3) a stabilizing group, and (4) optionally a linker group that cannot be degraded by TOP.
Wherein the oligopeptide is directly linked to the stabilizing group at a first binding site of the oligopeptide, and the oligopeptide is linked directly to the therapeutic agent or the oligopeptide Indirectly attached to the therapeutic agent through the linker group at the second binding site of
The method comprising administering to the patient a compound characterized by the stabilizing group preventing degradation of the compound by enzymes present in whole blood, and wherein the compound is degradable by TOP. 33. The method of claim 32, wherein said compound is administered intravenously. 33. The method of claim 32, wherein said patient is treated for a medical condition selected from the group consisting of cancer, neoplastic disease, tumor, inflammatory disease and infectious disease. 33. The method of claim 32, wherein said target cells are multidrug resistant. A method for designing a prodrug for administration to a patient,
(1) Formula (AA) _n -AA ⁴ -AA ³ -AA ² -AA ¹ :
Wherein each AA independently represents an amino acid,
n is an integer of 0 to 16,
AA ⁴ represents an amino acid that is not genetically encoded;
AA ³ represents any amino acid,
AA ² represents any amino acid, and AA ¹ represents any amino acid.]
(2) linking the oligopeptide at a first binding site of the oligopeptide to a stabilizing group that prevents degradation of the oligopeptide by enzymes present in whole blood;
(3) linking the oligopeptide directly or indirectly to a therapeutic agent at the second binding site of the oligopeptide;
Wherein steps (2) and (3) can be performed in any order or simultaneously, and further, the conjugate is formed by performing steps (1)-(3);
(4) testing whether the conjugate can be degraded by TOP, and (5) if the conjugate can be degraded by TOP, selecting the conjugate as a prodrug;
A method comprising: 37. The method of claim 36, wherein said first binding site is the N-terminus of said oligopeptide. 37. The method of claim 36, wherein said second binding site is the C-terminus of said oligopeptide. 37. The method of claim 36, wherein said first binding site is the C-terminus of said oligopeptide. 37. The method of claim 36, wherein said second binding site is the N-terminus of said oligopeptide. 37. A prodrug designed according to the method of claim 36. A method for treating resistance to a therapeutic agent in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of
(1) a therapeutic agent that can be inserted into target cells,
(2) Formula (AA) _n -AA ⁴ -AA ³ -AA ² -AA ¹ :
Wherein each AA independently represents an amino acid,
n is an integer of 0 to 16,
AA ⁴ represents an amino acid that is not genetically encoded;
AA ³ represents any amino acid,
AA ² represents any amino acid, and AA ¹ represents any amino acid.
(3) a stabilizing group, and (4) optionally a linker group that cannot be degraded by TOP.
Wherein the oligopeptide is directly linked to the stabilizing group at a first binding site of the oligopeptide, and the oligopeptide is linked directly to the therapeutic agent or the oligopeptide Indirectly attached to the therapeutic agent through the linker group at the second binding site of
Administering a compound characterized in that the stabilizing group prevents degradation of the compound by enzymes present in whole blood and that the compound can be degraded by TOP;
A method comprising: A method for reducing the toxicity of a therapeutic agent intended for administration to a patient,
Binding the oligopeptide that can be degraded by TOP to a stabilizing group at a first binding site of the oligopeptide and binding the therapeutic agent directly or indirectly at a second binding site of the oligopeptide. Thereby covalently form a prodrug that can be degraded by TOP, thereby providing reduced toxicity of the therapeutic agent when administered to a patient. 44. The method of claim 43, wherein the prodrug allows for administration of an increased dose of the therapeutic agent in prodrug form to the patient relative to the dose of the therapeutic agent in unconjugated form. The oligopeptide,
Formula ^{_{(AA) n -AA 4 -AA 3}} -AA 2 -AA 1:
Wherein each AA independently represents an amino acid,
n is an integer of 0 to 16,
AA ⁴ represents an amino acid that is not genetically encoded;
AA ³ represents any amino acid,
AA ² represents any amino acid, and AA ¹ represents any amino acid]. A product for diagnosis or assay,
(1) (a) a marker,
(B) Formula ^{_{(AA) n -AA 4 -AA 3}} -AA 2 -AA 1:
Wherein each AA independently represents an amino acid,
n is an integer of 0 to 16,
AA ⁴ represents an amino acid that is not genetically encoded;
AA ³ represents any amino acid,
AA ² represents any amino acid, and AA ¹ represents any amino acid.
(C) a stabilizing group, and (d) optionally a linker group that is not degradable by TOP.
Wherein the oligopeptide is directly linked to the stabilizing group at the first binding site of the oligopeptide, and the oligopeptide is linked directly to the marker, or Indirectly attached to the marker at said second binding site through said linker group;
The stabilizing group prevents degradation of the compound by enzymes present in whole blood, and the compound is characterized in that the compound can be degraded by TOP, and (2) optionally, at least one compound useful in the detection of the marker. One reagent,
A product comprising: A method for removing free therapeutic agent in the manufacture of a prodrug, comprising:
(1) coupling the optionally protected stabilizing group-oligopeptide conjugate with a free therapeutic agent;
(2) contacting the polymer resin binding free therapeutic agent remaining after step (1) with the reactant of step (1) to form a therapeutic agent-polymer resin complex; and A method comprising removing a therapeutic agent-polymer resin complex. The method according to claim 47, wherein the polymer resin is polystyrene methyl isocyanate or polystyrene sulfonyl chloride. Wherein said optionally protected stabilizing group-oligopeptide conjugate comprises:
Formula ^{_{(AA) n -AA 4 -AA 3}} -AA 2 -AA 1:
Wherein each AA independently represents an amino acid,
n is an integer of 0 to 16,
AA ⁴ represents an amino acid that is not genetically encoded;
AA ³ represents any amino acid,
AA ² represents any amino acid, and AA ¹ represents any amino acid]. (1) Alkyl or allyl ester protected stabilizing groups, oligonucleotides and therapeutics in the presence of an activating agent to produce an alkyl or allyl ester protected stabilizing group-oligopeptide-therapeutic agent conjugate Couple the agent,
(2) removing the uncoupled therapeutic agent remaining after the coupling step, and (3) protecting the alkyl or allyl ester to prepare a stabilizing group-oligopeptide-therapeutic agent prodrug compound. A method for producing a prodrug compound comprising the step of deprotecting a stabilized conjugate-oligopeptide-therapeutic agent conjugate. 51. The method of claim 50, wherein the alkyl or allyl ester protected stabilizing group-oligopeptide and the therapeutic agent are present in a 2: 1 to 1: 1 molar ratio. 52. The method of claim 51, wherein said molar ratio is between 1.75: 1 and 1.5: 1. 53. The method of claim 52, wherein said molar ratio is 1.66: 1. The coupling step comprises:
(A) combining an alkyl or allyl ester protected stabilizing group, an oligopeptide and a therapeutic agent in DMF,
(B) adding DIEA,
(C) reacting the alkyl or allyl ester protected stabilizing group, oligopeptide and therapeutic agent in the presence of an active agent to form a conjugate, and (d) brine to form a precipitate. 51. The method of claim 50, comprising precipitating the conjugate by adding a solution. 51. The method of claim 50, wherein said therapeutic agent is an anthracycline. 56. The method of claim 55, wherein said anthracycline is doxorubicin. 55. The method of claim 54, wherein said activator is HATU. 55. The method of claim 54, wherein the DIEA and the alkyl or allyl ester protected stabilizing group-oligopeptide are present in a molar ratio of 3: 1 to 1.5: 1. 59. The method of claim 58, wherein said molar ratio is between 2.5: 1 and 2: 1. 60. The method according to claim 59, wherein said molar ratio is 2.18: 1. 55. The method of claim 54, wherein said reacting step is performed at 0 <0> C. 55. The method of claim 54, wherein said reacting is performed for 30 minutes. 58. The method of claim 57, wherein the activator and the alkyl or allyl ester protected stabilizing group-oligopeptide are present in a molar ratio of 1.5: 1 to 1: 1. 64. The method of claim 63, wherein said molar ratio is 1.1: 1. 55. The method of claim 54, wherein the brine solution is 20% (w / v) to 40% (w / v) NaCl in water. 66. The method of claim 65, wherein the brine solution is 25% (w / v) to 35% (w / v) NaCl in water. 67. The method of claim 66, wherein the brine solution is 30% (w / v) NaCl in water. 55. The method of claim 54, wherein said precipitation step is performed at a pH of between 5.0 and 7.0. 69. The method of claim 68, wherein said pH is between 5.8 and 6.0. The removing step comprises:
(A) dissolving the conjugate in DMF,
(B) dissolving the scavenger resin in anhydrous DMF,
(C) adding an alkyl or allyl ester protected stabilizing group-oligopeptide-therapeutic agent conjugate formed in the coupling step to the scavenger resin to form a conjugate-resin mixture. And
(D) maintaining the mixture at 0 ° C. to 30 ° C. for 2 to 24 hours, wherein the uncoupled therapeutic agent reacts with the resin;
(E) removing the resin from the mixture and (f) adding a brine solution to form a precipitate of an alkyl or allyl ester protected stabilizing group-oligopeptide-therapeutic agent conjugate. 51. The method of claim 50 comprising precipitating the residue. 71. The method of claim 70, wherein said scavenger resin is selected from the group consisting of polystyrene isocyanate, polystyrene methyl isocyanate, polystyrene thioisocyanate, polystyrene methyl thioisocyanate, polystyrene sulfonyl chloride, polystyrene methyl sulfonyl chloride, and polystyrene benzaldehyde. 72. The method of claim 71, wherein said scavenger resin is polystyrene isocyanate. 51. The method of claim 50, wherein said deprotecting step further comprises deprotecting with an enzyme. 74. The method of claim 73, wherein said enzyme is an esterase. The esterases may be porcine liver esterase, Candida anartica B lipase, Candida rugosa lipase, Pseudomonas cepacia lipase, CLEC-B-lipase, CLEC-B-lipase, Candida rugosa lipase, Pseudomonas cepacia lipase. 75. The method of claim 74, wherein the method is selected from the group consisting of CLEC-CR (cross-linked Candida rugosa lipase), CLEC-PC (pseudomonas cepacia lipase), and chirazyme (esterase immobilized on Sepharose). 76. The method of claim 75, wherein said esterase is Candida antartica B lipase. 75. The method of claim 74, wherein said esterase is cross-linked or immobilized on a solid support. The step of releasing protection includes:
(A) washing the enzyme to remove free enzymes,
(B) adding the washed enzyme to an alkyl or allyl ester protected stabilizing group-oligopeptide-therapeutic agent conjugate;
(C) in order to create the stabilizing group-oligopeptide-therapeutic agent prodrug compound, the enzyme and the conjugate are at least at 15 ° C to 40 ° C at a pH of 5.0 to 8.0 Reacting for 18 hours and (d) separating the enzyme from the prodrug compound
78. The method of claim 77, comprising: 79. The method of claim 78, further comprising, after the step of reacting the enzyme with the conjugate, adding an additional enzyme cross-linked or immobilized on a tissue support.

Images