JP3554399B2 - Peptide derivatives - Google Patents

Description

【０００７】
上記式中の置換基について述べると、Ｑ は Ｄ−Ａｒｇ （Ｄ−アルギニン残基）またはＬ−Ａｒｇ （Ｌ− アルギニン残基）を意味し、Ｒ^１は水素原子またはＣ_１−６（炭素数１〜６個の）アルキル基を意味する。これらのうち、Ｑ が Ｄ−Ａｒｇであり、Ｒ^１が水素原子である化合物が好ましい。Ｒ^２は水素原子、Ｃ_１−６アルキル基、アリール基、Ｃ_１−６アルカノイル基、またはアリールカルボニル基を示す。アリール基、アルカノイル基、またはアリールカルボニル基としては、例えば、置換若しくは無置換のフェニル基、好ましくは無置換フェニル基などのアリール基；アセチル基やプロパノイル基などのアルカノイル基；又はベンゾイル基などのアリールカルボニル基を用いることができる。
【０００８】
Ｘ は−ＯＲ^３、−ＮＲ^４Ｒ^５、又は−ＮＲ^６−ＣＲ^７Ｒ^８Ｒ^９のいずれかを示す。Ｒ^３は水素原子またはＣ_１−６アルキル基を示し、Ｒ^４は水素原子またはＣ_１−６アルキル基を示す。Ｒ^５はＣ_１−６ヒドロキシアルキル基またはスルホン酸置換Ｃ_１−６アルキル基を示す。水酸基またはスルホン酸基はアルキル基のいかなる位置に置換していてもよいが、末端置換のアルキル基が好ましい。Ｒ^４及びＲ^５は一緒になってＲ^４及びＲ^５が置換する窒素原子と共に ５または６ 員含窒素飽和複素環基を示してもよく、該複素環は２個以上の窒素原子を含んでいてもよい。例えば、−ＮＲ^４Ｒ^５として１−ピペラジニル基、１−ピロリジニル基、又は１−ピペリジニル基などを用いることができる。
【０００９】
また、Ｘ がＮＲ^６−ＣＲ^７Ｒ^８Ｒ^９ を示す場合、Ｒ^６は水素原子、Ｃ_１−６アルキル基、又はフェニル基等のアリール基が置換したＣ_１−６アルキル基（アラルキル基）を示す。アラルキル基としては、例えば、ベンジル基など挙げることができる。Ｒ^７は水素原子；カルボキシル基；メトキシカルボニル、エトキシカルボニルなどのＣ_１−６アルコキシ基が置換したカルボニル基；置換若しくは無置換カルバモイル基；カルボキシル基が置換したＣ_１−６アルキル基；置換若しくは無置換カルバモイル基が置換したＣ_１−６アルキル基；またはＣ_１−６アルコキシ基が置換したカルボニル基を有するＣ_１−６アルキル基を示す。
【００１０】
Ｒ^８は水素原子；Ｃ_１−６アルキル基；アミノＣ_１−６アルキル基；アミジノ基が置換したＣ_１−６アルキル基；グアニジノ基が置換したＣ_１−６アルキル基、ヒドロキシＣ_１−６アルキル基；カルボキシル基が置換したＣ_１−６アルキル基、又は置換若しくは無置換カルバモイルが置換したＣ_１−６アルキル基を示す。あるいは、Ｒ^６及びＲ^８が一緒になってＲ^６が置換する窒素原子とともに環上にカルボキシル基を有する５ または６ 員の含窒素飽和複素環基を形成してもよい。このような複素環としては、２−カルボキシ−１− ピロリジニル基 （−Ｐｒｏ−ＯＨ）や３−カルボキシ−１− ピペリジニル基を挙げることができる。これらのうち、例えば、Ｒ^７がカルボキシエチル基またはカルバモイルエチル基であり、Ｒ^８が水素原子である組合せが好ましい。Ｒ^９は水素原子又はＣ_１−６アルキル基を示す。上記の各置換基において、アルキル基、アルコキシ基、又はアルカノイル基は直鎖または分枝のいずれでもよい。
【００１１】
上記の式Ｉで示される本発明の化合物は、Ｌ−チロシン残基及びＱ が示すＤ−Ａｒｇ またはＬ−Ａｒｇ 残基に由来する２個の不斉炭素の他、Ｑ に結合するフェニルアラニン残基に由来する不斉炭素、Ｒ^７およびＲ^８が置換する不斉炭素（ただしＲ^７およびＲ^８が同時に同一の置換基を示す場合を除く）、並びに上記の各置換基に任意に存在する１以上の不斉炭素を有する。Ｌ−Ｔｙｒ 、Ｄ−Ａｒｇ 、及びＬ−Ａｒｇ 残基に由来するもの以外の不斉炭素はＲ−またはＳ−のいずれの配置でもよい。また、本発明の式Ｉで示される化合物には、上記の式で示される任意の光学活性体またはラセミ体、ジアステレオ異性体またはそれらの任意の混合物もすべて包含される。
【００１２】
また本発明の化合物には、塩酸塩、酢酸塩、又はパラトルエンスルホン酸などの酸付加塩や、アンモニウム塩又は有機アミン塩などの塩基付加塩が含まれる。さらに上記の一般式で示される化合物の他、上記化合物の２量体ないし多量体である化合物、及びこれらの化合物のＣ−末端とＮ−末端が結合した環状の化合物が包含される。
【００１３】
本発明のペプチドは、モルヒネを凌駕する鎮痛効果を有する。鎮痛作用に伴うヒスタミン遊離作用や心拍数の低下作用がモルヒネに比して相対的に弱く、モルヒネとの交差耐性の程度も低いので、癌疼痛治療に使用するのに適することが予想される。投与経路としては静脈内投与、皮下投与、経口投与等が挙げられるが、経鼻吸収を含む粘膜吸収製剤および経皮吸収製剤も有用性が期待される。
【００１４】
本発明のペプチド誘導体は、ペプチド合成に通常用いられる固相法および液相法で合成することができる。例えば、固相法によりアミジノ基を有しないペプチド鎖を合成し、さらにＮ末端のチロシンのアミノ基にアミジノ基を導入することによって目的のペプチド誘導体を製造することができ、また、予めアミジノ基を導入した後Ｃ末端を修飾することもできる。
【００１５】
アミノ基等の保護基および縮合反応の縮合剤等は、優れたものが種々知られており、以下の実施例を参考に、また、例えば： 鈴木紘一編「タンパク質工学−基礎と応用」丸善（株）（１９９２）及びそこに引用された文献； Ｍ． Ｂｏｎｄａｎｓｚｋｙ， ｅｔ ａｌ．， “Ｐｅｐｔｉｄｅ Ｓｙｎｔｈｅｓｉｓ”， Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ， Ｎ．Ｙ．， １９７６； 並びに Ｊ．Ｍ． Ｓｔｅｗａｒｔ ａｎｄ Ｄ．Ｊ． Ｙｏｕｎｇ， “Ｓｏｌｉｄ Ｐｈａｓｅ Ｐｅｐｔｉｄｅ Ｓｙｎｔｈｅｓｉｓ”， Ｗ．Ｈ．Ｆｒｅｅｍａｎ ａｎｄ Ｃｏ．， Ｓａｎ Ｆｒａｎｃｉｓｃｏ， １９６９ 等を参照して適宜選択使用することができる。固相法では市販の各種ペプチド合成装置、例えばパーキン・エルマー・ジャパン製（Ｐｅｒｋｉｎ Ｅｌｍｅｒ Ｊａｐａｎ，旧社名 Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）のＭｏｄｅｌ ４３０Ａを利用するのが便利なこともある。合成に使用する樹脂、試薬等は市販品等を容易に入手でき、それらの例は実施例に示した。
【００１６】
【実施例】
以下に実施例により本発明をさらに具体的に説明するが、本発明はこれら実施例に限定されるものではない。本実施例を参照し、あるいは本実施例の方法を修飾・変更することによって、あるいは出発原料または反応試薬を適宜選択することにより、一般式Ｉに包含される本発明の所望の本発明ペプチド誘導体を容易に製造することができる。なお、実施例においては、アミノ酸基の意味は通常用いられているものと同様である。Ｄ−体とＬ−体があるアミノ酸が言及されている場合には、そのアミノ酸はＤ−と特に表示していない場合はＬ−アミノ酸を意味する。また、以下の略号を使うことがあり、特に示していない場合にも同様な略号を用いる場合がある。
【００１７】
Ｚ ：ベンジルオキシカルボニル基
ＯＴｃｅ ：トリクロロエチルエステル基
Ｂｏｃ ：ｔ−ブトキシカルボニル基
ＷＳＣＩ ：１−エチル−３−（３−ジメチルアミノプロピル）−カルボジイミド
ＴｏｓＯＨ ：パラトルエンスルホン酸
ＯＢｚｌ ：ベンジルオキシ基
ＭｅβＡｌａ 又は βＭｅＡｌａ ：Ｎ−メチル− β− アラニン
Ｈ−βＡｌａ−ｏｌ： ＮＨ_２ＣＨ_２ＣＨ_２ＣＨ_２ＯＨ
Ｆｍｏｃ ：９−フルオレニルメチルオキシカルボニル
Ｐｍｃ ：２，２，５，７，８−ペンタメチルクロマン−６−スルホニル
ｔ−Ｂｕ ：三級ブチル
ＮＭＰ ：Ｎ−メチルピロリドン
ＤＭＦ ：ジメチルホルムアミド
ＤＭＳＯ ：ジメチルスルホキシド
ＴＦＡ ：トリフルオロ酢酸
ＴＥＡ ：トリエチルアミン
ＤＣＭ ：ジクロロメタン
ＤＭＡＰ ：Ｎ，Ｎ−ジメチルアミノピリジン
ＤＩＰＥＡ ：Ｎ，Ｎ−ジイソプロピルエチルアミン
ＤＩＰＣＩ ：Ｎ，Ｎ−ジイソプロピルカルボジイミド
ＨＯＢｔ ：１−ヒドロキシベンゾトリアゾール
ＥＤＣ ：１−（３− ジメチルアミノプロピル）−３−エチル− カルボジイミド
ＨＢＴＵ ：２−（１Ｈ−ベンゾトリアゾール−１−イル）−１，１，３，３−テトラメチルウロニウム・ヘキサフルオロホスフェート
ＰｙＢｒｏｐ：ブロモトリス（ピロリジノ）ホスホニウム・ヘキサフルオロホスフェート
Ａｌｋｏ樹脂：ｐ−アルコキシベンジルアルコール樹脂 ［４−ヒドロキシメチルフェノキシメチルコポリスチレン １％ ジビニルベンゼン樹脂、Ｊ． Ａｍ． Ｃｈｅｍ． Ｓｏｃ．， ９５， １３２８ （１９７４）］， 渡辺化学工業
Ｆｍｏｃ−ＮＨ−ＳＡＬ 樹脂： ４−（２’，４’− ジメトキシフェニル−９−フルオレニルメトキシカルボニルアミノメチル）フェノキシ樹脂：渡辺化学工業
【００１８】
例１
Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−Ｐｈｅ−ＮＨＣＨ_２ＣＨ_２ＣＯＯＨ
Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ （ＡＢＩ）社の Ｍｏｄｅｌ ４３０Ａ ペプチド合成装置を用いて、上記ペプチドを固相法（Ｏｒｉｇｉｎａｌ Ａｕｔｏｐｒｏｇｒａｍ ｆｏｒ ｔｈｅ Ｆｍｏｃ／ＮＭＰ ｍｅｔｈｏｄ）により以下のように合成した。
【００１９】
Ｆｍｏｃ−β−Ａｌａ−Ａｌｋｏ 樹脂 ０．２５ ｍｍｏｌ／６７５ ｍｇ をＮＭＰ で１回洗浄し、２０％ ピペリジン含有ＮＭＰ で４分間、さらに同じく２０％ ピペリジン含有ＮＭＰ で１６分間処理した。ＮＭＰ で５回洗浄した後、６１分間 Ｆｍｏｃ−Ｐｈｅ−ＯＨと反応させた。次いでＮＭＰ にて４回洗浄し、４回目の洗浄液より樹脂を回収し、未反応のアミノ基は無水酢酸と反応させた。
【００２０】
以上の１サイクルを１２０ 分で行い、同様の操作を２サイクル目は Ｆｍｏｃ−Ｐｈｅ−ＯＨの代わりに Ｆｍｏｃ−Ｄ−Ａｒｇ（Ｐｍｃ）−ＯＨ を用いて、３サイクル目は Ｆｍｏｃ−Ｔｙｒ（ｔ−Ｂｕ）−ＯＨを用いて繰り返した。側鎖保護基は、Ｄ−Ａｒｇ に対してはＰｍｃ を、Ｔｙｒ に対してはｔ−Ｂｕを用いた。
【００２１】
上記の操作により得られた樹脂５００ ｍｇをフェノール（結晶）０．７５ ｇ、エタンジチオール ０．２５ ｍｌ、チオアニソール ０．５０ ｍｌ、水 ０．５０ ｍｌおよびＴＦＡ １０．０ ｍｌ の混合液中で室温にて３時間撹拌処理し、樹脂からペプチドを分離させると同時に保護基を除去した。次いで ３μｍ のフィルター（ＡＤＶＡＮＴＥＣ−Ｐｏｌｙｆｌｏｎ フィルター） で濾過し、濾液に冷ジエチルエーテル ２００ ｍｌ を加え、生じた沈殿を ３μｍ のフィルター（ＡＤＶＡＮＴＥＣ−Ｐｏｌｙｆｌｏｎ フィルター）で濾取した。フィルター上の沈殿は２ Ｎ 酢酸１０〜２０ ｍｌ に溶解し、凍結乾燥して粗ペプチドを得た。
【００２２】
粗ペプチド１３５ ｍｇを水 １３．５ ｍｌに溶解し、１ Ｍ のｏ−メチルイソウレア １３．５ ｍｌを加え、４℃で１４日間撹拌し、アミジノ化を行った。得られた粗アミジノ化ペプチド１０６ ｍｇを ２０ ｍｌの ０．１％ ＴＦＡ 水溶液に溶解した。０．０１Ｎ の塩酸でｐＨ ４〜５ に調整後、Ｇｉｌｓｏｎ ＨＰＬＣ システムでペプチドを精製した。ＨＰＬＣにはＣｏｓｍｏｓｉｌ Ｃ_１８カラムを用い、０．１％ ＴＦＡ水溶液および ７０％アセトニトリル含有 ０．１％ ＴＦＡ 水溶液の混合液の連続濃度勾配（混合比は開始時 ０％ から４５分後の６０％まで）で行い、流速は１０ ｍｌ／分とした。
【００２３】
精製されたペプチドのうち約 １００μｇ を加水分解管に採取し１ ｍｌの ０．２％ フェノールを含有する６ Ｎ 塩酸を加え、脱気封管後、１１０ ℃で２４時間加水分解した。室温冷却後４０℃で濃縮乾固した後、０．０１Ｎ 塩酸１ ｍｌに溶解し、溶液２０μｌ をアミノ酸分析計で解析した（分析計：日立 Ｌ−８５００ 、カラム：Ｈｉｔａｃｈｉ Ｃｕｓｔｏｍイオン交換樹脂＃２６２２、プレカラム： Ｈｉｔａｃｈｉ Ｃｕｓｔｏｍ イオン交換樹脂＃ ２６５０Ｌ、バッファ−： リチウムバッファー（０．０９ Ｎ−ｐＨ ２．８， ０．２５ Ｎ−ｐＨ ３．７， ０．７２ Ｎ−ｐＨ ３．６，１．００ Ｎ−ｐＨ ４．１， 再生 ０．２０Ｎ）、反応温度：１３５ ℃、バッファー・ポンプの流速：０．３０ ｍｌ／分、ニンヒドリン・ポンプの流速：０．３５ ｍｌ／ 分、検出器の波長：５７０ ｎｍ／４４０ ｎｍ、分析時間：１５０ 分）。その結果、アミノ酸分析の結果は上記の構造を支持していた。
【００２４】
精製ペプチド約１５０ μｇ を ２５０μｌ の５％酢酸に溶解し、溶液の１ μｌ をＬｉｑｕｉｄＳＩＭＳ にて質量分析した （ＭＳ及びＭＳ／ ＭＳ、セシウムイオンガン使用、分析計： Ｆｉｎｎｉｇａｎ ＴＳＱ ７００， Ｍａｔｒｉｘ： グリセロール−チオグリセロール（１：１） 、Ｃｏｌｌｉｓｉｏｎ Ｇａｓ： Ａｒ ガス ３〜５ ｍＴｏｒｒ， Ｃｏｌｌｉｓｉｏｎ Ｅｎｅｒｇｙ： −２０ ｅＶ， Ｅｌｅｃｔｒｏｎ Ｍｕｌｔｉｐｌｉｅｒ： １０００−１５００ Ｖ）。その結果は上記の構造を支持していた。
【００２５】
例２
Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−Ｐｈｅ−ＮＨＣＨ_２ＣＨ_２ＣＯＮＨ_２
上記ペプチド誘導体を、合成開始時にＦｍｏｃ−ＮＨ−ＳＡＬ 樹脂 ０．２５ ｍｍｏｌ／ ３８５ ｍｇを用い、１サイクル目に Ｆｍｏｃ−β−Ａｌａ−ＯＨ を、２サイクル目に Ｆｍｏｃ−Ｐｈｅ−ＯＨを、３サイクル目に Ｆｍｏｃ−Ｄ−Ａｒｇ（Ｐｍｃ）−ＯＨ を用いて、さらに４サイクル目に Ｆｍｏｃ−Ｔｙｒ（ｔ−Ｂｕ）−ＯＨを用いたこと以外は実施例１と同様な操作により得た。
【００２６】
アミジノ化ペプチド誘導体の合成および精製も、混合液を０．０５％ 蟻酸水溶液および ７０％アセトニトリル含有０．０５％ 蟻酸水溶液（混合比は ＨＰＬＣ 開始時 ０％ から４５分後の４０％ までの連続濃度勾配）としたこと以外は実施例１と同様に行った。アミノ酸組成分析および質量分析も実施例１と同様に行い、上記の構造を支持する結果を得た。
【００２７】
例３
Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−Ｐｈｅ−Ｎ（ＣＨ_３）ＣＨ_２ＣＨ_２ＣＯＯＨ
上記構造を有するテトラペプチド誘導体を、Ｆｍｏｃ／ＮＭＰ法による固相法で以下のように合成した。濾過にはグラスフィルターを用いた。
【００２８】
Ａｌｋｏ樹脂 ０．５００ ｇを ＤＭＦ ６ ｍｌ で膨潤させた後、Ｆｍｏｃ−Ｎ− メチル− β− アラニン （Ｆｍｏｃ− β−ＭｅＡｌａ−ＯＨ ）０．２２８ ｇ およびピリジン ０．０９３ ｍｌ を樹脂に加え、１分間振盪し、次いで塩化 ２，６− ジクロロベンゾイル ０．１４７ ｇを加えて２４時間振盪した。生成した Ｆｍｏｃ−β−ＭｅＡｌａ−Ａｌｋｏ 樹脂を ６ ｍｌ のＤＭＦ で３回、次いで ６ ｍｌ のメタノールで３回、さらに ６ ｍｌ のＤＣＭ で３回洗浄し、未反応のヒドロキシメチル基はＤＣＭ ６ ｍｌ中で塩化ベンゾイル ０．０８９１ ｍｌおよびピリジン ０．０８４７ ｍｌを加え１時間振盪しベンゾイル化した。さらにアミノ酸樹脂を６ ｍｌのＤＣＭ で３回、６ ｍｌのＤＭＦ で３ 回、 ６ ｍｌ のメタノールで３ 回順次洗浄し、水酸化カリウムデシケーター中で真空乾燥した。
【００２９】
Ｆｍｏｃ− β−ＭｅＡｌａ−Ａｌｋｏ 樹脂を １２ ｍｌのＤＭＦ で３回、次いで ２０ ％ ピペリジンを含むＤＭＦ １２ ｍｌ で３回、さらに １２ ｍｌのＤＭＦ で６回処理することにより Ｆｍｏｃ 基を除去し、Ｆｍｏｃ−Ｐｈｅ−ＯＨ ０．２６２ ｇ 、ＰｙＢｒｏｐ （渡辺化学工業）０．３１５ ｇ 、ＮＭＰ ６ ｍｌ、ＤＩＰＥＡ ０．２７３ ｍｌを加え、２４時間振盪し、Ｆｍｏｃ−Ｐｈｅ− β−ＭｅＡｌａ−Ａｌｋｏ 樹脂を生成させた。濾過し、６ ｍｌのＮＭＰ による洗浄後、未反応のアミノ基は １− アセチルイミダゾール ０．２４８ ｇ、ＤＩＰＥＡ ０．０７８４ ｍｌ を含むＤＭＦ ６ ｍｌ中で１ 時間処理しキャップした。次いで、得られた樹脂を ６ ｍｌ のＮＭＰ で洗浄した。
【００３０】
Ｆｍｏｃ−Ｐｈｅ− β−ＭｅＡｌａ−Ａｌｋｏ 樹脂から上記と同様な操作によりＦｍｏｃ基を除去し、Ｆｍｏｃ−Ｄ−Ａｒｇ（Ｐｍｃ）−ＯＨ ０．５５７ ｇ、ＨＯＢｔ ０．１２１ ｇ、ＨＢＴＵ ０．２９９ ｇ、ＤＩＰＥＡ ０．２７４ ｍｌを加え、１時間振盪しＦｍｏｃ−Ｄ−Ａｒｇ（Ｐｍｃ）−Ｐｈｅ−β−ＭｅＡｌａ−Ａｌｋｏ 樹脂を生成させた。次いで前項と同様に濾過、洗浄後、未反応のアミノ基をキャップした。
【００３１】
Ｆｍｏｃ−Ｄ−Ａｒｇ（Ｐｍｃ）−Ｐｈｅ−β−ＭｅＡｌａ−Ａｌｋｏ 樹脂から上記と同様な操作によりＦｍｏｃ基を除去し、Ｆｍｏｃ−Ｔｙｒ（ｔ−Ｂｕ）−ＯＨ ０．３１０ ｇ 、ＨＯＢｔ ０．１０３ ｇ、ＨＢＴＵ ０．２５６ ｇ、ＤＩＰＥＡ ０．２３５ ｍｌを加え、１時間振盪しＦｍｏｃ−Ｔｙｒ（ｔ−Ｂｕ）−Ｄ−Ａｒｇ（Ｐｍｃ）−Ｐｈｅ−β−ＭｅＡｌａ−Ａｌｋｏ 樹脂を生成させた。前項と同様に濾過、洗浄後、未反応のアミノ基をキャップした。
【００３２】
Ｆｍｏｃ−Ｔｙｒ（ｔ−Ｂｕ）−Ｄ−Ａｒｇ（Ｐｍｃ）−Ｐｈｅ−β−ＭｅＡｌａ−Ａｌｋｏ 樹脂から上記と同様な操作によりＦｍｏｃ基を除去し、Ｍａｔｓｕｅｄａらの方法（Ｔｈｅ Ｊｏｕｒｎａｌ ｏｆ Ｏｒｇａｎｉｃ Ｃｈｅｍｉｓｔｒｙ， ５７，２４９７〜２５０２， １９９２）に従って調製した １Ｈ−ピラゾール−１− カルボキサミジン塩酸塩 ０．９８９ ｇ、ＤＩＰＥＡ １．２９３ ｍｌ、ＤＭＦ ６ ｍｌを加えて１０〜６０℃、より好ましくは４０〜５０℃で １時間ないしは ４時間反応させてＮ末端のチロシンのアミノ基をアミジノ化した。次いで濾過、洗浄（ＮＭＰ ６ ｍｌで３回、さらにメタノール ６ ｍｌ で３回）し水酸化カリウムデシケータ中で真空乾燥を行った。
【００３３】
上記の操作により得られた樹脂 ５９６ ｍｇ をグラスフィルター上においてＴＦＡ 、フェノール、水の混合液（ＴＦＡ ：フェノール：水 ＝ ９３：２：５） ５ ｍｌ で１時間処理し濾過した。同様の操作を計２回行った。さらに樹脂をＴＦＡ ５ ｍｌで ５分処理し濾過した。同様の操作を計３回行った。各操作で得られた濾液を合して２０℃以下で溶媒を減圧留去した。残渣にジエチルエーテル ２０ ｍｌを加え白色沈殿とし、上清を捨てる操作を計３回行い、得られた白色粉末を水 ２０ ｍｌに溶解し、分液ロート中にてジエチルエーテル ５ ｍｌ で３回洗浄し、水層を凍結乾燥し、粗アミジノ化ペプチドを得た。
【００３４】
粗アミジノ化ペプチド ５６．２ ｍｇを ０．０５％ ＴＦＡ水溶液 １０ ｍｌに溶解し、島津ＨＰＬＣシステムにて精製した。ＨＰＬＣにＹＭＣ Ｄ−ＯＤＳ−５−ＳＴ Ｃ_１８カラムと ０．５％ アセトニトリル含有 ０．０５％ ＴＦＡ水溶液および ７０％アセトニトリル含有 ０．０５％ ＴＦＡ水溶液の混合液（混合比はＨＰＬＣ開始時０％から５０分後の９０％ までの連続濃度勾配で流速は １ ｍｌ／分）を用いた。アミノ酸組成分析および質量分析は実施例１と同様に行い、上記の構造を支持する結果を得た。
【００３５】
例４
Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−Ｐｈｅ−Ｎ（ＣＨ_２ＣＨ_３）ＣＨ_２ＣＨ_２ＣＯＯＨ
上記構造を有するテトラペプチド誘導体を、Ｆｍｏｃ／ＮＭＰ法による固相法で以下のように合成した。濾過にはグラスフィルターを用いた。
【００３６】
Ａｌｋｏ樹脂 １．０００ ｇをＮＭＰ １２ ｍｌ で膨潤させた後、Ｆｍｏｃ−Ｎ− エチル− β− アラニン（Ｆｍｏｃ− β−ＥｔＡｌａ−ＯＨ） ０．４７５ ｇおよびＤＭＡＰ ０．０１７ ｇを加え、１分間振盪し、次いでＤＩＰＣＩ ０．１７７ ｇ を添加して２４時間振盪した。生成した Ｆｍｏｃ−β−ＥｔＡｌａ−Ａｌｋｏ 樹脂をＮＭＰ １２ ｍｌ で３回、次いで１２ ｍｌ のメタノールで３回、さらにＤＣＭ １２ ｍｌ で３回洗浄し、未反応のヒドロキシメチル基はＤＣＭ １２ ｍｌ 中で塩化ベンゾイル ０．１７８ｍｌおよびピリジン０．１７０ ｍｌを加え１時間振盪しベンゾイル化した。さらにアミノ酸樹脂を １２ ｍｌのＤＣＭ で３回、１２ ｍｌ のＤＭＦ で３回、１２ ｍｌ のメタノールで３ 回順次洗浄し、水酸化カリウムデシケーター中で真空乾燥した。
【００３７】
Ｆｍｏｃ− β−ＥｔＡｌａ−Ａｌｋｏ 樹脂を ２０ ｍｌのＤＭＦ で３回、次いで ２０％ピペリジンを含むＤＭＦ １２ ｍｌ で３ 回、さらに１２ ｍｌ のＤＭＦ で６回処理することによりＦｍｏｃ基を除去し、Ｆｍｏｃ−Ｐｈｅ−ＯＨ ０．３８７ ｇ， ＰｙＢｒｏｐ ０．４６６ ｇ， ＮＭＰ １２ ｍｌ， 及びＤＩＰＥＡ ０．５２３ ｍｌを加え、２４時間振盪し、Ｆｍｏｃ−Ｐｈｅ− β−ＥｔＡｌａ−Ａｌｋｏ 樹脂を生成させた。濾過後、１２ ｍｌ のＮＭＰ で洗浄し、未反応のアミノ基は１−アセチルイミダゾール ０．５５１ ｇ、ＤＩＰＥＡ ０．１７４ ｍｌを含むＤＭＦ １２ ｍｌ 中で１時間処理しキャップした。次いで、得られた樹脂を再度 １２ ｍｌのＮＭＰ で洗浄した。
【００３８】
Ｆｍｏｃ−Ｐｈｅ− β−ＥｔＡｌａ−Ａｌｋｏ 樹脂から上記と同様な操作によりＦｍｏｃ基を除去した。樹脂にＦｍｏｃ−Ｄ−Ａｒｇ（Ｐｍｃ）−ＯＨ ０．７０７ ｇ、ＨＯＢｔ ０．１５３ ｇ、ＨＢＴＵ ０．３７９ ｇ、ＤＩＰＥＡ ０．３４８ ｍｌを加え、１ 時間振盪しＦｍｏｃ−Ｄ−Ａｒｇ（Ｐｍｃ）−Ｐｈｅ−β−ＥｔＡｌａ−Ａｌｋｏ 樹脂を生成させた。前項と同様に濾過、洗浄後、未反応のアミノ基をキャップした。
【００３９】
Ｆｍｏｃ−Ｄ−Ａｒｇ（Ｐｍｃ）−Ｐｈｅ−β−ＥｔＡｌａ−Ａｌｋｏ 樹脂から上記と同様な操作によりＦｍｏｃ基を除去し、Ｆｍｏｃ−Ｔｙｒ（ｔ−Ｂｕ）−ＯＨ ０．４６０ ｇ 、ＨＯＢｔ ０．１５３ ｇ、ＨＢＴＵ ０．３９９ ｇ、ＤＩＰＥＡ ０．３４８ ｍｌを加え、１時間振盪しＦｍｏｃ−Ｔｙｒ（ｔ−Ｂｕ）−Ｄ−Ａｒｇ（Ｐｍｃ）−Ｐｈｅ−β−ＥｔＡｌａ−Ａｌｋｏ 樹脂を生成させた。上記と同様に濾過、洗浄後、未反応のアミノ基をキャップした。
【００４０】
Ｆｍｏｃ−Ｔｙｒ（ｔ−Ｂｕ）−Ｄ−Ａｒｇ（Ｐｍｃ）−Ｐｈｅ−β−ＥｔＡｌａ−Ａｌｋｏ 樹脂から上記と同様な操作によりＦｍｏｃ基を除去し、実施例３と同様に調製した １Ｈ−ピラゾール−１− カルボキサミジン塩酸塩 ２．１９９ ｇ、ＤＩＰＥＡ ２．８７４ ｍｌ、ＤＭＦ ６ ｍｌを加え４０〜５０℃で１ 時間ないしは４ 時間反応させてＮ末端のチロシンのアミノ基をアミジノ化した。次いで濾過、洗浄（ＮＭＰ １２ ｍｌ で３回、さらにメタノール １２ ｍｌで３回）し水酸化カリウムデシケータ中で真空乾燥を行った。
【００４１】
上記の操作により得られた樹脂 １．５１４ ｇをグラスフィルター上においてＴＦＡ 、フェノール、及び水を含む混合液（ＴＦＡ ：フェノール：水＝９３：２ ：５） １０ ｍｌで１時間処理し濾過した。同様の操作を計２回行った。さらに樹脂をＴＦＡ １０ ｍｌ で５分処理し濾過した。同様の操作を計３回行った。各操作で得られた濾液を合して２０℃以下で溶媒を減圧留去した。残渣にジエチルエーテル ２０ ｍｌを加え白色沈殿とし、上清を捨てる操作を計３ 回行った。得られた白色粉末を水 ３０ ｍｌに溶解し、分液ロート中でジエチルエーテル ５ ｍｌ で３回洗浄し、水層を凍結乾燥し、粗アミジノ化ペプチドを得た。
【００４２】
粗アミジノ化ペプチド １００ ｍｇ を ０．０５％ ＴＦＡ水溶液 ２０ ｍｌに溶解し、実施例３と同様に精製した。アミノ酸組成分析および質量分析は実施例１と同様に行い、上記の構造を支持する結果を得た。
【００４３】
例５
Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−Ｐｈｅ−ＯＭｅ
Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−Ｐｈｅ−ＮＨＭｅ
Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−Ｐｈｅ− βＡｌａ−ｏｌ
Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−Ｐｈｅ−Ｇｌｙ−ＯＨ
Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−Ｐｈｅ−Ａｌａ−ＯＨ
Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−Ｐｈｅ−Ｍｅ βＡｌａ−ＯＥｔ
Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−Ｐｈｅ−（ｎ−Ｐｒ） βＡｌａ−ＯＨ
上記のペプチドの製造原料として、次式で表される保護ペプチド： Ｚ−ＨＮＣ（Ｎ−Ｚ）−Ｔｙｒ（Ｂｚｌ）−Ｄ−Ａｒｇ（Ｚ_２）−Ｐｈｅ−ＯＨを液相法により以下のようにして製造した。
【００４４】
出発原料である Ｚ−Ｐｈｅ−ＯＴｃｅ ２５４ ｇ を ２５％臭化水素−酢酸 ９００ ｍｌ で処理してＺ 基を脱離した後、氷浴下でＣＨ_２Ｃｌ_２ １０００ ｍｌに溶解した。この溶液に Ｂｏｃ−Ｄ−Ａｒｇ（Ｚ_２）−ＯＨ ２８８ ｇ， ＨＯＢｔ ８５ ｇを加え、ＴＥＡ ７７ ｍｌ で中和した後、 ＥＤＣ・ＨＣｌ １２１ ｇ により縮合しＢｏｃ−Ｄ−Ａｒｇ（Ｚ_２）−Ｐｈｅ−ＯＴｃｅとした。次いで、Ｂｏｃ−Ｄ−Ａｒｇ（Ｚ_２）−Ｐｈｅ−ＯＴｃｅ２４１ ｇを ４Ｎ 塩酸−酢酸エチルエステル １０００ ｍｌで処理して Ｂｏｃ基を脱離し、氷浴下で ＤＭＦ １３００ ｍｌに溶解した。この溶液にＢｏｃ−Ｔｙｒ（Ｂｚｌ）−ＯＨ １０８ ｇ， ＨＯＢｔ ４６ ｇを加え、ＴＥＡ ４２ ｍｌ で中和した後、 ＥＤＣ・ＨＣｌ ６５ ｇにより縮合し、次式で示される保護ペプチド：Ｂｏｃ−Ｔｙｒ（Ｂｚｌ）−Ｄ−Ａｒｇ（Ｚ_２）−Ｐｈｅ−ＯＴｃｅ を得た。
【００４５】
Ｂｏｃ−Ｔｙｒ（Ｂｚｌ）−Ｄ−Ａｒｇ（Ｚ_２）−Ｐｈｅ−ＯＴｃｅ ４８ ｇを４Ｎ塩酸−酢酸エチルエステル２５０ ｍｌで処理してＢｏｃ 基を脱離後、氷浴下で ＤＭＦ １５０ ｍｌ に溶解した。この溶液を ＴＥＡ７ ｍｌ で中和した後、Ｚ−ＨＮＣ（Ｎ−Ｚ）−Ｐｙｒａｚｏｌｅ （ アミジノ化試薬） １９ ｇを加え、室温で攪拌し、次式の保護ペプチド：Ｚ−ＨＮＣ（Ｎ−Ｚ）−Ｔｙｒ（Ｂｚｌ）−Ｄ−Ａｒｇ（Ｚ_２）−Ｐｈｅ−ＯＴｃｅ を得た。この保護ペプチド４２ ｇを水浴下酢酸に溶解し、その中に亜鉛末２１ ｇを加えて２時間攪拌し、反応液より亜鉛末を除き、減圧濃縮して保護ペプチド：Ｚ−ＨＮ−Ｃ（Ｎ−Ｚ）−Ｔｙｒ（Ｂｚｌ）−Ｄ−Ａｒｇ（Ｚ_２）−Ｐｈｅ−ＯＨを得た。
【００４６】
Ｚ−ＨＮＣ（Ｎ−Ｚ）−Ｔｙｒ（Ｂｚｌ）−Ｄ−Ａｒｇ（Ｚ_２）−Ｐｈｅ−ＯＨにＭｅＯＨ （ＥＤＣ−ＤＭＡＰ法）； ＮＨ_２Ｍｅ （ＥＤＣ−ＨＯＢｔ法）； Ｈ− βＡｌａ−ｏｌ （ＥＤＣ−ＨＯＢｔ法）； Ｈ−Ｇｌｙ−ＯＢｚｌ ・ＴｏｓＯＨ （ＥＤＣ−ＨＯＢｔ 法）； Ｈ−Ａｌａ− ＯＢｚｌ・ＴｏｓＯＨ （ＥＤＣ−ＨＯＢｔ 法）； Ｈ−Ｍｅ βＡｌａ−ＯＥｔ （ＥＤＣ−ＨＯＢｔ 法）；又はＨ−（ｎ−Ｐｒ）βＡｌａ−ＯＢｚｌ・ＴｏｓＯＨ （ＥＤＣ−ＨＯＢｔ 法） を縮合させた。生成物を水浴下酢酸に溶解してパラジウム炭素の存在下で接触還元し、触媒を除いて反応液を減圧濃縮し、さらに凍結乾燥を行って粉末の目的物ペプチドを得た。下記の条件のアミノ酸分析及び質量分析の結果はそれぞれのペプチド化合物の構造を支持していた。
【００４７】
アミノ酸分析は、ペプチドを約 ０．５ ｍｇ を加水分解管に採取し、１ ｍｌの ６Ｎ 塩酸を加え、脱気封管後、１１０ ℃で２４時間加水分解した。室温冷却後４０℃で濃縮乾固した後、残査を精製水５ ｍｌに溶解した。この溶解液より ５μｌ を採り減圧乾燥し、残査に ５０ ｍＭ ＮａＨＣＯ_３ 緩衝液（ｐＨ９．０） ２０μｌ 、ダブシルクロライド−アセトニトリル溶液 ４０ μｌ を加え、７０℃で１０分間加温した。反応液を減圧濃縮し、残査を ５０％エタノール １００μｌ に溶解し、その溶液 ２０ μｌ を液体クロマトグラフィーで解析した。質量分析は、ペプチド約 １５０μｇ を ２５０μｌ の ５％ 酢酸に溶解し、１ μｌ を Ｌｉｑｕｉｄ ＳＩＭＳにて解析した。
【００４８】
例６
Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−ＭｅＰｈｅ−Ｍｅ βＡｌａ−ＯＨ
Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−ＥｔＰｈｅ−Ｍｅ βＡｌａ−ＯＨ
上記ペプチドは、 ＴｏｓＯＨ・ＭｅβＡｌａ−ＯＢｚｌを出発原料として、Ｃ−末端より順次液相法により製造した。Ｂｏｃ−ＭｅＰｈｅ−ＯＨと ＴｏｓＯＨ・ＭｅβＡｌａ−ＯＢｚｌを ＥＤＣ−ＨＯＢｔ 法により縮合し、Ｂｏｃ−ＭｅＰｈｅ−ＭｅβＡｌａ−ＯＢｚｌを得て、Ｂｏｃ−ＭｅＰｈｅ−ＭｅβＡｌａ−ＯＢｚｌより ４Ｎ 塩酸−酢酸エチルエステルを用いて Ｂｏｃ基を脱離後、Ｂｏｃ−Ｄ−Ａｒｇ（Ｚ_２）−ＯＨとＥＤＣ−ＨＯＢｔ法により縮合して、Ｂｏｃ−Ｄ−Ａｒｇ（Ｚ_２）−ＭｅＰｈｅ−ＭｅβＡｌａ−ＯＢｚｌとした。次いで、Ｂｏｃ−Ｄ−Ａｒｇ（Ｚ_２）−ＭｅＰｈｅ−ＭｅβＡｌａ−ＯＢｚｌより ４Ｎ 塩酸−酢酸エチルエステルを用いて Ｂｏｃ基を脱離後、Ｂｏｃ−Ｔｙｒ（Ｂｚｌ）−ＯＨ とＥＤＣ−ＨＯＢｔ法により縮合して、保護ペプチド：Ｂｏｃ−Ｔｙｒ（Ｂｚｌ）−Ｄ−Ａｒｇ（Ｚ_２）−ＭｅＰｈｅ −ＭｅβＡｌａ−ＯＢｚｌを得た。
【００４９】
Ｂｏｃ−Ｔｙｒ（Ｂｚｌ）−Ｄ−Ａｒｇ（Ｚ_２）−ＭｅＰｈｅ−Ｍｅ βＡｌａ−ＯＢｚｌを ４Ｎ 塩酸−酢酸エチルエステルで処理して Ｂｏｃ基を脱離した後、Ｚ−ＨＮＣ（Ｎ−Ｚ）−Ｐｙｒａｚｏｌｅ （アミジノ化試薬）を加えて室温で攪拌し、保護ペプチド：Ｚ−ＨＮＣ（Ｎ−Ｚ）−Ｔｙｒ（Ｂｚｌ）−Ｄ−Ａｒｇ（Ｚ_２）−ＭｅＰｈｅ−ＭｅβＡｌａ−ＯＢｚｌを得た。この保護ペプチドを酢酸に溶解し、パラジウム炭素を加えて溶液に水素ガスを吹き込んで接触還元した。パラジウム炭素を除いた後、減圧濃縮し、凍結乾燥を行って粉末のペプチド Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−ＭｅＰｈｅ−ＭｅβＡｌａ−ＯＨを得た。このペプチドは、例５に記載したアミノ酸分析及び質量分析において上記の構造を支持する結果を与えた。
【００５０】
Ｂｏｃ−ＭｅＰｈｅ−ＯＨの代わりに Ｂｏｃ−ＥｔＰｈｅ−ＯＨ を用いて、上記と同様の方法により、ＴｏｓＯＨ・ＭｅβＡｌａ−ＯＢｚｌとＥＤＣ−ＨＯＢｔ法で縮合してＢｏｃ−ＥｔＰｈｅ−ＭｅβＡｌａ−ＯＢｚｌを製造した。次いで、上記と同様の方法により脱保護と縮合を繰り返し、次式で示される保護ペプチド：Ｚ−ＨＮＣ（Ｎ−Ｚ）−Ｔｙｒ（Ｂｚｌ）−Ｄ−Ａｒｇ（Ｚ_２）−ＥｔＰｈｅ−ＭｅβＡｌａ−ＯＢｚｌを得た。この保護ペプチドを酢酸に溶解してパラジウム炭素を加え、この溶液に水素ガスを吹き込んで接触還元した。パラジウム炭素を除いた後、減圧濃縮し、凍結乾燥を行って粉末のペプチド Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−ＥｔＰｈｅ−ＭｅβＡｌａ−ＯＨを得た。このペプチドは、例５に記載したアミノ酸分析及び質量分析により上記の構造を支持する結果を与えた。
【００５１】
例７
Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−Ｐｈｅ−Ｍｅ βＡｌａ−ＯＨ
ペプチド合成で通常用いられる液相法で以下のように合成した。 Ｚ−Ｐｈｅ−ＯＴｃｅ ２５４ ｇ を出発原料として、２５％ 臭化水素−酢酸 ９００ ｍｌ で Ｚ基を脱離後、氷浴下でＣＨ_２Ｃｌ_２ １０００ｍｌ に溶解した。この溶液にＢｏｃ−Ｄ−Ａｒｇ（Ｚ_２）−ＯＨ ２８８ ｇ、ＨＯＢｔ ８５ ｇ を加え、ＴＥＡ ７７ ｍｌ で中和した後、ＥＤＣ ・ＨＣｌ １２１ ｇ により縮合しＢｏｃ−Ｄ−Ａｒｇ（Ｚ_２）−Ｐｈｅ−ＯＴｃｅとした。次いで、Ｂｏｃ−Ｄ−Ａｒｇ（Ｚ_２）−Ｐｈｅ−ＯＴｃｅ ２４１ ｇを ４Ｎ 塩酸−酢酸エチルエステル １０００ ｍｌを用いて Ｂｏｃ基を脱離した後、氷浴下で ＤＭＦ １３００ ｍｌに溶解した。この溶液に Ｂｏｃ−Ｔｙｒ（Ｂｚｌ）−ＯＨ １０８ ｇ、ＨＯＢｔ ４６ ｇ を加え、ＴＥＡ ４２ ｍｌ で中和した後、 ＥＤＣ・ＨＣｌ ６５ ｇにより縮合して、次式で示される保護ペプチド：Ｂｏｃ−Ｔｙｒ（Ｂｚｌ）−Ｄ−Ａｒｇ（Ｚ_２）−Ｐｈｅ−ＯＴｃｅ を得た。
【００５２】
Ｂｏｃ−Ｔｙｒ（Ｂｚｌ）−Ｄ−Ａｒｇ（Ｚ_２）−Ｐｈｅ−ＯＴｃｅ ４８ ｇを ４Ｎ 塩酸−酢酸エチルエステル ２５０ｍｌ で処理して Ｂｏｃ基を脱離し、氷浴下でＤＭＦ １５０ ｍｌに溶解した。この溶液をＴＥＡ ７ ｍｌで中和した後、Ｚ−ＨＮＣ（Ｎ−Ｚ）−Ｐｙｒａｚｏｌｅ（アミジノ化試薬） １９ ｇを加え、室温で攪拌し、次式の保護ペプチド：Ｚ−ＨＮＣ（Ｎ−Ｚ）−Ｔｙｒ（Ｂｚｌ）−Ｄ−Ａｒｇ（Ｚ_２）−Ｐｈｅ−ＯＴｃｅを得た。この保護ペプチド ４２ ｇ を水浴下酢酸に溶解し、その中に亜鉛末 ２１ ｇ を加え、２時間攪拌した。反応液より亜鉛末を除いた後に減圧濃縮して保護ペプチド：Ｚ−ＨＮＣ（Ｎ−Ｚ）−Ｔｙｒ（Ｂｚｌ）−Ｄ−Ａｒｇ（Ｚ_２）−Ｐｈｅ−ＯＨを得た。
【００５３】
上記保護ペプチド： Ｚ−ＨＮＣ（Ｎ−Ｚ）−Ｔｙｒ（Ｂｚｌ）−Ｄ−Ａｒｇ（Ｚ_２）−Ｐｈｅ−ＯＨ １．１５ ｇ とＴｏｓＯＨ ・ＭｅβＡｌａ−ＯＢｚｌ ０．９３ ｇ を氷浴下 ＤＭＦ ３０ ｍｌ（ＤＭＳＯ １０％ 含有） で溶解し、この溶液に ＨＯＯＢｔ ０．２４ ｇ を加えてＴＥＡ ０．２１ ｍｌ で中和した後、 ＥＤＣ・ＨＣｌ ０．２５ ｇにより縮合し、次式で示される保護ペプチド：Ｚ−ＨＮＣ（Ｎ−Ｚ）−Ｔｙｒ（Ｂｚｌ）−Ｄ−Ａｒｇ（Ｚ_２）−Ｐｈｅ−ＭｅβＡｌａ−ＯＢｚｌを得た。この保護ペプチド ０．９０ ｇ を酢酸 １００ ｍｌ に溶解し、その中にパラジウム炭素 ０．９０ ｇ を加え、この溶液に水素ガスを吹き込んで接触還元した。パラジウム炭素を除いた後減圧濃縮し、凍結乾燥を行って粉末のペプチド： Ｈ_２ＮＣ（ＮＨ）−Ｔｙｒ−Ｄ−Ａｒｇ−Ｐｈｅ−Ｍｅ βＡｌａ−ＯＨを得た。このペプチドは実施例５に記載したアミノ酸分析及び質量分析において上記の構造を支持する結果を与えた。
【００５４】
以下に本発明のペプチド誘導体の物理化学的性状として、ＨＰＬＣの保持時間及び薄層クロマトグラフィーのＲｆ値を表１に示す。これらのペプチド誘導体はいずれも凍結乾燥品として得た。また、同様の方法により合成した化合物を表２に示す。
【００５５】

【００５６】
薄層クロマトグラフィーの条件：
Ｒｆ^ａ ：ｎ−ブタノール：酢酸：精製水＝４：１：５を混合し、その上層を展開溶媒として用いた。
Ｒｆ^ｂ ：ｎ−ブタノール：酢酸：精製水：ピリジン＝１５：３：１０：１２ を展開溶媒として用いた。
薄層板 ：シリカゲル（Ｍｅｒｃｋ Ｆ２５４）
【００５７】
【表１】

【００５８】
【表２】

【００５９】
試験例
本発明のペプチド誘導体の鎮痛効果を圧刺激法にて以下のように評価した。マウスの尾根部に １０ ｍｍＨｇ／ 秒の割合で圧刺激を加え、もがき、刺激部位への噛みつきなどの行動を示す圧力を測定し、これを疼痛反応閾値とした。実験には予め４０〜５０ ｍｍＨｇ の圧力に反応するマウスを用いた。また最大刺激圧は １００ ｍｍＨｇ とした。鎮痛効果は次式

（式中、Ｐｏは薬物処理前の疼痛反応閾値、Ｐｔは薬物処理ｔ分後の疼痛反応閾値、Ｐｃは最大刺激圧である）に従い、ｐｅｒｃｅｎｔ ｏｆ ｍａｘｉｍｕｍ ｐｏｓｓｉｂｌｅ ｅｆｆｅｃｔ （％ ｏｆ ＭＰＥ） として算出した。用量反応曲線から ５０ ％ の％ ｏｆＭＰＥ を与える薬物投与量をＥＤ_５０値として求め、薬物の鎮痛効力を比較した。皮下投与（背部皮下）及び経口投与の結果を表３に示す。
【００６０】
【表３】

【００６１】
【発明の効果】
本発明のペプチド誘導体は癌疼痛等の治療に使用することができるので有用である。[0001]
[Industrial applications]
The present invention relates to a peptide derivative which exerts a pharmacological action such as analgesia through an action on an opioid receptor or the like.
[0002]
[Prior art]
Opioid receptors to which opioids such as morphine bind were proven to exist in the early 1970's. Opioid receptors are currently roughly classified into three types: μ, δ, and κ. Morphine mainly acts as an agonist on the μ receptor, and exerts pharmacological effects such as analgesia, suppression of intestinal motility, and suppression of respiration.
[0003]
Since 1975, endogenous morphine-like substances that bind to opioid receptors have been discovered one after another. To date, all of these substances are peptides, collectively referred to as opioid peptides. The pharmacological effect of the opioid peptide is basically considered to be the same as that of morphine, and since it is a substance originally present in the living body, it is expected that the drug may be a drug having safety higher than that of morphine. However, natural opioid peptides have problems in pharmacokinetics, and have not yet been used as pharmaceuticals.
[0004]
In the 1980s, delmorphin containing D-alanine was isolated from frog skin. The analgesic effect of delmorphin was approximately 1,000 times stronger than that of morphine by intraventricular administration, and was found to be relatively stable in the body. Thereafter, a synthetic opioid peptide containing D-amino acids was produced. In particular, synthetic opioid peptides with high κ receptor selectivity are expected as analgesics without narcotic properties, and clinical trials have been conducted. However, their effects, side effects that may be caused by being κ agonists, and profitability Therefore, its potential as a pharmaceutical has been questioned.
[0005]
Furthermore, these synthetic opioid peptides are difficult to use as oral preparations, and cannot be used as a substitute for MS contin, which is a sustained-release oral preparation of morphine sulfate widely used in recent years as a therapeutic agent for cancer pain. . The daily dose of MS Contin increases to the gram unit, and it may be difficult to take it.In addition, it may cause itching or other side effects thought to be caused by histamine releasing action, and the administration must be discontinued. In some cases. Therefore, an alternative drug having safety and efficacy higher than that of morphine is desired.
[0006]
Problems to be Solved by the Invention and Means for Solving the Problems
The present inventors have diligently searched for an opioid peptide derivative having an excellent analgesic effect and oral absorbability in order to solve the above problems. As a result, they have found that an oligopeptide derivative having L-Tyr- (L or D) -Arg-Phe as a basic skeleton and having an amidino group at the N-terminus and a salt thereof retain such properties, and have completed the present invention. That is, the peptide derivative of the present invention can be represented by the following formula I.
Embedded image

[0007]
Describing the substituents in the above formula, Q means D-Arg (D-arginine residue) or L-Arg (L-arginine residue);¹Is a hydrogen atom or C_1-6It means an alkyl group (having 1 to 6 carbon atoms). Of these, Q is D-Arg and R¹Is preferably a hydrogen atom. R²Is a hydrogen atom, C_1-6Alkyl group, aryl group, C_1-6It represents an alkanoyl group or an arylcarbonyl group. The aryl group, alkanoyl group or arylcarbonyl group includes, for example, an aryl group such as a substituted or unsubstituted phenyl group, preferably an unsubstituted phenyl group; an alkanoyl group such as an acetyl group or a propanoyl group; or an aryl group such as a benzoyl group A carbonyl group can be used.
[0008]
X is -OR³, -NR⁴R⁵Or -NR⁶-CR⁷R⁸R⁹Indicates one of R³Is a hydrogen atom or C_1-6Represents an alkyl group;⁴Is a hydrogen atom or C_1-6Shows an alkyl group. R⁵Is C_1-6Hydroxyalkyl or sulfonic acid substituted C_1-6Shows an alkyl group. The hydroxyl group or the sulfonic acid group may be substituted at any position of the alkyl group, but a terminal-substituted alkyl group is preferred. R⁴And R⁵Is R together⁴And R⁵May represent a 5- or 6-membered nitrogen-containing saturated heterocyclic group together with the nitrogen atom to be substituted, and the heterocyclic ring may contain two or more nitrogen atoms. For example, -NR⁴R⁵A 1-piperazinyl group, 1-pyrrolidinyl group, 1-piperidinyl group or the like can be used.
[0009]
X is NR⁶-CR⁷R⁸R⁹  , R⁶Is a hydrogen atom, C_1-6C substituted by an aryl group such as an alkyl group or a phenyl group_1-6Shows an alkyl group (aralkyl group). Examples of the aralkyl group include a benzyl group. R⁷Is a hydrogen atom; a carboxyl group; C such as methoxycarbonyl and ethoxycarbonyl_1-6A carbonyl group substituted with an alkoxy group; a substituted or unsubstituted carbamoyl group; C substituted with a carboxyl group_1-6Alkyl group; substituted or unsubstituted carbamoyl group-substituted C_1-6An alkyl group; or C_1-6C having a carbonyl group substituted by an alkoxy group_1-6Shows an alkyl group.
[0010]
R⁸Is a hydrogen atom; C_1-6Alkyl group; amino C_1-6Alkyl group; C substituted by amidino group_1-6Alkyl group; C substituted by guanidino group_1-6Alkyl group, hydroxy C_1-6Alkyl group; C substituted by carboxyl group_1-6C substituted by an alkyl group or a substituted or unsubstituted carbamoyl_1-6Shows an alkyl group. Alternatively, R⁶And R⁸But together R⁶May form a 5- or 6-membered nitrogen-containing saturated heterocyclic group having a carboxyl group on the ring together with the nitrogen atom to be substituted. Examples of such a heterocyclic ring include a 2-carboxy-1-pyrrolidinyl group (-Pro-OH) and a 3-carboxy-1-piperidinyl group. Of these, for example, R⁷Is a carboxyethyl group or a carbamoylethyl group;⁸Is preferably a hydrogen atom. R⁹Is a hydrogen atom or C_1-6Shows an alkyl group. In each of the above substituents, the alkyl group, alkoxy group, or alkanoyl group may be linear or branched.
[0011]
The compound of the present invention represented by the above formula I has an L-tyrosine residue and two asymmetric carbons derived from D-Arg or L-Arg residue represented by Q 2, and a phenylalanine residue bonded to Q 2. An asymmetric carbon derived from⁷And R⁸Is substituted with an asymmetric carbon (R⁷And R⁸Have the same substituents at the same time) and one or more asymmetric carbon atoms optionally present in each of the above substituents. Asymmetric carbons other than those derived from L-Tyr, D-Arg, and L-Arg residues may be in either R- or S- configuration. Further, the compound represented by the formula I of the present invention also includes any optically active substance, a racemic form, a diastereoisomer or any mixture thereof represented by the above formula.
[0012]
The compounds of the present invention also include acid addition salts such as hydrochloride, acetate or paratoluenesulfonic acid, and base addition salts such as ammonium salt or organic amine salt. Further, in addition to the compounds represented by the above general formulas, compounds which are dimers or multimers of the above compounds, and cyclic compounds in which the C-terminal and the N-terminal of these compounds are bonded are included.
[0013]
The peptide of the present invention has an analgesic effect exceeding that of morphine. Since the histamine releasing action and the heart rate lowering action associated with the analgesic action are relatively weaker than morphine and the degree of cross-resistance with morphine is low, it is expected to be suitable for use in treating cancer pain. Examples of the administration route include intravenous administration, subcutaneous administration, and oral administration. Mucosal absorption preparations including nasal absorption and transdermal absorption preparations are also expected to be useful.
[0014]
The peptide derivative of the present invention can be synthesized by a solid phase method and a liquid phase method usually used for peptide synthesis. For example, a target peptide derivative can be produced by synthesizing a peptide chain having no amidino group by a solid phase method and further introducing an amidino group to the amino group of tyrosine at the N-terminus. After introduction, the C-terminus can be modified.
[0015]
Various excellent protecting groups such as an amino group and a condensing agent for a condensation reaction are known. For example, refer to the following Examples, for example: Koichi Suzuki, "Protein Engineering-Fundamentals and Applications," Maruzen ( (1992) and references cited therein; Bondanszky, et al. , "Peptide Synthesis", John Wiley & Sons, N.W. Y. J., 1976; M. Stewart and D. J. Young, "Solid Phase Peptide Synthesis", W.W. H. Freeman and Co. , San Francisco, 1969, etc., and can be selected and used as appropriate. In the solid phase method, it may be convenient to use various commercially available peptide synthesizers, for example, Model 430A manufactured by Perkin Elmer Japan (former company name Applied Biosystems). Commercially available products and the like can be easily obtained for the resins and reagents used in the synthesis, and examples thereof are shown in Examples.
[0016]
【Example】
Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples. By referring to the present example, or by modifying or changing the method of the present example, or by appropriately selecting starting materials or reaction reagents, the desired inventive peptide derivative of the present invention encompassed by general formula I Can be easily manufactured. In the examples, the meanings of the amino acid groups are the same as those usually used. When an amino acid having a D-form and an L-form is referred to, the amino acid means an L-amino acid unless otherwise indicated as D-. In addition, the following abbreviations may be used, and similar abbreviations may be used unless otherwise indicated.
[0017]
Z: benzyloxycarbonyl group
OTce: trichloroethyl ester group
Boc: t-butoxycarbonyl group
WSCI: 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide
TosOH: paratoluenesulfonic acid
OBzl: benzyloxy group
MeβAla or βMeAla: N-methyl-β-alanine
H-βAla-ol: NH₂CH₂CH₂CH₂OH
Fmoc: 9-fluorenylmethyloxycarbonyl
Pmc: 2,2,5,7,8-pentamethylchroman-6-sulfonyl
t-Bu: tertiary butyl
NMP: N-methylpyrrolidone
DMF: dimethylformamide
DMSO: dimethyl sulfoxide
TFA: trifluoroacetic acid
TEA: triethylamine
DCM: dichloromethane
DMAP: N, N-dimethylaminopyridine
DIPEA: N, N-diisopropylethylamine
DIPCI: N, N-diisopropylcarbodiimide
HOBt: 1-hydroxybenzotriazole
EDC: 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimide
HBTU: 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate
PyBrop: bromotris (pyrrolidino) phosphonium hexafluorophosphate
Alko resin: p-alkoxybenzyl alcohol resin [4-hydroxymethylphenoxymethyl copolystyrene 1% divinylbenzene resin; Am. Chem. Soc. , 95, 1328 (1974)], Watanabe Chemical Industry
Fmoc-NH-SAL resin: 4- (2 ', 4'-dimethoxyphenyl-9-fluorenylmethoxycarbonylaminomethyl) phenoxy resin: Watanabe Chemical Industry
[0018]
Example 1
H₂NC (NH) -Tyr-D-Arg-Phe-NHCH₂CH₂COOH
Using a Model 430A peptide synthesizer manufactured by Applied Biosystems (ABI), the above peptide was synthesized as follows by a solid-phase method (Original Autoprogram for the Fmoc / NMP method).
[0019]
0.25 mmol / 675 mg of Fmoc-β-Ala-Alko resin was washed once with NMP, and treated with NMP containing 20% piperidine for 4 minutes, and further treated with NMP containing 20% piperidine for 16 minutes. After washing 5 times with NMP, it was reacted with Fmoc-Phe-OH for 61 minutes. Next, the resultant was washed four times with NMP, and the resin was recovered from the fourth washing liquid, and unreacted amino groups were reacted with acetic anhydride.
[0020]
The above-described one cycle is performed in 120 minutes, and the same operation is performed in the second cycle using Fmoc-D-Arg (Pmc) -OH instead of Fmoc-Phe-OH, and in the third cycle using Fmoc-Tyr (t- Repeated with (Bu) -OH. As the side chain protecting group, Pmc was used for D-Arg, and t-Bu was used for Tyr.
[0021]
500 mg of the resin obtained by the above operation was mixed in a mixture of 0.75 g of phenol (crystal), 0.25 ml of ethanedithiol, 0.50 ml of thioanisole, 0.50 ml of water and 10.0 ml of TFA. The mixture was stirred at room temperature for 3 hours to separate the peptide from the resin and at the same time remove the protecting group. Then, the mixture was filtered through a 3 μm filter (ADVANTEC-Polyflon filter), 200 ml of cold diethyl ether was added to the filtrate, and the resulting precipitate was collected by filtration with a 3 μm filter (ADVANTEC-Polyflon filter). The precipitate on the filter was dissolved in 10-20 ml of 2N acetic acid and lyophilized to obtain a crude peptide.
[0022]
135 mg of the crude peptide was dissolved in 13.5 ml of water, 13.5 ml of 1 M o-methylisourea was added, and the mixture was stirred at 4 ° C. for 14 days to perform amidination. 106 mg of the obtained crude amidinated peptide was dissolved in 20 ml of 0.1% TFA aqueous solution. After adjusting the pH to 4-5 with 0.01 N hydrochloric acid, the peptide was purified on a Gilson HPLC system. Cosmosil C for HPLC₁₈Using a column, a continuous concentration gradient of a mixture of a 0.1% TFA aqueous solution and a 0.1% TFA aqueous solution containing 70% acetonitrile (mixing ratio from 0% at the start to 60% after 45 minutes) was performed. It was 10 ml / min.
[0023]
About 100 μg of the purified peptide was collected in a hydrolysis tube, added with 1 ml of 6N hydrochloric acid containing 0.2% phenol, degassed and sealed, and hydrolyzed at 110 ° C. for 24 hours. After cooling to room temperature and concentrating to dryness at 40 ° C., the residue was dissolved in 1 ml of 0.01N hydrochloric acid, and 20 μl of the solution was analyzed with an amino acid analyzer (analyzer: Hitachi L-8500; column: Hitachi Custom ion exchange resin # 2622; Pre-column: Hitachi Custom ion exchange resin # 2650 L, buffer: lithium buffer (0.09 N-pH 2.8, 0.25 N-pH 3.7, 0.72 N-pH 3.6, 1.00 N -PH 4.1, regeneration 0.20N), reaction temperature: 135 ° C, buffer pump flow rate: 0.30 ml / min, ninhydrin pump flow rate: 0.35 ml / min, detector wavelength: 570 nm / 440 nm, analysis time: 150 minutes). As a result, the results of amino acid analysis supported the above structure.
[0024]
About 150 μg of the purified peptide was dissolved in 250 μl of 5% acetic acid, and 1 μl of the solution was subjected to mass spectrometry by LiquidSIMS (MS and MS / MS, using a cesium ion gun; analyzer: Finnigan TSQ 700, Matrix: glycerol-thioglycerol) (1: 1), Collision Gas: Ar gas 3-5 mTorr, Collision Energy: -20 eV, Electron Multiplier: 1000-1500 V). The results supported the above structure.
[0025]
Example 2
H₂NC (NH) -Tyr-D-Arg-Phe-NHCH₂CH₂CONH₂
The above peptide derivative was prepared using Fmoc-NH-SAL resin (0.25 mmol / 385 mg) at the start of synthesis, Fmoc-β-Ala-OH in the first cycle, and Fmoc-Phe-OH in the second cycle and three cycles. The same procedure as in Example 1 was carried out except that Fmoc-D-Arg (Pmc) -OH was used for the eyes and Fmoc-Tyr (t-Bu) -OH was used for the fourth cycle.
[0026]
For the synthesis and purification of the amidinated peptide derivative, the mixed solution was prepared using a 0.05% formic acid aqueous solution and a 70% acetonitrile-containing 0.05% formic acid aqueous solution (mixing ratio: 0% at the start of HPLC to 40% after 45 minutes). The procedure was performed in the same manner as in Example 1 except that the gradient was changed to (Gradient). Amino acid composition analysis and mass spectrometry were performed in the same manner as in Example 1, and the results supporting the above structure were obtained.
[0027]
Example 3
H₂NC (NH) -Tyr-D-Arg-Phe-N (CH₃) CH₂CH₂COOH
The tetrapeptide derivative having the above structure was synthesized as follows by a solid phase method using the Fmoc / NMP method. A glass filter was used for filtration.
[0028]
After swelling 0.500 g of Alko resin with 6 ml of DMF, 0.228 g of Fmoc-N-methyl-β-alanine (Fmoc-β-MeAla-OH) and 0.093 ml of pyridine were added to the resin. After shaking for 1 minute, 0.147 g of 2,6-dichlorobenzoyl chloride was added and shaken for 24 hours. The resulting Fmoc-β-MeAla-Alko resin was washed three times with 6 ml of DMF, then three times with 6 ml of methanol, and three times with 6 ml of DCM, and unreacted hydroxymethyl groups were removed in 6 ml of DCM. Then, 0.0891 ml of benzoyl chloride and 0.0847 ml of pyridine were added, and the mixture was shaken for 1 hour to benzoylate. Further, the amino acid resin was washed three times with 6 ml of DCM, three times with 6 ml of DMF, and three times with 6 ml of methanol, and dried in a potassium hydroxide desiccator under vacuum.
[0029]
The Fmoc-group was removed by treating the Fmoc-β-MeAla-Alko resin three times with 12 ml of DMF, then three times with 12 ml of DMF containing 20% piperidine, and six times with 12 ml of DMF, to remove the Fmoc-group. 0.262 g of Phe-OH, 0.315 g of PyBrop (Watanabe Chemical Industry), 6 ml of NMP and 0.273 ml of DIPEA were added, and shaken for 24 hours to produce an Fmoc-Phe-β-MeAla-Alko resin. . After filtering and washing with 6 ml of NMP, unreacted amino groups were treated with 6 ml of DMF containing 0.248 g of 1-acetylimidazole and 0.0784 ml of DIPEA for 1 hour and capped. Then, the obtained resin was washed with 6 ml of NMP.
[0030]
The Fmoc group was removed from the Fmoc-Phe-β-MeAla-Alko resin by the same operation as above, and 0.557 g of Fmoc-D-Arg (Pmc) -OH, 0.121 g of HOBt, 0.299 g of HBTU, 0.274 ml of DIPEA was added, and the mixture was shaken for 1 hour to produce Fmoc-D-Arg (Pmc) -Phe-β-MeAla-Alko resin. Then, after filtration and washing as in the previous section, unreacted amino groups were capped.
[0031]
The Fmoc group was removed from the Fmoc-D-Arg (Pmc) -Phe-β-MeAla-Alko resin by the same operation as described above, and 0.310 g of Fmoc-Tyr (t-Bu) -OH and 0.103 g of HOBt were removed. , HBTU 0.256 g and DIPEA 0.235 ml were added and shaken for 1 hour to produce Fmoc-Tyr (t-Bu) -D-Arg (Pmc) -Phe-β-MeAla-Alko resin. After filtration and washing as in the previous section, unreacted amino groups were capped.
[0032]
The Fmoc group was removed from the Fmoc-Tyr (t-Bu) -D-Arg (Pmc) -Phe-β-MeAla-Alko resin by the same operation as described above, and the method of Matsudada et al. (The Journal of Organic Chemistry,570.989 g of 1H-pyrazole-1-carboxamidine hydrochloride, 1.293 ml of DIPEA, and 6 ml of DMF prepared according to the method described at 10-60 ° C, more preferably at 40-50 ° C. After reacting for 4 to 4 hours, the amino group of tyrosine at the N-terminal was amidinated. Then, the mixture was filtered, washed (3 times with 6 ml of NMP, and 3 times with 6 ml of methanol), and dried in a potassium hydroxide desiccator under vacuum.
[0033]
596 mg of the resin obtained by the above operation was treated on a glass filter with 5 ml of a mixed solution of TFA, phenol and water (TFA: phenol: water = 93: 2: 5) for 1 hour and filtered. The same operation was performed twice in total. The resin was further treated with 5 ml of TFA for 5 minutes and filtered. The same operation was performed three times. The filtrate obtained by each operation was combined and the solvent was distilled off under reduced pressure at 20 ° C. or lower. 20 ml of diethyl ether was added to the residue to form a white precipitate, and the operation of discarding the supernatant was performed three times in total. The obtained white powder was dissolved in 20 ml of water, and washed 3 times with 5 ml of diethyl ether in a separating funnel. Then, the aqueous layer was freeze-dried to obtain a crude amidinated peptide.
[0034]
56.2 mg of the crude amidinated peptide was dissolved in 10 ml of a 0.05% TFA aqueous solution and purified with a Shimadzu HPLC system. HPLC shows YMC D-ODS-5-ST C₁₈A mixture of the column and a 0.05% TFA aqueous solution containing 0.5% acetonitrile and a 0.05% TFA aqueous solution containing 70% acetonitrile (mixing ratio is a continuous concentration gradient from 0% at the start of HPLC to 90% after 50 minutes). The flow rate was 1 ml / min). Amino acid composition analysis and mass spectrometry were performed in the same manner as in Example 1, and results supporting the above structure were obtained.
[0035]
Example 4
H₂NC (NH) -Tyr-D-Arg-Phe-N (CH₂CH₃) CH₂CH₂COOH
The tetrapeptide derivative having the above structure was synthesized as follows by a solid phase method using the Fmoc / NMP method. A glass filter was used for filtration.
[0036]
After swelling 1.000 g of Alko resin with 12 ml of NMP, 0.475 g of Fmoc-N-ethyl-β-alanine (Fmoc-β-EtAla-OH) and 0.017 g of DMAP were added and shaken for 1 minute. Then, 0.177 g of DIPCI was added and shaken for 24 hours. The resulting Fmoc-β-EtAla-Alko resin was washed three times with 12 ml of NMP, then three times with 12 ml of methanol and three times with 12 ml of DCM, and unreacted hydroxymethyl groups were chlorinated in 12 ml of DCM. 0.178 ml of benzoyl and 0.170 ml of pyridine were added and shaken for 1 hour to benzoylate. Further, the amino acid resin was washed with 12 ml of DCM three times, with 12 ml of DMF three times, and with 12 ml of methanol three times, and dried in a potassium hydroxide desiccator under vacuum.
[0037]
The Fmoc group was removed by treating the Fmoc-β-EtAla-Alko resin three times with 20 ml of DMF, then three times with 12 ml of DMF containing 20% piperidine, and further six times with 12 ml of DMF to remove the Fmoc-group. 0.387 g of Phe-OH, 0.466 g of PyBrop, 12 ml of NMP, and 0.523 ml of DIPEA were added, and the mixture was shaken for 24 hours to produce Fmoc-Phe-β-EtAla-Alko resin. After filtration, the precipitate was washed with 12 ml of NMP, and unreacted amino groups were treated with 12 ml of DMF containing 0.551 g of 1-acetylimidazole and 0.174 ml of DIPEA for 1 hour and capped. Then, the obtained resin was washed again with 12 ml of NMP.
[0038]
The Fmoc group was removed from the Fmoc-Phe-β-EtAla-Alko resin by the same operation as described above. 0.707 g of Fmoc-D-Arg (Pmc) -OH, 0.153 g of HOBt, 0.379 g of HBTU and 0.348 ml of DIPEA were added to the resin, and the mixture was shaken for 1 hour and Fmoc-D-Arg (Pmc)- Phe-β-EtAla-Alko resin was produced. After filtration and washing as in the previous section, unreacted amino groups were capped.
[0039]
The Fmoc group is removed from the Fmoc-D-Arg (Pmc) -Phe-β-EtAla-Alko resin by the same operation as above, and 0.460 g of Fmoc-Tyr (t-Bu) -OH and 0.153 g of HOBt are removed. , HBTU 0.399 g and DIPEA 0.348 ml were added and shaken for 1 hour to produce Fmoc-Tyr (t-Bu) -D-Arg (Pmc) -Phe-β-EtAla-Alko resin. After filtration and washing as described above, unreacted amino groups were capped.
[0040]
1H-pyrazole-1 prepared in the same manner as in Example 3 by removing the Fmoc group from the Fmoc-Tyr (t-Bu) -D-Arg (Pmc) -Phe-β-EtAla-Alko resin by the same operation as described above. -2.199 g of carboxamidine hydrochloride, 2.874 ml of DIPEA and 6 ml of DMF were added, and the mixture was reacted at 40 to 50 ° C for 1 to 4 hours to amidinate the amino group of tyrosine at the N-terminal. Next, the mixture was filtered and washed (3 times with 12 ml of NMP and 3 times with 12 ml of methanol), and dried in a potassium hydroxide desiccator under vacuum.
[0041]
1.514 g of the resin obtained by the above operation was treated on a glass filter with 10 ml of a mixed solution containing TFA, phenol and water (TFA: phenol: water = 93: 2: 5) for 1 hour and filtered. The same operation was performed twice in total. The resin was further treated with 10 ml of TFA for 5 minutes and filtered. The same operation was performed three times. The filtrate obtained by each operation was combined and the solvent was distilled off under reduced pressure at 20 ° C. or lower. 20 ml of diethyl ether was added to the residue to form a white precipitate, and the operation of discarding the supernatant was performed three times in total. The obtained white powder was dissolved in 30 ml of water, washed three times with 5 ml of diethyl ether in a separating funnel, and the aqueous layer was lyophilized to obtain a crude amidinated peptide.
[0042]
The crude amidinated peptide (100 mg) was dissolved in a 0.05% TFA aqueous solution (20 ml) and purified in the same manner as in Example 3. Amino acid composition analysis and mass spectrometry were performed in the same manner as in Example 1, and results supporting the above structure were obtained.
[0043]
Example 5
H₂NC (NH) -Tyr-D-Arg-Phe-OMe
H₂NC (NH) -Tyr-D-Arg-Phe-NHMe
H₂NC (NH) -Tyr-D-Arg-Phe-βAla-ol
H₂NC (NH) -Tyr-D-Arg-Phe-Gly-OH
H₂NC (NH) -Tyr-D-Arg-Phe-Ala-OH
H₂NC (NH) -Tyr-D-Arg-Phe-MeβAla-OEt
H₂NC (NH) -Tyr-D-Arg-Phe- (n-Pr) βAla-OH
As a raw material for producing the above peptide, a protected peptide represented by the following formula: Z-HNC (NZ) -Tyr (Bzl) -D-Arg (Z₂) -Phe-OH was prepared by the liquid phase method as follows.
[0044]
254 g of Z-Phe-OTce, which is a starting material, is treated with 900 ml of 25% hydrogen bromide-acetic acid to remove the Z group, and then CH is cooled in an ice bath.₂Cl₂  Dissolved in 1000 ml. Boc-D-Arg (Z₂) -OH (288 g) and HOBt (85 g) were added, neutralized with TEA (77 ml), and condensed with EDC · HCl (121 g) to obtain Boc-D-Arg (Z₂) -Phe-OTce. Then, Boc-D-Arg (Z₂) -Phe-OTce (241 g) was treated with 4N hydrochloric acid-acetic acid ethyl ester (1000 ml) to remove the Boc group, and dissolved in DMF (1300 ml) in an ice bath. 108 g of Boc-Tyr (Bzl) -OH and 46 g of HOBt were added to this solution, neutralized with 42 ml of TEA, and then condensed with 65 g of EDC · HCl to obtain a protected peptide represented by the following formula: Boc-Tyr ( Bzl) -D-Arg (Z₂) -Phe-OTce was obtained.
[0045]
Boc-Tyr (Bzl) -D-Arg (Z₂) -Phe-OTce (48 g) was treated with 4N hydrochloric acid-ethyl acetate (250 ml) to remove the Boc group, and then dissolved in DMF (150 ml) in an ice bath. After neutralizing this solution with 7 ml of TEA, 19 g of Z-HNC (NZ) -Pyrazole (amidination reagent) was added, and the mixture was stirred at room temperature, and protected peptide of the following formula: Z-HNC (NZ) -Tyr (Bzl) -D-Arg (Z₂) -Phe-OTce was obtained. 42 g of this protected peptide was dissolved in acetic acid in a water bath, and 21 g of zinc dust was added thereto, and the mixture was stirred for 2 hours. -Z) -Tyr (Bzl) -D-Arg (Z₂) -Phe-OH was obtained.
[0046]
Z-HNC (NZ) -Tyr (Bzl) -D-Arg (Z₂) -Phe-OH with MeOH (EDC-DMAP method); NH₂Me (EDC-HOBt method); H-βAla-ol (EDC-HOBt method); H-Gly-OBzl · TosOH (EDC-HOBt method); H-Ala-OBzl · TosOH (EDC-HOBt method); Me βAla-OEt (EDC-HOBt method); or H- (n-Pr) βAla-OBzl · TosOH (EDC-HOBt method) was condensed. The product was dissolved in acetic acid in a water bath and catalytically reduced in the presence of palladium carbon. The catalyst was removed, and the reaction solution was concentrated under reduced pressure, followed by lyophilization to obtain a powdered target peptide. The results of amino acid analysis and mass spectrometry under the following conditions supported the structure of each peptide compound.
[0047]
For amino acid analysis, about 0.5 mg of the peptide was collected in a hydrolysis tube, 1 ml of 6N hydrochloric acid was added, and after degassing and sealing, the mixture was hydrolyzed at 110 ° C. for 24 hours. After cooling to room temperature and concentrating to dryness at 40 ° C., the residue was dissolved in 5 ml of purified water. Take 5 μl of this solution, dry it under reduced pressure, and add 50 mM NaHCO₃  20 μl of a buffer solution (pH 9.0) and 40 μl of dabsyl chloride-acetonitrile solution were added, and the mixture was heated at 70 ° C. for 10 minutes. The reaction solution was concentrated under reduced pressure, the residue was dissolved in 100 μl of 50% ethanol, and 20 μl of the solution was analyzed by liquid chromatography. For mass spectrometry, about 150 μg of the peptide was dissolved in 250 μl of 5% acetic acid, and 1 μl was analyzed by Liquid SIMS.
[0048]
Example 6
H₂NC (NH) -Tyr-D-Arg-MePhe-Me βAla-OH
H₂NC (NH) -Tyr-D-Arg-EtPhe-Me βAla-OH
The peptide was produced by TosOH.MeβAla-OBzl as a starting material and sequentially from the C-terminal by a liquid phase method. Boc-MePhe-OH and TosOH.MeβAla-OBzl are condensed by the EDC-HOBt method to obtain Boc-MePhe-MeβAla-OBzl. After desorption, Boc-D-Arg (Z₂) -OH and EDC-HOBt method to form a Boc-D-Arg (Z₂) -MePhe-MeβAla-OBzl. Then, Boc-D-Arg (Z₂) -MePhe-MeβAla-OBzl Boc group was eliminated from the mixture with 4N hydrochloric acid-ethyl acetate using 4N hydrochloric acid-ethyl acetate, and then condensed with Boc-Tyr (Bzl) -OH by EDC-HOBt method to obtain a protected peptide: Boc-Tyr (Bzl). ) -D-Arg (Z₂) -MePhe-MeβAla-OBzl was obtained.
[0049]
Boc-Tyr (Bzl) -D-Arg (Z₂) -MePhe-MeβAla-OBzl was treated with 4N hydrochloric acid-ethyl acetate to remove the Boc group, then Z-HNC (NZ) -Pyrazole (amidinating reagent) was added, and the mixture was stirred at room temperature. Protected peptide: Z-HNC (NZ) -Tyr (Bzl) -D-Arg (Z₂) -MePhe-MeβAla-OBzl was obtained. This protected peptide was dissolved in acetic acid, palladium carbon was added, and hydrogen gas was blown into the solution to perform catalytic reduction. After removing the palladium carbon, the mixture was concentrated under reduced pressure, freeze-dried, and powdered peptide H₂NC (NH) -Tyr-D-Arg-MePhe-MeβAla-OH was obtained. This peptide gave results supporting the above structure in amino acid analysis and mass spectrometry described in Example 5.
[0050]
Using Boc-EtPhe-OH in place of Boc-MePhe-OH, Boc-EtPhe-MeβAla-OBzl was condensed with TosOH.MeβAla-OBzl by the EDC-HOBt method in the same manner as described above. Then, deprotection and condensation are repeated in the same manner as described above to obtain a protected peptide represented by the following formula: Z-HNC (NZ) -Tyr (Bzl) -D-Arg (Z₂) -EtPhe-MeβAla-OBzl was obtained. This protected peptide was dissolved in acetic acid, palladium carbon was added, and hydrogen gas was blown into the solution to perform catalytic reduction. After removing the palladium carbon, the mixture was concentrated under reduced pressure, freeze-dried, and powdered peptide H₂NC (NH) -Tyr-D-Arg-EtPhe-MeβAla-OH was obtained. This peptide gave results supporting the above structure by amino acid analysis and mass spectrometry as described in Example 5.
[0051]
Example 7
H₂NC (NH) -Tyr-D-Arg-Phe-Me βAla-OH
It was synthesized as follows by a liquid phase method usually used for peptide synthesis. Starting from 254 g of Z-Phe-OTce as a starting material, the Z group was eliminated with 900 ml of 25% hydrogen bromide-acetic acid, and CH was removed in an ice bath.₂Cl₂  It was dissolved in 1000 ml. Boc-D-Arg (Z₂) -OH and 85 g of HOBt were added, neutralized with 77 ml of TEA, and then condensed with 121 g of EDC · HCl to obtain Boc-D-Arg (Z₂) -Phe-OTce. Then, Boc-D-Arg (Z₂241 g of-)-Phe-OTce was dissolved in 1300 ml of DMF in an ice bath after removing the Boc group using 1000 ml of 4N hydrochloric acid-ethyl acetate. To this solution were added 108 g of Boc-Tyr (Bzl) -OH and 46 g of HOBt, neutralized with 42 ml of TEA, and then condensed with 65 g of EDC · HCl to obtain a protected peptide represented by the following formula: Boc-Tyr (Bzl) -D-Arg (Z₂) -Phe-OTce was obtained.
[0052]
Boc-Tyr (Bzl) -D-Arg (Z₂48) g) -Phe-OTce was treated with 250 ml of 4N hydrochloric acid-ethyl acetate to remove the Boc group and dissolved in 150 ml of DMF in an ice bath. After neutralizing this solution with 7 ml of TEA, 19 g of Z-HNC (NZ) -Pyrazole (amidinating reagent) was added, and the mixture was stirred at room temperature to obtain a protected peptide of the following formula: Z-HNC (NZ) ) -Tyr (Bzl) -D-Arg (Z₂) -Phe-OTce was obtained. 42 g of this protected peptide was dissolved in acetic acid in a water bath, and 21 g of zinc powder was added thereto, followed by stirring for 2 hours. After removing zinc dust from the reaction solution, the mixture was concentrated under reduced pressure and the protected peptide: Z-HNC (NZ) -Tyr (Bzl) -D-Arg (Z₂) -Phe-OH was obtained.
[0053]
The above protected peptide: Z-HNC (NZ) -Tyr (Bzl) -D-Arg (Z₂) -Phe-OH (1.15 g) and TosOH.MeβAla-OBzl (0.93 g) were dissolved in 30 ml of DMF (containing 10% of DMSO) in an ice bath, and 0.24 g of HOOBt was added to this solution, and TEA was added. After neutralization with 21 ml, the mixture was condensed with 0.25 g of EDC · HCl and protected peptide represented by the following formula: Z-HNC (NZ) -Tyr (Bzl) -D-Arg (Z₂) -Phe-MeβAla-OBzl was obtained. 0.90 g of this protected peptide was dissolved in 100 ml of acetic acid, 0.90 g of palladium carbon was added thereto, and hydrogen gas was blown into the solution to perform catalytic reduction. After removing the palladium carbon, the mixture was concentrated under reduced pressure, freeze-dried, and the peptide of powder: H₂NC (NH) -Tyr-D-Arg-Phe-MeβAla-OH was obtained. This peptide gave results supporting the above structure in amino acid analysis and mass spectrometry described in Example 5.
[0054]
The retention time of HPLC and the Rf value of thin-layer chromatography are shown in Table 1 below as physicochemical properties of the peptide derivative of the present invention. All of these peptide derivatives were obtained as lyophilized products. Table 2 shows compounds synthesized by the same method.
[0055]

[0056]
Conditions for thin layer chromatography:
Rf^a        : N-butanol: acetic acid: purified water = 4: 1: 5, and the upper layer was used as a developing solvent.
Rf^b        : N-butanol: acetic acid: purified water: pyridine = 15: 3: 10: 12 was used as a developing solvent.
Thin plate: silica gel (Merck F254)
[0057]
[Table 1]

[0058]
[Table 2]

[0059]
Test example
The analgesic effect of the peptide derivative of the present invention was evaluated by the pressure stimulation method as follows. A pressure stimulus was applied to the ridge of the mouse at a rate of 10 mmHg / sec, and the pressure indicating a behavior such as struggling and biting the stimulus site was measured, and this was defined as a pain response threshold. In the experiment, mice that had previously responded to a pressure of 40 to 50 mmHg were used. The maximum stimulation pressure was 100 mmHg. The analgesic effect is

(Where Po is the pain response threshold before drug treatment, Pt is the pain response threshold t minutes after drug treatment, and Pc is the maximum stimulation pressure), and was calculated as percent of maximum possible effect (% of MPE). The dose of the drug giving 50%% ofMPE from the dose response curve is the ED₅₀The analgesic efficacy of the drugs was compared. Table 3 shows the results of subcutaneous administration (subcutaneous back) and oral administration.
[0060]
[Table 3]

[0061]
【The invention's effect】
The peptide derivative of the present invention is useful because it can be used for treating cancer pain and the like.

Claims (12)

The following formula:

Wherein R ¹ represents a hydrogen atom or a C _1-6 alkyl group; R ² represents a hydrogen atom, a C _1-6 alkyl group, an aryl group, a C _1-6 alkanoyl group, or an arylcarbonyl group; Represents D-Arg or L-Arg; X represents OR ³ (R ³ represents a hydrogen atom or a C _1-6 alkyl group), NR ⁴ R ⁵ (R ⁴ represents a hydrogen atom or a C _1-6 alkyl group) And R ⁵ represents a C _1-6 hydroxyalkyl group or a sulfonic acid-substituted C _1-6 alkyl group, or R ⁴ and R ⁵ together form a 5- or 6-membered nitrogen-containing saturated group together with the nitrogen atom which they substitute. A heterocycle), or NR ⁶ -CR ⁷ R ⁸ R ⁹ (R ⁶ represents a hydrogen atom, a C _1-6 alkyl group, or an aryl-substituted C _1-6 alkyl group, R ⁷ represents a hydrogen atom, a carboxyl group , C _1-6 alkoxycarbonyl group, a carboxy C _1-6 alkyl group, C _1-6 alkyl substituted or unsubstituted carbamoyl C _1-6 alkyl group or a C _1-6 aralkyl, Indicates alkoxycarbonyl C _1-6 alkyl group, R ⁸ is a hydrogen atom, C _1-6 alkyl, amino C _1-6 alkyl group, an amidino C _1-6 alkyl group, a guanidino C _1-6 alkyl, hydroxy C _{A 1-6} alkyl group, a carboxy C _1-6 alkyl group, or a C _1-6 alkyl- substituted or unsubstituted carbamoyl C _1-6 alkyl group, or R ⁶ and R ^{8 taken} together and substituted by R ⁶ A 5- or 6-membered carboxy-substituted nitrogen-containing saturated heterocyclic ring together with a nitrogen atom, and R ⁹ represents a hydrogen atom or a C _1-6 alkyl group)]; The compound according to claim ¹ , wherein R ¹ is a hydrogen atom, and a salt thereof. The compound according to claim 1 or 2, wherein R ⁶ is methyl or ethyl, and a salt thereof. The compound according to any one of claims 1 to 3, wherein R ⁷ is a carboxyl group or a carbamoyl group, and a salt thereof. The compound according to any one of claims 1 to 3 , wherein R ⁷ is a carboxymethyl group or a carbamoylmethyl group, and a salt thereof. The compound according to any one of claims 1 to 5, wherein Q is D-Arg, and a salt thereof. The compound according to claim 6, wherein R ¹ is a hydrogen atom, R ⁶ is a hydrogen atom, R ⁷ is a carboxymethyl group, and R ⁸ is a hydrogen atom. The compound according to claim 6, wherein R ¹ is a hydrogen atom, R ⁶ is a hydrogen atom, R ⁷ is a carbamoylmethyl group, and R ⁸ is a hydrogen atom. The compound according to claim 6, wherein R ¹ is a hydrogen atom, R ⁶ is a methyl group, R ⁷ is a carboxymethyl group, and R ⁸ is a hydrogen atom. The compound according to claim 6, wherein R ¹ is a hydrogen atom, R ⁶ is an ethyl group, R ⁷ is a carboxymethyl group, and R ⁸ is a hydrogen atom. A multimer or cyclic compound of the compound according to claim 1, and a salt thereof. The salt of the compound according to any one of claims 1 to 11, which is a hydrochloride or an acetate.