JP3786461B2 - New physiologically active substance - Google Patents

Description

【０００８】
（式中、Ｒ₁ は水素原子または低級アルキル基、Ｒ₂ はシアノ基またはカルボキシル基を意味する。）
より具体的には、一般式（Ｉ）において、Ｒ₁ がメチル基、Ｒ₂ がシアノ基であり、12Z、 14Eの立体配置を有する化合物（II）、Ｒ₁ がメチル基、Ｒ₂ がシアノ基であり、12Z、 14Zの立体配置を有する化合物(III) 、Ｒ₁が水素原子、Ｒ₂がシアノ基であり、12Z、 14Eの立体配置を有する化合物 (IV) 、Ｒ₁ がメチル基、Ｒ₂ がカルボキシル基であり、12Z、 14Eの立体配置を有する化合物（Ｖ）、それらの塩またはそれらの水和物、及びこれら化合物の製造方法、並びにこれら化合物を有効成分とする、血管新生の異常増殖を伴う各種疾患に対する予防および治療剤等として有効な医薬に関する。
【０００９】
これら化合物はボレリジンの類縁化合物であって、ボレリジンはストレプトミセス属の微生物の培養液から抗生物質として1949年単離構造決定された化合物である（Berger J. et al., Arch. Biochem. Biophys., 22, 476, 1949）。
【００１０】
【発明の実施の形態】
本発明化合物の抽出原料である微生物培養液の微生物種としては、ストレプトミセス属を選び、 Streptomyces albovinaceus，Streptomyces rochei およびStreptomyces sp. C 2989 などボレリジンを産生するいずれの微生物も使用することができる（Singh S.K. et al., Antimicrob. Agents. Chemother. 27，239, 1985；Berger J., et al., Arch. Biochem. Biophys.，22，476, 1949 ；Lumb M.et al., Nature, 206 ，263, 1965)。
その例として、例えばストレプトミセス・ロチェイ（Streputomyces rochei) ATCC 10739などが挙げられるが、特にボレリジンの生産力が高く好適な菌株として本発明者等が石垣島の土壌より分離した放線菌 Mer-N7167株が挙げられる。本菌株は以下の菌学的性質を有する。
【００１１】
形態；分岐し良く伸長する基生菌糸と、同じく良く伸長する気中菌糸とからなり、気中菌糸の先端は胞子化する。胞子鎖は10〜50個連なり、ゆるやかなラセン状を呈するが、曲状のものも見られる。胞子の表面は平滑で大きさは直径１μm ×１〜２μm 程度である。
【００１２】
菌体成分；ジアミノピメリン酸としては、LL−型を含み、糖はガラクトース、グルコース、マンノース、リボースを含む。
【００１３】
各種培地上での生育；
ISP-１ 生育は中程度で、薄く白色の気中菌糸を産する。培養裏面はわずかに黄色になる。
ISP-２ 生育は良好で、白色の気中菌糸上に灰色ないし灰白色（Light grayish reddish brown ）の胞子を多量に産する。培養裏面はわずかに黄褐色を呈する。
ISP-３ 生育は良好で、胞子の色は灰紫色（Grayish purple）、他はISP-２と同様。
ISP-４ 胞子の色は灰色（Medium gray)他はISP-２と同様。
ISP-５ 生育は弱く、白色の気中菌糸を少し産する。
改変チロシン培地 メラニン色素は産生しない。
なお、いずれの培地でも可溶性色素はほとんど見られない。
【００１４】
糖資化性；
（＋）グルコース、イノシトール、フラクトース、ラムノース、マンノース、アラビノース
（±）キシロース
（−）ラフィノース、シュクロース
（＋）；良く資化する （−）；資化しない （±）；その中間
同定；
本株の性状を同時に実施した Streptomyces rochei type strain IFO 12908の性状と比較すると、表１に示すような相違点が見られる。
【００１５】
【表１】

【００１６】
このような相違はあるものの種が異なるほどの差ではなく、ストレプトミセス・ロチェイ（ Streptomyces rochei )の種内変動の範囲であるので、本発明者らは本菌株をストレプトミセス・ロチェイ（ Streptomyces rochei ) Mer-N7167と命名した。なお、本菌株は、平成６年１１月２８日付けで工業技術院生命工学工業技術研究所に FERM P-14670 として寄託されている。
【００１７】
血管新生阻害活性のスクリーニング法として、ラット大動脈片をコラーゲンゲル内にて培養した場合に観察される管腔形成の阻害度を指標とした（ Nicosia R.F., Lab. Invest., 63, 115, 1990）。また、血管内皮細胞に対する増殖抑制作用及びスレオニン tＲＮＡ合成酵素阻害活性をも測定した。
【００１８】
ストレプトミセス属の微生物を通常の適切な培養条件にて培養後、培養液を清澄濾過したのちブタノールまたはメチルイソブチルケトンなどの有機溶媒を加え抽出し、有機溶媒層を減圧下濃縮する。次いでメタノールにて抽出し、石油エーテル（light petroleum)などで処理し粗抽出物を得る。次いでシリカゲルなどを用いる吸着クロマトグラフィー、LH-20 ゲルクロマトグラフィー、分配クロマトグラフィー、薄層クロマトグラフィー、ペーパークロマトグラフィーなどを適宜利用して分画し、活性スクリーニングにより活性画分を確認する。上記手法を適宜組み合わせることにより活性物質を単離することができる。吸着クロマトグラフィーに使用する溶媒としては、クロロホルム、メタノール、アセトン、ヘキサン、トルエンなど通常使用される有機溶媒を用い、適宜濃度を選択、組み合わせて使用することができる。結晶化の溶媒としてはクロロホルムとヘキサン、アセトン、四塩化炭素などの組み合わせを適宜選択して用いることができる。一つの手法として M. Lumbらの方法がある（Nature, 206, 263, 1965）。単離した化合物の構造解析は、元素分析、GC-MS、 NMR、融点、紫外・赤外吸収スペクトルなど常法の手法によって行うことができる。
【００１９】
本発明化合物は強力な血管新生阻害活性を有することから、異常な血管新生が観察されている疾患、例えばリウマチ性関節炎、固形癌、アテローム性動脈硬化症、糖尿病性網膜症、血管腫、乾せんなどの予防剤として、また治療薬として期待されるものである。
【００２０】
該化合物を各種疾患治療・予防剤として投与する場合、錠剤、散剤、顆粒剤、カプセル剤、シロップ剤などとして経口的に投与してもよいし、また噴霧剤、坐剤、注射剤、外用剤、点滴剤として非経口的に投与してもよい。投与量は症状の程度、年齢、肝疾患の種類などにより著しく異なるが、通常成人１日当たり約 1mg〜100mg を１日１〜数回にわけて投与する。
【００２１】
製剤化の際は通常の製剤担体を用い、常法により製造する。すなわち、経口用固形製剤を調製する場合は、主薬に賦形剤、更に必要に応じて結合剤、崩壊剤、滑沢剤、着色剤、矯味矯臭剤などを加えた後、常法により錠剤、被覆錠剤、顆粒剤、散剤、カプセル剤などとする。これらの錠剤、顆粒剤には糖衣、ゼラチン衣、その他必要により適宜コーティングすることは勿論差し支えない。
注射剤を調製する場合には、主薬に必要によりpH調整剤、緩衝剤、安定化剤、可溶化剤などを添加し、常法により皮下、筋肉内、静脈内用注射剤とする。
【００２２】
【実施例】
以下の実施例により本発明をさらに詳細に説明するが、本発明はこれらに限定されるものではない。
なお、例中の％は特記しない限り重量基準である。
【００２３】
実施例１．精製方法
種培地として、グリセロール 2.0％、グルコース 2.0％、大豆粉 2.0％、酵母エキス0.5 ％、塩化ナトリウム0.25％、炭酸カルシウム0.32％及び微量金属溶液（硫酸銅0.25％、塩化マンガン0.25％及び硫酸亜鉛0.25％の溶液を予め調製）0.2 ％の組成からなる培地を用いた。生産培地としては、種培地のグリセロール 2.0％のかわりにポテト澱粉 2.0％とし、他の組成は同じものを用いた。ジャー培養に際しては、消泡剤0.05％を添加した。殺菌前pHを 7.4に調整して使用した。前記の種培地 100mlを分注した 500ml容三角フラスコを 120℃で15分間殺菌し、これにMer-N 7167株の斜面寒天培養の１白金耳を接種し、28℃で３日間振盪培養して種培養とした。生産培地15Ｌを、各30Ｌ容ジャー・ファーメンター２基に分注して 120℃、20分間殺菌し、種培養を各 100mlずつ接種し、28℃で５日間通気（0.5vvm）、攪拌（300rpm）培養した。培養終了後遠心分離して上清を集め、pH７に調整して、ダイヤイオンHP-20 （三菱化成社製）３Ｌに吸着させ、カラムに充填し、水洗浄、20％メタノール洗浄後、80％アセトンで溶出させた。溶出液約３Ｌを、減圧下で濃縮してアセトンを除去し、水を加えて約１Ｌにして、酢酸エチル１Ｌで２回抽出した。抽出液を減圧下で濃縮乾固し、黒褐色の油状物質を得た。この油状物質を少量のメタノールに溶解し、セファデックス LH-20（ファルマシア・バイオテク社製）カラム（400ml ）の上部に載せ、メタノールで展開し、活性画分を濃縮乾固した。次に少量のクロロホルム：メタノール＝50：１に溶解し、シリカゲル60（メルク社製）カラム（150ml)の上部に載せ、クロロホルム：メタノール＝50：１、20：１及び10：１、各 500mlで溶出させた。クロロホルム：メタノール＝50：１〜20：１で溶出される活性画分を集め、濃縮乾固した。次に少量のアセトンに溶解し、少量のシリカゲル60と混ぜ、減圧下で濃縮乾固させ、シリカゲル60カラム（150ml)の上部に載せ、ヘキサン：アセトン＝３：１、２：１及び１：２、各 500mlで溶出させた。ヘキサン：アセトン＝３：１〜２：１で溶出される活性画分を集め、減圧下濃縮乾固した。この試料を少量のトルエン: アセトン＝５：１に溶解し、シリカゲル60カラム（50ml）の上部に載せ、トルエン：アセトン＝５：１、４：１及び２：１、各 200mlで溶出させた。トルエン：アセトン＝５：１で溶出される活性画分を集め、減圧下濃縮乾固した。この試料をCOSMOSIL 3C₁₈ カラム（3 μm, 4.6x100mm）、フォトダイオードアレイ検出器を用いて分析したところ、図１に示すように、ボレリジンと同一紫外吸収スペクトルを示す３種のピーク（それぞれＰ−１、Ｐ−２、Ｐ−３と記す。）を確認した（移動相：アセトニトリル:10mM りん酸二水素カリウム緩衝液、 pH3.5 ＝１：１、イソクラティック溶出、１ml/min、ＯＤ₂₅₄nm ）。なお、図１のメインピークはボレリジンである。
【００２４】
実施例２ Ｐ−１およびＰ−２の精製と構造決定
Mer-N7167 株のタンク培養液170 Ｌを遠心分離して上清140 Ｌを得、これをpH 7に調整してダイヤイオンHP-20 ７Ｌに吸着させた。HP-20 樹脂を集めカラムに充填し水洗後、80％アセトン水、20Ｌで溶出した。溶出液を減圧下濃縮してアセトンを留去し、水を加えて４Ｌにして、酢酸エチル４Ｌで２回抽出した。酢酸エチル相を併せ減圧下濃縮乾固し、黒褐色油状物質27g を得た。この油状物質をメタノールで調製したセファデックスLH-20 カラムに付し、メタノールで展開し、TLC （メルク社製シリカゲル60、No.5715、 展開溶媒：クロロホルム：メタノール＝９：１、硫酸発色、Rf値 0.42 ）にてモニターしながら、ボレリジンを含む画分を集め濃縮乾固した。得られた試料をシリカゲル60（750ml)のカラムに吸着させ、クロロホルム２Ｌで洗浄後、クロロホルム：メタノール＝49:1および19:1、各2.5 Ｌで溶出し、ボレリジンを含む画分を集め濃縮乾固した。得られた試料を更にシリカゲル60(750ml) のカラムに吸着させ、ヘキサン：アセトン＝4:1 、２Ｌで洗浄後、同じく 7:3の混合溶媒で溶出した。ボレリジンを含む画分を集め濃縮乾固し、5.95ｇの精製物を得た。生物活性を示すこの精製物をクロロホルム−ヘキサンの混合溶媒から結晶化し、ボレリジンの無色針状結晶4.29ｇを得た。母液を濃縮乾固し（1.58g)、少量のジメチルスルホキシドに溶解した後、アセトニトリル：10mMりん酸二水素カリウム緩衝液(pH3.5) ＝2:3 であらかじめ緩衝化しておいたYMC-GEL ODS-AM 120-S50（ YMC社製、600ml)のカラムに付した。同じ組成の混合溶媒２Ｌで洗浄後、9:11さらに1:1 の組成の混合溶媒各２Ｌで溶出し、溶出液をHPLCでモニターしながら、ボレリジンを純粋に含む画分と目的物質を含む画分とに分けた。両画分ともにアセトニトリルを留去し、酢酸エチルで抽出、酢酸エチルを留去後凍結乾燥して、それぞれ820mg と107mg の白色粉末を得た。目的物質を含む試料は、さらにHPLC( YMC-Pack D-ODS-S-5カラム、5 μm、20x250mm、 移動相、アセトニトリル：10mMりん酸二水素カリウム緩衝液 pH3.5＝11:9、 イソクラティック溶出、15ml/min、OD₂₅₄nm ）により分取し、同様に脱塩処理を行い、ボレリジン12mg、Ｐ−１ 59mg 、Ｐ−２ 11mg を純粋な白色粉末として単離した。
【００２５】
Ｐ−１の物理化学的性状
１）融点： 88-90℃
２）高分解能EI Mass スペクトル：測定の結果、m/z489.3087 にM⁺が観測され、分子式はＣ₂₈Ｈ₄₃ＮＯ₆ であると決定した。質量計算値はm/z489.3090 である。３）¹Ｈ核磁気共鳴スペクトル：図２に示した。
４）¹³Ｃ核磁気共鳴スペクトル：重クロロホルム中での測定結果を表２に示した。
【００２６】
Ｐ−２の物理化学的性状
１）融点： 88-91℃
２）高分解能EI Mass スペクトル：測定の結果、m/z489.3087 にM⁺が観測され、分子式はＣ₂₈Ｈ₄₃ＮＯ₆ であると決定した。質量計算値はm/z489.3090 である。
３）¹Ｈ核磁気共鳴スペクトル：図３に示した。
４）¹³Ｃ核磁気共鳴スペクトル：重クロロホルム中での測定結果を表２に示した。
【００２７】
【表２】

【００２８】
以上の結果から、Ｐ−１は下記式（II）で表される、ボレリジンの11位における立体異性体（化合物II）、Ｐ−２は下記式(III) で表される、ボレリジンの14および15位の二重結合に関する幾何異性体（化合物III)であると決定した。
【００２９】
【化３】

【００３０】
実施例３ Ｐ−３の分離と構造解析
図１に記載のHPLC解析クロマトに基づいて、Ｐ−３をHPLC−質量分析計（LC-Mass)にて分析した結果、ボレリジンより分子量が14少ない類縁体であり、図４に示すマススペクトル及び図５に示すマススペクトルの開裂様式より、下記式 (IV) で表される10−デスメチルボレリジン（化合物IV）であることが判明した。
【００３１】
【化４】

【００３２】
実施例４ Ｐ−４の単離と構造解析
Mer-N7167 株を、グリセロール2.0 ％、酵母エキス0.2 ％、コーンスティープリカー2.0 ％および塩化ナトリウム0.3 ％からなる培地にて培養し、得られた培養液の一部をそのまま等量の1-ブタノールで抽出し、濃縮乾固して得た粗抽出物を図１と同様の条件でHPLC分析したところ図６に示すごとく、これまでのＰ−１、２、３とは異なるＰ−４が存在することが確認された。そこで以下の方法によりＰ−４を単離した。全培養液15ＬをpH３に調整し、1-ブタノール10Ｌを加え、１時間攪拌後遠心分離し、1-ブタノール層を減圧下濃縮乾固した。得られた油状物質を酢酸エチル、水、各々１Ｌに分配し、酢酸エチル層を分離し、減圧下濃縮乾固して黒褐色油状物質4.8gを得た。次にこの粗抽出物を少量のジメチルスルホキシドに溶解し、YMC-GEL ODS-AM 120-S50のカラム（600ml)に付した。アセトニトリル：10mMりん酸二水素カリウム緩衝液 pH3.5＝２：３の混合溶媒２Ｌで洗浄後、17:25 さらに9:11の組成の混合溶媒各２Ｌで溶出し、溶出液をHPLCでモニターしながら、ボレリジンを純粋に含む画分とＰ−４を含む画分とに分け、脱塩処理後凍結乾燥して、それぞれ617mg と240mg の白色粉末を得た。Ｐ−４を含む画分は、さらにシリカゲル60カラム（150ml)に吸着させ、 TLC（展開溶媒、クロロホルム：メタノール＝４：１、Rf値＝0.11）にてモニターしながら、クロロホルム：メタノール＝49:1、 19:1および４：１の混合溶媒で溶出し、ボレリジンおよびＰ−４をそれぞれ60mgおよび98mgの純粋な白色粉末として単離した。Ｐ−４は、ヘキサン−アセトンの混合溶媒から結晶化し、無色結晶51mgを得た。
【００３３】
Ｐ−４の物理化学的性状
１）融点：108-111 ℃
２）高分解能EI Mass スペクトル：測定の結果、m/z508.3041 にM⁺が観測され、分子式はＣ₂₈Ｈ₄₄Ｏ₈ であると決定した。質量計算値はm/z508.3036 である。
３）紫外吸収スペクトル：
λ_maxMeOH 254nm(ε14,900)
λ_max0.01N HCl-MeOH 260nm(ε15,300)
λ_max0.01N NaOH-MeOH 249nm(ε16,500)
４）赤外吸収スペクトル：臭化カリウム中での拡散反射法による測定結果を図７に示した。
５） ¹Ｈ核磁気共鳴スペクトル：図８に示した。
６）¹³Ｃ核磁気共鳴スペクトル：重クロロホルム中での測定結果を表３に示した。
【００３４】
【表３】

【００３５】
以上の結果から、Ｐ−４は、Ｐ−１の12位のニトリル基がカルボキシル基に置き換わった、式（Ｖ）で表されるボレリジン類縁体（化合物Ｖ）であると決定した。
【００３６】
【化５】

【００３７】
実施例５．血管新生阻害活性
ラット大動脈片をコラーゲンゲル内にて培養し、観察される管腔形成の阻害度を血管新生阻害活性とした。Sprague-Dawley系雄ラット（８〜12週齢）より摘出した大動脈をハンクス液で洗浄しながら周辺の脂肪組織を丁寧に除去した。大動脈を切開し２mm角の切片を作成した後、24ウエルプレート内へ内皮細胞面を上にして静置する。次に、中性化したタイプＩコラーゲンゲル（Cellmatrix type Ｉ−Ａ：新田ゼラチン）500 μl を各ウエルへ注ぎクリーンベンチ内で室温下約20分間放置してゲルを固まらせた。ゲルが固まったことを確認した後 500μl の MCDB 131 （クロレラ工業社製）培地を各ウエルに加えCO₂ インキュベーター（５％CO₂)で 37 ℃下培養した。翌日試験検体を含むMCDB 131培地と培養液を交換し、さらに培養４日目に再度試験検体含有のMCDB 131培地と交換して培養を続けた。そして、試験検体添加後７日目の時点で、大動脈の周囲に形成された毛細血管数を顕微鏡を用いて測定した。
【００３８】
その結果は表４に示すごとく、Ｐ−１、Ｐ−２、Ｐ−４は濃度依存的に血管新生を阻害した。そのIC₅₀値はそれぞれ 8.4ng/ml、4.4ng/ml、2.8ng/ml であった。なお、阻害活性はサンプル無添加群の毛細血管数との比較で表した。
【００３９】
【表４】

【００４０】
実施例６．血管内皮細胞に対する増殖抑制作用
ヒト血管内皮細胞（HUVEC:クラボウ）を EGM培地（クラボウ）に30,000cells/mlの濃度で懸濁した後、96ウエルプレート中に 100μl ずつ添加する。37℃で一晩培養した後に、同じ培地で希釈した検体含有液を各ウエルに100 μl ずつ添加し３日間培養した。そして、50μl の0.33％ MTT液を各ウエルに加えて 2-3時間培養した後、培養液を吸引して除いた。次いで、100 μl のジメチルスルホキシドを加えて溶解し、540nm の吸光度をプレートリーダーを用いて測定した。阻害濃度は無処理群との比較によって算出した。
その結果、Ｐ−１、Ｐ−２、Ｐ−４のIC₅₀値はそれぞれ39ng/ml, 36ng/ml, 10μg/mlであり、前二者に強い増殖阻害作用が確認された。
【００４１】
実施例 ７．スレオニンtRNA合成酵素阻害作用
スレオニンtRNA合成酵素の調製は以下の方法で行った。１×10⁸ 個のヒト鼻咽喉癌(KB)細胞を集め PBSで１回洗浄後、10mlのlysis buffer(10ml Tris HCl pH7.5, 50mM KCl, 1mM MgCl₂, 0.1mM DTT, 20% glycerol, 1% NP-40) に懸濁し氷冷下30分間放置する。16000rpm(KUBOTA RK-2000T,RA2 rotor) で20分間遠心して得た上清を -20℃で保存した。スレオニンtRNA合成酵素阻害作用の測定は200 μl の反応液にて実施した。50mM HEPES pH8.2, 5mM ATP, 2.5mM CTP, 40mM KCl, 10mM MgCl₂, 0.1mM EDTA, 200 μg/ml Rabbit liver tRNA, 100 μM[³H]Threonine, 100 μg/ml酵素溶液に検体を加え30℃で45分間反応を行った。次に、反応液の100 μl を2.5ml の冷却しておいた10％ TCA溶液に加えてtRNAを沈殿させた。グラスフィルター（φ 2.1cm) を用いて沈殿を濾取し、10％ TCA(5ml×３), EtOH(２ml×２) の順に洗浄した。そして、液体シンチレーションカウンターにより沈殿したスレオニン量を測定した。
その結果、ボレリジン、Ｐ−１およびＰ−２は酵素阻害作用を示し、そのIC₅₀値はそれぞれ0.06μg/ml、 0.95 μg/ml、 0.75 μg/mlであった。
【図面の簡単な説明】
【図１】 実施例１で行ったボレリジン画分のHPLC 3C₁₈ カラムクロマトグラフィーの結果を示す図である。
【図２】 重クロロホルム中でのＰ−１の ¹Ｈ核磁気共鳴スペクトルである。
【図３】 重クロロホルム中でのＰ−２の ¹Ｈ核磁気共鳴スペクトルである。
【図４】 Ｐ−３のLC-Mass チャートである。
【図５】 Ｐ−３のマススペクトル開裂様式を示す図である。
【図６】 実施例４で行ったボレリジン画分のHPLC 3C₁₈ カラムクロマトグラフィーの結果を示す図である。
【図７】 臭化カリウム中でのＰ−４の赤外吸収スペクトルである。
【図８】 重クロロホルム中でのＰ−４の ¹Ｈ核磁気共鳴スペクトルである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel physiologically active substance effective for prevention and treatment of various diseases associated with abnormal growth of angiogenesis.
[0002]
[Prior art]
Angiogenesis is an essential biological phenomenon during fetal vascularization and the morphological and functional development of each organ, but occurs only in the female sexual cycle in mature individuals. However, in mature individuals, it is known that pathological increases in neovascularization are involved in the development or progression of various diseases. Specifically, cancer, rheumatoid arthritis, atherosclerosis, diabetic retinopathy, hemangioma, psoriasis and the like are listed as diseases accompanied by abnormal angiogenesis (Marsha A. et al., Biotechnology, 9 , 630). , 1991). In particular, the growth of solid tumors has been reported to depend on angiogenesis (Folkman J., J. Natl. Cancer Inst., 82 , 4, 1990). Angiogenesis inhibitors are a new treatment for refractory solid cancers. Expected to be a medicine.
[0003]
There have been reports on some angiogenesis inhibitors, but no effective substances have yet been found that can be put to practical use (published patent publication No. 3-109324, published patent publication No. 3-236324, published patent publication). Hei 3-2184).
[0004]
[Problems to be solved by the invention]
An object of the present invention is to provide a prophylactic and therapeutic agent for various diseases associated with abnormal growth of angiogenesis by isolating a novel substance having angiogenesis inhibitory activity.
[0005]
[Means for Solving the Problems]
In view of the above situation, the present inventors have started searching for new anti-angiogenic substances having stronger inhibitory activity and fewer side effects using microbial culture as a raw material. As a result, it was found that an angiogenesis inhibitory substance was produced in the culture solution of microorganisms belonging to the genus Streptomyces, and as a result of isolation and structural analysis of this active substance, it was found to be borrelidin. In the purification process of this substance, as a result of HPLC analysis using a photodiode array detector, four peaks showing the same ultraviolet absorption spectrum as borrelidin were confirmed. As a result of isolation and structure determination of these compounds, it was confirmed that they were novel substances and had angiogenesis inhibitory activity, and the present invention was completed.
[0006]
That is, the present invention relates to a compound represented by the general formula (I), a salt thereof or a hydrate thereof, a production method thereof, and a medicine containing them as an active ingredient.
[0007]
[Chemical 2]

[0008]
(In the formula, R ₁ represents a hydrogen atom or a lower alkyl group, and R ₂ represents a cyano group or a carboxyl group.)
More specifically, in the general formula (I), R ₁ is a methyl group, R ₂ is a cyano group, a compound (II) having a configuration of 12Z, 14E, R ₁ is a methyl group, and R ₂ is a cyano group. A compound (III) having a configuration of 12Z, 14Z, R ₁ is a hydrogen atom, R ₂ is a cyano group, and a compound (IV) having a configuration of 12Z, 14E, R ₁ is a methyl group, Compound (V) wherein R ₂ is a carboxyl group and has a configuration of 12Z, 14E, a salt thereof or a hydrate thereof, a method for producing these compounds, and an angiogenic agent comprising these compounds as active ingredients The present invention relates to a pharmaceutical effective as a preventive and therapeutic agent for various diseases accompanied by abnormal growth.
[0009]
These compounds are related compounds of borrelidin, and borrelidin is a compound whose structure was isolated in 1949 as an antibiotic from the culture solution of Streptomyces microorganisms (Berger J. et al., Arch. Biochem. Biophys. , 22 , 476, 1949).
[0010]
DETAILED DESCRIPTION OF THE INVENTION
As the microbial species of the microorganism culture solution that is the raw material for extraction of the compound of the present invention, Streptomyces genus is selected, and any microorganism that produces borrelidin such as Streptomyces albovinaceus, Streptomyces rochei and Streptomyces sp. C 2989 can be used (Singh SK et al., Antimicrob. Agents. Chemother. 27 , 239, 1985; Berger J., et al., Arch. Biochem. Biophys., 22 , 476, 1949; Lumb M. et al., Nature, 206 , 263 , 1965).
Examples include Streptomyces rochei ATCC 10739. As a suitable strain having high productivity of borrelidin, the present inventors have isolated the actinomycete Mer-N7167 strain isolated from the soil of Ishigaki Island. Is mentioned. This strain has the following mycological properties.
[0011]
Morphology: It consists of basic hyphae that branch and extend well, and aerial hyphae that elongate well, and the tip of the aerial hyphae is sporulated. There are 10 to 50 spore chains, which have a gentle spiral shape, but some are also bent. The spore surface is smooth and has a diameter of about 1 μm × 1 to 2 μm.
[0012]
Cell component: Diaminopimelic acid includes LL-type, and sugar includes galactose, glucose, mannose, and ribose.
[0013]
Growth on various media;
ISP-1 Growth is moderate, producing thin white aerial hyphae. The back side of the culture becomes slightly yellow.
ISP-2 Grows well and produces a large amount of light grayish reddish brown spores on white aerial mycelium. The rear surface of the culture is slightly yellowish brown.
ISP-3 Growth is good, spore color is grayish purple, others are the same as ISP-2.
ISP-4 The color of the spore is gray (Medium gray) and others are the same as ISP-2.
ISP-5 Growth is weak and produces a little white aerial hyphae.
Modified tyrosine medium No melanin pigment is produced.
In any medium, almost no soluble pigment is seen.
[0014]
Sugar utilization;
(+) Glucose, inositol, fructose, rhamnose, mannose, arabinose (±) xylose (−) raffinose, sucrose (+); well assimilated (−); not assimilated (±); intermediate identification;
When the properties of this strain were compared with those of Streptomyces rochei type strain IFO 12908, which were simultaneously performed, the differences shown in Table 1 were observed.
[0015]
[Table 1]

[0016]
Although there are such differences, the difference is not so great that the species is different, but is within the range of intraspecific variation of Streptomyces rochei, so we have identified this strain as Streptomyces rochei. It was named Mer-N7167. This strain was deposited as FERM P-14670 with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology on November 28, 1994.
[0017]
As a screening method for angiogenesis inhibitory activity, the degree of inhibition of lumen formation observed when rat aortic strips were cultured in collagen gel was used as an index (Nicosia RF, Lab. Invest., 63 , 115, 1990). . In addition, the growth inhibitory action on vascular endothelial cells and the threonine tRNA synthetase inhibitory activity were also measured.
[0018]
After culturing the microorganism belonging to the genus Streptomyces under normal and appropriate culture conditions, the culture solution is clarified and filtered, and then extracted with an organic solvent such as butanol or methyl isobutyl ketone, and the organic solvent layer is concentrated under reduced pressure. Next, extraction with methanol is performed, and a crude extract is obtained by treatment with petroleum ether or the like. Subsequently, fractionation is carried out by appropriately using adsorption chromatography using silica gel or the like, LH-20 gel chromatography, partition chromatography, thin layer chromatography, paper chromatography, etc., and the active fraction is confirmed by activity screening. The active substance can be isolated by appropriately combining the above methods. As a solvent used for the adsorption chromatography, a commonly used organic solvent such as chloroform, methanol, acetone, hexane, toluene and the like can be used by appropriately selecting and combining the concentrations. As a crystallization solvent, a combination of chloroform and hexane, acetone, carbon tetrachloride and the like can be appropriately selected and used. One method is the method of M. Lumb et al. (Nature, 206 , 263, 1965). The structural analysis of the isolated compound can be carried out by conventional methods such as elemental analysis, GC-MS, NMR, melting point, ultraviolet / infrared absorption spectrum.
[0019]
Since the compound of the present invention has a potent angiogenesis inhibitory activity, diseases in which abnormal angiogenesis is observed, such as rheumatoid arthritis, solid cancer, atherosclerosis, diabetic retinopathy, hemangioma, psoriasis, etc. It is expected as a preventive agent and as a therapeutic agent.
[0020]
When the compound is administered as a therapeutic or prophylactic agent for various diseases, it may be administered orally as tablets, powders, granules, capsules, syrups, etc., and sprays, suppositories, injections, and external preparations. Alternatively, it may be administered parenterally as an infusion. The dose varies significantly depending on the degree of symptom, age, type of liver disease, etc., but about 1 mg to 100 mg per adult is usually administered once to several times a day.
[0021]
In formulating, it is produced by a conventional method using an ordinary pharmaceutical carrier. That is, when preparing an oral solid preparation, after adding an excipient to the main ingredient, and further adding a binder, a disintegrant, a lubricant, a coloring agent, a flavoring agent, etc., if necessary, a tablet by a conventional method, Coated tablets, granules, powders, capsules, etc. Of course, these tablets and granules may be appropriately coated with sugar coating, gelatin coating, etc. as required.
When preparing an injection, a pH adjuster, a buffer, a stabilizer, a solubilizing agent, etc. are added to the main drug as necessary, and an injection for subcutaneous, intramuscular or intravenous use is prepared by a conventional method.
[0022]
【Example】
The following examples further illustrate the present invention in detail but are not to be construed to limit the scope thereof.
In the examples, “%” is based on weight unless otherwise specified.
[0023]
Example 1. Purification method As seed medium, glycerol 2.0%, glucose 2.0%, soy flour 2.0%, yeast extract 0.5%, sodium chloride 0.25%, calcium carbonate 0.32% and trace metal solution (copper sulfate 0.25%, manganese chloride 0.25% and zinc sulfate) A medium having a composition of 0.2% was used. As the production medium, potato starch 2.0% was used instead of glycerol 2.0% in the seed medium, and the other compositions were the same. In the jar culture, 0.05% of an antifoaming agent was added. The pH before sterilization was adjusted to 7.4. A 500 ml Erlenmeyer flask containing 100 ml of the above seed medium was sterilized at 120 ° C for 15 minutes, inoculated with 1 platinum ear of slope agar culture of Mer-N 7167 strain, and shake-cultured at 28 ° C for 3 days. A seed culture was used. Dispense 15 L of production medium into two 30 L jar fermenters, sterilize at 120 ° C. for 20 minutes, inoculate 100 ml each of seed culture, aerate (0.5 vvm), and stir (300 rpm) at 28 ° C. for 5 days. ) Cultured. After completion of the culture, the supernatant is collected by centrifugation, adjusted to pH 7, adsorbed on 3 L of Diaion HP-20 (Mitsubishi Kasei), packed in a column, washed with water, washed with 20% methanol, then 80% Elute with acetone. About 3 L of the eluate was concentrated under reduced pressure to remove acetone, water was added to make about 1 L, and extracted twice with 1 L of ethyl acetate. The extract was concentrated to dryness under reduced pressure to give a dark brown oily substance. This oily substance was dissolved in a small amount of methanol, placed on the top of a Sephadex LH-20 (Pharmacia Biotech) column (400 ml), developed with methanol, and the active fraction was concentrated to dryness. Next, dissolve in a small amount of chloroform: methanol = 50: 1 and place on the top of a silica gel 60 (Merck) column (150 ml). Chloroform: methanol = 50: 1, 20: 1 and 10: 1, 500 ml each Elute. The active fractions eluted with chloroform: methanol = 50: 1 to 20: 1 were collected and concentrated to dryness. It is then dissolved in a small amount of acetone, mixed with a small amount of silica gel 60, concentrated to dryness under reduced pressure, placed on top of a silica gel 60 column (150 ml), hexane: acetone = 3: 1, 2: 1, and 1: 2. And eluted with 500 ml each. The active fractions eluted with hexane: acetone = 3: 1 to 2: 1 were collected and concentrated to dryness under reduced pressure. This sample was dissolved in a small amount of toluene: acetone = 5: 1, placed on top of a silica gel 60 column (50 ml), and eluted with 200 ml each of toluene: acetone = 5: 1, 4: 1 and 2: 1. The active fractions eluted with toluene: acetone = 5: 1 were collected and concentrated to dryness under reduced pressure. When this sample was analyzed using a COSMOSIL 3C ₁₈ column (3 μm, 4.6 × 100 mm) and a photodiode array detector, as shown in FIG. 1, three types of peaks (each of P− 1 (denoted as P-2, P-3)) (mobile phase: acetonitrile: 10 mM potassium dihydrogen phosphate buffer, pH 3.5 = 1: 1, isocratic elution, 1 ml / min, OD ₂₅₄ nm). The main peak in FIG. 1 is borrelidin.
[0024]
Example 2 Purification and structure determination of P-1 and P-2
Centrifugation of the tank culture solution 170 L of Mer-N7167 strain gave 140 L of supernatant, which was adjusted to pH 7 and adsorbed to 7 L of Diaion HP-20. HP-20 resin was collected, packed in a column, washed with water, and eluted with 80 L of acetone and 20 L. The eluate was concentrated under reduced pressure, acetone was distilled off, water was added to make 4 L, and extracted twice with 4 L of ethyl acetate. The ethyl acetate phases were combined and concentrated to dryness under reduced pressure to obtain 27 g of a black brown oily substance. This oily substance was applied to a Sephadex LH-20 column prepared with methanol and developed with methanol. TLC (silica gel 60, No. 5715, Merck & Co., Inc., developing solvent: chloroform: methanol = 9: 1, sulfuric acid color, Rf While monitoring at 0.42), fractions containing borrelidin were collected and concentrated to dryness. The obtained sample was adsorbed on a column of silica gel 60 (750 ml), washed with 2 L of chloroform, and eluted with chloroform: methanol = 49: 1 and 19: 1, 2.5 L each, and fractions containing borrelidin were collected and concentrated to dryness. Solidified. The obtained sample was further adsorbed on a column of silica gel 60 (750 ml), washed with hexane: acetone = 4: 1, 2 L, and eluted with a mixed solvent of 7: 3. Fractions containing borrelidin were collected and concentrated to dryness to obtain 5.95 g of purified product. This purified product showing biological activity was crystallized from a mixed solvent of chloroform and hexane to obtain 4.29 g of colorless needle-like crystals of borrelidin. The mother liquor was concentrated to dryness (1.58 g), dissolved in a small amount of dimethyl sulfoxide, and then YMC-GEL ODS previously buffered with acetonitrile: 10 mM potassium dihydrogen phosphate buffer (pH 3.5) = 2: 3. -AM 120-S50 (YMC, 600 ml) column. After washing with 2 L of mixed solvent of the same composition, elute with 2 L each of 9:11 and 1: 1 mixed solvent, and monitor the eluate with HPLC, and fractions containing pure borrelidin and target substance Divided into minutes. Acetonitrile was distilled off from both fractions, extracted with ethyl acetate, and the ethyl acetate was distilled off and freeze-dried to obtain 820 mg and 107 mg of white powder, respectively. The sample containing the target substance was further analyzed by HPLC (YMC-Pack D-ODS-S-5 column, 5 μm, 20x250mm, mobile phase, acetonitrile: 10 mM potassium dihydrogen phosphate buffer, pH 3.5 = 11: 9, (Tick elution, 15 ml / min, OD ₂₅₄ nm), and desalted in the same manner, and 12 mg of borrelidin, 59 mg of P-1, and 11 mg of P-2 were isolated as pure white powder.
[0025]
Physicochemical properties of P-1 1) Melting point: 88-90 ° C
2) High resolution EI Mass spectrum: As a result of the measurement, M ⁺ was observed at m / z 489.3087, and the molecular formula was determined to be C ₂₈ H ₄₃ NO ₆ . The calculated mass is m / z 489.3090. 3) ¹ H nuclear magnetic resonance spectrum: shown in FIG.
4) ¹³ C nuclear magnetic resonance spectrum: Table 2 shows the measurement results in deuterated chloroform.
[0026]
Physicochemical properties of P-2 1) Melting point: 88-91 ° C
2) High resolution EI Mass spectrum: As a result of the measurement, M ⁺ was observed at m / z 489.3087, and the molecular formula was determined to be C ₂₈ H ₄₃ NO ₆ . The calculated mass is m / z 489.3090.
3) ¹ H nuclear magnetic resonance spectrum: shown in FIG.
4) ¹³ C nuclear magnetic resonance spectrum: Table 2 shows the measurement results in deuterated chloroform.
[0027]
[Table 2]

[0028]
From the above results, P-1 is represented by the following formula (II), stereoisomer at the 11th position of borrelidin (compound II), P-2 is represented by the following formula (III), 14 It was determined to be a geometric isomer (compound III) with respect to the double bond at position 15.
[0029]
[Chemical 3]

[0030]
Example 3 Separation and structural analysis of P-3 Based on the HPLC analysis chromatogram described in FIG. 1, P-3 was analyzed with an HPLC-mass spectrometer (LC-Mass). As a result, the molecular weight was 14 less than that of borrelidin. From the mass spectrum shown in FIG. 4 and the cleavage pattern of the mass spectrum shown in FIG. 5, it was found to be 10-desmethylborrelidin (compound IV) represented by the following formula (IV).
[0031]
[Formula 4]

[0032]
Example 4 Isolation and structural analysis of P-4
The Mer-N7167 strain is cultured in a medium consisting of glycerol 2.0%, yeast extract 0.2%, corn steep liquor 2.0% and sodium chloride 0.3%, and a portion of the resulting culture solution is directly used with an equal amount of 1-butanol. The crude extract obtained by extraction and concentration to dryness was analyzed by HPLC under the same conditions as in FIG. 1. As shown in FIG. 6, P-4 different from P-1, 2, and 3 existed so far. It was confirmed. Therefore, P-4 was isolated by the following method. 15 L of the whole culture solution was adjusted to pH 3, 10 L of 1-butanol was added, the mixture was stirred for 1 hour, centrifuged, and the 1-butanol layer was concentrated to dryness under reduced pressure. The obtained oily substance was partitioned between 1 L each of ethyl acetate and water, and the ethyl acetate layer was separated and concentrated to dryness under reduced pressure to obtain 4.8 g of a black brown oily substance. The crude extract was then dissolved in a small amount of dimethyl sulfoxide and applied to a YMC-GEL ODS-AM 120-S50 column (600 ml). Acetonitrile: 10 mM potassium dihydrogen phosphate buffer solution Washed with 2 L of mixed solvent of pH 3.5 = 2: 3, then eluted with 2 L of mixed solvent of 17:25 and 9:11, and the eluate was monitored by HPLC. However, it was divided into a fraction containing pure borrelidin and a fraction containing P-4, desalted and then freeze-dried to obtain 617 mg and 240 mg of white powder, respectively. The fraction containing P-4 was further adsorbed onto a silica gel 60 column (150 ml) and monitored with TLC (developing solvent, chloroform: methanol = 4: 1, Rf value = 0.11), while chloroform: methanol = 49: Eluting with a mixed solvent of 1, 19: 1 and 4: 1, borrelidin and P-4 were isolated as 60 mg and 98 mg pure white powder, respectively. P-4 was crystallized from a mixed solvent of hexane-acetone to obtain 51 mg of colorless crystals.
[0033]
Physicochemical properties of P-4 1) Melting point: 108-111 ° C
2) High-resolution EI Mass spectrum: As a result of the measurement, M ⁺ was observed at m / z508.3041, and the molecular formula was determined to be C ₂₈ H ₄₄ O ₈ . The calculated mass is m / z 508.3036.
3) Ultraviolet absorption spectrum:
λ _max MeOH 254 nm (ε14,900)
λ _max 0.01N HCl-MeOH 260nm (ε15,300)
λ _max 0.01N NaOH-MeOH 249nm (ε16,500)
4) Infrared absorption spectrum: FIG. 7 shows the measurement result by diffuse reflection method in potassium bromide.
5) ¹ H nuclear magnetic resonance spectrum: shown in FIG.
6) ¹³ C nuclear magnetic resonance spectrum: Table 3 shows the measurement results in deuterated chloroform.
[0034]
[Table 3]

[0035]
From the above results, P-4 was determined to be a borrelidin analog (compound V) represented by the formula (V) in which the nitrile group at the 12-position of P-1 was replaced with a carboxyl group.
[0036]
[Chemical formula 5]

[0037]
Embodiment 5 FIG. Angiogenesis inhibitory activity Rat aorta pieces were cultured in collagen gel, and the observed degree of inhibition of lumen formation was defined as angiogenesis inhibitory activity. The peripheral adipose tissue was carefully removed while washing the aorta excised from Sprague-Dawley male rats (8-12 weeks old) with Hanks' solution. The aorta is dissected and a 2 mm square section is prepared, and then placed in a 24-well plate with the endothelial cell surface facing up. Next, 500 μl of neutralized type I collagen gel (Cellmatrix type IA: Nitta Gelatin) was poured into each well and left in a clean bench at room temperature for about 20 minutes to solidify the gel. After confirming that the gel had hardened, 500 μl of MCDB 131 (Chlorella Kogyo) medium was added to each well and cultured at 37 ° C. in a CO ₂ incubator (5% CO ₂ ). On the next day, the MCDB 131 medium containing the test specimen was replaced with the culture solution, and the culture was continued by replacing with the MCDB 131 medium containing the test specimen again on the fourth day of culture. Then, on the seventh day after the addition of the test sample, the number of capillaries formed around the aorta was measured using a microscope.
[0038]
The results are shown in Table 4, and P-1, P-2, and P-4 inhibited angiogenesis in a concentration-dependent manner. The IC ₅₀ values were 8.4 ng / ml, 4.4 ng / ml and 2.8 ng / ml, respectively. The inhibitory activity was expressed by comparison with the number of capillaries in the sample-free group.
[0039]
[Table 4]

[0040]
Example 6 Growth inhibitory action on vascular endothelial cells Suspend human vascular endothelial cells (HUVEC: Kurabo Industries) in EGM medium (Kurabo) at a concentration of 30,000 cells / ml, and add 100 μl each in a 96-well plate. After culturing overnight at 37 ° C., 100 μl of the sample-containing solution diluted with the same medium was added to each well and cultured for 3 days. Then, 50 μl of 0.33% MTT solution was added to each well and incubated for 2-3 hours, and then the culture solution was aspirated off. Subsequently, 100 μl of dimethyl sulfoxide was added and dissolved, and the absorbance at 540 nm was measured using a plate reader. Inhibitory concentrations were calculated by comparison with the untreated group.
As a result, the IC ₅₀ values of P-1, P-2, and P-4 were 39 ng / ml, 36 ng / ml, and 10 μg / ml, respectively, and a strong growth inhibitory action was confirmed in the former two.
[0041]
Example 7 Threonine tRNA synthetase inhibitory action Threonine tRNA synthetase was prepared by the following method. 1 × 10 ⁸ human nasopharyngeal carcinoma (KB) cells were collected and washed once with PBS, then 10 ml lysis buffer (10 ml Tris HCl pH 7.5, 50 mM KCl, 1 mM MgCl ₂ , 0.1 mM DTT, 20% glycerol, Suspend in 1% NP-40) and leave under ice cooling for 30 minutes. The supernatant obtained by centrifugation at 16000 rpm (KUBOTA RK-2000T, RA2 rotor) for 20 minutes was stored at -20 ° C. The threonine tRNA synthetase inhibitory activity was measured in 200 μl of the reaction solution. 50mM HEPES pH8.2, 5mM ATP, 2.5mM CTP, 40mM KCl, 10mM MgCl 2, a specimen 0.1mM EDTA, 200 μg / ml Rabbit liver tRNA, 100 μM [3 H] Threonine, 100 μg / ml enzyme solution was added The reaction was performed at 30 ° C. for 45 minutes. Next, 100 μl of the reaction solution was added to 2.5 ml of a cooled 10% TCA solution to precipitate tRNA. The precipitate was collected using a glass filter (φ 2.1 cm), and washed with 10% TCA (5 ml × 3) and EtOH (2 ml × 2) in this order. Then, the amount of threonine precipitated was measured by a liquid scintillation counter.
As a result, borrelidin, P-1 and P-2 showed enzyme inhibitory activity, and their IC ₅₀ values were 0.06 μg / ml, 0.95 μg / ml and 0.75 μg / ml, respectively.
[Brief description of the drawings]
1 is a diagram showing the results of HPLC 3C ₁₈ column chromatography of a borrelidin fraction carried out in Example 1. FIG.
FIG. 2 is a ¹ H nuclear magnetic resonance spectrum of P-1 in deuterated chloroform.
FIG. 3 is a ¹ H nuclear magnetic resonance spectrum of P-2 in deuterated chloroform.
FIG. 4 is an LC-Mass chart of P-3.
FIG. 5 is a diagram showing a mass spectral cleavage mode of P-3.
6 is a diagram showing the results of HPLC 3C ₁₈ column chromatography of the borrelidin fraction performed in Example 4. FIG.
FIG. 7 is an infrared absorption spectrum of P-4 in potassium bromide.
FIG. 8 is a ¹ H nuclear magnetic resonance spectrum of P-4 in deuterated chloroform.

Claims (4)

Streptomyces rochei Mer-N7167 (FERM P-14670) is cultured in a nutrient medium, and the compound represented by the general formula (I), a salt thereof or a hydrate thereof is collected from the culture medium. A process for producing a compound represented by the general formula (I), a salt thereof or a hydrate thereof.

(In the formula, R ₁ represents a hydrogen atom or a lower alkyl group, and R ₂ represents a cyano group or a carboxyl group.) In the general formula of claim 1 wherein (I), R ₁ is a hydrogen atom, R ₂ is a cyano group, 12Z, that of compound having a configuration of 14E, a salt thereof or a hydrate thereof. In the general formula of claim 1 wherein (I), R ₁ is methyl, R ₂ is a carboxyl group, 12Z, that of compound having a configuration of 14E, a salt thereof or a hydrate thereof. A medicament comprising the compound according to claim 2 or 3 or a salt or hydrate thereof as an active ingredient.

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