JP3623266B2 - New compound F-10463a - Google Patents

Description

【００３０】
物理化学的性状
１） 性質；無色油状物質、
２） 溶解性；メタノール、酢酸エチル、クロロホルムなどの有機溶媒に可溶、水に不溶
３） 呈色試験；５０％ 硫酸、ヨウ素に陽性。
【００３１】
４） 分子式； Ｃ_２９Ｈ_４３ＮＯ_５
５） 分子量；４８５ （高分解能ＦＡＢＭＳ 法により ［Ｍ＋Ｈ］^＋ ４８６．３１９９ （測定値） ）（計算値：４８６．３２０４）
６） 比旋光度；［ α］ ＋６４．４４ （ｃ ０．０９ ｉｎ ＭｅＯＨ）
７） 紫外線吸収スペクトル；（メタノール中）３００ ｎｍ（ ε４１５００）
８） 赤外線吸収スペクトル；（クロロホルム中）
３４１０， １６５５， １６０８， １５０８ ｃｍ^−１
９） ^１Ｈ− 核磁気共鳴スペクトル；（ 重メタノール中：ＰＰＭ， ＴＭＳ 基準）
７．１５（ｍ， ２Ｈ）， ６．５３（ｄｄ， Ｊ＝１４．９， １０．７Ｈｚ， １Ｈ）， ６．２５（ｄｄ， Ｊ＝１４．９， １１．１Ｈｚ， １Ｈ），６．１５（ｄｄ， Ｊ＝１５．１， １０．７Ｈｚ， １Ｈ）， ６．０７（ｄｄ， Ｊ＝９．８， １．６Ｈｚ， １Ｈ）， ５．８９（ｄ， Ｊ＝１４．８Ｈｚ， １Ｈ）， ５．７０（ｄｄ， １５．１， ８．７Ｈｚ， １Ｈ）， ４．８（ＨＤＯ のシグナルの下に隠れている、 １Ｈ）， ４．０５（ｍ， １Ｈ）， ３．６７（ｄ， Ｊ＝３．９Ｈｚ， １Ｈ）， ３．５９（ｍ， １Ｈ）， ３．５２（ｄｄ， Ｊ＝１０．９， ５．１Ｈｚ， １Ｈ）， ３．４５（ｄｄ， Ｊ＝１０．９， ５．８Ｈｚ， １Ｈ）， ２．３５（ｍ， １Ｈ）， ２．２７（ｍ， １Ｈ），２．０８（ｄｄ， Ｊ＝１４．７， ３．６Ｈｚ， １Ｈ）， １．８９（ｍ， ２Ｈ）， １．７９（ｄｄ， １３．１， ７．１Ｈｚ， １Ｈ），１．５９（ｍ， １Ｈ）， １．５４（ｄ， Ｊ＝１．３Ｈｚ， ３Ｈ）， １．３３（ｍ， ２Ｈ）， １．１９（ｍ， １Ｈ），〜１．１（ｍ， １Ｈ）， １．００（ｄ， Ｊ＝６．７Ｈｚ， ３Ｈ）， ０．９１（ｄ， Ｊ＝６．６Ｈｚ， ３Ｈ）， ０．８６（ｔ， Ｊ＝７．４Ｈｚ， ３Ｈ）， ０．８３（ｄ， Ｊ＝６．５Ｈｚ， ３Ｈ）
１０） ^１３Ｃ−核磁気共鳴スペクトル；（ 重メタノール中：ＰＰＭ， 重メタノール基準）
２００．１（ｓ），１６９．０（ｓ），１４６．６（ｄ），１４６．４（ｄ），１４２．９（ｄ），１４１．９（ｄ），１３４．７（ｄ），１３４．０（ｓ）
１３２．５（ｄ），１３０．４（ｄ），１２９．９（ｄ），１２４．３（ｄ），７８．０（ｓ），６６．１（ｔ），５８．７（ｄ），５０．１（ｔ），
４９．８（ｄ），４８．５（ｄ），４５．７（ｔ），４０．３（ｔ），３６．７（ｄ），３５．９（ｄ），３２．２（ｔ），３０．０（ｄ），２２．４（ｑ）
２２．０（ｑ），２０．４（ｑ），１６．９（ｑ），１３．０（ｑ）
１１） 高速液体クロマトグラフグラフィー
保持時間：５．６５ 分
カラム： ナカライテスク製コスモシール ５ Ｃ１８−ＡＲ， ４．６φＸ１５０ｍｍ
溶媒： ７０％ アセトニトリル− 水
流速： １ｍｌ／ｍｌ
検出： ＵＶ ２１０ｎｍ
生産菌
本発明において用いられる Ｄａｓｙｓｃｙｐｈｕｓ 属に属する菌株としては、例えば Ｄａｓｙｓｃｙｐｈｕｓ ｍｏｌｌｉｓｓｉｍｕｓ（Ｌａｓｃｈ） Ｄｅｎｎｉｓ ＳＡＮＫ１３８９２株（ＦＥＲＭ ＢＰ−４４９１ ） を挙げることができ、この菌株の菌学的性状は次のとおりである。
【００３２】
ＳＡＮＫ１３８９２ は、１９９１年５ 月、青森県において採集された枯死した草本の茎から分離したものである。
【００３３】
子嚢盤は直径最大で０．２ｍｍ の無柄の平坦な円盤状で、灰橙色〜黄色を呈する。子嚢盤の周縁に毛を有し、縁は盛り上がらない。托外皮層は多角菌組織様を呈し、淡褐色で薄膜の細胞よりなる。托髄層は密に絡み合って外壁とやや平行な菌糸よりなり、絡み合い菌組織を呈する。毛は托外皮層の最外層の細胞より生じて真直ぐに伸長し、円筒形であり、薄膜、多数の隔壁を有し、最大長２００ μｍ 、太さ３．５ μｍ である。その先端はほとんど鈍頭である。表面はほとんど平滑で、樹脂状の不定形物質が散在・付着する。子嚢はクランプより生じ、円筒形、８ 胞子性で、大きさ５８−６６ ｘ ５−６．５ μｍ である。その先端はメルツァー試薬にて青変する。側糸は細い槍型であり、 大きさ７４．５−１００ ｘ ３．５−４μｍ 、子嚢より約２０μｍ 上に伸長する。子嚢胞子は桿形から紡錘形、単細胞、無色であり、大きさ１８−２６．５ ｘ ２．５−５ μｍ である。
【００３４】
ＰＤＡ上の生育は、２３℃、１０日で １．５ ｃｍ に達する。表面は淡澄色〜澄白色を呈し、コロニー中央部にてやや盛り上がる。気菌糸の発達はあまり旺盛でなく、絡み合って菌糸束を形成する。表面は粉状となる。裏面は、淡澄色〜澄白色を呈する。さらに培養を継続することによって、より濃色となる。分生子は観察されない。
【００３５】
以上のような菌学的特徴から、本菌に該当するものを検索したところ、Ｏｔａｎｉ（１９６７） Ｎｏｔｅｓ ｏｎ ｓｏｍｅ ｃｕｐ ｆｕｎｇｉ ｏｆ ｔｈｅ Ｈｙａｌｏｓｃｙｐｈａｃｅａｅ ｃｏｌｌｅｃｔｅｄ ｉｎ Ｈｏｋｋａｉｄｏ，Ｊａｐａｎ． Ｔｒａｎｓ．Ｍｙｃｏｌ． Ｓｏｃ． Ｊａｐａｎ ８：３３−４２．に記載されているＤａｓｙｓｃｙｐｈｕｓ ｍｏｌｌｉｓｓｉｍｕｓ （Ｌａｓｃｈ） Ｄｅｎｎｉｓ（ダシスキファス・モリシムス・ラッシュ・デニス）に一致した。よって本菌をＤａｓｙｓｃｙｐｈｕｓ ｍｏｌｌｉｓｓｉｍｕｓ （Ｌａｓｃｈ） Ｄｅｎｎｉｓと同定し、Ｄａｓｙｓｃｙｐｈｕｓ ｍｏｌｌｉｓｓｉｍｕｓ （Ｌａｓｃｈ） Ｄｅｎｎｉｓ ＳＡＮＫ１３８９２ と命名した。本菌は、１９９３年１２月１０日に通産省工業技術院生命工学工業技術研究所に国際寄託され、寄託番号 ＦＥＲＭ ＢＰ−４４９１ が付された。
【００３６】
培養法及び精製法
本発明の新菌株を分離するに際し使用される分離培地としては炭素源、窒素源、無機イオンおよび有機栄養源等より選択されたものを適宜含有する培地であれば合成または天然培地の何れでも使用可能である。Ｆ−１０４６３ａは ＳＡＮＫ１３８９２ 株を適当な培地で培養し、それから採取することによって得られる。栄養源としては、従来、真菌類の培養に利用されている公知のものが使用できる。例えば、炭素源としてはグルコース、シュクロース、澱粉、グリセリン、水飴、糖蜜、大豆油などが使用できる。また、窒素源としては大豆粉、コーンスチープリカー、イーストエキス、麦芽エキス、ジャガイモ、硫酸アンモニウム、硝酸ナトリウム等を使用しうる。このほか必要に応じて炭酸カルシウム、リン酸塩等の無機塩類を添加するほか、菌株の発育を助け、Ｆ−１０４６３ａの生産を促進するような有機および無機物を適当に添加することができる。培養法としては、一般の抗生物質を生産する方法と同じく液体培養法、特に深部培養法が最も適している。培養は、好気的条件下で行なわれ、培養に適当な温度は１５ー３０℃であるが、多くの場合２３℃付近で培養する。Ｆ−１０４６３ａの生産は、振盪培養で通常５−８ 日で最高値に達する。
【００３７】
培養終了後、培養液中の菌体あるいはロ液に存在するＦ−１０４６３ａを培養液の容量程度のアセトン、アセトニトリルのような有機溶媒を添加し混合することにより抽出する。抽出物中に存在する固形部分を珪藻土をろ過操作助剤とするろ過操作または遠心分離によって分別し、そのろ液または上清中に存在するＦ−１０４６３ａを、スフィンゴミエリナーゼ阻害活性を指標にして、その物理化学的性状を利用し抽出精製する。例えば、この抽出液中に存在するＦ−１０４６３ａは、まず濃縮操作で混在する有機溶媒を除去した後に、水と混和しない有機溶剤、例えばｎーブタノール、メチルエチルケトン、酢酸エチル、クロロホルム、塩化エチレン、塩化メチレンなどの単独または、それらの組み合わせにより抽出精製することができる。あるいは吸着剤として、例えば活性炭または吸着用樹脂であるアンバーライトＸＡＤ−２、ＸＡＤ−４（ローム・アンド・ハース社製）等や、ダイヤイオンＨＰー１０、ＨＰー２０、ＣＨＰー２０Ｐ、ＨＰー５０（三菱化成（株）製）等を使用する事ができる。Ｆ−１０４６３ａを含む液を上記のごとき吸着剤の層を通過させて不純物を吸着させて取り除くか、またはＦ−１０４６３ａを吸着させた後、メタノール水、アセトン水、ｎ−ブタノール水などを用いて溶出させることにより得られる。このようにして得られたＦ−１０４６３ａは、更にシリカゲル、フロリジルのような担体を用いた吸着カラムクロマトグラフィー、セファデックスＬＨ−２０（ファルマシア社製）などを用いた分配カラムクロマトグラフィ、セファデックスＧ−２５（ファルマシア製）などを用いたゲルろ過クロマトグラフィ、および順相、逆相カラムを用いた高速液体クロマトグラフィ等で精製することが出来る。
【００３８】
スフィンゴミエリナーゼ阻害活性は以下の方法で測定できる（ Ｊ． Ｌｉｐｉｄ Ｒｅｓ．２０， ４５６ （１９７９））。
【００３９】
即ち、先ず、基質溶液として１０μｌの［Ｎ−メチル−^１４Ｃ］スフィンゴミエリン（牛、５２ｍＣｉ／ｍｍｏｌ、２５μＣｉ／ｍｌ；アマシャム社）と２００μｌのスフィンゴミエリン（牛、２０ｍＭ、シグマ社）を窒素ガスで乾固させた後、２００μｌの１Ｍトリスー塩酸（ｐＨ７． ５）、４０μｌの１０％（ｖ／ｖ）トリトンＸ−１００、２０μｌの０． ５Ｍ ＭｇＣｌ_２ 、及び１． ２４ｍｌのＨ_２ Ｏを加えて４８℃、３０分のインキュベーションを行い、プローブ型超音波破砕装置で、２０Ｗの出力条件下、１５秒、２回の超音波処理を施し、［^１４Ｃ］スフィンゴミエリンを含む混合ミセル系を作成した。
【００４０】
スフィンゴミエリナーゼ反応は、この様にして用意した基質溶液１５０μｌに検体溶液１０μｌを混合し、ウイスターイマミチ系雄性ラット脳のミクロソーム画分（２５、０００×ｇ〜１００、０００×ｇ、蛋白質濃度３〜４ｍｇ／ｍｌ）４０μｌを酵素溶液として加えて３７℃、４０分インキュベーションすることにより行った。反応終了後、クロロホルム：メタノール（２：１、ｖ／ｖ）を５００μｌ加えて抽出操作を施し、得られた水層１５０μｌを３ｍｌのピコフローＴＭ４０（パッカードジャパン株式会社）と混合して反応物である［^１４Ｃ］ホスフォリルコリン量を液体シンチレーションカウンターで測定した。スフィンゴミエリナーゼ活性は、この測定値からスフィンゴミエリナーゼ反応に必要なＭｇＣｌ_２ を除いた場合での測定値を差し引いた値として計算される。
【００４１】
【実施例】
次に実施例をあげて本発明を更に具体的に説明するが、本発明はこれに限定されない。
【００４２】
実施例１． Ｆ−１０４６３ａ物質の精製
（１） 培養
Ｄａｓｙｓｃｙｐｈｕｓ ｍｏｌｌｉｓｓｉｍｕｓ（Ｌａｓｃｈ） Ｄｅｎｎｉｓ ＳＡＮＫ１３８９２ 株を無菌的に、滅菌した後述の組成の培養培地１００ ｍｌ を含む５００ ｍｌ の三角フラスコ（種フラスコ）に接種した。次いでこれを５日間、２００ｒｐｍ のロータリー振とう機で前培養を行った。
【００４３】
【表１】
培地組成
グリセロール ５０ｇ
ジャガイモ ５０ｇ
イ−スト・イクストラクト ５ｇ
マルト・イクストラクト ５ｇ
消泡剤（ＣＢ−４４２） ０．２ｇ
イオン交換水 １０００ｍｌ
ｐＨ 無調整
本培養は以下のように行った。滅菌した上述の組成の培養培地１００ ｍｌ を含む５００ ｍｌ の三角フラスコ １５ 本に種培養液をそれぞれ５ｍｌ入れ、２３℃で７日間、２００ｒｐｍ のロータリー振とう機で培養を行った。
【００４４】
（２）スフィンゴミエリナーゼ阻害活性の測定法
スフィンゴミエリナーゼの酵素標品の調製は、基本的には Ｇａｔｔ Ｓ．（Ｂｉｏｃｈｅｍ． ｂｉｏｐｈｙｓ． Ｒｅｓ． Ｃｏｍｍｕｎ． ６８， ２３５ （１９７６））の方法に従って行った。すなわち、スフィンゴミエリナーゼの酵素源としてラット脳を用い、先ず、以下の様にそのミクロソーム画分を調製した。１０匹のウイスターイマミチ系雄性ラット（９週齢）を頚動脈放血後、全脳を摘出した。迅速に、小脳を除去後、予め４℃に冷却したバッファーＡ（０． ２５Ｍ 蔗糖、１ｍＭ ＥＤＴＡ、１ｍＭ ＰＭＳＦ（Ｐｈｅｎｙｌｍｅｔｈｙｌｓｕｌｆｏｎｙｌ Ｆｌｕｏｒｉｄｅ ）、０． １ｍＭ ロイペプチン、５ｍＭ トリスー塩酸緩衝液、ｐＨ７． ４）１３０ｍｌを加え、４℃条件下、ポッターのホモジナイザーを用いて脳細胞の破砕を行った。次に、得られた細胞破砕液を４℃条件下、７００×ｇ、１０分間の遠心分離を行い、その上清を更に、４℃条件下、２５、０００×ｇで１０分間の遠心分離を行った。最後に、得られた上清を４℃条件下、１００、０００×ｇで６０分間の超遠心分離を行い、その沈澱物をミクロソーム画分とした。尚、この画分はスフィンゴミエリナーゼの活性測定時まで液体窒素下で凍結保存し、使用時にバッファーＡで蛋白質濃度３〜４ｍｇ／ｍｌ程度になる様に調製した。
【００４５】
スフィンゴミエリナーゼ活性は混合ミセル系を使って、Ｈｏｓｔｅｔｌｅｒ Ｋ．Ｙ． と Ｙａｚａｋｉ Ｐ．Ｊ． の方法（Ｊ． Ｌｉｐｉｄ Ｒｅｓ． ２０， ４５６ （１９７９））を参考にして、以下の様に測定した。即ち、先ず、混合した１０μｌの［Ｎ−メチル−^１４Ｃ］スフィンゴミエリン（牛、５２ｍＣｉ／ｍｍｏｌ、２５μＣｉ／ｍｌ；アマシャム社）と２００μｌのスフィンゴミエリン（牛、２０ｍＭ、シグマ社）を窒素ガスで乾固させた後、２００μｌの１Ｍトリスー塩酸（ｐＨ７． ５）、４０μｌの１０％（ｖ／ｖ）トリトンＸ−１００、２０μｌの０． ５Ｍ ＭｇＣｌ_２、及び１． ２４ｍｌの Ｈ_２Ｏを加えて４８℃、３０分のインキュベーションを行った。そして、プローブ型超音波破砕装置で、２０Ｗの出力条件下、１５秒、２回の超音波処理を施し、［^１４Ｃ］スフィンゴミエリンを含む混合ミセル系を作成した。
【００４６】
スフィンゴミエリナーゼ反応は、この様にして用意した基質溶液１５０μｌに検体溶液１０μｌを混合し、先に供述した酵素溶液４０μｌを加えて３７℃、４０分インキュベーションすることにより行った。反応終了後、クロロホルム：メタノール（２：１、ｖ／ｖ）を５００μｌ加えて抽出操作を施し、得られた水層１５０μｌを３ｍｌのピコフローＴＭ４０（パッカードジャパン株式会社）と混合して反応物である［^１４Ｃ］ホスフォリルコリン量を液体シンチレーションカウンターで測定した。スフィンゴミエリナーゼ活性は、この測定値からスフィンゴミエリナーゼ反応に必要なＭｇＣｌ_２ を除いた場合での測定値を差し引いた値として計算される。
【００４７】
この方法で測定した、スフィンゴミエリナーゼ反応を ５０％ 阻害するのに必要な Ｆ−１０４６３ａ の濃度は ２０ μｇ／ｍｌであった。
【００４８】
（３） 単離
三角フラスコ １５ 本分の培養液を ６Ｎ 塩酸で ｐＨ ３ に調整し、続いて ３０００ 回転、１０ 分、遠心分離を行った。得られた菌体に ８０％ アセトンを ５００ ｍｌ 加え、１ 時間攪拌した。この混合物を ３０００ 回転、１０ 分、遠心分離を行い、上清を減圧濃縮してアセトンを除去した。次にこれを等量の酢酸エチルで２回抽出した。酢酸エチル層を飽和食塩水で洗浄後、無水硫酸ナトリウムで乾燥し、減圧濃縮して褐色の油状物を ４３０ ｍｇ 得た。このうち ４００ｍｇ を少量の塩化メチレンーメタノール、１００：１ の溶媒に溶解し、同じ溶媒で平衡化した８０ｍｌ のシリカゲルカラムにチャージした。このカラムを ２４０ｍｌ の 塩化メチレンーメタノール、１００：１ の溶媒で溶出し、さらに ２４０ ｍｌ の ５０：１， ２０：１ 及び １０：１ の溶媒で溶出し、それぞれの溶出液を分画した。酵素阻害活性は ２０：１ の分画に認められたのでこの分画を濃縮し、７８ｍｇ の褐色の油状物質を得た。これを少量のメタノールに溶解し、以下のように調製用 ＨＰＬＣ で精製した。すなわち、約 ２０ｍｇ ずつ、７０％ アセトニトリル水溶液で平衡化した ＨＰＬＣ カラム（ナカライテスク製コスモシル５Ｃ１８−ＡＲ， ２０ φＸ２５０ｍｍ）にチャージし、同じ溶媒で １０ｍｌ／ｍｉｎ． の流速で展開した。２１０ ｎｍ の吸収を検出し、約 ３０ 分にでるピークを分取した。分取した溶液を減圧濃縮し、２２ｍｇ の Ｆ−１０４６３ａ 物質を得た。
【００４９】
【発明の効果】
本発明の新規化合物 Ｆ−１０４６３ａ は、抗ＨＩＶ剤、抗糖尿病剤、抗動脈硬化剤、抗骨粗しょう症剤、抗血栓剤、抗炎症剤、免疫抑制剤、利尿剤、そして、呼吸器系疾患、甲状腺疾患、アルツハイマー病、肝炎、腎炎、白血病、及びカケクシアに対する予防薬、治療薬として使用できる。[0001]
[Industrial application fields]
The present invention relates to a novel compound F-10463a that inhibits sphingomyelinase and is useful as various pharmaceuticals, and a method for producing the same.
[0002]
[Prior art]
Interleukin-1β (hereinafter referred to as “IL-1β”) has various biological actions and is generally considered to be a biological substance essential for maintaining the homeostasis of the living body. However, when an abnormality occurs in the IL-1β production regulatory function and IL-1β is excessively produced, it causes various diseases. Tumor necrosis factor-α (hereinafter referred to as “TNF-α”) has effects such as killing certain tumor cells and virus-infected cells, and enhancing the antibacterial action of granulocytes. If TNF-α is also produced in excess, it becomes a major etiology of several diseases.
[0003]
These two cytokines are the products of completely different genes, have no similar structure, and have their own receptors corresponding to each of them. However, their target cells and biological activities often overlap. For example, both cytokines are a major cause of septic shock caused by endotoxin (LPS) in vivo [Tracy K. et al. J. et al. et al. Science, 234, 470 (1986), Tracey K. et al. J. et al. et al. Nature (London), 330, 662 (1987)] and others, granulomas [Kobaashi K. et al. et al. J. et al. Immunol. , 134, 358 (1985)], Neisseria meningitidis meningitis and malaria infection [Curfs J. et al. H. A. et al. J. et al. Exp. Med. , 172, 1287 (1990)], etc., are closely related to infectious diseases derived from exogenous microorganisms, parasites and viruses. Each major stage in such an acute phase inflammatory reaction, that is, local infiltration of inflammatory cells [Gamble J. et al. R. et al. Proc. Natl. Acad. Sci. U. S. A. , 82, 8667 (1985), Masayuki Miyasaka et al. Annual Review Immunization '91, 57 Chugai Medical Co., Ltd. (1991)], fever [Dinarello C. et al. A. Lymphokines, 14, 1 (1987)], induction of acute phase proteins [Perimter D. et al. H. et al. J. et al. Clin. Invest. , 78, 1349 (1986)], prostanoids, especially PGE₂    For promoting production [Dayr J. et al. -M. et al. J. et al. Exp. Med. , 162, 2163 (1985), Turinsky J. et al. et al. Am. J. et al. Physiol. 262, E476 (1992), Ballou L. et al. R. et al. J. et al. Biol. Chem. 267, 20044 (1992)], both cytokines play an active role.
[0004]
IL-1β and TNF-α are involved in the development and progression of chronic inflammatory diseases such as rheumatoid arthritis (RA), activation of lymphocyte infiltration in synovial tissue, promotion of proliferation of synovial cells, And promotes bone resorption by destruction of chondrocytes and activation of osteoclasts [Mize S. et al. B. et al. Proc. Natl. Acad. Sci. U. S. A. 78, 2474 (1980), Miyasaka N. et al. et al. Arthritis Rheum. , 31, 480 (1988), Arend W., et al. P. and Dayer J .; -M. Arthritis Rheumatism. 33, 305 (1990)]. In addition, collagen disease, which is the same rheumatic disease [Tanaka Y. et al. et al. J. et al. Immunol. , 143, 1584 (1989)], Kawasaki disease mainly consisting of systemic vasculitis [Leung D. et al. Y. M.M. et al. J. et al. Exp. Med. , 164, 1958 (1986)] and is also known to be involved in chronic inflammation associated with granulomas and subsequent fibrosis [Le J. et al. and Vilseck J. et al. Lab. Invest. 56, 234 (1987)]. Glucocorticoids currently used as therapeutic agents for chronic inflammatory diseases are known to have a part of their action in suppressing the production of these cytokines [Lew W. et al. et al. J. et al. Immunol. , 140, 1895 (1988)], glucocorticoids have the disadvantage of inducing various serious side effects due to their various physiological actions.
[0005]
Furthermore, IL-1β and TNF-α adhere to monocytes to vascular endothelial cells and migrate to the subendothelium [Pober J. et al. S. et al. J. et al. Immunol. , 137, 1893 (1986), Nelken N., et al. A. et al. J. et al. Clin. Invest. 88, 1121 (1991)], promoting abnormal proliferation of vascular smooth muscle cells in the intima [Raines E. et al. W. et al. Science 243, 393 (1989)], involved in the development and progression of atherosclerosis.
[0006]
IL-1β and TNF-α also promote the production of platelet activating factor (PAF), induce tissue factor to the endothelial cell membrane surface, decrease thrombomodulin protein C, and suppress the production of plasminogen activator inhibitor 1. As a whole, platelet aggregation and blood coagulation are induced and cause thrombus formation [Bevilacqua M. et al. P. et al. Proc. Natl. Acad. Sci. U. S. A. , 83, 4533 (1986), Le J. et al. and Vilseck J. et al. Lab. Invest. , 56, 234 (1987), Atsushi Sato Contemporary Medicine, 23, 3163 (1991)].
[0007]
In insulin-dependent diabetes mellitus (IDDM), latent, chronic and autoimmune inflammation occurs in the islets, particularly pancreatic β cells, in the process leading to its onset, but IL-1β and TNF-α are involved in it. [Nerup J. et al. et al. Diabetes Care. , 11, 16 (1988). On the other hand, in non-insulin dependent diabetes mellitus (NIDDM), TNF-α acts on muscle and hepatocytes through production in adipocytes and is involved in exerting insulin resistance [Spiegelman B. et al. M.M. et al. J. et al. Biol. Chem. , 268, 6823 (1993)].
[0008]
IL-1β and TNF-α are deeply involved in the growth of mesangial cells, which are the main cause of the development of glomerulonephritis, and the growth of the substrate [Werber H. et al. I. et al. J. et al. Immunol. , 138, 3207 (1987), Baud L. et al. et al. Kidney Int. , 41, 600 (1992)].
[0009]
IL-1β and TNF-α stimulate the production of IL-2 from T cells, their secretion, and expression of their receptors, and also act on other immune cells to enhance their functions and thereby enhance their immunity [ Gilis S. and Mizel S. et al. B. Proc. Natl. Acad. Sci. U. S. A. 78, 1133 (1981), Schurich P. et al. et al. J. et al. Immunol. , 138, 1786 (1987)]. By this action, both cytokines contribute to the development of graft-versus-host disease (GVHD), which occurs during transplantation, for example.
[0010]
TNF-α causes anorexia by suppressing lipoprotein lipase activity of adipocytes in patients with chronic infections and cancers, thereby causing extreme weight loss and wasting (cachexia). Therefore, TNF-α is It is called cachectin [Butler B. et al. et al. Nature (London), 316, 552 (1985)].
[0011]
TNF-α activates transcription from HIV viral genome end: LTR: inserted into the chromosome via transcription factor NF-κB in HIV (human immunodeficiency virus) -infected cells. [Nabel G. et al. Nature, 326, 711 (1987), Schreck R., et al. et al. EMBO Journal, 10, 2247 (1991)].
[0012]
As other diseases based on overproduction of IL-1β and TNF-α, fulminant hepatitis [Mutto Y. et al. et al. Lancet II, 72 (1988)], asthma, idiopathic pulmonary fibrosis [Kelley J. et al. Am. Rev. Respir. Dis. , 141 (3), 765 (1990)], ARDS (adult respiratory distress syndrome) [Miller A. et al. et al. Lancet II, 712 (1989)] and other respiratory diseases, autoimmune thyroid diseases [Katsumi Eguchi et al. Approach 1 from the latest medicine From cytokines, Medical View, 38 (1991)], Lyme disease [Havict G. et al. S. et al. J. et al. Immunol. , 134, 3147 (1985)], Alzheimer's disease [Griffin W. et al. S. T.A. at al. Proc. Natl. Acad. Sci. U. S. A. , 86, 7611 (1989)], Crohn's disease [Ayumi Yakita, History of Medicine, 147, 375 (1988)], toxic shock syndrome [Ikejima T. et al. et al. J. et al. Clin. Invest. , 73, 1312 (1984)], osteoporosis [Pacifici R., et al. et al. Proc. Natl. Acad. Sci. U. S. A. , 86, 2398 (1989)], gout [Di Giovine F., et al. S. et al. J. et al. Immunol. , 138, 3213 (1987)], acute myeloid leukemia [Sakai K. et al. et al. J. et al. Exp. Med. , 166, 1587 (1987)], endometritis [Romero R., et al. , Et al. Am. J. et al. Obstet. Gynecol. 160, 1117 (1989)].
[0013]
Sphingomyelinase is an enzyme that decomposes this into ceramide and phosphorylcholine using sphingomyelin, which is one of the choline-containing lipids contained in the cell membrane system and nucleus in the living body, as a substrate. This enzyme was initially found as one of the lysosomal hydrolyzing enzymes having an optimum pH in the acidic range, but recently, the enzyme activity having an optimum pH in the neutral range has also been detected in the microsomal fraction and plasma membrane. It has been found [Allan D. et al. Biochim. Biophys. Acta. , 693, 53 (1982), T-Koizumi K. et al. and Kojima K.K. J. et al. Biochem. , 99, 1803 (1986), it is considered that these enzymes are actually involved in the metabolism of sphingomyelin in vivo.
[0014]
Ceramide, one of the products of the above reaction, is further hydrolyzed by ceramidase to produce fatty acids and sphingosine. And it has been confirmed by in vivo experiments that sphingomyelin is metabolized in the mammalian body to become ceramide and sphingosine [Schneider P. et al. B. and Kennedy E.M. P. J. et al. Lipid Res. , 9, 58 (1968)]. These ceramides and sphingosines, which are degradation products of sphingomyelin, have been shown to be involved in cell proliferation / differentiation and the information transfer control mechanism closely related to them [Kiyohide Kojima and Keiko Koizumi protein nucleic acid Enzyme, 36, 629 (1991)], this reaction pathway is called the sphingomyelin pathway.
[0015]
It has been shown that this sphingomyelin pathway is involved when IL-1β or TNF-α binds to a receptor of a target cell and is subsequently signaled in the cell [Dressler K. et al. A. et al. Science, 255, 1715 (1992), Mathias et al. Science, 259, 519 (1993)].
[0016]
Therefore, the signal transduction of these TNFα and IL1β can be blocked by an inhibitor of sphingomyelinase activity, and the pathological condition involving these cytokines can be improved.
[0017]
On the other hand, a sphingomyelinase reaction product activates cyclooxygenase, through which PGE is activated.₂It has been shown to promote production (Ballou LR et al. J. Biol. Chem. 267, 20044, (1992)).
[0018]
In addition, the sphingomyelinase reaction itself promotes the uptake of LDL and denatured LDL involved in the development of atherosclerosis into peripheral cells, and increases cholesterol ester synthesis and intracellular accumulation [Stein O. et al. et al. Biochim. Biochim. Acta. , 1126, 291 (1992), Chatterjee S .; J. et al. Biol. Chem. , 268, 3401 (1993)], is expected to be involved in the development of this disease state.
[0019]
Furthermore, activation of sphingomyelinase reduces the content of sphingomyelin in the apical membrane on the side of the sinusoid in the proximal tubule of the kidney and reduces the uptake of phosphate and sugar that function in a Na-dependent manner [Vrtovsnik F . et al. Kidney International. , 41, 983 (1992)].
[0020]
In addition, it was shown that the amount of ceramide was increased in CEM cells infected with HIV [Veldhoven P. et al. P. V. et al. Biochem. Biophys. Res. Comm. , 187, 209 (1992)], it is shown that sphingomyelinase is activated in HIV-infected cells in vivo.
[0021]
Based on the above facts, specific inhibitors for sphingomyelinase are anti-HIV agents, anti-diabetic agents, anti-arteriosclerotic agents, anti-osteoporosis agents, anti-thrombotic agents, anti-inflammatory agents, immunosuppressive agents, diuretics, And it can be used as a prophylactic or therapeutic agent for respiratory diseases, thyroid diseases, Alzheimer's disease, hepatitis, nephritis, leukemia, and cachexia.
[0022]
However, no specific and potent inhibitor against sphingomyelinase has been found to date.
[0023]
Conventionally, soluble IL-1 receptors and IL-1 receptor antagonists have been found as substances that specifically inhibit the action of IL-1β, and these have been used to improve symptoms in septic shock patients and RA patients. ing.
[0024]
In addition, clinical trials for endotoxin shock and acute GHVD using a soluble TNF receptor and an anti-TNF antibody that specifically inhibit TNF-α action have been carried out and its effectiveness has been observed [ Vincent J.H. -L. et al. Chest, 101, 810 (1992), Herve P. et al. et al. Blood, 79, 3362 (1992)].
[0025]
However, since these are all peptidic or high molecular weight substances, they have drawbacks in absorption into the body as drugs and stability in blood. From this viewpoint, a low molecular weight biologically active substance having specific inhibitory activity against sphingomyelinase has been desired.
[0026]
[Problems to be solved by the invention]
The present inventors searched for a substance having sphingomyelinase inhibitory activity from microbial metabolites, and inhibited sphingomyelinase in a culture solution of a Dasycyphylus mollismus (Lasch) Dennis strain belonging to the genus Dasycyphus isolated from a dead herbaceous stem. It was found that a novel compound F-10463a having activity was produced, and the present invention was completed.
[0027]
[Means for Solving the Problems]
F-10463a of the present invention has the following structural formula and properties.
[0028]
Structural formula
[0029]
[Chemical 2]

[0030]
Physicochemical properties
1) Properties: colorless oily substance,
2) Solubility; soluble in organic solvents such as methanol, ethyl acetate and chloroform, insoluble in water
3) Color test: Positive for 50% sulfuric acid and iodine.
[0031]
4) Molecular formula; C₂₉H₄₃NO₅
5) Molecular weight: 485 (by high resolution FABMS method [M + H]⁺  486.3199 (measured value)) (calculated value: 486.3204)
6) Specific rotation: [α] +64.44 (c 0.09 in MeOH)
7) Ultraviolet absorption spectrum; (in methanol) 300 nm (ε41500)
8) Infrared absorption spectrum; (in chloroform)
3410, 1655, 1608, 1508 cm^-1
9)¹H-nuclear magnetic resonance spectrum (in deuterated methanol: PPM, TMS standard)
7.15 (m, 2H), 6.53 (dd, J = 14.9, 10.7 Hz, 1H), 6.25 (dd, J = 14.9, 11.1 Hz, 1H), 6.15. (Dd, J = 15.1, 10.7 Hz, 1H), 6.07 (dd, J = 9.8, 1.6 Hz, 1H), 5.89 (d, J = 14.8 Hz, 1H), 5.70 (dd, 15.1, 8.7 Hz, 1H), 4.8 (hidden under HDO signal, 1H), 4.05 (m, 1H), 3.67 (d, J = 3.9 Hz, 1 H), 3.59 (m, 1 H), 3.52 (dd, J = 10.9, 5.1 Hz, 1 H), 3.45 (dd, J = 10.9, 5. 8 Hz, 1H), 2.35 (m, 1H), 2.27 (m, 1H), 2.08 (dd, J = 14.7, 3.6 Hz, 1H), 1.89 (m, 2H), 1.79 (dd, 13.1, 7.1 Hz, 1H), 1.59 (m, 1H), 54 (d, J = 1.3 Hz, 3H), 1.33 (m, 2H), 1.19 (m, 1H), to 1.1 (m, 1H), 1.00 (d, J = 6 0.7 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H), 0.83 (d, J = 6.5 Hz, 3H)
10)¹³C-nuclear magnetic resonance spectrum (in deuterated methanol: PPM, deuterated methanol reference)
200.1 (s), 169.0 (s), 146.6 (d), 146.4 (d), 142.9 (d), 141.9 (d), 134.7 (d), 134 .0 (s)
132.5 (d), 130.4 (d), 129.9 (d), 124.3 (d), 78.0 (s), 66.1 (t), 58.7 (d), 50 .1 (t),
49.8 (d), 48.5 (d), 45.7 (t), 40.3 (t), 36.7 (d), 35.9 (d), 32.2 (t), 30 0.0 (d), 22.4 (q)
22.0 (q), 20.4 (q), 16.9 (q), 13.0 (q)
11) High performance liquid chromatography
Retention time: 5.65 minutes
Column: Cosmo Seal 5 C18-AR, 4.6φX150mm, manufactured by Nacalai Tesque
Solvent: 70% acetonitrile-water
Flow rate: 1 ml / ml
Detection: UV 210nm
Producing bacteria
Examples of strains belonging to the genus Dasycyphus used in the present invention include, for example, Dasyscyhus mollismus (Lasch) Dennis SANK13892 (FERM BP-4491). The bacteriological properties of these strains are as follows.
[0032]
SANK13892 was isolated from dead stalks of herbs collected in May 1991 in Aomori Prefecture.
[0033]
The ascending disc is a flat, disk-shaped pattern with a maximum diameter of 0.2 mm and has a gray-orange to yellow color. There are hairs on the periphery of the cyst, and the edges do not rise.托 The outer skin layer has a multi-faceted fungus-like structure and consists of light brown, thin-film cells. The spinal cord layer is intertwined closely and consists of hyphae slightly parallel to the outer wall, and presents an intertwined fungus tissue. The hair is generated from the outermost cell of the wing skin layer and extends straight, has a cylindrical shape, has a thin film, a large number of partition walls, has a maximum length of 200 μm and a thickness of 3.5 μm. Its tip is almost blunt. The surface is almost smooth and resinous amorphous substances are scattered and adhered. The ascosm originates from the clamp, is cylindrical, 8-spore and is 58-66 x 5-6.5 μm in size. The tip turns blue with the Melzer reagent. The side thread is a thin cocoon-shaped and has a size of 74.5-100 × 3.5-4 μm and extends about 20 μm above the ascending sac. Ascospores are cocoon-shaped to spindle-shaped, single-cell, colorless, and have a size of 18-26.5 x 2.5-5 μm.
[0034]
Growth on PDA reaches 1.5 cm at 23 ° C. for 10 days. The surface is light to clear white and rises slightly in the center of the colony. The development of aerial hyphae is not very vigorous and entangles to form a hyphae bundle. The surface becomes powdery. The back surface is light to clear white. Furthermore, it becomes darker color by continuing culture | cultivation. Conidia are not observed.
[0035]
From the above bacteriological characteristics, those that correspond to this bacterium were searched for. Otani (1967) Notes on some cups of the Hyalocyphaceae collected in Hokaido, Japan. Trans. Mycol. Soc. Japan 8: 33-42. Accord with Dasycyphys mollissimus (Lasch) Dennis described in. Therefore, this bacterium was identified as Dasycyphys mollicus (Lasch) Dennis and named Dassycyphus mollicus (Lasch) Dennis SANK13892. This fungus was deposited internationally at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology on December 10, 1993, and was given the deposit number FERM BP-4491.
[0036]
Culture method and purification method
As a separation medium used for separating the new strain of the present invention, any medium that is appropriately selected from carbon sources, nitrogen sources, inorganic ions, organic nutrient sources, etc. can be used, either synthetic or natural media. Is possible. F-10463a is obtained by culturing the SANK13892 strain in an appropriate medium and then collecting it. As the nutrient source, known ones conventionally used for culturing fungi can be used. For example, glucose, sucrose, starch, glycerin, starch syrup, molasses, soybean oil and the like can be used as the carbon source. As the nitrogen source, soy flour, corn steep liquor, yeast extract, malt extract, potato, ammonium sulfate, sodium nitrate and the like can be used. In addition to the above, inorganic salts such as calcium carbonate and phosphate can be added as needed, and organic and inorganic substances that assist the growth of the strain and promote the production of F-10463a can be appropriately added. As the culture method, the liquid culture method, particularly the deep culture method, is most suitable, as is the method for producing general antibiotics. Cultivation is performed under aerobic conditions, and the temperature suitable for culturing is 15-30 ° C., but in many cases, culturing is performed at around 23 ° C. Production of F-10463a reaches its maximum in 5-8 days, usually with shaking culture.
[0037]
After completion of the culture, F-10463a present in the bacterial cells or broth in the culture solution is extracted by adding an organic solvent such as acetone or acetonitrile having a volume of the culture solution and mixing. The solid portion present in the extract is separated by filtration operation or centrifugation using diatomaceous earth as a filtration operation aid, and F-10463a present in the filtrate or supernatant is used as an index of sphingomyelinase inhibitory activity. Extraction and purification using the physicochemical properties. For example, F-10463a present in this extract is obtained by first removing the organic solvent mixed in by the concentration operation, and then immiscible with an organic solvent such as n-butanol, methyl ethyl ketone, ethyl acetate, chloroform, ethylene chloride, methylene chloride. Etc. alone or in combination thereof. Alternatively, as an adsorbent, for example, activated carbon or amberlite XAD-2 or XAD-4 (made by Rohm and Haas) which is an adsorbing resin, Diaion HP-10, HP-20, CHP-20P, HP- 50 (Mitsubishi Kasei Co., Ltd.) can be used. The liquid containing F-10463a is passed through the adsorbent layer as described above to adsorb and remove impurities, or after F-10463a is adsorbed, methanol water, acetone water, n-butanol water or the like is used. It is obtained by eluting. F-10463a thus obtained was further subjected to adsorption column chromatography using a carrier such as silica gel and Florisil, partition column chromatography using Sephadex LH-20 (Pharmacia), Sephadex G- It can be purified by gel filtration chromatography using 25 (Pharmacia) or the like, and high performance liquid chromatography using normal phase or reverse phase columns.
[0038]
Sphingomyelinase inhibitory activity can be measured by the following method (J. Lipid Res. 20, 456 (1979)).
[0039]
That is, first, 10 μl of [N-methyl-¹⁴C] Sphingomyelin (cow, 52 mCi / mmol, 25 μCi / ml; Amersham) and 200 μl of sphingomyelin (cow, 20 mM, Sigma) were dried with nitrogen gas, and then 200 μl of 1M Tris-HCl (pH 7.5). ), 40 μl of 10% (v / v) Triton X-100, 20 μl of 0.1%. 5M MgCl₂  And 1. 24 ml of H₂  O was added and incubated at 48 ° C. for 30 minutes. Using a probe-type ultrasonic crusher, sonication was performed twice for 15 seconds under an output condition of 20 W, [¹⁴C] A mixed micelle system containing sphingomyelin was prepared.
[0040]
In the sphingomyelinase reaction, 150 μl of the substrate solution prepared in this way is mixed with 10 μl of the sample solution, and the microsomal fraction of Wistar Imamichi male rat brain (25,000 × g to 100,000 × g, protein concentration of 3 (~ 4mg / ml) was added as an enzyme solution and incubated at 37 ° C for 40 minutes. After completion of the reaction, 500 μl of chloroform: methanol (2: 1, v / v) was added to perform extraction operation, and 150 μl of the obtained aqueous layer was mixed with 3 ml of Picoflow TM40 (Packard Japan Co., Ltd.) to obtain a reaction product. [¹⁴C] The amount of phosphorylcholine was measured with a liquid scintillation counter. The sphingomyelinase activity was calculated from the measured value using MgCl required for the sphingomyelinase reaction.₂  Calculated as the value obtained by subtracting the measured value excluding.
[0041]
【Example】
EXAMPLES Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.
[0042]
Example 1. Purification of substance F-10463a
(1) Culture
Dasycyphus mollissimus (Lasch) Dennis SANK13892 was aseptically inoculated into a 500 ml Erlenmeyer flask (seed flask) containing 100 ml of a sterilized culture medium having the composition described below. This was then precultured for 5 days on a rotary shaker at 200 rpm.
[0043]
[Table 1]
Medium composition
Glycerol 50g
Potato 50g
East Extract 5g
Mart Extract 5g
Antifoam (CB-442) 0.2g
Ion exchange water 1000ml
pH unadjusted
The main culture was performed as follows. 5 ml each of the seed culture solution was put into 15 500 ml Erlenmeyer flasks containing 100 ml of the sterilized culture medium having the above composition, and cultured at 23 ° C. for 7 days with a 200 rpm rotary shaker.
[0044]
(2) Method for measuring sphingomyelinase inhibitory activity
The preparation of sphingomyelinase enzyme preparation is basically as described in Gatt S. et al. (Biochem. Biophys. Res. Commun. 68, 235 (1976)). That is, rat brain was used as an enzyme source for sphingomyelinase, and the microsomal fraction was first prepared as follows. Ten Wistar Imamichi male rats (9 weeks old) were exsanguinated to the carotid artery, and then the whole brain was removed. After rapidly removing the cerebellum, 130 ml of buffer A (0.25 M sucrose, 1 mM EDTA, 1 mM PMSF (Phenylmethylsulfonyl Fluoride), 0.1 mM leupeptin, 5 mM Tris-HCl buffer, pH 7.4) previously cooled to 4 ° C. was added. Brain cells were disrupted using a Potter homogenizer under the condition of 4 ° C. Next, the obtained cell lysate is centrifuged at 700 × g for 10 minutes at 4 ° C., and the supernatant is further centrifuged at 25,000 × g for 10 minutes at 4 ° C. went. Finally, the obtained supernatant was subjected to ultracentrifugation at 100,000 × g for 60 minutes under 4 ° C., and the precipitate was used as a microsomal fraction. This fraction was frozen and stored under liquid nitrogen until the measurement of sphingomyelinase activity, and the protein concentration was adjusted to about 3 to 4 mg / ml with buffer A when used.
[0045]
Sphingomyelinase activity was determined using a mixed micellar system by Hostoster K. Y. And Yazaki P. J. et al. (J. Lipid Res. 20, 456 (1979)) was used for measurement as follows. First, 10 μl of mixed [N-methyl-¹⁴C] Sphingomyelin (cow, 52 mCi / mmol, 25 μCi / ml; Amersham) and 200 μl of sphingomyelin (cow, 20 mM, Sigma) were dried with nitrogen gas, and then 200 μl of 1M Tris-HCl (pH 7.5). ), 40 μl of 10% (v / v) Triton X-100, 20 μl of 0.1%. 5M MgCl₂And 1. 24 ml of H₂O was added and incubated at 48 ° C. for 30 minutes. Then, with a probe-type ultrasonic crusher, ultrasonic treatment was performed twice for 15 seconds under an output condition of 20 W, [¹⁴C] A mixed micelle system containing sphingomyelin was prepared.
[0046]
The sphingomyelinase reaction was carried out by mixing 10 μl of the sample solution with 150 μl of the substrate solution prepared in this way, adding 40 μl of the enzyme solution described above, and incubating at 37 ° C. for 40 minutes. After completion of the reaction, 500 μl of chloroform: methanol (2: 1, v / v) was added to perform extraction operation, and 150 μl of the obtained aqueous layer was mixed with 3 ml of Picoflow TM40 (Packard Japan Co., Ltd.) to obtain a reaction product. [¹⁴C] The amount of phosphorylcholine was measured with a liquid scintillation counter. The sphingomyelinase activity was calculated from the measured value using MgCl required for the sphingomyelinase reaction.₂  Calculated as the value obtained by subtracting the measured value excluding.
[0047]
The concentration of F-10463a required to inhibit the sphingomyelinase reaction by 50%, measured by this method, was 20 μg / ml.
[0048]
(3) Isolation
The culture solution for 15 Erlenmeyer flasks was adjusted to pH 3 with 6N hydrochloric acid, and then centrifuged at 3000 rpm for 10 minutes. To the obtained cells, 500 ml of 80% acetone was added and stirred for 1 hour. The mixture was centrifuged at 3000 rpm for 10 minutes, and the supernatant was concentrated under reduced pressure to remove acetone. This was then extracted twice with an equal volume of ethyl acetate. The ethyl acetate layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 430 mg of a brown oil. Of this, 400 mg was dissolved in a small amount of methylene chloride-methanol, 100: 1 solvent and charged to an 80 ml silica gel column equilibrated with the same solvent. This column was eluted with 240 ml of methylene chloride-methanol, 100: 1 solvent, and further eluted with 240 ml of 50: 1, 20: 1 and 10: 1 solvents, and the respective eluates were fractionated. Enzyme inhibitory activity was found in the 20: 1 fraction, so this fraction was concentrated to yield 78 mg of a brown oil. This was dissolved in a small amount of methanol and purified by preparative HPLC as follows. That is, about 20 mg each was charged into an HPLC column (Cosmosil 5C18-AR, 20φX250 mm, manufactured by Nacalai Tesque) equilibrated with 70% acetonitrile aqueous solution, and 10 ml / min. Deployed at a flow rate of. Absorption at 210 nm was detected, and a peak at about 30 minutes was collected. The separated solution was concentrated under reduced pressure to give 22 mg of F-10463a material.
[0049]
【The invention's effect】
The novel compound F-10463a of the present invention is an anti-HIV agent, anti-diabetic agent, anti-arteriosclerotic agent, anti-osteoporosis agent, anti-thrombotic agent, anti-inflammatory agent, immunosuppressive agent, diuretic agent, and respiratory disease. , Thyroid disease, Alzheimer's disease, hepatitis, nephritis, leukemia, and cyccia.

Claims (3)

Novel compound represented by the following formula F-10463a:

A method for producing F-10463a, which comprises culturing F-10463a-producing bacteria belonging to the genus Dasycyphyhus and collecting F-10463a from the culture. The production method according to claim 2, wherein the F-10463a-producing bacterium belonging to the genus Dasycyphus is Dasyscyhus mollismus (Lasch) Dennis SANK13892 (FERM BP-4491).