JP3851661B2 - Novel physiologically active substance and method for producing the same - Google Patents

Description

また、本発明は、ストレプトミセスに属し、式(I)で示される新規生理活性物質を産生及び製造するために用いることのできる微生物に関する。
このような微生物としては、具体的には本発明者らによって沖縄本島の土壌から分離採取されたストレプトミセス・スペクタビリス（Streptomyces spectabilis）ＳＮＦ4435株（FERM BP-5915）を挙げることができる。
また、本発明は、前記微生物を培養し、培養物中に前記新規生理活性物質を産生せしめ、これを採取することよりなる新規生理活性物質の製造法に関する。
また、本発明は、前記微生物を培養し、培養物中に類縁体のスペクチナビリンを産生し、このテトラエン部を閉環させて合成化学的な手段で新規生理活性物質を製造する方法に関する。
さらに、本発明は、前記式(I)で示される新規生理活性物質またはその薬理学的に許容される塩を有効成分とする医薬に関する。
本発明の医薬は、臓器移植による免疫拒絶反応あるいは自己免疫疾患の治療剤、アレルギー疾患の治療剤、感染症の抗菌剤、或いは制癌剤耐性克服剤等として用いられる。
すなわち、本発明の生理活性物質は、新規なニトロフェニルピロン系の化合物であって、その活性からみて臓器移植等による免疫拒絶反応の抑制剤、全身性エリテマトーデス、慢性関節リウマチ、ブドウ膜炎等の自己免疫疾患の治療剤、アトピー性皮膚炎等のアレルギー疾患の治療剤、各種微生物による感染症の抗菌剤、或いは制癌剤耐性克服剤等の医薬として有用である。
【図面の簡単な説明】
第１図は、本発明化合物ＳＮＦ4435Ｃのメタノール中（15.4μｇ／ml）での紫外線吸収スペクトルを示す。
第２図は、本発明化合物ＳＮＦ4435Ｃの臭化カリウム錠での赤外線吸収スペクトルを示す。
第３図は、本発明化合物ＳＮＦ4435Ｃの重クロロホルム溶液中での500MHz ¹ＨＮＭＲスペクトル（ＴＭＳ基準）を示す。
第４図は、本発明化合物ＳＮＦ4435Ｃの重クロロホルム溶液中での125MHz¹³ＣＮＭＲスペクトル（重クロロホルム基準）を示す。
第５図は、本発明化合物ＳＮＦ4435Ｄのメタノール中（18.0μｇ／ml）での紫外線吸収スペクトルを示す。
第６図は、本発明化合物ＳＮＦ4435Ｄの臭化カリウム錠での赤外線吸収スペクトルを示す。
第７図は、本発明化合物ＳＮＦ4435Ｄの重クロロホルム溶液中での500MHz ¹ＨＮＭＲスペクトル（ＴＭＳ基準）を示す。
第８図は、本発明化合物ＳＮＦ4435Ｄの重クロロホルム溶液中での125MHz¹³ＣＮＭＲスペクトル（重クロロホルム基準）を示す。
発明を実施するための最良の形態
本発明の新規生理活性物質（以下、本発明化合物という）は、微生物を培養することにより得られる。この時、用いる微生物としては、本発明化合物を産生する菌であれば特に限定されないが、好ましくはストレプトミセス属（Streptomyces sp.）に属する菌、特に好ましくはストレプトミセス・スペクタビリス（Streptomyces spectabilis）ＳＮＦ4435株を挙げることができる。このストレプトミセス・スペクタビリスＳＮＦ4435株は、本発明者らが、沖縄本島から採取した土壌より分離した菌株であり、通商産業省工業技術院生命工学工業技術研究所に受託番号ＦＥＲＭ P-15476として、平成８年２月27日に受託し、その後、この原寄託よりブダペスト条約に基づく寄託に移管し、受託番号ＦＥＲＭ BP-5915が付されている。
ストストレプトミセス・スペクタビリス（Streptomyces spectabilis）ＳＮＦ4435株の分離は、沖縄本島の土壌を放線菌の分離に通常用いられている方法、例えばGoodfellow, M.；Actinomycetologica Vol. 2, No.1 13〜29(1988)に記載の方法によって容易に実施することができる。
このようにして分離されたストレプトミセス・スペクタビリスＳＮＦ4435株の性状を示すと次のとおりである。
１．形態
気菌糸はうっすらと着生し、わずかに疑似輪生分岐が見られるが菌糸の分断は認められない。気菌糸は赤色系まれには白色系の色調を呈し、10個以上の胞子の連鎖が形成されて屈曲性の直線型をしている。胞子の表面はほぼ平滑で、大きさが0.4×0.8μm位の円柱状である。菌核、胞子のう、遊走子等の特殊な器官は見出されない。
２．各種培地における生育状態
各種寒天平板培地において、27℃、14日間培養した結果を表１に示す。尚、色の記載については、ＪＩＳ色名帳（JIS Z 8102準拠、日本規格協会、平成５年４月15日、第１版第３刷発行）に従った。

(1)生育温度範囲
イースト・麦芽寒天培地を用い、７℃、12℃、17℃、22℃、27℃、32℃、37℃、42℃及び47℃の各温度で試験した結果、７℃、42℃及び47℃を除き、そのいずれの温度でも生育した。最適生育温度は27℃〜32℃付近と思われる。
(2)ゼラチンの液化
グルコース・ペプトン・ゼラチン培地、27℃培養あるいは単純ゼラチン培地、20℃培養において、いずれの培地でも培養10日後頃より液化が始まり、21日後の観察での液化作用は中程度であった。
(3)スターチの加水分解
スターチ・無機塩寒天培地、27℃培養において、培養３日後頃より水解性が認められ、その作用は比較的強い。
(4)脱脂牛乳の凝固・ペプトン化
脱脂牛乳培地、37℃培養において、凝固は認められなかったが、培養７日後頃よりペプトン化が観察された。
(5)メラニン様色素の生成
トリプトン・イースト・ブロス培地、ペプトン・イースト・鉄寒天培地またはチロシン寒天培地を用いた27℃培養において、ペプトン・イースト・鉄寒天培地のみに培養３日後頃からメラニン様色素の生成が認められた。
(6)炭素源の利用性
プリドハム・ゴドリーブ寒天培地、27℃培養において、Ｄ−グルコース、Ｄ−キシロース、Ｄ−フラクトース、ラフィノース、イノシトール、Ｄ−マンニトール、マンノース、Ｄ−ガラクトースを利用し、Ｌ−アラビノース、シュクロース、Ｌ−ラムノースは利用しない。
以上の性状を要約すると、ＳＮＦ4435株の菌学的性状は、気菌糸が直線状で胞子のうは形成しないが、わずかに疑似輪生分岐が認められる。気菌糸の先端には10個程度の胞子を連鎖し、その表面は平滑である。種々の培地で、気菌糸の色調はピンクから赤色系を呈す。可溶性色素はペプトン・イースト・鉄寒天培地でメラニン様色素が生成され、またスターチの水解性及び蛋白の分解力は中程度である。
尚、細胞壁に含まれる2，6−ジアミノピメリン酸はＬＬ−型であった。これらの性状より、ＳＮＦ4435株はストレプトミセス（Streptomyces）属に属すると考えられ、「バージーズ・マニュアル・オブ・デターミナティブ・バクテリオロジー（Bergey's Manual of Determinative Bacteriology）」第８版、及びＩＳＰ報告「インターナショナル・ジャーナル・オブ・システマティック・バクテリオロジー（International Journal of Systematic Bacteriology）」第18巻、69頁、279頁（1968年）、同19巻、391頁（1969年）、同22巻、265頁（1972年）より検索した結果、ストレプトミセス・スペクタビリス（Streptomyces spectabilis）を近縁種として挙げられる。そこで、ＳＮＦ4435株とストレプトミセス・スペクタビリス（Streptomyces spectabilis IFO 13424）とを比較した。結果を第２表に示す。

この結果より、気菌糸の色調及び生育温度範囲等に若干の差異が認められる以外、気菌糸の形態、胞子の表面、生育状態やメラニン様色素を生成する点及び炭素源の利用性などで両者はよく一致する。従って、ＳＮＦ4435株をストレプトミセス・スペクタビリス（Streptomyces spectabilis）と同定した。
本発明で用いられる微生物は、Ｘ線、紫外線等の照射処理、又は亜硝酸、Ｎ−メチル−Ｎ’−ニトロ−Ｎ−ニトロソグアニン（ＮＴＧ）等の変異誘起剤による処理、形質転換、形質導入又は融合等の通常用いられる菌種変換処理方法により変異させた微生物であっても良い。
本発明化合物は、通常の微生物が利用しうる栄養源含有培地にこれらの本発明化合物の産生菌を接種して発育させることにより、その培養物中に産生され、あるいは化合物を製造するために用いられる微生物を接種して発育させその培養物を原料として有機合成により、目的とする本発明化合物を製造することができる。栄養源としては、放線菌の栄養源に利用されているのものであれば良く、合成培地、半合成培地、天然培地などいずれも使用できる。例えば、炭素源としてはグルコース、グリセロール、麦芽糖、デンプン、シュクロース、糖蜜、水飴又はデキストリンなどが単独又は混合物として用いられる。窒素源としては、大豆粉、コーングルテンミール、コーンスティープリカー、肉エキス、酵母エキス、綿実粕、ペプトン、小麦胚芽、魚粉、尿素などの有機窒素源、硫酸アンモニウム、硝酸ナトリウムなどの無機窒素源が単独又は混合物として用いられる。無機塩としては、炭酸カルシウム、塩化ナトリウム、塩化カリウム、硫酸マグネシウム又は各種リン酸塩等を使用することができ、さらに必要に応じて、鉄、銅、コバルト、モリブデン、マンガン又は亜鉛などの重金属塩を微量添加することもできる。又、培養中発泡の著しい時には、消泡剤として公知の各種消泡剤を適宜培地中に添加しても良い。この他に、該生産菌が利用し、本発明化合物の生産に有用な有機及び無機物を、適宜用いることができる。
菌株の培養方法としては、一般の微生物代謝産物の生産法と同様に行えば良く、固体培養でも液体培養でも良い。液体培養の場合は、静置培養、攪拌培養、振とう培養又は通気培養等のいずれを実施しても良いが、ストレプトミセス・スペクタビリスＳＮＦ4435株を培養する場合には、特に振とう培養又は深部通気攪拌培養が好ましい。又、培養条件によっても異なるが、好ましい培地のpHは４〜８の範囲で、培養温度は22〜37℃、好ましくは25〜30℃が適当である。また培養時間は48〜168時間、好ましくは96〜144時間である。
培養物から目的とする本発明新規生理活性物質を単離、製造するには、微生物の生産する代謝物を単離するのに通常使用される分離手段を適宜利用すれば良い。また、必要があれば合成化学的な手段を適宜利用すれば良い。
培養により生成した本発明化合物は、通常培養物中の菌体内及び菌体外の両方に蓄積されることが多いので、例えば遠心分離、濾過等の手段により培養濾液及び菌体に分離し、培養濾液及び菌体より通常の分離手段、例えば、透析法、溶媒抽出法、不純物との溶解度差を利用する方法、イオン交換樹脂法又は吸着もしくは分配クロマトグラフィー法及びゲル濾過法などを単独又は適宜組み合わせて、場合によっては反復使用することによって分離精製することができる。
合成化学的な手段を利用して採取される本発明化合物は、微生物を培養して培養液中に生成された本発明化合物の類縁体（ニトロフェニルピロン系）について上記分離手段を使用することによって分離精製し、合成化学的な手段、例えば触媒を用いた閉環反応により製造させることができる。反応に用いられる有機溶媒としては、例えばメタノール、エタノール、第３ブチルアルコール、テトラヒドロフラン、ジエチルエーテル、エチレングリコール、ジメチルエーテル、ジメチルホルムアミド、ジメチルスルホキシド、ベンゼン、トルエン、キシレン、ジオキサン、塩化メチレン、クロロホルム、ジクロロエタン又はアセトニトリル等が挙げられる。閉環反応を光を用いて行う場合は、通常200nm〜800nmの波長の光を用いるが、必要に応じてこれ以上またはこれ以下の波長を選択できる。閉環反応の反応温度は、通常−20〜400℃であり、必要に応じてこれ以上またはこれ以下の温度を選択できる。閉環反応の反応時の圧力は、通常１〜10atmで、必要に応じてこれ以上またはこれ以下の圧力を選択できる。閉環反応に用いられる触媒としては、例えば塩化アルミニウム、塩化スズ、フッ化ホウ素、フッ化ホウ素酸銅およびヨウ素等が挙げられる。閉環反応の反応時間は、通常30分から２日の範囲であるが、必要に応じてこれ以上またはこれ以下の時間を選択できる。
本発明におけるスペクチナビリンのテトラエン部を閉鎖させる反応式を示す。

上記反応条件により閉環反応を行った後、有機合成化学の分野における公知の方法、例えば溶媒抽出、再結晶、クロマトグラフィー、イオン交換樹脂、ＨＰＬＣ等を用いる方法によりＳＮＦ4435ＣおよびＳＮＦ4435Ｄを分離精製することができる。
得られた本発明化合物の性状を示す。尚、ＳＮＦ4435は平面構造（式(I)参照）は同一だが光学異性体が存在し、それぞれをＳＮＦ4435Ｃ又はＤとする。これらの光学異性体は、液体クロマトグラフィー等の常法の分離手段によってそれぞれの光学異性体に分離することができる。尚、医薬として用いる場合は光学異性体単体であっても良いし、ラセミ体であっても良い。
ＳＮＦ4435Ｃの物理化学的性状
（１）色及び性状：乳白色粉末
（２）分子式：Ｃ₂₈Ｈ₃₁ＮＯ₆
（３）マススペクトル（ＦＡＢ−ＭＳ）：ｍ／ｚ 478（Ｍ＋Ｈ）⁺
（４）比旋光度：［α］_D ²⁶−105.6°（C 0.10, CHCl₃）
（５）紫外線吸収スペクトル：図１に示す。
λ_max ^MeOHnm（ε）：271（19322）
（６）赤外線吸収スペクトル：図２に示す。
ν_max ^KBrcm^-1：2950，2850，1670，1600，1520，1350，1255，1165，1040
（７）¹Ｈ−核磁気共鳴スペクトル：図３に示す。（500MHz，CDCl₃）
δ(ppm)：8.19(d,J=8.7Hz,2H)，7.55(d,J=8.7Hz,2H)，5.58(d,J=1.4Hz,1H)，4.95(S,1H)，4.78(t,J=8.2Hz,1H)，4.32(d,J=9.8Hz, 1H)，3.97(d, J=9.8Hz,1H)，3.96(S,3H)，3.64(S,1H)，2.84(S,1H)，2.43(d, J=8.2Hz,2H)，1.89(S,3H)，1.84(S,3H)，1.74(S,3H)，1.72（d, J=1.4Hz,3H)，1.30(S,3H)
（８）¹³Ｃ−核磁気共鳴スペクトル：図４に示す。（125MHz，CDCl₃）
δ(ppm)：180.5(s)，162.2(s)，155.2(s)，146.9(s)，145.1(s)，131.0(s)，130.4(s)，129.1(d)，123.8(d)，123.6(d)，122.0(d)，119.6(s)，100.3(s)，73.5(d)，70.6(t)，63.7(d)，55.6(q)，51.8(s)，51.1(d)，46.4(t)，43.0(s)，30.4(q)，23.0(q)，22.2(q)，9.4(q)，7.0(q)
（９）溶解性：メタノール、クロロホルム、エタノール、アセトン、ピリジン、酢酸エチル、ジエチルエーテル、ジメチルスルホキシド等に可溶ヘキサン、水に不溶
（10）呈色反応：50％硫酸、ヨウ素に陽性
（11）ＨＰＬＣ：保持時間 14.6分
カラム；イナートシルＯＤＳ−２（φ4.6×250mm、ＧＬサイエンス社）
溶媒；80％メタノール・水、流速１ml／min、ＵＶ220nm
（12）酸性、中性、塩基性物質の区別：中性
ＳＮＦ4435Ｄの物理化学的性状
（１）色及び性状：乳白色粉末
（２）分子式：Ｃ₂₈Ｈ₃₁ＮＯ₆
（３）マススペクトル（ＦＡＢ−ＭＳ）：ｍ／ｚ 478（Ｍ＋Ｈ）⁺
（４）比旋光度：［α］_D ²⁶＋84.8°（C 0.10, CHCl₃）
（５）紫外線吸収スペクトル：図５に示す。
λ_max ^MeOHnm（ε）：270（21462）
（６）赤外線吸収スペクトル：図６に示す。
ν_max ^KBrcm^-1：2950，2850，1670，1600，1520，1350，1255，1165，1040
（７）¹Ｈ−核磁気共鳴スペクトル：図７に示す。（500MHz， CDCl₃）
δ(ppm)：8.15(d,J=8.7Hz,2H)，7.47(d,J=8.7Hz,2H)，5.70(S,1H)，4.94(dd,J=7.0, 9.5Hz,1H)，4.89(S,1H)，4.18(d,J=9.2Hz,1H)，3.84(d,J=9.2Hz,1H)，3.74(S,1H)，3.54(S,3H)，2.74(S,1H)，2.48(dd,J=13.1,9.5Hz,1H)，2.28(dd,J=13.1,7.0Hz,1H)，1.97(S,3H)，1.84(S,3H)，1.80（S,3H)，1.74(d,J=1.5Hz,3H)，1.31(S,3H)
（８）¹³Ｃ−核磁気共鳴スペクトル：図８に示す。（125MHz，CDCl₃）
δ(ppm)：180.5(s)，161.9(s)，154.9(s)，146.9(s)，144.0(s)，131.3(s)，131.2(s)，129.9(d)，124.4(d)，123.2(d)，121.9(d)，119.7(s)，100.0(s)，72.5(d)，70.6(t)，61.1(d)，55.2(d)，54.8(q)，51.1(s)，45.5(t)，43.0(s)，30.7(q)，22.2(q)，22.1(q)，9.4(q)，6.8(q)
（９）溶解性：メタノール、クロロホルム、エタノール、アセトン、ピリジン、酢酸エチル、ジエチルエーテル、ジメチルスルホキシド等に可溶ヘキサン、水に不溶
（10）呈色反応：50％硫酸、ヨウ素に陽性
（11）ＨＰＬＣ：保持時間 16.6分
カラム；イナートシルＯＤＳ−２（φ4.6×250mm、ＧＬサイエンス社）
溶媒；80％メタノール・水、流速１ml／min、ＵＶ220nm
（12）酸性、中性、塩基性物質の区別：中性
本発明の新規生理活性物質を医薬として用いる場合、薬理学的に許容される塩としても良い。薬理学的に許容される塩として、ナトリウム、カリウム等のアルカリ金属塩、或いはマグネシウム、カルシウム等のアルカリ土類金属塩、アルミニウムその他の金属塩、及びアルキルアミン塩、ピリジン塩等の有機アミン塩が挙げられる。この化合物又はその薬理学的に許容される塩は、ヒト及び動物に対し、医薬として経口的及び非経口的に安全に投与される。非経口的投与には、例えば静脈注射、筋肉内注射、皮下注射、腹腔内注射、経皮投与、経肺投与、経鼻投与、経腸投与、口腔内投与、経粘膜投与等が挙げられ、これらの製剤が投与される。例えば注射剤、坐剤、エアゾール剤、経皮吸収テープなどが挙げられる。又、経口投与製剤として例えば錠剤（糖衣錠、コーティング錠、バッカル錠を含む）、散剤、カプセル剤（ソフトカプセルを含む）、顆粒剤（コーティングした物、丸剤、トローチ剤、液剤、又はこれらの製剤学的に許容され得る徐放化製剤等）が挙げられる。経口投与用液剤には懸濁剤、乳剤、シロップ剤（ドライシロップを含む）、エリキシル剤などが挙げられる。
これらの製剤は公知の製剤学的製法に準じ、製剤として薬理学的に許容され得る担体、賦形剤、崩壊剤、滑沢剤、着色剤等と共に医薬組成物として投与される。これらの製剤に用いる担体や賦形剤としては、例えば乳糖、ブドウ糖、白糖、マンニトール、馬鈴薯デンプン、トウモロコシデンプン、炭酸カルシウム、リン酸カルシウム、硫酸カルシウム、結晶セルロース、カンゾウ末、ゲンチアナ末など、結合剤としては例えばデンプン、トラガントゴム、ゼラチン、シロップ、ポリビニルアルコール、ポリビニルエーテル、ポリビニルピロリドン、ヒドロキシプロピルセルロース、メチルセルロース、エチルセルロース、カルボキシメチルセルロースなど、崩壊剤としては例えばデンプン、寒天、ゼラチン末、カルボキシメチルセルロースナトリウム、カルボキシメチルセルロースカルシウム、結晶セルロース、炭酸カルシウム、炭酸水素ナトリウム、アルギン酸ナトリウムなど、滑沢剤としては例えばステアリン酸マグネシウム、タルク、水素添加植物油、マクロゴールなど、着色剤としては医薬品に添加することが許容されているものを、それぞれ用いることができる。錠剤、顆粒剤は必要に応じ白糖、ゼラチン、ヒドロキシプロピルセルロース、精製セラック、ゼラチン、グリセリン、ソルビトール、エチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドン、フタル酸セルロースアセテート、ヒドロキシプロピルメチルセルロースフタレート、メチルメタクリレート、メタアクリル酸重合体などで被膜しても良いし、２以上の層で被膜しても良い。さらにエチルセルロースやゼラチンのような物質のカプセルでも良い。又、注射剤を調製する場合は、主薬に必要に応じｐＨ調整剤、緩衝剤、安定化剤、可溶化剤などを添加して、常法により各注射剤とする。
本発明の新規生理活性物質を患者に投与する場合、症状の程度、患者の年齢、健康状態、体重などの条件によって異なり特に限定はされないが、成人１日当たり約10mg〜10gを経口或いは非経口的に１日１回若しくはそれ以上投与すれば良い。
急性毒性は、実施例６に示すようにほとんどみられなかった。
以下の実施例により本発明をより詳細に説明するが、これらは単に例示するのみであり、本発明はこれらにより何ら限定されるものではない。
〔実施例１〕
(1)微生物の分離
沖縄本島で採取した土壌を熱処理（60℃，10分間）することにより得られた乾燥土壌１gを10mlの滅菌水で懸濁した。該懸濁液を10^-3倍に希釈し、寒天平板培地（硫酸アンモニウム0.05％、リン酸一カリウム0.05％、塩化カルシウム0.01％、硫酸マグネシウム0.01％、尿素0.01％、グルコース0.05％、可溶性デンプン0.1％、クロラムフェニコール0.008％、硫酸第一鉄0.0005％、硫酸マンガン0.00016％、硫酸亜鉛七水和物0.00014％、塩化コバルト(II)0.0002％、寒天1.8％、pH6.0）で分離培養（27℃、10日間）を行った。出現した集落を上記寒天平板培地に画線塗抹、純粋分離を行って、生理活性物質ＳＮＦ4435を生産する放線菌ストレプトミセス・スペクタビリスＳＮＦ4435株（FERM BP-5915）を得た。
(2)微生物の培養
ストレプトミセス・スペクタビリスＳＮＦ4435株（FERM BP-5915）の斜面培地（グルコース・スターチ・アスパラギン寒天培地）からマットごと１cm角を切り出し、70mlの前培養培地（硫酸アンモニウム0.14％、リン酸一カリウム0.2％、塩化カルシウム0.03％、硫酸マグネシウム0.03％、尿素0.03％、ポリペプトン0.5％、酵母エキス0.1％、大豆粉３％、グルコース１％、可溶性デンプン0.5％、硫酸第一鉄0.0005％、硫酸マンガン0.00016％、硫酸亜鉛七水和物0.00014％、塩化コバルト（II）0.0002％、pH無調整）を入れた500ml容の三角フラスコ10本に接種し、27℃で４日間回転振盪機上で培養して前培養液を得た。この前培養液700mlを本培養培地（可溶性デンプン１％、グルコース２％、大豆粉2.5％、乾燥酵母0.4％、肉エキス0.1％、リン酸二カリウム0.005％、塩化ナトリウム0.2％、pH7.2）120Lを含む200L容タンクに接種して、27℃で５日間通気攪拌培養（通気量120L/分、攪拌200回転／分、内圧0.1kg/cm²）を行った。
〔実施例２〕
(1)培養物の精製による本発明化合物の製造法
実施例１(2)で得られた培養物120Lを連続遠心分離（17,000rpm、20L/h）によって遠心分離し、得られた菌体を、アセトン36Lで抽出した。アセトン抽出液は、減圧濃縮してアセトンを除去した後、等量の酢酸エチルで二回抽出した。酢酸エチル層を無水硫酸ナトリウムで乾燥し、減圧濃縮して粗抽出物70gを得た。これを少量のヘキサン−酢酸エチル（７：３）の溶媒に溶解し、同じ溶媒で平衡化した２Lのシリカゲルカラムに付した。このカラムを6Lのヘキサン−酢酸エチル（７：３）の溶媒で溶出した後、ヘキサン−酢酸エチル（１：１）で本発明化合物を溶出し、減圧濃縮して黄色粉末2.2gを得た。さらに、この粉末を少量のメタノールに溶解し、80％メタノール水溶液で平衡化したＨＰＬＣカラム（カプセルパックＣ18−ＳＧ120、φ20×250mm、資生堂社）に流速10ml／minで通し、同じ溶媒で溶出した。220nmの吸収を検出、本発明化合物ＳＮＦ4435の検出ピークを分取した。吸収が最初に大きくなる画分（保持時間約27分）をＳＮＦ4435Ｃとし、次いで吸収が大きくなる画分（保持時間約29分）をＳＮＦ4435Ｄとした。これらの画分を減圧濃縮し、化合物ＳＮＦ4435Ｃを18mg、ＳＮＦ4435Ｄを10mg、それぞれ乳白色粉末として得た。それらの粉末がＳＮＦ4435ＣおよびＳＮＦ4435Ｄであることは、それぞれの比旋光度を測定することによって確認され、前記した物理化学的性状を示した。
(2)合成による本発明化合物の製造法
実施例１(2)で得られた培養物４Ｌを遠心分離（5000rpm）し、得られた菌体をアセトン２Ｌで抽出した。アセトン抽出液は、減圧濃縮してアセトンを除去した後、等量の酢酸エチルで２回抽出、酢酸エチル層を無水硫酸ナトリウムで乾燥し、減圧濃縮して粗抽出物2.5gを得た。これを少量のクロロホルムに溶解して、同じ溶媒で平衡化した100mlのシリカゲルカラムに付し、300mlのクロロホルムで溶出、減圧濃縮して乾固させ、50mlのメタノールを加えて一晩静置、スペクチナビリンを49.5mg析出、沈殿させた。これを５mlのクロロホルムに溶解、2.0mgのヨウ素を加え、室温中4.5時間攪拌した。反応液に水を加え、酢酸エチルで抽出後、10％チオ硫酸ナトリウム水溶液、飽和塩化ナトリウム水溶液で順次洗浄し、有機層を乾燥させた。溶媒を留去して得られた油状残渣をシリカゲルカラムクロマトグラフィーに付し、ヘキサン−酢酸エチル（１：１）の混合液で処理、本発明化合物を溶出、減圧濃縮して黄色粉末30.5mgを得た。さらに、この粉末を少量のメタノールに溶解し、80％メタノール水溶液で平衡化したＨＰＬＣカラム（カプセルパックＣ18−ＳＧ120、φ20Ｘ250mm、資生堂社）に流速10ml/minで通し、同じ溶媒で溶出し、220nmの吸収を検出、本発明化合物ＳＮＦ4435のピークを分取した。吸収が最初に大きくなる画分（保持時間約27分）をＳＮＦ4435Ｃとし、次いで吸収が大きくなる画分（保持時間約29分）をＳＮＦ4435Ｄとした。これらの画分を減圧濃縮し、化合物ＳＮＦ4435Ｃを19.8mg、ＳＮＦ4435Ｄを2.2mg、それぞれ乳白色粉末として得た。それらの粉末がＳＮＦ4435ＣおよびＳＮＦ4435Ｄであることは、それぞれの比旋光度を測定することによって確認され、前記した物理化学的性状を示した。
〔実施例３〕
マイトジェンによる脾細胞の増殖反応に対する阻害活性
雄性ＢＡＬＢ／ｃマウスの脾細胞を、10％牛胎児血清を含むＲＰＭＩ 1640培地中に懸濁し、本発明化合物ＳＮＦ4435Ｃ及びＳＮＦ4435Ｄを様々な濃度で添加し、マイトジェン（コンカナバリンＡ又はＬＰＳ）添加又は無添加にて、37℃、５％炭酸ガス濃度の条件下で48時間培養、細胞増殖を³Ｈ−チミジンの細胞内取り込み率を基準として判定した。結果を第３表（ＳＮＦ4435Ｃ）及び第４表（ＳＮＦ4435Ｄ）に示す。なお、第３表及び第４表中のＴ／Ｃは細胞増殖率（（Mean±SD）/Control）を表す。

〔実施例４〕
細胞毒性
(1)10％の牛胎児血清を加えたＲＰＭＩ 1640培地中に、Ｋ562（ヒト白血病）細胞を１×10⁵個/ml接種し、37℃、５％CO₂インキュベーター内で24時間培養した後、これに本発明化合物ＳＮＦ4435Ｃ及びＳＮＦ4435Ｄを各々１mg／mlになるように添加してさらに培養を続けた。４８時間後、培養したＫ562細胞を顕微鏡で観察したが、本発明化合物ＳＮＦ4435Ｃ及びＳＮＦ4435Ｄのいずれを添加しても生育阻害は認められなかった。
(2)10％の牛胎児血清を加えたＲＰＭＩ 1640培地中に、ＫＢ（ヒト鼻咽腔癌）細胞を１×10⁵個／ml接種し、37℃、５％CO₂インキュベーター内で24時間培養した後、これに本発明化合物ＳＮＦ4435Ｃ及びＳＮＦ4435Ｄを各々１mg／mlになるように添加してさらに培養を続けた。48時間後、培養したＫＢ細胞を顕微鏡で観察したが、本発明化合物ＳＮＦ4435Ｃ及びＳＮＦ4435Ｄのいずれを添加しても生育阻害は認められなかった。
〔実施例５〕
抗微生物活性
本発明化合物ＳＮＦ4435Ｃ及びＳＮＦ4435Ｄの各種微生物に対する抗菌活性を、ペーパーディスク法（直径８mm）による阻止円の大きさ（直径）で観察した。生理活性物質ＳＮＦ4435Ｃ及びＳＮＦ4435Ｄは各々メタノールに溶解した100μgをペーパーディスクに染み込ませ測定に供した。結果を第５表に示す。

〔実施例６〕
制癌剤耐性克服効果
５％の牛胎児血清を加えたＲＰＭＩ 1640培地（ギブコ社）に、2780ＡＤ細胞（Rogan A M et al., Science, vol.224, pp994-996（1984））を1X10⁶個／mlとなるように懸濁して37℃、５％CO₂インキュベーター内で24時間培養した。
³Ｈ−ビンクリスチン（アマシャム社）含有ＲＰＭＩ 1640に培地置換した後、本発明化合物ＳＮＦ4435ＣあるいはＳＮＦ4435Ｄを種々の濃度で添加、さらに２時間培養を続けた後、ビンクリスチンの細胞内取り込みをscintillation system USER No.10（ベックマン社）により測定した。結果を第６表に示す。

〔実施例７〕
急性毒性
本発明化合物ＳＮＦ4435Ｃ及びＳＮＦ4435Ｄを、それぞれ６週齢の雄性ＢＡＬＢ／ｃマウスに静脈内投与してその毒性を調べたところ、25mg／kgで異常は見られなかった。
産業上の利用可能性
以上の結果から、本発明により新規な生理活性物質、その生理活性物質を産生及び製造をするために用いる微生物ならびにその製造方法が提供される。詳しくは、免疫抑制活性、抗菌活性及び制癌剤耐性克服効果を有する新規生理活性物質、該生理活性物質を製造するために用いる微生物、及びその微生物を用いることにより該生理活性物質を採取する該生理活性物質の製造方法が提供される。本発明新規生理活性物質は、その効果より臓器移植等による免疫拒絶反応の抑制剤、全身性エリテマトーデス、慢性関節リウマチ、ブドウ膜炎等の自己免疫疾患の治療剤、アトピー性皮膚炎等のアレルギー疾患の治療剤、各種微生物による感染症の抗菌剤、或いは制癌剤耐性克服剤として有用である。
微生物への言及
寄託機関：通商産業省工業技術院生命工学工業技術研究所
住所：日本国茨城県つくば市東１丁目１番３号
寄託日：平成８年２月２７日
（平成８年２月２７日に原寄託され、平成９年４月１４日にブダペスト条約に基づく国際寄託へ移管）
受託番号：ＦＥＲＭ ＢＰ−５９１５Technical field
The present invention relates to a novel nitrophenylpyrone-based physiologically active substance, a microorganism used for producing and producing the same, and a method for producing the novel physiologically active substance. Furthermore, the present invention provides a pharmaceutical comprising such a novel physiologically active substance as an active ingredient, particularly an inhibitor of immune rejection by organ transplantation, autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, uveitis and the like. The present invention relates to a therapeutic agent, a therapeutic agent for allergic diseases such as atopic dermatitis, an antibacterial agent for infectious diseases caused by various microorganisms, or an anticancer drug resistance overcoming agent.
Background art
An immunosuppressive agent is effective for preventive treatment of diseases and pathological conditions that require a decrease in immune response. For example, prevention of transplant rejection such as kidney, liver, bone marrow, heart and corneal transplant, treatment of autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, uveitis, allergic diseases such as atopic dermatitis, etc. Indicated as a treatment for many immune diseases. Since the US Food and Drug Administration (USFDA) approved cyclosporine in 1983, immunosuppressants have been actively developed, and drugs such as prograf and spanidine have been put to practical use. High reputation in the area. However, existing drugs point out new problems such as the frequent occurrence of kidney damage due to use and the generation of autoreactive lymphocytes, and their efficacy against autoimmune diseases that develop by generating autoantibodies. Was not always satisfactory.
Disclosure of the invention
In view of the above-mentioned situation, the present inventors have extensively searched for a substance having excellent immunosuppressive activity and less side effects in nature, and as a result of earnest search, have the activity in a culture of a strain belonging to the genus Streptomyces. A novel nitrophenylpyrone-based physiologically active substance has been found. Accordingly, an object of the present invention is to provide a novel nitrophenylpyrone-based physiologically active substance, a microorganism used for producing and producing the novel physiologically active substance, and a method for producing the novel physiologically active substance.
Specifically, a novel physiologically active substance having immunosuppressive activity, antiallergic activity, antibacterial activity, and anticancer drug resistance overcoming effect and less side effects, a microorganism used for producing and producing the physiologically active substance, and culturing the microorganism It is an object of the present invention to provide a method for producing the physiologically active substance to obtain the physiologically active substance.
Furthermore, this invention makes it a subject to provide the pharmaceutical which prevents or treats various diseases using the above physiological activities.
That is, the present invention relates to a novel physiologically active substance represented by the following formula (I) and a pharmacologically acceptable salt thereof.

The present invention also relates to a microorganism that belongs to Streptomyces and can be used to produce and produce a novel physiologically active substance represented by the formula (I).
As such microorganisms, specifically, Streptomyces spectabilis isolated and collected from the soil of the main island of Okinawa by the present inventors.(Streptomycesspectabilis) SNF4435 strain (FERM BP-5915).
The present invention also relates to a method for producing a novel physiologically active substance comprising culturing the microorganism, producing the novel physiologically active substance in the culture, and collecting the same.
The present invention also relates to a method for producing a novel physiologically active substance by synthetic chemical means by culturing the microorganism, producing an analog spectinavirin in the culture, and ring-closing the tetraene moiety.
Furthermore, the present invention relates to a pharmaceutical comprising as an active ingredient a novel physiologically active substance represented by the above formula (I) or a pharmacologically acceptable salt thereof.
The medicament of the present invention is used as a therapeutic agent for immune rejection caused by organ transplantation or an autoimmune disease, a therapeutic agent for allergic diseases, an antibacterial agent for infectious diseases, or an anticancer drug resistance overcomer.
That is, the physiologically active substance of the present invention is a novel nitrophenylpyrone-based compound, and in view of its activity, such as an inhibitor of immune rejection due to organ transplantation, systemic lupus erythematosus, rheumatoid arthritis, uveitis, etc. It is useful as a pharmaceutical agent such as a therapeutic agent for autoimmune diseases, a therapeutic agent for allergic diseases such as atopic dermatitis, an antibacterial agent for infectious diseases caused by various microorganisms, or an anticancer drug resistance overcomer.
[Brief description of the drawings]
FIG. 1 shows the ultraviolet absorption spectrum of the present compound SNF4435C in methanol (15.4 μg / ml).
FIG. 2 shows an infrared absorption spectrum of the compound SNF4435C of the present invention using a potassium bromide tablet.
FIG. 3 shows 500 MHz of the present compound SNF4435C in deuterated chloroform solution.¹1 shows an HNMR spectrum (TMS standard).
FIG. 4 shows 125 MHz of the present compound SNF4435C in deuterated chloroform solution.¹³C NMR spectrum (based on deuterated chloroform) is shown.
FIG. 5 shows an ultraviolet absorption spectrum of the present compound SNF4435D in methanol (18.0 μg / ml).
FIG. 6 shows an infrared absorption spectrum of the compound SNF4435D of the present invention using a potassium bromide tablet.
FIG. 7 shows 500 MHz of the present compound SNF4435D in deuterated chloroform solution.¹1 shows an HNMR spectrum (TMS standard).
FIG. 8 shows 125 MHz of the present compound SNF4435D in deuterated chloroform solution.¹³C NMR spectrum (based on deuterated chloroform) is shown.
BEST MODE FOR CARRYING OUT THE INVENTION
The novel physiologically active substance of the present invention (hereinafter referred to as the compound of the present invention) can be obtained by culturing microorganisms. At this time, the microorganism to be used is not particularly limited as long as it is a bacterium that produces the compound of the present invention. Preferably, the microorganism belongs to the genus Streptomyces (Streptomyces sp.), particularly preferably Streptomyces spectabilis(Streptomycesspectabilis) SNF4435 strain. This Streptomyces specterabilis strain SNF4435 is a strain isolated by the present inventors from soil collected from the main island of Okinawa, and has been assigned to the Institute of Biotechnology, Institute of Industrial Science and Technology as the reference number FERM P-15476. Deposited on February 27, 2008, then transferred from the original deposit to a deposit based on the Budapest Treaty, with the deposit number FERM BP-5915.
Streptomyces Specterbilis(Streptomycesspectabilis) Isolation of SNF4435 strain is facilitated by a method commonly used for isolation of actinomycetes from the main island of Okinawa, such as Goodfellow, M .; Actinomycetologica Vol. 2, No. 1 13-29 (1988). Can be implemented.
The properties of Streptomyces spectabilis SNF4435 strain thus isolated are as follows.
1. Form
The aerial hyphae grow slightly, and a slight pseudo-rotational branching is seen, but no division of the hyphae is observed. The aerial hyphae have a reddish color, rarely a whited color, and a chain of 10 or more spores is formed to form a flexible linear shape. The surface of the spore is almost smooth and has a cylindrical shape with a size of about 0.4 × 0.8 μm. Special organs such as mycorrhiza, spore and zoospore are not found.
2. Growth conditions in various media
Table 1 shows the results of culturing at 27 ° C. for 14 days in various agar plates. In addition, about the description of a color, it followed the JIS color name book (JIS Z8102 conformity, Japanese Standards Association, April 15, 1993, 1st edition 3rd printing issuance).

(1) Growth temperature range
As a result of testing at 7 ° C, 12 ° C, 17 ° C, 22 ° C, 27 ° C, 32 ° C, 37 ° C, 42 ° C and 47 ° C using yeast / malt agar medium, 7 ° C, 42 ° C and 47 ° C It grew at any temperature except for. The optimum growth temperature seems to be around 27 ° C to 32 ° C.
(2) Gelatin liquefaction
In the glucose / peptone / gelatin medium, 27 ° C. culture or simple gelatin medium, and 20 ° C. culture, the liquefaction started in about 10 days after the culture and the liquefaction effect in the observation after 21 days was moderate.
(3) Starch hydrolysis
In starch / inorganic salt agar medium, cultured at 27 ° C, hydrolyzability was observed around 3 days after cultivation, and its action was relatively strong.
(4) Coagulation and peptization of skim milk
Although coagulation was not observed in the defatted milk medium and cultured at 37 ° C., peptone formation was observed around 7 days after the culture.
(5) Formation of melanin-like pigment
In the culture at 27 ° C. using tryptone yeast broth medium, peptone yeast iron agar medium or tyrosine agar medium, the production of melanin-like pigment was observed only after about 3 days of culture in the peptone yeast iron agar medium alone.
(6) Use of carbon sources
L-arabinose, sucrose, L-rhamnose using D-glucose, D-xylose, D-fructose, raffinose, inositol, D-mannitol, mannose, D-galactose in culture at 27 ° C. Is not used.
Summarizing the above properties, the mycological properties of SNF4435 strain are straight aerial hyphae and do not form spores, but a slight pseudo-annular branching is observed. About 10 spores are linked to the tip of the aerial hyphae, and the surface is smooth. In various media, the color of aerial hyphae varies from pink to red. Soluble pigments produce melanin-like pigments in peptone-yeast-iron agar medium, and starch has a moderate hydrolyzability and protein degradability.
The 2,6-diaminopimelic acid contained in the cell wall was LL-type. From these properties, SNF4435 strain is Streptomyces (Streptomyces) "Bergey's Manual of Determinative Bacteriology" 8th edition and ISP report "International Journal of Systematic Bacteriology (International Journal) of Systematic Bacteriology), Vol. 18, 69, 279 (1968), 19, 19, 391 (1969), 22, 265 (1972).(Streptomyces  spectabilis)Is a related species. Therefore, SNF4435 stock and Streptomyces specterbilis(Streptomyces  spectabilisIFO 13424). The results are shown in Table 2.

From these results, there are some differences in the color tone and growth temperature range of the aerial hyphae, both in the form of the aerial hyphae, the surface of the spores, the growth state, the point of producing melanin-like pigments, and the availability of carbon sources Agree well. Therefore, SNF4435 strain was changed to Streptomyces Spectabilis.(Streptomyces  spectabilis)Was identified.
The microorganism used in the present invention may be treated by irradiation with X-rays, ultraviolet rays or the like, or treatment with a mutagen such as nitrous acid, N-methyl-N′-nitro-N-nitrosoguanine (NTG), transformation, or transduction. Alternatively, it may be a microorganism mutated by a commonly used bacterial species conversion treatment method such as fusion.
The compound of the present invention is produced in a culture by inoculating and growing a nutrient source-containing medium that can be used by ordinary microorganisms, and used to produce the compound. The target compound of the present invention can be produced by inoculating and growing the microorganism and growing the culture as a raw material by organic synthesis. Any nutrient source may be used as long as it is used as a nutrient source for actinomycetes, and any of synthetic media, semi-synthetic media, natural media, and the like can be used. For example, as the carbon source, glucose, glycerol, maltose, starch, sucrose, molasses, starch syrup or dextrin can be used alone or as a mixture. Nitrogen sources include organic nitrogen sources such as soy flour, corn gluten meal, corn steep liquor, meat extract, yeast extract, cottonseed meal, peptone, wheat germ, fish meal, urea, and inorganic nitrogen sources such as ammonium sulfate and sodium nitrate. Used alone or as a mixture. As the inorganic salt, calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate or various phosphates can be used, and if necessary, heavy metal salts such as iron, copper, cobalt, molybdenum, manganese or zinc Can also be added in a trace amount. Moreover, when foaming is remarkable during culture, various known antifoaming agents may be added to the medium as appropriate. In addition, organic and inorganic substances that are utilized by the producing bacteria and useful for the production of the compound of the present invention can be appropriately used.
The culture method of the strain may be the same as the production method of general microbial metabolites, and may be solid culture or liquid culture. In the case of liquid culture, static culture, agitation culture, shaking culture, aeration culture, or the like may be carried out. However, when Streptomyces spectabilis SNF4435 strain is cultured, particularly, shaking culture or deep aeration is used. Agitation culture is preferred. Further, although it depends on the culture conditions, the pH of the preferred medium is in the range of 4-8, and the culture temperature is 22-37 ° C, preferably 25-30 ° C. The culture time is 48 to 168 hours, preferably 96 to 144 hours.
In order to isolate and produce the target novel physiologically active substance of the present invention from the culture, a separation means usually used for isolating a metabolite produced by a microorganism may be appropriately used. If necessary, synthetic chemical means may be used as appropriate.
Since the compound of the present invention produced by culturing is usually accumulated both inside and outside the cells in the culture, it is separated into the culture filtrate and cells by means of, for example, centrifugation, filtration, etc. Ordinary separation means from the filtrate and bacterial cells, for example, dialysis method, solvent extraction method, method using difference in solubility with impurities, ion exchange resin method or adsorption or distribution chromatography method and gel filtration method alone or in combination as appropriate In some cases, it can be separated and purified by repeated use.
The compound of the present invention collected using synthetic chemical means is obtained by culturing microorganisms and using the above separation means for the analog (nitrophenylpyrone) of the compound of the present invention produced in the culture solution. Separation and purification can be carried out by synthetic chemical means, for example, a ring-closing reaction using a catalyst. Examples of the organic solvent used in the reaction include methanol, ethanol, tertiary butyl alcohol, tetrahydrofuran, diethyl ether, ethylene glycol, dimethyl ether, dimethylformamide, dimethyl sulfoxide, benzene, toluene, xylene, dioxane, methylene chloride, chloroform, dichloroethane or Examples include acetonitrile. When the ring-closing reaction is performed using light, light having a wavelength of 200 nm to 800 nm is usually used, but a wavelength longer or shorter can be selected as necessary. The reaction temperature of the ring closure reaction is usually −20 to 400 ° C., and a temperature higher or lower can be selected as necessary. The pressure during the ring-closing reaction is usually 1 to 10 atm, and a pressure higher or lower can be selected as necessary. Examples of the catalyst used for the ring closure reaction include aluminum chloride, tin chloride, boron fluoride, copper fluoborate and iodine. The reaction time of the ring-closing reaction is usually in the range of 30 minutes to 2 days, but a time longer than this can be selected as necessary.
The reaction formula which closes the tetraene part of spectinavirin in this invention is shown.

After carrying out the ring-closing reaction under the above reaction conditions, SNF4435C and SNF4435D can be separated and purified by a known method in the field of synthetic organic chemistry, for example, a method using solvent extraction, recrystallization, chromatography, ion exchange resin, HPLC or the like. it can.
The properties of the obtained compound of the present invention are shown. SNF4435 has the same planar structure (see formula (I)) but has optical isomers, which are designated as SNF4435C or D, respectively. These optical isomers can be separated into the respective optical isomers by a conventional separation means such as liquid chromatography. In addition, when using as a pharmaceutical, a single optical isomer may be sufficient and a racemic body may be sufficient.
Physicochemical properties of SNF4435C
(1) Color and properties: milky white powder
(2) Molecular formula: C₂₈H₃₁NO₆
(3) Mass spectrum (FAB-MS): m / z 478 (M + H)⁺
(4) Specific rotation: [α]_D ²⁶−105.6 ° (C 0.10, CHCl_Three)
(5) Ultraviolet absorption spectrum: as shown in FIG.
λ_max ^MeOHnm (ε): 271 (19322)
(6) Infrared absorption spectrum: as shown in FIG.
ν_max ^KBrcm^-1: 2950, 2850, 1670, 1600, 1520, 1350, 1255, 1165, 1040
(7)¹H-nuclear magnetic resonance spectrum: shown in FIG. (500MHz, CDCl_Three)
δ (ppm): 8.19 (d, J = 8.7Hz, 2H), 7.55 (d, J = 8.7Hz, 2H), 5.58 (d, J = 1.4Hz, 1H), 4.95 (S, 1H), 4.78 ( t, J = 8.2Hz, 1H), 4.32 (d, J = 9.8Hz, 1H), 3.97 (d, J = 9.8Hz, 1H), 3.96 (S, 3H), 3.64 (S, 1H), 2.84 ( S, 1H), 2.43 (d, J = 8.2Hz, 2H), 1.89 (S, 3H), 1.84 (S, 3H), 1.74 (S, 3H), 1.72 (d, J = 1.4Hz, 3H), 1.30 (S, 3H)
(8)¹³C-nuclear magnetic resonance spectrum: as shown in FIG. (125MHz, CDCl_Three)
δ (ppm): 180.5 (s), 162.2 (s), 155.2 (s), 146.9 (s), 145.1 (s), 131.0 (s), 130.4 (s), 129.1 (d), 123.8 (d), 123.6 (d), 122.0 (d), 119.6 (s), 100.3 (s), 73.5 (d), 70.6 (t), 63.7 (d), 55.6 (q), 51.8 (s), 51.1 (d), 46.4 (t), 43.0 (s), 30.4 (q), 23.0 (q), 22.2 (q), 9.4 (q), 7.0 (q)
(9) Solubility: Soluble in methanol, chloroform, ethanol, acetone, pyridine, ethyl acetate, diethyl ether, dimethyl sulfoxide, etc. Insoluble in hexane and water
(10) Color reaction: positive for 50% sulfuric acid and iodine
(11) HPLC: Retention time 14.6 minutes
Column; Inertsil ODS-2 (φ4.6 × 250mm, GL Sciences)
Solvent: 80% methanol / water, flow rate 1 ml / min, UV 220 nm
(12) Distinguishing between acidic, neutral and basic substances: neutral
Physicochemical properties of SNF4435D
(1) Color and properties: milky white powder
(2) Molecular formula: C₂₈H₃₁NO₆
(3) Mass spectrum (FAB-MS): m / z 478 (M + H)⁺
(4) Specific rotation: [α]_D ²⁶+ 84.8 ° (C 0.10, CHCl_Three)
(5) Ultraviolet absorption spectrum: as shown in FIG.
λ_max ^MeOHnm (ε): 270 (21462)
(6) Infrared absorption spectrum: as shown in FIG.
ν_max ^KBrcm^-1: 2950, 2850, 1670, 1600, 1520, 1350, 1255, 1165, 1040
(7)¹H-nuclear magnetic resonance spectrum: shown in FIG. (500MHz, CDCl_Three)
δ (ppm): 8.15 (d, J = 8.7Hz, 2H), 7.47 (d, J = 8.7Hz, 2H), 5.70 (S, 1H), 4.94 (dd, J = 7.0, 9.5Hz, 1H), 4.89 (S, 1H), 4.18 (d, J = 9.2Hz, 1H), 3.84 (d, J = 9.2Hz, 1H), 3.74 (S, 1H), 3.54 (S, 3H), 2.74 (S, 1H ), 2.48 (dd, J = 13.1, 9.5Hz, 1H), 2.28 (dd, J = 13.1, 7.0Hz, 1H), 1.97 (S, 3H), 1.84 (S, 3H), 1.80 (S, 3H) , 1.74 (d, J = 1.5Hz, 3H), 1.31 (S, 3H)
(8)¹³C-nuclear magnetic resonance spectrum: shown in FIG. (125MHz, CDCl_Three)
δ (ppm): 180.5 (s), 161.9 (s), 154.9 (s), 146.9 (s), 144.0 (s), 131.3 (s), 131.2 (s), 129.9 (d), 124.4 (d), 123.2 (d), 121.9 (d), 119.7 (s), 100.0 (s), 72.5 (d), 70.6 (t), 61.1 (d), 55.2 (d), 54.8 (q), 51.1 (s), 45.5 (t), 43.0 (s), 30.7 (q), 22.2 (q), 22.1 (q), 9.4 (q), 6.8 (q)
(9) Solubility: Soluble in methanol, chloroform, ethanol, acetone, pyridine, ethyl acetate, diethyl ether, dimethyl sulfoxide, etc. Insoluble in hexane and water
(10) Color reaction: positive for 50% sulfuric acid and iodine
(11) HPLC: Retention time 16.6 minutes
Column; Inertsil ODS-2 (φ4.6 × 250mm, GL Sciences)
Solvent: 80% methanol / water, flow rate 1 ml / min, UV 220 nm
(12) Distinguishing between acidic, neutral and basic substances: neutral
When the novel physiologically active substance of the present invention is used as a medicine, it may be a pharmacologically acceptable salt. Pharmacologically acceptable salts include alkali metal salts such as sodium and potassium, or alkaline earth metal salts such as magnesium and calcium, aluminum and other metal salts, and organic amine salts such as alkylamine salts and pyridine salts. Can be mentioned. This compound or a pharmacologically acceptable salt thereof is safely administered orally and parenterally as a pharmaceutical to humans and animals. Examples of parenteral administration include intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, transdermal administration, pulmonary administration, nasal administration, enteral administration, buccal administration, transmucosal administration, etc. These formulations are administered. For example, injections, suppositories, aerosols, transdermal tapes and the like can be mentioned. Oral preparations such as tablets (including sugar-coated tablets, coated tablets, buccal tablets), powders, capsules (including soft capsules), granules (coated products, pills, troches, liquids, or their pharmacology Sustained-release preparations and the like that are acceptable to the eye. Liquid preparations for oral administration include suspensions, emulsions, syrups (including dry syrups), elixirs and the like.
These preparations are administered as pharmaceutical compositions together with carriers, excipients, disintegrants, lubricants, colorants and the like that are pharmacologically acceptable as preparations in accordance with known pharmaceutical manufacturing methods. Carriers and excipients used in these formulations include, for example, lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, gentian powder, For example, starch, tragacanth gum, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose and the like, such as starch, agar, gelatin powder, sodium carboxymethyl cellulose, carboxymethyl cellulose calcium, Examples of lubricants are crystalline cellulose, calcium carbonate, sodium bicarbonate, sodium alginate, etc. Magnesium stearate, talc, hydrogenated vegetable oil, macrogol, those which are allowed to added to pharmaceuticals as coloring agents, can be used respectively. Tablets and granules are sucrose, gelatin, hydroxypropylcellulose, purified shellac, gelatin, glycerin, sorbitol, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, methyl methacrylate as required Further, it may be coated with a methacrylic acid polymer or the like, or may be coated with two or more layers. Furthermore, capsules of substances such as ethyl cellulose and gelatin may be used. Moreover, when preparing an injection, a pH adjuster, a buffer, a stabilizer, a solubilizing agent, etc. are added to the main drug as necessary, and each injection is prepared by a conventional method.
When the novel physiologically active substance of the present invention is administered to a patient, it varies depending on conditions such as the degree of symptoms, the patient's age, health condition, weight, etc., and is not particularly limited, but about 10 mg to 10 g per day for an adult is orally or parenterally. May be administered once or more daily.
Little acute toxicity was seen as shown in Example 6.
The present invention will be described in more detail with reference to the following examples, which are merely illustrative and the present invention is not limited thereto.
[Example 1]
(1) Microbial separation
1 g of dry soil obtained by heat-treating the soil collected on the main island of Okinawa (60 ° C., 10 minutes) was suspended in 10 ml of sterilized water. 10^-3Diluted twice, agar plate medium (ammonium sulfate 0.05%, monopotassium phosphate 0.05%, calcium chloride 0.01%, magnesium sulfate 0.01%, urea 0.01%, glucose 0.05%, soluble starch 0.1%, chloramphenicol 0.008%, Separation culture (27 ° C, 10 days) was performed with ferrous sulfate 0.0005%, manganese sulfate 0.00016%, zinc sulfate heptahydrate 0.00014%, cobalt (II) chloride 0.0002%, agar 1.8%, pH 6.0) . The emerged colonies were streaked and purely separated on the agar plate medium to obtain the actinomycetes Streptomyces spectabilis SNF4435 strain (FERM BP-5915) that produces the physiologically active substance SNF4435.
(2) Microbial culture
A 1 cm square was cut out from the slope medium (glucose, starch, asparagine agar medium) of Streptomyces spectaviris strain SNF4435 (FERM BP-5915), and 70 ml of preculture medium (0.14% ammonium sulfate, 0.2% monopotassium phosphate, chloride) 0.03% calcium, 0.03% magnesium sulfate, 0.03% urea, 0.5% polypeptone, 0.1% yeast extract, 3% soy flour, 1% glucose, 0.5% soluble starch, 0.0005% ferrous sulfate, 0.00016% manganese sulfate, zinc sulfate Inoculate 10 500ml Erlenmeyer flasks containing 0.00014% heptahydrate, 0.0002% cobalt (II) chloride, pH unadjusted) and incubate on a rotary shaker at 27 ° C for 4 days. Obtained. 700 ml of this preculture solution is used as the main culture medium (soluble starch 1%, glucose 2%, soybean flour 2.5%, dry yeast 0.4%, meat extract 0.1%, dipotassium phosphate 0.005%, sodium chloride 0.2%, pH 7.2) Inoculate a 200L tank containing 120L and aeration and agitation culture at 27 ° C for 5 days (aeration rate of 120L / min, agitation of 200rpm / min, internal pressure of 0.1kg / cm²)
[Example 2]
(1)Process for producing the compound of the present invention by purification of the culture
120 L of the culture obtained in Example 1 (2) was centrifuged by continuous centrifugation (17,000 rpm, 20 L / h), and the resulting cells were extracted with 36 L of acetone. The acetone extract was concentrated under reduced pressure to remove acetone, and then extracted twice with an equal amount of ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 70 g of a crude extract. This was dissolved in a small amount of hexane-ethyl acetate (7: 3) solvent and applied to a 2 L silica gel column equilibrated with the same solvent. The column was eluted with 6 L of hexane-ethyl acetate (7: 3), then the compound of the present invention was eluted with hexane-ethyl acetate (1: 1) and concentrated under reduced pressure to obtain 2.2 g of a yellow powder. Further, this powder was dissolved in a small amount of methanol, passed through an HPLC column (Capsule Pack C18-SG120, φ20 × 250 mm, Shiseido Co., Ltd.) equilibrated with 80% aqueous methanol solution, and eluted with the same solvent. Absorption at 220 nm was detected, and the detection peak of the present compound SNF4435 was fractionated. The fraction with the largest absorption (retention time of about 27 minutes) was designated as SNF4435C, and the fraction with the largest absorption (with a retention time of about 29 minutes) was designated as SNF4435D. These fractions were concentrated under reduced pressure to obtain 18 mg of compound SNF4435C and 10 mg of SNF4435D as milky white powders. These powders were confirmed to be SNF4435C and SNF4435D by measuring their specific rotations and exhibited the physicochemical properties described above.
(2)Method for producing the compound of the present invention by synthesis
4 L of the culture obtained in Example 1 (2) was centrifuged (5000 rpm), and the resulting cells were extracted with 2 L of acetone. The acetone extract was concentrated under reduced pressure to remove acetone, then extracted twice with an equal amount of ethyl acetate, the ethyl acetate layer was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 2.5 g of a crude extract. This is dissolved in a small amount of chloroform, applied to a 100 ml silica gel column equilibrated with the same solvent, eluted with 300 ml of chloroform, concentrated under reduced pressure to dryness, added with 50 ml of methanol and allowed to stand overnight, spectinavirin. Was precipitated and precipitated. This was dissolved in 5 ml of chloroform, 2.0 mg of iodine was added, and the mixture was stirred at room temperature for 4.5 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate, washed successively with 10% aqueous sodium thiosulfate and saturated aqueous sodium chloride, and the organic layer was dried. The oily residue obtained by evaporating the solvent was subjected to silica gel column chromatography, treated with a mixture of hexane-ethyl acetate (1: 1), the compound of the present invention was eluted, and concentrated under reduced pressure to obtain 30.5 mg of a yellow powder. Obtained. Further, this powder was dissolved in a small amount of methanol and passed through an HPLC column (Capsule Pack C18-SG120, φ20X250mm, Shiseido Co., Ltd.) equilibrated with an 80% aqueous methanol solution at a flow rate of 10 ml / min. Absorption was detected, and the peak of the compound SNF4435 of the present invention was collected. The fraction with the largest absorption (retention time of about 27 minutes) was designated as SNF4435C, and the fraction with the largest absorption (with a retention time of about 29 minutes) was designated as SNF4435D. These fractions were concentrated under reduced pressure to obtain 19.8 mg of compound SNF4435C and 2.2 mg of SNF4435D as milky white powders, respectively. These powders were confirmed to be SNF4435C and SNF4435D by measuring their specific rotations and exhibited the physicochemical properties described above.
Example 3
Inhibitory activity on proliferative response of splenocytes by mitogen
Spleen cells of male BALB / c mice are suspended in RPMI 1640 medium containing 10% fetal bovine serum, the compounds of the present invention SNF4435C and SNF4435D are added at various concentrations, and mitogen (concanavalin A or LPS) is added or not added. Incubate for 48 hours under conditions of 37 ° C and 5% carbon dioxide gas concentration.^ThreeDetermination was based on the intracellular uptake rate of H-thymidine. The results are shown in Table 3 (SNF4435C) and Table 4 (SNF4435D). In addition, T / C in Table 3 and Table 4 represents a cell proliferation rate ((Mean ± SD) / Control).

Example 4
Cytotoxicity
(1) 1 × 10 K562 (human leukemia) cells in RPMI 1640 medium supplemented with 10% fetal bovine serum^FiveIndividual / ml inoculation, 37 ° C, 5% CO₂After culturing in an incubator for 24 hours, the compounds of the present invention SNF4435C and SNF4435D were added to 1 mg / ml, and the culture was further continued. After 48 hours, the cultured K562 cells were observed with a microscope, but no growth inhibition was observed when any of the compounds SNF4435C and SNF4435D of the present invention was added.
(2) 1 x 10 KB (human nasopharyngeal carcinoma) cells in RPMI 1640 medium supplemented with 10% fetal bovine serum^FiveIndividual / ml inoculation, 37 ° C, 5% CO₂After culturing in an incubator for 24 hours, the compounds of the present invention SNF4435C and SNF4435D were added to 1 mg / ml, and the culture was further continued. After 48 hours, the cultured KB cells were observed with a microscope, and no growth inhibition was observed when any of the compounds SNF4435C and SNF4435D of the present invention was added.
Example 5
Antimicrobial activity
The antibacterial activity of the compounds SNF4435C and SNF4435D of the present invention against various microorganisms was observed by the size (diameter) of the inhibition circle by the paper disk method (diameter 8 mm). The physiologically active substances SNF4435C and SNF4435D were each soaked in 100 μg dissolved in methanol and used for measurement. The results are shown in Table 5.

Example 6
Overcoming anticancer drug resistance
In RPMI 1640 medium (Gibco) supplemented with 5% fetal bovine serum, 2780AD cells (Rogan A M et al., Science, vol. 224, pp994-996 (1984))⁶Suspended at 37 ° C, 5% CO₂The cells were cultured for 24 hours in an incubator.
^ThreeAfter replacing the medium with RPMI 1640 containing H-vincristine (Amersham), the compound SNF4435C or SNF4435D of the present invention was added at various concentrations, and further cultured for 2 hours, and then the intracellular uptake of vincristine was determined by scintillation system USER No. 10 (Beckman). The results are shown in Table 6.

Example 7
Acute toxicity
The compounds of the present invention, SNF4435C and SNF4435D, were intravenously administered to 6-week-old male BALB / c mice, respectively, and their toxicity was examined. No abnormality was observed at 25 mg / kg.
Industrial applicability
From the above results, the present invention provides a novel physiologically active substance, a microorganism used for producing and producing the physiologically active substance, and a method for producing the microorganism. Specifically, a novel physiologically active substance having an immunosuppressive activity, an antibacterial activity and an anticancer drug resistance overcoming effect, a microorganism used for producing the physiologically active substance, and the physiological activity of collecting the physiologically active substance by using the microorganism A method of manufacturing a material is provided. The novel physiologically active substance of the present invention is a suppressor of immune rejection due to organ transplantation due to its effect, a therapeutic agent for autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, uveitis, and allergic diseases such as atopic dermatitis It is useful as an antibacterial agent, an antibacterial agent for infectious diseases caused by various microorganisms, or an anticancer drug resistance overcomer.
Reference to microorganisms
Depositary: Institute of Biotechnology, Institute of Industrial Technology, Ministry of International Trade and Industry
Address: 1-3 1-3 Higashi, Tsukuba City, Ibaraki Prefecture, Japan
Deposit date: February 27, 1996
(The original deposit was made on February 27, 1996 and transferred to the international deposit based on the Budapest Treaty on April 14, 1997)
Accession Number: FERM BP-5915

Claims (13)

A novel physiologically active substance represented by the following formula (I), SNF4435 and a pharmacologically acceptable salt thereof.

The novel physiologically active substance, SNF4435C, and a pharmacologically acceptable salt thereof according to claim 1, wherein the specific light intensity by sodium D ray at 26 ° C is -105.6 ° (C 0.10, CHCl ₃ ). The novel physiologically active substance, SNF4435D and a pharmacologically acceptable salt thereof according to claim 1, wherein the specific light intensity by sodium D ray at 26 ° C is + 84.8 ° (C 0.10, CHCl ₃ ). A microorganism belonging to the genus Streptomyces and capable of producing the novel physiologically active substance according to any one of claims 1 to 3. The microorganism according to claim 4, wherein the microorganism belonging to the genus Streptomyces is Streptomyces spectabilis SNF4435 strain (FERM BP-5915) or a mutant thereof. The microorganism according to claim 4 or 5 belonging to the genus Streptomyces is cultured, the novel physiologically active substance according to any one of claims 1 to 3 is produced in the culture, and the microorganism is collected. A method for producing a novel physiologically active substance according to any one of claims 1 to 3. The method for producing a novel physiologically active substance according to any one of claims 1 to 3, wherein the tetraene moiety of spectinavirin is closed. The pharmaceutical which uses the novel bioactive substance or its pharmacologically acceptable salt as described in any one of Claims 1-3 as an active ingredient. The medicament according to claim 8, which is an immunosuppressant. The medicament according to claim 8, which is a therapeutic agent for allergic diseases. The medicament according to claim 8, which is a therapeutic agent for infectious diseases. The medicament according to claim 8, which is a therapeutic agent for autoimmune diseases. The medicament according to claim 8, which is an anticancer drug resistance overcomer.

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