JP2004210648A - Novel macrolide compound and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel antitumoral agent, antibacterial agent, or antimycotic agent. <P>SOLUTION: The antitumoral agent, antibacterial agent, or antimycotic agent contains as an active principle a compound represented by formula (1) or a salt thereof. In formula (1), R is a hydrogen atom or a group represented by formula (2). <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

［式中、Ｒは水素原子または式（２）を表す。］
【０００９】
【化４】

【００１０】
［２］前記１記載の式（１）で表される化合物又はその塩を有効成分とする抗腫瘍剤、抗菌剤又は抗真菌剤、
［３］ストレプトミセス属に属し、式（１）で表される化合物生産菌を培養して、その培養物から式（１）で表される化合物を採取することを特徴とする式（１）で表される化合物の製造方法、
［４］ストレプトミセス属に属し式（１）で表される化合物の生産菌がストレプトミセス・エスピー（Streptomyces sp.）ＳＰＡ−６９５２株又はそれらの誘導株である請求項３記載の製造方法、
［５］ストレプトミセス・エスピー（Streptomyces sp.）ＳＰＡ−６９５２株
に関する。
【００１１】
前記式（１）で表される化合物、又はその薬学上許容される塩は、ストレプトミセス属に属する放線菌ＳＰＡ−６９５２株の培養により製造することができる。該ＳＰＡ−６９５２株は大阪府内で採取した土壌から本発明者らによって単離されたものである。ＳＰＡ−６９５２株は次のような菌学的性質を有する。
【００１２】
（ａ）形態的性質
ＩＳＰ培地Ｎｏ．２〜５にて２７℃、２週間培養したときの形態的性質は、次のようなものである。基生菌糸はよく伸長し単純分岐をなし、気菌糸を形成する。気菌糸の先端には、２０個以上の胞子が連鎖した胞子鎖が形成される。胞子鎖の形状は曲状又はゆるい曲状である。胞子は、表面はしわ状で、大きさは直径０．５〜０．７μｍ、長さ０．８〜１．０μｍほどである。気菌糸及び基生菌糸の分断、及び胞子のう、鞭毛胞子、菌核等の特殊な構造は認められない。
（ｂ）培養的性質
ＩＳＰ培地Ｎｏ．２〜５にて２７℃、２週間培養したときのＳＰＡ−６９５２株の培養的性質を以下に示す。
【００１３】

【００１４】
（ｃ）生理学的性質
１．メラニン様色素の生成
ペプトン・イースト・鉄寒天培地（ＩＳＰ培地Ｎｏ．６） 陽性
チロシン寒天培地（ＩＳＰ培地Ｎｏ．７） 陽性
２．炭素源の利用性
Ｌ−アラビノース ＋＋
Ｄ−フルクトース ＋
Ｄ−グルコース ＋
イノシトール ＋＋
Ｄ−マンニトール −
ラフィノース ＋
Ｌ−ラムノース ＋
シュクロース ＋
Ｄ−キシロース ＋
【００１５】
３．化学分類学的性質
全菌体加水分解物中にＬＬ−ジアミノピメリン酸が検出された。
以上の菌学的性質より、ＳＰＡ−６９５２株はストレプトミセス属に属する放線菌であると同定し、ストレプトミセス・エスピー（Streptomyces sp.）ＳＰＡ−６９５２と命名し、独立行政法人産業技術総合研究所特許生物寄託センターに寄託した（受託番号ＦＥＲＭ Ｐ−１９１６４）。
上記放線菌の培養に使用される培地は液状でも固体でもよいが、通常は液体培地による振盪培養又は通気攪拌培養が有利である。使用する培地は、特に限定されるものではないが、該物質生産菌が資化しうる炭素源及び窒素源を含む栄養培地が用いられる。炭素源としては、例えばグルコース、ラクトース、グリセリン、デンプン、デキストリン、糖蜜等が挙げられる。窒素源としては、例えばポリペプトン、カザミノ酸等の蛋白質加水分解物、肉エキス、酵母エキス、大豆粕、コーンスティープリカー、アミノ酸類等の有機窒素源やアンモニウム塩や硝酸塩等の無機窒素源が挙げられる。その他、浸透圧調整、ｐＨ調整、微量成分の補給等のために、各種リン酸塩、硫酸マグネシウム、塩化ナトリウム、炭酸カルシウム等の無機塩類を培地に添加することも可能である。さらに菌の生育を促進する目的で、各種ビタミン類、核酸関連化合物等を培地に添加してもよい。なお、培養期間中に、シリコン、ポリプロピレングリコール誘導体、大豆油等の消泡剤を培地に添加することも可能である。
【００１６】
培養温度としては、好ましくは２０〜３７℃の範囲、更に好ましくは２５〜３０℃の範囲の温度が挙げられる。培養期間としては例えば、４〜８日間の範囲が挙げられる。培地のｐＨとして例えば、中性付近の範囲が挙げられる。
培養物から本発明の化合物を単離するには、微生物の生産する代謝物についての、通常使用される単離手段が使用できる。培養液上清中からの単離法としては、培養濾液からの通常の単離法、例えば溶媒抽出法、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及びゲル濾過クロマトグラフィー等が挙げられる。これらの単離法は単独又は組み合わせて行うことができる。また高速液体クロマトグラフィー（ＨＰＬＣ）や薄層クロマトグラフィーなどにより単離精製もできる。培養菌体から目的物を単離する場合は、濾過もしくは遠心分離等の手段で集めた菌体を、アセトンやメタノール等の親水性有機溶媒を用いて直接、菌体から抽出できる。抽出物からは、培養液上清からの単離精製法と同様の方法で、目的物を得ることができる。
【００１７】
本発明の化合物においては、それらの塩、好ましくは医薬的又は獣医薬的に許容される塩も本発明の範疇に含まれる。ここで、塩としては、例えば無機酸（例えば、塩酸、臭化水素、ヨウ化水素、硫酸、リン酸等）との塩、或いは有機酸（例えば、酢酸、プロピオン酸、乳酸、コハク酸、マレイン酸、酒石酸、クエン酸、グルコン酸、アスコルビン酸、安息香酸、メタンスルホン酸、ｐ−トルエンスルホン酸、ケイ皮酸、フマル酸、リンゴ酸、シュウ酸等）との塩などを挙げることができる。
本発明の化合物又はその塩には水やエタノール等の医薬品として許容される溶媒との溶媒和物も含まれる。
また、本発明化合物又はその塩は不斉炭素原子を含み立体異性体が存在するが、本発明の範疇にそれら異性体や、それらの混合物も含まれる。
【００１８】
本発明医薬は、経口的又は非経口的に投与することができる。すなわち通常用いられる投与形態、例えば粉末、顆粒、錠剤、カプセル剤、シロップ剤、懸濁液等の剤型で経口的に投与することができ、あるいは、例えば、その溶液、乳剤、懸濁液等の剤型にしたものを注射の型で非経口投与することができる。坐剤の型で直腸投与することもできる。前記の適当な投与剤型は、例えば、許容される通常の担体、賦型剤、結合剤、安定剤、希釈剤等に有効成分を配合することにより製造することができる。注射剤型で用いる場合には、例えば、薬学的に許容される緩衝剤、溶解補助剤、等張剤等を添加することもできる。
投与量及び投与回数は、投与法と患者の年齢、体重、病状等によって異なるが、経口投与の場合は、通常、成人の一日当たり投与量は０．１〜１０００ｍｇ、好ましくは１〜５００ｍｇの範囲で選択すればよい。
【００１９】
【実施例】以下実施例により本発明をさらに詳細に説明するが、本発明の技術的範囲はこれら実施例に限定されるものではない。
実施例１
ＳＰＡ−６９５２株の培養と活性物質の単離精製
Ｄ−グルコース１．５％、可溶性デンプン１．５％、綿実粉１．２％、酵母エキス０．０５％、塩化カリウム０．１％、硫酸マグネシウム７水和物０．００７％、炭酸カルシウム０．２％を含み、ｐＨ７．２に調整した培地７５ｍｌを５００ｍｌ容坂口フラスコに分注してオートクレーブで滅菌した。これに、斜面培養したストレプトミセス・エスピーＳＰＡ−６９５２株を１白金耳接種し、２７℃で４日間１３０ｒｐｍにて振盪培養して前培養とした。２リットル容坂口フラスコ２４本に同組成の培地を３００ｍｌずつ分注し、オートクレーブ滅菌後、上記前培養液を２％接種し、２７℃で７日間１１０ｒｐｍにて振盪培養した。培養終了後、培養液を２０℃、９０００ｒｐｍで１０分間遠心分離して、菌体画分と上清液画分とに分離した。
【００２０】
菌体画分に３リットルのアセトンを加え、室温にて１時間攪拌抽出後、抽出液を濾過により回収した。上清液画分は、等量の１−ブタノールを加え、室温にて１時間攪拌抽出後、静置分液により抽出液を回収した。両抽出液を減圧濃縮して褐色油状物８．５ｇを得た。これを５０ｍｌのメタノールに溶解し、ＴＯＹＯＰＥＡＲＬ（登録商標）ＨＷ−４０Ｆ（東ソー製）を用いるカラムクロマトグラフィー（φ３×５０ｃｍ）に付し、メタノールで溶出した。活性画分を減圧濃縮し、１．３ｇの褐色粉末の画分Ｉと４．２ｇの褐色粉末の画ＩＩとを得た。画分Ｉを２ｍｌのメタノールに溶解し、分取ＨＰＬＣに注入した。分取ＨＰＬＣの条件は、カラム：Ｗａｋｏｐａｋ（登録商標）Ｗａｋｏｓｉｌ−ＩＩ５Ｃ１８ＨＧ−Ｐｒｅｐ（φ３×１０ｃｍとφ３×２５ｃｍを連結、和光純薬工業製）、溶出液Ａ：１％ギ酸、同Ｂ：メタノール、グラジエント：Ｂ液濃度７０％から１００％へ４０分の直線グラジエント、流速：２０ｍｌ／分、検出：２２５ｎｍにおける紫外吸収、とした。保持時間２４分のピークを回収し、減圧濃縮することにより無色固体粗物質１６ｍｇを得た。
【００２１】
これを０．５ｍｌのメタノールに溶解し、再度分取ＨＰＬＣに注入した。分取ＨＰＬＣの条件は、カラム：Ｗａｋｏｐａｋ（登録商標）Ｗａｋｏｓｉｌ−ＩＩ５Ｃ１８ＲＳ（φ２×５ｃｍとφ２×２５ｃｍを連結、和光純薬工業製）、溶出液Ａ：１％ギ酸、同Ｂ：アセトニトリル、グラジエント：Ｂ液濃度５０％から５５％へ６０分の直線グラジエント、流速：７ｍｌ／分、検出：２２５ｎｍにおける紫外吸収、とした。保持時間１８分のピークを回収し、減圧濃縮乾固することにより無色固体のＳＰＡ−６９５２−１を５．９ｍｇ取得した。一方、画分ＩＩを４ｍｌのメタノールに溶解し、分取ＨＰＬＣに注入した。分取ＨＰＬＣの条件は、カラム：Ｗａｋｏｐａｋ（登録商標）Ｗａｋｏｓｉｌ−ＩＩ５Ｃ１８ＨＧ−Ｐｒｅｐ（φ５×１０ｃｍとφ５×２５ｃｍを連結、和光純薬工業製）、溶出液Ａ：１％ギ酸、同Ｂ：メタノール、グラジエント：Ｂ液濃度７０％から１００％へ４５分の直線グラジエント、流速：３０ｍｌ／分、検出：２２５ｎｍにおける紫外吸収、とした。保持時間４５分のピークを回収し、減圧濃縮することにより無色固体粗物質１８ｍｇを得た。これを０．５ｍｌのメタノールに溶解し、再度分取ＨＰＬＣに注入した。分取ＨＰＬＣの条件は、カラム：Ｗａｋｏｐａｋ（登録商標）Ｗａｋｏｓｉｌ−ＩＩ５Ｃ１８ＲＳ（φ２×５ｃｍとφ２×２５ｃｍを連結、和光純薬工業製）、溶出液Ａ：１％ギ酸、同Ｂ：アセトニトリル、グラジエント：Ｂ液濃度６０％から１００％へ７２分の直線グラジエント、流速：７ｍｌ／分、検出：２２５ｎｍにおける紫外吸収、とした。保持時間３５分のピークを回収し、減圧濃縮乾固することにより無色固体のＳＰＡ−６９５２−２を４．０ｍｇ取得した。
取得した各化合物の物理化学的性状は以下のようであった。
【００２２】
（ＳＰＡ−６９５２−１）
外観：無色固体
整数分子量：８９３
分子式：Ｃ_４９Ｈ_８３ＮＯ_１３
高速電子衝撃質量スペクトル(FAB-MS、小数点以下を四捨五入した整数値)
m/z（positive）：895(M+H)⁺
m/z（negative）：893(M-H)^-
高分解能高速電子衝撃質量スペクトル(HRFAB-MS)m/z(M+H)⁺：
実測値：894.5966
計算値：894.5943（Ｃ_４９Ｈ_８４ＮＯ_１３）
紫外可視吸収スペクトルλｍａｘ（メタノール中）ｎｍ（ε）：257(11300)
【００２３】
^１Ｈ―ＮＭＲ（ＣＤ_３ＯＤ）δｐｐｍ：
0.85(3H,d,6.7), 0.97(3H,d,7.0), 0.99(3H,t,7.3), 1.10(3H,s), 1.20〜2.15(27H,m), 1.33(3H,s), 1.35(3H,d,7.0), 1.35(3H,s), 1.38(3H,s), 2.20(1H,m), 2.27(1H,m), 2.49(1H,dd,14.4,2.2), 2.55(1H,d,14.4), 2.70(1H,dd,13.7,10.6),2.74(6H,s), 3.16(1H,m), 3.19(1H,brs), 3.75(1H,m), 3.80(1H,d,4.0), 3.80(1H,t,5.4), 3.88(1H,d,8.0), 4.03(1H,dt,10.4,2.4), 4.10(1H,ddd,11.3,4.3,1.8), 4.57(1H,m), 5.09(1H,dd,7.0,4.0), 5.17(1H,m), 5.24(1H,m), 6.02(1H,d,15.4), 6.14(1H,d,15.5), 6.98(1H,d,15.4)
^１３Ｃ―ＮＭＲ（ＣＤ_３ＯＤ）δｐｐｍ：
5.2, 7.9, 10.6, 15.3, 18.9, 20.1, 22.8, 24.2, 27.1, 27.9, 28.8, 28.9, 29.4, 29.9, 30.3, 31.9, 32.2, 33.4, 34.4, 35.7, 36.8, 37.7, 40.7, 42.7(2C), 44.3, 44.6, 64.3, 67.7, 68.1, 68.4, 70.8, 72.7, 75.5, 75.7, 76.7, 77.2, 79.6, 80.3, 84.9, 99.1, 99.1, 111.6, 120.3, 125, 126.8, 149.1, 153.4, 167.3
溶解性：水、ヘキサンに不溶、メタノール、ＤＭＳＯに可溶
【００２４】
これらからＳＰＡ−６９５２−１の構造式を、次式（１−ａ）と決定した。
【化５】

【００２５】
（ＳＰＡ−６９５２−２）
外観：無色固体
整数分子量：７５２
分子式：Ｃ_４１Ｈ_６８Ｏ_１２
高速電子衝撃質量スペクトル(FAB-MS、小数点以下を四捨五入した整数値)
m/z（positive）：753(M+H)⁺
m/z（negative）：751(M-H)^-
高分解能高速電子衝撃質量スペクトル(HRFAB-MS)m/z(M)⁺：
実測値：752.4724
計算値：752.4711（Ｃ_４１Ｈ_６８Ｏ_１２）
紫外可視吸収スペクトルλｍａｘ（メタノール中）ｎｍ（ε）：
206(11200)、257(12900)
【００２６】
^１Ｈ―ＮＭＲ（ＣＤ_３ＯＤ）δｐｐｍ：
0.85(3H,d,7.0), 0.93(3H,d,7.0), 0.99(3H,t,7.3), 1.18(3H,s), 1.20〜1.70(18H,m), 1.32(3H,s), 1.35(3H,s), 1.37(3H,s), 1.71(2H,m), 1.86(1H,m), 2.02(2H,m), 2.20(1H,m), 2.26(1H,m), 2.48(1H,dd,14.0,2.2), 2.54(1H,d,14.0), 2.69(1H,dd,13.7,10.6), 3.20(1H,brs), 3.72(1H,d,7.3), 3.76(1H,d,4.0), 3.77(1H,d,4.0), 3.77(1H,m), 3.85(1H,brt,10.3), 4.09(1H,ddd,9.4,3.0,1.8), 5.20(1H,m), 5.24(1H,m), 6.02(1H,d,15.8), 6.16(1H,d,15.5), 6.99(1H,d,15.8)^１３Ｃ―ＮＭＲ（ＣＤ_３ＯＤ）δｐｐｍ：
5.3, 8.0, 10.7, 20.1, 22.7, 23.7, 26.8, 28.8, 28.9, 29.3, 29.9, 30.8, 31.9, 32.3, 33.2, 34.3, 35.6, 36.7, 37.6, 39.9, 44.3, 44.6, 68.0, 68.3, 70.8, 71.8, 72.4, 76.1, 76.47, 76.52, 78.6, 80.1, 84.9, 99.2, 111.8, 120.5, 124.6, 127.1, 149.0, 153.1, 167.1
これらからＳＰＡ−６９５２−２の構造式を、次式（１−ｂ）と決定した。
【００２７】
【化６】

【００２８】
実施例２
ｉｎ ｖｉｔｒｏ抗腫瘍活性の測定
本発明化合物のｉｎ ｖｉｔｒｏ抗腫瘍活性をヒト前骨髄性白血病細胞株ＨＬ−６０を被験細胞として測定した。すなわち、ＨＬ−６０細胞を２×１０^５cell/mlの濃度で９６穴プレートに９０μｌずつ分注した。培地は牛胎児血清を１０％添加したＲＰＭＩ−１６４０培地を用いた。あらかじめ培地で希釈した本発明化合物１０μｌを、培養開始と同時に添加した。５％炭酸ガス培養器内で３７℃にて３日間培養した後、生細胞数を測定し、試料濃度と細胞増殖阻害率から、ＩＣ50値（５０％阻害のための濃度）を求めた。結果は以下の通りとなり、本発明化合物は強い抗腫瘍活性を示した。
被験化合物 ＩＣ50（μｇ／ｍｌ）
ＳＰＡ−６９５２−１ ０．５
ＳＰＡ−６９５２−２ ０．４
【００２９】
実施例３
抗菌・抗真菌活性の測定
本発明化合物の抗菌・抗真菌活性をBacillus subtilis ATCC 6633、Candida utilis NBRC 10707、Aspergillus niger JCM 10254を被験菌として測定した。すなわち、オートクレーブ後５０℃に冷却した寒天培地に本発明化合物を種々の濃度になるよう混合し、平板に固めた。これに上記被験菌を接種し、３０℃で１〜２日間インキュベートして生育の有無を観察した。培地は、細菌用には栄養寒天培地（ペプトン１％、肉エキス１％、ＮａＣｌ ０．５％、寒天１．５％、ｐＨ７．２）、真菌用にはサブロー寒天培地（Ｄ−グルコース４％、ポリペプトン１％、ｐＨ５．６）を用いた。生育阻止が認められた最小濃度をＭＩＣとして示した。結果は以下の通りとなり、本発明化合物は抗菌活性及び糸状菌に対する抗真菌活性を示した。

【００３０】
【発明の効果】
前記式（１）で表される新規マクロライド化合物又はその塩は、抗腫瘍活性及び抗菌、抗真菌活性を示し、抗腫瘍剤、抗菌剤又は抗真菌剤として有用である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel compound having antitumor activity, antibacterial activity, and antifungal activity, a microbiological production method of the compound, a medicament containing the compound as an active ingredient, and the like.
[0002]
[Prior art]
Conventionally, various antitumor agents, antibacterial agents, and antifungal agents have been developed and used for the treatment of cancer and infectious diseases.However, few are satisfactory in terms of efficacy and side effects, and new drugs There is a demand for development.
On the other hand, a 24-membered macrolide compound having a spiro ring side chain has been known as a group of physiologically active compounds found from microorganisms. As such 24-membered ring macrolide compounds, dunaimycins, ossamycins, NK154183s and the like have all been isolated from actinomycetes and are known (Non-Patent Documents 1, 2, and 3).
[0003]
[Non-patent document 1]
JE Hochlowski et al., Journal of Antibiotics vol. 44: 1318-1330, 1991
[0004]
[Non-patent document 2]
HAKirst et al., Journal of Antibiotics vol. 49: 162-167, 1996.
[0005]
[Non-Patent Document 3]
K. Tsuchiya et al., Journal of Antibiotics vol. 49: 1281-1283, 1996.
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a novel compound having antitumor, antibacterial, and antifungal activities, a microbiological production method of the compound, a medicament containing the compound as an active ingredient, and the like.
[0007]
[Means for Solving the Problems]
Based on these backgrounds, the present inventors have searched for a novel compound exhibiting antitumor, antibacterial or antifungal activity. As a result, the actinomycete SPA-6952 strain isolated by the present inventors has a novel 24-membered ring. It has been found that a macrolide compound is produced, and that the compound exhibits antitumor activity, antibacterial activity, and antifungal activity, thereby completing the present invention.
That is, the present invention
[0008]
[1] a compound represented by the following formula (1):
Embedded image

[In the formula, R represents a hydrogen atom or a formula (2). ]
[0009]
Embedded image

[0010]
[2] an antitumor agent, an antibacterial agent or an antifungal agent comprising as an active ingredient the compound represented by the formula (1) or a salt thereof according to the above 1;
[3] The formula (1), which belongs to the genus Streptomyces and comprises culturing a compound-producing bacterium represented by the formula (1), and collecting a compound represented by the formula (1) from the culture. A method for producing a compound represented by
[4] The production method according to claim 3, wherein the bacterium that belongs to the genus Streptomyces and produces the compound represented by the formula (1) is Streptomyces sp. SPA-6952 or a derivative thereof.
[5] The present invention relates to Streptomyces sp. SPA-6952 strain.
[0011]
The compound represented by the formula (1) or a pharmaceutically acceptable salt thereof can be produced by culturing an actinomycete SPA-6952 strain belonging to the genus Streptomyces. The SPA-6952 strain was isolated by the present inventors from soil collected in Osaka Prefecture. The SPA-6952 strain has the following mycological properties.
[0012]
(A) Morphological properties ISP medium No. The morphological properties when cultured at 27 ° C. for 2 weeks at 2 to 5 are as follows. The underlying mycelium elongates well and forms a simple branch to form an aerial mycelium. At the tip of the aerial hyphae, a spore chain in which 20 or more spores are linked is formed. The shape of the spore chain is curved or loosely curved. The spores are wrinkled on the surface, 0.5 to 0.7 μm in diameter and 0.8 to 1.0 μm in length. No division of the aerial hyphae and the base hyphae, and no special structures such as spores, flagella spores, sclerotia, etc. are observed.
(B) Cultural properties ISP medium No. The culture characteristics of the SPA-6952 strain when cultured at 27 ° C. for 2 weeks at 2 to 5 are shown below.
[0013]

[0014]
(C) Physiological properties Generation of melanin-like pigment Peptone yeast iron agar medium (ISP medium No. 6) Positive tyrosine agar medium (ISP medium No. 7) Positive 2. Utilization of carbon source L-arabinose ++
D-fructose +
D-glucose +
Inositol ++
D-mannitol-
Raffinose +
L-rhamnose +
Sucrose +
D-xylose +
[0015]
3. LL-Diaminopimelic acid was detected in the whole cell hydrolyzate.
From the above bacteriological properties, the SPA-6952 strain was identified as an actinomycete belonging to the genus Streptomyces, named Streptomyces sp. SPA-6952, and incorporated into the National Institute of Advanced Industrial Science and Technology. Deposited at the Patent Organism Depositary (Accession No. FERM P-19164).
The medium used for culturing the actinomycetes may be liquid or solid, but usually, shaking culture or aeration stirring culture using a liquid medium is advantageous. The medium used is not particularly limited, but a nutrient medium containing a carbon source and a nitrogen source that can be used by the substance-producing bacterium is used. Examples of the carbon source include glucose, lactose, glycerin, starch, dextrin, molasses and the like. Examples of the nitrogen source include protein hydrolysates such as polypeptone and casamino acids, meat extracts, yeast extracts, soybean meal, corn steep liquor, organic nitrogen sources such as amino acids, and inorganic nitrogen sources such as ammonium salts and nitrates. . In addition, inorganic salts such as various phosphates, magnesium sulfate, sodium chloride, and calcium carbonate can be added to the medium for osmotic pressure adjustment, pH adjustment, replenishment of trace components, and the like. Furthermore, various vitamins, nucleic acid-related compounds, and the like may be added to the medium for the purpose of promoting the growth of bacteria. During the culture period, an antifoaming agent such as silicon, a polypropylene glycol derivative, and soybean oil can be added to the medium.
[0016]
The culture temperature is preferably in the range of 20 to 37 ° C, more preferably in the range of 25 to 30 ° C. The culturing period includes, for example, a range of 4 to 8 days. Examples of the pH of the medium include a range around neutrality.
In order to isolate the compound of the present invention from the culture, commonly used isolation means for metabolites produced by microorganisms can be used. The isolation method from the culture supernatant may be a usual isolation method from the culture filtrate, for example, a solvent extraction method, an ion exchange resin method or adsorption or partition chromatography, and gel filtration chromatography. These isolation methods can be performed alone or in combination. It can also be isolated and purified by high performance liquid chromatography (HPLC) or thin layer chromatography. When the target substance is isolated from the cultured cells, the cells collected by means such as filtration or centrifugation can be directly extracted from the cells using a hydrophilic organic solvent such as acetone or methanol. From the extract, the desired product can be obtained in the same manner as in the isolation and purification method from the culture supernatant.
[0017]
In the compounds of the present invention, their salts, preferably, pharmaceutically or veterinarily acceptable salts are also included in the scope of the present invention. Here, as the salt, for example, a salt with an inorganic acid (eg, hydrochloric acid, hydrogen bromide, hydrogen iodide, sulfuric acid, phosphoric acid, etc.) or an organic acid (eg, acetic acid, propionic acid, lactic acid, succinic acid, maleic acid) Acid, tartaric acid, citric acid, gluconic acid, ascorbic acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid, cinnamic acid, fumaric acid, malic acid, oxalic acid, etc.).
The compound of the present invention or a salt thereof includes a solvate with a pharmaceutically acceptable solvent such as water or ethanol.
Further, the compound of the present invention or a salt thereof contains an asymmetric carbon atom and exists in stereoisomers, and the scope of the present invention includes those isomers and a mixture thereof.
[0018]
The medicament of the present invention can be administered orally or parenterally. That is, it can be orally administered in a commonly used dosage form such as a powder, granule, tablet, capsule, syrup, suspension or the like, or, for example, its solution, emulsion, suspension and the like. Can be administered parenterally in the form of injections. It can also be administered rectally in the form of suppositories. The above-mentioned suitable dosage form can be produced, for example, by mixing the active ingredient with an acceptable usual carrier, excipient, binder, stabilizer, diluent and the like. When used in the injection form, for example, pharmaceutically acceptable buffers, solubilizing agents, isotonic agents and the like can also be added.
The dose and the number of times of administration vary depending on the administration method and the age, weight, and medical condition of the patient. In the case of oral administration, the daily adult dose is usually 0.1 to 1000 mg, preferably 1 to 500 mg. You can select it with.
[0019]
The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to these examples.
Example 1
Culture of SPA-6952 strain and isolation and purification of active substance D-glucose 1.5%, soluble starch 1.5%, cottonseed flour 1.2%, yeast extract 0.05%, potassium chloride 0.1%, 75 ml of a medium containing 0.007% of magnesium sulfate heptahydrate and 0.2% of calcium carbonate and adjusted to pH 7.2 was dispensed into a 500 ml Sakaguchi flask and sterilized in an autoclave. A loopful of Streptomyces sp. SPA-6952 strain, which had been cultivated on a slope, was inoculated into the loop and precultured by shaking at 130 rpm at 27 ° C. for 4 days. 300 ml of the medium having the same composition was dispensed into 24 2-liter Sakaguchi flasks, sterilized in an autoclave, inoculated with 2% of the above precultured solution, and cultured at 27 ° C. for 7 days at 110 rpm with shaking. After completion of the culture, the culture solution was centrifuged at 9000 rpm at 20 ° C. for 10 minutes to separate a bacterial cell fraction and a supernatant liquid fraction.
[0020]
3 L of acetone was added to the cell fraction, and the mixture was extracted with stirring at room temperature for 1 hour, and the extract was collected by filtration. The supernatant fraction was added with an equal amount of 1-butanol, extracted with stirring at room temperature for 1 hour, and the extract was recovered by standing liquid separation. Both extracts were concentrated under reduced pressure to obtain 8.5 g of a brown oil. This was dissolved in 50 ml of methanol and subjected to column chromatography (φ3 × 50 cm) using TOYOPEARL (registered trademark) HW-40F (manufactured by Tosoh), and eluted with methanol. The active fraction was concentrated under reduced pressure to obtain 1.3 g of a brown powder fraction I and 4.2 g of a brown powder fraction II. Fraction I was dissolved in 2 ml of methanol and injected on preparative HPLC. The conditions for preparative HPLC were as follows: column: Wakopak (registered trademark) Wakosil-II5C18HG-Prep (connecting φ3 × 10 cm and φ3 × 25 cm, manufactured by Wako Pure Chemical Industries), eluent A: 1% formic acid, eluent B: methanol, Gradient: linear gradient from solution B concentration 70% to 100% for 40 minutes, flow rate: 20 ml / min, detection: ultraviolet absorption at 225 nm. The peak having a retention time of 24 minutes was collected and concentrated under reduced pressure to obtain 16 mg of a colorless solid crude substance.
[0021]
This was dissolved in 0.5 ml of methanol and injected again into preparative HPLC. The conditions for preparative HPLC were as follows: column: Wakopak (registered trademark) Wakosil-II5C18RS (connecting φ2 × 5 cm and φ2 × 25 cm, manufactured by Wako Pure Chemical Industries), eluent A: 1% formic acid, eluent B: acetonitrile, gradient: The linear gradient was 60 minutes from solution B concentration of 50% to 55%, flow rate: 7 ml / min, detection: ultraviolet absorption at 225 nm. The peak with a retention time of 18 minutes was collected, and concentrated under reduced pressure to dryness to obtain 5.9 mg of a colorless solid, SPA-695-1. Separately, fraction II was dissolved in 4 ml of methanol and injected into preparative HPLC. The conditions for preparative HPLC were as follows: column: Wakopak (registered trademark) Wakosil-II5C18HG-Prep (connecting φ5 × 10 cm and φ5 × 25 cm, manufactured by Wako Pure Chemical Industries), eluent A: 1% formic acid, eluent B: methanol, Gradient: linear gradient of solution B concentration from 70% to 100% for 45 minutes, flow rate: 30 ml / min, detection: ultraviolet absorption at 225 nm. The peak having a retention time of 45 minutes was collected and concentrated under reduced pressure to obtain 18 mg of a colorless solid crude substance. This was dissolved in 0.5 ml of methanol and injected again into preparative HPLC. The conditions for preparative HPLC were as follows: column: Wakopak (registered trademark) Wakosil-II5C18RS (connecting φ2 × 5 cm and φ2 × 25 cm, manufactured by Wako Pure Chemical Industries), eluent A: 1% formic acid, eluent B: acetonitrile, gradient: The linear gradient was 72 minutes from solution B concentration of 60% to 100%, flow rate: 7 ml / min, detection: ultraviolet absorption at 225 nm. A peak having a retention time of 35 minutes was collected and concentrated under reduced pressure to dryness to obtain 4.0 mg of a colorless solid, SPA-692-2.
The physicochemical properties of each compound obtained were as follows.
[0022]
(SPA-695-1)
Appearance: colorless solid integer molecular weight: 893
Molecular formula: C ₄₉ H ₈₃ NO ₁₃
Fast electron impact mass spectrum (FAB-MS, integer value rounded to the nearest decimal place)
m / z (positive): 895 (M + H) ⁺
m / z (negative): 893 (MH) ^-
High resolution fast electron impact mass spectrum (HRFAB-MS) m / z (M + H) ⁺ :
Obtained value: 894.5966
_{Calculated: 894.5943 (C 49 H 84 NO} 13)
UV-visible absorption spectrum λmax (in methanol) nm (ε): 257 (11300)
[0023]
¹ H-NMR (CD ₃ OD) δ ppm:
0.85 (3H, d, 6.7), 0.97 (3H, d, 7.0), 0.99 (3H, t, 7.3), 1.10 (3H, s), 1.20-2.15 (27H, m), 1.33 (3H, s), 1.35 (3H, d, 7.0), 1.35 (3H, s), 1.38 (3H, s), 2.20 (1H, m), 2.27 (1H, m), 2.49 (1H, dd, 14.4,2.2), 2.55 ( 1H, d, 14.4), 2.70 (1H, dd, 13.7,10.6), 2.74 (6H, s), 3.16 (1H, m), 3.19 (1H, brs), 3.75 (1H, m), 3.80 (1H, d, 4.0), 3.80 (1H, t, 5.4), 3.88 (1H, d, 8.0), 4.03 (1H, dt, 10.4,2.4), 4.10 (1H, ddd, 11.3,4.3,1.8), 4.57 (1H , m), 5.09 (1H, dd, 7.0,4.0), 5.17 (1H, m), 5.24 (1H, m), 6.02 (1H, d, 15.4), 6.14 (1H, d, 15.5), 6.98 (1H , d, 15.4)
¹³ C-NMR (CD ₃ OD) δ ppm:
5.2, 7.9, 10.6, 15.3, 18.9, 20.1, 22.8, 24.2, 27.1, 27.9, 28.8, 28.9, 29.4, 29.9, 30.3, 31.9, 32.2, 33.4, 34.4, 35.7, 36.8, 37.7, 40.7, 42.7 (2C) , 44.3, 44.6, 64.3, 67.7, 68.1, 68.4, 70.8, 72.7, 75.5, 75.7, 76.7, 77.2, 79.6, 80.3, 84.9, 99.1, 99.1, 111.6, 120.3, 125, 126.8, 149.1, 153.4, 167.3
Solubility: insoluble in water and hexane, soluble in methanol and DMSO
From these, the structural formula of SPA-6992-1 was determined as the following formula (1-a).
Embedded image

[0025]
(SPA-695-2-2)
Appearance: colorless solid integer molecular weight: 752
Molecular formula: C ₄₁ H ₆₈ O ₁₂
Fast electron impact mass spectrum (FAB-MS, integer value rounded to the nearest decimal place)
m / z (positive): 753 (M + H) ⁺
m / z (negative): 751 (MH) ^-
High-resolution fast electron impact mass spectrum (HRFAB-MS) m / z (M) ⁺ :
Obtained value: 752.4724
_{Calculated: 752.4711 (C 41 H 68 O} 12)
UV-visible absorption spectrum λmax (in methanol) nm (ε):
206 (11200), 257 (12900)
[0026]
¹ H-NMR (CD ₃ OD) δ ppm:
0.85 (3H, d, 7.0), 0.93 (3H, d, 7.0), 0.99 (3H, t, 7.3), 1.18 (3H, s), 1.20-1.70 (18H, m), 1.32 (3H, s), 1.35 (3H, s), 1.37 (3H, s), 1.71 (2H, m), 1.86 (1H, m), 2.02 (2H, m), 2.20 (1H, m), 2.26 (1H, m), 2.48 (1H, dd, 14.0,2.2), 2.54 (1H, d, 14.0), 2.69 (1H, dd, 13.7,10.6), 3.20 (1H, brs), 3.72 (1H, d, 7.3), 3.76 (1H, d, 4.0), 3.77 (1H, d, 4.0), 3.77 (1H, m), 3.85 (1H, brt, 10.3), 4.09 (1H, ddd, 9.4,3.0,1.8), 5.20 (1H, m), 5.24 (1H, m), 6.02 (1H, d, 15.8), 6.16 (1H, d, 15.5), 6.99 (1H, d, 15.8) ¹³ C-NMR (CD ₃ OD) δ ppm:
5.3, 8.0, 10.7, 20.1, 22.7, 23.7, 26.8, 28.8, 28.9, 29.3, 29.9, 30.8, 31.9, 32.3, 33.2, 34.3, 35.6, 36.7, 37.6, 39.9, 44.3, 44.6, 68.0, 68.3, 70.8, 71.8, 72.4, 76.1, 76.47, 76.52, 78.6, 80.1, 84.9, 99.2, 111.8, 120.5, 124.6, 127.1, 149.0, 153.1, 167.1
From these, the structural formula of SPA-6992-2 was determined as the following formula (1-b).
[0027]
Embedded image

[0028]
Example 2
Measurement of in vitro antitumor activity The in vitro antitumor activity of the compound of the present invention was measured using human promyelocytic leukemia cell line HL-60 as a test cell. That is, HL-60 cells were dispensed at a concentration of 2 × 10 ⁵ cells / ml into a 96-well plate at 90 μl each. The medium used was RPMI-1640 medium supplemented with 10% fetal calf serum. 10 μl of the compound of the present invention, which had been diluted with a medium, was added simultaneously with the start of the culture. After culturing at 37 ° C. for 3 days in a 5% carbon dioxide incubator, the number of living cells was measured, and the IC 50 value (concentration for 50% inhibition) was determined from the sample concentration and the cell growth inhibition rate. The results were as follows, and the compound of the present invention showed strong antitumor activity.
Test compound IC50 (μg / ml)
SPA-6992-1 0.5
SPA-6992-2 0.4
[0029]
Example 3
Measurement of antibacterial and antifungal activities The antibacterial and antifungal activities of the compounds of the present invention were measured using Bacillus subtilis ATCC 6633, Candida utilis NBRC 10707 and Aspergillus niger JCM 10254 as test bacteria. That is, the compound of the present invention was mixed at various concentrations with an agar medium cooled to 50 ° C. after autoclaving and solidified on a flat plate. This was inoculated with the test bacteria and incubated at 30 ° C. for 1 to 2 days to observe the growth. The culture medium is a nutrient agar medium (peptone 1%, meat extract 1%, NaCl 0.5%, agar 1.5%, pH 7.2) for bacteria, and a Sabouraud agar medium (D-glucose 4%) for fungi. , Polypeptone 1%, pH 5.6). The minimum concentration at which growth was inhibited was indicated as MIC. The results are as follows, and the compound of the present invention showed antibacterial activity and antifungal activity against filamentous fungi.

[0030]
【The invention's effect】
The novel macrolide compound represented by the formula (1) or a salt thereof exhibits antitumor activity and antibacterial and antifungal activities, and is useful as an antitumor agent, antibacterial agent or antifungal agent.

Claims (5)

A compound represented by the following formula (1).

[In the formula, R represents a hydrogen atom or a formula (2). ]

An antitumor, antibacterial or antifungal agent comprising the compound represented by the formula (1) according to claim 1 or a salt thereof as an active ingredient. The compound represented by the formula (1), which belongs to the genus Streptomyces and is characterized by culturing a compound-producing bacterium represented by the formula (1) and collecting a compound represented by the formula (1) from the culture. A method for producing a compound. 4. The production method according to claim 3, wherein the strain producing Streptomyces sp. And the compound producing the compound represented by the formula (1) is Streptomyces sp. Strain SPA-6952 or a derivative thereof. Streptomyces sp. SPA-6952 strain.