JP3875024B2 - Novel physiologically active substance that inhibits human immunodeficiency virus (HIV) growth - Google Patents
Novel physiologically active substance that inhibits human immunodeficiency virus (HIV) growth Download PDFInfo
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- JP3875024B2 JP3875024B2 JP2000557384A JP2000557384A JP3875024B2 JP 3875024 B2 JP3875024 B2 JP 3875024B2 JP 2000557384 A JP2000557384 A JP 2000557384A JP 2000557384 A JP2000557384 A JP 2000557384A JP 3875024 B2 JP3875024 B2 JP 3875024B2
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- hiv
- cells
- tat
- salt
- hydrate
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- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
- C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
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- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
技術分野
本発明はＨＩＶ（ヒト免疫不全ウイルス）Ｔａｔ分子の活性を阻害し、ＨＩＶ増殖を抑制するＨＩＶ感染症治療に有効な新規生理活性物質に関する。
従来技術
ＡＩＤＳはレトロウイルスＨＩＶ−１およびＨＩＶ−２により引き起こされる免疫系の病気であり、ほとんど患者の致死をもたらす。ＡＩＤＳの予防および症状の軽減のために、ウイルスの増殖を抑制する組成物の探索に興味がもたれている。
ＨＩＶは細胞に感染後、遺伝子ＲＮＡがウイルス粒子中に含まれる逆転写酵素によりＤＮＡに逆転写されＨＩＶインテグラーゼにより宿主細胞のＤＮＡに組み込まれる。組み込まれたＨＩＶ遺伝子の５’端にあるＬＴＲ中のプロモーターからＨＩＶ ＲＮＡが転写され遺伝子ＲＮＡとなると同時に、そのＲＮＡを鋳型としてウイルス蛋白質が合成される。合成されたウイルス蛋白質のいくつかはＨＩＶプロテアーゼにより切断されて成熟蛋白となり次世代のウイルス粒子を形成する。以上のウイルス生活環より、逆転写酵素阻害・インテグラーゼ阻害・ＨＩＶ ＬＴＲからの転写阻害・プロテアーゼ阻害などがＨＩＶ感染症治療薬の作用点として挙げられる。
これまでにＨＩＶ感染症治療薬としてヌクレオシド系および非ヌクレオシド系逆転写酵素阻害剤、プロテアーゼ阻害剤が実用化され治療に効果を上げている。しかし、既にこれらの抗ＨＩＶ剤に対する耐性株の出現が報告されており（Ｇｕｎｔｈａｒｄ Ｈ．ｅｔ ａｌ．，Ｊ．ｏｆ Ｖｉｒｏｌ．７２（３）：２４２２（１９９８））、新たな抗ＨＩＶ剤の開発が望まれている。特に、作用点の異なる抗ＨＩＶ剤は耐性化変異が交叉しないことから、既存の薬剤に耐性を獲得してしまったＨＩＶ感染の治療を可能にし、また既存の薬剤と併用することで耐性の出現を遅らせることが期待される。
ＨＩＶ Ｔａｔ分子はＨＩＶ ＲＮＡ５’端のＴＡＲ領域に結合して、ＨＩＶ ＬＴＲからの転写を促進する活性（Ｔａｔ活性）を持つことが知られている（Ｌａｓｐｉａ Ｍ．Ｆ．ｅｔ ａｌ．，Ｃｅｌｌ ５９：２８３（１９８９））。Ｔａｔを欠失したＨＩＶが増殖できない（Ｐｏｐｉｋ Ｗ．ｅｔ ａｌ．，Ｊ．ｏｆ Ｖｉｒｏｌ．６７（２）：１０９４（１９９３））ことから、Ｔａｔ活性を阻害する薬剤（Ｔａｔ阻害剤）はＨＩＶ増殖を抑制し、新規メカニズムのＨＩＶ感染症治療薬となることが期待される。
これまでＲｏ ５−３３３５およびラットでの腎毒性を軽減したＲｏ ２４−７４２９がＴａｔ活性を阻害しＨＩＶ増殖を抑制する化合物として報告されているが、Ｒｏ ５−３３３５は標準的な抗ＨＩＶ活性の評価系であるＭＴ−４急性感染系で効果を示さない（Ｗｉｔｖｒｏｕｗ Ｍ．ｅｔ ａｌ．，Ａｎｔｉｍｉｃｒｏｂ．Ａｇｅｎｔｓ ａｎｄ Ｃｈｅｍｏｔｈｅｒ．３６：２６２８（１９９２））など抗ＨＩＶ活性が充分でなく、また、臨床導入したＲｏ ２４−７４２９は効果が得られなかった（Ｈａｕｂｒｉｃｈ Ｒ．Ｈ．ｅｔ ａｌ．，Ｊ．ｏｆ Ｉｎｆｅｃｔ．Ｄｉｓｅａｓｅ １７２：１２４６（１９９５））。
また、ＴａｔのＴＡＲ領域と結合するドメインのアナログであるＡＬＸ４０−４Ｃも、細胞系でＴａｔ活性を阻害しているとの報告はなく、また報告されている抗ＨＩＶ活性も吸着阻害に基づくことを示唆している（Ｏ’Ｂｒｉｅｎ Ｗ．ｅｔ ａｌ，．Ｊ．ｏｆ Ｖｉｒｏｌ．７０（５）：２８２５（１９９６））。
発明の開示
本発明はＴａｔ活性を阻害し、ＨＩＶ増殖を抑制する新規物質を単離し、ＨＩＶ感染症の治療剤を提供することにある。
上記現状に鑑み、本発明者らは微生物培養液を原料として、Ｔａｔ阻害剤の探索スクリーニングを行った。その結果、ストレプトミセス属に属する微生物の培養液中にＴａｔ阻害剤が産生されることを見出し、この活性物質を単離・構造決定したところ新規活性物質であることが判明した。そしてこの活性物質は、Ｒｏ ５−３３３５が活性を示さないＭＴ−４急性感染系を含む各種の急性ＨＩＶ感染系においてＨＩＶ増殖を抑制することが示された。また、ＲＮＡへの転写過程を抑制するという作用機序から予想されるとおり、持続感染系においてもＨＩＶ増殖を抑制し、逆転写酵素阻害剤が無効なＨＩＶ遺伝子が宿主細胞のＤＮＡに組み込まれた後でも効果を発揮できる治療薬と成りうることが示された。
すなわち本発明は、下記一般式（Ｉ）：
で表わされる化合物、その塩又はその水和物、及びその製造方法、並びにそれを有効成分とする医薬に関する。
より具体的には、一般式（Ｉ）において、Ｒ１が下記一般式（ＩＩ）：
で表される基である化合物、その塩又はその水和物、
一般式（Ｉ）において、Ｒ１が下記式（ＩＩＩ）：
ストレプトミセス・エスピー・エムイーアール・２４８７（Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．Ｍｅｒ−２４８７，ＦＥＲＭ Ｐ−１６７１８）菌を栄養培地中で培養し、その培養液から一般式（Ｉ）で表される化合物、その塩又はその水和物を採取する事を特徴とする、一般式（Ｉ）で表される化合物、その塩又はその水和物の製造方法を提供する。
一般式（Ｉ）で表される化合物、その塩又はその水和物を有効成分とするヒト免疫不全ウイルス（ＨＩＶ）の増殖阻害剤に関する。
式（Ｉ）で表される化合物、その塩又はその水和物を有効量と薬理上許容される担体とを含む医薬組成物、式（Ｉ）で表される化合物、その塩又はその水和物をヒト免疫不全ウイルス（ＨＩＶ）の増殖阻害剤の製造に用いること、式（Ｉ）で表される化合物、その塩又はその水和物の有効量を人に投与してヒト免疫不全ウイルス（ＨＩＶ）の増殖を阻害する方法を提供する。よって、ヒト免疫不全ウイルス（ＨＩＶ）の増殖による病気を予防・治療する。
本明細書においては、一般式（Ｉ）で表される化合物のＲ１が一般式（ＩＩ）で表される基であり、一般式（ＩＩ）中のＲ２がメチル基である化合物をＥＭ ２４８７Ａ、Ｒ２がエチル基である化合物をＥＭ ２４８７Ｂ、一般式（Ｉ）で表される化合物のＲ１が式（ＩＩＩ）で表される基である化合物をＥＭ ２４８７Ｃと称する。
発明の詳細な説明
産生株の菌学的性状
本発明化合物の代表的な産生菌株として、長野県志賀高原林地の土壌より分離された放線菌で、本発明者らが、エムイーアール・２４８７（Ｍｅｒ−２４８７）菌株と番号を付した菌株が挙げられる。このＭｅｒ−２４８７菌株の菌学的性状は次の通りである。
（１）形態
基生菌糸より直鎖状（Ｒｅｃｔｉｆｌｅｘｉｂｉｌｅｓ）の気中菌糸を伸長する。成熟した気中菌糸の先に１０〜５０個の円筒形の胞子からなる胞子鎖を形成する。胞子のうは認められない。胞子の大きさは０．６〜０．８μｍ×１．０〜１．２μｍ程度であり、胞子の表面は平滑状（ｓｍｏｏｔｈ）を示し、鞭毛は認められない。
（２）各種培地における生育状態
培養は全て２８℃で行った。色調の記載はコンティナー・コーポレーション・オブ・アメリカのカラー・ハーモニー・マニュアル（Ｃｏｎｔａｉｎｅｒ Ｃｏｒｐｏｒａｔｉｏｎ ｏｆ ＡｍｅｒｉｃａのＣｏｌｏｒ Ｈａｒｍｏｎｙ Ｍａｎｕａｌ）の（）内に示す符号で表示する。
１）イースト・麦芽寒天培地
生育は中程度で、その表面に気中菌糸を着生し、赤色系（５ｃａ〜６ｅｃ）の胞子が見られる。培養裏面は深緑色〜焦げ茶である。茶色の溶解性色素を僅かに産生する。
２）オートミール寒天培地
生育は良好で、その表面に気中菌糸を着生し、赤色系（５ｄｃ）の胞子が見られる。培養裏面は焦げ茶である。茶色の溶解性色素を僅かに産生する。
３）スターチ・無機塩寒天培地
生育は良好で、その表面に気中菌糸を豊富に着生し、赤色系（５ｃａ〜６ｅｃ）の胞子が見られる。培養裏面は深緑色〜青紫色である。溶解性色素は産生しない。
４）グリセリン・アスパラギン寒天培地
生育は中程度で、その表面に気中菌糸を着生しない。培養裏面はクリーム色で、溶解性色素は産生しない。
５）チロシン寒天培地
生育は中程度で、その表面に気中菌糸を着生しない。培地中にメラニン色素を産生しない。培養裏面は深緑色である。
（３）各種炭素源の同化性
プリードハム・ゴトリーブ寒天培地に各種の炭素源を加え生育を見た。
１）Ｌ−アラビノース −
２）Ｄ−キシロース ＋
３）Ｄ−グルコース ＋
４）Ｄ−フルクトース ±
５）シュークロース ＋
６）イノシトール −
７）Ｌ−ラムノース −
８）Ｄ−マンニトール −
９）ラフィノース −
＋は同化する、±は多少同化する、−は同化しない
（４）細胞壁成分の性状
細胞を加水分解したものをセルロースの薄層クロマトグラフィーによって分析したところ、本菌の細胞壁成分のジアミノピメリン酸（ｄｉａｍｉｎｏ ｐｉｍｅｒｉｃ ａｃｉｄ）の異性体型はＬＬ型であった。
以上の菌学的性質から本菌はストレプトミセス（Ｓｔｒｅｐｔｏｍｙｃｅｓ）属の菌であると思われる。
本発明者は本菌をストレプトミセス・エスピー・エムイーアール２４８７（Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．Ｍｅｒ−２４８７）として工業技術院生命工学工業技術研究所（郵便番号３０５−８５６６日本国茨城県つくば市東１丁目１番３号）にＦＥＲＭ Ｐ−１６７１８の番号で１９９８年３月１９日に寄託している。また、ＦＥＲＭ Ｐ−１６７１８は、１９９９年６月１８日に国際寄託ＦＥＲＭ ＢＰ−６７６２へ移管した。
（培養条件）
本発明の生理活性物質ＥＭ ２４８７Ａ，Ｂ，Ｃは、上記菌株を栄養源含有培地に接種し、好気的に培養することにより製造される。生理活性物質ＥＭ２４８７の生産菌としては、ストレプトミセス属に属しＥＭ ２４８７Ａ，Ｂ，Ｃを生産する能力を有するものであれば、上記菌株に限らず全て本発明に利用できる。
上記微生物の培養方法は、原則的には一般微生物の培養方法に準ずるが、通常は液体培養による振とう培養、通気撹拌培養等の好気的条件下で実施するのが好ましい。培養に用いられる培地としては、ストレプトミセス属に属する微生物が利用できる栄養源を含有する培地であればよく、各種の合成、半合成培地、天然培地などいずれも利用可能である。培地組成としては炭素源としてのグルコース、シュークロース、フルクトース、グリセリン、デキストリン、澱粉、糖蜜等を単独又は組み合わせて用いることができる。窒素源としてはファーマメディア、ペプトン、肉エキス、大豆粉、カゼイン、アミノ酸、酵母エキス、尿素等の有機窒素源を単独又は組み合わせて用いることができる。その他例えば塩化ナトリウム、塩化カリウム、炭酸カルシウム、硫酸マグネシウム、燐酸ナトリウム、燐酸カリウム、塩化コバルト等の塩類、重金属塩類、ビタミンＢ及びビオチン等のビタミン類も必要に応じ添加使用することができる。なお、培養中発泡が著しい場合には、公知の各種消泡剤を適宜培地中に添加することもできる。消泡剤の添加にあたっては、目的物質の生産に悪影響を与えない濃度とする必要があり、例えば使用濃度としては０．０５％以下が望ましい。
培養条件は、該菌株が良好に生育して上記物質を生産し得る範囲内で適宜選択し得る。例えば培地のｐＨは５〜９程度、通常中性付近とするのが望ましい。培養温度は、通常２０〜４０℃、好ましくは２８〜３５℃に保つのがよい。培養日数は２〜８日程度で、通常３〜５日である。上述した各種の培養条件は、使用微生物の種類や特性、外部条件等に応じて適宜変更でき、最適条件を選択できるのはいうまでもない。培養液中に蓄積された本発明の生理活性物質ＥＭ２４８７は、濾過、遠心分離等の既知の通常の固液分離手段によって菌体を分離し、その菌体からの抽出により回収可能である。
分離・精製
生理活性物質ＥＭ ２４８７Ａ，Ｂ，Ｃの分離・精製は既知の種々の方法を選択、組み合わせて行うことができる。例えば、メタノール、酢酸エチル、アセトン、ｎ−ブタノール等を用いた溶媒抽出、活性炭、アンバーライトＸＡＤ（ローム・アンド・ハース社製）、ダイヤイオンＨＰ−２０（三菱化学社製）等への吸着と含水アルコール、含水アセトン等による溶出、セファデックスＬＨ−２０（ファルマシア社製）、バイオ・ゲルＰ−２（バイオ・ラッド社製）等によるゲル濾過、シリカゲル、アルミナ等によるカラム法あるいは薄層クロマトグラフィー、順相あるいは逆相カラムを用いた分取用高速液体クロマトグラフィー（分取ＨＰＬＣ）等を、単独あるいは適宜組み合わせ、場合により反復使用することで分離、精製する事ができる。
菌の培養液からの本発明化合物の精製は以下のようにして精製することができる。ストレプトミセス属の微生物を通常の適切な培養条件にて培養後、培養液を清澄濾過したのちブタノール又はメチルイソブチルケトンなどの有機溶媒を加え抽出し、有機溶媒層を減圧下濃縮する。次いでメタノールにて抽出し、石油エーテル（ｌｉｇｈｔ ｐｅｔｒｏｌｅｕｍ）などで処理し粗抽出物を得る。次いでシリカゲルなどを用いる吸着クロマトグラフィー、ＬＨ ２０ゲルクロマトグラフィ、分配クロマトグラフィー、薄層クロマトグラフィー、ペーパークロマトグラフィーなどを適宜利用して分画し、活性スクリーニングにより活性画分を確認する。上記手法を適宜組み合わせることにより活性物質を単離することができる。吸着クロマトグラフィーに使用する溶媒としては、クロロホルム、メタノール、アセトン、ヘキサン、トルエンなど通常使用される有機溶媒を用い、適宜濃度を選択、組み合わせて使用することができる。結晶化の溶媒としてはクロロホルムとヘキサン、又はクロロホルムと四塩化炭素などを用いることができる。一つの手法としてＭ．Ｌｕｍｂらの方法がある（Ｎａｔｕｒｅ．２０６，２６３，１９６５）。
単離した化合物の構造解析は、元素分析、ＧＣ−ＭＳ、ＮＭＲ、融点など常法の手法によって行うことができる。
医薬への応用
単離した新規物質はＴａｔ活性を阻害し、急性感染系および持続感染系においてＨＩＶ増殖を抑制して、ＨＩＶ感染症治療で引き起こされるエイズの治療に有用である。また、ヌクレオシド系及び非ヌクレオシド系逆転写酵素阻害剤、プロテアーゼ阻害剤と併用してその治療効果を高めるため、あるいは耐性ウイルスの出現を遅らせるために使用しても良い。
特にヌクレオシド系及び非ヌクレオシド系逆転写酵素阻害剤、プロテアーゼ阻害剤に対して耐性を獲得してしまったＨＩＶに感染している患者に有用である。
該化合物を各種疾患治療・予防剤として投与する場合、錠剤、散剤、顆粒剤、カプセル剤、シロップ剤などとして経口的に投与してもよいし、また噴霧剤、坐剤、注射剤、外用剤、点滴剤として非経口的に投与してもよい。投与量は症状の程度、年齢、肝疾患の種類などにより著しく異なるが、通常成人１日当たり約１ｍｇ〜１０００ｍｇを１日１〜数回にわけて投与する。
製剤化の際は通常の製剤担体を用い、常法により製造する。すなわち、経口用固形製剤を調製する場合は、生薬に賦形剤、更に必要に応じて結合剤、崩壊剤、滑沢剤、着色剤、矯味矯臭剤などを加えた後、常法により錠剤、被覆錠剤、顆粒剤、散剤、カプセル剤などとする。これらの錠剤、顆粒剤には糖衣、ゼラチン衣、その他必要により適宜コーティングすることは勿論差し支えない。
注射剤を調製する場合には、生薬に必要によりｐＨ調整剤、緩衝剤、安定化剤、可溶化剤などを添加し、常法により皮下、筋肉内、静脈内用注射剤とする。
実施例
以下に実施例を挙げて本発明を更に具体的に説明するが、本発明はこれらによって何等限定されるものではない。実施例において、パーセント（％）は特記しない限り、重量／容量パーセントを示す。
実施例１ ＥＭ ２４８７Ａ，Ｂ，Ｃの分離精製
Ｍｅｒ−２４８７株の斜面培地（ＩＳＰ−２）から１白金耳を５０ｍｌの種母培地（グリセリン２％、グルコース２％、大豆粉（エスサンミート：味の素（株）製）２％、酵母エキス０．５％、塩化ナトリウム０．２５％、炭酸カルシウム０．３２％、硫酸銅０．０００５％、塩化マンガン０．０００５％、硫酸亜鉛０．０００５％、ｐＨ７．４）を入れた５００ｍｌ容の三角フラスコに接種し、２８℃で２日間回転振とう機上で培養して種培養液を得た。この種培養液それぞれ１５０ｍｌを本培養培地（デキストリン３％、グルコース０．５％、大豆粉（エスサンミート：味の素（株）製）１．５％、コーン・スティープ・リカー０．５％、炭酸カルシウム０．５％、消泡剤０．０５％、ｐＨ７．２）１５Ｌを含む３０Ｌ容ジャーファーメンター２基に接種して、２８℃で７０時間通気撹拌培養（通気量７．５Ｌ／ｍｉｎ、撹拌１００〜２５０ｒ．ｐ．ｍ．）を行った。培養終了後、得られた培養液２４Ｌを連続遠心分離器にかけ、上清と菌体とに分離した。また、菌体を２０Ｌのメタノールで抽出し、得られたメタノール抽出物を、減圧下で濃縮し、メタノールを留去し、上清と混合した。
混合液に水で膨張したダイヤイオンＨＰ−２０ ２．４Ｌを加え、撹拌した後、樹脂をろ取してカラムに充填し、２０％メタノール６Ｌで洗浄した。続いて、８０％アセトン４１Ｌで溶出し、溶出液を減圧下で濃縮し、アセトンを留去した。残った水溶液を減圧下濃縮乾固して粗抽出物を得た。
このものを少量のジメチルスルホキシドに溶解し、アセトニトリル−１０ｍＭリン酸緩衝液（ｐＨ７．０）＝２：８の混合溶液で予め平衝化しておいたＹＭＣ−ＧＥＬ ＯＤＳ−ＡＭ １２０−Ｓ５０（ワイエムシィ社製）のカラム（内径６０ｍｍ、長さ９５０ｍｍ）に二度に分けて付した。同じ組成の混合溶媒で洗浄し、続いて、３５：６５の組成の混合溶媒で溶出した。
溶出液は高速液体クロマトグラフで分析し、ＥＭ ２４８７Ａ，Ｂ，Ｃ（カラム：Ｊ’ｓｐｈｅｒｅ ＯＤＳ−Ｈ８０、内径４．６ｍｍ、長さ７５ｍｍ、ワイエムシイ社製、移動層：アセトニトリルー２０ｍＭリン酸緩衝液（ｐＨ７．０）＝３５：６５、流速：１ｍＬ／ｍｉｎ、検出：２６０ｎｍにおける紫外吸収、保持時間；Ａ：２．５ｍｉｎ、Ｂ：６．１ｍｉｎ、Ｃ：６．３ｍｉｎ）を含む溶出液を集め、ＥＭ ２４８７ Ａ，Ｂ，Ｃを含むフラクションを得た。アセトニトリルを減圧下留去した後、水で膨張したダイヤイオンＨＰ−２０を加え、撹拌した後、樹脂をカラムに充填し、２０％メタノールで洗浄した。続いて、８０％アセトンで溶出し、溶出液を減圧下で濃縮し、アセトンを留去した。残った水溶液を減圧下濃縮乾固して粗抽出物を得た。
このものを少量のジメチルスルホキシドに溶解し、分取ＨＰＬＣ（カラム：Ｊ’ｓｐｈｅｒｅ ＯＤＳ−Ｈ８０、内径２０ｍｍ、長さ２５０ｍｍ、ワイエムシイ社製、移動層：アセトニトリル−２０ｍＭリン酸緩衝液（ｐＨ７．０）＝３５：６５、流速：１０ｍＬ／ｍｉｎ、検出：２６０ｎｍにおける紫外吸収、保持時間；Ａ：１２．０ｍｉｎ、Ｂ：２２．０ｍｉｎ、Ｃ：２６．０ｍｉｎ）に付した。この分取をくりかえし、ＥＭ ２４８７ Ａ，Ｂ，Ｃのピークを示す溶出液をそれぞれ併せ、アセトニトリルを減圧下留去した後、水で膨張したダイヤイオンＨＰ−２０を加え、撹拌した後、樹脂をカラムに充填し、２０％メタノールで洗浄した。続いて、８０％アセトンで溶出し、溶出液を減圧下で濃縮し、アセトンを留去した。残った水溶液を減圧下濃縮乾固してＥＭ ２４８７Ａ，Ｂ，Ｃの純粋な白色粉末をそれぞれ４３２ｍｇ，４４．８ｍｇ，２２．４ｍｇ得た。
ＥＭ ２４８７Ａ精製および構造解析データ
１．色および性状：白色粉末
２．分子式：Ｃ３２Ｈ５７Ｎ５Ｏ１６Ｐ２
３．マススペクトル（ＦＡＢ−ＭＳ）：ｍ／ｚ ８２８（Ｍ−Ｈ）−
４．比旋光度：［α］Ｄ ２２ −７．９（ｃ １．３、ＭｅＯＨ）
５．紫外部吸収スペクトル：
中性メタノール中：λｍａｘ ｎｍ（ε）：２６２（３４２００）
酸性メタノール中：λｍａｘ ｎｍ（ε）：２６２（３６１００）
塩基性メタノール中：λｍａｘ ｎｍ（ε）：２６０（２７１００）
６．赤外部吸収スペクトル：
ＫＢｒ粉末中で測定した。主な吸収を示す。（波数、ｃｍ−１）
２９２０，２８５０，１６８０，１４７０，１２４０，１０７０，８８０，７２０，５５０
７．１Ｈ ＮＭＲスペクトル、１３Ｃ ＮＭＲスペクトルおよびＨＭＢＣ ｃｏｒｒｅｌａｔｉｏｎｓ
重メタノール溶液中で測定した結果を表１に示す。表には、化合物の炭素の位置（ｐｏｓｉｔｉｏｎ）、その分析データすなわちＥＭ ２４８７Ａの化学シフト表（Ｃｈｅｍｉｃａｌ Ｓｈｉｆｔ Ｔａｂｌｅ ｏｆ ２４８７Ａ）、炭素間の相関（ＨＭＢＣ ｃｏｒｒｅｌａｔｉｏｎｓ）を参照値（Ｒｅｆｅｒｅｎｃｅ）とともに示した。
可溶：中性の水、メタノール、ジメチルスルホキシド
不溶：ヘキサン、酢酸エチル、クロロホルム
９．高速液体クロマトグラフィー：
カラム：Ｊ’ｓｐｈｅｒｅ ＯＤＳ−Ｈ８０、内径４．６ｍｍ、長さ７５ｍｍ（ワイエムシイ社製）
溶媒：アセトニトリル−２０ｍＭリン酸緩衝液（ｐＨ７．０）＝３５：６５
流速：１ｍＬ／ｍｉｎ
検出波長：２６０ｎｍにおける紫外吸収
保持時間：２．５ｍｉｎ
ＥＭ ２４８７Ｂ精製および構造解析データ
１．色および性状：白色粉末
２．分子式：Ｃ３３Ｈ５８Ｎ５Ｏ１６Ｐ２
３．マススペクトル（ＦＡＢ−ＭＳ）：ｍ／ｚ ８４２（Ｍ−Ｈ）−
４．１Ｈ ＮＭＲスペクトル
重メタノール溶液中で測定した結果を表２に示す。
可溶：中性の水、メタノール、ジメチルスルホキシド
不溶：ヘキサン、酢酸エチル、クロロホルム
６．高速液体クロマトグラフィー：
カラム：Ｊ’ｓｐｈｅｒｅ ＯＤＳ−Ｈ８０、内径４．６ｍｍ、長さ７５ｍｍ（ワイエムシイ社製）
溶媒：アセトニトリルー２０ｍＭリン酸緩衝液（ｐＨ７．０）＝３５：６５
流速：１ｍＬ／ｍｉｎ
検出波長：２６０ｎｍにおける紫外吸収
保持時間：６．１ｍｉｎ
ＥＭ ２４８７Ｃ精製および構造解析データ
１．色および性状：白色粉末
２．分子式：Ｃ３３Ｈ５８Ｎ５Ｏ１６Ｐ２
３．マススペクトル（ＦＡＢ−ＭＳ）：ｍ／ｚ ８４２（Ｍ−Ｈ）−
４．１Ｈ ＮＭＲスペクトル
重メタノール溶液中で測定した結果を表３に示す。
可溶：中性の水、メタノール、ジメチルスルホキシド
不溶：ヘキサン、酢酸エチル、クロロホルム
６．高速液体クロマトグラフィー：
カラム：Ｊ’ｓｐｈｅｒｅ ＯＤＳ−Ｈ８０、内径４．６ｍｍ、長さ７５ｍｍ（ワイエムシイ社製）
溶媒：アセトニトリルー２０ｍＭリン酸緩衝液（ｐＨ７．０）＝３５：６５
流速：１ｍＬ／ｍｉｎ
検出波長：２６０ｎｍにおける紫外吸収
保持時間：６．３ｍｉｎ
実施例２ Ｔａｔ阻害活性
（１）レポーター遺伝子の構築
Ｔａｔ活性を阻害する化合物をスクリーニングするために、ＴＡＲ領域を含むＨＩＶ ＬＴＲからの転写が測定できるレポーター遺伝子を構築した。後藤らが報告したＴＮＦ−αプロモーターの後に分泌型アルカリフォスファターゼＰＬＡＰ、続いてＮｅｏ耐性遺伝子を含むＴＮＦ−αレポータープラスミドＴＮＦ−ａ−ＰＬＡＰ−ＰＧＫ−ｎｅｏ（Ｇｏｔｏ Ｍ．ｅｔ ａｌ．，Ｍｏｌ．Ｐｈａｒｍａｃｏｌ．４９：８６０（１９９６））のＴＮＦ−αプロモーターを含むＸｂａＩ−ＨｉｎｄＩＩＩ部位に、Ｎａｔｉｏｎａｌ Ｉｎｓｔｉｔｕｔｅ ｏｆ Ａｌｌｅｒｇｙ ａｎｄ Ｉｎｆｅｃｔｉｏｕｓ Ｄｉｓｅａｓｅ ＡＩＤＳ Ｒｅｓｅａｒｃｈ ａｎｄ Ｒｅｆｅｒｅｎｃｅ Ｒｅａｇｅｎｔ Ｐｒｏｇｒａｍより入手したｐＵＣ−ＢＥＮＮ−ＣＡＴのＨＩＶ ＬＴＲおよび感染細胞由来配列を含むＸｂａＩ−ＨｉｎｄＩＩＩ部位を挿入してレポーター遺伝子ｐＰＧＫＦを構築した。
（２）Ｔａｔ発現遺伝子の構築
Ｔａｔの２ｎｄ．エクソンが細胞の染色体に組み込まれたＨＩＶ ＬＴＲからの転写活性化に必要であるとの報告（Ｊｅａｎｇ Ｋ．Ｔ．Ｊ．ｏｆ Ｂｉｏｌ．Ｃｈｅｍ．２６８（３３）：２４９４０（１９９３））があることから、Ｎａｔｉｏｎａｌ Ｉｎｓｔｉｔｕｔｅ ｏｆ Ａｌｌｅｒｇｙ ａｎｄ Ｉｎｆｅｃｔｉｏｕｓ Ｄｉｓｅａｓｅ ＡＩＤＳ Ｒｅｓｅａｒｃｈ ａｎｄ Ｒｅｆｅｒｅｎｃｅ Ｒｅａｇｅｎｔ Ｐｒｏｇｒａｍより入手したＴａｔ発現ベクターｐＳＶ２ Ｔａｔに２ｎｄ．エクソンの配列（Ｃｏｌｌｍａｎ Ｒ．ｅｔ ａｌ．，Ｊ．ｏｆ Ｖｉｒｏｌ．６６（１２）：７５１７（１９９２））を付加した。配列番号１と配列番号２の合成オリゴヌクレオチドをアニール後、Ｖｅｎｔ ｐｏｌｙｍｅｒａｓｅ（Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｃｈｅｍｉｓｔｒｙ社製）により伸長反応を行いＢａｍＨＩ，ＢｇｌＩＩ消化して、ｐＳＶ２ ＴａｔのＢａｍＨＩ−ＢｇｌＩＩ部位に挿入しｐＳＶ２ Ｔａｔ−ｌｏｎｇを構築した。
（３）エレクトロポレーション法による遺伝子の導入
導入したい遺伝子配列を含む１０から４０μｇのＤＮＡを、１ｍｌの無血清ＲＰＭＩ培地中に懸濁した３×１０６から１×１０７のヒト白血病細胞ＣＥＭと混合し、４℃で１０分間放置した後、ジーンパルサー（バイオラッド社製）にて３００Ｖ，１０００μＦの条件で通電した。その細胞懸濁液を直ちに１０％ ＦＣＳを含むＲＰＭＩ培地（１０％ ＦＣＳ／ＲＰＭＩ培地）に希釈し、ＣＯ２インキュベータ中で培養した。
（４）アルカリフォスファターゼ活性の測定
被験溶液中のＦＣＳに含まれるアルカリフォスファターゼを失活させるために６５℃で２０〜３０分処理した。化学発光測定用の９６穴プレートに、被験溶液１０μｌ、炭酸バッファー（０．２８Ｍ Ｍａ２ＣＯ３ｂｕｆｆｅｒ ｐＨ１０．０，８ｍＭ ＭｇＳＯ４）５０μｌおよびルミステイン（住友金属社製）５０μｌを加え１時間室温で放置後、マイクロプレートルミノメーター ＬＢ９６Ｐ（ベルトールド社製）にて化学発光を測定した。
（５）レポーター細胞株の樹立
Ｔａｔ活性を阻害する化合物をスクリーニングするために、レポーター遺伝子を発現するレポーター細胞株を樹立した。レポーター遺伝子ｐＰＧＫＦをエレクトロポレーション法によりヒト白血病細胞ＣＥＭ細胞に導入した。ＣＯ２インキュベータ中で一晩培養した後、０．８ｍｇ／ｍｌゲネチシン（Ｓｉｇｍａ社製）を含む１０％ ＦＣＳ／ＲＰＭＩ培地に懸濁し２４穴プレートで培養した。２週間後、細胞が増殖してきたウエルの培養上清中のアルカリフォスファターゼ活性を測定し、活性のあるウエルからＰＬＡＰ産生細胞を限外希釈法にてクローニングした。
クローニングした細胞を増殖させた後、Ｔａｔ発現遺伝子の導入、およびＴＮＦによりＰＬＡＰ産生の高まるクローンを選択した。選択したレポーター細胞は継代培養してＴａｔ阻害活性の測定に使用した。
（６）ＭＴＴ法による生細胞の測定
９６穴プレートに培養した細胞に、７．５ｍｇ／ｍｌのＭＴＴ溶液２０μｌを加えＣＯ２インキュベータ中で２時間培養した。細胞を吸い込まないように１５０μｌの上清を除き、１００μｌの１０％ ＴｒｉｔｏｎＸ−１００を含むイソプロパノールを加えてＭＴＴが還元されてできた色素（ホルマザン）を可溶化した。各ウエルの５４０ｎｍの吸光度をプレートリーダー（バイオラッドＭｏｄｅｌ ３５５０）を用いて測定し、生細胞の指標とした。
（７）細胞内で発現させたＴａｔに対するＥＭ ２４８７Ａ，Ｂ，Ｃ阻害活性
レポーター細胞にエレクトロポレーション法によりＴａｔ発現遺伝子ｐＳＶ２ Ｔａｔ−ｌｏｎｇ遺伝子を導入後、ＣＯ２インキュベータ中で培養した。また、Ｔａｔ発現遺伝子を導入しない細胞も同様に培養した。翌日、培養した細胞を１０００ｒｐｍ５分間の遠心で集め、１０％ ＦＣＳ／ＲＰＭＩ培地中に１．２５×１０５／ｍｌの濃度に調整して、細胞懸濁液とした。
被験検体はＤＭＳＯに溶解し、１０％ ＦＣＳ／ＲＰＭＩで１００倍に希釈して検体希釈液を調整した。
９６穴プレートに、検体希釈液２０μｌ、２０ｎｇ／ｍｌのＴＮＦを含むあるいは含まない１０％ ＦＣＳ／ＲＰＭＩ培地２０μｌ、細胞懸濁液１６０μｌを加え、ＣＯ２インキュベータ中で２日間培養した。培養上清１０μｌを採取しアルカリフォスファターゼ活性を測定した。また、ＭＴＴ法にて検体の細胞毒性を測定した。
表４に培養上清中のアルカリフォスファターゼ活性およびＭＴＴ法の吸光度を示した（括弧内は薬剤無添加のコントロールを１００％としたパーセント）。ＥＭ ２４８７Ａ，Ｂ，Ｃは１０μｇ／ｍｌの濃度でＴａｔ発現遺伝子を導入しない細胞のアルカリフォスファターゼの産生には影響を与えず、細胞毒性も示さなかった。Ｔａｔ発現遺伝子の導入によりアルカリフォスファターゼの産生量は約２０倍に促進されたが、ＥＭ ２４８７Ａ，Ｂ，ＣはＴａｔにより促進されたアルカリフォスファターゼの産生のみを抑制した。
また、ＴＮＦの存在下ではＴａｔ発現遺伝子導入および非導入細胞のアルカリフォスファターゼ産生は更に約３倍促進された。この場合においても、ＥＭ ２４８７Ａ，Ｂ，ＣはＴａｔ発現遺伝子を導入しない細胞のアルカリフォスファターゼの産生には影響を与えず、Ｔａｔ発現遺伝子の導入により促進されたアルカリフォスファターゼの産生のみを抑制した。
（８）細胞外から供給したＴａｔに対するＥＭ ２４８７Ａの阻害活性
レポーター細胞に無血清培地中２μｇ／ｍｌのＴａｔ（Ｉｍｍｕｎｏ Ｄｉａｇｎｏｓｔｉｃｓ社製），１ｍＭ Ｄｉｔｈｉｏｔｈｒａｔｏｌ，１００μＭ Ｃｈｌｏｒｏｑｕｉｎの存在下で２時間培養し、１０００ｒｐｍ ５分間遠心後１０％ ＦＣＳ／ＲＰＭＩ培地に懸濁し、ＣＯ２インキュベータ中で培養した。また、Ｔａｔ非存在下で同様に処理した細胞も培養した。翌日、培養した細胞を１０００ｒｐｍ ５分間の遠心で集め、１０％ ＦＣＳ／ＲＰＭＩ培地中に１．２５×１０５／ｍｌの濃度に調整して、細胞懸濁液とした。
９６穴プレートに、検体希釈液２０μｌ、２０ｎｇ／ｍｌのＴＮＦを含むあるいは含まない１０％ ＦＣＳ／ＲＰＭＩ培地２０μｌ、細胞懸濁液１６０μｌを加え、ＣＯ２インキュベータ中で２日間培養した。培養上清１０μｌを採取しアルカリフォスファターゼ活性を測定した。また、ＭＴＴ法にて検体の細胞毒性を測定した。
細胞外からＴａｔを供給することによりアルカリフォスファターゼの産生量は約３０倍に促進された。また、ＴＮＦ存在下ではアルカリフォスファターゼ産生は更に約３倍に促進された。表４に培養上清中のアルカリフォスファターゼ活性およびＭＴＴ法の吸光度を示した（括弧内は薬剤無添加のコントロールを１００％としたパーセント）。ＴＮＦの存在非存在にかかわらず、ＥＭ ２４８７Ａは１０μｇ／ｍｌの濃度でＴａｔを供給しない細胞のアルカリフォスファターゼの産生には影響を与えず、Ｔａｔ発現遺伝子の導入により促進されたアルカリフォスファターゼの産生のみを抑制した。無処置（Ｎｏｎｅ Ｔｒｅａｔ）とともに表４に結果を示した。
ヒト白血病細胞Ｍｏｌｔ−４にＨＩＶ−１ ＨＴＬＶ−ＩＩＩＢ株を感染させた。ＨＩＶ感染細胞および非感染細胞１×１０５ｃｅｌｌｓ／ｍｌを種々の濃度の薬剤とともに３７℃にて培養し、４日後に同じ濃度の薬剤を含む培養液にて１：５に希釈した。培養開始から７日目にＭＴＴ法にて生細胞数を測定し、ＥＭ ２４８７Ａ，Ｂ，Ｃの細胞傷害抑制活性（抗ＨＩＶ活性）と細胞毒性の判定を行った。
薬剤無添加のＨＩＶ非感染細胞の生細胞数を１００％とし、薬剤無添加のＨＩＶ感染細胞の生細胞数を０％として、ＨＩＶ感染細胞に対して５０％の細胞傷害抑制活性を示す薬剤濃度ＥＣ５０を求めた。
薬剤無添加の非感染細胞培養中の生細胞数を１００％とし、培地のみの中の生細胞数を０％として、非感染細胞に対して５０％の細胞傷害活性を示す薬剤濃度ＣＣ５０を求めた。
表５に示すようにＥＭ ２４８７Ａ，Ｂ，ＣはＭｏｌｔ−４急性感染系において、ＨＩＶ増殖において抗ＨＩＶ活性を示した。表には、５０％細胞傷害抑制濃度（ＥＣ５０（μＭ））、５０％細胞傷害濃度（ＣＣ５０（μＭ））、選択係数（ｓｅｌｅｃｔｉｖｉｔｙ ｉｎｄｅｘ）を示した。
ヒト白血病細胞ＭＴ−４にＨＩＶ−１ ＨＴＬＶ−ＩＩＩＢ株を感染させた。ＨＩＶ感染細胞および非感染細胞１×１０５ｃｅｌｌｓ／ｍｌを種々の濃度の薬剤とともに３７℃にて培養した。培養開始から４日目にＭＴＴ法にて生細胞を測定し、薬剤添加による細胞傷害抑制活性（抗ＨＩＶ活性）と細胞毒性の判定を行った。
表６に示すようにＲｏ ２４−７４２９はＭＴ−４急性感染系において効果を示さなかったが、ＥＭ ２４８７Ａには抗ＨＩＶ活性が認められた。
ＰＨＡで刺激したＰＢＭＣ １×１０５ｃｅｌｌｓ／ｗｅｌｌにＨＩＶ−１ ＨＴＬＶ−ＩＩＩＢ株を感染させ、種々の濃度の薬剤とともに３７℃にて培養した。培養開始から６日目にＥＬＩＳＡによりｐ２４抗原を測定してＨＩＶ量を定量するとともに、ＭＴＴ法にて生細胞を測定し、薬剤添加によるＨＩＶ産生抑制活性（抗ＨＩＶ活性）と細胞毒性の判定を行った。
薬剤無添加のｐ２４抗原量を１００％とし、５０％のＨＩＶ増殖抑制活性を示す薬剤濃度ＥＣ５０を求めた。
表７に示すようにＥＭ ２４８７ＡはＰＢＭＣ急性感染系において抗ＨＩＶ活性が認められた。
実施例６ ＯＭ１０．１持続感染系における抗ＨＩＶ活性
ＨＩＶ−１が感染した前骨髄球ＯＭ１０．１細胞 １×１０５ｃｅｌｌｓ／ｗｅｌｌを種々の濃度の薬剤にて２時間前処理し、その後１ｎｇ／ｍｌのＴＮＦ−αにて刺激、さらに薬剤存在下にて培養を続けた。培養開始から３日目にＥＬＩＳＡによりｐ２４抗原を測定してＨＩＶ量を定量するとともに、ＭＴＴ法にて生細胞を測定し、薬剤添加によるＨＩＶ産生抑制活性（抗ＨＩＶ活性）と細胞毒性の判定を行った。
表７に示すようにＥＭ ２４８７ＡはＯＭ１０．１持続感染系において抗ＨＩＶ活性が認められた。
逆転写酵素阻害剤、プロテアーゼ阻害剤と併用することで、ＨＩＶ増殖を更に抑制することが期待されることから、耐性ウイルスの出現を遅らせるために使用することができる。特に、作用点が異なり耐性が交叉しないことから、ヌクレオシド系及び非ヌクレオシド系逆転写酵素阻害剤、プロテアーゼ阻害剤に対して耐性を獲得してしまったＨＩＶに感染している患者に有用である。
【配列表】
The present invention relates to a novel physiologically active substance effective in the treatment of HIV infection that inhibits the activity of HIV (human immunodeficiency virus) Tat molecule and suppresses HIV proliferation.
Conventional technology
AIDS is a disease of the immune system caused by the retroviruses HIV-1 and HIV-2 and almost results in death of the patient. In order to prevent AIDS and reduce symptoms, there is an interest in searching for compositions that inhibit viral growth.
After HIV infects cells, the gene RNA is reverse transcribed into DNA by reverse transcriptase contained in the virus particle and incorporated into the host cell DNA by HIV integrase. HIV RNA is transcribed from the promoter in the LTR at the 5 'end of the incorporated HIV gene to become gene RNA, and at the same time, a viral protein is synthesized using the RNA as a template. Some of the synthesized viral proteins are cleaved by HIV protease to become mature proteins to form next generation virus particles. From the above viral life cycle, reverse transcriptase inhibition, integrase inhibition, transcription inhibition from HIV LTR, protease inhibition and the like are mentioned as the action points of drugs for treating HIV infection.
So far, nucleoside and non-nucleoside reverse transcriptase inhibitors and protease inhibitors have been put to practical use as therapeutic agents for HIV infection and are effective in treatment. However, the emergence of resistant strains against these anti-HIV agents has already been reported (Gunthard H. et al., J. of Virol. 72 (3): 2422 (1998)), and the development of new anti-HIV agents has been reported. It is desired. In particular, since anti-HIV agents with different action points do not cross resistance-resistant mutations, it is possible to treat HIV infections that have acquired resistance to existing drugs, and emerge resistance when used in combination with existing drugs. Is expected to delay.
HIV Tat molecules are known to bind to the TAR region at the 5 ′ end of HIV RNA and have activity (Tat activity) that promotes transcription from the HIV LTR (Laspia MF et al., Cell 59: 283 (1989)). Since HIV lacking Tat cannot proliferate (Popik W. et al., J. of Virol. 67 (2): 1094 (1993)), an agent that inhibits Tat activity (Tat inhibitor) inhibits HIV proliferation. It is expected to suppress and become a therapeutic agent for HIV infection with a novel mechanism.
So far, Ro 5-3335 and Ro 24-7429, which reduced nephrotoxicity in rats, have been reported as compounds that inhibit Tat activity and suppress HIV proliferation, while Ro 5-3335 exhibits standard anti-HIV activity. Anti-HIV activity such as MT-4 acute infection system, which is an evaluation system (Witvrouw M. et al., Antimicrob. Agents and Chemother. 36: 2628 (1992)) is not sufficient, and clinically introduced Ro 24-7429 had no effect (Haurich RH et al., J. of Infect. Disese 172: 1246 (1995)).
In addition, ALX40-4C, an analog of a domain that binds to the TAT region of Tat, has not been reported to inhibit Tat activity in cell lines, and the reported anti-HIV activity is based on adsorption inhibition. (O'Brien W. et al, J. of Virol. 70 (5): 2825 (1996)).
Disclosure of the invention
It is an object of the present invention to provide a therapeutic agent for HIV infection by isolating a novel substance that inhibits Tat activity and suppresses HIV proliferation.
In view of the above situation, the present inventors conducted a screening screening for a Tat inhibitor using a microorganism culture solution as a raw material. As a result, it was found that a Tat inhibitor was produced in the culture solution of microorganisms belonging to the genus Streptomyces, and when this active substance was isolated and structurally determined, it was found to be a novel active substance. This active substance has been shown to inhibit HIV proliferation in various acute HIV infection systems, including MT-4 acute infection systems where Ro 5-3335 does not show activity. In addition, as expected from the mechanism of action to suppress the transcription process to RNA, HIV gene was suppressed in persistent infection system, and reverse transcriptase inhibitor was incorporated into the host cell DNA. It has been shown that it can be a therapeutic drug that can be effective even later.
That is, the present invention provides the following general formula (I):
And a salt thereof or a hydrate thereof, a production method thereof, and a pharmaceutical comprising the same as an active ingredient.
More specifically, in the general formula (I), R1Is represented by the following general formula (II):
A compound represented by the following formula: salt, hydrate thereof,
In general formula (I), R1Is represented by the following formula (III):
Streptomyces sp.Mer-2487, FERM P-16718 is cultured in a nutrient medium, and the compound represented by the general formula (I), its salt or its A method for producing a compound represented by the general formula (I), a salt thereof or a hydrate thereof is characterized by collecting a hydrate.
The present invention relates to a growth inhibitor of human immunodeficiency virus (HIV) comprising a compound represented by formula (I), a salt thereof or a hydrate thereof as an active ingredient.
A pharmaceutical composition comprising an effective amount of a compound represented by the formula (I), a salt thereof or a hydrate thereof and a pharmaceutically acceptable carrier, a compound represented by the formula (I), a salt thereof or a hydration thereof Is used for the manufacture of a human immunodeficiency virus (HIV) growth inhibitor, and an effective amount of a compound represented by formula (I), a salt thereof or a hydrate thereof is administered to a human immunodeficiency virus ( A method of inhibiting the growth of HIV) is provided. Therefore, the disease caused by proliferation of human immunodeficiency virus (HIV) is prevented and treated.
In the present specification, R of the compound represented by the general formula (I)1Is a group represented by the general formula (II), and R in the general formula (II)2Is a methyl group, EM 2487A, R2EM 2487B is a compound in which is an ethyl group, R of the compound represented by the general formula (I)1A compound in which is a group represented by the formula (III) is referred to as EM 2487C.
Detailed Description of the Invention
Mycological properties of the production strain
As representative production strains of the compounds of the present invention, there are actinomycetes isolated from the soil of Shiga Kogen Forest, Nagano Prefecture, and the strains that the inventors have numbered as M-2487 (Mer-2487) strains. It is done. The mycological properties of this Mer-2487 strain are as follows.
(1) Form
A straight aerial hyphae is elongated from the basic hyphae. A spore chain consisting of 10 to 50 cylindrical spores is formed at the tip of a mature aerial mycelium. Spores are not allowed. The size of the spore is about 0.6 to 0.8 μm × 1.0 to 1.2 μm, the surface of the spore is smooth, and no flagella are observed.
(2) Growth state in various media
All cultures were performed at 28 ° C. The description of the color tone is indicated by the symbol shown in parentheses in the Color Harmony Manual of Container Corporation of America (Color Harmony Manual) of the Container Corporation of America.
1) Yeast / malt agar medium
Growth is moderate, aerial hyphae grow on the surface, and red (5ca-6ec) spores are seen. The back side of the culture is dark green to dark brown. A slight amount of brown soluble pigment is produced.
2) Oatmeal agar medium
The growth is good, and aerial hyphae are grown on the surface, and red (5 dc) spores are observed. The back of the culture is dark brown. A slight amount of brown soluble pigment is produced.
3) Starch and inorganic salt agar medium
It grows well, has abundant aerial hyphae on its surface, and has red (5ca-6ec) spores. The rear surface of the culture is dark green to blue-violet. No soluble pigment is produced.
4) Glycerin / asparagine agar medium
It grows moderately and does not form aerial hyphae on its surface. The back of the culture is creamy and does not produce soluble pigment.
5) Tyrosine agar medium
It grows moderately and does not form aerial hyphae on its surface. Does not produce melanin pigment in the medium. The back of the culture is dark green.
(3) Assimilation of various carbon sources
Growth was observed by adding various carbon sources to Preedham Gotleyve Agar.
1) L-arabinose-
2) D-xylose +
3) D-glucose +
4) D-fructose ±
5) Shoe close +
6) Inositol-
7) L-rhamnose-
8) D-mannitol-
9) Raffinose-
+ Is assimilated, ± is somewhat assimilated,-is not assimilated
(4) Properties of cell wall components
When the hydrolyzed cells were analyzed by thin layer chromatography on cellulose, the isomer form of diaminopimelic acid as the cell wall component of the bacterium was LL type.
From the above bacteriological properties, this bacterium seems to be a bacterium belonging to the genus Streptomyces.
The present inventor designated Streptomyces sp. Mer-2487 as Streptomyces sp. Mer-2487, Institute of Biotechnology, Institute of Industrial Science and Technology No.) was deposited on March 19, 1998 under the number FERM P-16718. In addition, FERM P-16718 was transferred to International Deposit FERM BP-6762 on June 18, 1999.
(Culture conditions)
The physiologically active substance EM 2487A, B, C of the present invention is produced by inoculating the above-mentioned strain in a nutrient source-containing medium and culturing aerobically. As the bacteria producing the biologically active substance EM2487, any bacteria can be used in the present invention as long as they belong to the genus Streptomyces and have the ability to produce EM2487A, B, C.
The method for culturing microorganisms is basically the same as the method for culturing general microorganisms, but usually it is preferably carried out under aerobic conditions such as shaking culture by liquid culture and aeration and agitation culture. The medium used for the culture may be a medium containing a nutrient source that can be used by microorganisms belonging to the genus Streptomyces, and any of various synthetic, semi-synthetic, natural, and other media can be used. As the medium composition, glucose, sucrose, fructose, glycerin, dextrin, starch, molasses and the like as a carbon source can be used alone or in combination. As the nitrogen source, organic nitrogen sources such as pharmamedia, peptone, meat extract, soybean powder, casein, amino acid, yeast extract, urea and the like can be used alone or in combination. In addition, for example, salts such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, sodium phosphate, potassium phosphate, and cobalt chloride, heavy metal salts, and vitamins such as vitamin B and biotin can be added and used as necessary. In addition, when foaming is remarkable during culture, various known antifoaming agents can be appropriately added to the medium. When adding the antifoaming agent, it is necessary to adjust the concentration so as not to adversely affect the production of the target substance. For example, the use concentration is preferably 0.05% or less.
The culture conditions can be appropriately selected within a range in which the strain can grow well and produce the substance. For example, it is desirable that the pH of the medium is about 5 to 9, usually around neutral. The culture temperature is usually 20 to 40 ° C, preferably 28 to 35 ° C. The culture days are about 2 to 8 days, usually 3 to 5 days. It goes without saying that the various culture conditions described above can be appropriately changed according to the type and characteristics of microorganisms used, external conditions, etc., and optimal conditions can be selected. The physiologically active substance EM2487 of the present invention accumulated in the culture solution can be recovered by separating the cells by known normal solid-liquid separation means such as filtration and centrifugation, and extracting from the cells.
Separation / Purification
Separation and purification of the physiologically active substance EM 2487A, B, C can be performed by selecting and combining various known methods. For example, solvent extraction using methanol, ethyl acetate, acetone, n-butanol, etc., adsorption to activated carbon, Amberlite XAD (Rohm & Haas), Diaion HP-20 (Mitsubishi Chemical) Elution with hydrous alcohol, hydrous acetone, etc., gel filtration with Sephadex LH-20 (Pharmacia), Bio-Gel P-2 (Bio-Rad), column method with silica gel, alumina, etc. or thin layer chromatography Separation and purification can be performed by using high-performance liquid chromatography (preparative HPLC) or the like using a normal phase or reverse phase column alone or in combination as appropriate, and optionally repeatedly.
The compound of the present invention can be purified from the culture solution of fungi as follows. After culturing a microorganism belonging to the genus Streptomyces under normal and appropriate culture conditions, the culture solution is clarified and filtered, and then extracted with an organic solvent such as butanol or methyl isobutyl ketone, and the organic solvent layer is concentrated under reduced pressure. Next, extraction with methanol and treatment with petroleum ether or the like give a crude extract. Subsequently, fractionation is carried out by appropriately using adsorption chromatography using silica gel or the like, LH 20 gel chromatography, partition chromatography, thin layer chromatography, paper chromatography, etc., and the active fraction is confirmed by activity screening. The active substance can be isolated by appropriately combining the above methods. As a solvent used for the adsorption chromatography, a commonly used organic solvent such as chloroform, methanol, acetone, hexane, toluene and the like can be used by appropriately selecting and combining the concentrations. As a crystallization solvent, chloroform and hexane, chloroform and carbon tetrachloride, or the like can be used. As one method, M.I. Lumb et al. (Nature. 206, 263, 1965).
The structural analysis of the isolated compound can be performed by conventional methods such as elemental analysis, GC-MS, NMR, and melting point.
Application to medicine
The isolated novel substance inhibits Tat activity, suppresses HIV growth in acute and persistent infection systems, and is useful for the treatment of AIDS caused by the treatment of HIV infection. Further, it may be used in combination with nucleoside and non-nucleoside reverse transcriptase inhibitors and protease inhibitors to enhance the therapeutic effect, or to delay the emergence of resistant viruses.
It is particularly useful for patients infected with HIV who have acquired resistance to nucleoside and non-nucleoside reverse transcriptase inhibitors and protease inhibitors.
When the compound is administered as an agent for treating or preventing various diseases, it may be administered orally as tablets, powders, granules, capsules, syrups, etc., and sprays, suppositories, injections, and external preparations. Alternatively, it may be administered parenterally as an infusion. The dose varies significantly depending on the degree of symptoms, age, type of liver disease, etc., but usually about 1 mg to 1000 mg per day for an adult is administered in 1 to several times a day.
In formulating, it is produced by a conventional method using an ordinary pharmaceutical carrier. That is, when preparing an oral solid preparation, after adding excipients, further binders, disintegrants, lubricants, coloring agents, flavoring agents and the like to herbal medicines, tablets by conventional methods, Coated tablets, granules, powders, capsules, etc. Of course, these tablets and granules may be appropriately coated with sugar coating, gelatin coating, etc. as required.
When preparing an injection, pH adjusters, buffers, stabilizers, solubilizing agents, etc. are added to the crude drug as necessary, and are used as subcutaneous, intramuscular and intravenous injections by conventional methods.
Example
The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples. In the examples, percent (%) indicates weight / volume percent unless otherwise specified.
Example 1 Separation and purification of EM 2487A, B, C
1 platinum loop from the slope medium (ISP-2) of Mer-2487 strain, 50 ml of seed mother medium (glycerin 2%, glucose 2%, soybean flour (Essan Meat: manufactured by Ajinomoto Co., Inc.) 2%, yeast extract 0. 500 ml Erlenmeyer flask containing 5%, sodium chloride 0.25%, calcium carbonate 0.32%, copper sulfate 0.0005%, manganese chloride 0.0005%, zinc sulfate 0.0005%, pH 7.4) And seeded at 28 ° C. for 2 days on a rotary shaker to obtain a seed culture. 150 ml of each of these seed cultures was added to the main culture medium (dextrin 3%, glucose 0.5%, soybean flour (Essan Meat: Ajinomoto Co., Inc.) 1.5%, corn steep liquor 0.5%, calcium carbonate Inoculate two 30L jar fermenters containing 15% of 0.5%, antifoam 0.05%, pH 7.2) and aeration and agitation culture at 28 ° C. for 70 hours (aeration rate 7.5 L / min, agitation) 100-250 rpm)). After completion of the culture, the obtained culture solution 24L was subjected to a continuous centrifuge to separate the supernatant and the cells. In addition, the bacterial cells were extracted with 20 L of methanol, and the obtained methanol extract was concentrated under reduced pressure, and the methanol was distilled off and mixed with the supernatant.
After adding 2.4 L of Diaion HP-20 swollen with water to the mixture and stirring, the resin was collected by filtration, filled into a column, and washed with 6 L of 20% methanol. Subsequently, elution was performed with 41 L of 80% acetone, the eluate was concentrated under reduced pressure, and acetone was distilled off. The remaining aqueous solution was concentrated to dryness under reduced pressure to obtain a crude extract.
This was dissolved in a small amount of dimethyl sulfoxide, and YMC-GEL ODS-AM 120-S50 (YMC Co., Ltd.) previously equilibrated with a mixed solution of acetonitrile-10 mM phosphate buffer (pH 7.0) = 2: 8. Column) (inner diameter 60 mm, length 950 mm). The mixture was washed with a mixed solvent having the same composition and subsequently eluted with a mixed solvent having a composition of 35:65.
The eluate was analyzed by a high performance liquid chromatograph, and EM 2487A, B, C (column: J'sphere ODS-H80, inner diameter 4.6 mm, length 75 mm, manufactured by YMC Co., Ltd., moving bed: acetonitrile-20 mM phosphate buffer) (PH 7.0) = 35: 65, flow rate: 1 mL / min, detection: ultraviolet absorption at 260 nm, retention time; A: 2.5 min, B: 6.1 min, C: 6.3 min) are collected. Fractions containing EM 2487 A, B, C were obtained. After acetonitrile was distilled off under reduced pressure, Diaion HP-20 swollen with water was added and stirred, and then the resin was packed into a column and washed with 20% methanol. Subsequently, elution was performed with 80% acetone, the eluate was concentrated under reduced pressure, and acetone was distilled off. The remaining aqueous solution was concentrated to dryness under reduced pressure to obtain a crude extract.
This was dissolved in a small amount of dimethyl sulfoxide, and preparative HPLC (column: J'sphere ODS-H80, inner diameter 20 mm, length 250 mm, manufactured by YMC Co., Ltd., moving bed: acetonitrile-20 mM phosphate buffer (pH 7.0) = 35:65, flow rate: 10 mL / min, detection: ultraviolet absorption at 260 nm, retention time; A: 12.0 min, B: 22.0 min, C: 26.0 min). This fractionation was repeated, and the eluates showing the peaks of EM 2487 A, B, and C were combined. Acetonitrile was distilled off under reduced pressure, then Diaion HP-20 swollen with water was added and stirred, and then the resin was added. The column was packed and washed with 20% methanol. Subsequently, elution was performed with 80% acetone, the eluate was concentrated under reduced pressure, and acetone was distilled off. The remaining aqueous solution was concentrated to dryness under reduced pressure to obtain 432 mg, 44.8 mg, and 22.4 mg of pure white powders of EM 2487A, B, and C, respectively.
EM 2487A purification and structural analysis data
1. Color and properties: white powder
2. Molecular formula: C32H57N5O16P2
3. Mass spectrum (FAB-MS): m / z 828 (MH)−
4). Specific rotation: [α]D 22 -7.9 (c 1.3, MeOH)
5. Ultraviolet absorption spectrum:
In neutral methanol: λmax nm (ε): 262 (34200)
In acidic methanol: λmax nm (ε): 262 (36100)
In basic methanol: λmax nm (ε): 260 (27100)
6). Red external absorption spectrum:
Measured in KBr powder. The main absorption is shown. (Wave number, cm-1)
2920, 2850, 1680, 1470, 1240, 1070, 880, 720, 550
7).1H NMR spectrum,13C NMR spectra and HMBC correlations
Table 1 shows the results of measurement in a deuterated methanol solution. The table shows the carbon position of the compound, its analytical data, that is, the chemical shift table of EM 2487A (Chemical Shift Table of 2487A), the correlation between carbons (HMBC correlations), and the reference value (Reference).
Soluble: neutral water, methanol, dimethyl sulfoxide
Insoluble: hexane, ethyl acetate, chloroform
9. High performance liquid chromatography:
Column: J'sphere ODS-H80, inner diameter 4.6 mm, length 75 mm (manufactured by YMC Corporation)
Solvent: acetonitrile-20 mM phosphate buffer (pH 7.0) = 35: 65
Flow rate: 1 mL / min
Detection wavelength: UV absorption at 260 nm
Holding time: 2.5 min
EM 2487B purification and structural analysis data
1. Color and properties: white powder
2. Molecular formula: C33H58N5O16P2
3. Mass spectrum (FAB-MS): m / z 842 (MH)−
4).1H NMR spectrum
Table 2 shows the results of measurement in deuterated methanol solution.
Soluble: neutral water, methanol, dimethyl sulfoxide
Insoluble: hexane, ethyl acetate, chloroform
6). High performance liquid chromatography:
Column: J'sphere ODS-H80, inner diameter 4.6 mm, length 75 mm (manufactured by YMC Corporation)
Solvent: Acetonitrile-20 mM phosphate buffer (pH 7.0) = 35: 65
Flow rate: 1 mL / min
Detection wavelength: UV absorption at 260 nm
Holding time: 6.1 min
EM 2487C purification and structural analysis data
1. Color and properties: white powder
2. Molecular formula: C33H58N5O16P2
3. Mass spectrum (FAB-MS): m / z 842 (MH)−
4).1H NMR spectrum
Table 3 shows the results of measurement in deuterated methanol solution.
Soluble: neutral water, methanol, dimethyl sulfoxide
Insoluble: hexane, ethyl acetate, chloroform
6). High performance liquid chromatography:
Column: J'sphere ODS-H80, inner diameter 4.6 mm, length 75 mm (manufactured by YMC Corporation)
Solvent: Acetonitrile-20 mM phosphate buffer (pH 7.0) = 35: 65
Flow rate: 1 mL / min
Detection wavelength: UV absorption at 260 nm
Holding time: 6.3 min
Example 2 Tat inhibitory activity
(1) Construction of reporter gene
To screen for compounds that inhibit Tat activity, a reporter gene was constructed that can measure transcription from the HIV LTR containing the TAR region. TNF-α reporter plasmid TNF-a-PLAP-PGK-neo containing a TNF-α promoter and secreted alkaline phosphatase PLAP followed by Neo resistance gene reported by Goto et al. (Goto M. et al., Mol. Pharmacol. 49: 860 (1996)) at the XbaI-HindIII site containing the TNF-α promoter, pUC obtained from the National Institute of Allergy and Infectious Disease AIDS Research and Reference Reagent Programmable B cells from ENC The XbaI-HindIII site containing it was inserted to construct the reporter gene pPGKF.
(2) Construction of Tat expression gene
Tat 2nd. Because there is a report that an exon is necessary for transcriptional activation from an HIV LTR integrated in a cell chromosome (Jiang KTJ of Biol. Chem. 268 (33): 24940 (1993)). The Tat expression vector pSV2 Tat obtained from the National Institute of Allergy and Infectious Disease AIDS Research and Reference Reagent Program 2nd. An exon sequence (Collman R. et al., J. of Virol. 66 (12): 7517 (1992)) was added. After annealing the synthetic oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2, it was subjected to an extension reaction with Vent polymerase (manufactured by New England Biochemistry), digested with BamHI and BglII, inserted into the BamHI-BglII site of pSV2 Tat, and pSV2 Tat-long-log Built.
(3) Gene introduction by electroporation
10 to 40 μg of DNA containing the gene sequence to be introduced was suspended in 1 ml of serum-free RPMI medium 3 × 106To 1 × 107The human leukemia cell CEM was mixed and allowed to stand at 4 ° C. for 10 minutes, and then energized under conditions of 300 V and 1000 μF with Gene Pulser (Bio-Rad). The cell suspension is immediately diluted in RPMI medium (10% FCS / RPMI medium) containing 10% FCS, and CO2Cultured in an incubator.
(4) Measurement of alkaline phosphatase activity
In order to inactivate alkaline phosphatase contained in FCS in the test solution, it was treated at 65 ° C. for 20 to 30 minutes. In 96-well plate for chemiluminescence measurement, 10 μl of test solution, carbonate buffer (0.28M Ma2CO3buffer pH 10.0, 8 mM MgSO4) 50 μl and Lumistein (Sumitomo Metals) 50 μl were added and allowed to stand at room temperature for 1 hour, and then chemiluminescence was measured with a microplate luminometer LB96P (Berthold).
(5) Establishment of reporter cell line
In order to screen for compounds that inhibit Tat activity, a reporter cell line expressing a reporter gene was established. The reporter gene pPGKF was introduced into human leukemia cells CEM cells by electroporation. CO2After overnight culture in an incubator, the suspension was suspended in a 10% FCS / RPMI medium containing 0.8 mg / ml geneticin (manufactured by Sigma) and cultured in a 24-well plate. Two weeks later, the alkaline phosphatase activity in the culture supernatant of the well in which the cells had grown was measured, and PLAP producing cells were cloned from the active wells by the limiting dilution method.
After the cloned cells were grown, clones with increased PLAP production by introduction of Tat-expressing gene and TNF were selected. The selected reporter cells were subcultured and used to measure Tat inhibitory activity.
(6) Measurement of living cells by MTT method
To cells cultured in a 96-well plate, add 20 μl of a 7.5 mg / ml MTT solution and add CO.2The cells were cultured for 2 hours in an incubator. 150 μl of the supernatant was removed so as not to suck cells, and 100 μl of isopropanol containing 10% Triton X-100 was added to solubilize the dye (formazan) formed by reducing MTT. The absorbance at 540 nm of each well was measured using a plate reader (Bio-Rad Model 3550), and used as an index of living cells.
(7) EM 2487A, B, C inhibitory activity against Tat expressed in cells
After introducing the Tat expression gene pSV2 Tat-long gene into the reporter cells by electroporation, CO2Cultured in an incubator. In addition, cells not transfected with a Tat expression gene were cultured in the same manner. The next day, the cultured cells were collected by centrifugation at 1000 rpm for 5 minutes and 1.25 × 10 in 10% FCS / RPMI medium.5The cell suspension was adjusted to a concentration of / ml.
The test sample was dissolved in DMSO and diluted 100-fold with 10% FCS / RPMI to prepare a sample dilution solution.
To a 96-well plate, add 20 μl of sample diluent, 20 μl of 10% FCS / RPMI medium with or without 20 ng / ml TNF, 160 μl of cell suspension, and add CO2The cells were cultured for 2 days in an incubator. 10 μl of the culture supernatant was collected and the alkaline phosphatase activity was measured. Further, the cytotoxicity of the specimen was measured by the MTT method.
Table 4 shows the alkaline phosphatase activity in the culture supernatant and the absorbance of the MTT method (in parentheses are percentages where the control with no drug added is 100%). EM 2487A, B, C did not affect the production of alkaline phosphatase in cells not transfected with the Tat-expressing gene at a concentration of 10 μg / ml, and showed no cytotoxicity. Although the amount of alkaline phosphatase produced by the introduction of the Tat-expressing gene was promoted about 20 times, EM 2487A, B, and C suppressed only the production of alkaline phosphatase promoted by Tat.
In addition, in the presence of TNF, alkaline phosphatase production in Tat-expressed gene introduced and non-introduced cells was further accelerated about 3 times. In this case as well, EM 2487A, B, and C did not affect the production of alkaline phosphatase in cells that did not introduce the Tat expression gene, but only suppressed the production of alkaline phosphatase that was promoted by the introduction of the Tat expression gene.
(8) Inhibitory activity of EM 2487A against Tat supplied from outside the cell
The reporter cells were cultured in serum-free medium for 2 hours in the presence of 2 μg / ml Tat (manufactured by Immuno Diagnostics), 1 mM Dithiothratrol, 100 μM Chloroquin, suspended in 10% FCS / RPMI medium after centrifugation at 1000 rpm for 5 minutes.2Cultured in an incubator. In addition, cells treated in the same manner in the absence of Tat were also cultured. The next day, cultured cells were collected by centrifugation at 1000 rpm for 5 minutes, and 1.25 × 10 in 10% FCS / RPMI medium.5The cell suspension was adjusted to a concentration of / ml.
To a 96-well plate, add 20 μl of sample diluent, 20 μl of 10% FCS / RPMI medium with or without 20 ng / ml TNF, 160 μl of cell suspension, and add CO2The cells were cultured for 2 days in an incubator. 10 μl of the culture supernatant was collected and the alkaline phosphatase activity was measured. Further, the cytotoxicity of the specimen was measured by the MTT method.
By supplying Tat from the outside of the cell, the production amount of alkaline phosphatase was promoted about 30 times. In the presence of TNF, alkaline phosphatase production was further accelerated about 3 times. Table 4 shows the alkaline phosphatase activity in the culture supernatant and the absorbance of the MTT method (in parentheses are percentages where the control with no drug added is 100%). Regardless of the presence or absence of TNF, EM 2487A does not affect the production of alkaline phosphatase in cells that do not supply Tat at a concentration of 10 μg / ml, only the production of alkaline phosphatase promoted by the introduction of the Tat expression gene. Suppressed. The results are shown in Table 4 together with no treatment (None Treat).
Human leukemia cell Molt-4 was infected with HIV-1 HTLV-IIIB strain. HIV infected cells and uninfected cells 1 × 105The cells / ml were cultured with various concentrations of drugs at 37 ° C., and after 4 days, diluted 1: 5 with a culture solution containing the same concentration of drugs. On the seventh day from the start of the culture, the number of viable cells was measured by the MTT method, and the cytotoxicity inhibitory activity (anti-HIV activity) and cytotoxicity of EM 2487A, B, C were determined.
Drug concentration that exhibits 50% cytotoxic activity against HIV-infected cells, with the number of living cells of HIV-uninfected cells without added drug being 100% and the number of living cells of HIV-infected cells without added drug being 0% EC50Asked.
Drug concentration CC showing 50% cytotoxic activity against non-infected cells, assuming that the number of living cells in non-infected cell culture without addition of drug is 100% and the number of living cells in the medium alone is 0%50Asked.
As shown in Table 5, EM 2487A, B and C showed anti-HIV activity in HIV proliferation in the Molt-4 acute infection system. The table shows the 50% cytotoxic inhibition concentration (EC50(ΜM)), 50% cytotoxic concentration (CC50(ΜM)), and the selectivity index (selectivity index).
Human leukemia cells MT-4 were infected with HIV-1 HTLV-IIIB strain. HIV infected cells and uninfected cells 1 × 105Cells / ml were cultured at 37 ° C. with various concentrations of drugs. On the 4th day from the start of culture, live cells were measured by the MTT method, and cytotoxicity-inhibiting activity (anti-HIV activity) and cytotoxicity due to drug addition were determined.
As shown in Table 6, Ro 24-7429 had no effect in the MT-4 acute infection system, but EM 2487A showed anti-HIV activity.
PBMC stimulated with PHA 1 × 105cells / well were infected with HIV-1 HTLV-IIIB strain and cultured at 37 ° C. with various concentrations of drugs. On the 6th day from the start of culture, the amount of HIV is measured by ELISA to determine the amount of HIV, and live cells are measured by the MTT method to determine HIV production inhibitory activity (anti-HIV activity) and cytotoxicity by adding drugs. went.
Drug concentration EC showing HIV growth inhibitory activity of 50% of HIV-free p24 antigen amount 100%50Asked.
As shown in Table 7, EM 2487A was found to have anti-HIV activity in the PBMC acute infection system.
Example 6 Anti-HIV activity in OM 10.1 persistent infection system
Promyelocytic OM10.1 cells infected with HIV-1 1 × 105Cells / well were pretreated with various concentrations of drugs for 2 hours, then stimulated with 1 ng / ml TNF-α, and further cultured in the presence of the drugs. On the third day from the start of culture, the amount of HIV is measured by ELISA and the amount of HIV is quantified, and live cells are measured by the MTT method to determine HIV production inhibitory activity (anti-HIV activity) and cytotoxicity by adding drugs. went.
As shown in Table 7, EM 2487A was found to have anti-HIV activity in the OM10.1 persistent infection system.
Since combined use with a reverse transcriptase inhibitor and a protease inhibitor is expected to further suppress HIV growth, it can be used to delay the emergence of resistant viruses. In particular, it is useful for patients who are infected with HIV who has acquired resistance to nucleoside and non-nucleoside reverse transcriptase inhibitors and protease inhibitors, since the point of action is different and resistance does not cross.
[Sequence Listing]
Claims (6)
で表わされる化合物、その塩又はその水和物。The following general formula (I):
Or a salt or hydrate thereof.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18179998 | 1998-06-29 | ||
| PCT/JP1999/003428 WO2000000631A1 (en) | 1998-06-29 | 1999-06-25 | Novel physiologically active substances inhibiting the proliferation of human immunodeficiency virus (hiv) |
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| Publication Number | Publication Date |
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| JP3875024B2 true JP3875024B2 (en) | 2007-01-31 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2000557384A Expired - Fee Related JP3875024B2 (en) | 1998-06-29 | 1999-06-25 | Novel physiologically active substance that inhibits human immunodeficiency virus (HIV) growth |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JP3875024B2 (en) |
| AU (1) | AU4289899A (en) |
| WO (1) | WO2000000631A1 (en) |
-
1999
- 1999-06-25 AU AU42898/99A patent/AU4289899A/en not_active Abandoned
- 1999-06-25 JP JP2000557384A patent/JP3875024B2/en not_active Expired - Fee Related
- 1999-06-25 WO PCT/JP1999/003428 patent/WO2000000631A1/en active Application Filing
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| WO2000000631A1 (en) | 2000-01-06 |
| AU4289899A (en) | 2000-01-17 |
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