JP2762522B2 - Angiogenesis inhibitor - Google Patents
Angiogenesis inhibitorInfo
- Publication number
- JP2762522B2 JP2762522B2 JP5336889A JP5336889A JP2762522B2 JP 2762522 B2 JP2762522 B2 JP 2762522B2 JP 5336889 A JP5336889 A JP 5336889A JP 5336889 A JP5336889 A JP 5336889A JP 2762522 B2 JP2762522 B2 JP 2762522B2
- Authority
- JP
- Japan
- Prior art keywords
- wf2015a
- column
- methanol
- angiogenesis inhibitor
- angiogenesis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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Description
【発明の詳細な説明】 産業上の利用分野 この発明は新規な血管新生阻害剤に関するものであ
る。さらに詳細には、この発明はWF2015Aおよび／また
はWF2015Bを有効成分として含有する血管新生阻害剤に
関するものである。The present invention relates to a novel angiogenesis inhibitor. More specifically, the present invention relates to an angiogenesis inhibitor containing WF2015A and / or WF2015B as an active ingredient.
従来の技術及び発明が解決しようとする問題点 従来、血管新生阻害剤としては種々のものが知られて
いるが、これらのものは医薬品として必ずしも満足され
る性質を備えているものではなかった。そこで、この発
明者等は新しいタイプの血管新生阻害剤の開発を企図し
た。2. Description of the Related Art Problems to be solved by the prior art and the invention Conventionally, various angiogenesis inhibitors have been known, but these do not always have properties that are satisfactory as pharmaceuticals. Thus, the present inventors have attempted to develop a new type of angiogenesis inhibitor.
発明の構成および効果 この発明の血管新生阻害剤の有効成分であるWF2015A
およびWF2015Bは下記一般式により示される （式中、Ｘが式 で示される基である化合物がWF2015Aを意味し、Ｘが式 で示される基である化合物がWF2015Bを意味する） 上記一般式（Ｉ）で示される化合物は例えば特願昭63
-262044号（特開平1-132390号）記載の方法、より具体
的には後記製造例で示す方法により製造することができ
る。Configuration and Effect of the Invention WF2015A which is an active ingredient of the angiogenesis inhibitor of the present invention
And WF2015B are represented by the following general formula (Where X is the formula The compound represented by the formula means WF2015A, and X is a group represented by the formula The compound represented by the formula represents WF2015B.) The compound represented by the above general formula (I) is described in, for example,
-262044 (Japanese Patent Application Laid-Open No. 1-132390), and more specifically, it can be produced by the method described in the following Production Examples.
後記製造例で得られるWF2015AおよびＢは下記の理化
学的性質を有する： （１）WF2015A ａ）分子量 410［FAB-MS:m/z411（Ｍ＋Ｈ）］ ｂ）元素分析 C 64.55:H 8.06;O 27.39（差として算出） ｃ）比旋光度 ▲［α］23 D▼＝−52°（Ｃ＝1.0,CH3OH） ｄ）UV吸収スペクトル 末端吸収（CHCl3中） ｅ）IR吸収スペクトル ｆ）1H NMR吸収スペクトル（CDCl3） δ:6.95（1H,dd,J＝15.5および4.5Hz）， 6.20（1H,dd,J＝15.5および2Hz）， 5.70（1H,広いｓ）， 5.20（1H,広いt,J＝7Hz）， 4.31（1H,m）， 3.90（1H,m）， 3.68（1H,dd,J＝11および3Hz）， 3.44（3H,s）， 2.98（1H,d,J＝4.2Hz）， 2.62（1H,dd,J＝6.5および6Hz）， 2.56（1H,d,J＝4.2Hz）， 2.36（1H,m）， 2.16（1H,m）， 2.10（1H,m）， 2.00（1H,m）， 1.98（1H,d,J＝11Hz）， 1.87（1H,m）， 1.74（3H,d,J＝1Hz）， 1.66（3H,d,J＝1Hz）， 1.21（3H,s）， 1.14（3H,d,J＝6.5Hz）， 1.07（1H,m）， ｇ）13C NMR吸収スペクトル（CDCl3） δ:165.8（ｓ）， 146.6（ｄ）， 135.0（ｓ）， 121.8（ｄ）， 118.3（ｄ）， 79.4（ｄ）， 74.5（ｄ）， 69.9（ｄ）， 66.3（ｄ）， 61.1（ｄ）， 59.4（ｓ）， 58.9（ｓ）， 56.7（ｑ）， 50.7（ｔ）， 48.2（ｄ）， 29.2（ｔ）， 27.2（ｔ）， 25.6（ｑ）， 25.6（ｔ）， 17.9（ｑ）， 17.1（ｑ）， 13.7（ｑ）， ｈ）溶解性 可溶：クロロホルム、メタノール、アセトン、エタノー
ル 不溶:n−ヘキサン、水 ｉ）呈色反応 陽性：沃素蒸気との反応 陰性：ニンヒドリン反応、モリッシュ反応、塩化第二鉄
反応 ｊ）物質の性質 中性物質 （２）WF2015B ａ）分子量 447［FAB-MS:m/z469（ｍ＋Na）］ ｂ）元素分析 C 58.03;H 7.94;Cl 7.31;O 26.72（差として算出） ｃ）比旋光度 ▲［α］23 D▼＝−248°（Ｃ＝2.0,CHCl3） ｄ）UV吸収スペクトル 末端吸収（CHCl3中） ｅ）IR吸収スペクトル ｆ）1H NMR（400MHz,CDCl3） δ:6.99（1H,dd,J＝５および15.5Hz）， 6.20（1H,dd,J＝２および15.5Hz）， 5.55（1H,m）， 5.18（1H,m）， 4.36（1H,m）， 3.98（1H,dq,J＝４および6Hz）， 3.87（1H,d,J＝11Hz）， 3.49（1H,d,J＝11Hz）， 3.31（1H,m）， 3.30（3H,s）， 2.97（1H,t,J＝6.5H,z）， 2.50-2.40（2H,m）， 2.17（1H,m）， 2.05（1H,m）， 1.90-1.75（2H,m）， 1.73（3H,s）， 1.66（3H,s）， 1.49（3H,s）， 1.40（1H,広いd,J＝14Hz）， 1.20（3H,d,J＝6Hz） ｇ）13C NMR（100MHz,CDCl3） δ:165.7（ｓ）， 146.3（ｄ）， 134.8（ｓ）， 122.4（ｄ）， 118.2（ｄ）， 78.7（ｄ）， 76.2（ｓ）， 74.5（ｄ）， 70.0（ｄ）， 66.1（ｄ）， 64.0（ｓ）， 62.3（ｄ）， 56.7（ｄ）， 50.5（ｔ）， 43.3（ｄ）， 29.1（ｔ）， 27.5（ｔ）， 25.8（ｑ）， 23.5（ｔ）， 22.2（ｑ）， 17.9（ｑ）， 17.6（ｑ） ｈ）溶解性 可溶：クロロホルム、メタノール、アセトン、エタノー
ル 不溶:n−ヘキサン、水 ｉ）呈色反応 陽性：沃素蒸気との反応 陰性：ニンヒドリン反応、モリッシュ反応、塩化第二鉄
反応 ｊ）物質の性質 中性物質 以上の理化学的性質および別途研究の結果から、WF20
15AおよびＢの化学構造は前記のように決定された。WF2015A and B obtained in Preparation Examples described below have the following physicochemical properties: (1) WF2015A a) Molecular weight 410 [FAB-MS: m / z411 (M + H)] b) Elemental analysis C 64.55: H 8.06; O 27.39 (Calculated as the difference) c) Specific rotation ▲ [α] 23 D ▼ = -52 ° (C = 1.0, CH 3 OH) d) UV absorption spectrum Terminal absorption (in CHCl 3 ) e) IR absorption spectrum f) 1 H NMR absorption spectrum (CDCl 3 ) δ: 6.95 (1H, dd, J = 15.5 and 4.5 Hz), 6.20 (1H, dd, J = 15.5 and 2 Hz), 5.70 (1H, wide s), 5.20 ( 1H, wide t, J = 7Hz), 4.31 (1H, m), 3.90 (1H, m), 3.68 (1H, dd, J = 11 and 3Hz), 3.44 (3H, s), 2.98 (1H, d, J = 4.2Hz), 2.62 (1H, dd, J = 6.5 and 6Hz), 2.56 (1H, d, J = 4.2Hz), 2.36 (1H, m), 2.16 (1H, m), 2.10 (1H, m ), 2.00 (1H, m), 1.98 (1H, d, J = 11 Hz), 1.87 (1H, m), 1.74 (3H, d, J = 1 Hz), 1.66 (3H, d, J = 1 Hz), 1.21 (3H, s), 1.14 (3H, d, J = 6.5 Hz), 1.07 (1H, m), g) 13 C NMR absorption spectrum (CDCl 3 ) δ: 165.8 (s), 146.6 (d), 135.0 ( s), 121.8 (d), 118.3 (d), 79.4 (d), 74.5 (d), 69.9 (d), 66.3 (d), 61.1 (d), 59.4 (s), 58.9 (s), 56.7 ( q), 50.7 (t), 48.2 (d), 29.2 (t), 27.2 (t), 25.6 (q), 25.6 (t), 17.9 ( q), 17.1 (q), 13.7 (q), h) Solubility Soluble: chloroform, methanol, acetone, ethanol Insoluble: n-hexane, water i) Color reaction Positive: reaction with iodine vapor Negative: ninhydrin reaction , Morish reaction, ferric chloride reaction j) Properties of substances Neutral substances (2) WF2015B a) Molecular weight 447 [FAB-MS: m / z469 (m + Na)] b) Elemental analysis C 58.03; H 7.94; Cl 7.31; O 26.72 (calculated as the difference) c) Specific rotation ▲ [α] 23 D ▼ = -248 ° (C = 2.0, CHCl 3 ) d) UV absorption spectrum Terminal absorption (in CHCl 3 ) e) IR absorption spectrum f) 1 H NMR (400 MHz, CDCl 3 ) δ: 6.99 (1 H, dd, J = 5 and 15.5 Hz), 6.20 (1 H, dd, J = 2 and 15.5 Hz), 5.55 (1 H, m), 5.18 ( 1H, m), 4.36 (1H, m), 3.98 (1H, dq, J = 4 and 6Hz), 3.87 (1H, d, J = 11Hz), 3.49 (1H, d, J = 11Hz), 3.31 (1H , m), 3.30 (3H, s), 2.97 (1H, t, J = 6.5H, z), 2.50-2.40 (2H, m), 2.17 (1H, m), 2.05 (1H, m), 1.90- 1.75 (2H, m), 1.73 (3H, s), 1.66 (3H, s), 1.49 (3H, s), 1.40 (1H, wide d, J = 14Hz), 1.20 (3H, d, J = 6Hz) g) 13 C NMR (100 MHz, CDCl 3 ) δ: 165.7 (s), 146.3 (d), 134.8 (s), 122.4 (d), 118.2 (d), 78.7 (d), 76.2 (s), 74.5 ( d), 70.0 (d), 66.1 (d), 64.0 (s), 62.3 (d), 56.7 (d), 50.5 (t), 43.3 (d), 29.1 (t), 27.5 (t), 25.8 ( q), 23.5 (t), 22.2 (q), 17.9 (q), 17.6 (q) h) Solubility Soluble: chloroform, methanol , Acetone, ethanol Insoluble: n-hexane, water i) Color reaction Positive: reaction with iodine vapor Negative: ninhydrin reaction, Morish reaction, ferric chloride reaction j) Properties of substances Neutral substances The above physicochemical properties and According to the results of a separate study, WF20
The chemical structures of 15A and B were determined as described above.
次に、W2015AおよびＢの生物学的な性質を下記試験例
により説明する。Next, the biological properties of W2015A and B are described by the following test examples.
試験例１ 人臍帯静脈内皮細胞に対するWF2015AおよびＢの作用 フィブロネクチンで被覆した96穴マイクロタイタープ
レートに、15％牛胎児血清100μg/ml ECGS（Endothelia
l cell growth supplement）および10μg/mlヘパリンを
含むMCDB151培地を加えた後、一定濃度のWF2015Aまたは
WF2015Bのメタノール溶液を加え種々の濃度の希釈液を
作製した。さらに、上記成分を含むMCDB151培地を用い
て人臍帯静脈内皮細胞液を調製し、２×103個の割合で
各穴（final volume200μｌ）に接種し、CO2インキュベ
ーター中で37℃,5日間培養した。培養後MTT法T.Mosman;
J.Immunol.Methods 65,55,1983）により細胞増殖度合を
測定し、WF2015AまたはWF2015Bに対する血管内皮細胞の
増殖抑制率を算出した。Test Example 1 Effect of WF2015A and B on human umbilical vein endothelial cells A 96-well microtiter plate coated with fibronectin was loaded with 15% fetal calf serum 100 μg / ml ECGS (Endothelia).
l cell growth supplement) and MCDB151 medium containing 10μg / ml heparin, and then add a certain concentration of WF2015A or
Methanol solutions of WF2015B were added to prepare dilute solutions of various concentrations. Furthermore, a human umbilical vein endothelial cell solution was prepared using the MCDB151 medium containing the above components, inoculated into each well (final volume 200 μl) at a rate of 2 × 10 3 cells, and cultured at 37 ° C. for 5 days in a CO 2 incubator. did. After culture MTT method T. Mosman;
J. Immunol. Methods 65 , 55, 1983), the degree of cell proliferation was measured, and the growth inhibition rate of vascular endothelial cells against WF2015A or WF2015B was calculated.
その結果、WF2015AおよびWF2015Bは血管内皮細胞の増
殖に対して強い抑制効果を示し、それらのIC50値は共に
２×102pg/mlであった。As a result, WF2015A and WF2015B showed a strong inhibitory effect on the proliferation of vascular endothelial cells, and their IC 50 values were both 2 × 10 2 pg / ml.
試験例２ 鶏受精卵の漿尿膜法によるWF2015Aの血管新生に対する
阻害作用： ３日令の鶏受精卵から無菌的に注射針で卵白を約1ml
抜き取り、歯科用ドリルで漿尿膜を傷つけないように卵
殻に傷をつけ、ピンセットで殻を破って約１cm2の窓を
開け、スキンクロージャーで窓をふさいで37℃で孵卵し
た。24時間後に、あらかじめ一定濃度のWF2015Aメタノ
ール溶液2.5μｌと0.1％メチルセルロース水溶液2.5μ
ｌを混合し、乾燥させて作製したサンプルデイスクを漿
尿膜上に置き、再び窓をスキンクロージャでふさいで37
℃で孵卵を続けた。さらに、３日後に漿尿膜に新生され
る血管の発達状態からWF2015Aの血管新生に対する阻害
作用を観察した。Test Example 2 Inhibitory effect of WF2015A on angiogenesis of chicken fertilized egg by chorioallantoic membrane method: Approximately 1 ml of egg white from a 3 day old chicken fertilized egg aseptically with a syringe needle
Sampling, scratching the eggshell so as not to damage the chorioallantoic membrane with a dental drill, open the windows of about 1cm 2 to beat the shell with a pair of tweezers, and incubation at 37 ℃ blocking the window in the skin closure. Twenty-four hours later, a fixed concentration of WF2015A methanol solution 2.5 μl and a 0.1% methylcellulose aqueous solution 2.5 μl
The sample disk prepared by mixing and drying was placed on the chorioallantoic membrane, and the window was closed again with a skin closure.
Incubation was continued at ℃. Further, after 3 days, the inhibitory effect of WF2015A on angiogenesis was observed from the state of development of the blood vessels that are formed in the chorioallantoic membrane.
その結果、WF2015Aの10μｇ投与群では、ほぼ完全な
（約100％）血管新生阻害作用が観察された。As a result, in the 10 μg administration group of WF2015A, almost complete (about 100%) angiogenesis inhibitory action was observed.
試験例３ 家兎角膜内でのWF2015Aの血管新生に対する阻害作用： 兎角膜内での血管新生作用は、M.A,Gimbroneらの方法
（Journal National Cancer Institute 52,413,1974）
により検討した。すなわち、兎（ニュージーランドホワ
イト種、雌９週令）を用いてネンブタール麻酔下、角膜
中央部にメスで約2mmの切れ目を入れ、その切れ目から
眼科用虹彩スパテルで角膜間質部にポケット状構造を作
った。そのポケット底に、あらかじめ作製しておいた一
定量のWF2015Aを含むElvax（ethylenevinyl acetate）
ペレット（1mm角）を挿入し、さらにFGF（Fibroblast G
rowth Factor,ウシ脳由来）１μｇを含有するElvaxペレ
ットを続いて挿入し、角膜輪部からFGFペレットに向か
って延びてくる新生血管を経日的に（２週間）観察し
た。その結果、WF2015AはFGFによって新生される血管を
200μｇ投与群では完全に（約100％）阻害した。Test Example 3 Inhibitory effect of WF2015A on angiogenesis in rabbit cornea: Angiogenic effect on rabbit cornea was determined by the method of MA, Gimbrone et al. (Journal National Cancer Institute 52 , 413, 1974).
It was examined by. That is, a rabbit (New Zealand White, female, 9 weeks old) underwent anesthesia with Nembutal to make a cut of about 2 mm in the center of the cornea with a scalpel, and from that cut, a pocket-like structure was formed in the corneal interstitium with an ophthalmic iris spatula. Had made. Elvax (ethylenevinyl acetate) containing a certain amount of WF2015A prepared in advance in the bottom of the pocket
Insert a pellet (1mm square) and add FGF (Fibroblast G
An Elvax pellet containing 1 μg of rowth Factor (from bovine brain) was subsequently inserted, and new blood vessels extending from the limbus toward the FGF pellet were observed daily (two weeks). As a result, WF2015A is able to
In the 200 μg administration group, the inhibition was complete (about 100%).
試験例４ WF2015AおよびＢの急性毒性 WF2015AまたはＢの生理食塩水溶液を、１日１回５日
間、ICR系マウス５匹（雌性、５週令）の腹腔内に投与
（１回投与量100mg/kg）した。その結果、マウスに異常
な症状は認められなかった。Test Example 4 Acute toxicity of WF2015A and B A physiological saline solution of WF2015A or B was intraperitoneally administered to 5 ICR mice (female, 5 weeks old) once a day for 5 days (single dose: 100 mg / kg) )did. As a result, no abnormal symptoms were observed in the mice.
以上の試験例から明らかなように、この発明の血管新
生阻害剤は顕著な血管新生阻害作用を有し、血管の異常
増殖によって引き起こされる疾患、例えば、糖尿病性網
膜症、リュウマチ性関節炎、未熟児網膜症、老人性黄班
部変性等の予防または治療に使用される。As is clear from the above test examples, the angiogenesis inhibitor of the present invention has a remarkable angiogenesis inhibitory action, and is caused by abnormal growth of blood vessels, for example, diabetic retinopathy, rheumatoid arthritis, premature infant It is used for prevention or treatment of retinopathy, senile macular degeneration and the like.
この発明の血管新生阻害剤の有効成分は医薬として許
容しうる担体と混合して、例えば、カプセル、錠剤、顆
粒剤、粉剤、口内錠、舌下錠、液剤などの製剤の形でヒ
トを含む哺乳動物に経口または非経口的に投与できる。The active ingredient of the angiogenesis inhibitor of the present invention is mixed with a pharmaceutically acceptable carrier and includes humans in the form of, for example, capsules, tablets, granules, powders, buccal tablets, sublingual tablets, liquid preparations and the like. It can be administered orally or parenterally to a mammal.
医薬として許容しうる担体としては、製薬の目的に慣
用される種々の有機または無機担体、たとえば賦形剤
（たとえば、スクロース、でん粉、マンニット、ソルビ
ット、ラクトース、グルコース、セルロース、タルク、
りん酸カルシウム、炭酸カルシウ等）、結合剤（セルロ
ース、メチルセルロース、ヒドロキシプロピルセルロー
ス、ポリプロピルピロリドン、ゼラチン、アラビアゴ
ム、ポリエチレングリコール、スクロース、でん粉
等）、崩壊剤（たとえば、でん粉、カルボキシメチルセ
ルロース、カルボキシメチルセルロースカルシウム、ヒ
ドロキシプロピルでん粉、グリコールでん粉ナトリウ
ム、重炭酸ナトリウム、りん酸カルシウム、くえん酸カ
ルシウム等）、滑沢剤（たとえば、ステアリン酸マグネ
シウム、エーロジル、タルク、ラウリル硫酸ナトリウム
等）、矯味剤（たとえば、くえん酸、メントール、グリ
シン、オレンジ粉末等）、保存剤（安息香酸ナトリウ
ム、重亜硫酸ナトリウム、メチルパラベン、プロピルバ
ラベン等）、安定剤（くえん酸、くえん酸ナトリウム、
酢酸等）、懸濁化剤（たとえば、メチルセルロース、ポ
リビニルピロリドン、ステアリン酸アルミニウム等）、
分散剤（たとえば、界面活性剤等）、水性希釈剤（たと
えば、水）、油類（たとえば、ごま油等）、基剤ワック
ス（たとえば、カカオ脂、ポリエチレングリコール、白
色ワセリン等）等が包含されうる。Pharmaceutically acceptable carriers include various organic or inorganic carriers commonly used for pharmaceutical purposes, such as excipients (eg, sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc,
Calcium phosphate, calcium carbonate, etc.), binders (cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch, etc.), disintegrants (eg, starch, carboxymethylcellulose, carboxymethylcellulose) Calcium, hydroxypropyl starch, glycol starch sodium, sodium bicarbonate, calcium phosphate, calcium citrate, etc., lubricants (eg, magnesium stearate, aerosil, talc, sodium lauryl sulfate, etc.), and flavoring agents (eg, kuen) Acid, menthol, glycine, orange powder, etc.), preservative (sodium benzoate, sodium bisulfite, methylparaben, propylparaben, etc.), stabilizer (kuen) , Sodium citrate,
Acetic acid, etc.), suspending agents (eg, methyl cellulose, polyvinylpyrrolidone, aluminum stearate, etc.),
Dispersants (eg, surfactants, etc.), aqueous diluents (eg, water), oils (eg, sesame oil, etc.), base waxes (eg, cocoa butter, polyethylene glycol, white petrolatum, etc.) and the like can be included. .
有効成分の用量は、疾患の種類、患者の体重および／
または年令、さらには投与経路等の種々の因子に応じて
変更すべきである。The dose of the active ingredient depends on the type of disease, the weight of the patient and / or
Alternatively, they should be changed according to various factors such as age, and furthermore, administration route.
WF2015AまたはＢの好ましい用量は、通常、注射の場
合には0.01〜10mg/kg/日、また経口投与の場合には0.5
〜50mg/kg/日の用量範囲から適宜選択される。The preferred dose of WF2015A or B is usually 0.01-10 mg / kg / day for injection and 0.5 for oral administration.
The dose is appropriately selected from a dose range of 5050 mg / kg / day.
以下の製造例は、この発明の血管新生阻害剤の有効成
分の具体的な製造法を説明するためのものである。The following production examples are for describing specific production methods of the active ingredient of the angiogenesis inhibitor of the present invention.
製造例１ 可溶性でん粉２％、とうもろこしでん粉１％、グルコ
ース１％、綿実粉１％、乾燥酵母１％、ペプトン0.5
％、コーンスチープリカー0.5％および炭酸カルシウム
0.2％を含有する水性培地（pH6.0）（160ml）を、500ml
容三角フラスコ19本に分注し、120℃で20分間滅菌し
た。スコレコバシデイウム・アレナリウムScolecobasid
ium arenarium）F-2015微工研条寄第1520号の斜面培養
物１白金耳を各培地に接種し、25℃で３日間振盪培養し
た。得られた培養物を、あらかじめ120℃で20分間滅菌
した200l容ジャーフアーメンター中の可溶性でん粉３
％、グルコース１％、小麦の胚芽１％、綿実粉0.5％お
よび炭酸カルシウム0.2％を含有する水性培地（120l）
に接種し、25℃で３日間培養した。Production Example 1 Soluble starch 2%, corn starch 1%, glucose 1%, cottonseed flour 1%, dry yeast 1%, peptone 0.5
%, Corn steep liquor 0.5% and calcium carbonate
500 ml of an aqueous medium (pH 6.0) (160 ml) containing 0.2%
The solution was dispensed into 19 Erlenmeyer flasks and sterilized at 120 ° C. for 20 minutes. Scolecobasidium arenalium
ium arenarium) One loopful of the slant culture of F-2015, Microtechnological Laboratory, No. 1520, was inoculated into each medium and cultured at 25 ° C. for 3 days with shaking. The resulting culture was washed with soluble starch 3 in a 200-liter jar arm mentor previously sterilized at 120 ° C. for 20 minutes.
%, Glucose 1%, wheat germ 1%, cottonseed powder 0.5% and calcium carbonate 0.2%
And cultured at 25 ° C. for 3 days.
こうして得た培養液を、珪藻土（20kg）を用いて濾過
した。濾液（95l）を酢酸エチル（95l）で抽出した。こ
の抽出操作を２回行い、抽出液を合せた。無水硫酸マグ
ネシウムで脱水後、酢酸エチル層を減圧下に濃縮した。
濃縮物をシリカゲルクロマトグラフィーカラム（１）
に付した。カラムをｎ−ヘキサン（3l）およびｎ−ヘキ
サン−酢酸エチル混液（1:1、3l）で洗った。活性画分
を酢酸エチル（4l）で溶出し、ついで減圧下に濃縮し
た。これをさらにシリカゲルカラム（400ml）に付し
た。クロロホルム（1.2l）およびクロロホルム−メタノ
ール混液（100:1、12l）でカラムを洗浄したのち、クロ
ロホルム−メタノール混液（75:1、50:1、および25:1）
で段階的に溶出した。活性画分を減圧下に濃縮し、残渣
を逆相シリカゲルODS逆カラムクロマトグラフィーに付
した。カラムをメタノール−水混液（1:1、300ml）で洗
浄したのち、メタノール−水混液（3:2および7:3、各30
0ml）で段階的に展開した。活性画分を減圧下に蒸発乾
固して、精製された無色油状のWF2015A（92mg）を得
た。The culture solution thus obtained was filtered using diatomaceous earth (20 kg). The filtrate (95l) was extracted with ethyl acetate (95l). This extraction operation was performed twice, and the extracts were combined. After dehydration with anhydrous magnesium sulfate, the ethyl acetate layer was concentrated under reduced pressure.
The concentrate is applied to a silica gel chromatography column (1)
Attached. The column was washed with n-hexane (3 l) and a mixture of n-hexane-ethyl acetate (1: 1, 3 l). The active fraction was eluted with ethyl acetate (4 l) and then concentrated under reduced pressure. This was further applied to a silica gel column (400 ml). After washing the column with chloroform (1.2 l) and a chloroform-methanol mixture (100: 1, 12 l), a chloroform-methanol mixture (75: 1, 50: 1, and 25: 1)
Eluted step by step. The active fraction was concentrated under reduced pressure, and the residue was subjected to reverse phase silica gel ODS reverse column chromatography. The column was washed with a methanol-water mixture (1: 1, 300 ml), and then methanol-water mixture (3: 2 and 7: 3, 30
0 ml). The active fraction was evaporated to dryness under reduced pressure to obtain purified colorless oily WF2015A (92 mg).
製造例２ スコレコバシディウム・アレナリウムF-2015（微工研
条寄第1520号）の斜面培養物の１白金耳を、可溶性でん
粉２％、とうもろこしでん粉１％、グリコース１％、綿
実粉１％、乾燥酵母１％、ペプトン0.5％、コーンスチ
ープリカー0.5％、NaCl 3％およびCaCO3 0.2％を含有す
る種培地（pH6.0）（160ml）を20本の500ml容三角フラ
スコの各々へ分注し常法通り滅菌したものに接種し、回
転振盪機を用いて250rpmにて25℃で72時間培養した。Production Example 2 One platinum loop of a slope culture of Scolecobacidium arenalium F-2015 (Jiko No. 1520) was added with 2% of soluble starch, 1% of corn starch, 1% of glucose and 1% of cottonseed powder. %, Dried yeast 1%, peptone 0.5%, corn steep liquor 0.5%, NaCl 3% and CaCO 3 0.2% (160 ml) were divided into each of 20 500 ml Erlenmeyer flasks. The mixture was poured and sterilized as usual, inoculated, and cultured at 25 ° C. for 72 hours at 250 rpm using a rotary shaker.
得られた種培養液を、ステンレス製200lジャーファー
メンター中に上記と同じ滅菌培地160l中へ接種し、ファ
ーメンターを200rpm、25℃で48時間攪拌した。こうして
得た種培養90lを、さらに、可溶性でん粉３％、グルコ
ース１％、綿実粉0.5％、小麦の胚芽１％ NaCl 3％およ
びCaCO3 0.2％を含有する4000l容鋼製ファーメンター中
の滅菌生産培地3000lに接種した。3000l/分の通気およ
び130rpmの攪拌のもとに、25℃で72時間培養を行なっ
た。The obtained seed culture was inoculated into 160 l of the same sterilized medium as above in a 200 l jar fermenter made of stainless steel, and the fermenter was stirred at 200 rpm at 25 ° C for 48 hours. 90 l of the seed culture thus obtained are further sterilized in a 4000 l steel fermenter containing 3% soluble starch, 1% glucose, 0.5% cottonseed flour, 1% wheat germ 3% NaCl and 0.2% CaCO 3. 3000 l of production medium were inoculated. The culture was performed at 25 ° C for 72 hours under aeration at 3000 l / min and stirring at 130 rpm.
培養液（2850l）を珪藻土（65kg）を用いて濾過し
た。濾液を、活性炭（600l）のカラムに通した。カラム
を脱イオン水180lで洗い、80％アセトン水（180l）で溶
出した。溶出液を減圧下に体積17lまで濃縮した。濃縮
物を酢酸エチル18lで抽出した。酢酸エチル層を分離
し、減圧乾固して、粉末（61.3g）を得た。この粗製粉
末をシリカゲル0.3lと混合した。混合物をシリカゲル
（3l）を用いるカラムクロマトグラフィーに付した。カ
ラムをヘキサン10lで、ついでヘキサン−酢酸エチル
（1:1）10lで洗浄し、酢酸エチル10lで溶出した。活性
画分を集め、蒸発乾固して粉末を得、この粉末をシリカ
ゲルと混合した。混合物を、シリカゲル（１）を用い
るカラムクロマトグラフィーに付した。カラムをクロロ
ホルム3l、ついでクロロホルム−メタノール（100:1）3
lで展開し、クロロホルム−メタノール（75:1）3lで溶
出した。溶出液を減圧乾固して粉末（16.4g）を得、こ
の粉末を逆相シリカゲルODS30mlと混合した。混合物を
逆相シリカゲルODS（600ml）カラムにかけ、メタノール
−水（1:1）2.4lで洗浄した。カラムをメタノール−水
（3:2）で溶出した。活性画分を集め、減圧下に濃縮し
て、油状のWF2015B（305mg）を得た。The culture solution (2850 l) was filtered using diatomaceous earth (65 kg). The filtrate was passed through a column of activated carbon (600 l). The column was washed with 180 l of deionized water and eluted with 80% aqueous acetone (180 l). The eluate was concentrated under reduced pressure to a volume of 17 l. The concentrate was extracted with 18 l of ethyl acetate. The ethyl acetate layer was separated and dried under reduced pressure to obtain a powder (61.3 g). This crude powder was mixed with 0.3 l of silica gel. The mixture was subjected to column chromatography on silica gel (3 l). The column was washed with 10 l of hexane and then with 10 l of hexane-ethyl acetate (1: 1) and eluted with 10 l of ethyl acetate. The active fractions were collected and evaporated to dryness to give a powder, which was mixed with silica gel. The mixture was subjected to column chromatography using silica gel (1). The column is made up of 3 l of chloroform, then chloroform-methanol (100: 1) 3
and eluted with 3 l of chloroform-methanol (75: 1). The eluate was dried under reduced pressure to obtain a powder (16.4 g), which was mixed with 30 ml of reverse-phase silica gel ODS. The mixture was applied to a reverse phase silica gel ODS (600 ml) column and washed with 2.4 l of methanol-water (1: 1). The column was eluted with methanol-water (3: 2). The active fraction was collected and concentrated under reduced pressure to obtain WF2015B (305 mg) as an oil.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 ＦＩ Ｃ１２Ｒ 1:645） (Ｃ１２Ｐ 17/16 Ｃ１２Ｒ 1:645） ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 6 Identification code FI C12R 1: 645) (C12P 17/16 C12R 1: 645)
Claims (1)
として含有する血管新生阻害剤。1. An angiogenesis inhibitor comprising WF2015A and / or WF2015B as an active ingredient.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5336889A JP2762522B2 (en) | 1989-03-06 | 1989-03-06 | Angiogenesis inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5336889A JP2762522B2 (en) | 1989-03-06 | 1989-03-06 | Angiogenesis inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02233610A JPH02233610A (en) | 1990-09-17 |
| JP2762522B2 true JP2762522B2 (en) | 1998-06-04 |
Family
ID=12940874
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5336889A Expired - Lifetime JP2762522B2 (en) | 1989-03-06 | 1989-03-06 | Angiogenesis inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2762522B2 (en) |
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-
1989
- 1989-03-06 JP JP5336889A patent/JP2762522B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH02233610A (en) | 1990-09-17 |
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