JP2887524B2 - Optical resolution method using microorganisms - Google Patents
Optical resolution method using microorganismsInfo
- Publication number
- JP2887524B2 JP2887524B2 JP33572290A JP33572290A JP2887524B2 JP 2887524 B2 JP2887524 B2 JP 2887524B2 JP 33572290 A JP33572290 A JP 33572290A JP 33572290 A JP33572290 A JP 33572290A JP 2887524 B2 JP2887524 B2 JP 2887524B2
- Authority
- JP
- Japan
- Prior art keywords
- compound
- ethyl
- pyrano
- indolizine
- ethylenedioxy
- 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.)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は微生物を利用した光学分割法に関し、更に詳
細には合成抗癌剤として有用であるカンプトテシン誘導
体の重要な合成中間体である次式（１） で表わされる（Ｓ）−４−エチル−6,6−（エチレンジ
オキシ）−7,8−ジヒドロ−４−ヒドロキシ−1H−ピラ
ノ〔3,4−ｆ〕インドリジン−3,10（4H）−ジオンを微
生物を利用した光学分割により製造する方法に関する。Description: FIELD OF THE INVENTION The present invention relates to an optical resolution method using a microorganism, and more particularly, to the following formula (1) which is an important synthetic intermediate of a camptothecin derivative useful as a synthetic anticancer agent. ) (S) -4-ethyl-6,6- (ethylenedioxy) -7,8-dihydro-4-hydroxy-1H-pyrano [3,4-f] indolizine-3,10 (4H) The present invention relates to a method for producing dione by optical resolution using microorganisms.
従来、次の式（３） で表わされるカンプトテシン誘導体は、合成抗癌剤とし
て有用であり、その合成中間体として上記化合物（１）
が重要であることが知られている（特開昭63−152382号
公報）。かかる化合物（１）の製造法としては、当該化
合物（１）のラセミ体をジアステレオマーに誘導後分別
結晶化による光学分割法（特開昭63−152382号公報）等
が知られている。Conventionally, the following equation (3) Is useful as a synthetic anticancer agent, and as a synthetic intermediate thereof, the compound (1)
Is known to be important (JP-A-63-152382). As a method for producing such a compound (1), an optical resolution method by inducing a racemate of the compound (1) into a diastereomer and then fractional crystallization (JP-A-63-152382) is known.
しかしながら、これら従来の光学分割法は操作が煩雑
である、生成物の収率が低い等の欠点を有し、工業的な
製法としては充分満足できるものではなかった。However, these conventional optical resolution methods have disadvantages such as complicated operation and low product yield, and have not been sufficiently satisfactory as an industrial production method.
かかる実状において、本発明者らは微生物を利用して
化合物（１）のみを選択的に得るべく種々探索したとこ
ろ、バチルス属に属する微生物がラセミ体である下記化
合物（２）のＳ体にのみ作用してアシル基を除去し、Ｒ
体には作用しないことを見出し、本発明を完成するに到
った。In this situation, the present inventors have conducted various searches to selectively obtain only the compound (1) using a microorganism, and found that the microorganism belonging to the genus Bacillus is only racemic in the S form of the following compound (2). Acting to remove the acyl group,
They found that they did not act on the body, and completed the present invention.
本発明は次の反応式によって示される。 The present invention is illustrated by the following reaction scheme.
（式中、Ｒはアルキル基を示す） すなわち、一般式（２）で表わされる４−アシルオキ
シ−４−エチル−6,6−（エチレンジオキシ）−7,8−ジ
ヒドロ−1H−ピラノ〔3,4−ｆ〕インドリジン−3,10（4
H）−ジオンのラセミ体に、バチルス属に属する微生物
の菌体、培養物又はこれらの分画物を作用せしめること
を特徴とする化合物（１）の製造法である。 (Wherein, R represents an alkyl group) That is, 4-acyloxy-4-ethyl-6,6- (ethylenedioxy) -7,8-dihydro-1H-pyrano [3 , 4-f] indolizine-3,10 (4
A process for producing the compound (1), which comprises reacting a racemic form of H) -dione with a cell, culture or a fraction thereof of a microorganism belonging to the genus Bacillus.
本発明方法の原料化合物を表わす上記一般式（２）
中、Ｒで示されるアルキル基としてはメチル基、エチル
基、ｎ−プロピル基等の低級アルキル基が好ましい。The above general formula (2) representing a starting compound of the method of the present invention.
Among them, the alkyl group represented by R is preferably a lower alkyl group such as a methyl group, an ethyl group, and an n-propyl group.
かかる化合物（２）のラセミ体（以下、単に化合物
（２）という）は、例えば特開昭63−152382号記載の方
法に従って得られた４−エチル−6,6−（エチレンジオ
キシ）−7,8−ジヒドロ−４−ヒドロキシ−1H−ピラノ
〔3,4−ｆ〕インドリジン−3,10（4H）−ジオンに、脂
肪酸無水物、脂肪酸ハライド等の脂肪酸の反応性誘導体
を反応させることにより製造される。Such a racemic compound (2) (hereinafter simply referred to as compound (2)) can be obtained, for example, from 4-ethyl-6,6- (ethylenedioxy) -7 obtained by the method described in JP-A-63-152382. , 8-dihydro-4-hydroxy-1H-pyrano [3,4-f] indolizine-3,10 (4H) -dione is reacted with a reactive derivative of a fatty acid such as a fatty acid anhydride or a fatty acid halide. Manufactured.
本発明方法を実施するには、まず化合物（２）を緩衝
液に懸濁し、次いでこれにバチルス属に属する微生物の
菌体、培養物又はこれらの分画物を静かに加えて撹拌す
ればよい。In order to carry out the method of the present invention, compound (2) is first suspended in a buffer solution, and then cells, cultures or fractions of microorganisms belonging to the genus Bacillus are gently added and stirred. .
用いられるバチルス属に属する微生物としては、バチ
ルス リケニホルミス（Bacillus licheniformis）、バ
チルス マイコデス（Bacillus mycoides）等が挙げら
れる。より具体的にはバチルス リケニホルミスNRIC−
1007（Bacillus licheniformis NRIC−1007,東京農業大
学より入手）、バチルスマイコデス IAM−1190（Bacil
lus mycoides IAM−1190,東京大学応用微生物研究所よ
り入手）等が挙げられる。これらの微生物の培養物とし
ては、当該微生物を適当な培地で培養したものを挙げる
ことができる。上記微生物の分画物としては菌体又は培
養物を水又は適当な緩衝液で抽出したもの；液体培地で
培養を行った場合には培養物の濾液、該抽出液又は濾液
に硫安又はアルコールを加えることにより得られる沈澱
物；該抽出液又は濾液をゲル濾過、限外濾過、イオン交
換クロマトグラフィー等を用いて分画したもの；菌体の
破砕物；及び該破砕物を前記の分画方法を用いて分画し
たものを挙げることができる。Examples of the microorganism belonging to the genus Bacillus include Bacillus licheniformis, Bacillus mycoides, and the like. More specifically, Bacillus licheniformis NRIC-
1007 (Bacillus licheniformis NRIC-1007, obtained from Tokyo University of Agriculture), Bacillus mycodes IAM-1190 (Bacil
lus mycoides IAM-1190, obtained from the Institute of Applied Microorganisms, The University of Tokyo). Cultures of these microorganisms include those obtained by culturing the microorganisms in an appropriate medium. As a fraction of the microorganism, a cell or culture is extracted with water or a suitable buffer; when cultured in a liquid medium, a filtrate of the culture, ammonium sulfate or alcohol is added to the extract or filtrate. A precipitate obtained by the addition; a fraction obtained by fractionating the extract or filtrate using gel filtration, ultrafiltration, ion exchange chromatography, or the like; a disrupted product of bacterial cells; and the fractionation method described above. Can be used.
反応は、通常20〜50℃、好ましくは30〜40℃、pH4〜
９好ましくは６〜７で24〜72時間程度行われる。反応に
使用する化合物（２）の濃度は、0.1〜１重量％の間で
可能であるが通常0.1〜0.2重量％程度で行うのが望まし
い。使用する菌体の量はとくに限定されないが、目安と
して原料に対して0.1ないし２重量培程度が一般的であ
るが反応条件によってはこれよりも少なくてもよい。反
応に用いられる緩衝液としては、リン酸緩衝液、クエン
酸緩衝液等が挙げられる。反応終了後反応液を濾過、減
圧濃縮、カラムクロマトグラフィー、晶析等の通常の精
製手段を用いて、化合物（１）を単離することができ
る。The reaction is usually carried out at 20 to 50 ° C, preferably 30 to 40 ° C, pH 4 to
9 and preferably 6 to 7 for about 24 to 72 hours. The concentration of the compound (2) used in the reaction can be in the range of 0.1 to 1% by weight, but it is usually desirable to carry out the reaction at about 0.1 to 0.2% by weight. The amount of cells to be used is not particularly limited, but is generally about 0.1 to 2 parts by weight based on the starting material, but may be smaller depending on the reaction conditions. Examples of the buffer used for the reaction include a phosphate buffer, a citrate buffer and the like. After completion of the reaction, the compound (1) can be isolated by using a usual purification means such as filtration, concentration under reduced pressure, column chromatography, and crystallization.
得られた化合物（１）は、例えば特開昭63−152382号
公報記載の方法により前記カンプトテシン誘導体
（３）、その他の合成抗癌剤に導くことができる。The obtained compound (1) can be converted to the camptothecin derivative (3) and other synthetic anticancer agents by the method described in, for example, JP-A-63-152382.
本発明の製造法は温和な条件下で反応を行うことがで
き、反応における副反応がほとんど無いため目的の化合
物（１）を高純度に製造することができる。従って本発
明の製造法は、化合物（１）の工業的製造方法として優
れたものである。According to the production method of the present invention, the reaction can be carried out under mild conditions, and since there are almost no side reactions in the reaction, the target compound (1) can be produced with high purity. Therefore, the production method of the present invention is excellent as an industrial production method of compound (1).
以下、本発明を更に実施例により説明するが、本発明
はこれにより限定されるものではない。Hereinafter, the present invention will be further described with reference to examples, but the present invention is not limited thereto.
参考例１ バチルス属に属する微生物は、普通ブイヨン培地（肉
エキス0.5％、ペプトン１％、食塩0.5％、pH7.0）に、
前培養菌体を接種し、30℃で１日培養した。培養後、遠
心分離した菌体に生理食塩水を加え、再び遠心して菌体
を得た。これを五酸化リン上にて減圧乾燥して乾燥菌体
を得た。Reference Example 1 Microorganisms belonging to the genus Bacillus can be cultured in a common bouillon medium (meat extract 0.5%, peptone 1%, salt 0.5%, pH 7.0).
The precultured cells were inoculated and cultured at 30 ° C. for 1 day. After the culture, physiological saline was added to the centrifuged cells, and the cells were centrifuged again to obtain the cells. This was dried under reduced pressure on phosphorus pentoxide to obtain dried cells.
参考例２ ４−アセトキシ−４−エチル−6,6−（エチレンジオキ
シ）−7,8−ジヒドロ−1H−ピラノ〔3,4−ｆ〕インドリ
ジン−3,10（4H）−ジオン ４−エチル−6,6−（エチレンジオキシ）−7,8−ジヒ
ドロ−４−ヒドロキシ−1H−ピラノ〔3,4−ｆ〕インド
リジン−3,10（4H）−ジオン3.07gを塩化メチレン10ml
に懸濁し氷冷撹拌下、ジメチルアミノピリジン124mg、
無水酢酸1.13mlを加え室温で４時間放置した。HPLCで反
応の終了を確認後、反応液を減圧下濃縮した。残渣をイ
ソプロピルアルコール20mlから結晶化し濾過した。結晶
を冷イソプロピルアルコールで洗浄後乾燥し標記化合物
3.41gを得た。Reference Example 2 4-acetoxy-4-ethyl-6,6- (ethylenedioxy) -7,8-dihydro-1H-pyrano [3,4-f] indolizine-3,10 (4H) -dione 4- 3.07 g of ethyl-6,6- (ethylenedioxy) -7,8-dihydro-4-hydroxy-1H-pyrano [3,4-f] indolizine-3,10 (4H) -dione and 10 ml of methylene chloride
Suspended under ice-cooling and stirring, dimethylaminopyridine 124 mg,
1.13 ml of acetic anhydride was added and left at room temperature for 4 hours. After confirming the completion of the reaction by HPLC, the reaction solution was concentrated under reduced pressure. The residue was crystallized from 20 ml of isopropyl alcohol and filtered. The crystals were washed with cold isopropyl alcohol and dried to give the title compound.
3.41 g was obtained.
IR ν max cm-1 1740,1660,1610（Ｃ＝Ｏ） FT−NMR（CDCl3中のδ値ppm） 0.87（3H,t,J＝7Hz） 2.07（2H,t,J＝7Hz） 2.11（3H,s） 2.42（2H,t,J＝7Hz） 4.0〜4.3（6H,m） 5.36（2H,ABquartet,J＝17Hz,45Hz） 6.07（1H,s） 実施例１ （Ｓ）−４−エチル−6,6−（エチレンジオキシ）−7,8
−ジヒドロ−４−ヒドロキシ−1H−ピラノ〔3,4−ｆ〕
インドリジン−3,10（4H）−ジオン ４−アセトキシ−４−エチル−6,6−（エチレンジオ
キシ）−7,8−ジヒドロ−1H−ピラノ〔3,4−ｆ〕インド
リジン−3,10（4H）−ジオン1.00gを0.1Mリン酸緩衝液
（pH7.0）500mlに懸濁し、次いでバチルス マイコデス
IAM−1190の乾燥菌体1.0gを加え、30℃で48時間撹拌し
た。HPLCで反応の終了を確認後、反応液を分液（水−ク
ロロホルム）し、セライト濾過により菌体を除いた。有
機層を水で洗浄後乾燥し濃縮した。残渣をシリカゲルク
ロマトグラフィー（クロロホルム−アセトン）で精製し
420mgの標題化合物と494mgの（Ｒ）−４−アセトキシ−
４−エチル−6,6−（エチレンジオキシ）−7,8−ジヒド
ロ−1H−ピラノ〔3,4−ｆ〕インドリジン−3,10（4H）
−ジオンを回収した。IR ν max cm −1 1740, 1660, 1610 (C = O) FT-NMR (δ value ppm in CDCl 3 ) 0.87 (3H, t, J = 7 Hz) 2.07 (2H, t, J = 7 Hz) 2.11 ( 3H, s) 2.42 (2H, t, J = 7Hz) 4.0-4.3 (6H, m) 5.36 (2H, ABquartet, J = 17Hz, 45Hz) 6.07 (1H, s) Example 1 (S) -4-ethyl -6,6- (ethylenedioxy) -7,8
-Dihydro-4-hydroxy-1H-pyrano [3,4-f]
Indolizine-3,10 (4H) -dione 4-acetoxy-4-ethyl-6,6- (ethylenedioxy) -7,8-dihydro-1H-pyrano [3,4-f] indolizine-3, 1.00 g of 10 (4H) -dione was suspended in 500 ml of 0.1 M phosphate buffer (pH 7.0), and then Bacillus mycodes
1.0 g of dry cells of IAM-1190 was added, and the mixture was stirred at 30 ° C. for 48 hours. After confirming the completion of the reaction by HPLC, the reaction solution was separated (water-chloroform), and the cells were removed by celite filtration. The organic layer was washed with water, dried and concentrated. The residue was purified by silica gel chromatography (chloroform-acetone).
420 mg of the title compound and 494 mg of (R) -4-acetoxy-
4-ethyl-6,6- (ethylenedioxy) -7,8-dihydro-1H-pyrano [3,4-f] indolizine-3,10 (4H)
-Dione was recovered.
得られた目的化合物の機器分析値は別途合成のそれと
完全に一致し光学純度は、光学異性体分離カラムによる
分析により96％e.e.であった。The instrumental analysis value of the obtained target compound completely coincided with that of the separately synthesized compound, and the optical purity was 96% ee by analysis with an optical isomer separation column.
FT−NMR（CDCl3中のδ値ppm） 0.98（3H,t,J＝7Hz） 1.76（2H,t,J＝7Hz） 2.41（2H,t,J＝7Hz） 4.0〜4.3（6H,m） 5.37（2H,ABquartet,J＝16Hz,43Hz） 6.56（1H,s） HPLC条件 カラム:ULTRON ES−OVM（親和化工社製） 移動相:2.5％エタノール（含20mMリン酸緩衝液pH6.
0） 流 速:1.0ml/min 波 長:254nm 実施例２ （Ｓ）−４−エチル−6,6−（エチレンジオキシ）−7,8
−ジヒドロ−４−ヒドロキシ−1H−ピラノ〔3,4−ｆ〕
インドリジン−3,10（4H）−ジオン ４−アセトキシ−４−エチル−6,6−（エチレンジオ
キシ）−7,8−ジヒドロ−1H−ピラノ〔3,4−ｆ〕インド
リジン−3,10（4H）−ジオン1.00gを0.1Mリン酸緩衝液
（pH7.0）500mlに懸濁し、次いでバチルス リケニホル
ミスNRIC−1007の乾燥菌体2.0gを加え、30℃で48時間撹
拌した。HPLCで反応の終了を確認後、実施例１と同様の
操作を行い415mgの標題化合物と498mgの（Ｒ）−４−ア
セトキシ−４−エチル−6,6−（エチレンジオキシ）−
7,8−ジヒドロ−1H−ピラノ〔3,4−ｆ〕インドリジン−
3,10（4H）−ジオンを回収した。FT-NMR (δ value ppm in CDCl 3 ) 0.98 (3H, t, J = 7 Hz) 1.76 (2H, t, J = 7 Hz) 2.41 (2H, t, J = 7 Hz) 4.0-4.3 (6H, m) 5.37 (2H, ABquartet, J = 16Hz, 43Hz) 6.56 (1H, s) HPLC conditions Column: ULTRON ES-OVM (manufactured by Affinity Chemical Co., Ltd.) Mobile phase: 2.5% ethanol (containing 20 mM phosphate buffer pH 6.
0) Flow rate: 1.0 ml / min Wavelength: 254 nm Example 2 (S) -4-ethyl-6,6- (ethylenedioxy) -7,8
-Dihydro-4-hydroxy-1H-pyrano [3,4-f]
Indolizine-3,10 (4H) -dione 4-acetoxy-4-ethyl-6,6- (ethylenedioxy) -7,8-dihydro-1H-pyrano [3,4-f] indolizine-3, 1.00 g of 10 (4H) -dione was suspended in 500 ml of 0.1 M phosphate buffer (pH 7.0), and then 2.0 g of dried Bacillus licheniformis NRIC-1007 cells were added, followed by stirring at 30 ° C for 48 hours. After confirming the completion of the reaction by HPLC, the same operation as in Example 1 was carried out, and 415 mg of the title compound and 498 mg of (R) -4-acetoxy-4-ethyl-6,6- (ethylenedioxy)-
7,8-dihydro-1H-pyrano [3,4-f] indolizine-
3,10 (4H) -dione was recovered.
得られた目的化合物の光学純度は、光学異性体分離カ
ラムによる分析により95％e.e.であった。The optical purity of the obtained target compound was 95% ee by analysis with an optical isomer separation column.
フロントページの続き (58)調査した分野(Int.Cl.6，ＤＢ名) C12P 41/00 ＣＡ（ＳＴＮ) ＲＥＧＩＳＴＲＹ（ＳＴＮ)Continuation of the front page (58) Field surveyed (Int. Cl. 6 , DB name) C12P 41/00 CA (STN) REGISTRY (STN)
Claims (1)
（エチレンジオキシ）−7,8−ジヒドロ−1H−ピラノ
〔3,4−ｆ〕インドリジン−3,10（4H）−ジオンのラセ
ミ体に、バチルス属に属する微生物の菌体、培養物又は
これらの分画物を作用せしめることを特徴とする、次式
（１） で表わされる（Ｓ）−４−エチル−6,6−（エチレンジ
オキシ）−7,8−ジヒドロ−４−ヒドロキシ−1H−ピラ
ノ〔3,4−ｆ〕インドリジン−3,10（4H）−ジオンの製
造法。1. The general formula (2) (Wherein R represents an alkyl group) 4-acyloxy-4-ethyl-6,6-
A racemic body of (ethylenedioxy) -7,8-dihydro-1H-pyrano [3,4-f] indolizine-3,10 (4H) -dione, a microorganism, a culture, or a microorganism belonging to the genus Bacillus. The following formula (1) is characterized by allowing these fractions to act. (S) -4-ethyl-6,6- (ethylenedioxy) -7,8-dihydro-4-hydroxy-1H-pyrano [3,4-f] indolizine-3,10 (4H) -A process for the production of diones.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33572290A JP2887524B2 (en) | 1990-11-30 | 1990-11-30 | Optical resolution method using microorganisms |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33572290A JP2887524B2 (en) | 1990-11-30 | 1990-11-30 | Optical resolution method using microorganisms |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04200393A JPH04200393A (en) | 1992-07-21 |
| JP2887524B2 true JP2887524B2 (en) | 1999-04-26 |
Family
ID=18291739
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP33572290A Expired - Fee Related JP2887524B2 (en) | 1990-11-30 | 1990-11-30 | Optical resolution method using microorganisms |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2887524B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SG103322A1 (en) * | 1996-10-30 | 2004-04-29 | Tanabe Seiyaku Co | S type 2-substituted hydroxy-2-indolidinylbutyric ester compounds and process for preparation thereof |
| CN103087072B (en) * | 2013-02-05 | 2014-02-26 | 复旦大学 | (+)-Tricyclic hydroxy lactone preparation method |
-
1990
- 1990-11-30 JP JP33572290A patent/JP2887524B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH04200393A (en) | 1992-07-21 |
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