WO1997027314A1 - Process for producing oleanolic acid analogs by culturing hairy root - Google Patents

Process for producing oleanolic acid analogs by culturing hairy root Download PDF

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WO1997027314A1
WO1997027314A1 PCT/JP1997/000127 JP9700127W WO9727314A1 WO 1997027314 A1 WO1997027314 A1 WO 1997027314A1 JP 9700127 W JP9700127 W JP 9700127W WO 9727314 A1 WO9727314 A1 WO 9727314A1
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oleanolic acid
plant
producing
hairy
hairy root
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PCT/JP1997/000127
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French (fr)
Japanese (ja)
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Kenji Katoh
Masami Matsumoto
Keiichiro Yasumi
Noboru Shirane
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Shionogi & Co., Ltd.
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Priority to AU13196/97A priority Critical patent/AU1319697A/en
Publication of WO1997027314A1 publication Critical patent/WO1997027314A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
    • C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
    • 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
    • C12P33/00—Preparation of steroids

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  • the present invention relates to a novel method for producing an oleanolic acid analog by hairy root culture. More specifically, the present invention relates to a method for culturing hairy roots induced by transformation with a plasmid capable of inducing hairy roots, The present invention relates to a novel method for producing analogs of oleanolic acid by collecting myricerol, 27-oxy-lactate fueroyl myricerol, myricerol, or 27-oxyl-yl phylloyl myrilone from glycerol.
  • Background art
  • Myricerol, 27-oxy-forced phenol oil myricerol, myricerone and 27-oxy-powered phenol oil myricerone, shown below, are useful as compounds that can be intermediates in the production of various compounds.
  • R 3 is hydrogen or a metabolic ester residue
  • R 4 is an optionally substituted aryl or an optionally substituted aromatic complex
  • one of X and Y is hydroxy and the other is Is hydrogen, or X and Y are oxo together, and Z is oxygen or two hydrogens.
  • R 3 is hydrogen or a metabolizable ester residue
  • R 6 is hydrogen or —R 6 —R 7
  • R 6 is —S 0 3 —, one CH 2 COO—, —COCOO— or
  • R 8 COO-(R 8 is lower alkylene or lower alkenylene) and R 7 is hydrogen or lower alkyl
  • Is very useful as an intermediate for producing the compound represented by Compounds (II) and (III) are known to be endothelin receptor antagonists (W092Z12991 and JP-A-7-53484), and various compounds caused by excessive secretion of endothelin are known. It is a compound useful as a therapeutic or prophylactic agent for diseases such as hypertension, cerebrovascular contraction, acute dysfunction, acute proliferative arrhythmia, acute myocardial infarction, intimal hyperplasia, and cerebral edema.
  • 27-hydroxyl phenol oil myricelone itself has a non-peptide endothelin receptor antagonist activity and exhibits an antihypertensive effect (FEBS letters, no. Vol., Pp. 411-44 (1992)).
  • 27-hydroxy phenol oil myricerol or 27-hydroxy phenol is extracted from the bark of milli ricacerifera (W092Z 12 9 9 1) or Merianthus. Extraction method from root bark of Comosath (Me lianthusc omo sus) (Journal of S outh African Chemical Institute, 1974) Vol. 27, pp. 131-136).
  • Myricerol and myricelone can be obtained by further alkaline hydrolysis of the thus obtained 27-hydroxyl fueroyl myricerol and 27-oxyl fueroyl mycelon, or by cod root bark, grape skin or It was obtained by a method of semi-synthetic production by chemically modifying oleanolic acid extracted from olive leaves as a raw material (Japanese Patent Application Laid-Open No. 7-316188).
  • the method of extracting from natural products has problems such as low yield and inconsistent content depending on the plant production area, and it has been difficult to supply a sufficient amount.
  • the method of semi-synthetic production from oleanolic acid was very difficult because it required about 10 steps of chemical reaction to synthesize mycelol or mycelone from oleanolic acid.
  • Methods for obtaining useful metabolites by hairy root cultivation include methods for producing tanshinones using tanzine in JP-A-2-231090, and lentils in JP-A-4-75595 and JP-A-4-177494.
  • a method for producing phyllosides by a family of family plants is described. Disclosure of the invention
  • the present inventors have conducted intensive studies with the aim of developing a new method for producing oleanolic acid analogs, and as a result, it has been found that the desired compound can be produced very easily in high yield by hairy root culture.
  • the present invention was completed.
  • the present invention provides a hairy root derived from a plant capable of producing an oleanolic acid derivative, which is transformed by a plasmid capable of leading the hairy root, and culturing the hairy root.
  • Another object of the present invention is to provide a method for producing an oleanolic acid derivative by collecting the oleanolic acid derivative from the obtained culture.
  • the present invention also provides a hairy root derived from a plant capable of producing an oleanolic acid analog, the hairy root being transformed by a plasmid capable of inducing a hairy root. .
  • the desired oleanolic acid analog can be obtained very simply and stably in high yield.
  • oleanolic acid analog refers to oleanolic acid, myricerol, 27-oxycaffeoyl myricerol, myriceron, 27-oxy-potassium fueoyl myricerone, and derivatives thereof.
  • the compounds to be obtained are included.
  • the “plant capable of producing an oleanolic acid analog” used in the present invention means an entire plant or a part thereof that produces the oleanolic acid analog.
  • Examples of a plant that produces the above-mentioned “oleanolic acid derivative” include, for example, Merianthus comomosus (Melianthuscomo sus) and Merianthus major (Me1ianthusmajor), and Bers ama abyssinica ), Millica such as White peach (My ricacerifera), Polygala such as Polygona japonica and Polygalasenega, and Astrobema A such as A stilbethunbergii.
  • the term "hairy root” refers to a differentiated or undifferentiated cultured tissue obtained by infecting a plant with bacteria and introducing a part of the plasmid retained by the bacteria into the plant. Means It is preferable that the undifferentiated culture has been sterilized by a method such as subculture in a medium containing an antibiotic.
  • plasmid capable of inducing hairy roots refers to the T-DNA region of Ri plasmid, which has all the necessary regions for hairy root formation. Any brasside that can be formed can be suitably used.
  • R i plasmid or recombinant plasmid of R i plasmid and T i brasmid can be mentioned.
  • Recombinant plasmid is the T-DNA region of Ri brasmid, which is the entire region required for hairy root formation transferred to the T-DNA region of Ti plasmid. Any one that has acquired the inductive ability can be used in the present invention.
  • hairy roots may be induced using the above-mentioned plant and the above-mentioned brasside by a commonly used method, and cultured and collected.
  • a sterile plant is prepared using the above plant by a conventional method.
  • the seeds, apical buds or stems of a plant are cut off, sterilized using a solution of sodium hypochlorite, ethanol, or the like, and then aseptically planted in a medium.
  • the medium used here is not limited in any way.
  • a synthetic medium or a medium containing an appropriate amount of nutrients such as a carbon source, a nitrogen source, inorganic ions and vitamins, and commonly used in plant tissue culture * is used. Any of natural media can be suitably used.
  • Examples of the carbon source include glucose, sucrose, maltose, invert sugar, and the like. One or more of these may be appropriately selected in consideration of plant assimilation. Select and use them in combination.
  • Nitrogen sources include, for example, ammonium ions (eg, ammonium nitrate, ammonium sulfate, etc.), nitrate ions (eg, potassium nitrate, ammonium nitrate, etc.), amino acids (eg, glycine, glutamine, glutamic acid), protein decomposition products such as peptone, and meat.
  • ammonium ions eg, ammonium nitrate, ammonium sulfate, etc.
  • nitrate ions eg, potassium nitrate, ammonium nitrate, etc.
  • amino acids eg, glycine, glutamine, glutamic acid
  • protein decomposition products such as peptone
  • meat include inorganic nitrogen compounds or organic nitrogen compounds such as extracts and yeast extracts. One or more of these may be appropriately selected and used from the viewpoint of plant assimilation.
  • inorganic ion examples include inorganic salts composed of cations such as potassium ion, calcium ion, magnesium ion, and iron ion and anions such as sulfate ion, phosphate ion, citrate ion, and chloride ion, for example, magnesium sulfate, Potassium phosphate, potassium nitrate, calcium carbonate, ferric chloride and the like are listed.
  • vitamins include thiamine, pyridoxine, nicotinamide, inositol and the like. One or more of these may be appropriately selected and used.
  • the medium include the Murashige and Skoog's medium, the Nitche and Nitce * s medium, or a modified medium thereof.
  • glucose sucrose
  • plant hormones such as auxin and cytokinin can be used alone or in an appropriate combination of two or more.
  • These media may be adjusted to pH 5 to 7, preferably around pH 5.7, and sterilized by a generally used method.
  • sterilization method include, for example, a method of applying an autoclave, or using an aseptic filter to filter under an aseptic environment.
  • the germ-free bacteria produced by the above method are inoculated with bacteria having a plasmid capable of inducing hairy roots.
  • the bacteria used here are not particularly limited, and any bacteria having the above-mentioned plasmid can be suitably used.
  • Agrobacterium rhizogenes (Ag robacteri um rhizogenes), Agrobacterium um tume faciens, Ri plasmid or recombinant plasmid is introduced into the cells, and preferably Agrobacterium umme faciens.
  • Murizogenes is preferred.
  • Agrobacterium rhizogenes which is a strain that produces an abnormal amino acid of the mykimopine type, is preferred because of its high hairy root induction efficiency.
  • Agrobacterium umme faciliens incorporating Ri Brasmid or recombinant plasmid into cells is described in Plant Physiology (P 1 ant Physio 1 ogy, 1994, Vol. 104, No. 801). -80 p. 2) can be used to introduce Ri brasmid or recombinant plasmid into cells.
  • the inoculation may be performed by a commonly used known method. For example, a method in which bacteria are attached to a sessile needle or the like and pierced into a sterile plant, a method in which a sterile plant is damaged and a bacterium is applied thereto, a method in which a bacterial suspension is dropped or sprayed on the wound, and a cultivation of bacteria There is a method of immersing a sterile plant in a liquid or a bacterium-containing liquid.
  • Hairy roots are formed when the plant inoculated with bacteria is allowed to stand on a suitable medium for about 1 to 6 weeks, preferably in the dark, and the culture temperature varies depending on the plant species. : ⁇ 30, preferably about 25 and ⁇ 28.
  • the medium used here is not particularly limited, and the same medium as described above can be used.
  • the hairy root obtained by the above method is sterilized according to a usual method.
  • a method of cutting out the tip including the growth point of plant tissue and culturing it several times, or using an antibiotic (cefotaxime, carpenicillin, etc.) with a final concentration of 0.5 mgZm 1 to 1 mgZm 1 A method of cultivating hairy roots cut out on a medium may be used.
  • the medium used here is the same as described above except for the case where an antibiotic is added, and is not particularly limited.
  • the culturing may be carried out under conditions of about 20 "C to about 30, preferably about 25" C to about 28X: preferably in a dark place.
  • those having a high growth rate and those having a large number of branches are selected, and are selected from about 20 to about 30 X :, preferably about 25 to about 25.
  • the medium used here is the same as above.
  • the oleanolic acid derivative is extracted from the hairy roots after the above culture by a commonly used extraction method.
  • the immersion liquid is first immersed in the solvent, the immersion liquid is filtered, and the solvent in the obtained extract is distilled off.
  • immersion in a solvent may be performed directly, or once a drying step such as freeze-drying is performed, immersion in the solvent may be performed.
  • the solvent may be any of water, a polar organic solvent and a non-polar solvent, but is preferably a solvent such as methanol or ethyl acetate. Methanol is particularly preferred because of its high extraction efficiency.
  • Any of the generally used methods can be suitably used for distilling off the solvent.
  • a method such as decompression can be used, and distillation by an evaporator is particularly preferable.
  • the obtained residue is purified and recrystallized by a commonly used method to obtain the desired compound.
  • the purification method include column chromatography, high-performance liquid chromatography, and the like.
  • a method of purifying with a column such as silica gel is preferable.
  • the solvent used for recrystallization may be alcohol (methanol, ethanol, etc.), benzene or a mixture thereof, and a 1: 1 mixture of ethanol and benzene is particularly preferred.
  • the oleanolic acid derivatives obtained by the method of the present invention are useful as various pharmaceuticals and intermediates for producing the same.
  • the oleanolic acid analog When used as a pharmaceutical or an intermediate for producing the same, it may be converted to a salt that can be formed as necessary.
  • salts that can be formed include salts with alkali metals (such as sodium, potassium and lithium), alkaline earth metals (such as calcium and magnesium), ammonium and organic bases (such as triethylamine and pyridine). I can do it. These salts can be formed by a commonly used method.
  • the compound obtained by hairy root culture * is 27-oxy-caffeoylmycerol
  • it is subjected to a hydrolysis reaction and a selective oxidation reaction at the 3-position to obtain mycelone.
  • 27-oxy-caffeoylmilycetone is obtained by hairy root ginger, it is hydrolyzed, and if myricerol is obtained, it is subjected to a selective oxidation reaction at the 3-position. Obtain myriceron.
  • R 5 is hydrogen or a R 6 - is R 7
  • R 6 is - S 0 3 -, - CH 2 COO-, one COCOO- or a COR 8 COO-
  • R 8 is a lower alkylene or lower Alkenylene
  • R 7 is hydrogen or lower alkyl
  • R 9 is t-butoxycarbonyl or hydrogen.
  • Murashige-Skoog medium (1900 mg of potassium sulfate, 50 mg of ammonium nitrate, 150 mg of agglomerated calcium 2) Hydrate 440 mg, magnesium sulfate heptahydrate 37 Omg, dipotassium hydrogen phosphate 17 Omg, ferrous sulfate heptahydrate 27.8 mg, ethylenediaminetetrahydroacetic acid disodium 37.3 mg , Manganese sulfate tetrahydrate 22.3 mg, zinc sulfate heptahydrate 8.6 mg, boric acid 6.2 mg, potassium iodide 0.83 mg, sodium molybdate dihydrate 0.25 mg , Copper sulfate pentahydrate 0.02 5 mg, cobalt chloride hexahydrate 0.02 5 mg 00ml).
  • Agrobacterium rhizogenes IFO 144554 was inoculated into the upper hypocotyl part of the obtained sterile seedling using a stalked needle, and 2 to 4 weeks later, the hairy root generated from the inoculated site was cut off.
  • the cells were inoculated in Murashige-Skoog medium solidified with 0.2% dielan gum, and cultured in the dark at 25 for 20 days. Then, the cells were transferred to Murashige-Skoog medium solidified with 0.2% dielan gum containing cefotaxime (O.C.O.SnigZm1), and subcultured * every week with a medium containing the same antibiotics. ⁇ 8 times repeated to get rid of hairy roots,
  • Murashige-Skoog's modified medium is used in the Murashige-Skoog mineral medium for 1 000 ml.
  • thiamine hydrochloride 0.5 mg of pyridoxine hydrochloride, 0.5 mg of nicotinic acid, 10 Omg of myo-inositol,
  • nicotinic acid 10 Omg of myo-inositol
  • Add 30 g of sucrose and 2.0 g of gellan gum then add distilled water to make up to 1 000 ml, adjust the pH to around 5.8, and dispense 40 ml in 1 000 m 1 Meyer.
  • the autoclave was sterilized for 15 minutes.
  • Hairy roots (40 g) of Merianthus comosas two weeks after the start of culture were extracted by immersing twice in methanol (80 ml) twice for three days.
  • the extract and 16 Oml of hexane were mixed, and liquid-liquid distribution was carried out in a separating funnel to remove the hexane fraction and remove low-polar substances such as lipids.
  • the remaining methanol fraction was concentrated under reduced pressure by an evaporator and evaporated to dryness, and then 100 ml each of ethyl ethyl phosphate and water were added thereto, followed by liquid-liquid distribution in a separating funnel. After removing the aqueous layer, the ethyl acetate layer was dried under reduced pressure to obtain a crude extract.
  • the crude extract was dissolved in a mixed solvent of chloroform and methanol (95: 5) to obtain a crude extract, which was then subjected to column chromatography (si1icagel 60, 230 # 400 mesh, Merck). The extract was added. First, elution was performed with a mixed solvent of chloroform and methanol (95: 5), and then elution was performed with a mixed solvent of chloroform and methanol (8: 2). After collecting and concentrating the yellow fraction, a part thereof was purified by high performance liquid chromatography to obtain about 17 mg (yield: about 1% of fresh weight) of a target compound.
  • the high-performance liquid chromatography column uses Deve 1 osi 1 ODS-HG-5 (20 mm x 250 mm) and a flow rate of 10 ml using a mixture of acetonitrile and water (8: 2). / min, temperature 20t: Purification was performed while monitoring with UV 330
  • Compound (III) was synthesized in the same manner as in JP-A-7-53484 and JP-A-7-3161888.
  • the mother liquor was chromatographed on ODS silica gel and eluted with a mixture of methanol-water (85:15).
  • the precipitated crystals were collected and recrystallized from methanol together with the above crystals to obtain 13.5 g of myristerol.
  • a solution of mycerol (10.19 g, 21.55 mmo 1) obtained in the first step in pyridine solution (80 ml) was placed in a nitrogen atmosphere at room temperature under anhydrous nitrogen (80 ml, 0.1 ml). 86 mo 1) was added dropwise over 10 minutes. After stirring for 3.5 hours in the room, the mixture was cooled to Ot: and methanol (69 m; 1.70 mO 1) was added dropwise over 5 minutes. After stirring at room temperature for 30 minutes, the mixture was poured into a mixture of ice-concentrated hydrochloric acid (90 ml), water (200 ml), and methylene hydride (400 ml).
  • an oleanolic acid derivative can be produced very easily and in a high yield as compared with the conventional method.
  • the obtained oleanolic acid analogs are very useful as various pharmaceuticals or intermediates for their production.

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Abstract

A process for producing oleanolic acid analogs which comprises inducing and culturing hairy roots originating in a plant capable of forming oleanolic acid analogs (such as myricerol, 27-hydroxy-caffeoylmyricerol, myricerone or 27-hydroxy-caffeoylmyriceron) by using plasmids such as Ri plasmid capable of inducing hairy roots and taking up the oleanolic acid analogs from the culture medium thus obtained.

Description

明細書 毛状根培蹇によるォレアノール酸類縁体の製造法 技術分野  Description Method for producing oleanolic acid analog by hairy root culture
本発明は、 毛状根培養によるォレアノール酸類縁体の新規な製造法に関する 詳しくは、 毛状根を锈導し得るプラスミ ドによって形質転換して誘導された毛 状根を培養し、 該培養物からミ リセロール、 2 7 —ォキシ一力フエオイルミリ セロール、 ミリセロンまたは 2 7 —ォキシ一力フエオイルミ リセロンを採取す ることによるォレアノール酸類縁体の新規な製造法に関する。 背景技術  The present invention relates to a novel method for producing an oleanolic acid analog by hairy root culture. More specifically, the present invention relates to a method for culturing hairy roots induced by transformation with a plasmid capable of inducing hairy roots, The present invention relates to a novel method for producing analogs of oleanolic acid by collecting myricerol, 27-oxy-lactate fueroyl myricerol, myricerol, or 27-oxyl-yl phylloyl myrilone from glycerol. Background art
下記に示されるミリセロール、 2 7 —ォキシ一力フエオイルミリセロール、 ミ リセロンおよび 2 7 —ォキシ一力フエオイルミ リセロンは様々な化合物の製 造中間体となり得る化合物として有用である。  Myricerol, 27-oxy-forced phenol oil myricerol, myricerone and 27-oxy-powered phenol oil myricerone, shown below, are useful as compounds that can be intermediates in the production of various compounds.
Figure imgf000003_0001
ミリセロール ミリセロン
Figure imgf000003_0001
Millicerol mycelone
=単結合 z^s =ー 結 = Single bond z ^ s =ー connection
Rl= -OH R l = -OH
R2= -H R2= -H R 2 = -HR 2 = -H
27-ォキシ-カフ; tオイルミリセ口-ル 27-才キシ -力フエ才イルミリセロン 27-oxy-cuff; t oil myrice mouth-
--^ =単結合 = =二重結合
Figure imgf000003_0002
例えば、 下記式 （ I I ) または （ I I I ) -^ = Single bond = = double bond
Figure imgf000003_0002
For example, the following formula (II) or (III)
Figure imgf000004_0001
Figure imgf000004_0001
(式中、 R 3は水素または代謝性エステル残基であり、 R4は置換されていても よいァリールまたは置換されていてもよい芳香族複素 であり、 Xと Yの一方 はヒ ドロキシで他方は水素、 または Xと Yが一緒になつてォキソであり、 Zは 酸素または 2個の水素である。 ） (Wherein, R 3 is hydrogen or a metabolic ester residue, R 4 is an optionally substituted aryl or an optionally substituted aromatic complex, and one of X and Y is hydroxy and the other is Is hydrogen, or X and Y are oxo together, and Z is oxygen or two hydrogens.)
Figure imgf000004_0002
Figure imgf000004_0002
(式中、 R 3は水素または代謝性エステル残基であり、 R6は水素または— R6 — R7であり、 R6は— S 03—、 一 CH2COO—、 — COCOO—または一 C O R 8 C O O - (R8は低級アルキレンまたは低級アルケニレン） であり、 R7 は水素または低級アルキルである） (Wherein R 3 is hydrogen or a metabolizable ester residue, R 6 is hydrogen or —R 6 —R 7 , R 6 is —S 0 3 —, one CH 2 COO—, —COCOO— or One COR 8 COO-(R 8 is lower alkylene or lower alkenylene) and R 7 is hydrogen or lower alkyl)
で示される化合物の製造中間体として非常に有用である。 化合物 （ I I ) およ び （ I I I ) は、 エンドセリン受容体アンタゴニス トであることが知られてお り （W092Z 1 299 1および特開平 7— 5 3484) 、 エンドセリンの過 剰分泌に起因する様々な疾患、 例えば高血圧、 脳血管収縮、 急性臂不全、 急性 増殖性胥症、 急性心筋硬塞、 血管内膜肥厚、 脳浮腫等の治療剤および予防剤と して有用な化合物である。 Is very useful as an intermediate for producing the compound represented by Compounds (II) and (III) are known to be endothelin receptor antagonists (W092Z12991 and JP-A-7-53484), and various compounds caused by excessive secretion of endothelin are known. It is a compound useful as a therapeutic or prophylactic agent for diseases such as hypertension, cerebrovascular contraction, acute dysfunction, acute proliferative arrhythmia, acute myocardial infarction, intimal hyperplasia, and cerebral edema.
また、 2 7—ォキシ一力フエオイルミ リセロンは、 それ自体が非ペプチド性 エンドセリ ン受容体アンタゴニスト活性を有し、 降圧作用を示すことが知られ ている （フエブス レタ—ズ （F E B S l e t t e r s ) 第 30 5巻， 第 4 1一 44頁 （ 1 992) ) 。 従来、 2 7—ォキシ一力フエオイルミリセロールまたは 2 7—ォキシ一カフ ェォイルミ リセロンは、 ミリ力 セリ フエラ （My r i c a c e r i f e r a、 シロコャマモモ） の樹皮から抽出する方法 （W092Z 1 2 9 9 1 ) また はメリアンサス コモサス （Me l i a n t h u s c omo s u s ) の根皮 から抽出する方法 （ジャーナル · ォブ *ザ ' サウス · アフリカン ' ケミカル - インスティチュー 卜 (J o u r n a l o f t h e S o u t h A f r i c a n C h em i c a l I n s t i t u t e) 1 974年、 第 2 7卷、 第 1 3 1〜 1 36頁） 等により得られていた。 In addition, it is known that 27-hydroxyl phenol oil myricelone itself has a non-peptide endothelin receptor antagonist activity and exhibits an antihypertensive effect (FEBS letters, no. Vol., Pp. 411-44 (1992)). Conventionally, 27-hydroxy phenol oil myricerol or 27-hydroxy phenol is extracted from the bark of milli ricacerifera (W092Z 12 9 9 1) or Merianthus. Extraction method from root bark of Comosath (Me lianthusc omo sus) (Journal of S outh African Chemical Institute, 1974) Vol. 27, pp. 131-136).
ミ リセロールおよびミリセロンは、 この様にして得られた 2 7—ォキシ一力 フエオイルミ リセロールおよび 2 7—ォキシ一力フエオイルミリセロンをさら にアルカリ加水分解する方法、 またはタラ根皮、 ブドウの皮もしくはォリーブ の葉から抽出したォレアノ一ル酸を原料として化学修飾を行ない、 半合成的に 製造する方法 （特開平 7— 3 1 6 1 8 8) により得ていた。  Myricerol and myricelone can be obtained by further alkaline hydrolysis of the thus obtained 27-hydroxyl fueroyl myricerol and 27-oxyl fueroyl mycelon, or by cod root bark, grape skin or It was obtained by a method of semi-synthetic production by chemically modifying oleanolic acid extracted from olive leaves as a raw material (Japanese Patent Application Laid-Open No. 7-316188).
しかし、 天然物から抽出する方法は収率が低い、 植物の産地により含量が一 定しない等の問題点があり、 充分な量の供給には困難な点があった。 また、 ォ レアノール酸から半合成的に製造する方法も、 ォレアノール酸からミリセロー ルまたはミリセロンを合成するためには約 1 0工程もの化学反応を必要とする ことから非常に困難であった。  However, the method of extracting from natural products has problems such as low yield and inconsistent content depending on the plant production area, and it has been difficult to supply a sufficient amount. Also, the method of semi-synthetic production from oleanolic acid was very difficult because it required about 10 steps of chemical reaction to synthesize mycelol or mycelone from oleanolic acid.
従って、 エンドリ ン受容体アンタゴニス トとして、 または化合物 （ I I ) お よび （ I I I ) の中間体として有用なォレアノール酸類縁体を簡便に高収率で 得る方法の開発が望まれていた。  Therefore, development of a method for easily obtaining a oleanolic acid analog useful as an endrin receptor antagonist or as an intermediate of compounds (II) and (III) has been desired.
毛状根培萎により有用代謝物を得る方法としては、 特開平 2— 23 1 090 にタンジンによるタンシノン類の製造方法、 特開平 4— 7 5 59 5および特開 平 4一 7 7494にリ ンドウ科植物によるフエ二ル配糖体の製造方法が記載さ れている。 発明の開示  Methods for obtaining useful metabolites by hairy root cultivation include methods for producing tanshinones using tanzine in JP-A-2-231090, and lentils in JP-A-4-75595 and JP-A-4-177494. A method for producing phyllosides by a family of family plants is described. Disclosure of the invention
本発明者らは、 ォレアノール酸類縁体の新規製造法の開発を目指して鋭意研 究を統けた結果、 毛状根培養により、 目的化合物が極めて容易に ¾収率で製造 される事を見出し、 本発明を完成した。 The present inventors have conducted intensive studies with the aim of developing a new method for producing oleanolic acid analogs, and as a result, it has been found that the desired compound can be produced very easily in high yield by hairy root culture. The present invention was completed.
即ち、 本発明は、 ォレアノール酸類緣体を生成し得る植物体由来の毛状根で あって、 毛状根を锈導し得るプラスミ ドによって形質転換して锈導された該毛 状根を培養し、 得られた培養物からォレアノール酸類緣体を採取することによ るォレアノール酸類緣体の製造法を提供するものである。 さらに、 ォレアノー ル酸類縁体を生成し得る植物体由来の毛状根であって、 毛状根を誘導し得るプ ラスミ ドによって形質転換して誘導された毛状根をも提供するものである。 本発明の製造法によれば、 目的とするォレアノール酸類縁体が極めて簡便に 、 高収率で安定的に得ることができる。  That is, the present invention provides a hairy root derived from a plant capable of producing an oleanolic acid derivative, which is transformed by a plasmid capable of leading the hairy root, and culturing the hairy root. Another object of the present invention is to provide a method for producing an oleanolic acid derivative by collecting the oleanolic acid derivative from the obtained culture. Furthermore, the present invention also provides a hairy root derived from a plant capable of producing an oleanolic acid analog, the hairy root being transformed by a plasmid capable of inducing a hairy root. . According to the production method of the present invention, the desired oleanolic acid analog can be obtained very simply and stably in high yield.
本発明において、 「ォレアノール酸類緣体」 とは、 ォレアノール酸、 ミ リセ ロール、 2 7—ォキシカフェオイルミリセロール、 ミ リセロン、 2 7—ォキシ 一力フエオイルミリセロンおよびこれらの化合物に誘導し得る化合物等を包含 する。 発明を実施するための最良の形態  In the present invention, the term “oleanolic acid analog” refers to oleanolic acid, myricerol, 27-oxycaffeoyl myricerol, myriceron, 27-oxy-potassium fueoyl myricerone, and derivatives thereof. The compounds to be obtained are included. BEST MODE FOR CARRYING OUT THE INVENTION
本発明に用いる 「ォレアノール酸類緣体を生成し得る植物体」 とは、 上記 Γ ォレアノール酸類縁体」 を生成する植物全体またはその一部分を意味する。 上 記 Γォレアノール酸類緣体」 を生成する植物体としては、 例えば、 メリアンサ ス コモサス （Me l i a n t h u s c omo s u s ) およびメリアンサス メージャー （Me 1 i a n t h u s m a j o r ) 等のメリアンサス厲、 ベル サマ アビシ二力 （B e r s ama a b y s s i n i c a) 等のベルサマ厲 、 シロコャマモモ （My r i c a c e r i f e r a ) 等のミリカ厲、 ヒメハ ギ（P o l y g a l a j a p o n i c a)およびセネガ（P o l y g a l a s e n e g a) 等のポリガラ厲、 ァカショウマ （A s t i l b e t h u n b e r g i i ) 等のァスチルべ厲、 ア トロキマ （A t r 0 X i m a ) 厲、 ィ レック ス ァスプレラ （ I l e x a s p r e l l a) 等のィ レックス （ I l e x) 厲、 ァカメガシヮ （Ma 1 1 o t u s j a p o n i c um) 等のマロタス厲 、 メロキア ビラミダ一夕 （Me l o c h i a p y r am i d a t a) 等の メロキア厲、 ロイプテリア チリアンサ （R h o i p t e l e a c h i l i a n t h a ) 等の口イブテリァ属植物が挙げられるが、 中でも 2 7—ォキシ一 カフェオイルミリセロールを生成し、 本発明方法により目的化合物が高含量で 得られるメリアンサス コモサスが特に好ましい。 これらの植物の種子， 胚、 葉、 茎、 根等いずれの部位においても本発明に好適に用いられることができ、 その一部を切り取って切片を用いればよい。 The “plant capable of producing an oleanolic acid analog” used in the present invention means an entire plant or a part thereof that produces the oleanolic acid analog. Examples of a plant that produces the above-mentioned “oleanolic acid derivative” include, for example, Merianthus comomosus (Melianthuscomo sus) and Merianthus major (Me1ianthusmajor), and Bers ama abyssinica ), Millica such as White peach (My ricacerifera), Polygala such as Polygona japonica and Polygalasenega, and Astrobema A such as A stilbethunbergii. X ima) 厲, I lexasprella, etc., Ilex lex, Ikamegashi (Ma 11 otusjaponic um), etc., Malotas 厲, Meloquia viramida, etc. (Melochiapyr am idata), etc. The Loup terrier chiliansa (R hoipteleachili Antha) and the like, but among them, Merianthus comosas, which produces 27-oxycaffeoyl myricerol and obtains the target compound in a high content by the method of the present invention, is particularly preferable. Any part of the seeds, embryos, leaves, stems, roots, etc. of these plants can be suitably used in the present invention, and a part thereof may be cut out and used.
本発明において 「毛状根」 とは、 細菌を植物体に感染させることによって、 細菌が保持しているプラスミ ドの一部を植物に導入し、 それによつて誘導され た分化あるいは未分化培養組織を意味する。 この未分化培赛組織は抗生物質を 含む培地で継代培養を行う等の方法で除菌されているものが好ましい。  In the present invention, the term "hairy root" refers to a differentiated or undifferentiated cultured tissue obtained by infecting a plant with bacteria and introducing a part of the plasmid retained by the bacteria into the plant. Means It is preferable that the undifferentiated culture has been sterilized by a method such as subculture in a medium containing an antibiotic.
「毛状根を誘導し得るプラスミ ド」 とは、 R i プラスミ ドの T一 D N A頜域 であって毛状根形成に必要な頜域を全て有しており、 植物体に毛状根を形成さ せ得るブラスミ ドであればいずれも好適に用いることができる。 例えば、 R i プラスミ ドまたは R i プラスミ ドと T i ブラスミ ドの組換えプラスミ ド等が举 げられる。 組換えプラスミ ドは、 R i ブラスミ ドの T一 D N A頜域であって毛 状根形成に必要な領域全てを T i プラスミ ドの T— D N A領域に移したもので あって、 毛状根の誘導能を獲得したものであればいずれでも本発明に利用が可 能である，  The term "plasmid capable of inducing hairy roots" refers to the T-DNA region of Ri plasmid, which has all the necessary regions for hairy root formation. Any brasside that can be formed can be suitably used. For example, R i plasmid or recombinant plasmid of R i plasmid and T i brasmid can be mentioned. Recombinant plasmid is the T-DNA region of Ri brasmid, which is the entire region required for hairy root formation transferred to the T-DNA region of Ti plasmid. Any one that has acquired the inductive ability can be used in the present invention.
目的とするォレアノール類縁体を得るには、 上記植物体と上記ブラスミ ドを 用い、 通常行なわれる方法により毛状根を誘導し、 それを培養して採取すれば よい。  In order to obtain the desired oleanol analog, hairy roots may be induced using the above-mentioned plant and the above-mentioned brasside by a commonly used method, and cultured and collected.
まず、 上記植物体を用いて、 常法により無菌植物体を作成する。 例えば、 植 物体の種子、 頂芽または茎部等を切り取り、 次亜塩素酸ナトリウム、 エタノー ル等の溶液を用いて滅菌し、 無菌的に培地に植え込む。  First, a sterile plant is prepared using the above plant by a conventional method. For example, the seeds, apical buds or stems of a plant are cut off, sterilized using a solution of sodium hypochlorite, ethanol, or the like, and then aseptically planted in a medium.
ここで用いる培地は何ら限定されるものではなく、 炭素源、 窒素源、 無機ィ オンおよびビタミン類等の栄養素を適当量含有し、 植物組織培 *において通常 使用されるものであれば合成培地または天然培地のいずれでも好適に用いるこ とができる。  The medium used here is not limited in any way.A synthetic medium or a medium containing an appropriate amount of nutrients such as a carbon source, a nitrogen source, inorganic ions and vitamins, and commonly used in plant tissue culture * is used. Any of natural media can be suitably used.
炭素源としては、 例えばグルコース、 スクロース、 マルトース、 転化糖等が 举げられ、 これらの中から植物の資化性を考慮して 1種または 2種以上を適宜 選択し、 組み合わせて用いればよい。 Examples of the carbon source include glucose, sucrose, maltose, invert sugar, and the like. One or more of these may be appropriately selected in consideration of plant assimilation. Select and use them in combination.
窒素源としては， 例えばアンモニゥムイオン （例えば硝酸アンモニゥム、 硫 酸アンモニゥム等） 、 硝酸イオン （例えば硝酸カリウム、 硝酸アンモニゥム等 ) 、 アミノ酸 （例えばグリシン、 グルタミン、 グルタミン酸） 、 ペプトン等の タンパク質の分解物、 肉エキス、 酵母エキス等の無機窒素化合物または有機窒 素化合物等が举げられる。 これらの中から植物の資化性を考慮して 1種または 2種以上を適宜選択して用いればよい。  Nitrogen sources include, for example, ammonium ions (eg, ammonium nitrate, ammonium sulfate, etc.), nitrate ions (eg, potassium nitrate, ammonium nitrate, etc.), amino acids (eg, glycine, glutamine, glutamic acid), protein decomposition products such as peptone, and meat. Examples include inorganic nitrogen compounds or organic nitrogen compounds such as extracts and yeast extracts. One or more of these may be appropriately selected and used from the viewpoint of plant assimilation.
無機イオンとしては、 カリウムイオン、 カルシウムイオン、 マグネシウムィ オン、 鉄イオンなどのカチオンと硫酸イオン、 リン酸イオン、 確酸イオン、 塩 素イオンなどのァニオンとから構成される無機塩類、 例えば硫酸マグネシウム 、 リ ン酸カリウム、 硝酸カリウム， 炭酸カルシウム、 塩化第二鉄等が举げられ 、 ビタミン類としては、 チアミン、 ピリ ドキシン、 ニコチンアミ ド、 イノシト ール等が例示される。 これらの内 1種または 2種以上を適宜選択して用いれば よい。  Examples of the inorganic ion include inorganic salts composed of cations such as potassium ion, calcium ion, magnesium ion, and iron ion and anions such as sulfate ion, phosphate ion, citrate ion, and chloride ion, for example, magnesium sulfate, Potassium phosphate, potassium nitrate, calcium carbonate, ferric chloride and the like are listed. Examples of vitamins include thiamine, pyridoxine, nicotinamide, inositol and the like. One or more of these may be appropriately selected and used.
好ましい培地の例としては、 ムラシゲ · スクーグ （Mu r a s h i g e a n d S k o o g ' s ) 培地、 ニッチェ 'ニッチェ （N i t c h e a n d N i t c e * s ) 培地またはそれらの改変培地等が挙げられる， ここに、 さら にグルコース、 スクロース、 マルトース等の適当な糖類を単独でまたは 2種以 上を適当に組合わせて適当量、 例えば 3 %程度添加してもよい。 また、 必要で あれば植物ホルモン、 例えばオーキシンやサイ トカイニンを単独でまたは 2種 以上を適当に組合わせて添加し、 用いることも可能である。  Preferred examples of the medium include the Murashige and Skoog's medium, the Nitche and Nitce * s medium, or a modified medium thereof. In addition, glucose, sucrose A suitable amount of sugars, such as maltose, alone or in combination of two or more, may be added in an appropriate amount, for example, about 3%. If necessary, plant hormones such as auxin and cytokinin can be used alone or in an appropriate combination of two or more.
これらの培地は p H 5〜 7、 好ましくは pH 5. 7前後に諷整し、 一般的に 行なわれる方法で滅菌して用いればよい。 滅菌法としては、 例えばォ— トクレ ーブにかける、 または無菌フィルターを用いて無菌環境下で濂過する等の方法 が例示される。  These media may be adjusted to pH 5 to 7, preferably around pH 5.7, and sterilized by a generally used method. Examples of the sterilization method include, for example, a method of applying an autoclave, or using an aseptic filter to filter under an aseptic environment.
上記の方法で作成した無菌植物体に毛状根を誘導し得るプラスミ ドを保持し ている細菌を接種する。  The germ-free bacteria produced by the above method are inoculated with bacteria having a plasmid capable of inducing hairy roots.
ここで用いる細菌は特に限定されず、 上記プラスミ ドを有するものであれば いずれでも好適に用いられる。 例えばァグロパクテリゥム リゾゲネス （Ag r o b a c t e r i um r h i z o g e n e s ) 、 ァグロノヽ'クテリウム ッ メ フ ァ シエンス （Ag r o b a c t e r i um t ume f a c i e n s ) 、 R i プラスミ ドまたは組換えプラスミ ドを菌体内に導入したァグロバクテリウ ム ッメファシエンスが好ましく、 その中でもァグロパクテリゥム リゾゲネ スが好ましい。 特にミキモピンタイプの異常アミノ酸を生産する系統のァグロ パクテリゥム リゾゲネスは毛状根の誘導効率が高く好ましい。 R iブラスミ ドまたは組換えプラスミ ドを菌体内に組み込んだァグロパクテリゥム ッメフ ァシエンスは、 プラントフイジィォロジー （P 1 a n t P h y s i o 1 o g y、 1 994年、 第 1 04巻、 第 80 1— 80 2頁） 等に記載の公知の方法を 用いれば R i ブラスミ ドまたは組換えプラスミ ドを菌体内に導入することがで さる， The bacteria used here are not particularly limited, and any bacteria having the above-mentioned plasmid can be suitably used. For example, Agrobacterium rhizogenes (Ag robacteri um rhizogenes), Agrobacterium um tume faciens, Ri plasmid or recombinant plasmid is introduced into the cells, and preferably Agrobacterium umme faciens. Murizogenes is preferred. In particular, Agrobacterium rhizogenes, which is a strain that produces an abnormal amino acid of the mykimopine type, is preferred because of its high hairy root induction efficiency. Agrobacterium umme faciliens incorporating Ri Brasmid or recombinant plasmid into cells is described in Plant Physiology (P 1 ant Physio 1 ogy, 1994, Vol. 104, No. 801). -80 p. 2) can be used to introduce Ri brasmid or recombinant plasmid into cells.
接種には通常用いられる公知の方法を用いればよい。 例えば、 細菌を有柄針 等に付着させ無菌植物体に突き刺す方法、 無菌植物体に傷を付けそこに細菌を 塗付する方法、 傷に細菌懸澳液を滴下または噴霧する方法、 および細菌培養液 または細菌含有液体中に無菌植物体を浸演する方法等がある。  The inoculation may be performed by a commonly used known method. For example, a method in which bacteria are attached to a sessile needle or the like and pierced into a sterile plant, a method in which a sterile plant is damaged and a bacterium is applied thereto, a method in which a bacterial suspension is dropped or sprayed on the wound, and a cultivation of bacteria There is a method of immersing a sterile plant in a liquid or a bacterium-containing liquid.
細菌を接種した植物体を適当な培地上で 1〜 6週閒程度、 好ましくは暗所下 で静置しておけば毛状根が発生する， 培養温度は植物種によって変化するが、 約 20X：〜 3 0で、 好ましくは約 2 5で〜 2 8 に設定すればよい。 ここで用 いる培地は特に限定されず、 前記と同様のものを用いることができる。  Hairy roots are formed when the plant inoculated with bacteria is allowed to stand on a suitable medium for about 1 to 6 weeks, preferably in the dark, and the culture temperature varies depending on the plant species. : ~ 30, preferably about 25 and ~ 28. The medium used here is not particularly limited, and the same medium as described above can be used.
上記の方法により得られた毛状根を通常の方法に従って無菌状態とする。 例 えば、 植物組織の生長点を含む先端部を切出して培養する操作を数回蠑り返す 方法、 または最終濃度 0. 5mgZm 1〜 1 mgZm 1の抗生物質 （セフオタ キシム、 カルペニシリン等） を含む培地上で切出した毛状根を培蹇する方法等 を用いればよい。  The hairy root obtained by the above method is sterilized according to a usual method. For example, a method of cutting out the tip including the growth point of plant tissue and culturing it several times, or using an antibiotic (cefotaxime, carpenicillin, etc.) with a final concentration of 0.5 mgZm 1 to 1 mgZm 1 A method of cultivating hairy roots cut out on a medium may be used.
ここで用いる培地も抗生物質を添加する埸合以外は前記と同様であり、 特に 限定されない。 培養は約 20"C〜約 30 、 好ましくは約 2 5 "C〜約 2 8 X：条 件下、 好ましくは暗所下で行なえばよい。  The medium used here is the same as described above except for the case where an antibiotic is added, and is not particularly limited. The culturing may be carried out under conditions of about 20 "C to about 30, preferably about 25" C to about 28X: preferably in a dark place.
こうして得られた無菌状態の毛状根の中で増殖速度が大きいものおよび分枝 数の多いものを選抜し、 それを約 2 0 〜約 3 0 X：、 好ましくは約 2 5で〜約 2 8 tで、 さらに好ましくは暗所下で培 ¾することにより、 生産効率のよい毛 伏根の大 S培養が可能となる。 Among the aseptic hairy roots thus obtained, those having a high growth rate and those having a large number of branches are selected, and are selected from about 20 to about 30 X :, preferably about 25 to about 25. By culturing at 28 t, more preferably in a dark place, large S culture of hairy roots with high production efficiency becomes possible.
ここで使用される培地も上記と同様である。  The medium used here is the same as above.
上記培養を終えた毛状根から、 通常用いられる抽出方法によりォレアノール 酸類緣体を抽出する。 例えば、 まず溶媒により浸漬し、 浸漬液を濂遢後、 得ら れた抽出液の溶媒を留去する。  The oleanolic acid derivative is extracted from the hairy roots after the above culture by a commonly used extraction method. For example, the immersion liquid is first immersed in the solvent, the immersion liquid is filtered, and the solvent in the obtained extract is distilled off.
浸濱の方法としては、 直接溶媒に浸清してもよく、 一旦凍結乾燥等の乾燥手 段を講じた後に溶媒に浸演してもよい。 溶媒としては水、 極性有機溶媒および 非極性溶媒のいずれでもよいが、 好ましくは、 メタノール、 酢酸ェチル等の溶 媒であり、 特にメタノールが抽出効率が高く好ましい。  As a method of immersion, immersion in a solvent may be performed directly, or once a drying step such as freeze-drying is performed, immersion in the solvent may be performed. The solvent may be any of water, a polar organic solvent and a non-polar solvent, but is preferably a solvent such as methanol or ethyl acetate. Methanol is particularly preferred because of its high extraction efficiency.
溶媒の留去には一般的に用いられている方法であればいずれでも好適に用い られる， 例えば、 滅圧濺縮等の方法が挙げられ、 特にエバボレーターによる留 去が好ましい。  Any of the generally used methods can be suitably used for distilling off the solvent. For example, a method such as decompression can be used, and distillation by an evaporator is particularly preferable.
得られた残留物を通常用いられる方法で精製 · 再結晶化して目的化合物を得 る。 精製方法としては例えばカラムクロマトグラフィ ー、 高速液体クロマトグ ラフィ一等が挙げられるが、 特にシリカゲル等のカラムで精製する方法が好ま しい。  The obtained residue is purified and recrystallized by a commonly used method to obtain the desired compound. Examples of the purification method include column chromatography, high-performance liquid chromatography, and the like. In particular, a method of purifying with a column such as silica gel is preferable.
再結晶に用いる溶媒はアルコール （メタノール、 エタノール等） 、 ベンゼン またはそれらの混合物等を用いればよく、 エタノールとベンゼンの 1 ： 1混合 物が特に好ましい。  The solvent used for recrystallization may be alcohol (methanol, ethanol, etc.), benzene or a mixture thereof, and a 1: 1 mixture of ethanol and benzene is particularly preferred.
本発明の方法により得られたォレアノ—ル酸類緣体は、 各種医薬品およびそ の製造中間体として有用である。  The oleanolic acid derivatives obtained by the method of the present invention are useful as various pharmaceuticals and intermediates for producing the same.
ォレアノール酸類縁体を医薬品およびその製造中間体として用いる場合、 必 要に応じて生成し得る塩に変換してもよい。 生成し得る塩の例としては、 アル カリ金属 （ナトリウム、 カリウム、 リチウム等） 、 アルカリ土類金属 （カルシ ゥム、 マグネシウム等） 、 アンモニゥムまたは有機塩基 （トリェチルァミン、 ピリジン等） 等との塩が举げられる。 これらの塩は通常行なわれる方法で生成 させることができる。  When the oleanolic acid analog is used as a pharmaceutical or an intermediate for producing the same, it may be converted to a salt that can be formed as necessary. Examples of salts that can be formed include salts with alkali metals (such as sodium, potassium and lithium), alkaline earth metals (such as calcium and magnesium), ammonium and organic bases (such as triethylamine and pyridine). I can do it. These salts can be formed by a commonly used method.
また、 ォレアノール酸類縁体を医薬品の製造中間体として用いる場合、 通常 の化学反応に付して類緣体を合成することができる。 When oleanolic acid analogs are used as intermediates in the manufacture of pharmaceuticals, The analog can be synthesized by the chemical reaction of
例えば、 毛状根培 *により得られた化合物が 2 7—ォキシ—カフェオイルミ リセロールである場合には、 これを加水分解反応および 3位の選択的酸化反応 に付してミリセロンを得る。 また、 毛状根培姜により 2 7—ォキシ一カフェォ イルミ リセ口ンが得られた場合にはこれを加水分解し、 ミリセロールが得られ た場合には 3位の選択的酸化反応に付してミリセロンを得る。  For example, if the compound obtained by hairy root culture * is 27-oxy-caffeoylmycerol, it is subjected to a hydrolysis reaction and a selective oxidation reaction at the 3-position to obtain mycelone. In addition, if 27-oxy-caffeoylmilycetone is obtained by hairy root ginger, it is hydrolyzed, and if myricerol is obtained, it is subjected to a selective oxidation reaction at the 3-position. Obtain myriceron.
次に、 毛状根培養または上記の方法により得たミ リセロンにジ低級アルキル ホスホノ酢酸を反応させ、 その後、 化合物 （ I V) ：  Next, dilower alkyl phosphonoacetic acid is reacted with hairy root culture or myricerone obtained by the above method, and then the compound (IV):
Figure imgf000011_0001
Figure imgf000011_0001
(式中、 R 5は水素または一 R 6— R 7であり、 R6は— S 03—、 - C H 2 C O O—、 一 COCOO—または一 COR8COO— （R 8は低級アルキレンまたは 低級アルケニレン） であり、 R 7は水素または低級アルキルであり、 R9は t一 ブ卜キシカルボニルまたは水素である） (Wherein, R 5 is hydrogen or a R 6 - is R 7, R 6 is - S 0 3 -, - CH 2 COO-, one COCOO- or a COR 8 COO- (R 8 is a lower alkylene or lower Alkenylene), R 7 is hydrogen or lower alkyl, and R 9 is t-butoxycarbonyl or hydrogen.
で示されるアルデヒドとホーナーエモンズ反応の反応条件下で縮合させ、 必要 であれば脱保護および または化学修飾を加えることにより、 式 （ I I I ) ： By condensation under the reaction conditions of the Horner-Emmons reaction with the aldehyde represented by the formula (1) and, if necessary, by deprotection and / or chemical modification, the formula (I I I):
Figure imgf000011_0002
Figure imgf000011_0002
(ここで R 3および R 5は前記と同義である） (Where R 3 and R 5 are as defined above)
で示される化合物を製造することが可能である， 実施例 It is possible to produce the compound represented by the formula
以下に本発明を実施例によりさらに詳細に説明するが、 本発明はこれら実施 例に限定されるものではない。  Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.
実施例 1 2 7—ォキシ一力フエオイルミ リセロールの製造 ( 1 ) メリアンサス コモサス毛状根の誘導 Example 1 Preparation of 2 7-ethoxy-Ichirofuehoimyriserol (1) Induction of Merianthus commosus hairy roots
公知の方法 （プラント ·ティ ッシュ ·カルチャー · レターズ （P 1 a n t T i s s u e C u l t u r e L e t t e r s ) 第 9巻、 第 1号、 第 1 0~ 1 4頁 （ 1 9 9 2年） ） によりメリアンサス コモサスの種子を 1 %次亜塩素酸 ナトリウムで滅菌し、 無菌水で洗浄した後、 0. 2 %ジエランガムで固化した ムラシゲ · スクーグ培地 （硫酸力リウム 1 900mg、 硝酸アンモニゥム 1 6 50 m g , 塊化カルシウム 2水和物 440 m g、 硫酸マグネシウム 7水和物 3 7 Omg, リ ン酸水素二カリウム 1 7 Omg、 硫酸第一鉄 7水和物 2 7. 8 m g , エチレンジアミンテトラヒドロ酢酸 2ナトリウム 3 7. 3 m g , 硫酸マン ガン 4水和物 2 2. 3 m g , 硫酸亜鉛 7水和物 8. 6mg、 ホウ酸 6. 2 m g 、 ヨウ化カリ 0. 8 3mg、 モリブデン酸ナトリウム 2水和物 0. 2 5mg、 硫酸銅 5水和物 0. 02 5 m g、 塩化コバルト 6水和物 0. 02 5 mgZ l O 00m l ) に植込んだ。 得られた無菌幼植物体の上胚軸部分に有柄針を用いて ァグロパクテリゥム リゾゲネス I F O 1 4 5 54を接種し、 2〜4週間後に 接種部位から発生した毛状根を切り取り、 0. 2 %ジエランガムで固化したム ラシゲ · スクーグ培地に植込み、 暗所下 2 5 で 20日間培養した。 次いで、 セフオタキシム （澳度 0. SnigZm 1 ) 含有の 0. 2 %ジエランガムで固化 したムラシゲ · スクーグ培地に移し、 1週間毎に同上の抗生物質を含む培地で 継代培 *を行ない、 この操作を 4~ 8回繰り返すことにより、 除菌された毛状 根を得た，  By the known method (Plant Tissue Culture Letters (P1ant T issue Culture Letters), Vol. 9, No. 1, pages 10-14 (1992)) Seeds are sterilized with 1% sodium hypochlorite, washed with sterile water, and then solidified with 0.2% dielan gum. Murashige-Skoog medium (1900 mg of potassium sulfate, 50 mg of ammonium nitrate, 150 mg of agglomerated calcium 2) Hydrate 440 mg, magnesium sulfate heptahydrate 37 Omg, dipotassium hydrogen phosphate 17 Omg, ferrous sulfate heptahydrate 27.8 mg, ethylenediaminetetrahydroacetic acid disodium 37.3 mg , Manganese sulfate tetrahydrate 22.3 mg, zinc sulfate heptahydrate 8.6 mg, boric acid 6.2 mg, potassium iodide 0.83 mg, sodium molybdate dihydrate 0.25 mg , Copper sulfate pentahydrate 0.02 5 mg, cobalt chloride hexahydrate 0.02 5 mg 00ml). Agrobacterium rhizogenes IFO 144554 was inoculated into the upper hypocotyl part of the obtained sterile seedling using a stalked needle, and 2 to 4 weeks later, the hairy root generated from the inoculated site was cut off. The cells were inoculated in Murashige-Skoog medium solidified with 0.2% dielan gum, and cultured in the dark at 25 for 20 days. Then, the cells were transferred to Murashige-Skoog medium solidified with 0.2% dielan gum containing cefotaxime (O.C.O.SnigZm1), and subcultured * every week with a medium containing the same antibiotics. ~ 8 times repeated to get rid of hairy roots,
(2) メリアンサス コモサス毛状根の培 ¾  (2) Cultivation of Merianthus Comosas hairy roots
メリアンサス コモサス毛状根をムラシゲ · スクーグ改変培地で培養した。 ムラシゲ · スクーグ改変培地は 1 000m l用のムラシゲ♦ スクーグの無機培 地に 0. 5mgの塩酸チアミン、 0. 5 m gの塩酸ピリ ドキシン、 0. 5mg のニコチン酸、 1 0 Omgのミオ一イノシトール、 30 gの庶糖および 2. 0 gのゲランガムを加え、 その後蒸留水を加えて 1 000m l にし、 pHを 5. 8付近に調整した後、 1 000 m 1 のマイヤーに 40 m 1ずつ分注し、 1 2 1 ■Cのオートクレープに 1 5分間かけて滅菌した。 冷却後、 除菌された毛状根 5 Omg (新鮮重量） をとり、 無菌状態下に各マイヤーに移植し、 2 7でで 3週 間、 往復振通培養機 （ 5 5往復 分） を用いて暗所下に培養した。 その結果毛 状根は培蹇 2週間で約 50 Omg (新鮮重） と約 1 0倍に増加した。 Hairy roots of Merianthus comosus were cultured in Murashige-Skoog modified medium. Murashige-Skoog's modified medium is used in the Murashige-Skoog mineral medium for 1 000 ml.0.5 mg of thiamine hydrochloride, 0.5 mg of pyridoxine hydrochloride, 0.5 mg of nicotinic acid, 10 Omg of myo-inositol, Add 30 g of sucrose and 2.0 g of gellan gum, then add distilled water to make up to 1 000 ml, adjust the pH to around 5.8, and dispense 40 ml in 1 000 m 1 Meyer. The autoclave was sterilized for 15 minutes. After cooling, take 5 Omg (fresh weight) of the sterilized hairy roots, transfer them to each Meyer under aseptic conditions, and use 27 for 3 weeks During the incubation, the cells were cultured in the dark using a reciprocating shaker (55 reciprocations). As a result, the hairy root increased by about 10 times to about 50 Omg (fresh weight) in two weeks of culture.
(3) 27一ォキシ一力フエオイルミ リセロールの抽出  (3) Extraction of 27-hydroxyl-hue oil mycerol
培赛開始後 2週間を経過したメリアンサス コモサスの毛状根 （40 g) を メタノール （80m l ) に 3日間ずつ 2回浸演して抽出を行なった。 抽出液と へキサン 1 6 Om l を混和し、 分液ロート中で液液分配してへキサン画分を除 去し、 脂質などの低極性物質を除去した。 次に、 残ったメタノール画分をエバ ポレー夕によって減圧濃縮、 乾固した後、 これに齚酸ェチルと水をそれぞれ 1 0 0m l加えて分液ロー ト中で液液分配を行なった。 水層を除去した後に齚酸 ェチル層を減圧乾固し、 これを粗抽出物とした。  Hairy roots (40 g) of Merianthus comosas two weeks after the start of culture were extracted by immersing twice in methanol (80 ml) twice for three days. The extract and 16 Oml of hexane were mixed, and liquid-liquid distribution was carried out in a separating funnel to remove the hexane fraction and remove low-polar substances such as lipids. Next, the remaining methanol fraction was concentrated under reduced pressure by an evaporator and evaporated to dryness, and then 100 ml each of ethyl ethyl phosphate and water were added thereto, followed by liquid-liquid distribution in a separating funnel. After removing the aqueous layer, the ethyl acetate layer was dried under reduced pressure to obtain a crude extract.
粗抽出物をクロ口ホルムとメタノールの混合溶媒 （9 5 : 5) に溶解して粗 抽出液とし、 カラムクロマトグラフィ ー （ s i 1 i c a g e l 6 0、 230 # 40 0me s h, Me r c k社製） に粗抽出液を添加した。 最初にクロロホ ルムとメタノ—ルの混合溶媒 （9 5 ： 5) で溶出を行ない、 次にクロ口ホルム とメタノールの混合溶媒 （8 ： 2) で溶出を行なった。 黄色を呈す画分を回収 して濃縮した後、 その一部を高速液体クロマトグラフィーを用いて精製し、 目 的化合物を約 1 7 mg (収率 ： 新鮮重量の約 1 %) 得た。 高速液体クロマトグ ラフィ一のカラムは D e v e 1 o s i 1 OD S -HG- 5 (2 0 mm X 2 50 mm) を用い、 ァセトニトリルと水の混合溶媒 （8 : 2) を用いて、 流速 1 0m l /m i n , 温度 2 0t:、 UV 330でモニターしつつ精製を行なった  The crude extract was dissolved in a mixed solvent of chloroform and methanol (95: 5) to obtain a crude extract, which was then subjected to column chromatography (si1icagel 60, 230 # 400 mesh, Merck). The extract was added. First, elution was performed with a mixed solvent of chloroform and methanol (95: 5), and then elution was performed with a mixed solvent of chloroform and methanol (8: 2). After collecting and concentrating the yellow fraction, a part thereof was purified by high performance liquid chromatography to obtain about 17 mg (yield: about 1% of fresh weight) of a target compound. The high-performance liquid chromatography column uses Deve 1 osi 1 ODS-HG-5 (20 mm x 250 mm) and a flow rate of 10 ml using a mixture of acetonitrile and water (8: 2). / min, temperature 20t: Purification was performed while monitoring with UV 330
(4) 27—ォキシ一力フエオイルミリセロールの同定 (4) Identification of 27-hydroxyl fueoyl myricerol
メリアンサス コモサス毛状根から得られた化合物の UV、 V I S, I R、 MSと NMRのスぺク トルデータが 2 7一ォキシ一力フエオイルミ リセロール の標準品と完全に一致した為、 得られた化合物が 2 7—ォキシ一力フエオイル ミ リセロールであると確認された。  The UV, VIS, IR, MS and NMR spectral data of the compound obtained from Merianthus commosus hairy roots were completely consistent with the standard of 27-hydroxy fueroyl myricerol. It was confirmed to be 2 7-oxy-Ichiroku Hueoil Myriserol.
(1)旋光度 ： [a] D +1 44. 0' (メタノール ； C 1. 4 3)  (1) Optical rotation: [a] D +1 44.0 '(methanol; C1.43)
(2) 1HNMR (C5D5N, 40 0 MH z ； TM S 内部樓準 ； δ ppm)(2) 1 HNMR (C5D5N, 400 MHz; TMS internal standard; δ ppm)
0. 8 2 1 ( 3 H, s , H - 2 9 ) , 0. 82 7 (3 H， s， H - 2 5) , 0 . 8 96 ( 3 H, s， H— 30) ， 0. 9 2 6 ( 3 H, s， H - 2 3) , 0. 9 7 8 ( 3 H, s， H - 2 6) , 1. 1 0 8 (3 H， s , H - 24) ， 3. 2 9 6 - 3. 324 ( 2 H， m, H - 1 8， 3 ) , 4. 48 2 ( 1 H, d , H - 2 7 ) , 4. 603 ( 1 H, d , H - 2 7) ， 5. 7 54， 1 H, t . H— 1 2 ) , 6. 58 7 ( 1 H, d , H— 2 ' ) , 7. 1 1 6 ( 2 H, s , H - 8 ' , 9 ' ) , 7. 498 ( 1 H, s , H - 5 ' ) , 7. 9 0 9 ( 1 H, d , H - 3 ' ) 0.82 1 (3H, s, H-29), 0.827 (3H, s, H-25), 0 8 96 (3H, s, H-30), 0.926 (3H, s, H-23), 0.978 (3H, s, H-26), 1. 10 8 (3 H, s, H-24), 3.29 6-3.324 (2 H, m, H-18, 3), 4.48 2 (1 H, d, H-2 7), 4.603 (1H, d, H-27), 5.754,1H, t.H—12), 6.587 (1H, d, H—2 '), 7.1 1 16 (2H, s, H-8 ', 9'), 7.498 (1H, s, H-5 '), 7.909 (1H, d, H-3') )
(3)13CNMR (C5D5N ； TMS 内部標準 ； δ pm) 表 1 (3) 13 CNMR (C5D5N; TMS internal standard; δ pm) Table 1
位置  Position
C一 1 3 8. 9 C--1 3 8.9
C - 2 2 8. 0  C-2 28.0
C一 3 7 7. 9  C-1 3 7 7. 9
C - 4 39. 3  C-4 39.3
C - 5 5 5. 7  C-5 5 5. 7
C - 6 1 8. 8  C-6 1 8.8
C一 7 3 3. 6  C-1 7 3 3.6
C一 8 40. 4  C-1 8 40. 4
C - 9 49. 0  C-9 49. 0
C一 1 0 3 7. 5  C-1 0 3 7.5
C一 1 1 24. 4  C-1 1 24. 4
C一 1 2 1 26. 9  C-1 1 2 1 26. 9
C - 1 3 1 38. 8  C-1 3 1 38. 8
C一 14 4 5. 9  C-1 4 4 5.9
C一 1 5 24. 1  C-1 5 24. 1
C - 2 ' 1 1 5. 3  C-2 '1 1 5.3
C - 3 ' 145. 7 C-3 '145.7
Figure imgf000015_0001
Figure imgf000015_0001
C一 4， 1 2 6. 8  C-1 4, 1 26.8
C - 5 ' 1 1 5. 8  C-5'1 15.8
C - 6 ' 1 4 7. 7  C-6 '1 4 7. 7
C - 7 ' 1 50. 4 実施例 2 化合物 （ I I I ) の合成 C-7 '1 50. 4 Example 2 Synthesis of Compound (III)
特開平 7— 53484、 特開平 7— 3 1 6 1 8 8と同搽にして化合物 （ I I I ) を合成した。 Compound (III) was synthesized in the same manner as in JP-A-7-53484 and JP-A-7-3161888.
Figure imgf000016_0001
Figure imgf000016_0001
(m-2) , (m-3)(=Na塩）  (m-2), (m-3) (= Na salt)
(第 1工程） ミ リセロールの合成 実施例 1で得た 2 7—ォキシ一力フエオイルミ リセロールをァセチル化した 化合物 （ 5 6 g ) を 2分し、 それぞれ 1 0 %のメタノール性水酸化力リウム （ 7 0 0 m l、 1 0 %含水） に溶解し、 アルゴン気流下に還流した。 7 2時間後 に反応を停止し、 水 （ 1 00m l ) を加えて減圧下にメタノールを流去し、 稀 塩酸で液性を p H 6に整え、 酢酸ェチル （ 3 0 0 m 1、 3回） で抽出した。 齚 酸ェチル曆を水洗し、 酢酸ェチルを流去した。 残留物にメタノールを加えて生 じた決勝を «取した。 母液を OD Sシリカゲルのクロマトグラフィーにかけ、 メタノール一水 （8 5 : 1 5) の混液で流出させた。 析出する結晶を濂取し、 上記の結晶とあわせてメタノールより再結晶を行い 1 3. 5 gのミ リセロール を得た。 (First step) Synthesis of myricerol The compound (56 g) obtained by acetylating the 27-oxy-one-force phenol oil mycerol obtained in Example 1 was divided into 2 minutes, and 10% methanolic lithium hydroxide (700 ml, 10% water-containing) was obtained. ) And refluxed under an argon stream. 72 After 2 hours, stop the reaction, add water (100 ml), remove methanol under reduced pressure, adjust the pH to 6 with dilute hydrochloric acid, and add ethyl acetate (300 ml, 3 ml). Times).ェ Ethyl acetate was washed with water, and ethyl acetate was washed away. The final was created by adding methanol to the residue. The mother liquor was chromatographed on ODS silica gel and eluted with a mixture of methanol-water (85:15). The precipitated crystals were collected and recrystallized from methanol together with the above crystals to obtain 13.5 g of myristerol.
(第 2工程） 化合物 （ 1 ) の合成  (Step 2) Synthesis of Compound (1)
第 1工程で得たミリセロール （ 1 0. 1 9 g、 2 1. 5 5 mmo 1 ) のピリ ジン溶液 （8 0m l ) に窒素雰囲気下、 室温で、 無水齚酸 （ 8 0m 1、 0. 8 6 mo 1 ) を 1 0分間かけて滴下した。 室 ϋで 3時間半撹拌した後、 Ot:に冷 却し、 メタノール （69mし 1. 7 0 m o 1 ) を 5分間かけて滴下した。 室 温で 30分間撹拌した後、 氷一濃塩酸 （90m l ) —水 （ 200 m l ) —埴化 メチレン （400m l ) の混合液にあけた。 有機層を分取し、 水暦を埴化メチ レン （400 m l ) で抽出した， それぞれの有機層を、 1 N塩酸 （ 300 m l ) 、 食塩水 （ 300 m 1 X 2 ) で洗浄した後、 無水硫酸マグネシウムで乾燥し 、 濃縮して化合物 （ 1 ) の粗生成物を得た。 収量 1 2. 0 g (2 1. 5 5 mm o 1 ) , 収率 1 00 %,  A solution of mycerol (10.19 g, 21.55 mmo 1) obtained in the first step in pyridine solution (80 ml) was placed in a nitrogen atmosphere at room temperature under anhydrous nitrogen (80 ml, 0.1 ml). 86 mo 1) was added dropwise over 10 minutes. After stirring for 3.5 hours in the room, the mixture was cooled to Ot: and methanol (69 m; 1.70 mO 1) was added dropwise over 5 minutes. After stirring at room temperature for 30 minutes, the mixture was poured into a mixture of ice-concentrated hydrochloric acid (90 ml), water (200 ml), and methylene hydride (400 ml). The organic layer was separated and the lunar calendar was extracted with methylene clay (400 ml). Each organic layer was washed with 1 N hydrochloric acid (300 ml) and brine (300 ml x 2). After drying over anhydrous magnesium sulfate and concentration, a crude product of compound (1) was obtained. Yield 12.0 g (2 1.55 mm o 1), Yield 100%,
(第 3工程） 化合物 （2) の合成  (Step 3) Synthesis of Compound (2)
化合物 （ 1 ) ( 1 2. 0 g、 2 1. 5 5mmo l ) に窒素雰囲気下、 室温で 5 % KOHのメタノール溶液 （5 %含水、 300 m l ) を加え、 室温で 2時間 提拌した， 0でに冷却し、 4N塩酸 （6 0m l ) で中和した後、 メタノールを 滅圧除去した。 残さを氷一水 （400m l ) —齚酸ェチル （ 600 m l ) に溶 解し、 有機層を分取した。 水 ®を酢酸ェチル （ 2 0 0 m l ) で抽出し、 それぞ れの有機層を食壞水 （400m l ) で洗浄した。 無水硫酸マグネシウムで乾燥 後、 濃縮した。 残さをテトラヒドロフラン （ 5 0m l ) に溶解し、 シリカゲル ( 5 0 g) を加えた。 再度濃縮し、 カラムクロマトグラフィーにより精製して 目的化合物 （2) を得た。 収量 ： 6. 38 g ( 1 2. 40 mm o 1 ) 、 収率 5 8 %。 To a compound (1) (12.0 g, 21.55 mmol) was added a 5% KOH methanol solution (5% water content, 300 ml) at room temperature under a nitrogen atmosphere, and the mixture was stirred at room temperature for 2 hours. After cooling to 0 and neutralizing with 4N hydrochloric acid (60 ml), methanol was removed under reduced pressure. The residue was dissolved in ice-water (400 ml) -ethyl acetate (600 ml), and the organic layer was separated. The water was extracted with ethyl acetate (200 ml), and each organic layer was washed with corrosive water (400 ml). After drying over anhydrous magnesium sulfate, the mixture was concentrated. Dissolve the residue in tetrahydrofuran (50 ml), silica gel (50 g) was added. It was concentrated again and purified by column chromatography to obtain the target compound (2). Yield: 6.38 g (12.40 mmo 1), yield 58%.
クロマトグラフィー条件： S i 02 500 g , 埴化メチレン—齚酸ェチルノ塩 化メチレン = 1 Z 4—酢酸ェチル Chromatography conditions: S i 0 2 500 g, methylene chlorinated-ethyl ethynyl chloride methylene chloride = 1 Z 4-ethyl acetate
(第 4工程） 化合物 （3) の合成  (Step 4) Synthesis of compound (3)
化合物 （2) ( 1 8. 1 3 g , 3 5. 23mo l ) にクロ口ホルム （600 m l ) を加え、 アセトン （ 300 m l ) で希釈した。 この溶液に窒素雰囲気下 、 室温でジヨーンズ試薬 （C r 03ZH2S04) (約 2. 43M硫酸溶液、 2 1. 7 5m l , 5 2. 8 5 mm o l ) を滴下した。 室温で 30分擾拌した後、 メタノール （42. 94m l , 1. 06 m o 1 ) を滴下し、 室温で 2 0分擾拌 した， 反応液を水 （400m l ) にあけ、 クロロホルム （ 800 m 1 X 2 ) で 抽出した。 有機屬を水洗し （400 m l ) 、 無水硫酸マグネシウムで乾燥した 後、 濃縮して粗生成物を得た。 収量 ： 1 7. 39 g ( 33. 92 mm o 1 ) 、 収率 9 6 %。 Compound (2) (18.13 g, 35.23 mol) was added to chloroform (600 ml) and diluted with acetone (300 ml). To this solution under a nitrogen atmosphere, it was added dropwise Jiyonzu reagent at room temperature (C r 0 3 ZH 2 S0 4) ( about 2. 43M sulfuric acid solution, 2 1. 7 5m l, 5 2. 8 5 mm ol). After stirring at room temperature for 30 minutes, methanol (42.94 ml, 1.06 mol) was added dropwise, and the mixture was stirred at room temperature for 20 minutes. The reaction solution was poured into water (400 ml), and chloroform (800 ml) was added. 1 X 2). The organics were washed with water (400 ml), dried over anhydrous magnesium sulfate, and concentrated to obtain a crude product. Yield: 17.39 g (33.92 mmo1), 96% yield.
(第 5工程） ミ リセロンの合成  (Fifth step) Synthesis of myricelone
化合物 （3〉 ( 1 7. 38 g , 3 3. 9mmo 1 ) に窆素雰囲気下室温で 5 %KOHのメタノール溶液 （ 5 %含水、 8 7 0 m l ) を加え、 6 5 で 3時間 *拌した。 室温まで冷却し、 メタノールを滅圧除去して白色結晶が析出するま で濃縮した， 残さに氷一 4 N堪酸 （ 200 m l ) —塩化メチレン （ 6 00 m l ) を加えて酸性とし、 有機層を分取した。 水層を塩化メチレン （30 0 m 1 X 2) で抽出し、 それぞれの有機層を水 （400 m l X 2) で洗浄した。 無水硫 酸マグネシウムで乾燥後、 澳縮し、 カラムクロマトグラフィーにより精製して ミリセロンを得た.収量 1 3. 0 g ( 27. 6 2 mmo 1 ) 、 収率 8 1 %, クロマトグラフィー条件： S i 02 140 g, 塩化メチレン—齚酸ェチル /塩 化メチレン = 1ノ 9→ 1 6~» 1 4 To a compound (3) (17.38 g, 33.9 mmo 1) was added a 5% KOH methanol solution (5% water content, 870 ml) at room temperature under a nitrogen atmosphere, and the mixture was stirred at 65 for 3 hours *. The mixture was cooled to room temperature, concentrated under reduced pressure to remove methanol, and concentrated until white crystals were precipitated. The residue was acidified with 4N ice-acid (200 ml) —methylene chloride (600 ml), The organic layer was separated, the aqueous layer was extracted with methylene chloride (300 ml x 2), and each organic layer was washed with water (400 ml x 2), dried over anhydrous magnesium sulfate, and dried. . to give a Miriseron was purified by column chromatography yields 1 3. 0 g (27. 6 2 mmo 1), 8 1% yield, chromatographic conditions: S i 0 2 140 g, methylene chloride -齚Ethyl acid / methylene chloride = 1 no 9 → 1 6 ~ »1 4
(第 6工程） 化合物 （4) の合成  (Step 6) Synthesis of compound (4)
ジメチルホスホノ酢酸 （7. 0 7 g , 42. 1 mmo 1 ) の塩化メチレン溶 液 （ 1 00m l ) に、 窒素雰囲気下室温で塩化チォニル （ 9. 2 1 m l , 1 2 6 mm ο 1 ) を加えた。 室温で 4時間 «拌した後に濃縮して酸クロリ ド （ 7. 8 5 g ) を得た。 ミ リセロン （6. 6 0 g , 1 4. 0 mm o 1 ) の塩化メチレ ン溶液 （ 7 0m l ) に、 窒素雰囲気下、 — 7 8 でピリジン （4. 5 3 m U 5 6 mm 0 1 ) を滴下した。 このとき内温は一 7 3でまで上昇した。 ついで合 成した酸クロリ ド （ 7. 8 5 g, 1 4. 0 mm o 1 ) の塩化メチレン溶液 （ 7 Om l ) を 2 5分かけて滴下した， このときの内温は一 6 8¾：まで上昇した。 一 7 5 で 40分間 «拌した後、 溶媒を減圧除去した。 残さを THF (84 m 1 ) に懸¾させ、 0 に冷却した後、 2N 水酸化ナトリウム （ 1 4n ， 2To a methylene chloride solution (100 ml) of dimethylphosphonoacetic acid (7.07 g, 42.1 mmol) was added thionyl chloride (9.21 ml, 12) at room temperature under a nitrogen atmosphere. 6 mm ο 1) was added. After stirring at room temperature for 4 hours, the mixture was concentrated to obtain acid chloride (7.85 g). To a methylene chloride solution (70 ml) of myricelone (6.60 g, 14.0 mmo 1) in a nitrogen atmosphere, —78 with pyridine (4.53 m U 56 mm 0 1) ) Was added dropwise. At this time, the internal temperature rose to 1-73. Then, a methylene chloride solution (7 Oml) of the synthesized acid chloride (7.85 g, 14.0 mmo1) was added dropwise over 25 minutes, and the internal temperature was 168 6: Up. After stirring at 175 for 40 minutes, the solvent was removed under reduced pressure. The residue was suspended in THF (84 ml), cooled to 0, and then 2N sodium hydroxide (14n, 2
8 mmo 1 ) を加え， 0 で 1時間撹拌した。 反応液を氷一 1 N塩酸 （ 50m 1 ) 一酢酸ェチル （ 200 m l ) にあけ、 有機層を分取した。 水層を酢酸ェチ ル （ 1 50m l X 2) で抽出し、 それぞれの有機 Sを食塩水 （ 1 00 m 1 X 2 ) で洗浄した， 有機履をあわせ、 無水硫酸マグネシウムで乾燥し、 濃縮した。 カラムクロマトグラフィーにより生成して化合物 （4) を得た。 8 mmo1) was added, and the mixture was stirred at 0 for 1 hour. The reaction solution was poured into ice-cold 1 N hydrochloric acid (50 ml) and ethyl acetate (200 ml), and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (150 ml x 2), and each organic S was washed with saline (100 ml x 2). The organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated. did. Compound (4) was obtained by column chromatography.
クロマトグラフィー条件： s i 02 l 50 g, 酢酸ェチルノへキサン = 1 1→ 酢酸ェチル→クロ口ホルムノメタノール = 1 00/ 1→50/ 1 -*20/ 1. 収量 7. 43 g ( 1 1. 97 mm o 1 , 8 5 %) . Chromatographic conditions: si 0 2 l 50 g, ethyl ethyl acetate = 1 1 → ethyl acetate → formaldehyde methanol = 100/1 → 50/1-* 20 / 1. Yield 7.43 g (1 1 97 mm o 1, 85%).
(第 7工程） 化合物 （ I I I — 1 ) の合成  (Step 7) Synthesis of Compound (I I I-1)
化合物 （4) 6 2 1 mg ( 1 mmo 1 ) と参考例 1で得た化合物 （5) 2 9 Compound (4) 62 1 mg (1 mmo 1) and the compound obtained in Reference Example 1 (5) 2 9
9 m g ( 1. 2 mm o 1 ) を DMF 6m l に溶解した。 そこに DBU (ジァザ ビシクロウンデセン） 0. 3 5 8 m l , 堪化リチウム 9 3mgを加え室温で 19 mg (1.2 mmo 1) was dissolved in 6 ml of DMF. Then add DBU (diaza bicycloundecene) 0.35 8 ml, Lithium fluoride 93 mg, add 1 at room temperature
. 5時間浸拌した。 反応液を酢酸ェチル抽出し、 シリカゲルクロマトグラフィ 一 (クロ口ホルム Zメタノール） にかけ、 化合物 （ I I I— 1 ) 6 70mg ( 90 %収率） を得た。 Stir for 5 hours. The reaction mixture was extracted with ethyl acetate and subjected to silica gel chromatography (form Z methanol) to obtain 70 mg (90% yield) of the compound (II-1).
(第 8工程） 化合物 （ I I I一 2) 、 （ I I I一 3) の合成  (Eighth Step) Synthesis of Compounds (II-1) and (II-1)
化合物 （ I I 1— 1 ) 5mg (6. 6 mo l ) のメタノール 300 /^ 1溶 液に 1 N水酸化ナトリウム 1 00 1 を加えた。 室温で 1. 5時間挽拌後酢酸 ェチル抽出した。 抽出液を減圧濃縮し、 残さをシリカゲルクロマトグラフィー To a solution of 5 mg (6.6 mol) of compound (II-1) in 300 / ^ 1 methanol was added 1N sodium hydroxide 10001. After stirring at room temperature for 1.5 hours, the mixture was extracted with ethyl acetate. The extract is concentrated under reduced pressure, and the residue is subjected to silica gel chromatography.
(酢酸ェチル ： 酢酸 ： 水、 30 : 1 : 1 ) にて精製し、 化合物 （ I I I一 2 ) を 3. 5 mg (収率 7 3 %) 得た。 さらに、 この化合物 2 1. 9 m g (0. 0 3 mm o 1 ) を水 1. 2m l に懸濁し、 0. 1 N水酸化ナトリウム 6 00 / 1 を加えた。 生じた溶液を凍結乾燥し、 2ナトリウム塊 （化合物 I I I一 3 ) を 2 1. 5 mg (93 %収率） 得た。 (Ethyl acetate: acetic acid: water, 30: 1: 1) to give 3.5 mg (yield: 73%) of compound (III-12). Furthermore, the compound 21.9 mg (0.0 3 mmo 1) was suspended in 1.2 ml of water, and 0.1 N sodium hydroxide 600/1 was added. The resulting solution was freeze-dried to obtain 21.5 mg (93% yield) of a disodium mass (Compound III-13).
参考例 1 化合物 5 ) の合成 Reference Example 1 Synthesis of compound 5)
CHCOOMe
Figure imgf000020_0001
CHCOOMe
Figure imgf000020_0001
染井ら （C h em. P h a rm. B u i 1. 28 2 5 1 5 ( 1 9 80 ) ) の方法を応用して化合物 （ 5) を合成した。  Compound (5) was synthesized by applying the method of Somei et al. (Chem. Pharm. Bui 1.28 25 15 (1980)).
1 gのヒドロキシニトロペンズアルデヒド （6) を 2 Om lの酢酸一水 （ 1 1 g of hydroxynitropenzaldehyde (6) in 2 Oml of acetic acid monohydrate (1
： 1 ) 混液に溶解し、 三塩化チタン水溶液 2 5m l を加えた。 室温で 1 0分間 擾拌後、 水一酢酸ェチルにあけた。 水屬を炭酸ナトリウム水溶液で pH 8とし 、 酢酸ェチルで抽出した。 抽出液を無水硫酸マグネシウムで乾燥後、 約 2 0m: 1) The mixture was dissolved in a mixed solution, and 25 ml of an aqueous titanium trichloride solution was added. After stirring at room temperature for 10 minutes, the mixture was poured into water monoethyl acetate. The pH was adjusted to pH 8 with aqueous sodium carbonate and extracted with ethyl acetate. After drying the extract over anhydrous magnesium sulfate, about 20m
1 まで渙縮し、 （ 7 ) の酢酸溶液を得た。 この溶液に氷冷下ピリジン 0. 46 m 1 を加え、 そこに 3—メ トキシカルポニルァクリル酸クロリ ド 442mgを 加えた。 0でで 30分擾拌後、 酢酸ェチルで抽出した。 抽出液を濃縮し、 析出 した固体をろ過し、 化合物 （ 5) の粉末 43 2 mg (2 9 %収率） を得た。 発明の効果 Lysis to 1 gave an acetic acid solution of (7). To this solution, 0.46 ml of pyridine was added under ice cooling, and 442 mg of 3-methoxycarponylacrylic acid chloride was added thereto. After stirring at 0 for 30 minutes, the mixture was extracted with ethyl acetate. The extract was concentrated, and the precipitated solid was filtered to obtain 432 mg (29% yield) of powder of compound (5). The invention's effect
本発明の方法によれば、 ォレアノ—ル酸類緣体が従来の方法に比べて極めて 容易に、 かつ高収率で製造できる。 得られたォレアノール酸類縁体は各種医薬 品またはその製造中間体として非常に有用である，  According to the method of the present invention, an oleanolic acid derivative can be produced very easily and in a high yield as compared with the conventional method. The obtained oleanolic acid analogs are very useful as various pharmaceuticals or intermediates for their production.

Claims

請求の範囲 The scope of the claims
1. ォレアノール酸類縁体を生成し得る植物体由来の毛状根であって、 毛状根 を誘導し得るプラスミ ドによって形 K転換して锈導された該毛状根を培養し、 得られた培義物からォレアノール酸類縁体を採取することによるォレアノール 酸類縁体の製造法。 1. A hairy root derived from a plant capable of producing an oleanolic acid analog, which is obtained by culturing the hairy root induced by transformation into a K with a plasmid capable of inducing a hairy root. A method for producing an oleanolic acid analog by collecting the oleanolic acid analog from the culture medium obtained.
2. 毛状根の誘導方法が、 細菌を植物体に感染させることによって細菌が保持 しているブラスミ ドの一部を植物に導入する方法によるものであることを特徴 とする、 請求の範囲第 1項記載のォレアノール酸類縁体の製造法。  2. The method for inducing hairy roots is by introducing a part of a plasmid carried by the bacteria into the plant by infecting the plant with the bacteria. 2. The method for producing the oleanolic acid analog according to claim 1.
3. 該細菌がァグロパクテリゥム リゾゲネスまたはァグロパクテリゥム ッ メファシエンスである、 請求の範囲第 2項記載のォレアノール酸類縁体の製造 法，  3. The method for producing an oleanolic acid analog according to claim 2, wherein the bacterium is Agrobacterium rhizogenes or Agrobacterium mefaciens.
4. プラスミ ドが R i ブラスミ ドである請求の範囲第 1項または第 2項記載の 製造法。  4. The method according to claim 1 or 2, wherein the plasmid is R i brasmid.
5. 植物体がメリアンサス （Me 1 i a n t h u s ) 属、 ベルサマ （B e r s a m a ) 厲、 ミ リ力 （My r i c a ) 厲、 ポリガラ （P o 1 y g a 1 a) 属、 ァスチルぺ （A s t i 1 b e ) 厲、 アトロキマ （A t r o x i ma) 厲、 ィレ ックス （ I l e x) 厲、 マロタス （Ma i l o t u s ) 厲、 メロキア （Me l o c h i a) 属またはロイプテリア （Rh o i p t e l e a) 属植物体である 請求の範囲第 1項記載の製造法。  5. When the plant is of the genus Merianthus (Me 1 ianthus), versama (Bersama) 厲, Myrica 厲, polygala (Po 1 yga 1a), astyr ぺ (Asti 1 be) 厲, The production according to claim 1, which is a plant belonging to the genus Atroxima 厲, Ilex 厲, Marothus Ma, Melochia or Melopia, or Rhoiptelea. Law.
6. ォレアノール酸類縁体がミリセロール、 2 7—ォキシ—カフェオイルミ リ セロール、 ミ リセロンまたは 2 7—ォキシカフェオイルミ リセロンである請求 の範囲第 1項記載の製造法，  6. The process according to claim 1, wherein the oleanolic acid analogue is myricerol, 27-oxycaffeoylmycerol, myricerone or 27-oxycaffeoylmyricerone.
7. ォレアノール酸類縁体を生成し得る植物体由来の毛状根であって、 毛伏根 を誘導し得るブラスミ ドによって形質転換して誘導された毛状根。  7. Hairy roots derived from a plant capable of producing oleanolic acid analogs, the hairy roots being induced by transformation with a brassmid capable of inducing hairy roots.
8. 請求の範囲第 1項記載の製造法により得られたォレアノール酸類緣体を、 必要であればアル力リ加水分解反応およびノまたは 3位の選択的な酸化反応に 付した後、 ジ低級アルキルホスホノ醉酸を反応させ、 化合物 （ I V) ： OR" 8. If necessary, subject the oleanolic acid derivative obtained by the production method described in claim 1 to a hydrolysis reaction and a selective oxidation reaction at the 3- or 3-position to obtain a di-lower Reacting the alkylphosphonosuccinic acid to give compound (IV): OR "
(IV)  (IV)
OHC OHC
NHR。 NHR.
(式中、 1?6は水素またはー1?6—尺 7でぁり、 R6は一 S 03—、 — CH2CO O—、 一 COCOO—または一 COR8COO— （R 8は低級アルキレンまたは 低級アルケニレン） であり、 R 7は水素または低級アルキルであり、 R9は t一 ブトキシカルボニルまたは水素である） (Wherein, 1 6 hydrogen Matawa 1 6 -?? Length 7 Deari, R 6 an S 0 3 -, - CH 2 CO O-, one COCOO- or a COR 8 COO- (R 8 is Lower alkylene or lower alkenylene), R 7 is hydrogen or lower alkyl, and R 9 is t-butoxycarbonyl or hydrogen.
で示されるアルデヒ ドとホーナーエモンズ反応の反応条件下で縮合させ、 必要 であれば脱保護および または化学修飾を加えることによる、 式 （ I I I ) ： By condensation under the reaction conditions of the aldehyde and the Horner-Emmons reaction represented by the formula (III) by deprotection and / or chemical modification if necessary.
Figure imgf000022_0001
Figure imgf000022_0001
(ここで R 3は水素または代謝性エステル残基を表し、 R 5は前記と同義である(Where R 3 represents hydrogen or a metabolic ester residue, and R 5 is as defined above.
) )
で示される化合物の製造法 * Method for producing compound represented by *
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WO2005090285A1 (en) * 2004-03-24 2005-09-29 Shionogi & Co., Ltd. Novel crystal of triterpene derivative
WO2006077801A1 (en) * 2005-01-18 2006-07-27 Shionogi & Co., Ltd. Method for preparing myricerone derivative
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KR100900177B1 (en) * 2002-03-22 2009-06-02 시오노기세이야쿠가부시키가이샤 Process for producing triterpene derivative
WO2003080643A1 (en) * 2002-03-22 2003-10-02 Shionogi & Co., Ltd. Process for producing triterpene derivative
US7223882B2 (en) 2002-03-22 2007-05-29 Shionogi & Co., Ltd. Process for producing triterpene derivative
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WO2005090285A1 (en) * 2004-03-24 2005-09-29 Shionogi & Co., Ltd. Novel crystal of triterpene derivative
US7612225B2 (en) 2004-03-24 2009-11-03 Shionogi & Co., Ltd. Crystal of triterpene derivative
JP4707115B2 (en) * 2004-03-24 2011-06-22 塩野義製薬株式会社 New crystals of triterpene derivatives
WO2006077801A1 (en) * 2005-01-18 2006-07-27 Shionogi & Co., Ltd. Method for preparing myricerone derivative
JPWO2006077801A1 (en) * 2005-01-18 2008-06-19 塩野義製薬株式会社 Method for producing myricelon derivative
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WO2011116115A1 (en) 2010-03-16 2011-09-22 Novartis Ag Aliskiren composition comprising a medium chain fatty acid, their process of manufacturing

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