WO1997027314A1

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

Info

Publication number: WO1997027314A1
Application number: PCT/JP1997/000127
Authority: WO
Inventors: Kenji Katoh; Masami Matsumoto; Keiichiro Yasumi; Noboru Shirane
Original assignee: Shionogi & Co., Ltd.
Priority date: 1996-01-23
Filing date: 1997-01-22
Publication date: 1997-07-31
Also published as: AU1319697A

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

Description Method for producing oleanolic acid analog by hairy root culture

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.

Millicerol mycelone

= Single bond ^z ^ ^{s =}ー connection

R ^l = -OH

R ² = -HR ² = -H

27-oxy-cuff; t oil myrice mouth-

-^ = Single bond = = double bond

For example, the following formula (II) or (III)

(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.)

(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)

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.

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).

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).

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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. 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.

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.

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.

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.

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. 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.

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. 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.

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.

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.

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. 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.

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.

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.

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.

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.

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.

When oleanolic acid analogs are used as intermediates in the manufacture of pharmaceuticals, The analog can be synthesized by the chemical reaction of

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.

Next, dilower alkyl phosphonoacetic acid is reacted with hairy root culture or myricerone obtained by the above method, and then the compound (IV):

(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.

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):

(Where R ³ and R ⁵ are as defined above)

It is possible to produce the compound represented by the formula

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Preparation of 2 7-ethoxy-Ichirofuehoimyriserol (1) Induction of Merianthus commosus hairy roots

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) Cultivation of Merianthus Comosas hairy roots

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) Extraction of 27-hydroxyl-hue oil mycerol

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.

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) Identification of 27-hydroxyl fueoyl myricerol

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) Optical rotation: [a] D +1 44.0 '(methanol; C1.43)

(2) ¹ HNMR (C5D5N, 400 MHz; TMS internal standard; δ ppm)

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) ¹³ CNMR (C5D5N; TMS internal standard; δ pm) Table 1

Position

C--1 3 8.9

C-2 28.0

C-1 3 7 7. 9

C-4 39.3

C-5 5 5. 7

C-6 1 8.8

C-1 7 3 3.6

C-1 8 40. 4

C-9 49. 0

C-1 0 3 7.5

C-1 1 24. 4

C-1 1 2 1 26. 9

C-1 3 1 38. 8

C-1 4 4 5.9

C-1 5 24. 1

C-2 '1 1 5.3

C-3 '145.7

C-1 4, 1 26.8

C-5'1 15.8

C-6 '1 4 7. 7

C-7 '1 50. 4 Example 2 Synthesis of Compound (III)

Compound (III) was synthesized in the same manner as in JP-A-7-53484 and JP-A-7-3161888.

(m-2), (m-3) (= Na salt)

(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.

(Step 2) Synthesis of Compound (1)

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%,

(Step 3) Synthesis of Compound (2)

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%.

Chromatography conditions: S i 0 ₂ 500 g, methylene chlorinated-ethyl ethynyl chloride methylene chloride = 1 Z 4-ethyl acetate

(Step 4) Synthesis of compound (3)

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.

(Fifth step) Synthesis of myricelone

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

(Step 6) Synthesis of compound (4)

To 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 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.

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%).

(Step 7) Synthesis of Compound (I I I-1)

Compound (4) 62 1 mg (1 mmo 1) and the compound obtained in Reference Example 1 (5) 2 9

9 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

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).

(Eighth Step) Synthesis of Compounds (II-1) and (II-1)

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.

(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).

Reference Example 1 Synthesis of compound 5)

CHCOOMe

Compound (5) was synthesized by applying the method of Somei et al. (Chem. Pharm. Bui 1.28 25 15 (1980)).

1 g of hydroxynitropenzaldehyde (6) in 2 Oml of acetic acid monohydrate (1

: 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

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

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

The scope of the claims

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. 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. The method for producing an oleanolic acid analog according to claim 2, wherein the bacterium is Agrobacterium rhizogenes or Agrobacterium mefaciens.

4. The method according to claim 1 or 2, wherein the plasmid is R i brasmid.

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. The process according to claim 1, wherein the oleanolic acid analogue is myricerol, 27-oxycaffeoylmycerol, myricerone or 27-oxycaffeoylmyricerone.

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. 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)

OHC

NHR.

(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.

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.

(Where R ³ represents hydrogen or a metabolic ester residue, and R ⁵ is as defined above.

)

Method for producing compound represented by *