WO2001007040A1 - Multidrug-resistant cancer overcoming agents and process for producing the same - Google Patents

Multidrug-resistant cancer overcoming agents and process for producing the same Download PDF

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Publication number
WO2001007040A1
WO2001007040A1 PCT/JP2000/005036 JP0005036W WO0107040A1 WO 2001007040 A1 WO2001007040 A1 WO 2001007040A1 JP 0005036 W JP0005036 W JP 0005036W WO 0107040 A1 WO0107040 A1 WO 0107040A1
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compound
esterified
etherified
hydrogen atom
compounds
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PCT/JP2000/005036
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French (fr)
Japanese (ja)
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Masayoshi Ando
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Kobe Natural Products & Chemicals Co., Ltd.
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Priority to AU63155/00A priority Critical patent/AU6315500A/en
Publication of WO2001007040A1 publication Critical patent/WO2001007040A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D493/00Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/08Bridged systems
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/21Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/13Coniferophyta (gymnosperms)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/34Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/02Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen
    • C07C69/12Acetic acid esters
    • C07C69/14Acetic acid esters of monohydroxylic compounds
    • C07C69/145Acetic acid esters of monohydroxylic compounds of unsaturated alcohols
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/02Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen
    • C07C69/12Acetic acid esters
    • C07C69/18Acetic acid esters of trihydroxylic compounds
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/612Esters of carboxylic acids having a carboxyl group bound to an acyclic carbon atom and having a six-membered aromatic ring in the acid moiety
    • C07C69/618Esters of carboxylic acids having a carboxyl group bound to an acyclic carbon atom and having a six-membered aromatic ring in the acid moiety having unsaturation outside the six-membered aromatic ring
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00Systems containing at least three condensed rings
    • C07C2603/02Ortho- or ortho- and peri-condensed systems
    • C07C2603/04Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/36Ortho- or ortho- and peri-condensed systems containing three rings containing eight-membered rings
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00Systems containing at least three condensed rings
    • C07C2603/02Ortho- or ortho- and peri-condensed systems
    • C07C2603/04Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/38Ortho- or ortho- and peri-condensed systems containing three rings containing rings with at least nine members

Definitions

  • the present invention relates to an agent for overcoming cancer, particularly to an agent for overcoming a multidrug-resistant cancer administered to cancer cells (multidrug-resistant cancer cells) that have acquired resistance to a plurality of anticancer agents, and a method for producing the same.
  • multidrug-resistant cancer-overcoming drugs have been developed that are administered to cancer cells (multidrug-resistant cancer cells) that have acquired resistance (multidrug-resistant) to multiple anticancer drugs that have no similar chemical structure or action point with each other.
  • the calcium antagonist Verapamil has the effect of increasing the accumulation of multiple types of anticancer drugs (anticancer substances) such as vincristine in multidrug-resistant cancer cells (excretion inhibitory action). ), That is, it is recognized that it has a multidrug-resistant cancer overcoming action (multidrug-resistant overcoming action).
  • the multidrug-resistant cancer-overcoming agent is required to satisfy conditions such as having a strong multidrug-resistant cancer-overcoming effect and having few side effects.
  • conditions such as having a strong multidrug-resistant cancer-overcoming effect and having few side effects.
  • a multidrug-resistant cancer-overcoming agent that sufficiently satisfies the above conditions has not yet been found.
  • verapamil has a blood pressure lowering effect as a side effect, and thus has not been put to practical use.
  • the taxane-related compounds described in Heterocycles have a multidrug-resistant cancer overcoming effect similar to that of verapamil, it is still unknown whether it is practical or not.
  • characteristics such as low cell killing properties and low in vivo degradation properties may be required as a multidrug-resistant cancer-overcoming agent. Therefore, the search for a novel drug that overcomes multidrug-resistant cancer is particularly necessary from the viewpoint that it has the potential to satisfy these complex and diverse needs and expand the scope of application of the drug that overcomes multidrug-resistant cancer. Have been.
  • the main object of the present invention is to solve the above-mentioned conventional problems, and to provide a novel cancer-overcoming agent, particularly a cancer-cancelling agent to be administered to cancer cells, particularly cancer cells that have acquired resistance to a plurality of anticancer agents (multidrug-resistant cancer cells).
  • An object of the present invention is to provide an agent for overcoming a multidrug-resistant cancer and a method for producing the same.
  • the multidrug-resistant cancer-overcoming agent according to claim 1 of the present invention is characterized by containing at least one compound selected from a group of compounds represented by a specific chemical formula in order to solve the above problems.
  • a cancer-overcoming agent or a multidrug-resistant cancer-overcoming agent comprising at least one compound selected from the above compound group can be suitably administered to the cancer cells. That is, the present invention
  • R 1 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified
  • R 3 represents a hydrogen atom
  • R 4 may be a hydrogen atom or a hydroxyl group, or R 3 and R 4 may be combined together to form a single bond or —O—, or R 3 and R 6 may be combined together and —O— CH 2 — may be formed,
  • R 5 represents a hydrogen atom or a hydroxyl group which may be esterified or esterified
  • R 6 is a methyl group, a hydroxymethyl group, or a hydroxyl group which may be esterified or etherified,
  • R 7 is a hydroxyl group which may be esterified or etherified
  • R 8 is a hydrogen atom or a hydroxyl group which may be esterified or etherified
  • R 9 is a hydrogen atom or a hydroxyl group which may be esterified or etherified
  • R 1 Q may be taken together with a hydrogen atom or R 4 to represent a single bond
  • R 11 may be methyl or esterified or etherified A methyl group having a hydroxyl group
  • R 12 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified
  • R 13 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified
  • R 14 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified
  • R 15 represents a hydroxyl group which may be esterified or etherified
  • R 16 represents a hydrogen atom
  • R 17 may be a methyl group, or R 16 and R 17 may be taken together to form —O—CH 2 —,
  • R 18 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified
  • R 19 represents a hydroxyl group which may be esterified or etherified, and represents a double bond or a single bond
  • R 2 Q represents a hydroxyl group which may be esterified or etherified
  • R 21 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified
  • R 22 represents a hydrogen atom
  • R 23 is a hydrogen atom or a hydroxyl group which may be esterified or etherified
  • R 24 may be a hydrogen atom
  • R 25 is
  • R 26 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified
  • R 26 represents a hydrogen atom.
  • R 27, R 28, R 29, R 3Q, R 31 and R 32 are each independently hydrogen were identical or respectively or be esterified or etherified represents an hydroxy group.
  • a medicament characterized by:
  • a method for treating cancer which comprises administering the compound according to (1) or (2) and an anticancer substance to a cancer patient in combination,
  • a carcinostatic agent comprising a compound represented by the formula:
  • Taxus cuspi data sieb. Et Zucc. A method for producing the compound according to (2), wherein the compound according to (2) is collected from callus derived from the tissue; and (11) a taxin skeleton, a xinine skeleton or A drug having a Abjön skeleton and containing a compound having an anticancer substance accumulation enhancing action or an anticancer substance discharge inhibitory action;
  • the compounds represented by the chemical formulas (6) to (14) are taxin compounds, and the compounds represented by the chemical formulas (15) to (29) are taxinine compounds.
  • the compound represented by the chemical formula (23) is 2'-desacetylaustrospicatine.
  • the compound represented by the chemical formula (24) is 2-desacethoxy taxinine B, and the compound represented by the chemical formula (25) is taxinine E.
  • the compounds represented by the chemical formulas (17), (22), (27), (28) and (29) are novel xinine compounds.
  • the compound represented by the chemical formula (30) is taxuyunnanin C; the compound represented by the chemical formula (31) is 2 ⁇ , 5,10 / 3-triacetoxy-1 14) 3-propioni Roxy-taxa 4 (20), 11-gen; the compound represented by the chemical formula (32) is yunnanxane; the compound represented by the chemical formula (33) is 2 ⁇ , 5,10 / 3-triacetoxy—1 4 3— (2, —methyl) butylyl xy-taxa 4 (20), 11—gen; the compound represented by the chemical formula (34) is 2 ⁇ , 5,10 / 3-triacetoxy—14) 3-isobutylyloxy-mixer 4 (20), 11-gene; The compound represented by the chemical formula (35) is 2 ⁇ -hydroxy-5a, 10) 3, 14 3-triacetoxy-taxa 4 (20), 11-Gen (2-hydroxy-5, 10/3, 14 / 3-triacetoxy-taxa-4 (20), 11-diene); Chemical formula The evening xinine compound represented by (36) 5, 1 3a—Dia
  • the compound represented by the chemical formula (41) is 10-deacetyl taxin.
  • the compound is a taxinine compound; the compound represented by the chemical formula (44) is 2-desacetoxy taxinine E; the compound represented by the chemical formula (45) is taxin NA-2 (taxicin NA-2)
  • All of the compounds of the chemical formulas (6) to (47) can be collected or isolated (hereinafter simply referred to as “collection”) from Nippon Ichi Taxus cuspidata Sieb. Et Zucc. 6) Chemical conversion of compounds belonging to (47) into compounds belonging to other chemical formulas (6) to (47) Can be.
  • the chemical reaction for such a chemical transformation may be a traditional chemical reaction commonly known to chemists or pharmacists.
  • a reduction reaction for converting a carbonyl group into a hydroxyl group by catalytic reduction using a reduction catalyst for example, an esterification reaction for introducing an acetyl group to a cinnamoyl group into a hydroxyl group. Therefore, it is obvious for a chemist or a pharmacist that the compounds represented by the chemical formulas (6) to (47) have the same general formulas (1) to (5). It can be easily converted.
  • the present inventors have conducted intensive studies on the above problems, and as a result, when a compound represented by any of the above formulas (1) to (47) is administered to a cancer patient together with an anticancer substance, the anticancer substance enters the cancer cells.
  • cancer cells or multidrugs from cancer cells or multidrug-resistant cancer cells of anticancer agents whose accumulation in multidrug-resistant cancer cells is enhanced or incorporated into the cancer cells or multidrug-resistant cancer cells Suppressing the excretion of resistant cancer cells out of the cell, and consequently improving the effect of the anticancer substance as an anticancer agent.
  • the action of enhancing the accumulation of a substance in cancer cells or multidrug-resistant cancer cells (hereinafter sometimes simply referred to as cancer cells) or excluding it from cancer cells.
  • a substance hereinafter sometimes simply referred to as an anticancer substance
  • cancer cells multidrug-resistant cancer cells
  • it was useful as a cancer overcoming agent or multidrug resistant cancer overcome agent (sometimes simply referred to as cancer overcoming agent or less). That is, the present invention provides a medicament to be administered by combining the compounds represented by the above formulas (1) to (47) with another anticancer substance.
  • the present inventors have found that the compounds represented by the formulas (1) to (47) It has also been found that an anticancer substance other than the compounds represented by the formulas (1) to (47) is not necessarily required because it acts as a cancer agent.
  • the present inventors have found that the compounds represented by the above formulas (6) to (47) are derived from tissues such as needles of Japanese yew, Taxus cuspidata Sieb. Et Zucc. Can be removed from the callus, and a cancer-overcoming agent suitable for administration to cancer cells in this way can be used efficiently and efficiently while preserving the environment. It was found that it can be manufactured industrially.
  • the compound used as a cancer-surviving agent or a multidrug-resistant cancer-surviving agent in medicine is a compound represented by any of the general formulas (1) to (5).
  • the etherified hydroxyl group can be represented by the formula
  • the group represented by z C- is an acyl group.
  • groups represented by zi and z 2 can be a substituent for any such chemical modifications unless contrary to the spirit of the present invention, in the pharmaceutical field ester forming group or ether forming groups (or ester residue Or an ether residue) is well known in the patent literature or the scientific literature, and therefore, in the present invention, such a substituent for a modification known per se can be used, Such an ester-forming group or an ether-forming group may be performed according to a conventionally known method.
  • Groups represented by and Z 2 specifically, for example, carbon number 1 to 50 of about saturated or unsaturated, linear, branched or cyclic alkyl group, C 7 -C 2 o about Ararukiru group , etc. Ariru group of about C 6 -C 15 and the like.
  • substituents are commonly used for the chemical modification of active ingredients of ordinary pharmaceuticals such as, for example, a hydroxyl group, an amino group, a carbonyl group, a sulfonyl group (for example, a methylsulfonyl group), a halogen atom, a nitro group, and an alkoxy group. It may be further substituted by a substituent.
  • a method for producing the compound of the present invention and the like will be described.
  • These compounds which are taxane-related compounds such as taxin compounds or evening xinine compounds (generic name: evening xoids), are sufficient for, for example, cancer cells (multidrug-resistant cancer cells) that have acquired resistance to multiple anticancer agents.
  • Multi-drug resistance Has cancer-overcoming action.
  • the agent for overcoming a multidrug-resistant cancer is at least one selected from the group of compounds represented by the above chemical formulas (11), (15), (16), (17), (22), (24), and (26). More preferably, it contains one kind of compound. These seven compounds have a stronger effect of overcoming multidrug-resistant cancer. Then, they are represented by the above chemical formulas (17), (22), (27), (28), and (29).
  • the five types of xinine compounds are new substances.
  • the multidrug-resistant cancer-overcoming agent is capable of transferring various anticancer agents incorporated into cancer cells (multidrug-resistant cancer cells) that have acquired resistance to a plurality of anticancer agents (multidrug-resistant cancer cells) out of the multidrug-resistant cancer cells It has the effect of inhibiting any of a plurality of action mechanisms that emit.
  • an action mechanism for excreting an anticancer drug out of a multidrug-resistant cancer cell specifically, for example, various P-glycoproteins present or expressed in the multidrug-resistant cancer cell are incorporated into the multidrug-resistant cancer cell.
  • a mechanism that binds to various anticancer drugs and discharges them by active transport is exemplified. The above chemical formula
  • Each of the compounds represented by (6) to (29) alone has at least a function as an inhibitor of the active transport of an anticancer agent by P-glycoprotein (hereinafter, referred to as a P-glycoprotein inhibitor). It can be a multidrug-resistant cancer-overcoming agent according to the present invention.
  • P-glycoprotein inhibitor P-glycoprotein inhibitor
  • the fact that the above-mentioned compound has a multidrug-resistant cancer overcoming action is newly found by the present inventors.
  • the compounds represented by the chemical formulas (6) to (29) are referred to as compounds (6) to (29), respectively. the same).
  • At least one of the above compounds is extracted from needles of Japanese yew (Taxus cuspidata Sieb. Et Zucc.) Using, for example, an organic solvent. After that, the obtained extract is treated with an acid and or a base, followed by fractionation by liquid chromatography and a step of taking out the extract, thereby producing the extract.
  • the needle portion of the Japanese yew is relatively easy to obtain, and it is distributed, for example, from the subtropics of Hokkaido, Honshu, Shikoku, and Kyushu to temperate zones.
  • the “needle portion” includes “leaf”, “stalk”, and “twig” having a plurality of the “leaf (leaf + stalk)”. (The primordium may be used.) ⁇ “Sprouts” with the “sprout” shall be included.
  • the compounds (6) to (29) from the needle portion of yew tree, ie, taxane The specific method of extracting the related compound is not particularly limited, but a method of performing extraction is preferable, and a method of performing extraction using an organic solvent is most suitable.
  • a specific method for performing the extraction for example, there is a method in which a needle part (raw needle part) is pulverized as necessary, and then immersed in an organic solvent for several days to several weeks.
  • the extraction conditions are not particularly limited, but the extraction temperature is more preferably 30 or less.
  • organic solvent examples include, but are not limited to, methyl alcohol, ethyl alcohol, isopropyl alcohol, chloroform, n-hexane, ethyl acetate, various ethers, and toluene. Not something.
  • One of these organic solvents may be used alone, or two or more thereof may be used in combination.
  • methyl alcohol and ethyl acetate are more preferred.
  • the needle portion may be washed with an organic solvent in order to remove (degrease) oil contained in the needle portion.
  • organic solvents exemplified above, n-hexane is suitable for removing oil contained in needles.
  • the extract from which the taxane-related compound has been extracted is preferably treated with an acid and / or a base in order to separate and remove components such as an alloid-derivative and a phenol derivative.
  • a method of treating the extract with an acid and / or a base specifically, a method of washing the extract with acidic water and Z or basic water can be mentioned.
  • acids include, for example, inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid; and organic acids such as formic acid and acetic acid, but are not particularly limited. These acids may be used alone or in combination of two or more. Therefore, examples of the acidic water include aqueous solutions of these inorganic acids and organic acids.
  • the pH of the acidic water is particularly limited, However, it is preferably 5 or less, and more preferably 4 or less.
  • taxane-related compounds have an oxetane skeleton in the molecular structure that is easily cleaved (decomposed) by an acid. Therefore, it is generally considered that the treatment with an acid causes ring cleavage of the oxenone skeleton to destroy the taxane-related compound.
  • the present inventors have studied and found that the taxane-related compound did not undergo ring cleavage of the oxetane skeleton even when treated with an acid. Therefore, by treating with an acid, a taxane-related compound and components such as alkaloid derivatives can be separated.
  • the above base include: inorganic bases such as ammonia, sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate: organic bases such as ammonium compounds; It is not limited. One of these bases may be used alone, or two or more may be used in combination. Accordingly, examples of the basic water include aqueous solutions of these inorganic bases and / or organic bases.
  • the pH of the basic water is not particularly limited, but is preferably 9 or more, more preferably 10 or more, and particularly preferably 11 or more.
  • the taxane-related compound has an ester which is easily hydrolyzed by a base in its molecular structure. Therefore, it is generally believed that treatment with a base will hydrolyze the ester and destroy the taxane-related compound.
  • the inventors of the present application have examined that taxane-related compounds can undergo ester hydrolysis even when treated with a base, particularly a strong base. Turned out not to be. Therefore, by treating with a base, it is possible to separate a taxane-related compound from components such as a phenol derivative.
  • the extract after the treatment with an acid and / or a base is more preferably washed with water until neutral. It is more preferable that the extract after washing with water is concentrated (removing the organic solvent) so that the taxane-related compound can be easily isolated. By concentrating the extract, a neutral fraction is obtained.
  • the specific method for isolating and purifying the taxane-related compounds from the neutral fraction, that is, the individual compounds (6) to (29) belonging to the compound group, is not particularly limited.
  • a method employing lithography is preferred.
  • Specific examples of the liquid chromatography include silica gel column chromatography, reversed-phase or normal-phase high-performance liquid chromatography (HPLC), and centrifugal liquid-liquid distribution chromatography (CPC). There is no particular limitation. In the above-described high performance liquid chromatography, for example, it is sufficient to select the reversed phase or the normal phase in consideration of the pH of the mobile phase.
  • the stationary phase includes, but are not particularly limited to, silica gel, alumina, and ODS (octyl decylsilyl) -based compounds.
  • ODS octyl decylsilyl
  • stationary phases when employing reversed-phase high performance liquid chromatography or the like, ODS compounds are more preferable.
  • liquids suitable for use as a mobile phase (carrier, eluent) include, for example, methyl alcohol, ethyl alcohol, chloroform, n-hexane, ethyl acetate, various ethers, toluene, acetonitrile, Although water is mentioned, it is not particularly limited.
  • One of these liquids may be used alone, or two or more of them may be used in combination.
  • silica gel column chromatography and normal phase When using high performance liquid chromatography, etc., a mixed solution of n-hexane / ethyl acetate is more preferable, and when using reversed phase high performance liquid chromatography, etc., a mixed solution of methyl alcohol acetonitrile is preferred. More preferably, when employing centrifugal liquid-liquid distribution chromatography or the like, a mixed solution of n-hexanenomethyl alcohol is more preferable.
  • the pH of the liquid in the reversed-phase high-performance liquid chromatography that is, the pH of the mobile phase is adjusted to be acidic by a buffer solution. That is, the isolation and purification using the reversed-phase ODS column are more preferably performed under acidic conditions.
  • the buffer solution is not particularly limited as long as the pH of the mobile phase can be adjusted to 5.5 or less.
  • Examples of the buffer solution include, for example, aqueous ammonia acetate. Liquid and the like.
  • the combination of the above liquid and buffer solution is not particularly limited.
  • the resolution of the liquid chromatography is further improved, so that the taxane-related compound can be more selectively isolated.
  • the pH of the mobile phase is near neutral, the separation / removal of the alkyloid derivative may not be performed sufficiently.
  • the component (peak) to be separated / removed increases, or the component (peak) becomes broad and the separation ability is low. In some cases, the component (peak) cannot be specified (analyzed) due to a drop or a change in the retention time.
  • Separation of the neutral fraction using liquid chromatography and isolation and purification of taxane-related compounds include: (1) silica gel column chromatography using a silica gel flash column, Centrifugal liquid-liquid partition chromatography, n-hexane acetic acid Using a methyl-based mixed solution (eluent), the neutral fraction is separated and a fraction with a moderate polarity (fraction) is taken out. Next, (2) normal phase high performance liquid chromatography is used. The fraction was separated using a mixed solution (carrier) of n-hexane Z-ethyl acetate, and (3) methyl alcohol was added to 0.05 M by using reversed-phase high-performance liquid chromatography.
  • the fraction obtained in (1) above was further separated.
  • a method of extracting a fraction containing a taxane-related compound may be mentioned.
  • the taxane-related compound can be separated from the components such as the alkyloid derivative and the phenol derivative, so that the taxane-related compound can be purified. If necessary, the separation / purification operation of the above 1.3 is repeated to further improve the purity of the taxane-related compounds, ie, the individual compounds (6) to (29) belonging to the compound group. Can be done.
  • the above-described liquid chromatography may be employed, and the analysis operation may be performed under the same conditions as the above-mentioned isolation and purification conditions. That is, the analysis is more preferably performed under acidic conditions.
  • the resolution of liquid chromatography is further improved, so that taxane-related compounds can be more accurately analyzed. If the pH of the mobile phase is near neutral, the number of components (peaks) to be separated and removed increases, or the components (peaks) become broad and the separation ability decreases, and the retention time changes. May not be able to identify (analyze) the component (peak).
  • the compounds (6) to (29) are obtained by performing a series of extraction operations such as the above extraction.
  • Each of the compounds (6) to (29) is a multidrug against cancer cells (multidrug-resistant cancer cells) that have acquired resistance to a plurality of anticancer drugs. Has the effect of overcoming resistant cancer. Therefore, the multidrug-resistant cancer-overcoming agent comprising at least one compound selected from the group of compounds represented by the chemical formulas (6) to (29) can be suitably administered to the cancer cells.
  • the compounds (6) to (29) are stable to acids or bases as described in the above description (the description of the method for removing from needles), and are therefore decomposed in vivo, for example. Degradation by gastric juice, decomposition by bile, etc.) is low. Therefore, the multidrug-resistant cancer-overcoming agent according to the present invention comprising at least one compound selected from compounds (6) to (29) has, for example, a small restriction on its administration method and administration place, and Application is expected to be possible.
  • compounds (11), (15) to (17), (22), (24), and (26) are highly active compounds that have a multidrug-resistant cancer-surpassing action superior to verapamil. By itself, it can be a particularly effective (new) drug for overcoming multidrug-resistant cancer.
  • the compounds (6) to (29) can be taken out from the needle portion of the yew tree. That is, according to the production method of the present invention, the needles regenerated every year are used, so that the compounds (6) to (29) can be produced from Nippon yew while preserving the environment. Thus, a multidrug-resistant cancer-overcoming agent suitable for administration to cancer cells can be produced while preserving the environment.
  • the multidrug-resistant cancer-overcoming agent according to the present invention may further comprise, as necessary, an antibody capable of specifically binding to a specific site (or P-glycoprotein) of a cancer cell, an anticancer agent, and the like. It may be.
  • an antibody capable of specifically binding to a specific site (or P-glycoprotein) of a cancer cell an anticancer agent, and the like. It may be.
  • the administration methods of these multidrug-resistant cancer-surviving agents are particularly limited. Instead, if an optimal drug delivery system is adopted, it should be based on the properties of the substances contained in the multidrug-resistant cancer-overcoming agent (refer to the compounds (6) to (29), antibodies, anticancer agents, etc.) Good.
  • the compounds represented by the above chemical formulas (30) to (39) are each independently used as an inhibitor of at least the active transport of an anticancer agent by P-glycoprotein (hereinafter referred to as a P-glycoprotein inhibitor). It has a function and can be a multidrug-resistant cancer overcoming agent according to the present invention.
  • a P-glycoprotein inhibitor P-glycoprotein inhibitor
  • the compounds represented by (33) and (36) have remarkably higher action of overcoming multidrug-resistant cancer than verapamil which is a conventional multidrug-resistant cancer-overcoming agent, and are hereinafter referred to as highly active compounds. More preferably, the multidrug-resistant cancer-overcoming agent according to the present invention contains at least one of these highly active compounds.
  • the taxinin compound represented by the chemical formula (36) is a novel xinine compound according to the present invention, and the abyssinine compound represented by the chemical formulas (37) to (39). Are all novel aviethane compounds according to the present invention.
  • the compounds represented by the chemical formulas (30) to (39) will be referred to as compounds (30) to (39) as needed.
  • the multidrug-resistant cancer-overcoming agent according to the present invention further includes, as necessary, for example, an antibody capable of specifically binding to a specific site (or P-glycoprotein) of a cancer cell, an anticancer agent, and the like. May be configured. As a result, an effect of reliably supplying a multidrug-resistant cancer-overcoming agent to cancer cells to be targeted (multidrug-resistant) and an effect of enabling simultaneous supply of an anticancer agent to the cancer cells can be realized. I can do it.
  • the administration method of these multidrug-resistant cancer-overcoming agents is not particularly limited, and includes the substances contained in the multidrug-resistant cancer-overcoming agents (compounds (30) to (39), antibodies, anticancer agents, etc. Optimal drug delivery according to the physical properties of —You can adopt a system.
  • the method for producing a multidrug-resistant cancer-overcoming agent according to the present invention includes a method for preparing at least one of the compounds (30) to (39) contained in the multidrug-resistant cancer-overcoming agent described above (hereinafter simply referred to as a compound, (3) to (3 9) only when the compound refers to the compound of (1) Taxus cuspidata Sie. Et Zucc.) It is.
  • the “tissue of Japanese yew” refers to a tissue derived from Japanese yew, more specifically, a plant body (each of the tissues forming the root, stem, and leaf, that is, the entire vegetative organ). And the tissues that make up the flowers, pollen, and fruits (reproductive organs).
  • the term “leaf” as used herein refers to a unit composed of “leaf” and “stalk”, and “fruit” refers to “temporary seed garments (aryl) J and“ It is a concept that includes "seed.”
  • the “needle part” is compared in terms of A) the proliferative ability of cultured cells, that is, callus induction, and B) the collection of tissues. It is more suitable because it is easy to target and does not cause significant damage to the plant.
  • the above “needle part” is “leaf”, “petiole”, “twig” with a plurality of “leaves”, and “sprout (or its primordium)”.
  • young shoot with the “sprout” is also included.
  • seedle parts “young shoots” (green young shoots with a diameter of about 2 mm to 3 mm) collected in the autumn and winter are particularly excellent in callus inducibility, and in some generations. It is particularly preferable to carry out subculture over a long period, since the production amount of the compound does not decrease. Also, young tissues such as shoots and seedlings can be suitably used as tissues for callus induction.
  • Japan yew tissue There is no particular limitation on the timing of the callus-inducing “Japanese yew tissue” (hereinafter sometimes referred to as an explant), but if the above tissue is obtained from a plant, it must be collected before harvesting. Average monthly temperature of 18 X: below, better Preferably, they are collected after being grown in an environment that is less than or equal to 5. This environment may be artificially created.
  • Induction of callus from explants and their cultivation can be performed by a general method without using special additives (additives) or culturing conditions.
  • additives additives
  • plant culture media such as agar media (solid media)
  • auxin can be easily implemented.
  • shaking culture may be performed using a liquid medium containing auxin.
  • a section of an explant is sterilized with a 70% by weight aqueous solution of ethyl alcohol and the like, and then the above section contains agar powder and further contains auxin in a predetermined manner.
  • the cells are placed on a solid medium such as a modified Gamborg's medium containing the same concentration, and then cultured in a dark place at 25 to 27 (static culture). Not something.
  • the cultured cells are selected by a simple method using the above solid medium, for example, every 30 days to 50 days, more preferably every 40 days.
  • the callus can be cultured in large quantities with good reproducibility. That is, since the callus can be cultured in a large amount and contains the compound in a relatively large amount, the compound can be efficiently produced.
  • auxin examples include 1-naphthylacetic acid, 2-naphthylacetic acid (NAA; also known as naphthaleneacetic acid), 2,4-dichlorophenoxyacetic acid, indoleacetic acid, and 4-chloro-indoleacetic acid (4 -C1 IAA), indolebutyric acid and the like, but are not particularly limited.
  • NAA 2-naphthylacetic acid
  • 2-naphthylacetic acid also known as naphthaleneacetic acid
  • 2,4-dichlorophenoxyacetic acid 2,4-dichlorophenoxyacetic acid
  • indoleacetic acid examples of the auxins exemplified above, 1-naphthylacetic acid, 2-naphthylacetic acid, 2,4-Dichlorophenoxyacetic acid, and 4-chloroindoleacetic acid are more preferred.
  • the auxin content in the solid medium is preferably more than
  • Omg / L more preferably about 0. SmgZL. If the auxin content exceeds 1. OmgZL, callus growth may gradually decrease as the subculture is repeated. (4) The solid medium containing one-dose indole acetic acid can further improve the productivity of the compound as a secondary metabolite in cultured cells.
  • the solid medium further contains additives such as a browning inhibitor such as cytokinin and poly (N-vinyl-2-pyrrolidone); an elicita such as methyl jasmonate; an oligosaccharide; and various vitamins. It is good.
  • the solid medium containing methyl jasmonate can further improve the productivity of the compound in cultured cells.
  • the content of the additive in the solid medium may be set according to the type and combination of the additive.
  • Specific examples of the oligosaccharides include disaccharides to pentasaccharides obtained by hydrolyzing viscous polysaccharides extracted and purified from edible okra fruits (or their plants) with an acid.
  • KTOS oligosaccharides
  • the oligosaccharide is composed of, for example, monosaccharides such as rhamnose, galactose, galacturonic acid, and glucose.
  • the above-mentioned KT OS has an action of suppressing aging of cultured cells due to passage. For this reason, by using a solid medium containing KTOS, the subculture can be repeated while suppressing senescence, so that the cultured cells can be kept growing. Therefore, the solid medium containing KTOS can further improve the productivity of the compound with respect to cultured cells.
  • the solid medium (or liquid medium) contains 4-cloth indoleacetic acid and Z or oligosaccharide, and further contains, if necessary, 1-naphthylacetic acid and ⁇ or 2-naphthylacetic acid. Is particularly preferred.
  • the specific method of extracting the above compound from the callus is not particularly limited, but a method of performing extraction is preferable, and a method of performing extraction using an organic solvent is most suitable.
  • a specific method for performing the extraction for example, a method in which cocoa is dried by freeze-drying or the like, crushed if necessary, and then immersed in an organic solvent for several minutes to several hours can be mentioned.
  • the extraction conditions are not particularly limited, but the extraction temperature is preferably 30 or less. Then, the above compound can be isolated and purified by treating the obtained extract with an acid and / or a base, if necessary, and then fractionating by liquid chromatography.
  • the dried callus (hereinafter, dried) Callus) is crushed as necessary, immersed in a low-polar organic solvent such as n-hexane and extracted, and then the resulting extract is roughly separated by column chromatography using silica gel. After that, it can be isolated using normal phase high performance liquid chromatography (HPLC).
  • HPLC normal phase high performance liquid chromatography
  • the dried callus after the extraction of the compounds (30) to (36) is The extract is immersed in a highly polar organic solvent such as ethyl acetate, black form, ether, etc. for extraction, and then the resulting extract is treated with an acid and a base to remove coexisting alloid phenols. After being roughly separated by column chromatography using silica gel, it can be isolated using normal phase high performance liquid chromatography (HPLC).
  • HPLC normal phase high performance liquid chromatography
  • organic solvent examples include, for example, methyl alcohol and ethyl alcohol.
  • One of these organic solvents may be used alone, or two or more thereof may be used in combination.
  • methyl alcohol, n-hexane, and ethyl acetate are more preferred.
  • the extract from which the above compounds have been extracted may be treated with an acid and Z or a base, if necessary, to separate and remove basic components such as alloid derivatives and acidic components such as phenol derivatives contained as impurities. Processing is more preferred.
  • a method of treating the extract with an acid and Z or a base specifically, a method of washing the extract with acidic water and Z or a basic water can be mentioned.
  • the extract after the treatment with an acid and / or a base is more preferably washed with water until neutral.
  • the above-mentioned acids include, for example, inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid; and organic acids such as formic acid and acetic acid, but are not particularly limited. These acids may be used alone or in combination of two or more. Accordingly, the acidic water includes aqueous solutions of these inorganic acids and Z or organic acids.
  • the pH of the acidic water is not particularly limited, but is preferably 5 or less, and more preferably 4 or less.
  • the above base include: inorganic bases such as ammonia, sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate; and organic bases such as ammonium compounds. It is not limited. One of these bases may be used alone, or two or more may be used in combination. Accordingly, examples of the basic water include aqueous solutions of these inorganic bases and / or organic bases. PH of the basic water Is not particularly limited, but is preferably 9 or more, more preferably 10 or more, and particularly preferably 11 or more. By washing the extract with basic water, acidic components such as phenol derivatives can be separated and removed from the extract. It has been confirmed that all of the above compounds (30) to (39) are stable to acids or bases.
  • the extract from which the compound has been extracted is more preferably concentrated (to remove the organic solvent) so that the compound can be easily isolated. By concentrating the extract, a neutral fraction is obtained.
  • the specific method for isolating and purifying the above compound from the neutral fraction is not particularly limited, but a method employing liquid chromatography is preferred.
  • Specific examples of the liquid chromatography include silica gel column chromatography, reversed-phase or normal-phase high-performance liquid chromatography, and centrifugal liquid-liquid distribution chromatography (CPC). It is not something to be done.
  • the reversed phase or the normal phase may be selected in consideration of the pH of the mobile phase.
  • the stationary phase includes, but are not particularly limited to, silica gel, alumina, and ODS (octyl decylsilyl) -based compounds.
  • ODS octyl decylsilyl
  • stationary phases when employing reversed-phase high performance liquid chromatography or the like, ODS compounds are more preferable.
  • liquids suitable for use as a mobile phase (carrier, eluent) include, for example, methyl alcohol, ethyl alcohol, chloroform, n-hexane, ethyl acetate, various ethers, toluene, acetonitrile , Water and the like, but are not particularly limited.
  • One of these liquids may be used alone, or two or more of them may be used in combination.
  • silica gel column chromatography and normal phase When using high performance liquid chromatography, etc., a mixed solution of n-hexane and ethyl acetate is more preferable, and when using reverse phase high performance liquid chromatography, etc., a mixed solution of methyl alcohol / acetonitrile is preferable.
  • a mixed solution of n-hexane / methyl alcohol is more preferable.
  • the pH of the liquid in the reversed-phase high-performance liquid chromatography that is, the pH of the mobile phase is adjusted to be acidic by a buffer solution. That is, the isolation and purification using the reversed-phase ODS column are more preferably performed under acidic conditions.
  • the buffer solution may be any solution that can adjust the pH of the mobile phase to 5.5 or less, and is not particularly limited. Specific examples of the buffer solution include an aqueous solution of ammonium acetate. The combination of the above liquid and buffer solution is not particularly limited.
  • the resolution of the liquid chromatography is further improved, so that the above compound can be more selectively isolated. If the pH of the mobile phase is near neutral, the separation / removal of the alkyloid derivative may not be performed sufficiently. In addition, since the addition of a proton occurs reversibly on the nitrogen atom of the alkaloid derivative, the number of components (peaks) to be separated / removed increases, or the components (peaks) become broad and the separation ability decreases. In some cases, the component (peak) cannot be specified (analyzed) due to the change in the retention time.
  • the neutral fraction is separated by liquid chromatography to isolate and purify the above compounds.
  • specific methods include: (1) silica gel column chromatography using a silica gel flash column; Using liquid partition chromatography, n-hexaneethyl acetate Using a mixed solution (eluent) of the above, the neutral fraction is separated and a fraction (fraction) having a moderate polarity is taken out.
  • (2) normal phase high performance liquid chromatography The fraction was separated using a mixed solution (carrier) of n-hexane Z-ethyl acetate, and (3) reverse-phase high-performance liquid chromatography.
  • the fraction obtained in (1) above is further separated to contain the above compound
  • a method of extracting the fraction By performing the above separation / purification operations, the compound can be separated from a basic component such as an alkaloid derivative or an acidic component such as a phenol derivative, so that the compound can be purified. If necessary, the purity of the above compound can be further improved by repeating the above steps (2) and (3) for separation and purification. In other words, by performing the above separation and purification operations, the above compound can be efficiently extracted and separated.
  • the analysis operation may be performed under the same conditions as the above-mentioned isolation / purification conditions, using the above-mentioned liquid chromatography. That is, the analysis is more preferably performed under acidic conditions.
  • the resolution of liquid chromatography is further improved, so that the above compound can be analyzed more accurately. If the pH of the mobile phase is near neutral, the number of components (peaks) to be separated / removed increases, or the components (peaks) become broad, resulting in a decrease in resolution and a change in retention time. In some cases, the component (peak) cannot be specified (analyzed).
  • the method for analyzing the above compounds is not particularly limited.
  • Compounds (30) to (39) are obtained by performing a series of extraction operations such as the above extraction. All of the compounds (30) to (39) are plural It has a multidrug-resistant cancer overcoming effect on cancer cells that have acquired resistance to anticancer drugs (multidrug-resistant cancer cells). Therefore, a multidrug-resistant cancer-overcoming agent containing at least one compound selected from the group of compounds represented by the above chemical formulas (30) to (39) can be suitably administered to the cancer cells.
  • the multidrug-resistant cancer-overcoming agent according to the present invention comprising at least one compound selected from the compounds (30) to (39) has, for example, little restrictions on its administration method and administration place, and has a wide range. It is expected that a green application is possible.
  • the compounds (31), (33), and (36) are highly active compounds having a multidrug-resistant cancer-overcoming action superior to verapamil, and are therefore particularly effective (novel) multidrugs by themselves. It can be a drug for overcoming resistant cancer.
  • the above compounds (30) to (39) can be extracted from calli obtained by culturing Japanese yew tissues.
  • the above calli are derived from a small amount of explants and can be cultured in large quantities with simple equipment with good reproducibility.
  • the callus contains a relatively large amount of the above compound having a multidrug-resistant cancer overcoming effect. That is, according to the production method of the present invention, the above-mentioned compounds (30) to (39) can be efficiently produced from Nippon yew while preserving the environment. This makes it possible to efficiently produce a multidrug-resistant cancer-overcoming agent suitable for administration to cancer cells.
  • the compounds represented by the above chemical formulas (40) to (47) are each independently used as an inhibitor of at least the active transport of an anticancer agent by P-glycoprotein (hereinafter referred to as P-glycoprotein inhibitor). It has a function and can be a multidrug-resistant cancer overcoming agent according to the present invention. Further, among the above compounds, the chemical formula (40), The compounds represented by (41), (43), (44), (45), (46), and (47) are more effective than the conventional multidrug-resistant cancer-overcoming drug verapamil. Highly effective in overcoming resistant cancer and is hereinafter referred to as a highly active compound. More preferably, the multidrug-resistant cancer-overcoming agent according to the present invention contains at least one of these highly active compounds.
  • the multidrug-resistant cancer-overcoming agent according to the present invention further includes, as necessary, for example, an antibody capable of specifically binding to a specific site (or P-glycoprotein) of a cancer cell, an anticancer agent, and the like. May be configured.
  • an antibody capable of specifically binding to a specific site (or P-glycoprotein) of a cancer cell an anticancer agent, and the like. May be configured.
  • an effect of reliably supplying a multidrug-resistant cancer-overcoming agent to cancer cells to be targeted (multidrug-resistant) and an effect of enabling simultaneous supply of an anticancer agent to the cancer cells can be realized. I can do it.
  • the administration method of these multidrug-resistant cancer-overcoming agents is not particularly limited, and the substances contained in the multidrug-resistant cancer-overcoming agents (refer to the compounds (40) to (47), antibodies, anticancer agents, etc.) An optimal drug delivery system according to physical properties may be adopted.
  • the method for producing a multidrug-resistant cancer-overcoming agent according to the present invention comprises the step of introducing the compound (40) contained in the above-described multidrug-resistant cancer-overcoming agent from "a tissue of Nippon yew Taxus cuspidata Sieb. Et Zucc.). (Hereinafter sometimes referred to as production method A). Further, the method for producing another multidrug-resistant cancer-overcoming agent according to the present invention comprises A method that includes a step of removing at least one of the compounds (41) to (47) contained in the agent from the "needle portion" of Nippon Ichi (hereinafter sometimes referred to as Production Method B) .
  • the “tissue of Japanese yew” refers to a tissue derived from Japanese yew, more specifically, a plant body (each of the tissues forming the root, stem, and leaf, that is, the entire vegetative organ). And the tissues that make up the flowers, pollen, and fruits (reproductive organs).
  • the term “leaf” used herein refers to a unit composed of “leaf” and “stalk”, and “fruit” refers to “temporary garments” It is a concept that includes "seed.”
  • the “needle part” is compared in terms of A) the proliferative ability of cultured cells, that is, callus induction, and B) the collection of tissues. It is more suitable because it is easy to target and does not cause significant damage to the plant.
  • the above “needle part” is “leaf”, “petiole”, “twig” with a plurality of “leaves”, and “sprout (or its primordium)”.
  • young shoot with the “sprout” is also included.
  • seedle parts “young shoots” (green young shoots with a diameter of about 2 mm to 3 mm) collected in the autumn and winter are particularly excellent in callus inducibility, and in some generations. It is particularly preferable to carry out subculture over the whole, since the production of the compound (40) does not decrease.
  • young tissues such as shoots and seedlings can be suitably used as tissues for callus induction.
  • callus can be easily induced from the explant, so that large-scale culture can be performed with simple equipment, and the compound (40) can be extracted from the callus in a large amount.
  • a method for forming callus specifically, for example, a section of an explant is sterilized with 70% by weight (mass) aqueous solution of ethyl alcohol or the like, and then, the above-mentioned section contains agar powder.
  • a solid medium such as a modified gamborg medium containing auxin at a predetermined concentration, and then cultured in a dark place at 25 to 27 (static culture).
  • a solid medium such as a modified gamborg medium containing auxin at a predetermined concentration
  • the cultured cells are selected by a simple method using the above solid medium, for example, every 30 days to 50 days, more preferably every 40 days.
  • the callus can be cultured in large quantities with good reproducibility. That is, since the callus can be cultured in a large amount and contains the compound (40) in a relatively large amount, the compound (40) can be efficiently produced.
  • auxin examples include, for example, 1-naphthyl acetic acid, 2-naphthyl acetic acid (NAA; also known as naphthylene acetic acid), 2,4-dichlorophenoxy acetic acid, indole acetic acid, and 4-chloro-indole acetic acid (4-C1 IAA), indolebutyric acid and the like, but are not particularly limited.
  • NAA also known as naphthylene acetic acid
  • 2-chloro-indole acetic acid 4-chloro-indole acetic acid (4-C1 IAA), indolebutyric acid and the like, but are not particularly limited.
  • 1-naphthylacetic acid, 2-naphthylacetic acid, 2,4-dichlorophenoxyacetic acid, and 4-chloroindoleacetic acid are more preferred.
  • the auxin content in solid media is greater than 0 and 1.0. mgZL or less is preferable, and about 0 SmgZL is more preferable. If the auxin content exceeds 1. OmgZL, the callus growth may gradually decrease as the subculture is repeated.
  • the solid medium containing 4-cloth indole acetic acid can further improve the productivity of the compound (40), which is a secondary metabolite, in cultured cells.
  • the solid medium further contains additives such as cytokinin and poly (N-vinyl-2-pyrrolidone) as anti-browning agents; elicitas such as methyl jasmonate; oligosadride; various vitamins; It is good.
  • the solid medium containing methyl jasmonate can further improve the productivity of the compound (40) in cultured cells.
  • the content of the additive in the solid medium may be set according to the type and combination of the additive.
  • oligosaccharides include disaccharides to pentasaccharides obtained by hydrolyzing viscous polysaccharides extracted and purified from edible okra fruits (or their plants) with an acid.
  • a mixture of oligosaccharides (hereinafter abbreviated as KTOS) is mentioned.
  • the oligosaccharide is composed of, for example, monosaccharides such as rhamnose, galactose, galacturonic acid, and glucose.
  • the above-mentioned KT OS has an action of suppressing aging of cultured cells due to passage. For this reason, by using a solid medium containing KTOS, the subculture can be repeated while suppressing senescence, so that the cultured cells can be kept growing. Therefore, the solid medium containing KTOS can further improve the productivity of the compound (40) in cultured cells.
  • the solid medium contains 4-monoindoleacetic acid and / or oligosaccharide, and further contains 1-naphthylacetic acid and / or 2-naphthylacetic acid as necessary.
  • the specific method of extracting the compound (40) from the callus is not particularly limited, but a method of performing extraction is preferable, and a method of performing extraction using an organic solvent is most suitable.
  • a specific method for performing extraction for example, a method in which calli are dried by freeze-drying or the like, pulverized as necessary, and then immersed in an organic solvent for several minutes to several hours, may be mentioned.
  • the extraction conditions are not particularly limited, but the extraction temperature is more preferably 3 O: or less. Then, the compound (40) can be isolated and purified by treating the obtained extract with an acid and / or a base as necessary, and then fractionating by liquid chromatography.
  • the organic solvent include methyl alcohol, ethyl alcohol, isopropyl alcohol, chloroform, n-hexane, ethyl acetate, various ethers, and toluene, but are not particularly limited. Not something. One of these organic solvents may be used alone, or two or more thereof may be used in combination. Of the organic solvents exemplified above, methyl alcohol, n-hexane, and ethyl acetate are more preferred.
  • the extract from which the compound (40) has been extracted is, if necessary, separated and removed from a basic component such as an alkyloid derivative and an acidic component such as a phenol derivative contained as impurities. More preferably, treatment with an acid and / or a base.
  • a method of treating the extract with an acid and / or a base specifically, a method of washing the extract with acidic water and Z or basic water can be mentioned.
  • the above-mentioned acids include, for example, inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid; and organic acids such as formic acid and acetic acid, but are not particularly limited. These acids may be used alone or in combination of two or more. Therefore, as the acidic water, these inorganic acids and / or Is an aqueous solution of an organic acid.
  • the pH of the acidic water is not particularly limited, but is preferably 5 or less, and more preferably 4 or less.
  • taxane-related compounds have an oxetane skeleton in the molecular structure that is easily cleaved (decomposed) by an acid. Therefore, it is generally considered that the treatment with an acid causes ring cleavage of the oxenone skeleton, thereby destroying the taxane-related compound.
  • studies by the present inventors have revealed that the taxane-related compound of the present invention does not undergo ring cleavage of the oxetane skeleton even when treated with an acid. Therefore, by treating with an acid, the taxane-related compound of the present invention can be separated from components such as alkaloid derivatives.
  • the base include: inorganic bases such as ammonia, sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate; and organic bases such as ammonium compounds. It is not limited. One of these bases may be used alone, or two or more may be used in combination. Accordingly, examples of the basic water include aqueous solutions of these inorganic bases and / or organic bases.
  • the pH of the basic water is not particularly limited, but is preferably 9 or more, more preferably 10 or more, and particularly preferably 11 or more.
  • taxane-related compounds have an ester which is easily hydrolyzed by a base in a molecular structure. Therefore, it is generally believed that treatment with a base will hydrolyze the ester and destroy the taxane-related compound.
  • the inventors of the present application have studied and found that the present invention It was found that the taxane-related compound did not undergo ester hydrolysis even when treated with a base. Therefore, by treating with a base, the taxane-related compound of the present invention can be separated from components such as phenol derivatives.
  • the extract after the treatment with an acid and / or a base is more preferably washed with water until neutral.
  • the extract from which the compound (40) has been extracted (if washed with water, after washing) is concentrated so that the compound (40) can be easily isolated (by removing the organic solvent). More preferably). By concentrating the extract, a neutral fraction is obtained.
  • the specific method for isolating and purifying the compound (40) from the neutral fraction is not particularly limited, but a method employing liquid chromatography is preferred.
  • Specific examples of the liquid chromatography include silica gel column chromatography, reversed-phase or normal-phase high-performance liquid chromatography, and centrifugal liquid-liquid distribution chromatography (CPC), but are not particularly limited. Not something. In the above-described high performance liquid chromatography, for example, it is sufficient to select the reverse phase or the normal phase in consideration of the pH of the mobile phase.
  • the stationary phase includes, but are not particularly limited to, silica gel, alumina, and ODS (octyl decylsilyl) -based compounds.
  • ODS octyl decylsilyl
  • ⁇ DS compounds are more preferred.
  • liquids suitable for use as a mobile phase (carrier, eluent) include, for example, methyl alcohol, ethyl alcohol, chloroform, n-hexane, ethyl acetate, various ethers, toluene, acetonitrile, Although water is mentioned, it is not particularly limited.
  • One of these liquids may be used alone, or two or more of them may be used in combination.
  • silica gel column chromatography or normal phase high performance liquid chromatography is used among the liquids exemplified above, a mixed solution of n-hexane / ethyl acetate is more preferable.
  • a mixed solution of methyl alcohol / acetonitrile is more preferable, and when centrifugal liquid-liquid distribution chromatography or the like is employed, a mixed solution of n-hexanemethyl alcohol is more preferable.
  • the pH of the liquid in the reversed-phase high-performance liquid chromatography that is, the pH of the mobile phase is adjusted to be acidic by a buffer solution.
  • the buffer solution is not particularly limited as long as it can adjust the pH of the mobile phase to 5.5 or less.
  • Specific examples of the buffer solution include an aqueous solution of ammonium acetate.
  • the combination of the above liquid and buffer solution is not particularly limited.
  • the resolution of the liquid chromatography is further improved, so that the compound (40) can be more selectively isolated. .
  • the pH of the mobile phase is near neutral, the separation / removal of the alkyloid derivative may not be performed sufficiently.
  • proton addition to the nitrogen atom of the alkaloid derivative occurs reversibly, so that components (peaks) to be separated / removed increase or the components (peaks) are broadened, resulting in low resolution. In some cases, the component (peak) cannot be specified (analyzed) due to a drop or a change in the retention time.
  • the neutral fraction is separated by liquid chromatography, and the above compound (40) is isolated and purified.
  • specific methods include: (1) silica gel column chromatography using silica gel flash column. One, Separation of the neutral fraction using a mixed solution of n-hexane / ethyl acetate (eluent) using centrifugal liquid-liquid partition chromatography and fractionation with a moderate polarity (fraction) ), And then (2) normal-phase high-performance liquid chromatography is used, the fraction is separated using a mixed solution (carrier) of n-hexane Z-ethyl acetate, and (3) reverse Using high-performance liquid chromatography, a mixed solution (carrier) consisting of a mixture of methyl alcohol, a 0.05 M aqueous solution of ammonium acetate (PH 4.8) and acetonitrile in a volume ratio of 1: 2: 2 is obtained.
  • a mixed solution (carrier) consisting of a mixture of methyl alcohol
  • the compound (40) can be separated from a basic component such as an alkyloid derivative or an acidic component such as a phenol derivative. ) Can be purified.
  • the purity of the compound (40) can be further improved by repeating the above steps (1) and (3) as required. That is, by performing the above separation and purification operations, the compound (40) can be efficiently extracted and separated.
  • the above-mentioned “needle portion” of Nippon yew is powder-framed as necessary.
  • the obtained extract is treated with an acid and a base or a base, and then fractionated by liquid chromatography to obtain the above compound. It is.
  • the steps of extraction with an organic solvent, treatment of the extract with an acid and / or a base, and fractionation by liquid chromatography are performed in the same manner as in the case of removing the above compound (40) from callus. Therefore, detailed description thereof will be omitted.
  • the oil contained in the needle portion is The needle portion may be washed with an organic solvent for the purpose of removing (degreasing) the components.
  • This organic solvent may be the same as the organic solvent used for extracting the needle leaf ground material.
  • n-hexane is suitable for removing oil contained in the needle leaf. is there. Oil removal is required, especially when using raw needles.
  • the above-described liquid chromatography is employed, and analysis is performed under the same conditions as the above-mentioned separation / purification conditions. You just need to do the operation. That is, the analysis is more preferably performed under acidic conditions.
  • the resolving power of the liquid chromatography is further improved, so that the compounds (40) to (47) can be more accurately analyzed. If the pH of the mobile phase is near neutral, the number of components (peaks) to be separated / removed will increase, or the components (peaks) will be broadened and the resolution will decrease, or the retention time will change. As a result, it may not be possible to identify (analyze) the component (peak).
  • the method for analyzing the compounds (40) to (47) is not particularly limited.
  • Compounds (40) to (47) are obtained by performing a series of extraction operations such as the above extraction. All of the compounds (40) to (47) have a multidrug-resistant cancer overcoming effect on cancer cells (multidrug-resistant cancer cells) that have acquired resistance to a plurality of anticancer agents. Therefore, a multidrug-resistant cancer-overcoming agent comprising at least one compound selected from the group of compounds represented by the chemical formulas (40) to (47) can be suitably administered to the cancer cells.
  • compound (40) to (47) are stable to acids or bases as described in the above description (description of the method of removing from callus or needle), and therefore, for example, Less likely to be degraded in the body (decomposition by gastric juice, bile fluid, etc.). Therefore, compound (40) (47)
  • the multidrug-resistant cancer-overcoming agent according to the present invention comprising at least one compound selected from (47), is expected to be applicable to a wide range of applications, for example, with few restrictions on its administration method and administration location. Is done.
  • compounds (40), (41), (43), (44), (45), (46), and (47) are highly active compounds that have a multidrug-resistant cancer-surpassing action superior to verapamil Therefore, it alone can be a particularly effective (new) drug for overcoming multidrug-resistant cancer.
  • the compound (40) can be extracted from calli obtained by culturing the tissue of Yew Tree.
  • the callus is derived from a small amount of explants and can be cultured in large quantities with simple equipment with good reproducibility.
  • the callus contains a relatively large amount of the above compound having a multidrug-resistant cancer overcoming effect. That is, according to the production method A according to the present invention, the above-mentioned compound (40) can be produced efficiently from Nippon yichi, while preserving the environment. This makes it possible to efficiently produce a multidrug-resistant cancer-overcoming agent suitable for administration to cancer cells.
  • the compounds (41) to (47) can be extracted from the needle portion of yew tree. That is, according to the production method B of the present invention, the needles regenerated every year are used, so that the compounds (41) to (47) can be produced from Nippon Ichi, while preserving the environment. . This makes it possible to produce a multidrug-resistant cancer-overcoming agent suitable for administration to cancer cells while preserving the environment.
  • the compounds represented by the formulas (6) to (47) are, for example, an acetyl group, a compound represented by the formula
  • the hydroxyl group is easily produced by a known chemical modification reaction such as esterification or etherification of OH in the same manner as described above.
  • the hydrolysis reaction, esterification reaction, etherification reaction and reduction reaction for this purpose may be performed according to a conventional method. By these operations, the hydroxyl group is
  • the compounds represented by the formulas (1) to (47) are useful as a cancer-surviving agent, particularly a multidrug-resistant cancer-surviving agent, based on the above-mentioned anticancer substance accumulation enhancing action or anticancer substance excretion inhibitory action. is there.
  • the multidrug-resistant cancer-overcoming agent comprises a plurality of cancer cells having multidrug resistance (multidrug-resistant cancer cells) that excrete various anticancer active substances (anticancer agents) taken out of the multidrug-resistant cancer cells. It has the effect of inhibiting any of the mechanisms of action.
  • the compounds of formulas (1) to (47) exhibit the same mechanism of action on cancer cells that are not multidrug resistant but resistant to ordinary anticancer agents (the same applies hereinafter). Further, the compounds represented by the formulas (1) to (47) exhibit the same mechanism of action not only on resistant cancer cells but also on ordinary cancer cells (the same applies hereinafter).
  • the present invention contributes to excellent cancer treatment by such an action mechanism.
  • Each of the compounds represented by the above chemical formulas (1) to (47) alone functions as an inhibitor of at least the active transport of an anticancer agent by P-glycoprotein (hereinafter referred to as a P-glycoprotein inhibitor). It can be a cancer-surviving agent or a multidrug-resistant cancer-surviving agent according to the present invention.
  • a P-glycoprotein inhibitor P-glycoprotein inhibitor
  • many of the above compounds for example, many of the compounds represented by formulas (40) to (47), are compared with verapamil which is a conventional drug-resistant cancer-overcoming agent. It has a high ability to overcome multidrug-resistant cancer, and is hereinafter referred to as a highly active compound. More preferably, the agent for overcoming multidrug resistance cancer according to the present invention contains at least one of these highly active compounds.
  • the agent for overcoming the multidrug-resistant cancer according to the present invention may be, for example, an antibody capable of specifically binding to a specific site (or P-glycoprotein) of a cancer cell, an anticancer substance (an anticancer agent) Etc. may be further included.
  • an anticancer substance an anticancer agent Etc.
  • an effect of reliably supplying a multidrug-resistant cancer-overcoming agent to target (multidrug-resistant) cancer cells, an effect of enabling simultaneous supply of an anticancer agent to the cancer cells, and the like are realized. be able to.
  • the administration method of these multidrug-resistant cancer-overcoming agents is not particularly limited, and the substances contained in the multidrug-resistant cancer-overcoming agents (compounds represented by the above formulas (1) to (47), antibodies, An appropriate drug delivery system known per se may be adopted depending on the physical properties of the drug.
  • anticancer substance other than the compounds (1) to (47) used in the present invention include, for example, cyclophosphamide (CPM, Cyclophosphamide), Tablets — Oxides (HN 2 -oxide, Nitrogen-mustard-N-oxide, Inosfamide), Mezolefaran (L-PAM, Melphalan), Chlorambutyl (CHL, Chlorambucil), Imidazo Chloroethyl-amine compounds such as lecarpoxamide (DTIC, Imidazole carboxamide); for example, Azilizin (Azilizin) such as triethylene phosphalamide (Thi-TEPA, Tri-ethylene-phospharamide) and carpocon (CQ, Carboquone) ) -Based compounds; for example, sulfonic acid ester-based compounds such as busulfan (BUS, Buslfun) and improsulfant silicate (864-T, Improsulfan-tosilate);
  • Pyrimidine antagonists for example, mercaptopurine (6_MP, Purine antagonists such as 6-Mercaptopurine) and thioinosine (6MPR, Thioinosine); for example, vincristine (VCR, Vincristine), vinblastine (VLB, Vinblastine), vindesine (VDS, Vindestine) or the like of the plant Al Caroids: For example, mitomycin C (MMC, Mitomycin C), doxorubicin (Doxorubicin ADR; ADM, Adriamycin), aclarubicin (ACR, Aclarubicine), bleomycin (BLM, Bleomycin), ⁇ preomycin (PEP, Pepleomycin), chromomycin A3 ( Anticancer antibiotics (Antibiotics) such as CHRM, chromomycine, dactinomycin, neocarzinostatin (NCS, neocarzinostatin), and neosuramycin (NTM, neothr
  • the compounds represented by the formulas (1) to (47) also have an anticancer effect in addition to an anticancer active substance accumulation enhancing action or an anticancer active substance excretion inhibitory action. Combination or coexistence of substances is not necessary.
  • the medicament of the present invention can exert a therapeutic effect on cancer by administering a compound represented by any one of the formulas (1) to (47) and, if desired, the other anticancer active substance to a cancer patient.
  • a pharmaceutical preparation containing the compounds of formulas (1) to (47) together with another anticancer agent is administered.
  • a preparation containing the compounds of the formulas (1) to (47) and a preparation containing the other anticancer agent may be separately prepared, and they may be administered simultaneously or separately.
  • the anticancer substance other than the compounds represented by the formulas (1) to (47) may be a single type or a plurality of types. In the case of plural types, different preparations may be used. It may be administered at the same time or separately.
  • the medicament of the present invention includes, as anticancer agents, non-solid cancers such as leukemia, malignant lymphoma, and myeloma, or stomach cancer, esophagus cancer, large intestine, rectum, Teng's cancer, liver cancer, kidney cancer, bladder cancer, lung cancer, It can be used to treat solid cancers such as uterine cancer, ovarian cancer, breast cancer, prostate cancer, skin cancer, and brain tumor cancer.
  • non-solid cancers such as leukemia, malignant lymphoma, and myeloma
  • stomach cancer esophagus cancer
  • large intestine large intestine
  • rectum Teng's cancer
  • liver cancer liver cancer
  • kidney cancer bladder cancer
  • lung cancer It can be used to treat solid cancers such as uterine cancer, ovarian cancer, breast cancer, prostate cancer, skin cancer, and brain tumor cancer.
  • the above substance as an active ingredient that is, the above formula (1)
  • a compound thereof and an anticancer agent may be administered as it is, but generally, the above-mentioned substance as an active ingredient is further combined with one or more pharmaceutical additives. It is desirable to administer it in the form of a pharmaceutical composition containing the same.
  • a pharmaceutical composition can be manufactured according to a method known per se or a conventional method in the field of pharmaceuticals.
  • the drug of the present invention in the form of a pharmaceutical composition may optionally contain one or more active ingredients of other drugs other than those described above.
  • the medicament of the present invention can be applied to mammals including humans.
  • the administration route of the medicament of the present invention is not particularly limited, and the most effective administration route for treatment and / or prevention can be appropriately selected from either oral or parenteral administration.
  • Parenteral administration can include routes of administration such as intratracheal, rectal, subcutaneous, intramuscular, and intravenous.
  • routes of administration such as intratracheal, rectal, subcutaneous, intramuscular, and intravenous.
  • preparations suitable for oral administration include, for example, tablets, granules, powders, syrups, solutions, capsules, and suspensions.
  • preparations suitable for parenteral administration include: Mucosal absorbents and the like can be mentioned.
  • liquid preparations suitable for oral administration for example, sugars such as water, sucrose, sorbitol, fructose; glycols such as polyethylene glycol and propylene glycol; oils such as sesame oil, olive oil and soybean oil; — Lumber additives such as preservatives such as hydroxybenzoic acid esters can be used.
  • excipients such as lactose, glucose, sucrose, mannitol
  • disintegrants such as starch and sodium alginate
  • Lubricants such as gum and talc
  • binders such as polyvinyl alcohol, hydroxypropyl cellulose and gelatin
  • surfactants such as fatty acid esters
  • plasticizers such as glycerin
  • preparations suitable for parenteral administration can be prepared preferably using an aqueous medium isotonic with human blood.
  • an injection may be prepared as a solution, suspension, or dispersion using an aqueous medium selected from a salt solution, a glucose solution, or a mixture of a salt solution and a glucose solution together with appropriate auxiliaries according to a conventional method. it can.
  • Suppositories for enteral administration can be prepared using carriers such as cocoa butter, hydrogenated fats or hydrogenated carboxylic acids.
  • Sprays can be prepared using a simple substance which does not irritate the human oral and respiratory tract mucosa and which can promote absorption by dispersing the above-mentioned substance as an active ingredient as fine particles.
  • a carrier for example, lactose or dalserin can be used. It can be prepared as a preparation in the form of an aerosol or dry powder depending on the properties of the above-mentioned substance as an active ingredient and the carrier used.
  • parenteral preparations for example, one or more preparation additives selected from diluents, flavors, preservatives, excipients, lubricants, binders, surfactants, plasticizers, etc. Things can be used.
  • the form of the medicament of the present invention and the method for producing the medicament are limited to those specifically described above. It is not specified.
  • the administration fee and administration frequency of the medicament of the present invention are not particularly limited, and the kind of the substance as the active ingredient, the kind of the condition to be treated, the administration route, the age and weight of the patient, the symptoms, and the severity of the disease According to various conditions such as the above, it is possible to appropriately select according to a doctor's instruction.
  • the amount of the compound represented by any of the formulas (1) to (47) or the other anticancer substance is 0.01 to 200 mg / kg per day for an adult (preferably 5 to 50 mg L).
  • Kg / d) can be administered once a day or once every few days to once every several weeks, but the dosage and frequency of administration are not limited to this particular example.
  • the medicament of the present invention can be used in combination with other anticancer agents (anticancer agents, antitumor agents), and generally used in combination with several kinds of anticancer agents having different mechanisms of action. preferable.
  • the compounds of formulas (1) to (47) show antibacterial activity, for example, a minimum inhibitory concentration of ⁇ 1 000 r / cc against Bacillus Subtilis and the like.
  • it can be used as an ointment, for example, a skin external preparation for treating eczema, pimples, boils, and acne.
  • the action of overcoming the multidrug-resistant cancer (in this case, the function as a P-glycoprotein inhibitor) of some compounds belonging to the compound group was measured by the bioassay method shown below.
  • the bioassay method comprises 2780 AD cells (multidrug-resistant cancer cells established from human ovarian cancer cells A 2780), vincristine (anti- A reaction solution containing a predetermined amount of a cancer drug) and a candidate substance of a P-glycoprotein inhibitor (corresponding to a compound belonging to the above-mentioned compound group) is applied to each well, and is kept constant under a predetermined temperature condition. The reaction was performed by reacting for hours.
  • a comparative experiment using verapamil as a control drug was also performed to evaluate compounds (highly active compounds) having a multidrug-resistant cancer-surpassing action superior to verapamil.
  • the comparison with verapamil as a control drug refers to a comparative experiment performed using verapamil at the same concentration as the P-glycoprotein inhibitor candidate substance in each reaction solution.
  • the criterion is ⁇ one '' when the total amount of pinklistin uptake in each measurement test is 90% or less of the total pinklistin uptake in the control, and more than 90% to 110% If it is less than or equal to ⁇ soil '', it is more than 110% and less than 300%, ⁇ ten '', if it is more than 300% and less than 500%, it is ⁇ ten '', ⁇ +++ '' when it is more than 500% and less than 100%, ⁇ +++ '' when it is more than 100% and less than 200%
  • the case where the value exceeds 0% is defined as seven levels of “++++++”.
  • ⁇ P '' indicates that a drug-overcoming effect of verapamil or higher is recognized
  • ⁇ N '' indicates that no drug-overcoming effect of verapamil or higher is detected.
  • the candidate substance of the P-glycoprotein inhibitor determined to be in the above “ten” stage clearly has a function as a P-glycoprotein inhibitor (multidrug-resistant cancer overcoming action). Is determined.
  • the candidate substance of the P-glycoprotein inhibitor determined to be in the “soil” stage does not clearly recognize the function as the P-glycoprotein inhibitor.
  • the candidate substance for a P-glycoprotein inhibitor evaluated as “P” is judged to be a highly active compound.
  • the pH was adjusted to 9.0 by adding 29% aqueous ammonia to the above 0.5M sulfuric acid after washing, and then extracted three times (3 ⁇ 200 ml) with 200 ml of chloroform.
  • 1.08 g (0.087% of the same) of the phenol fraction was obtained from the above-mentioned 2 M aqueous sodium hydroxide solution after washing.
  • the crude neutral fraction was subjected to gradient elution using n-hexane, ethyl acetate, and methyl alcohol as solvents, by employing open column chromatography using silica gel.
  • Fi eluate to F 8 are acetate Echiru: hexane n- capacity ratio 2: mixed solution prepared by mixing 3, eluant F 9 is acetic Echiru, eluant F 10 is Mechiruaru call Met.
  • the yield of each fraction is Fi SS Omg. Fa ZZS Smg, F 3 722 mg, F 4 405 mg, F 5 3 1 1 mg in order from 1st (low polarity side) to 10th (high polarity side). , F 6 208 mg, F 7 1 77 mg, F 8 144 mg, F 9 237 mg, Fi 0 205 1 mg.
  • the second fraction (F 2 ) was applied to a silica gel stainless steel column (INERTSIL PREP-SIL, GL Science, 250 ⁇ 10 mm id, UV Using a normal-phase high-performance liquid chromatography with 254 nm), a mixed solution of ethyl acetate and n-hexane mixed at a volume ratio of 3: 7 (carrier, flow rate 5.0 ml / min. ) And 962 mg of a fraction with a retention time (tR) of 12.0 to 45.2 minutes was collected. This was further separated under the same conditions have use the column, retention time 0-1 7.0 minute fractions F 2.
  • INERTSIL PREP-SIL silica gel stainless steel column
  • fractionation F 2 - was obtained 3 from the compound (26) 1. 4mg. Therefore, 16.4 mg of compound (26) (0.0013% based on the amount of needles) was obtained from the crude neutral fraction.
  • 6.1 mg of the compound (26) was obtained from the crude alkaloid fraction. Therefore, 22.5 mg (0.0018%) of the compound (26) was obtained from the crude neutral fraction and the crude alkaloid fraction.
  • fraction F 2 - was aliquoted 5 (peak) 14. 5 mg - 3 retention time from 26.4 min fractions F 2. This was further separated under the same conditions using the above-mentioned column, and then isolated and purified, thereby obtaining 4.8 mg of the compound (17) (retention time: 40.3 minutes).
  • fractionation F 2 - 2 from the compound (1-7) 1. 4 mg were obtained. Therefore, 6.2 mg of the compound (17) (0.0005% based on the amount of needles) was obtained from the crude neutral fraction.
  • the third fraction (F 3 ) was subjected to normal-phase high-performance liquid chromatography using a silica gel stainless steel column (INERTSIL PREP-SIL, GL Science, 250 ⁇ 20 mm id, UV 254 nm) to obtain ethyl acetate. And n-hexane at a volume ratio of 1: 1 using a mixed solution (carrier, flow rate 15 ml / min.), And the retention time is 18.0 to 22.0 minutes. Fraction 1 64.7 mg was dispensed. They were separated and 2 (peak) 1 5 4. 4 mg - which fractions F 3 further separated, retention time 1 7.5 minutes at the same conditions using the above column.
  • INERTSIL PREP-SIL silica gel stainless steel column
  • fractions F 3 - a 2, ODS stainless steel column employs a reverse phase high performance liquid chromatography using methyl alcohol one le and 0 A 0.05M ammonium acetate buffer (pH 4.8) and acetonitrile were mixed and mixed at a volume ratio of 1: 1: 2 (carrier, flow rate 5.0 ml / min.) And purified. . This gave 4.4 mg of compound (22) (retention time: 9.8 minutes, 0.0004% based on the amount of needles).
  • the fifth fraction (F 5 ) was subjected to normal-phase high-performance liquid chromatography using a silica gel stainless steel column (INERTSIL PREP-SIL, GL Science, 250 ⁇ 10 mm id, UV 254 nm) to obtain ethyl acetate. And n-hexane were mixed at a volume ratio of 1: 1 using a mixed solution (carrier, flow rate 5.0 ml / min.), And the retention time was 18.0 to 35.0 minutes. Fraction Fs-i 1 58. lmg was collected.
  • INERTSIL PREP-SIL silica gel stainless steel column
  • the pH of the aqueous layer after washing was adjusted to 3.0 by adding 0.5 M sulfuric acid (80 Om1) to the aqueous 2M sodium hydroxide solution (pH 7.5). Extracted 6 times with l (6 X 500 ml). Further, the extracted liquid was dried with anhydrous sodium sulfate, and the outlet form was removed. This gave 22.5 g of crude acid fraction (0.21% based on the amount of needles).
  • Eluate acetate Echiru: hexane n- capacity ratio of 1: is a mixed solution obtained by mixing with 1, F 3 - the second eluate equal volume ratio of 3: be a mixed solution prepared by mixing 2 , F 3 - 3 of eluate equal volume ratio of 7:.. a mixed solution ing mixed with 3, F 3 4 eluates are ⁇ Echiru, F 3 5 eluate methyl alcohol Met.
  • the second fraction (F 3. 2) 4.
  • a mixed solution prepared by mixing with 1, F 3 - 2 - 6 eluate is acetic acid Echiru, F 3 -2 7 eluate of a methyl alcohol
  • fraction F 3-2-2 1 65 1 mg was subjected to reverse-phase high-performance liquid chromatography using an ODS stainless steel column (INERTSIL PREP-ODS, GL Science, 250 X 2.0 mm id, UV 254 nm).
  • the crude 7-epitaxol was converted to ethyl acetate and n- by normal phase high performance liquid chromatography using a silica gel stainless steel column (INERTSIL PREP-SIL, GL Science, 250 x 2.0 mm id, UV 254 nm). Purification was performed using a mixed solution (carrier, flow rate: 10.0 ml / min.) Obtained by mixing xanthane with xanthone at a volume ratio of 1: 1. This resulted in 25.7 mg of 7-epitaxol (retention time 17.1 minutes) and 40.6 mg of crude 7-epi-cephalomanine (retention time 18.6 minutes).
  • This crude 7-epipsephalomannine was purified by reverse phase high performance liquid chromatography using an ODS stainless steel column (Shodex C18-10E, 250 x 1.0 mm id, UV 254 nm) to obtain methyl alcohol and 0.05M Purification was performed using a mixed solution (carrier, flow rate: 5.0 ml / min.) Obtained by mixing ammonium acetate buffer (PH4.8) and acetonitrile at a volume ratio of 1: 2: 2. This gave 12.4 mg (retention time 14.2 minutes) of crude compound (29) and 16.5 mg (retention time 17.7 minutes) of 7-epi-cephalomanine.
  • the crude compound (29) was converted to ethyl acetate and n- by normal phase high performance liquid chromatography using a silica gel stainless steel column (INERTSIL PREP-SIL, GL Science, 250 X 0.46 mm id, UV 254 nm). Purification was performed using a mixed solution (carrier, flow rate: 2.0 ml / min.) Consisting of a mixture of xane and in a volume ratio of 2: 3. This gives 5.8 mg of compound (29) (retention time 6.7 min, 0.00000% based on needle volume) and 116.9 mg of 5-cinnamoyl-10-acetyl quinine (retention time 9.7 minutes).
  • IR 36 12, 3000, 1 730, 1 670, 1 640,
  • the above components were dissolved in 5 Om 1 of a mixed solution obtained by mixing methyl alcohol and ethyl acetate at a volume ratio of 1: 4. Next, the solution was washed three times (3 ⁇ 15 ml) with 15 ml of 0.5 M sulfuric acid as an acidic aqueous solution, and then washed with 15 ml of a 2 M aqueous sodium hydroxide solution as a basic aqueous solution. Washed twice (2 X 15 ml). Further, the above solution (oil layer) was washed four times (4 ⁇ 2 Om 1) with saturated saline 2 Om 1, and dried over anhydrous sodium sulfate to remove methyl alcohol and ethyl acetate. As a result, 1.86 g of a crude neutral fraction (0.71% based on the amount of needles) was obtained.
  • the extract was dried over anhydrous sodium sulfate, and the form at the outlet was removed. This gave 0.88 g of the crude acidic fraction (0.33% based on the amount of needles).
  • the above crude neutral fraction was subjected to flash column chromatography using 55.8 g of silica gel (Silica gel 60 MERK, 230 to 400 mesh), and n-hexane and ethyl acetate were used as solvents. by performing Gurajuento elution with methyl alcohol, and Fi fractionated into 10 fractions (less polar side) ⁇ F 10 (high polarity) (rough division).
  • the first fraction (F 1002.5 mg) was subjected to normal-phase high-performance liquid chromatography using a silica gel gel column (INERTSIL PREP-SIL, GL Science, 250 X 1.0 mm id, UV 254 nm). Then, it was isolated and purified using a mixed solution (carrier, flow rate 5.0 ml / min.) Obtained by mixing ethyl acetate and n-hexane at a volume ratio of 3: 7. 27.2 mg of crude xinine (retention time: 17.4 min) and 1.2 mg of compound (15) (retention time: 33.2 min, 0005% based on needle volume) Was.
  • INERTSIL PREP-SIL silica gel gel column
  • the crude alkaloid fraction was subjected to open column chromatography using 920 g of activated alumina (neutral, activity I), using n-hexane, ethyl acetate and methyl alcohol as solvents. By performing gradient gradient elution, It was fractionated (roughly divided) into 17 17 fractions.
  • the eluate of Fi is a mixed solution of ethyl acetate: n-hexane mixed at a volume ratio of 3: 7, and the eluate of F 2 ⁇ F 3 is the same volume ratio
  • the eluate of F 4 ⁇ F 5 is a mixed solution obtained by mixing at the same volume ratio of 7: 3.
  • the eluate of F 6 -F 7 is ethyl acetate.
  • Ah is, eluate F 8 is acetate
  • Echiru methyl alcohol volume ratio of 9: 1 is a mixed solution composed engaged mixed in
  • eluant F 9 is the volume ratio 4: mixed solution prepared by mixing 1
  • the eluate F 10 is equal volume ratio 7: mixed-solution prepared by mixing 3
  • eluant F u is the volume ratio of 1: is a mixed solution obtained by mixing with 1, F 12 eluate to F 17 were methyl alcohol.
  • the yield of each fraction was Fi 82.5 mg, F 2 103.6 mg, F 3 108.0 mg, F 4, in order from the first (low polarity side) to the 17th (high polarity side).
  • the 1-9-th fraction (Fi to F 9), employs ODS stearyl Nresukaramu (INERTSIL PREP ODS, GL Science, 250 X 20mm id, UV 254nm) reverse-phase high performance liquid chromatography using, A single solution (carrier, flow rate 15 ml / min.) was prepared by mixing methyl alcohol, 0.05M ammonium acetate buffer (pH 4.8) and acetonitrile at a volume ratio of 1: 1: 2. Separated and purified.
  • ODS stearyl Nresukaramu INERTSIL PREP ODS, GL Science, 250 X 20mm id, UV 254nm
  • taxin 1189 Omg (retention time: 8.9 min, 0.021 1% based on the amount of needles), which is the main component of the alkyloid fraction, and compound (6) 182 lmg ( With a retention time of 6.8 minutes, 0.043 1%) was taken out based on the amount of needles.
  • the compound (24) could be taken out from the needle portion of the yew tree (raw needle portion) at a rate of 0.0008% based on the amount of the needle portion.
  • the measurements of iH-NMR and i3C-NMR of compound (24) are summarized in Tables 19 and 20. Thus, the structure of compound (24) was analyzed. In addition, as a result of measuring physical properties and the like of the compound (24),
  • Tables 21 to 26 The results of measuring the effect of overcoming multidrug-resistant cancer are summarized in Tables 21 to 26.
  • dose concentration indicates the concentration (gZml) of the above compound or veravamil in the reaction solution
  • average VCR accumulation amount indicates the pin concentration in 2780 AD cells in each well. Shows the average value of the accumulated amount of Christin (VCR) (dpm / well).
  • VCR ratio indicates the results of comparison with a comparative experiment using verapamil as a control drug, and (maximum verapamil ratio) and (concentration) in the “Evaluation” section are compared with those of verapamil. And the ratio and concentration at which the effect was highest. Re-measurement was performed for compounds with high ability to overcome multidrug-resistant cancer (Tables 25 and 26).
  • the compounds (11), (15) to (17), (22), (24), (2 6) is a highly active (new) compound, which is a highly active compound that has a multidrug-resistant cancer-overcoming action and a multidrug-resistant cancer-overcoming action that surpasses verapamil. It turns out that it can be a drug-resistant cancer overcoming agent.
  • modified Gamborg's solid medium a modified Gamborg's medium having the composition shown in Table 28 below.
  • the above-mentioned solid medium was allowed to stand under 25: 5 place.
  • the cultured cells were allowed to stand and cultured to obtain calluses of Japanese yew.
  • a callus with good growth potential was selected about every 450 days, and the callus was passaged using a medium of the same composition.
  • the strain was established by subculture.
  • the concentration of agar powder, NAA, methyl jasmonate (JM), and oligosaccharide (KTOS) were adjusted to 10 g / L, 1.0 mg / L, 100 M, and 0.3 mg ZL, respectively.
  • the callus was placed on the added modified gamborg culture medium (hereinafter referred to as production medium 1), and cultured statically for 25 days in the dark for 25 days. As a result, a fresh weight of 831.9 g of callus (referred to as fresh callus) was obtained.
  • production medium 1 modified gamborg culture medium
  • the compounds (30), (31), (33) to (35), and the compound (36) as a novel taxinine compound were extracted using the fresh callus.
  • 83.19 g of fresh callus was collected and freeze-dried to obtain 86.97 g of dry callus.
  • the dried callus was stirred in 1.3 L of n-hexane for 1 hour to perform an extraction operation. This stirring / extraction operation was repeated three times.
  • n-hexane was distilled off from the n-hexane layer (extract) to obtain 1,847 mg of a crude n-hexane extract.
  • the compounds (30), (31), (33) to (36) were isolated from the n-hexane layer. More specifically, compound (35) contained 10.0 mg (0.012% by weight based on dry callus, retention time (tR) 21.6 minutes) and compound (36) contained 15 Omg (0.017% by weight based on dry callus, tR 28.1 min) Isolated. In addition, compound (30) and its 14-hydroxyl homologue compounds (31), (33), and (34) were sequentially converted to 377 mg (0.433% by weight based on dry callus, t R 1 Five.
  • the compound (35) and the compound (36) among the above compounds were induced using a production medium 26 having the following composition instead of the production medium 1. It was also isolated from calli that had been woven.
  • the production medium 2 is a modified gamborg medium to which agar powder, NAA, and methyl jasmonate are added in order of 10 gZL, 0.1 Smg / L, and 100 mM, respectively.
  • the isolation yields of the compound (35) and the compound (36) were 0 mg and 19.2 mg (0.026% by weight based on dry callus), respectively.
  • the production medium 3 is agar powder, NAA, methyl jasmonate, phenylalanine, and acetic acid, in that order: 10 g ZL, 0.5 mg ZL, 100 mM, 0.26 g / L, 1 m 1 / L
  • the isolated yield of compound (35) and compound (36) in production medium 3 was 0 mg and 19.2 mg (in dry callus, respectively). 0.026% by weight).
  • the production medium 4 is such that agar powder, NAA, phenylalanine, and vinegar become 10 gZL, 0.5 mg / L, 0.263 g / L, 1 ml / L, respectively.
  • the isolated yield of the compound (35) and the compound (36) in the production medium 4 was 58.8 mg (0.15% with respect to the dry callus) in the production medium 4 in this order. Wt%), Omg.
  • the production medium 5 is a modified gambog medium in which agar powder, NAA, and chitosan are added in such a manner as to give 10 gZL, 0.5 mg / L, and 1.25 gZL, respectively.
  • the isolation yields of the compound (35) and the compound (36) in 5 were 58.8 mg (0.105% by weight based on dry callus) and Omg, respectively.
  • the production medium 6 is a modified Gamborg's medium to which agar powder, ⁇ , ⁇ -cyclodextrin is added in order of 10 gZL, 0.5 mgZL, and 10 mM, respectively.
  • the isolation yields of the compound (35) and the compound (36) were 7.5 mg (0.015% by weight based on dry callus) and Omg, respectively. (Example 6)
  • the callus induction and tissue culture were carried out under the same conditions and in the same medium as in Example 1 above, using the young stems (needles) of the Japanese yew (@ATUS cuswWa a Sieb. Et Zuc) as explants. Subsequently, the agar powder, NAA, phenylalanine, and acetic acid were added in order to obtain a concentration of 10 gZL, 0.5 mg / L, 0.263 g / L, and 1.0 ml ZL. The calli obtained in a Gamborg's medium were placed on a bed, and static culture was performed at 25 under a place for 60 days. As a result, a fresh weight of 605.5 g of callus (referred to as fresh callus) was obtained.
  • the compounds (30) to (35) and taxol-related compounds were extracted using the fresh callus.
  • 605.5 g of fresh callus was collected and freeze-dried to obtain 55.9 g of dry callus.
  • extraction operations from the dried calli were sequentially performed using n-hexane, ethyl acetate, and methyl alcohol as organic solvents. That is, after performing 1 hour of stirring and extraction operation (repeated three times) using 1 L of n-hexane, stirring and 1 hour of extraction and extraction operation (repeated three times) using 1 L of ethyl acetate, Using 1 L of methyl alcohol, the mixture was stirred and extracted for 1 hour (repeated three times).
  • n-hexane was distilled off from the n-hexane layer (extract) to obtain 1061.7 mg of a crude n-hexane extract.
  • Ethyl acetate was distilled off from the ethyl acetate layer (extracted liquid) to obtain 856.2 mg of a crude ethyl acetate extract.
  • the crude ethyl acetate extract is dissolved in a mixed solution of methyl alcohol and ethyl acetate (mixture amount: 10 ml: 40 ml), and then a) washed with 0.5 M aqueous sulfuric acid solution (2 Om1) and subjected to alkaloid fractionation.
  • methyl alcohol was removed from the methyl alcohol layer (extract) to obtain a crude methyl alcohol extract.
  • This crude methyl alcohol extract is dissolved in a mixed solution of methyl alcohol Z water (mixing volume, 50 ml: 40 ml), and then extracted three times with a black form 10 Om1. The form was removed to obtain 98.3 mg of a crude black-mouthed form extract.
  • This crude form-form extract is dissolved in a mixed solution of methyl alcohol and ethyl acetate (mixture volume: 10 ml: 40 ml), and then a) washed with a 0.5 M aqueous sulfuric acid solution to remove alkaloids (basic components).
  • the above compounds (30), (31), (33) to (35) were isolated. More specifically, the compound (33) was 200.3 mg (retention time (tR) 11.0 minutes), the compound (34) was 17.0 mg (tR 12.9 minutes), the compound (3 1) at 24.6 mg (tR 16.5 min), compound (30) at 30.7 mg (tR 22.5 min), compound (35) at 29.5 mg (tR3 1.2 min) ) Isolated. In addition, 19.7 mg (t R 28.9 min) Isolated.
  • Fractions fr 1 to fr 6 are fractions obtained using an eluent obtained by mixing n-hexane and ethyl acetate at a volume ratio of 5: 5
  • fractions fr 7 to fr 9 are: This is a fraction obtained using an eluent obtained by mixing n-hexane and ethyl acetate in a volume ratio of 7: 3.
  • Fractions fr 10 and frll are fractions obtained using ethyl ethyl acetate as an eluent.
  • the fraction fr 12 is a fraction obtained using methyl alcohol as an eluent.
  • compound (33) was 40.8 mg (retention time (tR) 8.6 minutes)
  • compound (34) was 2.7 mg (tR 9.5 minutes)
  • compound (31) was 3.7 mg (tR11.8 min)
  • compound (30) was isolated at 59.1 mg (tR16.9 min)
  • compound (35) was isolated at 6.1 mg (tR22.1 min).
  • 2.7 mg (tR19.3 min) of taxusin was isolated.
  • 7-epitaxol (7-epitaxol) was 0.6 mg (retention time (tR) 10.5 minutes)
  • taxol C taxol C
  • 1.6 mg (tR 29.8 min) was isolated.
  • Fractions Fr 1 to Fr 13 are fractions obtained using an eluent obtained by mixing n-hexane and ethyl acetate at a volume ratio of 5: 5, and the fractions F rl4, F r15 is the fraction obtained using ethyl acetate as the eluent, and fractions Fr16 to Fr18 are the fractions obtained using methyl alcohol as the eluent.
  • fractions Fr7 to Frl3 were subjected to reverse-phase high-performance liquid chromatography (INERTSIL PREP ODS; 25 X 1 cm; using an ODS stainless steel column as a reverse-phase ODS column).
  • GL Science; Solvent Methyl alcohol: ammonium acetate buffer (pH 4.8): acetonitrile 1: 2: 2 mixed solution; flow rate: 5.Om 1 / min).
  • 7-epitaxol (7-epitaxol) was 0.5 mg (retention time (tR) 8.5 minutes), and taxol C (taxol C) was 0.5 mg (tR 8.6 minutes). 1.5 mg (tR 6.3 min) and 2.2 mg (tR 5.9 min) of taxol were isolated.
  • the compound (30) had a total of 455.5 mg (0.806% by weight based on the dry callus), and the compound (31) had a total of 30.9 mg ( 0.055 weight of dried callus %), Compound (32) totaling 22.3 mg (0.040% by weight based on dry callus), compound (33) totaling 272.3 mg (0.487% by weight based on dry callus), compound (34) was obtained in a total of 27.7 mg (0.050% by weight based on dry callus).
  • VCRf Verapamil ratio indicates the results of comparison with a comparative experiment using verapamil as a control drug, and (maximum verapamil ratio) and (concentration) in the “Evaluation” section are compared with verapamil, respectively. Then, the ratio and concentration when the effect is the highest is shown. Re-measurements were performed for compounds with a high effect of overcoming multidrug-resistant cancer (Table 33). Dosing concentration VCRf Verapamil ratio
  • the solid medium was allowed to stand at 25 in the dark.
  • the cultured cells were allowed to stand and cultured to obtain calluses of Japanese yew.
  • about 40 every 50 days calli having good proliferation were selected, and subculture was performed using a medium having the same composition to establish a strain.
  • the callus was placed on a modified gamborg medium (production medium) to which agar powder, ⁇ , and ⁇ -cyclodextrin were added in order to obtain a concentration of lOgZL, 0.5 mgZL, and 100 mM.
  • static culture was performed for 60 days under the same location.
  • a fresh weight of 471.4 g of callus (referred to as fresh callus) was obtained.
  • n-hexane was distilled off from the n-hexane layer (extract) to obtain 297.5 mg of a crude n-hexane extract.
  • Ethyl acetate was distilled off from the ethyl acetate layer (extract) to obtain 505.8 mg of a crude ethyl acetate extract.
  • the crude ethyl acetate extract is dissolved in a mixed solution of methyl alcohol / ethyl acetate (mixing volume: 10 ml: 40 ml), and then a) washed three times with a 0.5 M aqueous sulfuric acid solution 15 ml 1 The components were removed, and then b) washing was performed three times with 15 ml of a 2 M aqueous sodium hydroxide solution to remove acidic components (phenol derivatives). Subsequently, the obtained ethyl acetate layer was dried over anhydrous sodium hydrogen sulfate, and the solvent (ethyl acetate) was removed. Thus, 373.6 mg (0.79% by weight based on dry callus) of the ethyl acetate extract was obtained.
  • methyl alcohol was removed from the methyl alcohol layer (extract) to obtain a crude methyl alcohol extract.
  • the crude methyl alcohol extract was dissolved in a mixed solution of methyl alcohol and water (mixing volume, 50 ml: 200 ml), and then extracted three times with 100 ml of black form, followed by removal of form. As a result, 1,851.7 mg of a crude black-mouthed form extract was obtained.
  • the crude black-mouthed form extract was dissolved in a mixed solution of methyl alcohol Z and ethyl acetate (mixture amount: 10 ml: 40 ml), and then a) washed three times with a 0.5 M sulfuric acid aqueous solution 15 ml 1 times.
  • the substrate was washed three times with 15 ml of a 2 M aqueous sodium hydroxide solution to remove acidic components (phenol derivatives). Subsequently, the obtained ethyl acetate layer was dried over anhydrous sodium hydrogen sulfate, and the solvent (ethyl acetate) was removed to remove 91.49 mg of a black-mouthed form extract (1.94% by weight of the dried callus). %).
  • Fractions f1 to f3 are fractions obtained using an eluent obtained by mixing n-hexane and ethyl acetate at a volume ratio of 5: 5, and fraction f4 is a fraction obtained by mixing ethyl ethyl acetate. This is the fraction obtained as eluent, and fraction f5 is the fraction obtained using methyl alcohol as eluent.
  • Identification and structure determination of the isolated compound (37) should be carried out by analyzing the measurement results of PFG-COSY, PFG-HMQC, PFG-HMBC, etc. using 50 MHZ NMR equipped with an inverse probe. I went by. The results of these measurements are summarized in Tables 35 and 36. Thus, the structure of compound (37) was analyzed. In addition, as a result of measuring physical properties and the like of the compound (37),
  • the solid medium was allowed to stand for 25 t in a dark place.
  • the cultured cells were allowed to stand and cultured to obtain calluses of Japanese yew.
  • a callus with good growth was selected about every 40 to 50 days, and subculture was performed using a medium of the same composition to establish a strain.
  • a modified Gamborg's medium production medium
  • 4-C1 IAA 4-auxin indoleacetic acid
  • This callus was placed on the ground, and static culture was performed at 25 under the place for 60 days.
  • the subculture was performed once during the stationary culture period of 60 days. That is, two subcultures were performed.
  • 849.9 g of fresh calli (referred to as fresh calli) were obtained.
  • a compound (39) as a novel abiene compound was extracted using the above fresh calli.
  • 849.9 g of fresh callus was collected and freeze-dried to obtain 77.5 g of dry callus.
  • the extraction operation was performed in order. That is, the mixture was stirred for 1 hour with 1 L of n-hexane and extracted (repeated three times), and then temporarily stirred and extracted with 1 L of ethyl acetate (repeated three times). .
  • n-hexane was distilled off from the n-hexane layer (extract) to obtain 577.3 mg of a crude n-hexane extract.
  • Ethyl acetate was distilled off from the ethyl acetate layer (extract) to obtain 775.2 mg of crude ethyl acetate extract.
  • Fraction F (1) is a fraction obtained using an eluent obtained by mixing n-hexane and ethyl acetate in a volume ratio of 8: 2.
  • the fractions F (2) to F (5) are fractions obtained using an eluent obtained by mixing n-hexane and ethyl acetate in a volume ratio of 7: 3, and the fraction F ( 6) and F (7) are fractions obtained using an eluent obtained by mixing n-hexane and ethyl acetate in a volume ratio of 4: 6.
  • Fraction obtained with ethyl acetate as eluent, F (9) is the fraction obtained with methyl alcohol as eluent. It is.
  • Example 10 Callus induction and tissue culture were performed using young shoots (needles) of Nipponbari (73 ⁇ 4ATi ⁇ ci5 W'i3 ⁇ 4 Sieb. Et Zucc.) As explants. 40) was taken out. The young shoots as the explants were collected in winter (at an average temperature of 5 during the first month before collection).
  • sucrose sucrose
  • agar powder sucrose
  • NAA which is an auxin
  • the slices were placed on a modified Gamborg's medium (hereinafter, referred to as a modified Gamborg's solid medium) added to have a concentration. That is, the modified Gamborg's solid medium in this example is a solid medium having the composition shown in Table 40 below.
  • the solid medium was allowed to stand at 25 in the dark.
  • the cultured cells were allowed to stand and cultured to obtain calluses of Japanese yew.
  • about 40 A callus with good growth was selected every 50 days, and subculture was performed using a medium of the same composition to establish a strain.
  • agar powder, NAA, phenylalanine, and acetic acid were added in that order: 10 gZL, 0.5 mg / L, 0.2
  • the callus obtained was placed on a modified gump medium supplemented with a concentration of 63 g / L and 1.0 ml ZL, and the resulting calli were cultured statically at 25 in the dark for 60 days. As a result, 605.5 g of callus having a fresh weight (referred to as fresh callus) was obtained.

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Abstract

Agents for overcoming multidrug-resistant cancer which are to be administered to cancer cells acquiring resistance against a multiple number of anticancer drugs (multidrug-resistant cancer cells). These agents for overcoming multidrug-resistant cancer contain compounds having a taxine skeleton, a taxinine skeleton or an abietane skeleton which are taken out from, for examples, needles of Taxus cuspidata Sieb. et Zucc.

Description

明 細 書 多剤耐性癌克服剤およびその製造方法 技術分野  Description Agent for overcoming multidrug-resistant cancer and method for producing the same
本発明は、 癌克服剤、 特に、 複数の抗癌剤に対する耐性を獲得した癌 細胞 (多剤耐性癌細胞) に投与する多剤耐性癌克服剤およびその製造方 法に関するものである。 背景技術  The present invention relates to an agent for overcoming cancer, particularly to an agent for overcoming a multidrug-resistant cancer administered to cancer cells (multidrug-resistant cancer cells) that have acquired resistance to a plurality of anticancer agents, and a method for producing the same. Background art
従来より、 互いに化学構造や作用点に類似性の無い複数の抗癌剤に対 する耐性 (多剤耐性) を獲得した癌細胞 (多剤耐性癌細胞) に投与する 多剤耐性癌克服剤が種々開発されており、 有望な薬剤が幾つか報告され ている。例えば、カルシウム拮抗薬であるべラバミル (Verapamil) には、 ビンクリスチン (Vincristine) をはじめとする複数種の抗癌剤 (抗癌作用 物質) の、 多剤耐性癌細胞内への蓄積増強作用 (排出抑制作用) がある こと、 つまり、 多剤耐性癌克服作用 (多剤耐性克服作用) があることが 認められている。 また、 Heterocycles, 47(2), 1111-1133 (1998) には、 日 本ィチイ ( Taxus cuspidata Sieb. et Zucc.) の針葉部から取り出された 数種類のタキサン関連化合物 (総称; 夕キソイド) に、 上記べラパミル と同程度の多剤耐性癌克服作用があることが報告されている。 さらに、 Bioorganic & Medicinal Chemistry Lett., 7(4), 393-398 (1997)、 同 8, 1555-1558 (1998)、 同 9, 389-394 (1999)、 および Chem. Pharm. Bull., 46(7), 1135- 1139 (1998) には、 タキサン関連化合物のバイオアツセィ (Bioassay)に関する検討結果が報告されている。 発明の開示 Conventionally, various multidrug-resistant cancer-overcoming drugs have been developed that are administered to cancer cells (multidrug-resistant cancer cells) that have acquired resistance (multidrug-resistant) to multiple anticancer drugs that have no similar chemical structure or action point with each other. And several promising drugs have been reported. For example, the calcium antagonist Verapamil has the effect of increasing the accumulation of multiple types of anticancer drugs (anticancer substances) such as vincristine in multidrug-resistant cancer cells (excretion inhibitory action). ), That is, it is recognized that it has a multidrug-resistant cancer overcoming action (multidrug-resistant overcoming action). Heterocycles, 47 (2), 1111-1133 (1998) reported that several taxane-related compounds (general name: evening xoid) extracted from needles of Japanese yew (Taxus cuspidata Sieb. Et Zucc.). It has been reported that the drug has the same multidrug-resistant cancer-overcoming effect as verapamil. Furthermore, Bioorganic & Medicinal Chemistry Lett., 7 (4), 393-398 (1997), 8, 1555-1558 (1998), 9, 389-394 (1999), and Chem. Pharm. Bull., 46 (7), 1135-1139 (1998) reports the results of studies on the bioassay of taxane-related compounds. Disclosure of the invention
多剤耐性癌克服剤としては例えば、 強い多剤耐性癌克服作用を備えて いること、 及び、 副作用が少ないこと等の条件を満たすことが要求され る。 しかしながら、 上記の条件を充分に満たす多剤耐性癌克服剤は見出 されているとは未だいい難いのが現状である。 例えば、 上記べラパミル には、 副作用として血圧降下作用があることが認められており、 それゆ え、 実用化には至っていない。 また、 上記 Heterocyclesに記載されてい るタキサン関連化合物には、 ベラパミルと同程度の多剤耐性癌克服作用 があるものの、 実用可能であるかは未だ不明なところが多い。  The multidrug-resistant cancer-overcoming agent is required to satisfy conditions such as having a strong multidrug-resistant cancer-overcoming effect and having few side effects. However, it is still difficult to say that a multidrug-resistant cancer-overcoming agent that sufficiently satisfies the above conditions has not yet been found. For example, it has been recognized that verapamil has a blood pressure lowering effect as a side effect, and thus has not been put to practical use. In addition, although the taxane-related compounds described in Heterocycles have a multidrug-resistant cancer overcoming effect similar to that of verapamil, it is still unknown whether it is practical or not.
さらにまた、 多剤耐性癌克服剤として、 上記の条件に加え、 低殺細胞 性や、生体内での低分解性等の特性が必要とされる場合もある。従って、 新規な多剤耐性癌克服剤の探索は、 これら複雑多岐にわたるニーズを満 足させ、 多剤耐性癌克服剤の適用範囲を拡大しうる可能性を秘めている という観点から、 特に必要とされている。  Furthermore, in addition to the above-mentioned conditions, characteristics such as low cell killing properties and low in vivo degradation properties may be required as a multidrug-resistant cancer-overcoming agent. Therefore, the search for a novel drug that overcomes multidrug-resistant cancer is particularly necessary from the viewpoint that it has the potential to satisfy these complex and diverse needs and expand the scope of application of the drug that overcomes multidrug-resistant cancer. Have been.
本発明の主要な目的は、 上記従来の問題点に鑑み、 その目的は、 癌細 胞特に複数の抗癌剤に対する耐性を獲得した癌細胞 (多剤耐性癌細胞) に投与する新規な癌克服剤特に多剤耐性癌克服剤、 その製造方法等を提 供することにある。  The main object of the present invention is to solve the above-mentioned conventional problems, and to provide a novel cancer-overcoming agent, particularly a cancer-cancelling agent to be administered to cancer cells, particularly cancer cells that have acquired resistance to a plurality of anticancer agents (multidrug-resistant cancer cells). An object of the present invention is to provide an agent for overcoming a multidrug-resistant cancer and a method for producing the same.
本発明の請求の範囲第 1項記載の多剤耐性癌克服剤は、 上記の課題を 解決するために、 特定の化学式で表される化合物群より選ばれる少なく とも一種の化合物を含むことを特徴としている。  The multidrug-resistant cancer-overcoming agent according to claim 1 of the present invention is characterized by containing at least one compound selected from a group of compounds represented by a specific chemical formula in order to solve the above problems. And
上記化合物は何れも、 通常の癌細胞または複数の抗癌剤に対する耐性 を獲得した癌細胞(多剤耐性癌細胞)に対する癌克服作用を有している。 それゆえ、 前記化合物群より選ばれる少なくとも一種の化合物を含む癌 克服剤乃至多剤耐性癌克服剤は、 該癌細胞に好適に投与することができ る。 すなわち、 本発明は All of the above compounds have a cancer overcoming effect on normal cancer cells or cancer cells that have acquired resistance to a plurality of anticancer drugs (multidrug-resistant cancer cells). Therefore, a cancer-overcoming agent or a multidrug-resistant cancer-overcoming agent comprising at least one compound selected from the above compound group can be suitably administered to the cancer cells. That is, the present invention
( 1 ) 式 (1 ) (1) Equation (1)
Figure imgf000004_0001
Figure imgf000004_0001
(式中、  (Where
R1は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基を、 R 1 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified,
R 2は水素原子を、 または R1と R2とが一緒になつて =0を、 R 2 represents a hydrogen atom, or R 1 and R 2 together form = 0,
R 3は水素原子を、 R 3 represents a hydrogen atom,
R 4は水素原子もしくは水酸基を、 または R 3と R4とが一緒になつて、 単結合または— O—を形成してもよく、 または R3と R6が一緒になつて —O— CH2—を形成してもよく、 R 4 may be a hydrogen atom or a hydroxyl group, or R 3 and R 4 may be combined together to form a single bond or —O—, or R 3 and R 6 may be combined together and —O— CH 2 — may be formed,
R 5は水素原子またはエステル化もしくはェ一テル化されていてもよい 水酸基を、 R 5 represents a hydrogen atom or a hydroxyl group which may be esterified or esterified,
R6はメチル基、 ヒドロキシメチル基、 またはエステル化もしくはエーテ ル化されていてもよい水酸基、 R 6 is a methyl group, a hydroxymethyl group, or a hydroxyl group which may be esterified or etherified,
R 7はエステル化またはエーテル化されていてもよい水酸基、 R 7 is a hydroxyl group which may be esterified or etherified,
R 8は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基、 R 8 is a hydrogen atom or a hydroxyl group which may be esterified or etherified,
R 9は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基、 R 9 is a hydrogen atom or a hydroxyl group which may be esterified or etherified,
R1 Qは水素原子または R 4と一緒になつて単結合を表してもよい、R 1 Q may be taken together with a hydrogen atom or R 4 to represent a single bond,
R11はメチル基またはエステル化もしくはエーテル化されていてもよい 水酸基を有するメチル基、 R 11 may be methyl or esterified or etherified A methyl group having a hydroxyl group,
R 12は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基を、 R 12 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified,
R 13は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基を、 R 13 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified,
R 14は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基を示す) で表される化合物、 R 14 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified)
式 (2) Equation (2)
Figure imgf000005_0001
Figure imgf000005_0001
(式中、 R 15はエステル化もしくはエーテル化されていてもよい水酸基 を、 (Wherein, R 15 represents a hydroxyl group which may be esterified or etherified,
R 16は水素原子を、 R 16 represents a hydrogen atom,
R 17はメチル基を、 または R16と R17とが一緒になつて— O— CH2— を形成してもよく、 R 17 may be a methyl group, or R 16 and R 17 may be taken together to form —O—CH 2 —,
R 18は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基を、 R 18 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified,
R 19はエステル化もしくはエーテル化されていてもよい水酸基を、 は二重結合または単結合を示す)で表される化合物、 R 19 represents a hydroxyl group which may be esterified or etherified, and represents a double bond or a single bond),
式 (3)
Figure imgf000006_0001
Equation (3)
Figure imgf000006_0001
(式中、 R 2 Qはエステル化またはエーテル化されていてもよい水酸基を、 R 21は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基を、 R22は水素原子を、 または R21と R22とが一緒になつて =0 を形成してもよく、 (Wherein, R 2 Q represents a hydroxyl group which may be esterified or etherified, R 21 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified, R 22 represents a hydrogen atom, or R 21 And R 22 may together form = 0,
R 23は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基を、 R24は水素原子を、 または R23と R24とが一緒になつて =0 を形成してもよく、 R 25は水素原子またはエステル化もしくはエーテル 化されていてもよい水酸基を、 R26は水素原子を示す。 もっとも R25と R26とが一緒になつて =0を形成してもよい) で表される化合物、 式 (4) R 23 is a hydrogen atom or a hydroxyl group which may be esterified or etherified, R 24 may be a hydrogen atom, or R 23 and R 24 may together form = 0, and R 25 is R 26 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified, and R 26 represents a hydrogen atom. R 25 and R 26 may combine to form = 0), a compound represented by the formula (4)
Figure imgf000006_0002
Figure imgf000006_0002
(式中、 R27、 R28、 R29、 R3Q、 R31および R32はそれぞれ同一ま たは異なって水素原子、 またはエステル化もしくはエーテル化されてい てもよい水酸基を示す。 R33、 R34は水素原子を示すか R32と R33ま たは R28と R34とが一緒になつて =0を形成してもよい) で表される化 合物、 または 式 (5) (Wherein, R 27, R 28, R 29, R 3Q, R 31 and R 32 are each independently hydrogen were identical or respectively or be esterified or etherified represents an hydroxy group. R 33,, R 34 represents a hydrogen atom, or R 32 and R 33 or R 28 and R 34 can be taken together to form = 0), or Equation (5)
Figure imgf000007_0001
Figure imgf000007_0001
(式中、 R35、 R36、 R37、 R38および R39はそれぞれ同一または異 なって、 エステル化またはエーテル化されていてもよい水酸基を示す) で表される化合物またはその塩を含有することを特徴とする医薬、 (Wherein, R 35 , R 36 , R 37 , R 38 and R 39 are the same or different and each represents a hydroxyl group which may be esterified or etherified) or a salt thereof. A medicament characterized by:
(2) 化合物が式  (2) the compound has the formula
Figure imgf000007_0002
Figure imgf000007_0002
Figure imgf000007_0003
Figure imgf000007_0004
Figure imgf000008_0001
Figure imgf000007_0003
Figure imgf000007_0004
Figure imgf000008_0001
Figure imgf000008_0002
Figure imgf000008_0002
Figure imgf000008_0003
Figure imgf000008_0003
(12)
Figure imgf000008_0004
(12)
Figure imgf000008_0004
Figure imgf000008_0005
Figure imgf000009_0001
Figure imgf000009_0002
Figure imgf000009_0003
Figure imgf000009_0004
Figure imgf000009_0005
Figure imgf000010_0001
Figure imgf000010_0002
Figure imgf000010_0003
Figure imgf000008_0005
Figure imgf000009_0001
Figure imgf000009_0002
Figure imgf000009_0003
Figure imgf000009_0004
Figure imgf000009_0005
Figure imgf000010_0001
Figure imgf000010_0002
Figure imgf000010_0003
Figure imgf000010_0004
Figure imgf000010_0004
(23)
Figure imgf000010_0005
01
(twenty three)
Figure imgf000010_0005
01
Figure imgf000011_0001
Figure imgf000011_0002
Figure imgf000011_0001
Figure imgf000011_0002
Figure imgf000011_0003
Figure imgf000011_0004
Figure imgf000011_0005
Figure imgf000011_0003
Figure imgf000011_0004
Figure imgf000011_0005
01  01
9C0S0/00df/X3d OtO O/IO OAV
Figure imgf000012_0001
Figure imgf000012_0002
Figure imgf000012_0003
9C0S0 / 00df / X3d OtO O / IO OAV
Figure imgf000012_0001
Figure imgf000012_0002
Figure imgf000012_0003
Figure imgf000012_0004
Figure imgf000012_0004
9C0S0/00df/X3d OtO O/IO OAV
Figure imgf000013_0001
Figure imgf000013_0002
9C0S0 / 00df / X3d OtO O / IO OAV
Figure imgf000013_0001
Figure imgf000013_0002
Figure imgf000013_0003
Figure imgf000013_0003
(36) (36)
AcO OAc CO
Figure imgf000014_0001
AcO OAc CO
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000015_0002
Figure imgf000015_0003
Figure imgf000015_0004
Figure imgf000015_0005
Figure imgf000016_0001
Figure imgf000015_0001
Figure imgf000015_0002
Figure imgf000015_0003
Figure imgf000015_0004
Figure imgf000015_0005
Figure imgf000016_0001
または Or
Figure imgf000016_0002
Figure imgf000016_0002
(式中、 Acはァセチル基を、 B zはベンゾィル基を、 Meはメチル基 を、 P hはフエ二ル基を示す) で表される化合物を含有することを特徴 とする前記 (1) 記載の医薬、  (Wherein Ac represents an acetyl group, B z represents a benzoyl group, Me represents a methyl group, and Ph represents a phenyl group). The medicament described,
(3) 癌克服剤である前記 (1) または (2) に記載の医薬、  (3) the medicament according to the above (1) or (2), which is an agent for overcoming cancer;
(4) 多剤耐性癌克服剤である前記 (1) または (2) に記載の医薬、 (4) the medicament according to the above (1) or (2), which is a multidrug-resistant cancer-overcoming agent;
(5) 抗癌作用物質蓄積増強剤である前記 ( 1) 〜 (4) のいずれかに 記載の医薬、 (5) The medicament according to any of (1) to (4), which is an anticancer substance accumulation enhancer.
(6) さらに抗癌作用物質を含有する前記 (1) 〜 (5) のいずれかに 記載の医薬、  (6) The medicament according to any of (1) to (5), further comprising an anticancer substance.
(7) 前記 ( 1) または (2) に記載の化合物と抗癌作用物質とを組み 合わせて癌患者に投与することを特徴とする癌の治療方法、  (7) A method for treating cancer, which comprises administering the compound according to (1) or (2) and an anticancer substance to a cancer patient in combination,
(8) 式  Equation (8)
Figure imgf000016_0003
CSJ GO
Figure imgf000016_0003
CSJ GO
CM CM CM CM
CD CO CD CO
Figure imgf000017_0001
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000018_0001
AcO OAc AcO OAc
(42)  (42)
H OAc 20  H OAc 20
または Or
Figure imgf000019_0001
Figure imgf000019_0001
(式中、 Acはァセチル基を、 Meはメチル基を、 P hはフエ二ル基を 示す) で表される化合物、 (In the formula, Ac represents an acetyl group, Me represents a methyl group, and Ph represents a phenyl group.)
(9) 式  Equation (9)
Figure imgf000019_0002
Figure imgf000019_0002
または Or
Figure imgf000019_0003
Figure imgf000019_0003
(式中、 Acはァセチル基を、 P hはフエ二ル基を示す) で表される化 合物を含有することを特徴とする制癌剤、  (Wherein Ac represents an acetyl group and Ph represents a phenyl group), a carcinostatic agent comprising a compound represented by the formula:
( 1 0) 日本イチィ Taxus cusp i data Sieb. et Zucc. ) の組織または その組織より誘導されるカルスから前記 (2) に記載の化合物を採取す ることを特徴とする前記 (2) に記載された化合物の製造方法、 および ( 1 1) タキシン骨格、 夕キシニン骨格またはアビェ夕ン骨格を有し、 抗癌作用物質蓄積増強作用または抗癌作用物質排出抑制作用のある化合 物を含有する医薬、 (10) Taxus cuspi data sieb. Et Zucc. A method for producing the compound according to (2), wherein the compound according to (2) is collected from callus derived from the tissue; and (11) a taxin skeleton, a xinine skeleton or A drug having a Abjön skeleton and containing a compound having an anticancer substance accumulation enhancing action or an anticancer substance discharge inhibitory action;
に関する。 化学式 (6) 〜 (14) で表される化合物はタキシン化合物であり、 化学式( 1 5)〜(2 9) で表される化合物はタキシニン化合物である。 尚、 化学式 (23) で表される化合物は 2'-desacetylaustrospicatineで ある。 また、 化学式(24) で表される化合物は 2-desacethoxy taxinine Bであり、 化学式 (2 5) で表される化合物は taxinine Eである。 About. The compounds represented by the chemical formulas (6) to (14) are taxin compounds, and the compounds represented by the chemical formulas (15) to (29) are taxinine compounds. The compound represented by the chemical formula (23) is 2'-desacetylaustrospicatine. The compound represented by the chemical formula (24) is 2-desacethoxy taxinine B, and the compound represented by the chemical formula (25) is taxinine E.
なお、 化学式 (1 7)、 (2 2), (2 7)、 (2 8) および (2 9) で表 される化合物は新規夕キシニン化合物である。  The compounds represented by the chemical formulas (17), (22), (27), (28) and (29) are novel xinine compounds.
化学式(3 0)で表される化合物は夕クスユンナニン C (taxuyunnanin C) ;化学式 (3 1) で表される化合物は 2 α, 5 , 1 0 /3—トリァセ トキシ一 1 4 )3—プロピオ二ロキシ—タキサ— 4 (2 0), 1 1—ジェ ン;化学式 (32) で表される化合物はユンナキサン (yunnanxane) ; 化学式 (3 3) で表される化合物は 2 α, 5 , 1 0 /3—トリァセトキ シ— 1 4 3— (2, —メチル) ブチリ口キシ—タキサ— 4 (2 0), 1 1 —ジェン;化学式 (34) で表される化合物は 2 α, 5 , 1 0 /3—ト リアセトキシ— 14 )3—イソプチリロキシ—夕キサ— 4 (2 0), 1 1一 ジェン;化学式 (3 5) で表される化合物は 2 α—ヒドロキシー 5 a, 1 0 )3, 1 4 3 -トリァセトキシータキサ— 4 (20), 1 1—ジェン(2 — hydroxy— 5 , 1 0 /3, 1 4 /3― triacetoxy - taxa― 4 (2 0), 1 1 -diene) ;化学式 (3 6) で表される夕キシニン化合物は 5 , 1 3 a—ジァセトキシ一 9 a—シンナモイルォキシ— 1 0 /3—ヒドロキシ —夕キサ— 4 (2 0), 1 1一ジェン (5ひ, 1 3 a -diacetoxy- 9 ― cinnamoyloxy― 1 0 β— nydroxy— taxa— 4 2 0 1 1— diene) ; 化学式 (3 7) で表されるアビェ夕ン化合物は、 3 α, 7 , 1 1—ト リヒドロキシ一 1 2—メトキシ一 3, 2 0—エポキシ一アビエタ一 8, 1 1, 1 3—トリェン (3 α, 7 a, 1 1— trihydroxy— 1 2 - methoxy — 3, 20 -epoxy-abieta- 8 , 1 1, 1 3—triene) ;化学式 ( 3 8 ) で表されるアビェ夕ン化合物は、 3 /3, 1 2—ジヒドロキシ—アビエタ — 6, 8, 1 1, 1 3—テトラエン ( 3 /3, 1 2 - dihydroxy - abieta - 6, 8, 1 1, 1 3— tetraene);化学式 (3 9) で表されるアビェ夕ン 化合物は 3 α—ヒドロキシ— 9 ( 1 0→20)アベオアビエタ— 1, 5, 8, 1 0 (2 0), 1 3—ペン夕ェン— 7, 1 1, 1 2— trione;化学式 (40) で表される化合物は夕クスシン (taxusin) ;化学式 (4 1) で 表される夕キシニン化合物は 1 0—デァセチル 夕キシニン ( 1 0— deacetyl taxinine);化学式 (42) および化学式 (43) で表される化 合物はタキシニン化合物;化学式 (44) で表される化合物は 2—デス ァセトキシ タキシニン E (2 - desacetoxy taxinine E);化学式(45) で表される化合物はタキシン NA— 2 (taxicin NA-2) ;化学式 (46) で表される化合物は 2 —デスァセトキシ 夕キシニン J ( 2 — desacetoxy taxinine J);化学式 (47) で表される化合物は 5—シンナ モイルー 1 0—ァセチルータキシン I I ( 5—cinnamoyl— 1 0—acetyl 一 taxicin II) である。 The compound represented by the chemical formula (30) is taxuyunnanin C; the compound represented by the chemical formula (31) is 2α, 5,10 / 3-triacetoxy-1 14) 3-propioni Roxy-taxa 4 (20), 11-gen; the compound represented by the chemical formula (32) is yunnanxane; the compound represented by the chemical formula (33) is 2α, 5,10 / 3-triacetoxy—1 4 3— (2, —methyl) butylyl xy-taxa 4 (20), 11—gen; the compound represented by the chemical formula (34) is 2α, 5,10 / 3-triacetoxy—14) 3-isobutylyloxy-mixer 4 (20), 11-gene; The compound represented by the chemical formula (35) is 2α-hydroxy-5a, 10) 3, 14 3-triacetoxy-taxa 4 (20), 11-Gen (2-hydroxy-5, 10/3, 14 / 3-triacetoxy-taxa-4 (20), 11-diene); Chemical formula The evening xinine compound represented by (36) 5, 1 3a—Diacetoxy-1 9a—Cinnamoyloxy—10 / 3-Hydroxy—Sixa—4 (20), 1 1-Jen (5, 13a-diacetoxy-9—Cinnamoyloxy—10β — Nydroxy— taxa— 4 2 0 1 1— diene); The abyssinane compound represented by the chemical formula (3 7) is 3α, 7,11 1-trihydroxy-12-methoxy-1,3,20 —Epoxy-1,8,11,13,3-triene (3α, 7a, 11-trihydroxy—12-methoxy—3,20-epoxy-abieta-8,11,13-triene); The abiene compound represented by the chemical formula (38) is 3 / 3,12-dihydroxy-avieta-6,8,11,13-tetraene (3 / 3,12-dihydroxy-abieta-6 , 8,11,13-tetraene); The compound represented by the chemical formula (39) is 3α-hydroxy-9 (10 → 20) aveoavieta-1,5,8,10 (2 0), 1 3—Penhuen—7, 11, 1, 12—trione; represented by chemical formula (40) Is a compound represented by the chemical formula (42) and the chemical formula (43). The compound represented by the chemical formula (41) is 10-deacetyl taxin. The compound is a taxinine compound; the compound represented by the chemical formula (44) is 2-desacetoxy taxinine E; the compound represented by the chemical formula (45) is taxin NA-2 (taxicin NA-2) The compound represented by the chemical formula (46) is 2—desacetoxy taxinine J; the compound represented by the chemical formula (47) is 5—cinnamoyl-10—acetyrutaxin II (5— cinnamoyl—10-acetyl-taxicin II).
化学式(6)〜(47)の化合物はいずれも日本イチィ Taxus cuspidata Sieb. et Zucc.) より採取または単離 (以下単に採取と略称する) するこ とができるが、 このように採取した化学式 (6) 〜 (47) に属する化 合物は他の化学式 (6) 〜 (47) に属する化合物に化学変換すること ができる。 そのような化学変換のための化学反応は通常化学者または薬 学者が周知する伝統的な化学反応であってよい。具体的には例えば酸(例 えば塩酸) または塩基 (例えば水酸化ナトリウム) を使用してァセチル 基やシンナモイル基を水酸基に変換する加水分解反応;例えばナトリウ ムポロンハイドライドを使用する化学還元や水素ガスと還元触媒を使用 する接触還元などによるカルボ二ル基を水酸基に変換する還元反応;例 えば水酸基に例えばァセチル基ゃシンナモイル基を導入するエステル化 反応等を挙げることができる。 従って化学者または薬学者にとっては自 明のことであるが化学式 (6 ) 〜 (4 7 ) で表される化合物は、 同一一 般式 (1 ) 〜 (5 ) のそれぞれの範囲内において化学的に容易に変換可 能である。 All of the compounds of the chemical formulas (6) to (47) can be collected or isolated (hereinafter simply referred to as “collection”) from Nippon Ichi Taxus cuspidata Sieb. Et Zucc. 6) Chemical conversion of compounds belonging to (47) into compounds belonging to other chemical formulas (6) to (47) Can be. The chemical reaction for such a chemical transformation may be a traditional chemical reaction commonly known to chemists or pharmacists. Specifically, for example, a hydrolysis reaction for converting an acetyl group or a cinnamoyl group to a hydroxyl group using an acid (for example, hydrochloric acid) or a base (for example, sodium hydroxide); for example, chemical reduction using sodium borohydride or hydrogen gas And a reduction reaction for converting a carbonyl group into a hydroxyl group by catalytic reduction using a reduction catalyst; for example, an esterification reaction for introducing an acetyl group to a cinnamoyl group into a hydroxyl group. Therefore, it is obvious for a chemist or a pharmacist that the compounds represented by the chemical formulas (6) to (47) have the same general formulas (1) to (5). It can be easily converted.
本発明者等は、 上記課題について鋭意検討した結果、 上記式 (1 ) 〜 ( 4 7 ) で表される化合物を抗癌作用物質と共に癌患者に投与すると、 抗癌作用物質の癌細胞内への、 特に多剤耐性癌細胞内への蓄積が増強さ れるもしくは癌細胞もしくは多剤耐性癌細胞に取り込まれた抗癌作用物 質の癌細胞もしくは多剤耐性癌細胞内から癌細胞もしくは多剤耐性癌細 胞外への排出が抑制されること、 そしてその結果抗癌作用物質の抗癌剤 としての作用効果が向上すること、 つまり、 上記式の化合物は抗癌作用 物質もしくは抗多剤耐性癌作用物質 (以下単に抗癌作用物質と略称する こともある) が癌細胞もしくは多剤耐性癌細胞 (以下単に癌細胞と略称 することもある) 内に蓄積するのを増強する作用もしくは癌細胞から排 出されるのを抑制するため、 癌克服剤もしくは多剤耐性癌克服剤 (以下 単に癌克服剤と略称することもある) として有用であることを見出した。 すなわち、 本発明は、 上記式 ( 1 ) 〜 (4 7 ) で表される化合物と他の 抗癌作用物質とを組み合わせて、 投与される医薬を提供する。  The present inventors have conducted intensive studies on the above problems, and as a result, when a compound represented by any of the above formulas (1) to (47) is administered to a cancer patient together with an anticancer substance, the anticancer substance enters the cancer cells. In particular, cancer cells or multidrugs from cancer cells or multidrug-resistant cancer cells of anticancer agents whose accumulation in multidrug-resistant cancer cells is enhanced or incorporated into the cancer cells or multidrug-resistant cancer cells Suppressing the excretion of resistant cancer cells out of the cell, and consequently improving the effect of the anticancer substance as an anticancer agent. The action of enhancing the accumulation of a substance (hereinafter sometimes simply referred to as an anticancer substance) in cancer cells or multidrug-resistant cancer cells (hereinafter sometimes simply referred to as cancer cells) or excluding it from cancer cells. To be issued Therefore, it was useful as a cancer overcoming agent or multidrug resistant cancer overcome agent (sometimes simply referred to as cancer overcoming agent or less). That is, the present invention provides a medicament to be administered by combining the compounds represented by the above formulas (1) to (47) with another anticancer substance.
また、 本発明者等は、 式 (1 ) 〜 (4 7 ) で表される化合物自体が抗 癌作用物質として作用するから、 式 (1 ) 〜 (47) で表される化合物 以外の抗癌作用物質は必ずしも必要でないことも知見した。 Further, the present inventors have found that the compounds represented by the formulas (1) to (47) It has also been found that an anticancer substance other than the compounds represented by the formulas (1) to (47) is not necessarily required because it acts as a cancer agent.
また、 本発明者等は、 上記式 (6) 〜 (47) で表される化合物が日 本ィチイ 、Taxus cuspidata Sieb. et Zucc.) の例えば針葉部等の組織ま たはその組織より誘導されるカルスから取り出しうることができること, そしてこのようにして癌細胞に投与するのに好適な癌克服剤を、 毎年再 生される針葉部を使用する等、 環境を保全しながら効率的かつ工業的有 利に製造することができることを知見した。  In addition, the present inventors have found that the compounds represented by the above formulas (6) to (47) are derived from tissues such as needles of Japanese yew, Taxus cuspidata Sieb. Et Zucc. Can be removed from the callus, and a cancer-overcoming agent suitable for administration to cancer cells in this way can be used efficiently and efficiently while preserving the environment. It was found that it can be manufactured industrially.
また、 本発明者等は、 上記式 (1) 〜 (5) の範囲に属し、 上記式 (6) 〜 (47) で表される化合物以外の化合物は、 上記式 (6) 〜 (47) で表される化合物を日本ィチイから採取し、 これを例えば上記したと同 様の加水分解反応、 還元反応、 エステル化反応、 エーテル化反応等自体 公知の手段に付することにより容易に製造されうることを知見した。 発明を実施するための最良の形態  In addition, the present inventors have found that compounds other than the compounds represented by the above formulas (6) to (47) belong to the range of the above formulas (1) to (5), and the compounds represented by the above formulas (6) to (47) Can be easily produced by collecting the compound represented by the formula (I) from Nippon yew and subjecting it to a means known per se such as a hydrolysis reaction, a reduction reaction, an esterification reaction, an etherification reaction, etc. as described above. I found that. BEST MODE FOR CARRYING OUT THE INVENTION
本発明は医薬において癌克服剤または多剤耐性癌克服剤として使用さ れる化合物は一般式 (1) 〜 (5) で表される化合物である。  In the present invention, the compound used as a cancer-surviving agent or a multidrug-resistant cancer-surviving agent in medicine is a compound represented by any of the general formulas (1) to (5).
式 (1 ) 〜 (5) 中、 エステル化された水酸基はたとえば式  In the formulas (1) to (5), the esterified hydroxyl group is represented by the formula
0  0
II II
Figure imgf000023_0001
Figure imgf000023_0001
で表すことができ、 エーテル化された水酸基としては式 The etherified hydroxyl group can be represented by the formula
-o- z 2 -o- z 2
で表される。 そして、 式中 It is represented by And in the formula
0  0
II  II
z C - で表される基はァシル基である。 z iおよび z 2で表される基は本発明の精神に反しない限りどのような 化学修飾のための置換基であってもよいが、 医薬品分野でエステル形成 基もしくはエーテル形成基(またはエステル残基もしくはエーテル残基) として常用される置換基が特許文献上または学術文献上もよく知られて いるので、 本発明においてもこのような自体公知の修飾のための置換基 を採用することができ、 かかるエステル形成基もしくはエーテル形成基 は従来公知の方法に従って行われてよい。 The group represented by z C-is an acyl group. groups represented by zi and z 2 can be a substituent for any such chemical modifications unless contrary to the spirit of the present invention, in the pharmaceutical field ester forming group or ether forming groups (or ester residue Or an ether residue) is well known in the patent literature or the scientific literature, and therefore, in the present invention, such a substituent for a modification known per se can be used, Such an ester-forming group or an ether-forming group may be performed according to a conventionally known method.
および Z2で表される基は、 具体的には例えば炭素数 1〜50程度 の飽和または不飽和の直鎖状、 分枝状または環状のアルキル基、 C7〜C 2o程度のァラルキル基、 C6〜C15程度のァリール基等が挙げられる。 これらの置換基は例えば、 水酸基、 アミノ基、 力ルポキシル基、 スルホ ニル基 (例えばメチルスルホニル基)、 ハロゲン原子、 ニトロ基、 程度のアルコキシ基等の通常医薬の有効成分の化学修飾に常用される置 換基によってさらに置換されていてもよい。 ついで、 本発明化合物の製造方法等について説明する。 Groups represented by and Z 2, specifically, for example, carbon number 1 to 50 of about saturated or unsaturated, linear, branched or cyclic alkyl group, C 7 -C 2 o about Ararukiru group , etc. Ariru group of about C 6 -C 15 and the like. These substituents are commonly used for the chemical modification of active ingredients of ordinary pharmaceuticals such as, for example, a hydroxyl group, an amino group, a carbonyl group, a sulfonyl group (for example, a methylsulfonyl group), a halogen atom, a nitro group, and an alkoxy group. It may be further substituted by a substituent. Next, a method for producing the compound of the present invention and the like will be described.
説明は化合物 (6) 〜 (29) と化合物 (30) 〜 (39) と化合物 (40) 〜 (47) に分けて行う。  The description will be made separately for compounds (6) to (29), compounds (30) to (39), and compounds (40) to (47).
タキシン化合物または夕キシニン化合物等のタキサン関連化合物 (総 称; 夕キソイド) であるこれら化合物は、 例えば、 複数の抗癌剤に対す る耐性を獲得した癌細胞 (多剤耐性癌細胞) に対して、 充分な多剤耐性 癌克服作用がある。 多剤耐性癌克服剤は、 上記化学式 ( 1 1), (1 5), ( 16), ( 1 7), (22), (24), (26) で表される化合物群より選 ばれる少なくとも一種の化合物を含んでいることが、 より好ましい。 こ れら 7種類の化合物は、 より強い多剤耐性癌克服作用を備えている。 そ して、 上記化学式 (1 7), (22), (2 7), (28), (29) で表され る 5種類の夕キシニン化合物は、 新規物質である。 These compounds, which are taxane-related compounds such as taxin compounds or evening xinine compounds (generic name: evening xoids), are sufficient for, for example, cancer cells (multidrug-resistant cancer cells) that have acquired resistance to multiple anticancer agents. Multi-drug resistance Has cancer-overcoming action. The agent for overcoming a multidrug-resistant cancer is at least one selected from the group of compounds represented by the above chemical formulas (11), (15), (16), (17), (22), (24), and (26). More preferably, it contains one kind of compound. These seven compounds have a stronger effect of overcoming multidrug-resistant cancer. Then, they are represented by the above chemical formulas (17), (22), (27), (28), and (29). The five types of xinine compounds are new substances.
該多剤耐性癌克服剤は、 複数の抗癌剤に対する耐性 (多剤耐性) を獲 得した癌細胞 (多剤耐性癌細胞) 内に取り込まれた様々な抗癌剤を、 該 多剤耐性癌細胞外に排出する複数の作用機構の何れかを阻害する作用を 有するものである。 抗癌剤を多剤耐性癌細胞外に排出する作用機構とし て、 具体的には、 例えば、 多剤耐性癌細胞において存在または発現する P—糖蛋白質が、 多剤耐性癌細胞内に取り込まれた様々な抗癌剤と結合 し、 これらを能動輸送により排出する機構等が挙げられる。 上記化学式 The multidrug-resistant cancer-overcoming agent is capable of transferring various anticancer agents incorporated into cancer cells (multidrug-resistant cancer cells) that have acquired resistance to a plurality of anticancer agents (multidrug-resistant cancer cells) out of the multidrug-resistant cancer cells It has the effect of inhibiting any of a plurality of action mechanisms that emit. As an action mechanism for excreting an anticancer drug out of a multidrug-resistant cancer cell, specifically, for example, various P-glycoproteins present or expressed in the multidrug-resistant cancer cell are incorporated into the multidrug-resistant cancer cell. A mechanism that binds to various anticancer drugs and discharges them by active transport is exemplified. The above chemical formula
( 6 ) 〜 (2 9 ) で表される化合物は、 それぞれ単独で、 P—糖蛋白質 による抗癌剤の能動輸送の阻害剤 (以下、 P—糖蛋白質阻害剤と称する) としての機能を少なくとも有し、 本発明にかかる多剤耐性癌克服剤と成 り得る。 上記の化合物が多剤耐性癌克服作用を有することは、 本願発明 者が新たに見い出したことである。 尚、 以下の説明においては、 必要に 応じて、 例えば上記化学式 (6 ) 〜 (2 9 ) で表される化合物を、 順に、 化合物 (6 ) 〜 (2 9 ) と称する (他の化学式についても同じ)。 Each of the compounds represented by (6) to (29) alone has at least a function as an inhibitor of the active transport of an anticancer agent by P-glycoprotein (hereinafter, referred to as a P-glycoprotein inhibitor). It can be a multidrug-resistant cancer-overcoming agent according to the present invention. The fact that the above-mentioned compound has a multidrug-resistant cancer overcoming action is newly found by the present inventors. In the following description, if necessary, for example, the compounds represented by the chemical formulas (6) to (29) are referred to as compounds (6) to (29), respectively. the same).
本発明にかかる多剤耐性癌克服剤の製造方法は、 上記化合物のうちの 少なくとも 1つを、 日本ィチイ ( Taxus cuspidata Sieb. et Zucc.) の針 葉部から、 例えば、 有機溶媒を用いて抽出した後、 得られた抽出液を酸 および または塩基で処理し、 次いで液体クロマトグラフィーによって 分画し、 取り出す工程を行うことによって製造する方法である。 日本ィ チイの針葉部は、 入手が比較的容易であり、 該日本イチィは、 例えば北 海道、本州、 四国、 九州の亜熱帯から温帯にわたって分布している。 尚、 本発明における 「針葉部」 には、 「葉身」、 「葉柄」、 並びに、 該 「葉部 (葉 身 +葉柄)」 が複数枚付いた 「小枝」、 さらには、 「新芽 (その原基であつ ても良い)」 ゃ該 「新芽」 が付いた 「若茎」 が含まれることとする。  In the method for producing a multidrug-resistant cancer-overcoming agent according to the present invention, at least one of the above compounds is extracted from needles of Japanese yew (Taxus cuspidata Sieb. Et Zucc.) Using, for example, an organic solvent. After that, the obtained extract is treated with an acid and or a base, followed by fractionation by liquid chromatography and a step of taking out the extract, thereby producing the extract. The needle portion of the Japanese yew is relatively easy to obtain, and it is distributed, for example, from the subtropics of Hokkaido, Honshu, Shikoku, and Kyushu to temperate zones. In the present invention, the “needle portion” includes “leaf”, “stalk”, and “twig” having a plurality of the “leaf (leaf + stalk)”. (The primordium may be used.) ゃ “Sprouts” with the “sprout” shall be included.
日本ィチイの針葉部から前記化合物 (6 ) 〜 (2 9 )、 即ち、 タキサン 関連化合物を取り出す具体的な方法は、特に限定されるものではないが、 抽出を行う方法が好適であり、 有機溶媒を用いて抽出を行う方法が最適 である。 そして、 抽出を行う具体的な方法としては、 例えば、 針葉部 (生 針葉部) を、 必要に応じて粉砕した後、 有機溶媒に数日間から数週間浸 漬する方法が挙げられる。抽出条件は、特に限定されるものでは いが、 抽出温度は 3 0で以下であることがより好ましい。 The compounds (6) to (29) from the needle portion of yew tree, ie, taxane The specific method of extracting the related compound is not particularly limited, but a method of performing extraction is preferable, and a method of performing extraction using an organic solvent is most suitable. As a specific method for performing the extraction, for example, there is a method in which a needle part (raw needle part) is pulverized as necessary, and then immersed in an organic solvent for several days to several weeks. The extraction conditions are not particularly limited, but the extraction temperature is more preferably 30 or less.
上記の有機溶媒としては、 具体的には、 例えば、 メチルアルコール、 エチルアルコール、 イソプロピルアルコール、 クロ口ホルム、 n—へキ サン、 酢酸ェチル、 各種エーテル、 トルエン等が挙げられるが、 特に限 定されるものではない。これら有機溶媒は、一種類のみを用いてもよく、 また、 二種類以上を併用してもよい。 上記例示の有機溶媒のうち、 メチ ルアルコールおよび酢酸ェチルがより好ましい。  Specific examples of the organic solvent include, but are not limited to, methyl alcohol, ethyl alcohol, isopropyl alcohol, chloroform, n-hexane, ethyl acetate, various ethers, and toluene. Not something. One of these organic solvents may be used alone, or two or more thereof may be used in combination. Of the organic solvents exemplified above, methyl alcohol and ethyl acetate are more preferred.
また、 上記抽出操作を行う前に、 針葉部に含まれる油分を除去 (脱脂) するために、 該針葉部を有機溶媒で洗浄してもよい。 上記例示の有機溶 媒のうち、 n—へキサンが、 針葉部に含まれる油分を除去するのに好適 である。  In addition, before performing the extraction operation, the needle portion may be washed with an organic solvent in order to remove (degrease) oil contained in the needle portion. Among the organic solvents exemplified above, n-hexane is suitable for removing oil contained in needles.
タキサン関連化合物が抽出された抽出液は、 アル力ロイド誘導体ゃフ ェノール誘導体等の成分を分離 ·除去するために、 酸および または塩 基で処理することが好ましい。 抽出液を酸および/または塩基で処理す る方法としては、 具体的には、 抽出液を酸性水および Zまたは塩基性水 で洗浄する方法が挙げられる。  The extract from which the taxane-related compound has been extracted is preferably treated with an acid and / or a base in order to separate and remove components such as an alloid-derivative and a phenol derivative. As a method of treating the extract with an acid and / or a base, specifically, a method of washing the extract with acidic water and Z or basic water can be mentioned.
上記の酸としては、 具体的には、 例えば、 塩酸、 硫酸、 リン酸等の無 機酸;ギ酸、 酢酸等の有機酸;が挙げられるが、 特に限定されるもので はない。 これら酸は、 一種類のみを用いてもよく、 また、 二種類以上を 併用してもよい。 従って、 酸性水としては、 これら無機酸およびノまた は有機酸の水溶液が挙げられる。 該酸性水の P Hは、 特に限定されるも のではないが、 5以下がより好ましく、 4以下がさらに好ましい。 抽出 液を酸性水で洗浄することによって、 該抽出液から、 アルカロイド誘導 体等の成分を分離 ·除去することができる。 Specific examples of the above-mentioned acids include, for example, inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid; and organic acids such as formic acid and acetic acid, but are not particularly limited. These acids may be used alone or in combination of two or more. Therefore, examples of the acidic water include aqueous solutions of these inorganic acids and organic acids. The pH of the acidic water is particularly limited, However, it is preferably 5 or less, and more preferably 4 or less. By washing the extract with acidic water, components such as alkaloid derivatives can be separated and removed from the extract.
尚、 タキサン関連化合物のうちの幾つかの化合物は、 酸で環開裂 (分 解) し易いォキセタン骨格を分子構造に有している。 従って、 一般に、 酸で処理を行うと、 該ォキセ夕ン骨格の環開裂が生じてタキサン関連化 合物が破壊されてしまうと考えられている。 ところが、 本願発明者等が 検討したところ、 タキサン関連化合物は、 酸で処理を行っても、 ォキセ タン骨格の環開裂が生じないことが判明した。 それゆえ、 酸で処理を行 うことによって、 タキサン関連化合物と、 アルカロイド誘導体等の成分 とを分離することができる。  Some of the taxane-related compounds have an oxetane skeleton in the molecular structure that is easily cleaved (decomposed) by an acid. Therefore, it is generally considered that the treatment with an acid causes ring cleavage of the oxenone skeleton to destroy the taxane-related compound. However, the present inventors have studied and found that the taxane-related compound did not undergo ring cleavage of the oxetane skeleton even when treated with an acid. Therefore, by treating with an acid, a taxane-related compound and components such as alkaloid derivatives can be separated.
また、 上記の塩基としては、 具体的には、 例えば、 アンモニア、 水酸 化ナトリウム、 水酸化カリウム、 炭酸ナトリウム、 炭酸カリウム等の無 機塩基:アンモニゥム化合物等の有機塩基;が挙げられるが、 特に限定 されるものではない。 これら塩基は、一種類のみを用いてもよく、 また、 二種類以上を併用してもよい。 従って、 塩基性水としては、 これら無機 塩基および/または有機塩基の水溶液が挙げられる。 該塩基性水の P H は、 特に限定されるものではないが、 9以上がより好ましく、 1 0以上 がさらに好ましく、 1 1以上が特に好ましい。 抽出液を塩基性水で洗浄 することによって、 該抽出液から、 フエノール誘導体等の成分を分離 · 除去することができる。  Specific examples of the above base include: inorganic bases such as ammonia, sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate: organic bases such as ammonium compounds; It is not limited. One of these bases may be used alone, or two or more may be used in combination. Accordingly, examples of the basic water include aqueous solutions of these inorganic bases and / or organic bases. The pH of the basic water is not particularly limited, but is preferably 9 or more, more preferably 10 or more, and particularly preferably 11 or more. By washing the extract with basic water, components such as the phenol derivative can be separated and removed from the extract.
尚、 タキサン関連化合物は、 塩基で加水分解され易いエステルを分子 構造に有している。 従って、 一般に、 塩基で処理を行うと、 該エステル の加水分解が生じてタキサン関連化合物が破壊されてしまうと考えられ ている。 ところが、 本願発明者等が検討したところ、 タキサン関連化合 物は、 塩基、 特に強塩基で処理を行っても、 エステルの加水分解が生じ ないことが判明した。 それゆえ、 塩基で処理を行うことによって、 タキ サン関連化合物と、 フエノール誘導体等の成分とを分離することができ る。 The taxane-related compound has an ester which is easily hydrolyzed by a base in its molecular structure. Therefore, it is generally believed that treatment with a base will hydrolyze the ester and destroy the taxane-related compound. However, the inventors of the present application have examined that taxane-related compounds can undergo ester hydrolysis even when treated with a base, particularly a strong base. Turned out not to be. Therefore, by treating with a base, it is possible to separate a taxane-related compound from components such as a phenol derivative.
酸および または塩基で処理した後の抽出液は、 中性になるまで水洗 することがより好ましい。 また、 水洗後の抽出液は、 タキサン関連化合 物の単離が容易となるように、 濃縮する (有機溶媒を除去する) ことが より好ましい。 抽出液を濃縮することにより、 中性分画が得られる。 中性分画からタキサン関連化合物、 即ち、 前記化合物群に属する個々 の化合物 (6 ) 〜 (2 9 ) を単離 ·精製する具体的な方法は、 特に限定 されるものではないが、 液体クロマトグラフィーを採用する方法が好適 である。 該液体クロマトグラフィーとしては、 具体的には、 例えば、 シ リカゲルカラムクロマトグラフィー、 逆相または順相の高速液体クロマ トグラフィー (H P L C )、 遠心液液分配クロマトグラフィー (C P C ) 等が挙げられるが、 特に限定されるものではない。 上記の高速液体クロ マトグラフィ一は、 例えば、 移動相の p Hを考慮して、 逆相か順相かを 選択すればよい。  The extract after the treatment with an acid and / or a base is more preferably washed with water until neutral. It is more preferable that the extract after washing with water is concentrated (removing the organic solvent) so that the taxane-related compound can be easily isolated. By concentrating the extract, a neutral fraction is obtained. The specific method for isolating and purifying the taxane-related compounds from the neutral fraction, that is, the individual compounds (6) to (29) belonging to the compound group, is not particularly limited. A method employing lithography is preferred. Specific examples of the liquid chromatography include silica gel column chromatography, reversed-phase or normal-phase high-performance liquid chromatography (HPLC), and centrifugal liquid-liquid distribution chromatography (CPC). There is no particular limitation. In the above-described high performance liquid chromatography, for example, it is sufficient to select the reversed phase or the normal phase in consideration of the pH of the mobile phase.
固定相 (充填剤) としては、 具体的には、 例えば、 シリカゲル、 アル ミナ、 O D S (ォク夕デシルシリル) 系化合物等が挙げられるが、 特に 限定されるものではない。 上記例示の固定相のうち、 逆相高速液体クロ マトグラフィ一等を採用する場合には、 O D S系化合物がより好ましい。 移動相 (キャリア, 溶離液) として用いるのに好適な液体としては、 具体的には、 例えば、 メチルアルコール、 エチルアルコール、 クロロホ ルム、 n—へキサン、 酢酸ェチル、 各種エーテル、 トルエン、 ァセトニ トリル、 水等が挙げられるが、 特に限定されるものではない。 これら液 体は、一種類のみを用いてもよく、 また、二種類以上を併用してもよい。 上記例示の液体のうち、 シリカゲルカラムクロマトグラフィーや順相高 速液体クロマトグラフィー等を採用する場合には、 n—へキサン/酢酸 ェチル系の混合溶液がより好ましく、 逆相高速液体クロマトグラフィー 等を採用する場合には、 メチルアルコール アセトニトリル系の混合溶 液がより好ましく、 遠心液液分配クロマトグラフィー等を採用する場合 には、 n—へキサンノメチルアルコール系の混合溶液がより好ましい。 逆相高速液体クロマトグラフィーにおける上記液体の p H、 即ち、 移 動相の p Hは、 緩衝溶液によって酸性に調節されていることがより好ま しい。 つまり、 逆相 O D Sカラムを用いた単離 ·精製は、 酸性条件下で 行われることがより好ましい。 緩衝溶液は、 移動相の p Hを 5 . 5以下 に調節することができる溶液であればよく、 特に限定されるものではな レ 該緩衝溶液としては、 具体的には、 例えば、 酢酸アンモニゥム水溶 液等が挙げられる。 尚、 上記の液体と緩衝溶液との組み合わせは、 特に 限定されるものではない。 Specific examples of the stationary phase (filler) include, but are not particularly limited to, silica gel, alumina, and ODS (octyl decylsilyl) -based compounds. Among the stationary phases exemplified above, when employing reversed-phase high performance liquid chromatography or the like, ODS compounds are more preferable. Examples of liquids suitable for use as a mobile phase (carrier, eluent) include, for example, methyl alcohol, ethyl alcohol, chloroform, n-hexane, ethyl acetate, various ethers, toluene, acetonitrile, Although water is mentioned, it is not particularly limited. One of these liquids may be used alone, or two or more of them may be used in combination. Among the liquids exemplified above, silica gel column chromatography and normal phase When using high performance liquid chromatography, etc., a mixed solution of n-hexane / ethyl acetate is more preferable, and when using reversed phase high performance liquid chromatography, etc., a mixed solution of methyl alcohol acetonitrile is preferred. More preferably, when employing centrifugal liquid-liquid distribution chromatography or the like, a mixed solution of n-hexanenomethyl alcohol is more preferable. More preferably, the pH of the liquid in the reversed-phase high-performance liquid chromatography, that is, the pH of the mobile phase is adjusted to be acidic by a buffer solution. That is, the isolation and purification using the reversed-phase ODS column are more preferably performed under acidic conditions. The buffer solution is not particularly limited as long as the pH of the mobile phase can be adjusted to 5.5 or less. Examples of the buffer solution include, for example, aqueous ammonia acetate. Liquid and the like. The combination of the above liquid and buffer solution is not particularly limited.
逆相液体クロマトグラフィーにおける移動相の P Hを酸性に調節する ことにより、 該液体クロマトグラフィーの分離能がより向上するので、 タキサン関連化合物をより一層選択的に単離することができる。 移動相 の p Hが中性付近であると、 アル力ロイド誘導体の分離 ·除去が充分に 行われない場合がある。 また、 アルカロイド誘導体が有する窒素原子に 対してプロトン付加が可逆的に起こるので、 分離 ·除去すべき成分 (ピ —ク) が増加したり、 該成分 (ピーク) がブロードになって分離能が低 下したり、 保持時間が変化したりして、 成分 (ピーク) を特定 (解析) することができなくなる場合がある。  By adjusting the pH of the mobile phase in reverse phase liquid chromatography to acidic, the resolution of the liquid chromatography is further improved, so that the taxane-related compound can be more selectively isolated. If the pH of the mobile phase is near neutral, the separation / removal of the alkyloid derivative may not be performed sufficiently. In addition, since the protonation reversibly occurs on the nitrogen atom of the alkaloid derivative, the component (peak) to be separated / removed increases, or the component (peak) becomes broad and the separation ability is low. In some cases, the component (peak) cannot be specified (analyzed) due to a drop or a change in the retention time.
中性分画を液体クロマトグラフィーを採用して分離し、 タキサン関連 化合物を単離 ·精製する具体的な方法としては、 例えば、 先ず、 ①シリ 力ゲルフラッシュカラムを用いたシリカゲルカラムクロマトグラフィー や、 遠心液液分配クロマトグラフィーを採用して、 n—へキサン 酢酸 ェチル系の混合溶液 (溶離液) を用いて、 中性分画を分離して中程度の 極性を有する分画 (フラクション) を取り出し、 次に、 ②順相高速液体 クロマトグラフィーを採用して、 n—へキサン Z酢酸ェチル系の混合溶 液 (キャリア) を用いて、 該分画を分離し、 さらに、 ③逆相高速液体ク 口マトグラフィーを採用して、 メチルアルコールと 0 . 0 5 M酢酸アン モニゥム水溶液 (p H 4 . 8 ) とァセトニトリルとを容量比 1 : 2 : 2 で混合してなる混合溶液 (キャリア) を用いて、 上記②で得た分画をさ らに分離してタキサン関連化合物を含む分画を取り出す方法が挙げられ る。 上記の分離 ·精製操作を行うことにより、 タキサン関連化合物と、 アル力ロイド誘導体やフエノール誘導体等の成分とを分離することがで きるので、 タキサン関連化合物を精製することができる。 そして、 必要 に応じて、 上記② .③の分離 ·精製操作を繰り返して行うことにより、 タキサン関連化合物、 即ち、 前記化合物群に属する個々の化合物 (6 ) 〜 (2 9 ) の純度をさらに向上させることができる。 Separation of the neutral fraction using liquid chromatography and isolation and purification of taxane-related compounds include: (1) silica gel column chromatography using a silica gel flash column, Centrifugal liquid-liquid partition chromatography, n-hexane acetic acid Using a methyl-based mixed solution (eluent), the neutral fraction is separated and a fraction with a moderate polarity (fraction) is taken out. Next, (2) normal phase high performance liquid chromatography is used. The fraction was separated using a mixed solution (carrier) of n-hexane Z-ethyl acetate, and (3) methyl alcohol was added to 0.05 M by using reversed-phase high-performance liquid chromatography. Using a mixed solution (carrier) obtained by mixing an aqueous solution of ammonium acetate (pH 4.8) and acetonitrile at a volume ratio of 1: 2: 2, the fraction obtained in (1) above was further separated. A method of extracting a fraction containing a taxane-related compound may be mentioned. By performing the above separation / purification operations, the taxane-related compound can be separated from the components such as the alkyloid derivative and the phenol derivative, so that the taxane-related compound can be purified. If necessary, the separation / purification operation of the above ①.③ is repeated to further improve the purity of the taxane-related compounds, ie, the individual compounds (6) to (29) belonging to the compound group. Can be done.
得られたタキサン関連化合物を分析する際には、 上記液体クロマトグ ラフィーを採用して、 上記単離 ·精製条件と同様の条件で分析操作を行 えばよい。つまり、分析は、酸性条件下で行われることがより好ましい。 移動相の p Hを酸性に調節することにより、 液体クロマトグラフィーの 分離能がより向上するので、 タキサン関連化合物をより一層正確に分析 することができる。 移動相の p Hが中性付近であると、 分離,除去すベ き成分 (ピーク) が増加したり、 該成分 (ピーク) がブロードになって 分離能が低下したり、 保持時間が変化したりして、 成分 (ピーク) を特 定 (解析) することができなくなる場合がある。  When the obtained taxane-related compound is analyzed, the above-described liquid chromatography may be employed, and the analysis operation may be performed under the same conditions as the above-mentioned isolation and purification conditions. That is, the analysis is more preferably performed under acidic conditions. By adjusting the pH of the mobile phase to be acidic, the resolution of liquid chromatography is further improved, so that taxane-related compounds can be more accurately analyzed. If the pH of the mobile phase is near neutral, the number of components (peaks) to be separated and removed increases, or the components (peaks) become broad and the separation ability decreases, and the retention time changes. May not be able to identify (analyze) the component (peak).
上記抽出等の一連の取り出し操作を行うことにより、 前記化合物 (6 ) 〜 (2 9 ) が得られる。 前記化合物 (6 ) 〜 (2 9 ) は何れも、 複数の 抗癌剤に対する耐性を獲得した癌細胞 (多剤耐性癌細胞) に対する多剤 耐性癌克服作用を有している。 それゆえ、 前記化学式 (6) 〜 (29) で表される化合物群より選ばれる少なくとも一種の化合物を含む多剤耐 性癌克服剤は、 該癌細胞に好適に投与することができる。 The compounds (6) to (29) are obtained by performing a series of extraction operations such as the above extraction. Each of the compounds (6) to (29) is a multidrug against cancer cells (multidrug-resistant cancer cells) that have acquired resistance to a plurality of anticancer drugs. Has the effect of overcoming resistant cancer. Therefore, the multidrug-resistant cancer-overcoming agent comprising at least one compound selected from the group of compounds represented by the chemical formulas (6) to (29) can be suitably administered to the cancer cells.
該化合物 (6) 〜 (29) は、 前記説明 (針葉部からの取り出し方法 の説明) において述べたように、 酸または塩基に対して安定であり、 そ れゆえ、 例えば生体内で分解 (胃液による分解、 胆液による分解等) さ れるおそれが少ない。 従って、 化合物 (6) 〜 (29) より選ばれる少 なくとも一種の化合物を含んでなる本発明にかかる多剤耐性癌克服剤は、 例えば、 その投与方法や投与場所等の制限が少なく、 広範囲な適用が可 能であると期待される。  The compounds (6) to (29) are stable to acids or bases as described in the above description (the description of the method for removing from needles), and are therefore decomposed in vivo, for example. Degradation by gastric juice, decomposition by bile, etc.) is low. Therefore, the multidrug-resistant cancer-overcoming agent according to the present invention comprising at least one compound selected from compounds (6) to (29) has, for example, a small restriction on its administration method and administration place, and Application is expected to be possible.
特に、 化合物 (1 1), (1 5) 〜 ( 1 7), (22), (24), (26) は、 ベラパミルを上回る多剤耐性癌克服作用を有する高活性化合物であ ることから、 それ単独で特に有効な (新規な) 多剤耐性癌克服剤となり 得る。  In particular, compounds (11), (15) to (17), (22), (24), and (26) are highly active compounds that have a multidrug-resistant cancer-surpassing action superior to verapamil. By itself, it can be a particularly effective (new) drug for overcoming multidrug-resistant cancer.
また、 前記化合物 (6) 〜 (29) は、 日本ィチイの針葉部から取り 出すことができる。 即ち、 本発明にかかる製造方法によれば、 毎年再生 される針葉部を使用するので、 環境を保全しながら、 日本ィチイから前 記化合物 (6) 〜 (29) を製造することができる。 これにより、 癌細 胞に投与するのに好適な多剤耐性癌克服剤を、 環境を保全しながら製造 することができる。  Further, the compounds (6) to (29) can be taken out from the needle portion of the yew tree. That is, according to the production method of the present invention, the needles regenerated every year are used, so that the compounds (6) to (29) can be produced from Nippon yew while preserving the environment. Thus, a multidrug-resistant cancer-overcoming agent suitable for administration to cancer cells can be produced while preserving the environment.
尚、 本発明にかかる多剤耐性癌克服剤は、 必要に応じて、 例えば、 癌 細胞の特定部位 (または P—糖蛋白質) と特異的に結合可能な抗体や、 抗癌剤等をさらに含んで構成されていてもよい。 これにより、 標的とす べき (多剤耐性) 癌細胞への多剤耐性癌克服剤の供給を確実にする効果 や、 該癌細胞への抗癌剤の同時供給が可能となる効果等を実現すること ができる。 これら多剤耐性癌克服剤の投与方法は、 特に限定されるもの ではなく、 多剤耐性癌克服剤に含まれる物質 (上記化合物 (6 ) 〜 (2 9 ) や、 抗体、 抗癌剤等を指す) の物性等に応じた最適なドラッグデリ バリ一システムを採用すればよい。 The multidrug-resistant cancer-overcoming agent according to the present invention may further comprise, as necessary, an antibody capable of specifically binding to a specific site (or P-glycoprotein) of a cancer cell, an anticancer agent, and the like. It may be. As a result, it is possible to achieve an effect of reliably supplying a multidrug-resistant cancer-overcoming agent to cancer cells to be targeted (multidrug resistance), an effect of enabling simultaneous supply of an anticancer agent to the cancer cells, and the like. Can be. The administration methods of these multidrug-resistant cancer-surviving agents are particularly limited. Instead, if an optimal drug delivery system is adopted, it should be based on the properties of the substances contained in the multidrug-resistant cancer-overcoming agent (refer to the compounds (6) to (29), antibodies, anticancer agents, etc.) Good.
上記の化学式 (3 0 ) 〜 (3 9 ) で表される化合物は、 それぞれ単独 で、 少なくとも P—糖蛋白質による抗癌剤の能動輸送の阻害剤 (以下、 P—糖蛋白質阻害剤と称する) としての機能を有し、 本発明にかかる多 剤耐性癌克服剤となりうる。また、上記の化合物のうち、化学式(3 1 ), The compounds represented by the above chemical formulas (30) to (39) are each independently used as an inhibitor of at least the active transport of an anticancer agent by P-glycoprotein (hereinafter referred to as a P-glycoprotein inhibitor). It has a function and can be a multidrug-resistant cancer overcoming agent according to the present invention. In addition, among the above compounds, the chemical formula (31),
( 3 3 ), ( 3 6 ) で表される化合物は、 従来の多剤耐性癌克服剤である ベラパミルと比較して多剤耐性癌克服作用が著しく高く、 以下、 高活性 化合物と称する。 本発明にかかる多剤耐性癌克服剤は、 これらの高活性 化合物の少なくとも一つを含んでいることがより好ましい。 The compounds represented by (33) and (36) have remarkably higher action of overcoming multidrug-resistant cancer than verapamil which is a conventional multidrug-resistant cancer-overcoming agent, and are hereinafter referred to as highly active compounds. More preferably, the multidrug-resistant cancer-overcoming agent according to the present invention contains at least one of these highly active compounds.
上記の化合物が多剤耐性癌克服作用を有することは、 本願発明者が新 たに見出したことである。 また、 上記の化学式 (3 6 ) で表されるタキ シニン化合物は、 本発明にかかる新規な夕キシニン化合物であり、 上記 の化学式 (3 7 ) 〜 (3 9 ) で表されるアビェ夕ン化合物はいずれも、 本発明にかかる新規なアビエタン化合物である。 尚、 以下、 必要に応じ て、 上記化学式 (3 0 ) 〜 (3 9 ) で表される化合物を順に、 化合物 (3 0 ) 〜 (3 9 ) と称する。  The fact that the above-mentioned compound has a multidrug-resistant cancer overcoming effect is newly found by the present inventors. The taxinin compound represented by the chemical formula (36) is a novel xinine compound according to the present invention, and the abyssinine compound represented by the chemical formulas (37) to (39). Are all novel aviethane compounds according to the present invention. Hereinafter, the compounds represented by the chemical formulas (30) to (39) will be referred to as compounds (30) to (39) as needed.
尚、 本発明にかかる多剤耐性癌克服剤は必要に応じて、 例えば、 癌細 胞の特定部位 (または P—糖蛋白質) と特異的に結合可能な抗体や、 抗 癌剤等をさらに含んで構成されていてもよい。 これにより、 標的とすべ き(多剤耐性)癌細胞への多剤耐性癌克服剤の供給を確実とする効果や、 該癌細胞への抗癌剤の同時供給が可能となる効果等を実現することがで きる。 これら多剤耐性癌克服剤の投与方法は特に限定されるものではな く、 多剤耐性癌克服剤に含まれる物質 (上記化合物 (3 0 ) 〜 (3 9 ) や、 抗体、 抗癌剤等を指す) の物性等に応じた最適なドラッグデリバリ —システムを採用すればよい。 The multidrug-resistant cancer-overcoming agent according to the present invention further includes, as necessary, for example, an antibody capable of specifically binding to a specific site (or P-glycoprotein) of a cancer cell, an anticancer agent, and the like. May be configured. As a result, an effect of reliably supplying a multidrug-resistant cancer-overcoming agent to cancer cells to be targeted (multidrug-resistant) and an effect of enabling simultaneous supply of an anticancer agent to the cancer cells can be realized. I can do it. The administration method of these multidrug-resistant cancer-overcoming agents is not particularly limited, and includes the substances contained in the multidrug-resistant cancer-overcoming agents (compounds (30) to (39), antibodies, anticancer agents, etc. Optimal drug delivery according to the physical properties of —You can adopt a system.
本発明にかかる多剤耐性癌克服剤の製造方法は、 上記説明の多剤耐性 癌克服剤に含まれる化合物 (3 0 ) 〜 (3 9 ) の少なくとも一つ (以下、 単に化合物と称し、 特定の化合物を指す場合のみ (3 0 ) 〜 (3 9 ) の 番号を付すものとする)を、日本イチィ i Taxus cuspidata Sie . et Zucc.) の組織より誘導されるカルスから取り出す工程を含む製造方法である。 本発明において 「日本ィチイの組織」 とは、 日本ィチイに由来する組 織、 より具体的には、 植物体 (根部、 茎部、 葉部を成す各組織、 すなわ ち栄養器官全体を指す)、 並びに、 その花、 花粉、 及び果実 (生殖器官) を成す組織のことを指す。 尚、 ここでいう 「葉部」 とは、 「葉身」 と 「葉 柄」 とから構成される単位を指し、 また、 「果実」 とは、 「仮種衣 (ァリ ル) J および 「種子」 を含む概念である。 これら日本ィチイの組織の中で も、 特にその 「針葉部」 は、 A ) 培養細胞の増殖性、 即ち、 カルスの誘 導性に特に優れている点、及び、 B )組織の採取が比較的容易である上、 植物体に大きなダメージを与えることがない点等で、 より好適である。 上記 「針葉部」 とは、 「葉身」、 「葉柄」、 並びに、 「葉部」 が複数枚付い た 「小枝」、 さらには、 「新芽 (その原基であっても良い)」 ゃ該 「新芽」 が付いた 「若茎」 も含まれるものとする。 これら 「針葉部」 のうち、 特 に、 秋季並びに冬季に採取した 「若茎」 (直径 2 mm〜 3 mm程度の緑色 若茎部) が、 カルスの誘導性に特に優れると共に、 何代にもわたる継代 培養を行っても、上記化合物の生産量が低下しないので、特に好ましい。 また、 シュートや芽生え等の若い組織も、 カルス誘導のための組織とし て好適に使用することができる。  The method for producing a multidrug-resistant cancer-overcoming agent according to the present invention includes a method for preparing at least one of the compounds (30) to (39) contained in the multidrug-resistant cancer-overcoming agent described above (hereinafter simply referred to as a compound, (3) to (3 9) only when the compound refers to the compound of (1) Taxus cuspidata Sie. Et Zucc.) It is. In the present invention, the “tissue of Japanese yew” refers to a tissue derived from Japanese yew, more specifically, a plant body (each of the tissues forming the root, stem, and leaf, that is, the entire vegetative organ). And the tissues that make up the flowers, pollen, and fruits (reproductive organs). The term “leaf” as used herein refers to a unit composed of “leaf” and “stalk”, and “fruit” refers to “temporary seed garments (aryl) J and“ It is a concept that includes "seed." Among these Japanese yew tissues, in particular, the “needle part” is compared in terms of A) the proliferative ability of cultured cells, that is, callus induction, and B) the collection of tissues. It is more suitable because it is easy to target and does not cause significant damage to the plant. The above “needle part” is “leaf”, “petiole”, “twig” with a plurality of “leaves”, and “sprout (or its primordium)”. The term "young shoot" with the "sprout" is also included. Among these “needle parts”, “young shoots” (green young shoots with a diameter of about 2 mm to 3 mm) collected in the autumn and winter are particularly excellent in callus inducibility, and in some generations. It is particularly preferable to carry out subculture over a long period, since the production amount of the compound does not decrease. Also, young tissues such as shoots and seedlings can be suitably used as tissues for callus induction.
カルス誘導用の 「日本ィチイの組織」 (以下、 場合によっては外植体と 称する) の採取時期は特に限定されるものではないが、 上記組織を植物 体から得る場合には、 採取前 1力月間の平均気温が 1 8 X:以下、 より好 ましく 5で以下である環境下で生育された後に採取されることが好まし い。 尚、 この環境は、 人工的に作られるものであってもよい。 There is no particular limitation on the timing of the callus-inducing “Japanese yew tissue” (hereinafter sometimes referred to as an explant), but if the above tissue is obtained from a plant, it must be collected before harvesting. Average monthly temperature of 18 X: below, better Preferably, they are collected after being grown in an environment that is less than or equal to 5. This environment may be artificially created.
外植体からのカルス誘導およびその培養は、 特別な添加剤 (添加物) や培養条件を用いることなく、 一般的な手法で以て、 例えば、 寒天培地 等の市販の植物培養培地 (固体培地) とオーキシンとを用いることによ つて、 容易に実施することができる。 また、 場合によっては、 ォーキシ ンを含んでなる液体培地を用いて振盪培養を行ってもよい。 このように、 外植体から容易にカルスを誘導することができるので、 簡便な設備で以 て大量培養することができ、 該カルスから上記化合物を大量に取り出す ことができる。 カルスの形成方法としては、 具体的には、 例えば、 外植 体の切片を 7 0重量%エチルアルコール水溶液等で滅菌処理した後、 上 記の切片を、 寒天粉末を含み、 さらにオーキシンを所定の濃度で含む改 変ガンボーグ培地等の固体培地に置床し、 次いで、 2 5で〜 2 7で程度 の暗所に静置して培養 (静置培養) する方法が挙げられるが、 特に限定 されるものではない。  Induction of callus from explants and their cultivation can be performed by a general method without using special additives (additives) or culturing conditions. For example, commercially available plant culture media such as agar media (solid media) ) And auxin can be easily implemented. In some cases, shaking culture may be performed using a liquid medium containing auxin. As described above, calli can be easily induced from the explant, so that large-scale culture can be performed with simple equipment, and the compound can be extracted from the callus in a large amount. As a method for forming callus, specifically, for example, a section of an explant is sterilized with a 70% by weight aqueous solution of ethyl alcohol and the like, and then the above section contains agar powder and further contains auxin in a predetermined manner. In this method, the cells are placed on a solid medium such as a modified Gamborg's medium containing the same concentration, and then cultured in a dark place at 25 to 27 (static culture). Not something.
そして、 培養細胞は、 上記固体培地を用いた簡便な方法で以て、 例え ば 3 0日〜 5 0日毎、 より好ましくは 4 0日毎に増殖性に優れている力 ルスを選抜、 継代培養することによって、 該カルスを再現性良く、 大量 培養することができる。 すなわち、 上記カルスは、 大量培養可能である とともに上記化合物を比較的多量に含んでいるため、 該化合物を効率的 に製造することができる。  Then, the cultured cells are selected by a simple method using the above solid medium, for example, every 30 days to 50 days, more preferably every 40 days. By doing so, the callus can be cultured in large quantities with good reproducibility. That is, since the callus can be cultured in a large amount and contains the compound in a relatively large amount, the compound can be efficiently produced.
上記のオーキシンとしては、具体的には、例えば、 1—ナフチル酢酸、 2 —ナフチル酢酸 (N A A;別名、 ナフタレン酢酸)、 2 , 4—ジクロロ フエノキシ酢酸、 インドール酢酸、 4—クロローインドール酢酸 (4-C1 IAA)、インドール酪酸等が挙げられるが、特に限定されるものではない。 上記例示のオーキシンのうち、 1—ナフチル酢酸、 2—ナフチル酢酸、 2, 4ージクロロフエノキシ酢酸、 および 4—クロ口インドール酢酸が より好ましい。固体培地におけるオーキシンの含有量は、 0を越えて 1. Omg/L以下が好ましく、 0. SmgZL程度がより好ましい。 ォー キシンの含有量が 1. OmgZLを越えると、 継代培養を繰り返すにつ れて、 カルスの増殖性が次第に低下する場合がある。 4一クロ口インド ール酢酸を含む固体培地は、 培養細胞における、 2次代謝物である上記 化合物の生産性をより向上させることができる。 Specific examples of the auxin include 1-naphthylacetic acid, 2-naphthylacetic acid (NAA; also known as naphthaleneacetic acid), 2,4-dichlorophenoxyacetic acid, indoleacetic acid, and 4-chloro-indoleacetic acid (4 -C1 IAA), indolebutyric acid and the like, but are not particularly limited. Among the auxins exemplified above, 1-naphthylacetic acid, 2-naphthylacetic acid, 2,4-Dichlorophenoxyacetic acid, and 4-chloroindoleacetic acid are more preferred. The auxin content in the solid medium is preferably more than 0 and not more than 1. Omg / L, more preferably about 0. SmgZL. If the auxin content exceeds 1. OmgZL, callus growth may gradually decrease as the subculture is repeated. (4) The solid medium containing one-dose indole acetic acid can further improve the productivity of the compound as a secondary metabolite in cultured cells.
固体培地は、 必要に応じて、 サイトカイニン、 ポリ (N—ビニル— 2 —ピロリドン) 等の褐変防止剤;ジャスモン酸メチル等のエリシタ ;ォ リゴサッカライド ;各種ビタミン;等の添加剤をさらに含んでいてもよ い。 ジャスモン酸メチルを含む固体培地は、 培養細胞における、 上記化 合物の生産性をより向上させることができる。 固体培地における添加剤 の含有量は、 該添加剤の種類や組み合わせに応じて設定すればよい。 上記オリゴサッカライドとしては、 具体的には、 例えば、 食用のォク ラの果実 (またはその植物体) から抽出 ·精製された粘性多糖を酸で加 水分解してなる、 2糖〜 5糖のオリゴ糖の混合物 (以下、 KTOSと略 記す) が挙げられる。 該オリゴ糖は、 例えば、 ラムノース、 ガラクト一 ス、 ガラクッロン酸、 グルコース等の単糖で構成されている。 上記 KT OSは、 継代による培養細胞の老化を抑制する作用を備えている。 この ため、 KTOSを含む固体培地を用いることにより、 老化を抑制しなが ら継代培養を繰り返すことができるので、 培養細胞を増殖させ続けるこ とができる。 従って、 KTOSを含む固体培地は、 培養細胞に対して、 上記化合物の生産性をより向上させることができる。  The solid medium further contains additives such as a browning inhibitor such as cytokinin and poly (N-vinyl-2-pyrrolidone); an elicita such as methyl jasmonate; an oligosaccharide; and various vitamins. It is good. The solid medium containing methyl jasmonate can further improve the productivity of the compound in cultured cells. The content of the additive in the solid medium may be set according to the type and combination of the additive. Specific examples of the oligosaccharides include disaccharides to pentasaccharides obtained by hydrolyzing viscous polysaccharides extracted and purified from edible okra fruits (or their plants) with an acid. A mixture of oligosaccharides (hereinafter abbreviated as KTOS) is mentioned. The oligosaccharide is composed of, for example, monosaccharides such as rhamnose, galactose, galacturonic acid, and glucose. The above-mentioned KT OS has an action of suppressing aging of cultured cells due to passage. For this reason, by using a solid medium containing KTOS, the subculture can be repeated while suppressing senescence, so that the cultured cells can be kept growing. Therefore, the solid medium containing KTOS can further improve the productivity of the compound with respect to cultured cells.
そして、 固体培地 (または液体培地) は、 4—クロ口インドール酢酸 および Zまたはオリゴサッカライドを含むと共に、 必要に応じて、 1一 ナフチル酢酸および κまたは 2—ナフチル酢酸をさらに含んでいること が、 特に好ましい。 The solid medium (or liquid medium) contains 4-cloth indoleacetic acid and Z or oligosaccharide, and further contains, if necessary, 1-naphthylacetic acid and κ or 2-naphthylacetic acid. Is particularly preferred.
カルスから上記化合物を取り出す具体的な方法は、 特に限定されるも のではないが、 抽出を行う方法が好適であり、 有機溶媒を用いて抽出を 行う方法が最適である。 抽出を行う具体的な方法としては、 例えば、 力 ルスを凍結乾燥等によって乾燥させ、 必要に応じて粉砕した後、 有機溶 媒に数分間から数時間浸漬する方法が挙げられる。 抽出条件は、 特に限 定されるものではないが、 抽出温度は 30で以下であることがより好ま しい。 そして、 得られた抽出液を、 必要に応じて酸および または塩基 で処理し、次いで液体クロマトグラフィーによって分画することにより、 上記化合物を単離 ·精製することができる。  The specific method of extracting the above compound from the callus is not particularly limited, but a method of performing extraction is preferable, and a method of performing extraction using an organic solvent is most suitable. As a specific method for performing the extraction, for example, a method in which cocoa is dried by freeze-drying or the like, crushed if necessary, and then immersed in an organic solvent for several minutes to several hours can be mentioned. The extraction conditions are not particularly limited, but the extraction temperature is preferably 30 or less. Then, the above compound can be isolated and purified by treating the obtained extract with an acid and / or a base, if necessary, and then fractionating by liquid chromatography.
さらに具体的には、 上記化合物 (30) 〜 (36) は、 アビェ夕ン化 合物である化合物 (37) 〜 (39) と比較して極性が低いので、 乾燥 させたカルス (以下、 乾燥カルスと記す) を、 必要に応じて粉砕した後、 n—へキサン等の低極性の有機溶媒に浸潰して抽出し、 次いで、 得られ た抽出液をシリカゲルを用いたカラムクロマトグラフィーで粗分けした 後、 順相高速液体クロマトグラフィー (HPLC) を用いて単離するこ とができる。  More specifically, since the compounds (30) to (36) have a lower polarity than the compounds (37) to (39) which are abyssin compounds, the dried callus (hereinafter, dried) Callus) is crushed as necessary, immersed in a low-polar organic solvent such as n-hexane and extracted, and then the resulting extract is roughly separated by column chromatography using silica gel. After that, it can be isolated using normal phase high performance liquid chromatography (HPLC).
一方、 上記化合物 (37) 〜 (39) は、 化合物 (30) 〜 (36) と比較して極性が高いので、 該化合物 (30) 〜 (36) の抽出が終了 した後の乾燥カルスを、 酢酸ェチルやクロ口ホルム、 エーテル等の高極 性の有機溶媒に浸潰して抽出し、 次いで、 得られた抽出液を酸,塩基処 理することによって共存アル力ロイドゃフエノール類を除去し、 シリカ ゲルを用いたカラムクロマトグラフィーで粗分けした後、 順相高速液体 クロマトグラフィー (HPLC) を用いて単離することができる。  On the other hand, since the compounds (37) to (39) have higher polarity than the compounds (30) to (36), the dried callus after the extraction of the compounds (30) to (36) is The extract is immersed in a highly polar organic solvent such as ethyl acetate, black form, ether, etc. for extraction, and then the resulting extract is treated with an acid and a base to remove coexisting alloid phenols. After being roughly separated by column chromatography using silica gel, it can be isolated using normal phase high performance liquid chromatography (HPLC).
以下、 カルスから上記化合物を取り出す方法について、 詳述する。 上 記の有機溶媒としては、 具体的には、 例えば、 メチルアルコール、 ェチ ルアルコール、イソプロピルアルコール、 クロ口ホルム、 n—へキサン、 酢酸ェチル、 各種エーテル、 トルエン等が挙げられるが、 特に限定され るものではない。 これら有機溶媒は、一種類のみを用いてもよく、 また、 二種類以上を併用してもよい。 上記例示の有機溶媒のうち、 メチルアル コール、 n—へキサン、 および酢酸ェチルがより好ましい。 Hereinafter, a method for extracting the above compound from callus will be described in detail. Specific examples of the above-mentioned organic solvent include, for example, methyl alcohol and ethyl alcohol. Alcohol, isopropyl alcohol, chloroform, n-hexane, ethyl acetate, various ethers, toluene, and the like, but are not particularly limited. One of these organic solvents may be used alone, or two or more thereof may be used in combination. Among the organic solvents exemplified above, methyl alcohol, n-hexane, and ethyl acetate are more preferred.
上記化合物が抽出された抽出液は、 必要に応じて、 不純物として含ま れるアル力ロイド誘導体等の塩基性成分や、 フエノール誘導体等の酸性 成分を分離 ·除去するために、 酸および Zまたは塩基で処理することが より好ましい。抽出液を酸および Zまたは塩基で処理する方法としては、 具体的には、 抽出液を酸性水および Zまたは塩基性水で洗浄する方法が 挙げられる。 酸および または塩基で処理した後の抽出液は、 中性にな るまで水洗することがより好ましい。  The extract from which the above compounds have been extracted may be treated with an acid and Z or a base, if necessary, to separate and remove basic components such as alloid derivatives and acidic components such as phenol derivatives contained as impurities. Processing is more preferred. As a method of treating the extract with an acid and Z or a base, specifically, a method of washing the extract with acidic water and Z or a basic water can be mentioned. The extract after the treatment with an acid and / or a base is more preferably washed with water until neutral.
上記の酸としては、 具体的には、 例えば、 塩酸、 硫酸、 リン酸等の無 機酸;ギ酸、 酢酸等の有機酸;が挙げられるが、 特に限定されるもので はない。 これら酸は、 一種類のみを用いてもよく、 また、 二種類以上を 併用してもよい。 従って、 酸性水としては、 これら無機酸および Zまた は有機酸の水溶液が挙げられる。 該酸性水の P Hは、 特に限定されるも のではないが、 5以下がより好ましく、 4以下がさらに好ましい。 抽出 液を酸性水で洗浄することによって、 該抽出液から、 アルカロイド誘導 体等の塩基性成分を分離 ·除去することができる。  Specific examples of the above-mentioned acids include, for example, inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid; and organic acids such as formic acid and acetic acid, but are not particularly limited. These acids may be used alone or in combination of two or more. Accordingly, the acidic water includes aqueous solutions of these inorganic acids and Z or organic acids. The pH of the acidic water is not particularly limited, but is preferably 5 or less, and more preferably 4 or less. By washing the extract with acidic water, basic components such as alkaloid derivatives can be separated and removed from the extract.
また、 上記の塩基としては、 具体的には、 例えば、 アンモニア、 水酸 化ナトリウム、 水酸化カリウム、 炭酸ナトリウム、 炭酸カリウム等の無 機塩基; アンモニゥム化合物等の有機塩基;が挙げられるが、 特に限定 されるものではない。 これら塩基は、一種類のみを用いてもよく、 また、 二種類以上を併用してもよい。 従って、 塩基性水としては、 これら無機 塩基および または有機塩基の水溶液が挙げられる。 該塩基性水の p H は、 特に限定されるものではないが、 9以上がより好ましく、 1 0以上 がさらに好ましく、 1 1以上が特に好ましい。 抽出液を塩基性水で洗浄 することによって、 該抽出液から、 フエノール誘導体等の酸性成分を分 離 -除去することができる。 尚、 上記化合物 (3 0 ) 〜 (3 9 ) はいず れも、 酸または塩基に対して安定であることが確認されている。 Specific examples of the above base include: inorganic bases such as ammonia, sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate; and organic bases such as ammonium compounds. It is not limited. One of these bases may be used alone, or two or more may be used in combination. Accordingly, examples of the basic water include aqueous solutions of these inorganic bases and / or organic bases. PH of the basic water Is not particularly limited, but is preferably 9 or more, more preferably 10 or more, and particularly preferably 11 or more. By washing the extract with basic water, acidic components such as phenol derivatives can be separated and removed from the extract. It has been confirmed that all of the above compounds (30) to (39) are stable to acids or bases.
上記化合物が抽出された抽出液は、 該化合物の単離が容易となるよう に、 濃縮する (有機溶媒を除去する) ことがより好ましい。 抽出液を濃 縮することにより、 中性分画が得られる。  The extract from which the compound has been extracted is more preferably concentrated (to remove the organic solvent) so that the compound can be easily isolated. By concentrating the extract, a neutral fraction is obtained.
中性分画から上記化合物を単離 ·精製する具体的な方法は、 特に限定 されるものではないが、 液体クロマトグラフィーを採用する方法が好適 である。 該液体クロマトグラフィーとしては、 具体的には、 例えば、 シ リカゲルカラムクロマトグラフィー、 逆相または順相の高速液体クロマ トグラフィー、 遠心液液分配クロマトグラフィー (C P C ) 等が挙げら れるが、 特に限定されるものではない。 上記の高速液体クロマトグラフ ィ一は、 例えば、 移動相の p Hを考慮して、 逆相か順相かを選択すれば よい。  The specific method for isolating and purifying the above compound from the neutral fraction is not particularly limited, but a method employing liquid chromatography is preferred. Specific examples of the liquid chromatography include silica gel column chromatography, reversed-phase or normal-phase high-performance liquid chromatography, and centrifugal liquid-liquid distribution chromatography (CPC). It is not something to be done. In the above-described high performance liquid chromatography, for example, the reversed phase or the normal phase may be selected in consideration of the pH of the mobile phase.
固定相 (充填剤) としては、 具体的には、 例えば、 シリカゲル、 アル ミナ、 O D S (ォク夕デシルシリル) 系化合物等が挙げられるが、 特に 限定されるものではない。 上記例示の固定相のうち、 逆相高速液体クロ マトグラフィ一等を採用する場合には、 O D S系化合物がより好ましい。 移動相 (キャリア, 溶離液) として用いるのに好適な液体としては、 具体的には、 例えば、 メチルアルコール、 エチルアルコール、 クロロホ ルム、 n—へキサン、 酢酸工チル、 各種エーテル、 トルエン、 ァセトニ トリル、 水等が挙げられるが、 特に限定されるものではない。 これら液 体は、一種類のみを用いてもよく、 また、二種類以上を併用してもよい。 上記例示の液体のうち、 シリカゲルカラムクロマトグラフィーや順相高 速液体クロマトグラフィー等を採用する場合には、 n—へキサン 酢酸 ェチル系の混合溶液がより好ましく、 逆相高速液体クロマトグラフィ一 等を採用する場合には、 メチルアルコール/ァセトニトリル系の混合溶 液がより好ましく、 遠心液液分配クロマトグラフィ一等を採用する場合 には、 n—へキサン/メチルアルコール系の混合溶液がより好ましい。 逆相高速液体クロマトグラフィーにおける上記液体の p H、 即ち、 移 動相の p Hは、 緩衝溶液によって酸性に調節されていることがより好ま しい。 つまり、 逆相 O D Sカラムを用いた単離 ·精製は、 酸性条件下で 行われることがより好ましい。 緩衝溶液は、 移動相の p Hを 5 . 5以下 に調節することができる溶液であればよく、 特に限定されるものではな レ 。 該緩衝溶液としては、 具体的には、 例えば、 酢酸アンモニゥム水溶 液等が挙げられる。 尚、 上記の液体と緩衝溶液との組み合わせは、 特に 限定されるものではない。 Specific examples of the stationary phase (filler) include, but are not particularly limited to, silica gel, alumina, and ODS (octyl decylsilyl) -based compounds. Among the stationary phases exemplified above, when employing reversed-phase high performance liquid chromatography or the like, ODS compounds are more preferable. Examples of liquids suitable for use as a mobile phase (carrier, eluent) include, for example, methyl alcohol, ethyl alcohol, chloroform, n-hexane, ethyl acetate, various ethers, toluene, acetonitrile , Water and the like, but are not particularly limited. One of these liquids may be used alone, or two or more of them may be used in combination. Among the liquids exemplified above, silica gel column chromatography and normal phase When using high performance liquid chromatography, etc., a mixed solution of n-hexane and ethyl acetate is more preferable, and when using reverse phase high performance liquid chromatography, etc., a mixed solution of methyl alcohol / acetonitrile is preferable. In the case where centrifugal liquid-liquid distribution chromatography or the like is employed, a mixed solution of n-hexane / methyl alcohol is more preferable. More preferably, the pH of the liquid in the reversed-phase high-performance liquid chromatography, that is, the pH of the mobile phase is adjusted to be acidic by a buffer solution. That is, the isolation and purification using the reversed-phase ODS column are more preferably performed under acidic conditions. The buffer solution may be any solution that can adjust the pH of the mobile phase to 5.5 or less, and is not particularly limited. Specific examples of the buffer solution include an aqueous solution of ammonium acetate. The combination of the above liquid and buffer solution is not particularly limited.
逆相液体クロマトグラフィーにおける移動相の P Hを酸性に調節する ことにより、 該液体クロマトグラフィーの分離能がより向上するので、 上記化合物をより一層選択的に単離することができる。 移動相の p Hが 中性付近であると、 アル力ロイド誘導体の分離 ·除去が充分に行われな い場合がある。 また、 アルカロイド誘導体が有する窒素原子に対してプ 口トン付加が可逆的に起こるので、 分離 ·除去すべき成分 (ピーク) が 増加したり、該成分(ピーク)がブロードになって分離能が低下したり、 保持時間が変化したりして、 成分 (ピーク) を特定 (解析) することが できなくなる場合がある。  By adjusting the pH of the mobile phase in reverse phase liquid chromatography to be acidic, the resolution of the liquid chromatography is further improved, so that the above compound can be more selectively isolated. If the pH of the mobile phase is near neutral, the separation / removal of the alkyloid derivative may not be performed sufficiently. In addition, since the addition of a proton occurs reversibly on the nitrogen atom of the alkaloid derivative, the number of components (peaks) to be separated / removed increases, or the components (peaks) become broad and the separation ability decreases. In some cases, the component (peak) cannot be specified (analyzed) due to the change in the retention time.
中性分画を液体クロマトグラフィーを採用して分離し、 上記化合物を 単離 ·精製する具体的な方法としては、 例えば、 先ず、 ①シリカゲルフ ラッシュカラムを用いたシリカゲルカラムクロマトグラフィーや、 遠心 液液分配クロマトグラフィーを採用して、 n—へキサンノ酢酸ェチル系 の混合溶液 (溶離液) を用いて、 中性分画を分離して中程度の極性を有 する分画 (フラクション) を取り出し、 次に、 ②順相高速液体クロマト グラフィ一を採用して、 n—へキサン Z酢酸ェチル系の混合溶液 (キヤ リア) を用いて、 該分画を分離し、 さらに、 ③逆相高速液体クロマ.トグ ラフィーを採用して、 メチルアルコールと 0 . 0 5 M酢酸アンモニゥム 水溶液 (P H 4 . 8 ) とァセトニトリルとを容量比 1 : 2 : 2で混合し てなる混合溶液 (キャリア) を用いて、 上記②で得た分画をさらに分離 して上記化合物を含む分画を取り出す方法が挙げられる。 上記の分離 · 精製操作を行うことにより、 上記化合物と、 アルカロイド誘導体等の塩 基性成分やフェノール誘導体等の酸性成分とを分離することができるの で、 上記化合物を精製することができる。 そして、 必要に応じて、 上記 ② ·③の分離 ·精製操作を繰り返して行うことにより、 上記化合物の純 度をさらに向上させることができる。 つまり、 上記の分離 ·精製操作を 行うことにより、 上記化合物を効率的に抽出 ·分離することができる。 得られた上記化合物を分析する際には、 例えば、 上記液体クロマトグ ラフィーを採用して、 上記単離 ·精製条件と同様の条件で分析操作を行 えばよい。つまり、分析は、酸性条件下で行われることがより好ましい。 移動相の p Hを酸性に調節することにより、 液体クロマトグラフィーの 分離能がより向上するので、 上記化合物をより一層正確に分析すること ができる。移動相の P Hが中性付近であると、 分離 ·除去すべき成分(ピ ーク) が増加したり、 該成分 (ピーク) がブロードになって分離能が低 下したり、 保持時間が変化したりして、 成分 (ピーク) を特定 (解析) することができなくなる場合がある。 尚、 上記化合物の分析方法は、 特 に限定されるものではない。 The neutral fraction is separated by liquid chromatography to isolate and purify the above compounds. For example, specific methods include: (1) silica gel column chromatography using a silica gel flash column; Using liquid partition chromatography, n-hexaneethyl acetate Using a mixed solution (eluent) of the above, the neutral fraction is separated and a fraction (fraction) having a moderate polarity is taken out. Next, (2) normal phase high performance liquid chromatography The fraction was separated using a mixed solution (carrier) of n-hexane Z-ethyl acetate, and (3) reverse-phase high-performance liquid chromatography. Using a mixed solution (carrier) obtained by mixing an aqueous solution of ammonium acetate (PH 4.8) and acetonitrile in a volume ratio of 1: 2: 2, the fraction obtained in (1) above is further separated to contain the above compound There is a method of extracting the fraction. By performing the above separation / purification operations, the compound can be separated from a basic component such as an alkaloid derivative or an acidic component such as a phenol derivative, so that the compound can be purified. If necessary, the purity of the above compound can be further improved by repeating the above steps (2) and (3) for separation and purification. In other words, by performing the above separation and purification operations, the above compound can be efficiently extracted and separated. When the obtained compound is analyzed, for example, the analysis operation may be performed under the same conditions as the above-mentioned isolation / purification conditions, using the above-mentioned liquid chromatography. That is, the analysis is more preferably performed under acidic conditions. By adjusting the pH of the mobile phase to acidic, the resolution of liquid chromatography is further improved, so that the above compound can be analyzed more accurately. If the pH of the mobile phase is near neutral, the number of components (peaks) to be separated / removed increases, or the components (peaks) become broad, resulting in a decrease in resolution and a change in retention time. In some cases, the component (peak) cannot be specified (analyzed). The method for analyzing the above compounds is not particularly limited.
上記抽出等の一連の取り出し操作を行うことにより、 化合物 (3 0 ) 〜 (3 9 ) が得られる。 前記化合物 (3 0 ) 〜 (3 9 ) は何れも、 複数 の抗癌剤に対する耐性を獲得した癌細胞 (多剤耐性癌細胞) に対する多 剤耐性癌克服作用を有している。 それゆえ、 上記化学式 (3 0 ) 〜 ( 3 9 ) で表される化合物群より選ばれる少なくとも一種の化合物を含む多 剤耐性癌克服剤は、 該癌細胞に好適に投与することができる。 Compounds (30) to (39) are obtained by performing a series of extraction operations such as the above extraction. All of the compounds (30) to (39) are plural It has a multidrug-resistant cancer overcoming effect on cancer cells that have acquired resistance to anticancer drugs (multidrug-resistant cancer cells). Therefore, a multidrug-resistant cancer-overcoming agent containing at least one compound selected from the group of compounds represented by the above chemical formulas (30) to (39) can be suitably administered to the cancer cells.
これらの化合物 (3 0 ) 〜 (3 9 ) は、 上記説明 (カルスからの取り 出し方法の説明) において述べたように、 酸または塩基に対して安定で あり、 それゆえ、 例えば生体内で分解 (胃液による分解、 胆液による分 解等) されるおそれが少ない。 従って、 化合物 (3 0 ) 〜 (3 9 ) より 選ばれる少なくとも一種の化合物を含んでなる本発明にかかる多剤耐性 癌克服剤は、 例えば、 その投与方法や投与場所等の制限が少なく、 広範 囲な適用が可能であると期待される。  These compounds (30) to (39) are stable to acids or bases as described in the above description (description of the method for removing from callus), and are therefore decomposed in vivo, for example. (Decomposition by gastric juice, decomposition by bile fluid, etc.) Therefore, the multidrug-resistant cancer-overcoming agent according to the present invention comprising at least one compound selected from the compounds (30) to (39) has, for example, little restrictions on its administration method and administration place, and has a wide range. It is expected that a green application is possible.
特に、 化合物 (3 1 ), (3 3 ), ( 3 6 ) は、 ベラパミルを上回る多剤 耐性癌克服作用を有する高活性化合物であることから、 それ単独で特に 有効な (新規な) 多剤耐性癌克服剤となり得る。  In particular, the compounds (31), (33), and (36) are highly active compounds having a multidrug-resistant cancer-overcoming action superior to verapamil, and are therefore particularly effective (novel) multidrugs by themselves. It can be a drug for overcoming resistant cancer.
また、 上記化合物 (3 0 ) 〜 (3 9 ) は、 日本ィチイの組織を培養し て得られるカルスから取り出すことができる。 上記カルスは少量の外植 体より誘導され、 簡便な設備で以て再現性良く大量培養することができ る。 加えて、 該カルスは、 多剤耐性癌克服作用を有する上記化合物を比 較的多量に含んでいる。 即ち、 本発明にかかる製造方法によれば、 環境 を保全しながら、 日本ィチイから上記化合物 (3 0 ) 〜 (3 9 ) を効率 的に製造することができる。 これにより、 癌細胞に投与するのに好適な 多剤耐性癌克服剤を、 効率的に製造することができる。  In addition, the above compounds (30) to (39) can be extracted from calli obtained by culturing Japanese yew tissues. The above calli are derived from a small amount of explants and can be cultured in large quantities with simple equipment with good reproducibility. In addition, the callus contains a relatively large amount of the above compound having a multidrug-resistant cancer overcoming effect. That is, according to the production method of the present invention, the above-mentioned compounds (30) to (39) can be efficiently produced from Nippon yew while preserving the environment. This makes it possible to efficiently produce a multidrug-resistant cancer-overcoming agent suitable for administration to cancer cells.
上記の化学式 (4 0 ) 〜 (4 7 ) で表される化合物は、 それぞれ単独 で、 少なくとも P—糖蛋白質による抗癌剤の能動輸送の阻害剤 (以下、 P —糖蛋白質阻害剤と称する) としての機能を有し、 本発明にかかる多 剤耐性癌克服剤となりうる。また、上記の化合物のうち、化学式(4 0 ), (4 1), (43), (44), (45), (46), (4 7) で表される化合物 は、 従来の多剤耐性癌克服剤であるべラパミルと比較して多剤耐性癌克 服作用が高く、 以下、 高活性化合物と称する。 本発明にかかる多剤耐性 癌克服剤は、 これらの高活性化合物の少なくとも一つを含んでいること がより好ましい。 The compounds represented by the above chemical formulas (40) to (47) are each independently used as an inhibitor of at least the active transport of an anticancer agent by P-glycoprotein (hereinafter referred to as P-glycoprotein inhibitor). It has a function and can be a multidrug-resistant cancer overcoming agent according to the present invention. Further, among the above compounds, the chemical formula (40), The compounds represented by (41), (43), (44), (45), (46), and (47) are more effective than the conventional multidrug-resistant cancer-overcoming drug verapamil. Highly effective in overcoming resistant cancer and is hereinafter referred to as a highly active compound. More preferably, the multidrug-resistant cancer-overcoming agent according to the present invention contains at least one of these highly active compounds.
上記の化合物が多剤耐性癌克服作用を有することは、 本願発明者が新 たに見出したことである。 また、 上記の化学式 (4 1) 〜 (43) で表 される 3種の夕キシニン化合物はいずれも、 本発明にかかる新規なタキ シニン化合物である。 尚、 以下、 必要に応じて、 上記化学式 (40) 〜 (47) で表される化合物を順に、 化合物 (40) 〜 (47) と称する。 また、 化合物 (40) 〜 (47) を総称する場合には、 単に、 本発明の タキサン関連化合物と記載する場合もある。  The fact that the above-mentioned compound has a multidrug-resistant cancer overcoming effect is newly found by the present inventors. Further, all of the three kinds of xinine compounds represented by the above chemical formulas (41) to (43) are novel taxinine compounds according to the present invention. Hereinafter, the compounds represented by the above chemical formulas (40) to (47) will be referred to as compounds (40) to (47) as needed. When the compounds (40) to (47) are collectively referred to, they may be simply described as the taxane-related compound of the present invention.
尚、 本発明にかかる多剤耐性癌克服剤は必要に応じて、 例えば、 癌細 胞の特定部位 (または P—糖蛋白質) と特異的に結合可能な抗体や、 抗 癌剤等をさらに含んで構成されていてもよい。 これにより、 標的とすべ き(多剤耐性)癌細胞への多剤耐性癌克服剤の供給を確実とする効果や、 該癌細胞への抗癌剤の同時供給が可能となる効果等を実現することがで きる。 これら多剤耐性癌克服剤の投与方法は特に限定されるものではな く、 多剤耐性癌克服剤に含まれる物質 (上記化合物 (40) 〜 (47) や、 抗体、 抗癌剤等を指す) の物性等に応じた最適なドラッグデリバリ 一システムを採用すればよい。  The multidrug-resistant cancer-overcoming agent according to the present invention further includes, as necessary, for example, an antibody capable of specifically binding to a specific site (or P-glycoprotein) of a cancer cell, an anticancer agent, and the like. May be configured. As a result, an effect of reliably supplying a multidrug-resistant cancer-overcoming agent to cancer cells to be targeted (multidrug-resistant) and an effect of enabling simultaneous supply of an anticancer agent to the cancer cells can be realized. I can do it. The administration method of these multidrug-resistant cancer-overcoming agents is not particularly limited, and the substances contained in the multidrug-resistant cancer-overcoming agents (refer to the compounds (40) to (47), antibodies, anticancer agents, etc.) An optimal drug delivery system according to physical properties may be adopted.
本発明にかかる多剤耐性癌克服剤の製造方法は、 上記説明の多剤耐性 癌克服剤に含まれる化合物 (40) を、 「日本ィチイ Taxus cuspidata Sieb. et Zucc.) の組織」 より誘導されるカルスから取り出す工程を含む 方法 (以下、 製造方法 Aと称する場合がある) である。 また、 本発明に かかる他の多剤耐性癌克服剤の製造方法は、 上記説明の多剤耐性癌克服 剤に含まれる化合物 (4 1 ) 〜 (4 7 ) の少なくとも一つを、 日本イチ ィの 「針葉部」 から取り出す工程を含む方法 (以下、 製造方法 Bと称す る場合がある) である。 以下、 はじめに上記製造方法 Aについて説明を 行い、 製造方法 Bに関しては、 製造方法 Aとの相違点のみを記載する。 本発明において 「日本ィチイの組織」 とは、 日本ィチイに由来する組 織、 より具体的には、 植物体 (根部、 茎部、 葉部を成す各組織、 すなわ ち栄養器官全体を指す)、 並びに、 その花、 花粉、 及び果実 (生殖器官) を成す組織のことを指す。 尚、 ここでいう 「葉部」 とは、 「葉身」 と 「葉 柄」 とから構成される単位を指し、 また、 「果実」 とは、 「仮種衣 (ァリ ル)」 および 「種子」 を含む概念である。 これら日本ィチイの組織の中で も、 特にその 「針葉部」 は、 A ) 培養細胞の増殖性、 即ち、 カルスの誘 導性に特に優れている点、及び、 B )組織の採取が比較的容易である上、 植物体に大きなダメージを与えることがない点等で、 より'好適である。 上記 「針葉部」 とは、 「葉身」、 「葉柄」、 並びに、 「葉部」 が複数枚付い た 「小枝」、 さらには、 「新芽 (その原基であっても良い)」 ゃ該 「新芽」 が付いた 「若茎」 も含まれるものとする。 これら 「針葉部」 のうち、 特 に、 秋季並びに冬季に採取した 「若茎」 (直径 2 mm〜 3 mm程度の緑色 若茎部) が、 カルスの誘導性に特に優れると共に、 何代にもわたる継代 培養を行っても、 上記化合物 (4 0 ) の生産量が低下しないので、 特に 好ましい。 また、 シュートや芽生え等の若い組織も、 カルス誘導のため の組織として好適に使用することができる。 The method for producing a multidrug-resistant cancer-overcoming agent according to the present invention comprises the step of introducing the compound (40) contained in the above-described multidrug-resistant cancer-overcoming agent from "a tissue of Nippon yew Taxus cuspidata Sieb. Et Zucc.). (Hereinafter sometimes referred to as production method A). Further, the method for producing another multidrug-resistant cancer-overcoming agent according to the present invention comprises A method that includes a step of removing at least one of the compounds (41) to (47) contained in the agent from the "needle portion" of Nippon Ichi (hereinafter sometimes referred to as Production Method B) . Hereinafter, the above-mentioned production method A will be described first, and with respect to production method B, only differences from production method A will be described. In the present invention, the “tissue of Japanese yew” refers to a tissue derived from Japanese yew, more specifically, a plant body (each of the tissues forming the root, stem, and leaf, that is, the entire vegetative organ). And the tissues that make up the flowers, pollen, and fruits (reproductive organs). The term “leaf” used herein refers to a unit composed of “leaf” and “stalk”, and “fruit” refers to “temporary garments” It is a concept that includes "seed." Among these Japanese yew tissues, in particular, the “needle part” is compared in terms of A) the proliferative ability of cultured cells, that is, callus induction, and B) the collection of tissues. It is more suitable because it is easy to target and does not cause significant damage to the plant. The above “needle part” is “leaf”, “petiole”, “twig” with a plurality of “leaves”, and “sprout (or its primordium)”. The term "young shoot" with the "sprout" is also included. Among these “needle parts”, “young shoots” (green young shoots with a diameter of about 2 mm to 3 mm) collected in the autumn and winter are particularly excellent in callus inducibility, and in some generations. It is particularly preferable to carry out subculture over the whole, since the production of the compound (40) does not decrease. In addition, young tissues such as shoots and seedlings can be suitably used as tissues for callus induction.
カルス誘導用の 「日本ィチイの組織」 (以下、 場合によっては外植体と 称する) の採取時期は特に限定されるものではないが、 上記組織を植物 体から得る場合には、 採取前 1力月間の平均気温が 1 8で以下、 より好 ましく 5で以下である環境下で生育された後に採取されることが好まし い。 尚、 この環境は、 人工的に作られるものであってもよい。 外植体からのカルス誘導およびその培養は、 特別な添加剤 (添加物) や培養条件を用いることなく、 一般的な手法で以て、 例えば、 寒天培地 等の市販の植物培養培地 (固体培地) とオーキシンとを用いることによ つて、 容易に実施することができる。 また、 場合によっては、 ォーキシ ンを含んでなる液体培地を用いて振盪培養を行ってもよい。 このように、 外植体から容易にカルスを誘導することができるので、 簡便な設備で以 て大量培養することができ、 該カルスから上記化合物 (4 0 ) を大量に 取り出すことができる。 カルスの形成方法としては、 具体的には、 例え ば、 外植体の切片を 7 0重量 (質量) %エチルアルコール水溶液等で滅 菌処理した後、 上記の切片を、 寒天粉末を含み、 さらにオーキシンを所 定の濃度で含む改変ガンボーグ培地等の固体培地に置床し、 次いで、 2 5で〜 2 7で程度の暗所に静置して培養 (静置培養) する方法が挙げら れるが、 特に限定されるものではない。 There is no particular limitation on the timing of the callus-inducing “Japanese yew tissue” (hereinafter sometimes referred to as an explant), but if the above tissue is obtained from a plant, it must be collected before harvesting. It is preferably collected after growing in an environment where the average monthly temperature is below 18 and more preferably below 5. This environment may be artificially created. Induction of callus from explants and their cultivation can be performed by a general method without using special additives (additives) or culturing conditions. For example, commercially available plant culture media such as agar media (solid media) ) And auxin can be easily implemented. In some cases, shaking culture may be performed using a liquid medium containing auxin. As described above, callus can be easily induced from the explant, so that large-scale culture can be performed with simple equipment, and the compound (40) can be extracted from the callus in a large amount. As a method for forming callus, specifically, for example, a section of an explant is sterilized with 70% by weight (mass) aqueous solution of ethyl alcohol or the like, and then, the above-mentioned section contains agar powder. There is a method in which the cells are placed on a solid medium such as a modified gamborg medium containing auxin at a predetermined concentration, and then cultured in a dark place at 25 to 27 (static culture). However, there is no particular limitation.
そして、 培養細胞は、 上記固体培地を用いた簡便な方法で以て、 例え ば 3 0日〜 5 0日毎、 より好ましくは 4 0日毎に増殖性に優れている力 ルスを選抜、 継代培養することによって、 該カルスを再現性良く、 大量 培養することができる。 すなわち、 上記カルスは、 大量培養可能である とともに上記化合物 (4 0 ) を比較的多量に含んでいるため、 該化合物 ( 4 0 ) を効率的に製造することができる。  Then, the cultured cells are selected by a simple method using the above solid medium, for example, every 30 days to 50 days, more preferably every 40 days. By doing so, the callus can be cultured in large quantities with good reproducibility. That is, since the callus can be cultured in a large amount and contains the compound (40) in a relatively large amount, the compound (40) can be efficiently produced.
上記のオーキシンとしては、具体的には、例えば、 1 一ナフチル酢酸、 2—ナフチル酢酸 (N A A ;別名、 ナフ夕レン酢酸)、 2 , 4—ジクロロ フエノキシ酢酸、ィンドール酢酸、 4 _クロ口インドール酢酸 (4-C1 IAA)、 インドール酪酸等が挙げられるが、 特に限定されるものではない。 上記 例示のオーキシンのうち、 1 一ナフチル酢酸、 2—ナフチル酢酸、 2, 4—ジクロロフエノキシ酢酸、 および 4—クロ口インドール酢酸がより 好ましい。 固体培地におけるオーキシンの含有量は、 0を越えて 1 . 0 mgZL以下が好ましく、 0. SmgZL程度がより好ましい。 ォーキ シンの含有量が 1. OmgZLを越えると、 継代培養を繰り返すにつれ て、 カルスの増殖性が次第に低下する場合がある。 4—クロ口インドー ル酢酸を含む固体培地は、 培養細胞における、 2次代謝物である上記化 合物 (40) の生産性をより向上させることができる。 Examples of the auxin include, for example, 1-naphthyl acetic acid, 2-naphthyl acetic acid (NAA; also known as naphthylene acetic acid), 2,4-dichlorophenoxy acetic acid, indole acetic acid, and 4-chloro-indole acetic acid (4-C1 IAA), indolebutyric acid and the like, but are not particularly limited. Of the auxins exemplified above, 1-naphthylacetic acid, 2-naphthylacetic acid, 2,4-dichlorophenoxyacetic acid, and 4-chloroindoleacetic acid are more preferred. The auxin content in solid media is greater than 0 and 1.0. mgZL or less is preferable, and about 0 SmgZL is more preferable. If the auxin content exceeds 1. OmgZL, the callus growth may gradually decrease as the subculture is repeated. The solid medium containing 4-cloth indole acetic acid can further improve the productivity of the compound (40), which is a secondary metabolite, in cultured cells.
固体培地は、 必要に応じて、 サイトカイニン、 ポリ (N—ビニルー 2 —ピロリドン) 等の褐変防止剤; ジャスモン酸メチル等のエリシタ ;ォ リゴサッ力ライド ;各種ビタミン;等の添加剤をさらに含んでいてもよ い。 ジャスモン酸メチルを含む固体培地は、 培養細胞における、 上記化 合物 (40) の生産性をより向上させることができる。 固体培地におけ る添加剤の含有量は、 該添加剤の種類や組み合わせに応じて設定すれば よい。  The solid medium further contains additives such as cytokinin and poly (N-vinyl-2-pyrrolidone) as anti-browning agents; elicitas such as methyl jasmonate; oligosadride; various vitamins; It is good. The solid medium containing methyl jasmonate can further improve the productivity of the compound (40) in cultured cells. The content of the additive in the solid medium may be set according to the type and combination of the additive.
上記オリゴサッカライドとしては、 具体的には、 例えば、 食用のォク ラの果実 (またはその植物体) から抽出 ·精製された粘性多糖を酸で加 水分解してなる、 2糖〜 5糖のオリゴ糖の混合物 (以下、 KTOSと略 記す) が挙げられる。 該オリゴ糖は、 例えば、 ラムノース、 ガラクト一 ス、 ガラクッロン酸、 グルコース等の単糖で構成されている。 上記 KT OSは、 継代による培養細胞の老化を抑制する作用を備えている。 この ため、 KTOSを含む固体培地を用いることにより、 老化を抑制しなが ら継代培養を繰り返すことができるので、 培養細胞を増殖させ続けるこ とができる。 従って、 KTOSを含む固体培地は、 培養細胞における、 上記化合物 (40) の生産性をより向上させることができる。  Specific examples of the oligosaccharides include disaccharides to pentasaccharides obtained by hydrolyzing viscous polysaccharides extracted and purified from edible okra fruits (or their plants) with an acid. A mixture of oligosaccharides (hereinafter abbreviated as KTOS) is mentioned. The oligosaccharide is composed of, for example, monosaccharides such as rhamnose, galactose, galacturonic acid, and glucose. The above-mentioned KT OS has an action of suppressing aging of cultured cells due to passage. For this reason, by using a solid medium containing KTOS, the subculture can be repeated while suppressing senescence, so that the cultured cells can be kept growing. Therefore, the solid medium containing KTOS can further improve the productivity of the compound (40) in cultured cells.
そして、 固体培地 (または液体培地) は、 4一クロ口インドール酢酸 および またはオリゴサッカライドを含むと共に、 必要に応じて、 1一 ナフチル酢酸および または 2—ナフチル酢酸をさらに含んでいること が、 特に好ましい。 カルスから上記化合物 (4 0 ) を取り出す具体的な方法は、 特に限定 されるものではないが、 抽出を行う方法が好適であり、 有機溶媒を用い て抽出を行う方法が最適である。 抽出を行う具体的な方法としては、.例 えば、カルスを凍結乾燥等によって乾燥させ、必要に応じて粉砕した後、 有機溶媒に数分間から数時間浸漬する方法が挙げられる。 抽出条件は、 特に限定されるものではないが、 抽出温度は 3 O :以下であることがよ り好ましい。 そして、 得られた抽出液を、 必要に応じて酸およびノまた は塩基で処理し、 次いで液体クロマトグラフィーによって分画すること により、 上記化合物 (4 0 ) を単離 ·精製することができる。 It is particularly preferable that the solid medium (or the liquid medium) contains 4-monoindoleacetic acid and / or oligosaccharide, and further contains 1-naphthylacetic acid and / or 2-naphthylacetic acid as necessary. . The specific method of extracting the compound (40) from the callus is not particularly limited, but a method of performing extraction is preferable, and a method of performing extraction using an organic solvent is most suitable. As a specific method for performing extraction, for example, a method in which calli are dried by freeze-drying or the like, pulverized as necessary, and then immersed in an organic solvent for several minutes to several hours, may be mentioned. The extraction conditions are not particularly limited, but the extraction temperature is more preferably 3 O: or less. Then, the compound (40) can be isolated and purified by treating the obtained extract with an acid and / or a base as necessary, and then fractionating by liquid chromatography.
以下、 カルスから上記化合物 (4 0 ) を取り出す方法について、 詳述 する。 上記の有機溶媒としては、 具体的には、 例えば、 メチルアルコー ル、 エチルアルコール、 イソプロピルアルコール、 クロ口ホルム、 n— へキサン、 酢酸ェチル、 各種エーテル、 トルエン等が挙げられるが、 特 に限定されるものではない。 これら有機溶媒は、 一種類のみを用いても よく、 また、二種類以上を併用してもよい。上記例示の有機溶媒のうち、 メチルアルコール、 n—へキサン、 および酢酸ェチルがより好ましい。 上記化合物 (4 0 ) が抽出された抽出液は、 必要に応じて、 不純物と して含まれるアル力ロイド誘導体等の塩基性成分や、 フエノール誘導体 等の酸性成分を分離 ·除去するために、 酸および/または塩基で処理す ることがより好ましい。 抽出液を酸および または塩基で処理する方法 としては、 具体的には、 抽出液を酸性水および Zまたは塩基性水で洗浄 する方法が挙げられる。  Hereinafter, a method for extracting the compound (40) from the callus will be described in detail. Specific examples of the organic solvent include methyl alcohol, ethyl alcohol, isopropyl alcohol, chloroform, n-hexane, ethyl acetate, various ethers, and toluene, but are not particularly limited. Not something. One of these organic solvents may be used alone, or two or more thereof may be used in combination. Of the organic solvents exemplified above, methyl alcohol, n-hexane, and ethyl acetate are more preferred. The extract from which the compound (40) has been extracted is, if necessary, separated and removed from a basic component such as an alkyloid derivative and an acidic component such as a phenol derivative contained as impurities. More preferably, treatment with an acid and / or a base. As a method of treating the extract with an acid and / or a base, specifically, a method of washing the extract with acidic water and Z or basic water can be mentioned.
上記の酸としては、 具体的には、 例えば、 塩酸、 硫酸、 リン酸等の無 機酸;ギ酸、 酢酸等の有機酸;が挙げられるが、 特に限定されるもので はない。 これら酸は、 一種類のみを用いてもよく、 また、 二種類以上を 併用してもよい。 従って、 酸性水としては、 これら無機酸および/また は有機酸の水溶液が挙げられる。 該酸性水の p Hは、 特に限定されるも のではないが、 5以下がより好ましく、 4以下がさらに好ましい。 抽出 液を酸性水で洗浄することによって、 該抽出液から、 アルカロイド誘導 体等の塩基性成分を分離 ·除去することができる。 Specific examples of the above-mentioned acids include, for example, inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid; and organic acids such as formic acid and acetic acid, but are not particularly limited. These acids may be used alone or in combination of two or more. Therefore, as the acidic water, these inorganic acids and / or Is an aqueous solution of an organic acid. The pH of the acidic water is not particularly limited, but is preferably 5 or less, and more preferably 4 or less. By washing the extract with acidic water, basic components such as alkaloid derivatives can be separated and removed from the extract.
尚、 タキサン関連化合物の中には、 酸で環開裂 (分解) し易いォキセ タン骨格を分子構造に有するものがある。 従って、 一般に、 酸で処理を 行うと、 該ォキセ夕ン骨格の環開裂が生じてタキサン関連化合物が破壊 されてしまうと考えられている。 ところが、 本願発明者等が検討したと ころ、 本発明のタキサン関連化合物は、 酸で処理を行ってもォキセタン 骨格の環開裂が生じないことが判明した。 それゆえ、 酸で処理を行うこ とによって、 本発明のタキサン関連化合物と、 アルカロイド誘導体等の 成分とを分離することができる。  Some taxane-related compounds have an oxetane skeleton in the molecular structure that is easily cleaved (decomposed) by an acid. Therefore, it is generally considered that the treatment with an acid causes ring cleavage of the oxenone skeleton, thereby destroying the taxane-related compound. However, studies by the present inventors have revealed that the taxane-related compound of the present invention does not undergo ring cleavage of the oxetane skeleton even when treated with an acid. Therefore, by treating with an acid, the taxane-related compound of the present invention can be separated from components such as alkaloid derivatives.
また、 上記の塩基としては、 具体的には、 例えば、 アンモニア、 水酸 化ナトリウム、 水酸化カリウム、 炭酸ナトリウム、 炭酸カリウム等の無 機塩基;アンモニゥム化合物等の有機塩基;が挙げられるが、 特に限定 されるものではない。 これら塩基は、一種類のみを用いてもよく、 また、 二種類以上を併用してもよい。 従って、 塩基性水としては、 これら無機 塩基および/または有機塩基の水溶液が挙げられる。 該塩基性水の p H は、 特に限定されるものではないが、 9以上がより好ましく、 1 0以上 がさらに好ましく、 1 1以上が特に好ましい。 抽出液を塩基性水で洗浄 することによって、 該抽出液から、 フエノール誘導体等の酸性成分を分 離 ·除去することができる。  Specific examples of the base include: inorganic bases such as ammonia, sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate; and organic bases such as ammonium compounds. It is not limited. One of these bases may be used alone, or two or more may be used in combination. Accordingly, examples of the basic water include aqueous solutions of these inorganic bases and / or organic bases. The pH of the basic water is not particularly limited, but is preferably 9 or more, more preferably 10 or more, and particularly preferably 11 or more. By washing the extract with basic water, acidic components such as phenol derivatives can be separated and removed from the extract.
尚、 タキサン関連化合物の中には、 塩基で加水分解され易いエステル を分子構造に有するものがある。従って、一般に、塩基で処理を行うと、 該エステルの加水分解が生じてタキサン関連化合物が破壊されてしまう と考えられている。 ところが、 本願発明者等が検討したところ、 本発明 のタキサン関連化合物は、 塩基で処理を行ってもエステルの加水分解が 生じないことが判明した。 それゆえ、 塩基で処理を行うことによって、 本発明のタキサン関連化合物と、 フエノール誘導体等の成分とを分離す ることができる。 Some taxane-related compounds have an ester which is easily hydrolyzed by a base in a molecular structure. Therefore, it is generally believed that treatment with a base will hydrolyze the ester and destroy the taxane-related compound. However, the inventors of the present application have studied and found that the present invention It was found that the taxane-related compound did not undergo ester hydrolysis even when treated with a base. Therefore, by treating with a base, the taxane-related compound of the present invention can be separated from components such as phenol derivatives.
酸および/または塩基で処理した後の抽出液は、 中性になるまで水洗 することがより好ましい。 また、 上記化合物 (4 0 ) が抽出された抽出 液 (水洗する場合には水洗後のもの) は、 該化合物 (4 0 ) の単離が容 易となるように、濃縮する (有機溶媒を除去する) ことがより好ましい。 抽出液を濃縮することにより、 中性分画が得られる。  The extract after the treatment with an acid and / or a base is more preferably washed with water until neutral. The extract from which the compound (40) has been extracted (if washed with water, after washing) is concentrated so that the compound (40) can be easily isolated (by removing the organic solvent). More preferably). By concentrating the extract, a neutral fraction is obtained.
中性分画から上記化合物 (4 0 ) を単離 ·精製する具体的な方法は、 特に限定されるものではないが、 液体クロマトグラフィーを採用する方 法が好適である。 該液体クロマトグラフィーとしては、 具体的には、 例 えば、 シリカゲルカラムクロマトグラフィー、 逆相または順相の高速液 体クロマトグラフィー、 遠心液液分配クロマトグラフィー (C P C ) 等 が挙げられるが、 特に限定されるものではない。 上記の高速液体クロマ トグラフィ一は、 例えば、 移動相の p Hを考慮して、 逆相か順相かを選 択すればよい。  The specific method for isolating and purifying the compound (40) from the neutral fraction is not particularly limited, but a method employing liquid chromatography is preferred. Specific examples of the liquid chromatography include silica gel column chromatography, reversed-phase or normal-phase high-performance liquid chromatography, and centrifugal liquid-liquid distribution chromatography (CPC), but are not particularly limited. Not something. In the above-described high performance liquid chromatography, for example, it is sufficient to select the reverse phase or the normal phase in consideration of the pH of the mobile phase.
固定相 (充填剤) としては、 具体的には、 例えば、 シリカゲル、 アル ミナ、 O D S (ォク夕デシルシリル) 系化合物等が挙げられるが、 特に 限定されるものではない。 上記例示の固定相のうち、 逆相高速液体クロ マトグラフィ一等を採用する場合には、 〇D S系化合物がより好ましい。 移動相 (キャリア, 溶離液) として用いるのに好適な液体としては、 具体的には、 例えば、 メチルアルコール、 エチルアルコール、 クロロホ ルム、 n—へキサン、 酢酸ェチル、 各種エーテル、 トルエン、 ァセトニ トリル、 水等が挙げられるが、 特に限定されるものではない。 これら液 体は、一種類のみを用いてもよく、 また、二種類以上を併用してもよい。 上記例示の液体のうち、 シリカゲルカラムクロマトグラフィーや順相高 速液体クロマトグラフィー等を採用する場合には、 n—へキサン/酢酸 ェチル系の混合溶液がより好ましく、 逆相高速液体クロマトグラフィー 等を採用する場合には、 メチルアルコール/ァセトニトリル系の混合溶 液がより好ましく、 遠心液液分配クロマトグラフィー等を採用する場合 には、 n—へキサン メチルアルコール系の混合溶液がより好ましい。 逆相高速液体クロマトグラフィーにおける上記液体の p H、 即ち、 移 動相の p Hは、 緩衝溶液によって酸性に調節されていることがより好ま しい。 つまり、 逆相 O D Sカラムを用いた単離 ·精製は、 酸性条件下で 行われることがより好ましい。 緩衝溶液は、 移動相の p Hを 5 . 5以下 に調節することができる溶液であればよく、 特に限定されるものではな い。 該緩衝溶液としては、 具体的には、 例えば、 酢酸アンモニゥム水溶 液等が挙げられる。 尚、 上記の液体と緩衝溶液との組み合わせは、 特に 限定されるものではない。 Specific examples of the stationary phase (filler) include, but are not particularly limited to, silica gel, alumina, and ODS (octyl decylsilyl) -based compounds. When reversed phase high performance liquid chromatography or the like is employed among the stationary phases exemplified above, ΔDS compounds are more preferred. Examples of liquids suitable for use as a mobile phase (carrier, eluent) include, for example, methyl alcohol, ethyl alcohol, chloroform, n-hexane, ethyl acetate, various ethers, toluene, acetonitrile, Although water is mentioned, it is not particularly limited. One of these liquids may be used alone, or two or more of them may be used in combination. When silica gel column chromatography or normal phase high performance liquid chromatography is used among the liquids exemplified above, a mixed solution of n-hexane / ethyl acetate is more preferable. When employed, a mixed solution of methyl alcohol / acetonitrile is more preferable, and when centrifugal liquid-liquid distribution chromatography or the like is employed, a mixed solution of n-hexanemethyl alcohol is more preferable. More preferably, the pH of the liquid in the reversed-phase high-performance liquid chromatography, that is, the pH of the mobile phase is adjusted to be acidic by a buffer solution. That is, the isolation and purification using the reversed-phase ODS column are more preferably performed under acidic conditions. The buffer solution is not particularly limited as long as it can adjust the pH of the mobile phase to 5.5 or less. Specific examples of the buffer solution include an aqueous solution of ammonium acetate. The combination of the above liquid and buffer solution is not particularly limited.
逆相液体クロマトグラフィーにおける移動相の p Hを酸性に調節する ことにより、 該液体クロマトグラフィーの分離能がより向上するので、 上記化合物 (4 0 ) をより一層選択的に単離することができる。 移動相 の p Hが中性付近であると、 アル力ロイド誘導体の分離 ·除去が充分に 行われない場合がある。 また、 アルカロイド誘導体が有する窒素原子に 対してプロトン付加が可逆的に起こるので、 分離 *除去すべき成分 (ピ ーク) が増加したり、 該成分 (ピーク) がブロードになって分離能が低 下したり、 保持時間が変化したりして、 成分 (ピーク) を特定 (解析) することができなくなる場合がある。  By adjusting the pH of the mobile phase in reverse phase liquid chromatography to acidic, the resolution of the liquid chromatography is further improved, so that the compound (40) can be more selectively isolated. . If the pH of the mobile phase is near neutral, the separation / removal of the alkyloid derivative may not be performed sufficiently. In addition, proton addition to the nitrogen atom of the alkaloid derivative occurs reversibly, so that components (peaks) to be separated / removed increase or the components (peaks) are broadened, resulting in low resolution. In some cases, the component (peak) cannot be specified (analyzed) due to a drop or a change in the retention time.
中性分画を液体クロマトグラフィーを採用して分離し、上記化合物(4 0 ) を単離 ·精製する具体的な方法としては、 例えば、 先ず、 ①シリカ ゲルフラッシュカラムを用いたシリカゲル力ラムクロマトグラフィ一や、 遠心液液分配クロマトグラフィーを採用して、 n —へキサン/酢酸ェチ ル系の混合溶液 (溶離液) を用いて、 中性分画を分離して中程度の極性 を有する分画 (フラクション) を取り出し、 次に、 ②順相高速液体クロ マトグラフィーを採用して、 n—へキサン Z酢酸ェチル系の混合溶液(キ ャリア) を用いて、 該分画を分離し、 さらに、 ③逆相高速液体クロマト グラフィーを採用して、 メチルアルコールと 0 . 0 5 M酢酸アンモニゥ ム水溶液 (P H 4 . 8 ) とァセトニトリルとを容量比 1 : 2 : 2で混合 してなる混合溶液 (キャリア) を用いて、 上記②で得た分画をさらに分 離して上記化合物 (4 0 ) を含む分画を取り出す方法が挙げられる。 上 記の分離 ·精製操作を行うことにより、 上記化合物 (4 0 ) と、 アル力 ロイド誘導体等の塩基性成分やフエノール誘導体等の酸性成分とを分離 することができるので、 上記化合物 (4 0 ) を精製することができる。 そして、 必要に応じて、 上記② ·③の分離 ·精製操作を繰り返して行う ことにより、 上記化合物 (4 0 ) の純度をさらに向上させることができ る。 つまり、 上記の分離 ·精製操作を行うことにより、 上記化合物 (4 0 ) を効率的に抽出 ·分離することができる。 The neutral fraction is separated by liquid chromatography, and the above compound (40) is isolated and purified. For example, specific methods include: (1) silica gel column chromatography using silica gel flash column. One, Separation of the neutral fraction using a mixed solution of n-hexane / ethyl acetate (eluent) using centrifugal liquid-liquid partition chromatography and fractionation with a moderate polarity (fraction) ), And then (2) normal-phase high-performance liquid chromatography is used, the fraction is separated using a mixed solution (carrier) of n-hexane Z-ethyl acetate, and (3) reverse Using high-performance liquid chromatography, a mixed solution (carrier) consisting of a mixture of methyl alcohol, a 0.05 M aqueous solution of ammonium acetate (PH 4.8) and acetonitrile in a volume ratio of 1: 2: 2 is obtained. A method of further separating the fraction obtained in the above (1) and extracting a fraction containing the compound (40). By performing the above separation and purification operations, the compound (40) can be separated from a basic component such as an alkyloid derivative or an acidic component such as a phenol derivative. ) Can be purified. The purity of the compound (40) can be further improved by repeating the above steps (1) and (3) as required. That is, by performing the above separation and purification operations, the compound (40) can be efficiently extracted and separated.
一方、 上記製造方法 Bにおいて、 化合物 (4 1 ) 〜 (4 7 ) の少なく とも一つを取り出す工程では、 例えば、 上記説明の日本ィチイの 「針葉 部」 を必要に応じて粉枠し、 有機溶媒を用いて針葉部粉碎物の抽出を行 つた後、 得られ抽出液を酸およびノまたは塩基で処理し、 次いで液体ク 口マトグラフィ一によつて分画することにより、 上記化合物が取り出さ れる。 有機溶媒を用いた抽出、 抽出液に対する酸および または塩基で の処理、 並びに、 液体クロマトグラフィーによる分画、 の各工程は、 上 記化合物 (4 0 ) をカルスから取り出す場合と同様にして行うことがで きるため、 その詳細な説明を省略する。  On the other hand, in the above-mentioned production method B, in the step of taking out at least one of the compounds (41) to (47), for example, the above-mentioned “needle portion” of Nippon yew is powder-framed as necessary. After extraction of the needle leaf ground material using an organic solvent, the obtained extract is treated with an acid and a base or a base, and then fractionated by liquid chromatography to obtain the above compound. It is. The steps of extraction with an organic solvent, treatment of the extract with an acid and / or a base, and fractionation by liquid chromatography are performed in the same manner as in the case of removing the above compound (40) from callus. Therefore, detailed description thereof will be omitted.
尚、 製造方法 Bでは、 上記抽出操作を行う前に、 針葉部に含まれる油 分を除去 (脱脂) する目的で、 該針葉部を有機溶媒で洗浄してもよい。 この有機溶媒は、 針葉部粉碎物の抽出に用いられる有機溶媒と同様のも のを使用すればよく、 なかでも n—へキサンが、 針葉部に含まれる油分 を除去するのに好適である。 油分の除去は、 特に生針葉部を使用する場 合に必要とされる。 In addition, in the production method B, the oil contained in the needle portion is The needle portion may be washed with an organic solvent for the purpose of removing (degreasing) the components. This organic solvent may be the same as the organic solvent used for extracting the needle leaf ground material. Among them, n-hexane is suitable for removing oil contained in the needle leaf. is there. Oil removal is required, especially when using raw needles.
上記何れかの方法により得られた上記化合物 (4 0 ) 〜 (4 7 ) を分 析する際には、 例えば、 上記液体クロマトグラフィーを採用して、 上記 分離 ·精製条件と同様の条件で分析操作を行えばよい。つまり、分析は、 酸性条件下で行われることがより好ましい。 移動相の P Hを酸性に調節 することにより、液体クロマトグラフィ一の分離能がより向上するので、 上記化合物(4 0 ) 〜 (4 7 ) をより一層正確に分析することができる。 移動相の p Hが中性付近であると、 分離 '除去すべき成分 (ピーク) が 増加したり、該成分(ピーク)がブロードになって分離能が低下したり、 保持時間が変化したりして、 成分 (ピーク) を特定 (解析) することが できなくなる場合がある。 尚、 上記化合物 (4 0 ) 〜 (4 7 ) の分析方 法は、 特に限定されるものではない。  When the compounds (40) to (47) obtained by any of the above methods are analyzed, for example, the above-described liquid chromatography is employed, and analysis is performed under the same conditions as the above-mentioned separation / purification conditions. You just need to do the operation. That is, the analysis is more preferably performed under acidic conditions. By adjusting the pH of the mobile phase to acidic, the resolving power of the liquid chromatography is further improved, so that the compounds (40) to (47) can be more accurately analyzed. If the pH of the mobile phase is near neutral, the number of components (peaks) to be separated / removed will increase, or the components (peaks) will be broadened and the resolution will decrease, or the retention time will change. As a result, it may not be possible to identify (analyze) the component (peak). The method for analyzing the compounds (40) to (47) is not particularly limited.
上記抽出等の一連の取り出し操作を行うことにより、 化合物 (4 0 ) 〜 (4 7 ) が得られる。 前記化合物 (4 0 ) 〜 (4 7 ) は何れも、 複数 の抗癌剤に対する耐性を獲得した癌細胞 (多剤耐性癌細胞) に対する多 剤耐性癌克服作用を有している。 それゆえ、 上記化学式 (4 0 ) 〜 (4 7 ) で表される化合物群より選ばれる少なくとも一種の化合物を含む多 剤耐性癌克服剤は、 該癌細胞に好適に投与することができる。  Compounds (40) to (47) are obtained by performing a series of extraction operations such as the above extraction. All of the compounds (40) to (47) have a multidrug-resistant cancer overcoming effect on cancer cells (multidrug-resistant cancer cells) that have acquired resistance to a plurality of anticancer agents. Therefore, a multidrug-resistant cancer-overcoming agent comprising at least one compound selected from the group of compounds represented by the chemical formulas (40) to (47) can be suitably administered to the cancer cells.
これらの化合物 (4 0 ) 〜 (4 7 ) は、 上記説明 (カルスまたは針葉 部からの取り出し方法の説明) において述べたように、 酸または塩基に 対して安定であり、 それゆえ、 例えば生体内で分解 (胃液による分解、 胆液による分解等) されるおそれが少ない。 従って、 化合物 (4 0 ) 〜 (47) より選ばれる少なくとも一種の化合物を含んでなる本発明にか かる多剤耐性癌克服剤は、 例えば、 その投与方法や投与場所等の制限が 少なく、 広範囲な適用が可能であると期待される。 These compounds (40) to (47) are stable to acids or bases as described in the above description (description of the method of removing from callus or needle), and therefore, for example, Less likely to be degraded in the body (decomposition by gastric juice, bile fluid, etc.). Therefore, compound (40) (47) The multidrug-resistant cancer-overcoming agent according to the present invention, comprising at least one compound selected from (47), is expected to be applicable to a wide range of applications, for example, with few restrictions on its administration method and administration location. Is done.
特に、化合物(40), (41), (43), (44), (45), (46), (4 7) は、 ベラパミルを上回る多剤耐性癌克服作用を有する高活性化合物 であることから、 それ単独で特に有効な (新規な) 多剤耐性癌克服剤と なり得る。  In particular, compounds (40), (41), (43), (44), (45), (46), and (47) are highly active compounds that have a multidrug-resistant cancer-surpassing action superior to verapamil Therefore, it alone can be a particularly effective (new) drug for overcoming multidrug-resistant cancer.
また、 上記化合物 (40) は、 日本ィチイの組織を培養して得られる カルスから取り出すことができる。 上記カルスは少量の外植体より誘導 され、簡便な設備で以て再現性良く大量培養することができる。加えて、 該カルスは、 多剤耐性癌克服作用を有する上記化合物を比較的多量に含 んでいる。 即ち、 本発明にかかる製造方法 Aによれば、 環境を保全しな がら、 日本ィチイから上記化合物 (40) を効率的に製造することがで きる。 これにより、 癌細胞に投与するのに好適な多剤耐性癌克服剤を、 効率的に製造することができる。  In addition, the compound (40) can be extracted from calli obtained by culturing the tissue of Yew Tree. The callus is derived from a small amount of explants and can be cultured in large quantities with simple equipment with good reproducibility. In addition, the callus contains a relatively large amount of the above compound having a multidrug-resistant cancer overcoming effect. That is, according to the production method A according to the present invention, the above-mentioned compound (40) can be produced efficiently from Nippon yichi, while preserving the environment. This makes it possible to efficiently produce a multidrug-resistant cancer-overcoming agent suitable for administration to cancer cells.
一方、 上記化合物 (41) 〜 (47) は、 日本ィチイの針葉部から取 り出すことができる。 即ち、 本発明にかかる製造方法 Bによれば、 毎年 再生される針葉部を使用するので、 環境を保全しながら、 日本イチィか ら上記化合物 (41) 〜 (47) を製造することができる。 これにより、 癌細胞に投与するのに好適な多剤耐性癌克服剤を、 環境を保全しながら 製造することができる。  On the other hand, the compounds (41) to (47) can be extracted from the needle portion of yew tree. That is, according to the production method B of the present invention, the needles regenerated every year are used, so that the compounds (41) to (47) can be produced from Nippon Ichi, while preserving the environment. . This makes it possible to produce a multidrug-resistant cancer-overcoming agent suitable for administration to cancer cells while preserving the environment.
このようにして製造される式 (6) 〜 (47) で表される化合物以外 の化合物で式 (1) 〜 (5) の範囲に属する化合物の詳細な製造方法を 下記する。  The following is a detailed method for producing compounds other than the compounds represented by the formulas (6) to (47) and belonging to the formulas (1) to (5).
式 (6) 〜 (47) で表される化合物は例えばァセチル基、 式 The compounds represented by the formulas (6) to (47) are, for example, an acetyl group, a compound represented by the formula
又はOr
Figure imgf000053_0001
Figure imgf000053_0001
Figure imgf000053_0002
Figure imgf000053_0002
等で示されるァシル基を加水分解で脱ァシル化して水酸基とし、 所望に よりァシル化反応で異なるァシル基を導入することにより水酸基をエス テル化するか、 あるいは脱ァシル化生成した水酸基をエーテル化するか あるいは、 エポキシ基を開裂して水酸基を生成させてこれを上記のよう にエステル化またはエーテル化するか、 あるいは Is converted to a hydroxyl group by hydrolysis to give a hydroxyl group, and if desired, a different acyl group is introduced in the acylation reaction to esterify the hydroxyl group, or etherify the deoxylated hydroxyl group. Alternatively, the epoxy group is cleaved to form a hydroxyl group, which is Esterification or etherification to
0  0
II  II
一 C一 を還元して  Reducing one C1
H H
— C——  — C——
OH とし、 この水酸基を上記と同様ににエステル化あるいはエーテル化する 等自体公知の化学修飾反応によって、 容易に製造される。 このための加 水分解反応、 エステル化反応、 エーテル化反応還元反応は常法に従って 行われてよい。 これらの操作によって、 水酸基は上記した  The hydroxyl group is easily produced by a known chemical modification reaction such as esterification or etherification of OH in the same manner as described above. The hydrolysis reaction, esterification reaction, etherification reaction and reduction reaction for this purpose may be performed according to a conventional method. By these operations, the hydroxyl group is
0  0
II  II
- 0-C-Z! 、 一 O— Z 2等の置換基に変換される、 式 (6 ) 〜 (4 7 ) 以外の式 ( 1 ) 〜 (5 ) で表される化合物が容易に製造できる。 -! 0-CZ, is converted into a substituent such as one O-Z 2, (6) - (4 7) other than the Formula (1) compounds represented by - (5) can be easily produced.
式 (1 ) 〜 (4 7 ) で表される化合物は、 上記した抗癌作用物質蓄積 増強作用または抗癌作用物質排出抑制作用に基づき、 癌克服剤、 特に多 剤耐性癌克服剤として有用である。 該多剤耐性癌克服剤は、 多剤耐性を 有する癌細胞 (多剤耐性癌細胞) 内に取り込まれた様々な抗癌作用物質 (抗癌剤) を、 多剤耐性癌細胞外に排出する複数の作用機構のいずれか を阻害する作用を有するものである。 多剤耐性でなくて、 通常の抗癌作 用物質に耐性を有する癌細胞に対しても、 式 ( 1 ) 〜 (4 7 ) の化合物 は同じ作用機構を示す (以下同じ)。 また、 耐性癌細胞に対してだけでは なくして、 通常の癌細胞に対しても、 式 (1 ) 〜 (4 7 ) で表される化 合物は同じ作用機構を示す (以下同じ)。  The compounds represented by the formulas (1) to (47) are useful as a cancer-surviving agent, particularly a multidrug-resistant cancer-surviving agent, based on the above-mentioned anticancer substance accumulation enhancing action or anticancer substance excretion inhibitory action. is there. The multidrug-resistant cancer-overcoming agent comprises a plurality of cancer cells having multidrug resistance (multidrug-resistant cancer cells) that excrete various anticancer active substances (anticancer agents) taken out of the multidrug-resistant cancer cells. It has the effect of inhibiting any of the mechanisms of action. The compounds of formulas (1) to (47) exhibit the same mechanism of action on cancer cells that are not multidrug resistant but resistant to ordinary anticancer agents (the same applies hereinafter). Further, the compounds represented by the formulas (1) to (47) exhibit the same mechanism of action not only on resistant cancer cells but also on ordinary cancer cells (the same applies hereinafter).
多剤耐性癌細胞外に排出する作用機構として、 具体的には、 例えば、 多剤耐性癌細胞において存在または発現する P —糖蛋白質が、 多剤耐性 癌細胞内に取り込まれた様々な抗癌剤と結合し、 これらを能動輸送によ り排出する機構等を挙げることができるが特にこれに限定されるもので はない。 本発明はこのようは作用機構によって、 優れた癌治療に貢献す る。 As an action mechanism of excretion out of multidrug-resistant cancer cells, specifically, for example, Mechanisms exist in which P-glycoprotein present or expressed in multidrug-resistant cancer cells binds to various anticancer drugs incorporated into multidrug-resistant cancer cells and excretes them by active transport. It is not particularly limited to this. The present invention contributes to excellent cancer treatment by such an action mechanism.
上記の化学式 (1 ) 〜 (4 7 ) で表される化合物は、 それぞれ単独で、 少なくとも P—糖蛋白質による抗癌剤の能動輸送の阻害剤 (以下、 P— 糖蛋白質阻害剤と称する) としての機能を有し、 本発明にかかる癌克服 剤もしくは多剤耐性癌克服剤となりうる。 また、 上記の化合物のうち多 くの化合物、 例えば式 (4 0 ) 〜 (4 7 ) の化合物等で表される多くの 化合物は、 従来の多剤耐性癌克服剤であるベラパミルと比較して多剤耐 性癌克服作用が高く、 以下、 高活性化合物と称する。 本発明にかかる多 剤耐性癌克服剤は、 これらの高活性化合物の少なくとも一つを含んでい ることがより好ましい。  Each of the compounds represented by the above chemical formulas (1) to (47) alone functions as an inhibitor of at least the active transport of an anticancer agent by P-glycoprotein (hereinafter referred to as a P-glycoprotein inhibitor). It can be a cancer-surviving agent or a multidrug-resistant cancer-surviving agent according to the present invention. In addition, many of the above compounds, for example, many of the compounds represented by formulas (40) to (47), are compared with verapamil which is a conventional drug-resistant cancer-overcoming agent. It has a high ability to overcome multidrug-resistant cancer, and is hereinafter referred to as a highly active compound. More preferably, the agent for overcoming multidrug resistance cancer according to the present invention contains at least one of these highly active compounds.
本発明に使用される化合物が多剤耐性癌克服作用を有することは、 本 願発明者が新たに見出したことである。 また、 式 (6 ) 〜 (4 7 ) で示 される化合物を化合物 (6 ) 〜 (4 7 ) と称することもある。  The fact that the compound used in the present invention has a multidrug-resistant cancer overcoming action is newly found by the present inventors. Further, the compounds represented by the formulas (6) to (47) may be referred to as compounds (6) to (47).
尚、 本発明にかかる多剤耐性癌克服剤は必要に応じて、 例えば、 癌細 胞の特定部位 (または P —糖蛋白質) と特異的に結合可能な抗体や、 抗 癌作用物質 (抗癌剤) 等をさらに含んで構成されていてもよい。 これに より、 標的とすべき (多剤耐性) 癌細胞への多剤耐性癌克服剤の供給を 確実とする効果や、 該癌細胞への抗癌剤の同時供給が可能となる効果等 を実現することができる。 これら多剤耐性癌克服剤の投与方法は特に限 定されるものではなく、 多剤耐性癌克服剤に含まれる物質 (上記式 (1 ) 〜 (4 7 ) で表される化合物や、 抗体、 抗癌作用物質を指す) の物性等 に応じた自体公知の最適なドラッグデリバリーシステムを採用すればよ い' 本発明で使用される化合物 ( 1 ) 〜 (4 7 ) 以外の抗癌作用物質とし ては具体的には例えば、シクロホスフアミド(CPM, Cyclophosphamide) , ナ イ ト ロ ジ ェ ン マ ス タ 一 ド ー Ν — ォ キ シ ド ( HN2-oxide, Nitrogen-mustard-N-oxide 、 イノォ フアミ r (Ifosfamide)、 メゾレフ ァラン (L-PAM, Melphalan)、 クロラムブチル (CHL, Chlorambucil)、 イミダゾ一ルカルポキサミ ド (DTIC, Imidazole carboxamide) 等のク ロル—ェチル—アミン系化合物;例えばトリエチレンフォスファラミ ド ( Thio-TEPA, Tri-ethylene-phospharamide ) 、 カルポコ ン ( CQ, Carboquone) 等のアジリジン (Azilizin) 系化合物;例えばブスルファ ン ( BUS, Buslfun )、 ィ ムプロスルフ ァ ン ト シレー ト ( 864-T, Improsulfan-tosilate) 等のスルフォン酸エステル系化合物;例えばミ ト ブロニトール (DBM, Mitobronitol) 等のエポキシド化合物;例えばニム スチン (ACNU, Nimustine-HCl) 等のニトロソ ·ウレァ系化合物;例え ばカルムスチン(BCNU, Carmustine)、口ムスチン(CCNU, lomustine)、 セムスチン (methyl-CCNU, semustine)、 G A N U等のその他のアルキ ル化剤;例えばァメプテリン (MTX, Amepterin) 等の葉酸拮抗剤;例え ば 5—フルォロウラシル(5-FU, 5-Fluoro-uracil)、テガフール(Tegafur)、 カルモフール (Carmofur)、 シタラビン (Ara'C, Cytarabin)、 エノシ夕 ビン (Enocitabine)、 テガフール · ゥラシル (Tegafururacil)、 アンシ 夕 ビン ( Ancitabine ) 等の ピ リ ミ ジン代謝拮抗剤 ( Pyrimidine antagonist) ;例えばメルカプトプリン (6_MP, 6-Mercaptopurine)、 チ ォイノシン (6MPR, Thioinosine ) 等のプリ ン代謝拮抗剤 (Purine antagonist);例えばビンクリスチン ( VCR, Vincristine)、 ビンブラスチ ン (VLB, Vinblastine)、 ビンデシン (VDS, Vindestine) 等の植物アル カロイド;例えばマイトマイシン C (MMC, Mitomycin C)、 ドキソルビ シン (Doxorubicin ADR;ADM, Adriamycin) , アクラルビシン (ACR, Aclarubicine) , ブレオマイシン (BLM, Bleomycin)、 ぺプレオマイシン (PEP, Pepleomycin)、 クロモマイシン A3 (CHRM, chromomycine)、 ダクチノマイシン (dactinomycin)、 ネオカルチノスタチン (NCS, neocarzinostatin)、 ネオスラマイシン (NTM, neothramycin) 等の抗癌 性抗生物質 (Antibiotics);例えばフォスフェストロール (Fosfestrol)、 ェピチォスタノ一ル (Epitiostanol)、 メピチォスタン (Mepitiostane)、 ェチェルエストラジオール (Ethinylestradiol)、 エストラムスチン (Estramustine) 等の女性ホルモン ; 例えばフルォキシメステロン ( Fluoxymesterone )、 プロピオン酸テストステロン ( Testosterone propionate )、 プロピオン酸 ドロモス夕ノ ロン ( Dromostanolone propionate) 等の男性ホルモン;例えば夕モキシフェン (Tamoxifen) 等のエステロゲン拮抗剤;例えばシスブラチン (Cisplatinum)、 C BD C A ( JM-8, Carboplatin)、 CH I P (JM9)、 TNO— 6等の DNAキ レート剤;例えばィンターフェロン ( I FN、 Interferon;例えば I FN - , I F N— )3、 I F N—ァ) 等の B RM ( Biological response modifires) が挙げられる。 The agent for overcoming the multidrug-resistant cancer according to the present invention may be, for example, an antibody capable of specifically binding to a specific site (or P-glycoprotein) of a cancer cell, an anticancer substance (an anticancer agent) Etc. may be further included. As a result, an effect of reliably supplying a multidrug-resistant cancer-overcoming agent to target (multidrug-resistant) cancer cells, an effect of enabling simultaneous supply of an anticancer agent to the cancer cells, and the like are realized. be able to. The administration method of these multidrug-resistant cancer-overcoming agents is not particularly limited, and the substances contained in the multidrug-resistant cancer-overcoming agents (compounds represented by the above formulas (1) to (47), antibodies, An appropriate drug delivery system known per se may be adopted depending on the physical properties of the drug. Specific examples of the anticancer substance other than the compounds (1) to (47) used in the present invention include, for example, cyclophosphamide (CPM, Cyclophosphamide), Tablets — Oxides (HN 2 -oxide, Nitrogen-mustard-N-oxide, Inosfamide), Mezolefaran (L-PAM, Melphalan), Chlorambutyl (CHL, Chlorambucil), Imidazo Chloroethyl-amine compounds such as lecarpoxamide (DTIC, Imidazole carboxamide); for example, Azilizin (Azilizin) such as triethylene phosphalamide (Thi-TEPA, Tri-ethylene-phospharamide) and carpocon (CQ, Carboquone) ) -Based compounds; for example, sulfonic acid ester-based compounds such as busulfan (BUS, Buslfun) and improsulfant silicate (864-T, Improsulfan-tosilate); for example, mitobronitol (DBM, Mitobronit) ol) or the like; for example, nitroso-urea compounds such as nimustine (ACNU, Nimustine-HCl); for example, carmustine (BCNU, Carmustine), oral mucin (CCNU, lomustine), semustine (methyl-CCNU, semustine) And other alkylating agents such as GANU; folate antagonists such as amapterin (MTX, Amepterin); for example, 5-FU, 5-Fluoro-uracil, Tegafur, Carmofur , Cytarabine (Ara'C, Cytarabin), Enocitabine, Tegafururacil, Tegafururacil, Ancitabine, etc. Pyrimidine antagonists; for example, mercaptopurine (6_MP, Purine antagonists such as 6-Mercaptopurine) and thioinosine (6MPR, Thioinosine); for example, vincristine (VCR, Vincristine), vinblastine (VLB, Vinblastine), vindesine (VDS, Vindestine) or the like of the plant Al Caroids: For example, mitomycin C (MMC, Mitomycin C), doxorubicin (Doxorubicin ADR; ADM, Adriamycin), aclarubicin (ACR, Aclarubicine), bleomycin (BLM, Bleomycin), ぺ preomycin (PEP, Pepleomycin), chromomycin A3 ( Anticancer antibiotics (Antibiotics) such as CHRM, chromomycine, dactinomycin, neocarzinostatin (NCS, neocarzinostatin), and neosuramycin (NTM, neothramycin); for example, Fosfestrol, Epithiostano Female hormones such as Epitiostanol, Mepitiostane, Ethinylestradiol, and Estramustine; for example, Fluoxymesterone, Testosterone propionate, Dromose propionate Dromostanolon male hormones such as epropionate); estrogen antagonists such as tamoxifen; DNA masks such as cisplatin (Cisplatinum), CBDCA (JM-8, Carboplatin), CHIP (JM9), and TNO-6 Rate agents; for example, BRM (biological response modifires) such as interferon (IFN, Interferon; for example, IFN-, IFN-) 3, IFN-a).
もっとも式 ( 1) 〜 (47) で示される化合物は上記したように、 抗 癌作用物質蓄積増強作用または抗癌作用物質排出抑制作用以外に抗癌作 用をも示すから、 上記の抗癌作用物質の併用もしくは共存は必ずしも必 要ではない。  However, as described above, the compounds represented by the formulas (1) to (47) also have an anticancer effect in addition to an anticancer active substance accumulation enhancing action or an anticancer active substance excretion inhibitory action. Combination or coexistence of substances is not necessary.
従って、 本発明の医薬は式 ( 1) 〜 (47) で示される化合物と所望 によりそれ以外の上記の抗癌作用物質とを癌患者に投与することによつ て、 癌治療効果を奏する事ができる。 投与に際しては、 式 (1) 〜 (4 7) の化合物とそれ以外の抗癌作用物質を一緒に含む医薬製剤を投与し てもよいし、 式 ( 1 ) 〜 (4 7 ) の化合物を含む製剤とそれ以外の抗癌 作用物質を含む製剤を別々に製造しておき、 それらを同時にあるいは 別々に投与するようにしてもよい。 もちろん、 式 ( 1 ) 〜 (4 7 ) で示 される化合物以外の抗癌作用物質は単一種類でもよいし、 複数種類でも よい。 複数種類の場合は、 それぞれ別の製剤であってもよい。 それは同 時に投与されてもよいし、 別々に投与されてもよい。 Therefore, the medicament of the present invention can exert a therapeutic effect on cancer by administering a compound represented by any one of the formulas (1) to (47) and, if desired, the other anticancer active substance to a cancer patient. Can be. Upon administration, a pharmaceutical preparation containing the compounds of formulas (1) to (47) together with another anticancer agent is administered. Alternatively, a preparation containing the compounds of the formulas (1) to (47) and a preparation containing the other anticancer agent may be separately prepared, and they may be administered simultaneously or separately. Good. Of course, the anticancer substance other than the compounds represented by the formulas (1) to (47) may be a single type or a plurality of types. In the case of plural types, different preparations may be used. It may be administered at the same time or separately.
本発明の医薬は、 抗癌剤として、 例えば、 白血病、 悪性リンパ腫、 骨 髄腫などの非固形癌、 あるいは胃がん、 食道癌、 大腸、 直腸、 滕臓癌、 肝臓癌、 腎臓癌、 膀胱癌、 肺癌、 子宮癌、 卵巣癌、 乳癌、 前立腺癌、 皮 膚癌、 脳腫瘍癌などの固形癌の治療に用いることができる。  The medicament of the present invention includes, as anticancer agents, non-solid cancers such as leukemia, malignant lymphoma, and myeloma, or stomach cancer, esophagus cancer, large intestine, rectum, Teng's cancer, liver cancer, kidney cancer, bladder cancer, lung cancer, It can be used to treat solid cancers such as uterine cancer, ovarian cancer, breast cancer, prostate cancer, skin cancer, and brain tumor cancer.
本発明の医薬としては、有効成分である上記物質(すなわち上記式( 1 ) As the medicament of the present invention, the above substance as an active ingredient (that is, the above formula (1)
〜 (4 7 ) で示される化合物あるいはそれと抗癌作用物質) をそのまま 投与してもよいが、 一般的には、 有効成分である上記の物質とさらに 1 または 2以上の製剤用添加物とを含む医薬組成物の形態で投与すること が望ましい。 このような医薬組成物は、 それ自体製剤学の分野で周知ま たは慣用の方法に従って製造することが可能である。 また、 医薬組成物 の形態の本発明の医薬には、 所望により、 上記した以外の他の医薬の有 効成分が 1または 2以上含まれていてもよい。 なお、 本発明の医薬は、 ヒトを含む哺乳類動物に摘要可能である。 (47) or a compound thereof and an anticancer agent) may be administered as it is, but generally, the above-mentioned substance as an active ingredient is further combined with one or more pharmaceutical additives. It is desirable to administer it in the form of a pharmaceutical composition containing the same. Such a pharmaceutical composition can be manufactured according to a method known per se or a conventional method in the field of pharmaceuticals. The drug of the present invention in the form of a pharmaceutical composition may optionally contain one or more active ingredients of other drugs other than those described above. The medicament of the present invention can be applied to mammals including humans.
本発明の医薬の投与経路は特に限定されず、 経口または非経口投与の いずれかから治療及び/または予防のために最も効果的な投与経路を適 宜選択することができる。非経口投与としては、 気道内、 直腸内、 皮下、 筋肉内、 及び静脈内などの投与経路を挙げることができる。 経口投与に 適する製剤の例としては、 例えば、 錠剤、 顆粒剤、 散剤、 シロップ剤、 溶液剤、 カプセル剤、 または懸濁剤などを挙げることができ、 非経口投 与に適する製剤の例と経粘膜吸収剤などを挙げることができる。 経口投与に適する液体製剤の製造には、 例えば、 水、 蔗糖、 ソルビッ 卜、 果糖などの糖類;ポリエチレングリコール、 プロピレングリコール などのグリコール類; ごま油、 オリ一ブ油、 大豆油などの油類; P —ヒ ドロキシ安息香酸エステル類などの防腐剤などの製材用添加物を用いる ことができる。 また、 カプセル剤、 錠剤、 散剤、 または顆粒剤などの固 形製剤の製造には、 例えば、 乳糖、 ブドウ糖、 蔗糖、 マンニットなどの 賦形剤;澱粉、 アルギン酸ソーダなどの崩壊剤;ステアリン酸マグネシ ゥム、 タルクなどの潤沢剤;ポリビニルアルコール、 ヒドロキシプロピ ルセルロース、 ゼラチンなどの結合剤;脂肪酸エステルなどの界面活性 剤;グリセリンなどの可塑剤を用いることができる。 The administration route of the medicament of the present invention is not particularly limited, and the most effective administration route for treatment and / or prevention can be appropriately selected from either oral or parenteral administration. Parenteral administration can include routes of administration such as intratracheal, rectal, subcutaneous, intramuscular, and intravenous. Examples of preparations suitable for oral administration include, for example, tablets, granules, powders, syrups, solutions, capsules, and suspensions. Examples of preparations suitable for parenteral administration include: Mucosal absorbents and the like can be mentioned. For the production of liquid preparations suitable for oral administration, for example, sugars such as water, sucrose, sorbitol, fructose; glycols such as polyethylene glycol and propylene glycol; oils such as sesame oil, olive oil and soybean oil; — Lumber additives such as preservatives such as hydroxybenzoic acid esters can be used. For the production of solid preparations such as capsules, tablets, powders, or granules, for example, excipients such as lactose, glucose, sucrose, mannitol; disintegrants such as starch and sodium alginate; Lubricants such as gum and talc; binders such as polyvinyl alcohol, hydroxypropyl cellulose and gelatin; surfactants such as fatty acid esters; plasticizers such as glycerin can be used.
非経口投与に適する製剤のうち注射剤や点滴剤などの血管内投与用製 剤は、 好ましくはヒト血液と等張の水性媒体を用いて調製することがで きる。 例えば、 注射剤は、 塩溶液, ブドウ糖溶液, または塩水とブドウ 糖溶液の混合物から選ばれる水性媒体を用い、 常法に従って適当な助剤 と共に溶液、 懸濁液、 または分散液として調製することができる。 腸内 投与のための座剤は、 例えばカカオ脂、 水素化脂肪または水素化カルボ ン酸などの担体を用いて調製することができる。 噴霧剤は、 ヒトの口腔 及び気道粘膜を刺激せず、 かつ有効成分である上記の物質を微細な粒子 として分散させて吸収を促進することのできる単体を用いて調製するこ とができる。 このような担体として、 例えば、 乳糖またはダルセリンな どのを用いることができる。 有効成分である上記物質及び用いる担体の 性質により、 エアロゾルやドライパウダーなどの形態の調剤として調製 することができる。 非経口用の製剤の製造には、 例えば、 希釈剤、香料、 防腐剤、 賦形剤、 滑沢剤、 結合剤、 界面活性剤、 可塑剤などから選択さ れる 1または 2以上の調剤用添加物を用いることができる。 なお、 本発 明の医薬の形態及びその製造方法は、 上記に具体的に説明したものに限 定されることはない。 Among preparations suitable for parenteral administration, preparations for intravascular administration such as injections and drops can be prepared preferably using an aqueous medium isotonic with human blood. For example, an injection may be prepared as a solution, suspension, or dispersion using an aqueous medium selected from a salt solution, a glucose solution, or a mixture of a salt solution and a glucose solution together with appropriate auxiliaries according to a conventional method. it can. Suppositories for enteral administration can be prepared using carriers such as cocoa butter, hydrogenated fats or hydrogenated carboxylic acids. Sprays can be prepared using a simple substance which does not irritate the human oral and respiratory tract mucosa and which can promote absorption by dispersing the above-mentioned substance as an active ingredient as fine particles. As such a carrier, for example, lactose or dalserin can be used. It can be prepared as a preparation in the form of an aerosol or dry powder depending on the properties of the above-mentioned substance as an active ingredient and the carrier used. For the production of parenteral preparations, for example, one or more preparation additives selected from diluents, flavors, preservatives, excipients, lubricants, binders, surfactants, plasticizers, etc. Things can be used. The form of the medicament of the present invention and the method for producing the medicament are limited to those specifically described above. It is not specified.
本発明の医薬の投与料及び投与頻度は特に限定されず、 有効成分であ る上記物質の種類、 治療すべき病態の種類、 投与経路、 患者の年齢及び 体重、 症状、 及び疾患の重篤度などの種々の条件に応じて医師の指示に 従って適宜選択することが可能である。 例えば、 式 (1) ~ (47) で示 される化合物またはそれ以外の抗癌作用物質の使用量については、 成人 一日あたり 0. 0 1〜200mg/k g (好ましくは 5〜: L 50 m gノ k g) 程度を一日一回または数日から数週間に一回の割合で投与するこ とができるが、 投与量及び投与頻度はこの特定の例に限定されることは ない。 また、 本発明の医薬は他の抗癌作用物質 (抗癌剤、 抗腫瘍剤) と 組み合わせて用いることができ、 一般的には、 作用機序の異なる数種類 の抗癌作用物質と組み合わせて用いることが好ましい。  The administration fee and administration frequency of the medicament of the present invention are not particularly limited, and the kind of the substance as the active ingredient, the kind of the condition to be treated, the administration route, the age and weight of the patient, the symptoms, and the severity of the disease According to various conditions such as the above, it is possible to appropriately select according to a doctor's instruction. For example, the amount of the compound represented by any of the formulas (1) to (47) or the other anticancer substance is 0.01 to 200 mg / kg per day for an adult (preferably 5 to 50 mg L). (Kg / d) can be administered once a day or once every few days to once every several weeks, but the dosage and frequency of administration are not limited to this particular example. In addition, the medicament of the present invention can be used in combination with other anticancer agents (anticancer agents, antitumor agents), and generally used in combination with several kinds of anticancer agents having different mechanisms of action. preferable.
また、 式 (1) 〜 (47) の化合物は抗菌作用を示し、 例えば Bacillus Subtilis等に対して、 ≤ 1 000 r/c cの最小発育阻止濃度 (Minimum Inhibitory Concentration) を示すので、 これを常法により、 軟膏とし て例えば湿疹、 吹出物、 おでき、 にきび治療のための皮膚外用剤とする ことができる。  In addition, the compounds of formulas (1) to (47) show antibacterial activity, for example, a minimum inhibitory concentration of ≤1 000 r / cc against Bacillus Subtilis and the like. As a result, it can be used as an ointment, for example, a skin external preparation for treating eczema, pimples, boils, and acne.
〔実施例〕 〔Example〕
以下、 実施例により、 本発明をさらに詳細に説明するが、 本発明はこ れらにより何ら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 thereto.
〔多剤耐性癌克服作用の測定方法〕  (Measuring method of overcoming multidrug-resistant cancer)
前記化合物群に属する幾つかの化合物の多剤耐性癌克服作用 (ここで は、 P—糖蛋白質阻害剤としての機能) を、 以下に示すバイオアツセィ 法にて測定した。 該バイオアツセィ法は、 2780 AD細胞 (ヒト卵巣 癌細胞 A 2780より樹立された多剤耐性癌細胞)、 ビンクリスチン (抗 癌剤の一種)、 および、 P—糖蛋白質阻害剤の候補物質 (前記化合物群に 属する化合物に相当) を、 それぞれ所定量含んでなる反応溶液をゥエル にアプライし、 所定の温度条件下で一定時間反応させることにより行つ た。 The action of overcoming the multidrug-resistant cancer (in this case, the function as a P-glycoprotein inhibitor) of some compounds belonging to the compound group was measured by the bioassay method shown below. The bioassay method comprises 2780 AD cells (multidrug-resistant cancer cells established from human ovarian cancer cells A 2780), vincristine (anti- A reaction solution containing a predetermined amount of a cancer drug) and a candidate substance of a P-glycoprotein inhibitor (corresponding to a compound belonging to the above-mentioned compound group) is applied to each well, and is kept constant under a predetermined temperature condition. The reaction was performed by reacting for hours.
2 7 8 0 A D細胞にビンクリスチンを作用させると、 ビンクリスチン は該細胞に一旦は取り込まれるものの、 細胞膜に存在或いは発現する P 一糖蛋白質により細胞外へ排出されて、 その総取り込み量 (蓄積量) は 低い値を示す。 しかし、 P—糖蛋白質阻害剤としての機能を有する物質 をさらに加えると、 ビンクリスチンの 2 7 8 0 A D細胞への総取り込み 量が増加する。 上記の測定方法は、 この現象を利用したものである。 各測定試験のコントロールとしては、 上記反応溶液より P —糖蛋白質 阻害剤の候補物質を除いたものを使用し、 該コントロールと比較するこ とにより、 前記化合物群に属する幾つかの化合物の多剤耐性癌克服作用 を判定した。 また、 コントロールとの比較に加えてベラパミルを対照薬 剤とした比較実験も併せて行い、 ベラパミルを上回る多剤耐性癌克服作 用を有する化合物 (高活性化合物) の評価を行った。 尚、 ベラパミルを 対照薬剤とした比較とは、 各反応溶液において、 P —糖蛋白質阻害剤の 候補物質に代えて、 これと同濃度のベラパミルを用いて行われる比較実 験のこどを指す。  2780 When vincristine is allowed to act on AD cells, vincristine is once taken up by the cells, but is excreted extracellularly by P-glycoprotein present or expressed in the cell membrane, and the total uptake (accumulated amount) Indicates a low value. However, the addition of a substance that functions as a P-glycoprotein inhibitor increases the total uptake of vincristine into 279 AD cells. The above measurement method utilizes this phenomenon. As a control for each measurement test, a reaction solution excluding the candidate substance of the P-glycoprotein inhibitor from the above reaction solution was used, and by comparing with the control, a multi-drug of several compounds belonging to the compound group was obtained. The effect of overcoming resistant cancer was determined. In addition to the comparison with the control, a comparative experiment using verapamil as a control drug was also performed to evaluate compounds (highly active compounds) having a multidrug-resistant cancer-surpassing action superior to verapamil. The comparison with verapamil as a control drug refers to a comparative experiment performed using verapamil at the same concentration as the P-glycoprotein inhibitor candidate substance in each reaction solution.
尚、上記の測定方法に関する参考文献としては、例えば、 Rogan, A.M., et al. Reversal oi adriamycin resistance by verapamil in human ovarian cancer. Science, 224: 994-996, 1984や、 Broxterman, H.J., et al. Increase of daunorubicin and vincristine accumulation in multidrug resistant human ovarian carcinoma cells by a monoclonal antibody reacting with P-glycoprotein. Biochemical Pharmacology, 37: 2389-2393, 1988力挙げられる。 判定基準は、 各測定試験におけるピンクリスチンの総取り込み量が、 そのコントロールにおけるピンクリスチンの総取り込み量に対して 9 0 %以下である場合を 「一」、 9 0 %を超えて 1 1 0 %以下である場合を 「土」、 1 1 0 %を超えて 3 0 0 %以下である場合を 「十」、 3 0 0 %を 超えて 5 0 0 %以下である場合を 「十十」、 5 0 0 %を超えて 1 0 0 0 % 以下である場合を 「+ + +」、 1 0 0 0 %を超えて 2 0 0 0 %以下である 場合を 「+ + + +」、 2 0 0 0 %を超える場合を 「+ + + + +」 の 7段階 とした。 そして、 上記判定基準に基づく評価基準としては、 ベラパミル 以上の多剤耐性癌克服作用が認められる場合を 「P」、 ベラパミル以上の 多剤耐性癌克服作用が認められない場合を 「N」 とした。 In addition, as a reference about the above-mentioned measurement method, for example, Rogan, AM, et al. Reversal oi adriamycin resistance by verapamil in human ovarian cancer.Science, 224: 994-996, 1984, Broxterman, HJ, et al. Increase of daunorubicin and vincristine accumulation in multidrug resistant human ovarian carcinoma cells by a monoclonal antibody reacting with P-glycoprotein. Biochemical Pharmacology, 37: 2389-2393, 1988. The criterion is `` one '' when the total amount of pinklistin uptake in each measurement test is 90% or less of the total pinklistin uptake in the control, and more than 90% to 110% If it is less than or equal to `` soil '', it is more than 110% and less than 300%, `` ten '', if it is more than 300% and less than 500%, it is `` ten '', `` +++ '' when it is more than 500% and less than 100%, `` +++ '' when it is more than 100% and less than 200% The case where the value exceeds 0% is defined as seven levels of “++++++”. And, as the evaluation criteria based on the above judgment criteria, `` P '' indicates that a drug-overcoming effect of verapamil or higher is recognized, and `` N '' indicates that no drug-overcoming effect of verapamil or higher is detected. .
即ち、 上記 「十」 以上の段階であると判定された P—糖蛋白質阻害剤 の候補物質は、 P—糖蛋白質阻害剤としての機能 (多剤耐性癌克服作用) を明らかに有していると判断される。 一方、 上記 「土」 の段階であると 判定された P —糖蛋白質阻害剤の候補物質は、 P—糖蛋白質阻害剤とし ての機能が明確には認められないと判断される。 また、 上記 「P」 と評 価された P —糖蛋白質阻害剤の候補物質は、 高活性化合物であると判断 される。  That is, the candidate substance of the P-glycoprotein inhibitor determined to be in the above “ten” stage clearly has a function as a P-glycoprotein inhibitor (multidrug-resistant cancer overcoming action). Is determined. On the other hand, it is judged that the candidate substance of the P-glycoprotein inhibitor determined to be in the “soil” stage does not clearly recognize the function as the P-glycoprotein inhibitor. In addition, the candidate substance for a P-glycoprotein inhibitor evaluated as “P” is judged to be a highly active compound.
〔実施例 1〕 (Example 1)
樹高 2 mの日本ィチイの針葉部 (生針葉部) からタキサン関連化合物 である化合物 (1 7 )、 化合物 (1 8 )、 化合物 (2 2 ) および化合物 (2 6 ) を取り出した。 即ち、 上記の針葉部 1 2 4 8 gを n—へキサン 8 L に 1週間浸漬することにより脱脂した後、 酢酸ェチル 8 Lに浸漬した。 1週間浸漬した後、針葉部を濾別し、濾液である抽出液を得た。次いで、 抽出液から酢酸ェチルを室温で減圧除去し、 該酢酸ェチルに可溶な成分 (粗酢酸ェチル抽出物) 1 9 . 6 9 gを得た。 上記の成分を、 メチルアルコールと酢酸ェチルとを容量比 1 : 3で混 合してなる混合溶液 40 Om 1に溶解した。 次に、 該溶液を、 酸性水溶 液である 0. 5 M硫酸 10 Om lで 3回 (3 X 100m l ) 洗浄し、 続 いて、 塩基性水溶液である 2 M水酸化ナトリウム水溶液 1 0 Om 1で 2 回 (2 X 1 0 Om 1 ) 洗浄した。 さらに、 上記の溶液 (油層) を飽和食 塩水で洗浄した後、 無水硫酸ナトリウムで乾燥させ、 メチルアルコール および酢酸ェチルを除去した。 これにより、 粗中性分画 (テルペン分画) 7. 49 g (針葉部の量を基準として 0. 600 %) を得た。 Compounds (17), (18), (22) and (26), which are taxane-related compounds, were extracted from the needles (raw needles) of Nippon yew with a height of 2 m. That is, 1248 g of the needle portion was degreased by dipping in 8 L of n-hexane for 1 week, and then dipped in 8 L of ethyl acetate. After soaking for one week, the needle portion was separated by filtration to obtain an extract as a filtrate. Then, ethyl acetate was removed from the extract at reduced pressure at room temperature to obtain 19.69 g of a component (crude ethyl acetate extract) soluble in the ethyl acetate. The above components were dissolved in 40 Om 1 of a mixed solution obtained by mixing methyl alcohol and ethyl acetate at a volume ratio of 1: 3. Next, the solution was washed three times (3 x 100 ml) with 0.5 M sulfuric acid (10 Oml), which is an acidic aqueous solution, and subsequently, a basic aqueous solution (2 M aqueous sodium hydroxide solution, 10 Om1). 2 times (2 X 10 Om 1). Further, the above solution (oil layer) was washed with a saturated saline solution, and then dried over anhydrous sodium sulfate to remove methyl alcohol and ethyl acetate. This gave 7.49 g of a crude neutral fraction (terpene fraction) (0.600% based on the amount of needles).
一方、 洗浄後の上記 0. 5M硫酸に 29 %アンモニア水を添加して p Hを 9. 0とした後、 クロ口ホルム 200m lで 3回 (3 X 200m l ) 抽出した。 これにより、 粗アルカロイド分画 3. 65 g (針葉部の量を 基準として 0. 292 %) を得た。 また、 洗浄後の上記 2 M水酸化ナト リウム水溶液からフエノール分画 1. 08 g (同 0. 087 %) を得た。 次に、 上記の粗中性分画を、 シリカゲルを用いたオープンカラムクロ マトグラフィーを採用して、 溶媒として n—へキサンと酢酸ェチルとメ チルアルコールとを用いたグラジュェント溶出を行うことにより、 〜F10の 1 0個のフラクションに分画 (荒分け) した。 Fi 〜F8 の溶出 液は酢酸ェチル: n—へキサンが容量比 2 : 3で混合してなる混合溶液 であり、 F9 の溶出液は酢酸ェチルであり、 F10の溶出液はメチルアル コールであった。 各フラクションの収量は、 1番目 (低極性側) から 1 0番目(高極性側)に向かって順に、 Fi S S Omg. Fa Z Z S Smg, F3 722 mg, F4 405mg, F5 3 1 1 mg, F6 208mg, F 7 1 77mg, F8 144mg, F9 237mg, Fi0 205 1mgであ つた。 On the other hand, the pH was adjusted to 9.0 by adding 29% aqueous ammonia to the above 0.5M sulfuric acid after washing, and then extracted three times (3 × 200 ml) with 200 ml of chloroform. This gave 3.65 g of crude alkaloid fraction (0.292% based on the amount of needles). Further, 1.08 g (0.087% of the same) of the phenol fraction was obtained from the above-mentioned 2 M aqueous sodium hydroxide solution after washing. Next, the crude neutral fraction was subjected to gradient elution using n-hexane, ethyl acetate, and methyl alcohol as solvents, by employing open column chromatography using silica gel. It was fractionated (roughly divided) into 10 fractions of F10. Fi eluate to F 8 are acetate Echiru: hexane n- capacity ratio 2: mixed solution prepared by mixing 3, eluant F 9 is acetic Echiru, eluant F 10 is Mechiruaru call Met. The yield of each fraction is Fi SS Omg. Fa ZZS Smg, F 3 722 mg, F 4 405 mg, F 5 3 1 1 mg in order from 1st (low polarity side) to 10th (high polarity side). , F 6 208 mg, F 7 1 77 mg, F 8 144 mg, F 9 237 mg, Fi 0 205 1 mg.
そして、 上記 2番目のフラクション (F2 ) を、 シリカゲルステンレ スカラム (INERTSIL PREP-SIL, GL Science, 250 X 10mm i.d., UV 254nm ) を用いた順相高速液体クロマトグラフィーを採用して、 酢酸ェ チルと n—へキサンとを容量比 3 : 7で混合してなる混合溶液 (キヤリ ァ, 流速 5. 0 ml/min.) を用いて分離し、 保持時間 ( t R) 1 2. 0 〜45. 2分の分画 962mgを分取した。 これを上記カラムを用 いて同一条件でさらに分離し、 保持時間 0〜 1 7. 0分の分画 F2.2 26 9mg、 および保持時間 1 7. 0〜2 1. 8分の分画 F2-3 479. 2 m gを分取した。分画 F2-2 を上記カラムを用いて同一条件でさらに分離し、 保持時間 0〜 14. 0分の分画 F2-4 48. Omgを分取した。 Then, the second fraction (F 2 ) was applied to a silica gel stainless steel column (INERTSIL PREP-SIL, GL Science, 250 × 10 mm id, UV Using a normal-phase high-performance liquid chromatography with 254 nm), a mixed solution of ethyl acetate and n-hexane mixed at a volume ratio of 3: 7 (carrier, flow rate 5.0 ml / min. ) And 962 mg of a fraction with a retention time (tR) of 12.0 to 45.2 minutes was collected. This was further separated under the same conditions have use the column, retention time 0-1 7.0 minute fractions F 2. 2 26 9 mg, and retention time 1 7.0 to 2 1.8 min fractions F 2 - 3 was collected in the 479. 2 mg. Fraction F 2 - 2 was further separated under the same conditions using the above column, retention time 0 to 14.0 min fractions F 2 - 4 were separated and 48. Omg.
得られた分画 F 2.4 を、 O D Sステンレスカラム ( INERTSIL PREP-ODS, GL Science, 250 X 10mm i.d., UV 254nm )を用いた逆相高 速液体クロマトグラフィーを採用して、 メチルアルコールと 0. 05M 酢酸アンモニゥム緩衝液(PH4. 8) とァセトニトリルとを容量比 1 : 1 : 2で混合してなる混合溶液 (キャリア, 流速 5. 0 ml/min.) を用 いて単離 ·精製した。 これにより、 化合物 (26) 1 5. Omg (保持 時間 1 5. 1分) を取り出した。 Fractions F 2. 4 obtained, employ ODS stainless steel column (INERTSIL PREP-ODS, GL Science , 250 X 10mm id, UV 254nm) reverse-phase high-performance liquid chromatography with a methyl alcohol 0 Isolation and purification were performed using a mixed solution (carrier, flow rate 5.0 ml / min.) Obtained by mixing .05M ammonium acetate buffer (PH4.8) and acetonitrile at a volume ratio of 1: 1: 2. As a result, compound (26) 15.Omg (retention time 15.1 minutes) was obtained.
また、同様の操作を行うことにより、分画 F2-3から化合物(26) 1. 4mgを得た。 従って、 粗中性分画から化合物 (26) 1 6. 4mg (針 葉部の量を基準として 0. 00 1 3 %) を得た。 一方、 同様の操作を行 うことにより、 粗アルカロイド分画から化合物 (26) 6. lmgを得 た。 従って、 粗中性分画および粗アルカロイド分画から化合物 (26) 22. 5mg (同 0. 00 18 %) を得た。 Further, by performing the same operation, fractionation F 2 - was obtained 3 from the compound (26) 1. 4mg. Therefore, 16.4 mg of compound (26) (0.0013% based on the amount of needles) was obtained from the crude neutral fraction. On the other hand, by performing the same operation, 6.1 mg of the compound (26) was obtained from the crude alkaloid fraction. Therefore, 22.5 mg (0.0018%) of the compound (26) was obtained from the crude neutral fraction and the crude alkaloid fraction.
さらにまた、 上記分画 F2-3 から保持時間 26. 4分の分画 F2-5 (ピ ーク) 14. 5mgを分取した。 これを上記カラムを用いて同一条件で さらに分離し、 単離 ·精製することにより、 化合物 ( 1 7) 4. 8mg (保持時間 40. 3分) を取り出した。 Furthermore, the fraction F 2 - was aliquoted 5 (peak) 14. 5 mg - 3 retention time from 26.4 min fractions F 2. This was further separated under the same conditions using the above-mentioned column, and then isolated and purified, thereby obtaining 4.8 mg of the compound (17) (retention time: 40.3 minutes).
また、同様の操作を行うことにより、分画 F2-2から化合物(1 7) 1. 4mgを得た。 従って、 粗中性分画から化合物 (1 7) 6. 2mg (針 葉部の量を基準として 0. 0005 %) を得た。 Further, by performing the same operation, fractionation F 2 - 2 from the compound (1-7) 1. 4 mg were obtained. Therefore, 6.2 mg of the compound (17) (0.0005% based on the amount of needles) was obtained from the crude neutral fraction.
また、 前記 3番目のフラクション (F3 ) を、 シリカゲルステンレス カラム (INERTSIL PREP-SIL, GL Science, 250 X 20mm i.d., UV 254nm ) を用いた順相高速液体クロマトグラフィーを採用して、 酢酸ェ チルと n—へキサンとを容量比 1 : 1で混合してなる混合溶液 (キヤリ ァ, 流速 1 5 ml/min.) を用いて分離し、 保持時間 1 8. 0〜22. 0 分の分画 1 64. 7mgを分取した。 これを上記カラムを用いて同 一条件でさらに分離し、 保持時間 1 7. 5分の分画 F3-2 (ピーク) 1 5 4. 4mgを分取した。 次いで、 分画 F3-2 を、 ODSステンレスカラム (INERTSIL PREP-ODS, GL Science, 250 X 10mm i.d., UV 254nm ) を用いた逆相高速液体クロマトグラフィーを採用して、 メチルアルコ一 ルと 0. 05M酢酸アンモニゥム緩衝液 (pH4. 8) とァセトニトリ ルとを容量比 1 : 1 : 2で混合してなる混合溶液 (キャリア, 流速 5. 0 ml/min.) を用いて単離 ·精製した。 これにより、 化合物 (22) 4. 4mg (保持時間 9. 8分、 針葉部の量を基準として 0. 0004 %) を得た。 Further, the third fraction (F 3 ) was subjected to normal-phase high-performance liquid chromatography using a silica gel stainless steel column (INERTSIL PREP-SIL, GL Science, 250 × 20 mm id, UV 254 nm) to obtain ethyl acetate. And n-hexane at a volume ratio of 1: 1 using a mixed solution (carrier, flow rate 15 ml / min.), And the retention time is 18.0 to 22.0 minutes. Fraction 1 64.7 mg was dispensed. They were separated and 2 (peak) 1 5 4. 4 mg - which fractions F 3 further separated, retention time 1 7.5 minutes at the same conditions using the above column. Then, fractions F 3 - a 2, ODS stainless steel column (INERTSIL PREP-ODS, GL Science , 250 X 10mm id, UV 254nm) employs a reverse phase high performance liquid chromatography using methyl alcohol one le and 0 A 0.05M ammonium acetate buffer (pH 4.8) and acetonitrile were mixed and mixed at a volume ratio of 1: 1: 2 (carrier, flow rate 5.0 ml / min.) And purified. . This gave 4.4 mg of compound (22) (retention time: 9.8 minutes, 0.0004% based on the amount of needles).
また、 前記 5番目のフラクション (F5 ) を、 シリカゲルステンレス カラム (INERTSIL PREP-SIL, GL Science, 250 X 10mm i.d., UV 254nm ) を用いた順相高速液体クロマトグラフィーを採用して、 酢酸ェ チルと n—へキサンとを容量比 1 : 1で混合してなる混合溶液 (キヤリ ァ, 流速 5. 0 ml/min.) を用いて分離し、 保持時間 1 8. 0〜3 5. 0分の分画 Fs-i 1 58. lmgを分取した。 これを、 ODSステンレス カラム (INERTSIL PREP-ODS, GL Science, 250 X 10mm i.d., UV 254nm ) を用いた逆相高速液体クロマトグラフィーを採用して、 メチル アルコールと 0. 0 5M酢酸アンモニゥム緩衝液 (pH4. 8) とァセ トニトリルとを容量比 1 : 2 : 2で混合してなる混合溶液(キャリア, 流 速 5. 0 ml/min.) を用いて単離 ·精製した。 これにより、 化合物 (1 8) 3. 3mg (保持時間 1 5. 4分、 針葉部の量を基準として 0. 0 003 %) を得た。 The fifth fraction (F 5 ) was subjected to normal-phase high-performance liquid chromatography using a silica gel stainless steel column (INERTSIL PREP-SIL, GL Science, 250 × 10 mm id, UV 254 nm) to obtain ethyl acetate. And n-hexane were mixed at a volume ratio of 1: 1 using a mixed solution (carrier, flow rate 5.0 ml / min.), And the retention time was 18.0 to 35.0 minutes. Fraction Fs-i 1 58. lmg was collected. This was purified by reversed-phase high-performance liquid chromatography using an ODS stainless steel column (INERTSIL PREP-ODS, GL Science, 250 × 10 mm id, UV 254 nm), and methyl alcohol and 0.05 M ammonium acetate buffer (pH 4 . 8) Isolation and purification were performed using a mixed solution (carrier, flow rate: 5.0 ml / min.) Obtained by mixing tonitrile with a volume ratio of 1: 2: 2. As a result, 3.3 mg of compound (18) (retention time: 15.4 minutes, 0.0003% based on the amount of needles) was obtained.
単離された化合物の同定並びに構造決定は、 インバースプローブを装 着した 500 MH zの NMRを用い、 PFG— COSY、 P FG— HM QC、 P F G-HMB C等の測定結果を解析することによって行った。 これら測定結果のうち、 新規物質である化合物 (1 7) の iH— NM Rおよび i3C— NMRの測定結果を第 1表 ·第 2表にまとめて示す。 こ れにより、 化合物 ( 1 7) の構造を解析した。 また、 該化合物 ( 1 7) の物性値等を測定した結果、 Identification and structure determination of the isolated compound are performed by analyzing the measurement results of PFG-COSY, PFG-HMQC, PFG-HMBC, etc. using 500 MHz NMR equipped with an inverse probe. went. Among these measurement results, the measurement results of iH—NMR and i 3 C—NMR of compound (17), which is a novel substance, are summarized in Tables 1 and 2. Thus, the structure of the compound (17) was analyzed. In addition, as a result of measuring physical properties and the like of the compound (17),
77ナ : し 33H4。リ 8 77 Name: to 33H 4. Re 8
分子量 : 5 64. 2 7 20 (測定値)、 564. 2 72 3 (計算値) 融 点 : 9 8で〜 1 00で  Molecular weight: 564.2720 (measured value), 564.2723 (calculated value) Melting point: 98 to 100
C α ] D 20: + 97. 3° (c = 0. 45, CHC " ) C α] D 20: + 97. 3 ° (c = 0. 45, CHC ")
I R : 36 1 0, 1 745, 1 7 14, 1 672, 1 239 c
Figure imgf000066_0001
IR: 36 1 0, 1 745, 1 7 14, 1 672, 1 239 c
Figure imgf000066_0001
であった。 Met.
17 17
14a 14a
Figure imgf000067_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000068_0001
拿 1) Multiplicities were determined by DEPT.  Na 1) Multiplicities were determined by DEPT.
*2) Connections were determined by HMQC and multiplicities and coupling constants in Hz are in (35 parentheses.  * 2) Connections were determined by HMQC and multiplicities and coupling constants in Hz are in (35 parentheses.
*3) Determined by PFG-COSY.  * 3) Determined by PFG-COSY.
*4) Correlations from C to the indicated protons.  * 4) Correlations from C to the indicated protons.
*5) NOESY cross peaks.  * 5) NOESY cross peaks.
被¾ 2 また、 新規物質である化合物 (22) の iH— NMRおよび i3C— N MRの測定結果を第 3表 ·第 4表にまとめて示す。 これにより、 化合物 (22) の構造を解析した。 また、 該化合物 (22) の物性値等を測定 した結果、 Covered 2 Tables 3 and 4 summarize the results of iH-NMR and i3C-NMR measurements of compound (22), a new substance. Thus, the structure of compound (22) was analyzed. In addition, as a result of measuring the physical properties and the like of the compound (22),
分卞式 ·· し 35H42リ 10  Byebyne formula ··· 35H42 10
分子量 : 622. 2775 (測定値)、 622. 2778 (計算値) 融 点 : 95 :〜 96  Molecular weight: 622.2775 (measured value), 622.2778 (calculated value) Melting point: 95: ~ 96
〔ひ〕 D 20 : + 22. 1 0° (c = 0. 285, CHC 13 )  (H) D 20: +22.1 0 ° (c = 0.285, CHC 13)
I R : 3600, 1 742, 1 7 12, 1 640, 1236 c
Figure imgf000069_0001
IR: 3600, 1742, 1712, 1640, 1236 c
Figure imgf000069_0001
であった。 Met.
薩 R Sp e c t r a l Da a o c omp. ( 22 ) i n CDC " Sat R Sp e c t r a l Da a o c omp. (22) in CDC
位置 13C '· 1 conected lH *2 H-H COSY ·3 HMBC ·* NOES Y*5 Position 13 C '· 1 conected l H * 2 HH COSY · 3 HMBC · * NOES Y * 5
1 79.12 (s) H2, 14a. β, 16. 17, 1-OH  1 79.12 (s) H2, 14a.β, 16.17, 1-OH
2 79.19 (d) 6.14 (d, 2.0) Η14ί HI ί H17, 19  2 79.19 (d) 6.14 (d, 2.0) {14} HI ί H17, 19
3 61.45 (d) H2, 5. 7J5. 12. 20a, b. 19  3 61.45 (d) H2, 5.7J 5. 12.20a, b. 19
4 140.92 (s) H2, 5. 20a. b  4 140.92 (s) H2, 5.20a.b
5 76.31 (d) 5.64 (br t, 9.0) H6ff. P H6a, 20a, b H6J5. 20b  5 76.31 (d) 5.64 (br t, 9.0) H6ff.P H6a, 20a, b H6J5. 20b
/)) 2.20 On) H5. 6a. 7a, J3 H5, 7β  /)) 2.20 On) H5.6a.7a, J3 H5, 7β
6 25.77 U no  6 25.77 U no
e) 1.82 (ra) H5. 6β, 7a, J9 H7な e) 1.82 (ra) H5.6β, 7a, J9 H7
) 1.78 (ra) H6fl. , 7fl H6JJ, 7a. 9 ) 1.78 (ra) H6fl., 7fl H6JJ, 7a. 9
7 31.10 Ct H9, IS 7 31.10 Ct H9, IS
a) 1.16 (m) H6e. H. 7i H6o, 7ί  a) 1.16 (m) H6e. H. 7i H6o, 7ί
8 45.20 (s) H2, 9, 10. 19  8 45.20 (s) H2, 9, 10.19
9 82.20 (d) . 19 Η7 , 17. 19  9 82.20 (d). 19 Η7, 17.19
10 79.59 (d) 5.74 (br d p mo H10 10 79.59 (d) 5.74 (br d p mo H10
, 10.0) H9 H9  , 10.0) H9 H9
o  o
11 56.12 (s) H9, 10, 12, 16, 17, 18  11 56.12 (s) H9, 10, 12, 16, 17, 18
CD  CD
12 51.58 (d) 3.35 (q, 7.1) H18 H10, 18 H18  12 51.58 (d) 3.35 (q, 7.1) H18 H10, 18 H18
13 212.89 (s) H12. 14a. 18  13 212.89 (s) H12.14a. 18
a) 2.89 (d. 20.5) Π丄 iJEJ, ^Ua  a) 2.89 (d. 20.5) Π 丄 iJEJ, ^ Ua
14 46.23 (t) H2, 1-OH  14 46.23 (t) H2, 1-OH
jS) 2.43 (br d. 20.5) H2, 14a H14 . 16  jS) 2.43 (br d. 20.5) H2, 14a H14. 16
15 45.11 (s) H10, 12. 14a, 16. 17  15 45.11 (s) H10, 12.14a, 16.17
16 23.30 (q) 1.11 Cs) H17 H14jff. 17, 18  16 23.30 (q) 1.11 Cs) H17 H14jff. 17, 18
17 22.53 (q) 1.64 (s) H16 H2, 9, 16, 19  17 22.53 (q) 1.64 (s) H16 H2, 9, 16, 19
18 15.79 (q) 1.30 (d, 7.1) H12 H12 H12, 16  18 15.79 (q) 1.30 (d, 7.1) H12 H12 H12, 16
19 26.39 (q) 1.29 (s) H9 H9, 17  19 26.39 (q) 1.29 (s) H9 H9, 17
a) 5.88 (s) H20b  a) 5.88 (s) H20b
20 129.75 (t) H5  20 129.75 (t) H5
b) 5.72 (d. 0.7) H5, 20a  b) 5.72 (d.0.7) H5, 20a
¾ 〔第 4表〕 ¾ (Table 4)
Figure imgf000071_0001
Figure imgf000071_0001
化合物 (26) の iH— NMRおよび i3C— NMRの測定結果を第 5 表 -第 6表にまとめて示す。 これにより、 化合物 (26) の構造を解析 した。 また、 該化合物 (26) の物性値等を測定した結果、 The measurement results of iH-NMR and i3C-NMR of compound (26) are summarized in Tables 5 to 6. This analyzes the structure of compound (26) did. In addition, as a result of measuring physical properties and the like of the compound (26),
分子: λ ·· 35Η44Ο9  Molecule: λ35Η44Ο9
分子量 : 608. 2993 (測定値)、 608. 2986 (計算値) 融 点 : 98で〜9 9で  Molecular weight: 608. 2993 (measured value), 608. 2986 (calculated value) Melting point: 98 to 99
〔ひ〕 D 20 : + 65. 5° (c = l. 1 54, CHC " )  [H] D 20: + 65.5 ° (c = l. 154, CHC ")
I R : 36 16, 1 740, 1640 cm-i (CHC " ) であった。 IR: 36 16, 1740, 1640 cm-i (CHC ").
NMR S e c r a l Da t a o f c omp. ( 26 ) i n CDC 1 NMR S e cr a l Da t a o f c omp. (26) in CDC 1
位置 18 C *' conected H-H COSY HMBC " NOES Y Position 18 C * 'conected HH COSY HMBC "NOES Y
I 51.22 (d) 2.09 (br d, 10.0) H2r 14i H3, 14a, β, 16, 17 H2, 14/1. 16. 17 I 51.22 (d) 2.09 (br d, 10.0) H2 r 14i H3, 14a, β, 16, 17 H2, 14/1. 16. 17
2 70.32 (d) 4.24(ddd, 6.5, 6.5, 2.0) HI. 3, 2-QH HI. 3, 14a. JJ HI, 9 17. 19  2 70.32 (d) 4.24 (ddd, 6.5, 6.5, 2.0) HI. 3, 2-QH HI. 3, 14a.JJ HI, 9 17.19
3 45.64 (d) 3.23 (br d, 6.5) H2. 20a HI. 5, 19, 20a, b H7a, 14a  3 45.64 (d) 3.23 (br d, 6.5) H2.20a HI. 5, 19, 20a, b H7a, 14a
4 143.55 (s) H3, 5. 20a, b  4 143.55 (s) H3, 5.20a, b
5 78.53 (d) 5.46 (br t. 3.0) Η6α, β H3, 20a, b Η6α, β, 20a  5 78.53 (d) 5.46 (br t.3.0) Η6α, β H3, 20a, b Η6α, β, 20a
J!) 1.91 (ddd, 13.0, 6.0. H5, 6a. 7a, β H5, 7β  J!) 1.91 (ddd, 13.0, 6.0.H5, 6a.7a, β H5, 7β
6 29 18 (t) 5.0, 3.0) H7  6 29 18 (t) 5.0, 3.0) H7
a) 1.78 (m) H5, 6fi, 7a, β H5  a) 1.78 (m) H5, 6fi, 7a, β H5
H6な, β  H6, β
7 27.55 (t) H3 5 9 19 H3, 10  7 27.55 (t) H3 5 9 19 H3, 10
H6a, β H6 19  H6a, β H6 19
8 44.43 (s) ト o  8 44.43 (s) G o
e H3 6J5. 7i. 9. 19  e H3 6J5. 7i. 9.19
9 76.69 (d) 5.89 (d. 10.5) H10 H10. 19 H2, 17, 19  9 76.69 (d) 5.89 (d.10.5) H10 H10.19 H2, 17, 19
10 72.38 (d) 6.06 (d, 10.5) H9 H9 Η7β, 18  10 72.38 (d) 6.06 (d, 10.5) H9 H9 Η7β, 18
u 133.57 (s) HI, 10. 16, 17. 18 to u 133.57 (s) HI, 10. 16, 17. 18 to
12 136.59 (s) H10, 13, 14j9. 18 12 136.59 (s) H10, 13, 14j 9.18
13 70.52 (d) 5.85 (br dd. 9.5. 7.0) Η14β, 18 HI, 14o, β, 18 Η14ί, 16  13 70.52 (d) 5.85 (br dd.9.5.7.0) Η14β, 18 HI, 14o, β, 18 Η14ί, 16
(Ϊ) 2.66 (ddd, 15.0, Η1, 14α HI. 13, 14a. 16  (Ϊ) 2.66 (ddd, 15.0, Η1, 14α HI. 13, 14a. 16
14 10.0. 9.5) HI 3  14 10.0.9.5) HI 3
a) 1.32 (dd, 15.0, 7.0) H14]S H3, 14β  a) 1.32 (dd, 15.0, 7.0) H14] S H3, 14β
15 37.73 (s) HI, 10. 14<r, 16, 17  15 37.73 (s) HI, 10.14 <r, 16, 17
16 31.73 (q) 1.15 (s) H17 HI, 13. 14β, 17  16 31.73 (q) 1.15 (s) H17 HI, 13.14β, 17
17 26.88 (q) 1.70 (s) H16 HI. 2, 9, 6  17 26.88 (q) 1.70 (s) H16 HI. 2, 9, 6
18 15.33 (q) 2.29 (br d, 1.0) H13 H10. 2'  18 15.33 (q) 2.29 (br d, 1.0) H13 H10. 2 '
19 17.91 (q) 0.96 (s) H3. 7β, 9 H2. 7β. 9  19 17.91 (q) 0.96 (s) H3.7β, 9 H2.7.7β. 9
a) 5.58 (t. 1.5) Η3, 20b H5. 20b  a) 5.58 (t.1.5) Η3, 20b H5.20b
20 119.43 (t) H3, 5  20 119.43 (t) H3, 5
b) 5.51 (s) H20a H20a b) 5.51 (s) H20a H20a
〔第 6表〕 (Table 6)
Figure imgf000074_0001
Figure imgf000074_0001
また、 化合物 (1 8 ) の物性値等を測定した結果, 分子式 : C39H46015 分子量 : 754. 2835 (測定値)、 754. 2838 (計算値) 融 点 : 222で〜 223 (CDC l 3 ) In addition, as a result of measuring the physical properties of the compound (18), the molecular formula: C 39 H 46 0 15 Molecular weight: 754. 2835 (measured value), 754.2838 (calculated) Melting point: 222 ~ 223 (CDC l 3)
〔ひ〕 D 20 : — 24. 80° (c = 0. 254, CHC 13 ) [Shed] D 20: - 24. 80 ° ( c = 0. 254, CHC 1 3)
I R : 3450, 1 732, 1642, 1 252 cmi (CH C 1 a )  I R: 3450, 1 732, 1642, 1 252 cmi (CH C 1 a)
であった。 Met.
化合物 (1 7)、 化合物 ( 1 8)、 化合物 (22) および化合物 (26) の多剤耐性癌克服作用については、 後段にて詳述する。 〔実施例 2〕  The action of compound (17), compound (18), compound (22) and compound (26) to overcome multidrug-resistant cancer will be described in detail later. (Example 2)
日本ィチイの針葉部 (生針葉部) からタキサン関連化合物である化合 物 (27)、 化合物 (28) および化合物 (29) を取り出した。 即ち、 上記の針葉部 1 0. 47 k gを n—へキサン 66. 0 Lに 1週間浸漬す ることにより脱脂した後、 酢酸ェチル 66. 0 Lに浸漬した。 1週間浸 漬した後、 針葉部を濾別し、 濾液である抽出液を得た。 次いで、 抽出液 から酢酸ェチルを室温で減圧除去し、 該酢酸ェチルに可溶な成分 (粗酢 酸ェチル抽出物) 143. 3 g (針葉部の量を基準として 1. 37 %) を得た。  Compounds (27), (28) and (29), taxane-related compounds, were extracted from the needles (raw needles) of the yew tree. That is, 10.47 kg of the needle portion was degreased by immersing it in 66.0 L of n-hexane for 1 week, and then immersed in 66.0 L of ethyl acetate. After soaking for one week, the needle portion was separated by filtration to obtain an extract as a filtrate. Then, ethyl acetate was removed from the extract under reduced pressure at room temperature to obtain 143.3 g of a component (crude ethyl acetate extract) soluble in the ethyl acetate (1.37% based on the amount of needles). Was.
上記の成分を、 メチルアルコールと酢酸ェチルとを容量比 1 : 4で混 合してなる混合溶液 2500m 1に溶解した。 次に、 該溶液を、 酸性水 溶液である 0. 5M硫酸 600m lで 3回 (3 X 600m l ) 洗浄し、 続いて、 塩基性水溶液である 2 M水酸化ナトリウム水溶液 50 Om lで 2回 (2 X 500m l ) 洗浄した。 さらに、 上記の溶液 (油層) を飽和 食塩水 200m lで 4回 (4 X 200m l ) 洗浄した後、 無水硫酸ナト リウムで乾燥させ、 メチルアルコールおよび酢酸ェチルを除去した。 こ れにより、 粗中性分画 73. 1 g (針葉部の量を基準として 0. 70 %) を得た。 The above-mentioned components were dissolved in 2500 ml of a mixed solution obtained by mixing methyl alcohol and ethyl acetate at a volume ratio of 1: 4. Next, the solution was washed three times (3 x 600 ml) with 600 ml of 0.5 M sulfuric acid as an acidic aqueous solution, and then twice with 50 Oml of a 2 M aqueous sodium hydroxide solution as a basic aqueous solution. (2 X 500 ml) Washed. Further, the above solution (oil layer) was washed four times (4 × 200 ml) with 200 ml of saturated saline, and dried over anhydrous sodium sulfate to remove methyl alcohol and ethyl acetate. This gives a crude neutral fraction of 73.1 g (0.70% based on the amount of needles) I got
一方、 洗浄後の水層である上記 0. 5M硫酸 (pH l. 5) に 29 % アンモニア水 800m 1を添加して pHを 1 0. 0とした後、 クロロホ ルム 500m lで 6回 (6 X 500m l ) 抽出した。 さらに、 該抽出液 を無水硫酸ナトリウムで乾燥させ、 クロ口ホルムを除去した。 これによ り、 粗塩基性分画 1 3. 3 g (針葉部の量を基準として 0. 1 3 %) を 得た。  On the other hand, 800 ml of 29% aqueous ammonia was added to the above 0.5M sulfuric acid (pH 1.5), which was the aqueous layer after washing, to adjust the pH to 10.0, and then 6 times with 500 ml of chloroform (6 times). X 500 ml) extracted. Further, the extract was dried over anhydrous sodium sulfate to remove the clos por form. As a result, 13.3 g of the crude basic fraction (0.13% based on the amount of needles) was obtained.
また、洗浄後の水層である上記 2 M水酸化ナトリゥム水溶液( p H 7. 5) 〖こ 0. 5 M硫酸 80 Om 1を添加して pHを 3. 0とした後、 クロ 口ホルム 500m lで 6回 (6 X 500m l ) 抽出した。 さらに、 該抽 出液を無水硫酸ナトリウムで乾燥させ、 クロ口ホルムを除去した。 これ により、 粗酸性分画 22. 5 g (針葉部の量を基準として 0. 2 1 %) を得た。  The pH of the aqueous layer after washing was adjusted to 3.0 by adding 0.5 M sulfuric acid (80 Om1) to the aqueous 2M sodium hydroxide solution (pH 7.5). Extracted 6 times with l (6 X 500 ml). Further, the extracted liquid was dried with anhydrous sodium sulfate, and the outlet form was removed. This gave 22.5 g of crude acid fraction (0.21% based on the amount of needles).
次に、 上記の粗中性分画を、 シリカゲル (Silica gel 60 MERK, 70〜 230 mesh) 800 gを用いたオープンカラムクロマトグラフィーを採用 して、 溶媒として n—へキサンと酢酸ェチルとメチルアルコールとを用 いたグラジュェント溶出を行うことにより、 Fi (低極性側) 〜 F9 (高 極性側) の 9個のフラクションに分画 (荒分け) した。 〜F6 の溶出 液は酢酸ェチル: n—へキサンが容量比 3 : 2で混合してなる混合溶液 であり、 F7 · F8 の溶出液は酢酸ェチルであり、 F9 の溶出液はメチル アルコールであった。 Next, the above crude neutral fraction was subjected to open column chromatography using 800 g of silica gel (Silica gel 60 MERK, 70 to 230 mesh), and n-hexane, ethyl acetate and methyl alcohol were used as solvents. by performing Gurajuento elution had use bets and Fi 9 pieces fraction fraction (rough division) of the (low-polarity side) ~ F 9 (high polarity). ~F eluate 6 acetate Echiru: hexane n- capacity ratio of 3: a mixed solution prepared by mixing 2, eluent F 7 · F 8 is acetic Echiru, eluant F 9 is It was methyl alcohol.
そして、 上記 3番目のフラクション (F3 ) 8. 6 gを、 シリカゲル (Silicagel 60 MERK, 230-400 mesh ASTM) 650 gを用いたフラッシ ユカラムクロマトグラフィーを採用して、 溶媒として n—へキサンと酢 酸ェチルとメチルアルコールとを用いたグラジュェント溶出を行うこと により、 F3-i (低極性側) 〜F3-5 (高極性側) の 5個のフラクション に分画した。 の溶出液は酢酸ェチル: n—へキサンが容量比 1 : 1 で混合してなる混合溶液であり、 F3-2 の溶出液は同容量比 3 : 2で混合 してなる混合溶液であり、 F3-3 の溶出液は同容量比 7 : 3で混合してな る混合溶液であり、 F3.4 の溶出液は齚酸ェチルであり、 F3.5 の溶出液 はメチルアルコールであった。 Then, 8.6 g of the third fraction (F 3 ) was subjected to flash column chromatography using 650 g of silica gel (Silicagel 60 MERK, 230-400 mesh ASTM), and n-hexane was used as a solvent. and by performing Gurajuento elution with the acetic acid Echiru and methyl alcohol, F 3 -i (low polarity) to F 3 - 5 pieces of fraction 5 (high polarity) Fractionated. Eluate acetate Echiru: hexane n- capacity ratio of 1: is a mixed solution obtained by mixing with 1, F 3 - the second eluate equal volume ratio of 3: be a mixed solution prepared by mixing 2 , F 3 - 3 of eluate equal volume ratio of 7:.. a mixed solution ing mixed with 3, F 3 4 eluates are齚酸Echiru, F 3 5 eluate methyl alcohol Met.
次に、 上記 2番目のフラクション (F3.2 ) 4. 5 gを、 シリカゲル (Silicagel 60 MERK, 230〜400 mesh ASTM) 250 gを用いたフラッシ ユカラムクロマトグラフィーを採用して、 溶媒として n—へキサンと酢 酸ェチルとメチルアルコールとを用いたグラジュェント溶出を行うこと により、 Fan (低極性側) 〜F 3.2.7 (高極性側) の 7個のフラクショ ンに分画した。 Fa.^ · F 3-2-2 の溶出液は酢酸ェチル: n—へキサンが 容量比 3 : 2で混合してなる混合溶液であり、 F3-2-3 〜F3.2-5 の溶出液 は同容量比 4 : 1で混合してなる混合溶液であり、 F3-2-6 の溶出液は酢 酸ェチルであり、 F3-2.7 の溶出液はメチルアルコールであった。 Next, the second fraction (F 3. 2) 4. The 5 g, silica gel employing a flash Yu column chromatography using (Silicagel 60 MERK, 230~400 mesh ASTM ) 250 g, n as a solvent -.. by performing Gurajuento elution with a hexane and acetic acid Echiru and methyl alcohol to, Fan (low polarity) to F 3 2 7 was fractionated into seven Furakusho down (high polarity). . Fa ^ · F eluate 3-2-2 acetate Echiru: hexane n- capacity ratio of 3:. A mixed solution prepared by mixing at 2, F 3 -2- 3 ~F 3 2 - 5 the eluate same volume ratio of 4:. a mixed solution prepared by mixing with 1, F 3 - 2 - 6 eluate is acetic acid Echiru, F 3 -2 7 eluate of a methyl alcohol Was.
得られた分画 F 3-2-2 1 6 5 1 m gを、 OD Sステンレスカラム (INERTSIL PREP-ODS, GL Science, 250 X 2.0mm i.d., UV 254nm) を用いた逆相高速液体クロマトグラフィーを採用して、 メチルアルコー ルと 0. 05M酢酸アンモニゥム緩衝液 (pH4. 8) とァセトニトリ ルとを容量比 1 : 2 : 2で混合してなる混合溶液(キャリア, 流速 1 0. 0 ml/min.) を用いて単離 '精製した。 これにより、 粗 1 0—脱ァセチ ル— 7—ェピタキソ一ル 1 3 1. 3mg (保持時間 8. 9分)、 および、 粗 7—ェピタキソール 1 24. 7mg (保持時間 1 0. 1分) を取り出 した。  The obtained fraction F 3-2-2 1 65 1 mg was subjected to reverse-phase high-performance liquid chromatography using an ODS stainless steel column (INERTSIL PREP-ODS, GL Science, 250 X 2.0 mm id, UV 254 nm). A mixed solution consisting of methyl alcohol, 0.05M ammonium acetate buffer (pH 4.8), and acetonitrile at a volume ratio of 1: 2: 2 (carrier, flow rate: 10.0 ml / min) .) Was isolated and purified. This gives crude 10-deacetyl-7-epitaxol 131.3 mg (retention time 8.9 minutes) and crude 7-epitaxol 124.7 mg (retention time 10.1 minutes). I took it out.
次いで、 上記粗 1 0—脱ァセチル— 7—ェピタキソールを、 OD Sス テンレスカラム ( INERTSIL PREP ODS, GL Science, + Shodex C18-10E, 250 X 1.0mm i.d., UV 254nm)を用いた逆相高速液体クロマト グラフィーを採用して、 メチルアルコールと 0. 0 5M酢酸アンモニゥ ム緩衝液 (PH4. 8) とァセトニトリルとを容量比 1 : 2 : 2で混合 してなる混合溶液 (キャリア) を用いて精製した。 その後、 さらに、 シ リカゲルステンレスカラム (INERTSIL PREP-SIL, GL Science, 250 X 0.46mm i.d., UV 254nm ) を用いた順相高速液体クロマトグラフィーを 採用して、 酢酸ェチルと n—へキサンとを容量比 2 : 3で混合してなる 混合溶液 (キャリア, 流速 2. 0 ml/min.) を用いて精製した。 これに より、 1 0—脱ァセチル— 7—ェピ夕キソ一ルの他に、 化合物 (27) 1. 9mg (保持時間 9. 7分、 針葉部の量を基準として 0. 0000 18 %)、 および化合物 (28) 3. 2mg (保持時間 1 0. 8分、 同 0. 00003 1 %) を得た。 Then, the above crude 10-deacetyl-7-epitaxol was added to an ODS stainless column (INNERTSIL PREP ODS, GL Science, + Shodex C18-10E, 250 X 1.0 mm id, UV 254 nm) using a reversed-phase high-performance liquid. Chromat Purification was carried out using a mixed solution (carrier) obtained by mixing methyl alcohol, 0.05 M ammonium acetate buffer (PH4.8) and acetonitrile at a volume ratio of 1: 2: 2. After that, normal-phase high-performance liquid chromatography using silica gel stainless steel column (INERTSIL PREP-SIL, GL Science, 250 x 0.46 mm id, UV 254 nm) was adopted, and the volume of ethyl acetate and n-hexane was changed. Purification was performed using a mixed solution (carrier, flow rate: 2.0 ml / min.) Mixed at a ratio of 2: 3. As a result, in addition to 10-deacetyl-7-episoquinol, 1.9 mg of compound (27) (retention time: 9.7 minutes, 0.0018% based on the amount of needles) ), And 3.2 mg of compound (28) (retention time: 10.8 minutes, 0.000031%).
また、 上記粗 7—ェピタキソールを、 シリカゲルステンレスカラム (INERTSIL PREP-SIL, GL Science, 250 X 2.0mm i.d., UV 254nm) を 用いた順相高速液体クロマトグラフィーを採用して、 酢酸ェチルと n— へキサンとを容量比 1 : 1で混合してなる混合溶液 (キャリア, 流速 1 0. 0 ml/min.) を用いて精製した。 これにより、 7—ェピタキソール 25. 7mg (保持時間 1 7. 1分)、 および粗 7—ェピ—セファロマ二 ン 40. 6mg (保持時間 1 8. 6分) を得た。  The crude 7-epitaxol was converted to ethyl acetate and n- by normal phase high performance liquid chromatography using a silica gel stainless steel column (INERTSIL PREP-SIL, GL Science, 250 x 2.0 mm id, UV 254 nm). Purification was performed using a mixed solution (carrier, flow rate: 10.0 ml / min.) Obtained by mixing xanthane with xanthone at a volume ratio of 1: 1. This resulted in 25.7 mg of 7-epitaxol (retention time 17.1 minutes) and 40.6 mg of crude 7-epi-cephalomanine (retention time 18.6 minutes).
この粗 7—ェピ一セファロマニンを、 OD Sステンレスカラム (Shodex C18-10E, 250 X 1.0mm i.d., UV 254nm)を用いた逆相高速液 体クロマトグラフィーを採用して、 メチルアルコールと 0. 05M酢酸 アンモニゥム緩衝液(PH4. 8) とァセトニトリルとを容量比 1 : 2 : 2で混合してなる混合溶液 (キャリア,流速 5. 0 ml/min.) を用いて精 製した。 これにより、 粗製の化合物 (29) 1 2. 4mg (保持時間 1 4. 2分)、 および 7—ェピ—セファロマニン 1 6. 5mg (保持時間 1 7. 7分) を得た。 上記粗製の化合物(29)を、シリカゲルステンレスカラム(INERTSIL PREP-SIL, GL Science, 250 X 0.46mm i.d., UV 254nm ) を用いた順相 高速液体クロマトグラフィーを採用して、 酢酸ェチルと n—へキサンと を容量比 2: 3で混合してなる混合溶液(キャリア, 流速 2. 0 ml/min.) を用いて精製した。 これにより、 化合物 (29) 5. 8mg (保持時間 6. 7分、 針葉部の量を基準として 0. 000055 %)、 および 5—シ ンナモイル— 1 0—ァセチル夕キシン 116. 9mg (保持時間 9. 7分) を得た。 This crude 7-epipsephalomannine was purified by reverse phase high performance liquid chromatography using an ODS stainless steel column (Shodex C18-10E, 250 x 1.0 mm id, UV 254 nm) to obtain methyl alcohol and 0.05M Purification was performed using a mixed solution (carrier, flow rate: 5.0 ml / min.) Obtained by mixing ammonium acetate buffer (PH4.8) and acetonitrile at a volume ratio of 1: 2: 2. This gave 12.4 mg (retention time 14.2 minutes) of crude compound (29) and 16.5 mg (retention time 17.7 minutes) of 7-epi-cephalomanine. The crude compound (29) was converted to ethyl acetate and n- by normal phase high performance liquid chromatography using a silica gel stainless steel column (INERTSIL PREP-SIL, GL Science, 250 X 0.46 mm id, UV 254 nm). Purification was performed using a mixed solution (carrier, flow rate: 2.0 ml / min.) Consisting of a mixture of xane and in a volume ratio of 2: 3. This gives 5.8 mg of compound (29) (retention time 6.7 min, 0.00000% based on needle volume) and 116.9 mg of 5-cinnamoyl-10-acetyl quinine (retention time 9.7 minutes).
新規物質である化合物 (27) の 1H— NMRおよび we— NMRの 測定結果を第 7表 '第 8表にまとめて示す。 これにより、 化合物 (27) の構造を解析した。 また、 該化合物(27) の物性値等を測定した結果、 分子式 : 33H40O9  The 1H-NMR and we-NMR measurements of compound (27), a new substance, are summarized in Tables 7 and 8. Thus, the structure of compound (27) was analyzed. In addition, as a result of measuring the physical properties and the like of the compound (27), the molecular formula: 33H40O9
分子量 : 580. 2679 (測定値)、 580. 2673 (計算値) 融 点 : 2 1 5で〜 2 17で  Molecular weight: 580. 2679 (measured value), 580. 2673 (calculated value) Melting point: 215 to 217
〔ひ〕 D 20 : + 1 1 3. 27° (c = 0. 1 1 3, CHC 13 )  [H] D 20: + 1 1 3.27 ° (c = 0.13, CHC 13)
I R : 36 12, 3000, 1 730, 1 670, 1 640,
Figure imgf000079_0001
IR: 36 12, 3000, 1 730, 1 670, 1 640,
Figure imgf000079_0001
であった。 Met.
NMR Sp e c t r a l Da a o c omp. ( 27 ) i n CDC 13 NMR Sp e c t r a l Da ao c omp. (27) in CDC 13
位匿 13C *l conected lH H-H COSY*3 HMB C NOESY " 13 C * l conected l H HH COSY * 3 HMB C NOESY "
1 51 70 (d) 2.38 (br dd, 6.8 1.7) H2 14(5 H3, 148 16. 17 H2(m .14fi(m).16(m).17(m)  1 51 70 (d) 2.38 (br dd, 6.8 1.7) H2 14 (5 H3, 148 16.17 H2 (m .14fi (m) .16 (m) .17 (m)
2 6750 Cd") 4.39 On) Hi, 3, 2 - OH Hi, 3. i o, S HKnO, 9(m), 17(ra), 19(m)  2 6750 Cd ") 4.39 On) Hi, 3, 2-OH Hi, 3.io, S HKnO, 9 (m), 17 (ra), 19 (m)
3 4343 (d) 3.15 (d, 6.1) H2, 20a HI, 19, 20a, b H7(ra), 14i(m). 18(m)  3 4343 (d) 3.15 (d, 6.1) H2, 20a HI, 19, 20a, b H7 (ra), 14i (m) .18 (m)
4 141,05 (s) H3,  4 141,05 (s) H3,
5 77, 30 (d) 5.35 (br d, 4.1) Η6β. β H3. 6 20a. b H6a(ra), 6 m), 20b (w)  5 77, 30 (d) 5.35 (br d, 4.1) Η6β.β H3.6.20a.b H6a (ra), 6m), 20b (w)
6 35.22 (t) a) 2.09 (m) H5, 6¾ H7 H5(n0. 6i(s). 7(m)  6 35.22 (t) a) 2.09 (m) H5, 6¾ H7 H5 (n0.6 i (s) .7 (m)
i) 1.78 (br ddd, 15.6. H5, 60, 7 H5(m), 6o(s), 19(m)  i) 1.78 (br ddd, 15.6.H5, 60, 7 H5 (m), 6o (s), 19 (m)
11.5. 4.1)  11.5. 4.1)
7 69.88 (d) 5.34 (dd, 11.5, 5.6) H6a. ί H3, 6β, 9. 19 H3(m).60 (m), 10(m).18(m)  7 69.88 (d) 5.34 (dd, 11.5, 5.6) H6a.ί H3, 6β, 9.19 H3 (m) .60 (m), 10 (m) .18 (m)
8 47.65 (s) H3, 6 7. 19  8 47.65 (s) H3, 6 7.19
g 77.58 (d) 5.73 n t (d, 10.3) H10 H10. 19 H2(m), 17(m). 19(m)  g 77.58 (d) 5.73 n t (d, 10.3) H10 H10.19 H2 (m), 17 (m) .19 (m)
10 7072 (d) 5.28(br dd, 10.3. 5.2) H9, 10-OH H9, 10-OH H7(m). 18(m)  10 7072 (d) 5.28 (br dd, 10.3.5.2) H9, 10-OH H9, 10-OH H7 (m) .18 (m)
11 15440 (s) rr o c Hi, 10, 16 17, 18  11 15440 (s) rr o c Hi, 10, 16 17, 18
12 135 68 (s) H10. 14j} to 12 135 68 (s) H10.14j} to
13 199.80 (s) HI, 14a. H, 18 13 199.80 (s) HI, 14a.H, 18
14 35.83 (t) a) 2.22 (d, 20.0) 卿 HI, 2 H3(n0. 14 s)  14 35.83 (t) a) 2.22 (d, 20.0) Sir HI, 2 H3 (n0.14 s)
a 2.87 (dd. 20,0. 6.8) HI, 14s Hl(m), 14a(s), 16(m) a 2.87 (dd. 20,0.6.8) HI, 14s Hl (m), 14a (s), 16 (m)
15 37,81 (s) HI, 10, 14ί.16, 17 15 37,81 (s) HI, 10, 14ί.16, 17
16 37.86 (q) 1.26 (s) H17 H17 HKm), 14Km 17(w)  16 37.86 (q) 1.26 (s) H17 H17 HKm), 14Km 17 (w)
17 25.46 (q) 1.76 (s) HI 6 H16 HI On), 2(ra), 9(m),  17 25.46 (q) 1.76 (s) HI 6 H16 HI On), 2 (ra), 9 (m),
160), 19(w)  160), 19 (w)
18 14.10 (q) 2.22 (s) H3(w),7(m).10(ui)(2' (m) 18 14.10 (q) 2.22 (s) H3 (w), 7 (m) .10 (ui) ( 2 '(m)
19 13.35 (q) 1.05 (s) H3, 7 H20). 6 m), 9(m)  19 13.35 (q) 1.05 (s) H3, 7 H20) .6 m), 9 (m)
20 119.62 (t) H3. 20b H3  20 119.62 (t) H3. 20b H3
H20a  H20a
o o
2 - OH 1.72 Cbr d, 8,6) Hz 2-OH 1.72 Cbr d, 8,6) Hz
10 - OH 2.00 (d, 5.2) HlO  10-OH 2.00 (d, 5.2) HlO
OAc 170.00 (s) H7. 7-0Ac( e)  OAc 170.00 (s) H7.7-0 (Ac)
170.71 (s) H9. 9 - OAc (Me)  170.71 (s) H9.9.9-OAc (Me)
21.49 (q) 2.09 (s)  21.49 (q) 2.09 (s)
20.95 (q) 2.13 (s)  20.95 (q) 2.13 (s)
i,  i,
1 166.21 (s) Hz , 3  1 166.21 (s) Hz, 3
2' 117.31 (d) 6.43 (d, 15.9) H3' H3, H18(m), o-Ph(m)  2 '117.31 (d) 6.43 (d, 15.9) H3' H3, H18 (m), o-Ph (m)
3, 146.29 (d) 7.67 (d, 15.9) H2' o-Ph o-Ph(m)  3, 146.29 (d) 7.67 (d, 15.9) H2 'o-Ph o-Ph (m)
q-Ph 134.40 (s) H2', m-Ph  q-Ph 134.40 (s) H2 ', m-Ph
cv o一 128.52 (d) 7.77 (m) m-Ph H3', p-Ph H2'(m), 3' n), cv o 128.52 (d) 7.77 (m) m-Ph H3 ', p-Ph H2' (m), 3 'n),
m-Ph(m), p-Ph(m)  m-Ph (m), p-Ph (m)
m - 128.91 (d) 7.43 (m) P-Ph P-Ph o-Ph(m). p-Ph(w)  m-128.91 (d) 7.43 (m) P-Ph P-Ph o-Ph (m) .p-Ph (w)
p- 130.43 (d) 7.43 Cm) m-Ph o-Ph o-Ph(m). m-Ph(w)  p- 130.43 (d) 7.43 Cm) m-Ph o-Ph o-Ph (m) .m-Ph (w)
00 00
〇 ies and coupling constants in Hz are in
Figure imgf000081_0001
〇 ies and coupling constants in Hz are in
Figure imgf000081_0001
被¾ 8 MRの測定結果を第 9表 ·第 1 0表にまとめて示す。 これにより、 化合 物 (28) の構造を解析した。 また、 該化合物 (28) の物性値等を測 定した結果、 Covered 8 Tables 9 and 10 summarize the MR measurement results. Thus, the structure of the compound (28) was analyzed. In addition, as a result of measuring the physical properties and the like of the compound (28),
77ナ式 : し 33H40リ 9  77-type: 33H40 9
分子量 : 580. 2646 (測定値)、 580. 2673 (計算値) 融 点 : 9 1で〜 93で  Molecular weight: 580. 2646 (measured value), 580. 2673 (calculated value) Melting point: 91 to 93
C α D 20: - 60. 33° (c = 0. 1 79, CHC 13 ) C α D 20 :-60. 33 ° (c = 0.179, CHC 13)
I R : 36 1 2, 3464, 3000, 1 7 1 8, 1638,
Figure imgf000082_0001
IR: 3612, 3464, 3000, 1718, 1638,
Figure imgf000082_0001
であった。 Met.
NMR S ec t ra l Da ta of c omp. (28 ) in CDC 1 NMR Sec tra l Da ta of c omp. (28) in CDC 1
位置 18C * 1 conected 'H " H-H COSY*3 H BC " NOESY " Position 18 C * 1 conected 'H "HH COSY * 3 H BC" NOESY "
1 ΛΟ 7Λ (A) 1.65(br dd.8.2, 2.8) H \iS H14fl Ifi 17 H9( 5 \R(m , ^^(m)  1 ΛΟ 7Λ (A) 1.65 (br dd.8.2, 2.8) H \ iS H14fl Ifi 17 H9 (5 \ R (m, ^^ (m)
2 68 16 Cd) 4.60 (br d. 9.3) Hi 202- OH HI 20 2-0H m) SiC ) 17 m) 20  2 68 16 Cd) 4.60 (br d. 9.3) Hi 202- OH HI 20 2-0H m) SiC) 17 m) 20
3 3536 ft) a) 2.70 (d. 15.3) H3b HI 9, 20 H2Cra) 3b(s) 17 Cm) I9(m)  3 3536 ft) a) 2.70 (d. 15.3) H3b HI 9, 20 H2Cra) 3b (s) 17 Cm) I9 (m)
b) 1.99 (d. 15.3) H3a. 5. 20 H3a(m). 68(m). 19(m)  b) 1.99 (d. 15.3) H3a. 5.20 H3a (m). 68 (m). 19 (m)
4 130.38 (s) K3a, b, 5  4 130.38 (s) K3a, b, 5
5 eg 79 (d) 5.74 (br d. 6.7) H3b. 6i, 20 H3a. b. 6fl HfiflCm). 65 Cm)  5 eg 79 (d) 5.74 (br d.6.7) H3b.6i, 20 H3a.b.6fl HfiflCm) .65 Cm)
6 3252 (t) ) 2.05 (br dd, Η6β, 7 H5 H5Cm). 6iCs). 7 Cm)  6 3252 (t)) 2.05 (br dd, Η6β, 7 H5 H5Cm) .6iCs) .7 Cm)
14.8, 3.3)  14.8, 3.3)
ί) 2.26 (ddd. 14.8, H5, 6α. 7 HShfm). 5 fm). 6a Cs) 19 Cm")  ί) 2.26 (ddd. 14.8, H5, 6α. 7 HShfm) .5 fm) .6a Cs) 19 Cm ")
7 7096 (d) 5.20 (dd.12.5. 3,3) K6(t JS H3a. 6c. ί 19 H6a(m) 10 Cm) 18(n)  7 7096 (d) 5.20 (dd.12.5.3,3) K6 (t JS H3a. 6c.ί 19 H6a (m) 10 Cm) 18 (n)
Hf^ h fifl fi 7, 1Q  Hf ^ h fifl fi 7, 1Q
Q o  Q o
in 7689 (d) 5.40 (d. 2.9) 10 OH 00  in 7689 (d) 5.40 (d.2.9) 10 OH 00
to to
11 13204 (s) HI.10 16 17 18 11 13 204 (s) HI.10 16 17 18
12 135.84 (s) H10, 18  12 135.84 (s) H10, 18
13 69.76 u 5.41 (br d, 10.7) H14a, 18 HI, 14な, jo nl fi iiw, lbCin). 18 w),  13 69.76 u 5.41 (br d, 10.7) H14a, 18 HI, 14, na, jo nl fi iiw, lbCin) .18 w),
13-0Ac(Me)(w)  13-0Ac (Me) (w)
14 26.48 (t) な) 1,88 (dd.16.0.2.8) H14i. 13 HI Hl4 (s), 20 (in)  14 26.48 (t)) 1,88 (dd.16.0.2.8) H14i. 13 HI Hl4 (s), 20 (in)
β 2.68 (br ddd. HI, 13, 14a Hl(s). 13(m). 14な(s), 16(m)  β 2.68 (br ddd.HI, 13, 14a Hl (s). 13 (m). 14 (s), 16 (m)
16.0. 10.7, 8.2)  16.0. 10.7, 8.2)
15 37.56 (s) HI.10, 14j8,16,17  15 37.56 (s) HI.10, 14j8,16,17
16 32.77 (q) 1.19 (s) H17 H17 Hl(m), 13(ra), 14H(w). 17(ro)  16 32.77 (q) 1.19 (s) H17 H17 Hl (m), 13 (ra), 14H (w) .17 (ro)
17 24.79 (q) 1.14 (s) H16 H16 HKra). 2(m), 3a(m),  17 24.79 (q) 1.14 (s) H16 H16 HKra) .2 (m), 3a (m),
10-0HCm), 16(m)  10-0HCm), 16 (m)
¾ 9 O ¾ 9 O
oo ω oo ω
Figure imgf000084_0001
Figure imgf000084_0001
O  O
2 Two
MRの測定結果を第 1 1表 ·第 1 2表にまとめて示す。 これにより、 化 合物 (29) の構造を解析した。 また、 該化合物 (29) の物性値等を 測定した結果、 The measurement results of MR are summarized in Tables 11 and 12. As a result, the structure of the compound (29) was analyzed. In addition, as a result of measuring the physical property values and the like of the compound (29),
分子式 : し 3lH38〇7 Molecular formula: shi 3lH38〇 7
分子量 : 522. 262 1 (測定値)、 522. 26 18 (計算値) 融 点 : 1 36 :〜 1 37  Molecular weight: 522.262 1 (measured value), 522.26 18 (calculated value) Melting point: 136: 1137
C α ) D 20: + 144. 80° (c = 0. 433, CHC " ) C α) D 20 : + 144.80 ° (c = 0.433, CHC ")
I R : 3668, 2996, 1 7 14, 1 668, 1642, 1234 c m i (CHC 13 ) IR: 3668, 2996, 1 7 14, 1 668, 1642, 1234 cmi (CHC 1 3)
であった。 Met.
NMR Sp e c t r a l Da t a o f c omp. ( 29 ) i n CDC 1 s NMR Spectrum Da taofc omp. (29) in CDC 1 s
Figure imgf000086_0001
Figure imgf000086_0001
位度 18 c conected 】H * H-H COSY HMB C " NOESY Degree 18 c conected】 H * HH COSY HMB C "NOESY
1 51.54 (d〉 2.38 (dd, 6.9, 2.2) H2, 14i H3, 14a, β, 16, 17 H2(m), 14 s), 16 On), 17(m)  1 51.54 (d) 2.38 (dd, 6.9, 2.2) H2, 14i H3, 14a, β, 16, 17 H2 (m), 14 s), 16 On), 17 (m)
2 68.18 (d) 4.30 (br ddd, 7.8, HI.3, 2-OH HI, 3. 14OJ, 2-0H Hl(m), 2-0H(w), 9(m), 17(m),  2 68.18 (d) 4.30 (br ddd, 7.8, HI.3, 2-OH HI, 3.14OJ, 2-0H Hl (m), 2-0H (w), 9 (m), 17 (m),
6.6, 2.2) 19(in)  6.6, 2.2) 19 (in)
3 45.06 (d) 3.26 (br d, 6.6) H2, 20a HI, 7ί, 9, 19, H7(r(m). 14ff(ra), 18(ff)  3 45.06 (d) 3.26 (br d, 6.6) H2, 20a HI, 7ί, 9, 19, H7 (r (m) .14ff (ra), 18 (ff)
20a. b, 2-OH  20a.b, 2-OH
4 143.66 (s) H3. 5, 6e, 20a. b  4 143.66 (s) H3. 5, 6e, 20a.b
5 78.14 (d) 5.34 (»r 2,8) H6a, . 20b H3, 6a, β. 20a. i H6e(ra). 6 m). 20b (n)  5 78.14 (d) 5.34 (»r 2,8) H6a, .20b H3, 6a, β.20a.i H6e (ra) .6 m) .20b (n)
6 28.73 (t) 1.99 On) H5.6D.7a, β H7a, β 師), 6 s). 7fl(m). 7J(m)  6 28.73 (t) 1.99 On) H5.6D.7a, β H7a, β teacher), 6 s) .7fl (m) .7J (m)
1.74 (m) H5.6α.7a, β H5(ra), 6a(s). 19 (m)  1.74 (m) H5.6α.7a, β H5 (ra), 6a (s) .19 (m)
7 27.62 (t) a) 1.69 On) Ma. β, 7β H3, 5, 6α. 9. 19 H3(m), 6a (in). 7 (m),  7 27.62 (t) a) 1.69 On) Ma.β, 7β H3, 5, 6α. 9.19 H3 (m), 6a (in) .7 (m),
10(s), 18(w)  10 (s), 18 (w)
i) 1.74 On) H6ff, β, 7a H6 ra). 7fl(m)  i) 1.74 On) H6ff, β, 7a H6 ra) .7fl (m)
8 44.65 (s) H2. 3. 6α, β, Ία,  8 44.65 (s) H2.3.6α, β, Ία,
00 9, 19. 20a, b  00 9, 19.20a, b
9 79.04 (d) 5.69 (d, 9.9) H10 Η7β, ί, 10. 19 H2(s). I0-0»( ).17(m),19(m)  9 79.04 (d) 5.69 (d, 9.9) H10 Η7β, ί, 10.19 H2 (s) .I0-0 »() .17 (m), 19 (m)
10 72.05 (d) 5.02(dd, 9.9, 5.3) H9 H7a(s), 18(m)  10 72.05 (d) 5.02 (dd, 9.9, 5.3) H9 H7a (s), 18 (m)
111 1丄 Cvl β 111 1 丄 Cvl β
o  o
X HI. 10. 16, 17, 18  X HI. 10. 16, 17, 18
12 135.18 (s) H10, 14β, 13  12 135.18 (s) H10, 14β, 13
13 199.94 (s) HI. 1 β, 18  13 199.94 (s) HI. 1 β, 18
14 35.77 (t) a) 2.27 (d. 20.0) ΗΗβ o 1 HI. 2 H3(m), 14l(s). 2- 0H(ra)  14 35.77 (t) a) 2.27 (d.20.0) ΗΗβ o 1 HI.2 H3 (m), 14l (s) .2-0H (ra)
i) 2.87 (dd, 20.0. HI, 14a HKs), 1 (s), 16(m)  i) 2.87 (dd, 20.0. HI, 14a HKs), 1 (s), 16 (m)
6.9)  6.9)
15 37.75 (s)  15 37.75 (s)
16 38.02 (q) 1.28 (s) H17 HI 7 Hl(m), 14 m), 17(m)  16 38.02 (q) 1.28 (s) H17 HI 7 Hl (m), 14 m), 17 (m)
17 25.50 (q) 1.76 (s) H16 HI, 16 HKw), 2(m), 9(m), 16 Cm)  17 25.50 (q) 1.76 (s) H16 HI, 16 HKw), 2 (m), 9 (m), 16 Cm)
Bird
18 13.96 (q) 2.12 (s) H3(w), 7a (w). 10 On) 18 13.96 (q) 2.12 (s) H3 (w), 7a (w) .10 On)
19 17.71 (q) 0.97 (s) H3, 7a, i, 9 H2(m), 6 m). 9(m)  19 17.71 (q) 0.97 (s) H3, 7a, i, 9 H2 (m), 6 m) .9 (m)
20 118.01 (t) a) 5.44 (br s) H3 H3, 5 2-0H(m) t  20 118.01 (t) a) 5.44 (br s) H3 H3, 52-0H (m) t
b)5. 3 (br t, 1.2) H5 H5(w) %  b) 5.3 (br t, 1.2) H5 H5 (w)%
2-OH 1.83 (d, 7.8) H2( ), 14d(ra), 20a (m)  2-OH 1.83 (d, 7.8) H2 (), 14d (ra), 20a (m)
10-OH 2.10 (d, 5.3) H9(w)  10-OH 2.10 (d, 5.3) H9 (w)
OAc 171.74 (s) H9. 9-0Ac( e)  OAc 171.74 (s) H9.9.0-0Ac (e)
21.02 Cq) 2.18 Cs)  21.02 Cq) 2.18 Cs)
Γ 166.33 (s) H5. 2'. 3'  Γ 166.33 (s) H5. 2 '. 3'
2' 117.79 (d) 6. 2 (d, 15.9) H3' H3' o-Ph(m)  2 '117.79 (d) 6.2 (d, 15.9) H3' H3 'o-Ph (m)
3' 145.77 (d) 7.65 (d, 15.9) H2' , o-Ph H2 , o-Ph o-Ph(m)  3 '145.77 (d) 7.65 (d, 15.9) H2', o-Ph H2, o-Ph o-Ph (m)
q-Ph 134.50 (s) H2', 3'. m-Ph  q-Ph 134.50 (s) H2 ', 3'.m-Ph
o - 128.48 (d) 7.76 On) H3" , m-Ph H3' p-Ph H2'(m), 3' (m), m-Ph(ra)  o-128.48 (d) 7.76 On) H3 ", m-Ph H3 'p-Ph H2' (m), 3 '(m), m-Ph (ra)
m- 128.95 (d) 7.43 On) o-Ph, p-Ph o-Ph, p-Ph o-Ph(w), p-Ph(w)  m- 128.95 (d) 7.43 On) o-Ph, p-Ph o-Ph, p-Ph o-Ph (w), p-Ph (w)
p- 130.38 (d) 7. 3 On) m-Ph o-Ph m-Ph( )  p- 130.38 (d) 7.3 On) m-Ph o-Ph m-Ph ()
00 OS  00 OS
*1) Multiplicities were determined by DEPT.  * 1) Multiplicities were determined by DEPT.
ネ 2) Connections were determined ¾y HMQC and multiplicities and coupling constants in Hz are in F 2) Connections were determined ¾y HMQC and multiplicities and coupling constants in Hz are in
parentheses.  parentheses.
*3) Determined by PFG-COSY. * 3) Determined by PFG-COSY.
ネ 4) Correlations from C to the indicated protons. D) Correlations from C to the indicated protons.
*5) NOESY cross peaks. (s)=strong; On) -medium; (w)=weak * 5) NOESY cross peaks. (S) = strong; On) -medium; (w) = weak
〔実施例 3〕 (Example 3)
日本ィチイの針葉部 (生針葉部) からタキサン関連化合物である化合 物 (1 5) および化合物 ( 1 6) を取り出した。 即ち、 上記の針葉部 2 648を11ーへキサン1. 6 Lに 1週間浸漬することにより脱脂した後、 酢酸ェチル 1. 6 Lに浸漬した。 1週間浸漬した後、 針葉部を濾別し、 濾液である抽出液を得た。 次いで、 抽出液から酢酸ェチルを室温で減圧 除去し、 該酢酸ェチルに可溶な成分 (粗酢酸ェチル抽出物) 3. 6 1 g (針葉部の量を基準として 1. 3 7 %) を得た。 Compounds (15) and (16), which are taxane-related compounds, were extracted from the needle portion (raw needle portion) of Yew Tree. That is, after degreased by immersing 1 week needles 2 64 8 above hexane 1. 6 L to 11-2, and immersed in acetic acid Echiru 1. 6 L. After soaking for one week, the needle portion was separated by filtration to obtain an extract as a filtrate. Then, ethyl acetate was removed from the extract under reduced pressure at room temperature, and 3.61 g (1.37% based on the amount of needles) of a component soluble in the ethyl acetate (crude ethyl acetate extract) was added. Obtained.
上記の成分を、 メチルアルコールと酢酸ェチルとを容量比 1 : 4で混 合してなる混合溶液 5 Om 1に溶解した。 次に、 該溶液を、 酸性水溶液 である 0. 5M硫酸 1 5m lで 3回 (3 X 1 5m l ) 洗浄し、 続いて、 塩基性水溶液である 2 M水酸化ナトリゥム水溶液 1 5m 1で 2回 (2 X 1 5m l ) 洗浄した。 さらに、 上記の溶液 (油層) を飽和食塩水 2 Om 1で 4回(4 X 2 Om 1 )洗浄した後、無水硫酸ナトリゥムで乾燥させ、 メチルアルコールおよび酢酸ェチルを除去した。 これにより、 粗中性分 画 1. 8 6 g (針葉部の量を基準として 0. 7 1 %) を得た。  The above components were dissolved in 5 Om 1 of a mixed solution obtained by mixing methyl alcohol and ethyl acetate at a volume ratio of 1: 4. Next, the solution was washed three times (3 × 15 ml) with 15 ml of 0.5 M sulfuric acid as an acidic aqueous solution, and then washed with 15 ml of a 2 M aqueous sodium hydroxide solution as a basic aqueous solution. Washed twice (2 X 15 ml). Further, the above solution (oil layer) was washed four times (4 × 2 Om 1) with saturated saline 2 Om 1, and dried over anhydrous sodium sulfate to remove methyl alcohol and ethyl acetate. As a result, 1.86 g of a crude neutral fraction (0.71% based on the amount of needles) was obtained.
一方、 洗浄後の水層である上記 0. 5M硫酸 (pH l. 5) に 29 % アンモニア水 5 Om 1を添加して pHを 1 0. 0とした後、 クロ口ホル ム 20m lで 5回 (5 X 20m l ) 抽出した。 さらに、 該抽出液を無水 硫酸ナトリウムで乾燥させ、 クロ口ホルムを除去した。 これにより、 粗 塩基性分画 0. 36 g (針葉部の量を基準として 0. 14%) を得た。 また、洗浄後の水層である上記 2 M水酸化ナトリゥム水溶液( p H 7. 5) に 0. 5M硫酸 5 Om 1を添加して pHを 3. 0とした後、 クロ口 ホルム 20m lで 3回 (3 X 20m l ) 抽出した。 さらに、 該抽出液を 無水硫酸ナトリウムで乾燥させ、クロ口ホルムを除去した。これにより、 粗酸性分画 0. 88 g (針葉部の量を基準として 0. 33 %) を得た。 次に、 上記の粗中性分画を、 シリカゲル (Silica gel 60 MERK, 230〜 400 mesh) 55. 8 gを用いたフラッシュカラムクロマトグラフィーを 採用して、 溶媒として n—へキサンと酢酸ェチルとメチルアルコールと を用いたグラジュェント溶出を行うことにより、 Fi (低極性側) 〜 F10 (高極性側)の 10個のフラクションに分画(荒分け)した。 Fi 〜F5 の 溶出液は酢酸ェチル: n—へキサンが容量比 3 : 2で混合してなる混合 溶液であり、 F6 · F7 の溶出液は酢酸ェチルであり、 F8 〜F10の溶出 液はメチルアルコールであった。 On the other hand, 29% ammonia water (5 Om1) was added to the 0.5M sulfuric acid (pH 1.5), which was the aqueous layer after washing, to adjust the pH to 10.0. Extracted 5 times (5 X 20 ml). Further, the extract was dried over anhydrous sodium sulfate to remove the form. This gave 0.36 g of the crude basic fraction (0.14% based on the amount of needles). Also, 0.5M sulfuric acid (5Om1) was added to the 2M aqueous sodium hydroxide solution (pH 7.5), which was the aqueous layer after washing, to adjust the pH to 3.0. Extracted three times (3 X 20 ml). Further, the extract was dried over anhydrous sodium sulfate, and the form at the outlet was removed. This gave 0.88 g of the crude acidic fraction (0.33% based on the amount of needles). Next, the above crude neutral fraction was subjected to flash column chromatography using 55.8 g of silica gel (Silica gel 60 MERK, 230 to 400 mesh), and n-hexane and ethyl acetate were used as solvents. by performing Gurajuento elution with methyl alcohol, and Fi fractionated into 10 fractions (less polar side) ~ F 10 (high polarity) (rough division). Fi to F eluate 5 acetate Echiru: hexane n- capacity ratio of 3: a mixed solution prepared by mixing 2, eluent of F 6 · F 7 is acetic acid Echiru, F 8 to F 10 The eluate was methyl alcohol.
そして、 上記 1番目のフラクション (F 1 002. 5mgを、 シリ 力ゲルステンレスカラム (INERTSIL PREP-SIL, GL Science, 250 X 1.0mm i.d., UV 254nm) を用いた順相高速液体クロマトグラフィーを採 用して、 酢酸ェチルと n—へキサンとを容量比 3 : 7で混合してなる混 合溶液 (キャリア, 流速 5. 0 ml/min.) を用いて単離 '精製した。 こ れにより、 粗夕キシニン 2 7. 2mg (保持時間 1 7. 4分)、 および、 化合物 (1 5) 1. 2mg (保持時間 33. 2分、 針葉部の量を基準と して 0005 %) を取り出した。  The first fraction (F 1002.5 mg) was subjected to normal-phase high-performance liquid chromatography using a silica gel gel column (INERTSIL PREP-SIL, GL Science, 250 X 1.0 mm id, UV 254 nm). Then, it was isolated and purified using a mixed solution (carrier, flow rate 5.0 ml / min.) Obtained by mixing ethyl acetate and n-hexane at a volume ratio of 3: 7. 27.2 mg of crude xinine (retention time: 17.4 min) and 1.2 mg of compound (15) (retention time: 33.2 min, 0005% based on needle volume) Was.
次いで、 上記粗夕キシニンを、 シリカゲルステンレスカラム (INERTSIL PREP-SIL, GL Science, 250 X 1.0mm i.d., UV 254nm) を 用いた順相高速液体クロマトグラフィーを採用して、 酢酸ェチルと n— へキサンとを容量比 1 : 4で混合してなる混合溶液(キャリア, 流速 5. 0 ml/min.) を用いて精製した。 これにより、 タキシニン 2 1. 8 m g (保持時間 35. 0分)、 および、 化合物 ( 16) 1. 6mg (保持時間 40. 5分、 針葉部の量を基準として 0. 0006 %) を得た。  Then, the crude xinine was subjected to normal-phase high-performance liquid chromatography using a silica gel stainless steel column (INERTSIL PREP-SIL, GL Science, 250 × 1.0 mm id, UV 254 nm) to obtain ethyl acetate and n-hexane. Was purified using a mixed solution (carrier, flow rate: 5.0 ml / min.) Obtained by mixing and at a volume ratio of 1: 4. This gave 21.8 mg of taxinine (retention time 35.0 minutes) and 1.6 mg of compound (16) (retention time 40.5 minutes, 0.0006% based on the amount of needles). Was.
化合物 (1 5) の iH— NMRおよび 13C— NMRの測定結果を第 1 3表 ·第 14表にまとめて示す。 これにより、 化合物 ( 1 5) の構造を 解析した。 また、 該化合物 (1 5) の物性値等を測定した結果、 分卞;! : C 33H40O8 The measurements of iH-NMR and 13C-NMR of compound (15) are summarized in Tables 13 and 14. Thus, the structure of the compound (15) was analyzed. In addition, as a result of measuring physical properties and the like of the compound (15), Min Byon;!: C 33H40O8
分子量 : 564. 2723 (測定値)、 564. 2724 (計算値) 融 点 : 58で〜 60で  Molecular weight: 564.2723 (measured value), 564.2724 (calculated value) Melting point: 58 to 60
〔ひ〕 D 20 : + 87. 00° (c = 0. 1 0, CHC " ) [H] D 20 : + 87.00 ° (c = 0.10, CHC ")
I R : 3628, 2936, 1 734, 1 7 1 8, 167 2, 1 642, 1248 cm-i (CHC " )  I R: 3628, 2936, 1 734, 1 7 1 8, 167 2, 1642, 1248 cm-i (CHC ")
であった。 Met.
NMR Spe c t r a l Da t a o f c omp. ( 15 ) i n CDC 1 a NMR Spe c t r a l Da t a o f c omp. (15) in CDC 1 a
位置 conected Ή " H-H COSY " HMBC ·4 NOESY " Position conected Ή "HH COSY" HMBC · 4 NOESY "
1 48.68 (d 2.16 (m) H2 14j3 H3, 14な, 16, 17 H2 m . 14p in).16Cm). 17 Cm) CO 1 48.68 (d 2.16 (m) H2 14j3 H3, 14, na, 16, 17 H2 m .14p in) .16Cm) .17 Cm) CO
2 69 76 d 5.49 (dd. 6.2. 2.0) Hl 3 HI, 3, 14α, β Hluo), 9 s , I7 n), I9(m) 2 69 76 d 5.49 (dd. 6.2. 2.0) Hl 3 HI, 3, 14α, β Hluo), 9 s, I7 n), I9 (m)
3 43.13 o) 3.39 (br d, 6.2) H2 20av b HI, 19 20a, b 3 43.13 o) 3.39 (br d, 6.2) H2 20a v b HI, 19 20a, b
4 142.34 (s) H3> 6a  4 142.34 (s) H3> 6a
0 78.5o ί W 5.35 (br t. 2.7) ποα. β noff \M) · 6β W , 20b tw  0 78.5o ί W 5.35 (br t. 2.7) ποα.β noff \ M) 6β W, 20b tw
b a) 1.99 (br ddd, 13.7, no, iff, β tlfな,  b a) 1.99 (br ddd, 13.7, no, iff, β tlf,
)5) 1.75 (br dddd, ttot 7 ) 5) 1.75 (br dddd, tto t 7
な, fa-, p ΰ HDW S , JpW, 19 W  Na, fa-, p ΰ HDW S, JpW, 19 W
13.7. 11.0, 4.9, 2.7)  13.7. 11.0, 4.9, 2.7)
ύΚ).11 リ i) 1.56 (n) ΠΟ(Ζ> ffi no, OHt 7i)f 1 fifm  ύΚ) .11 l i) 1.56 (n) ΠΟ (Ζ> ffi no, OHt 7i) f 1 fifm
i) 1.88 (br ddd. 13.3. πού!, in 7  i) 1.88 (br ddd.13.3.πού !, in 7
4.9, 2.7)  4.9, 2.7)
1 Q  1 Q
o  o
75.70 4.32 (br d, 9.6) til r n n  75.70 4.32 (br d, 9.6) til r n n
H2 S , 17tSA 13 W  H2 S, 17tSA 13 W
10 nc 5.83 (d. 9.6) H3 H9 H了な n l W O 10 nc 5.83 (d.9.6) H3 H9 H n n W O
11 151.25 (s) H10, 16, 17.18 11 151.25 (s) H10, 16, 17.18
12 137.71 (s) H10, 18  12 137.71 (s) H10, 18
13 199.53 (s) HI, 14な, β, 18  13 199.53 (s) HI, 14, na, β, 18
14 36.08 (t) a) 2.43 (d, 19.8) Η14ί H2 H3(m), 14t(s)  14 36.08 (t) a) 2.43 (d, 19.8) Η14ί H2 H3 (m), 14t (s)
11)2.83 (dd. 19.8, 7.1) HI, 14a Hl(m)' 14a(s), 16(w)  11) 2.83 (dd.19.8, 7.1) HI, 14a Hl (m) '14a (s), 16 (w)
15 37.77 (s) H10, 14α, 16.17  15 37.77 (s) H10, 14α, 16.17
16 37.14 (q) 1.15 (s) H17 HI 7 HI ( , 14i(w). 17(m)  16 37.14 (q) 1.15 (s) H17 HI 7 HI (, 14i (w). 17 (m)
17 25.58 (q) 1.65 (s) H16 H16 HlCw), 20n), 9(s), 16(m)  17 25.58 (q) 1.65 (s) H16 H16 HlCw), 20n), 9 (s), 16 (m)
18 14.00 (q) 2.29 (S) H編  18 14.00 (q) 2.29 (S) H
19 17.67 (q) 1.14 (s) H3 9 H2Cm). 6jS(m 9(m) 19 17.67 (q) 1.14 (s) H3 9 H2Cm) .6jS (m 9 (m)
) It¾ctG ^H NMR 3- l— ) It¾ctG ^ H NMR 3- l—
20 116.78 (t) a) 5.33 (br s) H3, 20b H3 H20b(s) 20 116.78 (t) a) 5.33 (br s) H3, 20b H3 H20b (s)
b) 4.87 (br s) H3, 20a H5(w), 20a (s)  b) 4.87 (br s) H3, 20a H5 (w), 20a (s)
OAc 170.12 (s) H2. 2-0AcGJe  OAc 170.12 (s) H2.2.2-0AcGJe
169.73 (s) H10, 10-OAc(Me)  169.73 (s) H10, 10-OAc (Me)
π> 21.18 (q) 2.16 (s) π> 21.18 (q) 2.16 (s)
21.45 (q) 2.07 (s)  21.45 (q) 2.07 (s)
Γ 166.40 (s) H5, 2 3'  Γ 166.40 (s) H5, 2 3 '
2' 117.98 (d) 6.44 (d, 15.9) H3, H3' o-PhCm)  2 '117.98 (d) 6.44 (d, 15.9) H3, H3' o-PhCm)
3' 145.62 (d) 7.66 (d, 15.9) H2, o-Ph o-Ph(m)  3 '145.62 (d) 7.66 (d, 15.9) H2, o-Ph o-Ph (m)
q-Ph 134.57 (s) H2'. m-Ph  q-Ph 134.57 (s) H2'.m-Ph
o- 128.48 (d) 7.76 On) m-Ph H3'. p-Ph H2'(m), 3'(w), in-Ph(ni)  o- 128.48 (d) 7.76 On) m-Ph H3'.p-Ph H2 '(m), 3' (w), in-Ph (ni)
m - 128.94 (d) 7.43 Cm) P-Ph o-Ph. p-Ph o-Ph(ra). p-Ph(m)  m-128.94 (d) 7.43 Cm) P-Ph o-Ph.p-Pho o-Ph (ra) .p-Ph (m)
P一 130.32 (d) 7.43 (m) m-Ph o-Ph m-Ph(m)  P-130.32 (d) 7.43 (m) m-Ph o-Ph m-Ph (m)
*1) Multiplicities were determined by DEPT.  * 1) Multiplicities were determined by DEPT.
CO  CO
*2) Connections were determined by HMQC and multiplicities and coupling constants in Hz are in  * 2) Connections were determined by HMQC and multiplicities and coupling constants in Hz are in
parentheses.  parentheses.
*3) Determined by PFG-COSY.  * 3) Determined by PFG-COSY.
*4) Correlations from C to the indicated protons.  * 4) Correlations from C to the indicated protons.
*5) NOESY cross peaks. (s)=strong; (m) -medium; (w)=weak * 5) NOESY cross peaks. (S) = strong; (m) -medium; (w) = weak
を第 1 5表 ·第 1 6表にまとめて示す。 これにより、 化合物 (1 6) の 構造を解析した。 また、 該化合物 (1 6) の物性値等を測定した結果、 分子式 : し 35H420K) Are summarized in Tables 15 and 16. Thus, the structure of the compound (16) was analyzed. Also, the physical properties of the compound (16) were measured, and the molecular formula was: 35H420K)
分子量 : 622. 2783 (測定値)、 622. 2779 (計算値) 融 点 : 2 1 6T:〜 2 1 8  Molecular weight: 622. 2783 (measured value), 622. 2779 (calculated value) Melting point: 216 T: ~ 2 18
〔ひ〕 D 20 : + 2 1. 73° (c = 0. 092, CHC 13 ) [Shed] D 20: + 2 1. 73 ° (c = 0. 092, CHC 1 3)
I R : 2936, 1 750, 1 7 1 8, 1640, 1240 c
Figure imgf000093_0001
IR: 2936, 1750, 1718, 1640, 1240 c
Figure imgf000093_0001
であった。 Met.
NMR Sp e c t r a l Da t a o f c omp. ( 16 ) i n CDC 1 a NMR Sp e c t r a l Da t a o f c omp. (16) in CDC 1 a
皲 位匿 13C ·* 1 conected 'H H-H COSY " ΗΜΒ C ** NOE S Y " 皲 concealed 13 C * 1 conected 'H HH COSY "ΗΜΒ C ** NOE SY"
1 51.07 (d) 1.95 (br d. 8, 4. 1 .6) H2. L4j! Η3, 14α. 16.17 H2(n0. U m).16(m), 17(m)  1 51.07 (d) 1.95 (br d. 8, 4.1.6) H2. L4j! Η3, 14α. 16.17 H2 (n0. U m) .16 (m), 17 (m)
2 69.87 (d) 5.75 (br dd. 5,2, 1.6) HI, 3 HI, 3, 14α, jS HKm). 9(m). 17(m), 19(w)  2 69.87 (d) 5.75 (br dd. 5,2, 1.6) HI, 3 HI, 3, 14α, jS HKm) .9 (m) .17 (m), 19 (w)
3 43.11 (d) 3.12 (br d. 5.2) H2, 20a, b HI, 19, 20a. b H14 (m). 18(m)  3 43.11 (d) 3.12 (br d. 5.2) H2, 20a, b HI, 19, 20a.b H14 (m) .18 (m)
4 140.72 Cs) H3, 20a  4 140.72 Cs) H3, 20a
5 78.45 (d) 5.50 (br s) H20a. b H6o(m). 6j9(m)  5 78.45 (d) 5.50 (br s) H20a.b H6o (m) .6j9 (m)
6 27.72 (t) a) 2.01 (m) H5, 6ί, 7a, β H5(m), 6 s)  6 27.72 (t) a) 2.01 (m) H5, 6ί, 7a, β H5 (m), 6 s)
01.84 (m) H5, 6α. 7a, β H5(m). 6ff(s)  01.84 (m) H5, 6α.7a, β H5 (m) .6ff (s)
7 26.95 (t) e) 1.84 (m) Η6α. β, 7β H9, 19 H10(m)  7 26.95 (t) e) 1.84 (m) Η6α.β, 7β H9, 19 H10 (m)
β) 1.84 (m) Η6ιι, }, 7α  β) 1.84 (m) Η6ιι,}, 7α
8 43.76 (s) H2, 3. 9, 19  8 43.76 (s) H2, 3. 9, 19
g 77.00 Cd) 5.98 (d, 10· 7) H10 H10, 19 H2(m), 17(m), 19(m)  g 77.00 Cd) 5.98 (d, 10 ・ 7) H10 H10, 19 H2 (m), 17 (m), 19 (m)
10 71.86 (d) 5.39 ( 10.7) Η9 ΗΘ H7ff(m), 18(m)  10 71.86 (d) 5.39 (10.7) Η9 ΗΘ H7ff (m), 18 (m)
11 64.29 Cs) 鼠 16. 17.18  11 64.29 Cs) Rat 16.17.18
12 59,33 H10, W, 18 CD  12 59,33 H10, W, 18 CD
(S) OO (S) OO
13 208.21 (s) HI, 14s. β. 18 13 208.21 (s) HI, 14s.β. 18
14 QQ fid C† Λ (A Λ ヽ J1114 QQ fid C † Λ (A Λ ヽ J11
)2.69 (dd. 20.4. 8.4) HI. 14β HKm), 14o(s)  ) 2.69 (dd.20.4.8.4) HI.14β HKm), 14o (s)
15 38.34 (s) H10. 14c.16.17  15 38.34 (s) H10.14c.16.17
16 28.87 (q) 0.83 (s) H17 H17 HI (in). 17(ra)  16 28.87 (q) 0.83 (s) H17 H17 HI (in) .17 (ra)
17 25.27 (q) 1.85 (s) H16 HI. H16 HKm). 2(ra), 9 On), 16(m)  17 25.27 (q) 1.85 (s) H16 HI. H16 HKm) .2 (ra), 9 On), 16 (m)
18 15.75 (q) 2.00 (s) H3(m). 10(m)  18 15.75 (q) 2.00 (s) H3 (m) .10 (m)
19 17.97 (q) 0.99 (s) H3, 9 H2(w), 9(m)  19 17.97 (q) 0.99 (s) H3, 9 H2 (w), 9 (m)
20 119.90 (t) a) 5.51 (br s) Η3. 20b H3 H20b(s)  20 119.90 (t) a) 5.51 (br s) Η 3.20b H3 H20b (s)
b) 5.22 (br s) Η3, 20a H20a(s) b) 5.22 (br s) Η3, 20a H20a (s)
〔第 1 6表〕 (Table 16)
Figure imgf000095_0001
化合物 (1 5) および化合物 (1 6) の多剤耐性癌克服作用について は、 後段にて詳述する。 〔実施例 4〕
Figure imgf000095_0001
Effect of compound (15) and compound (16) on overcoming multidrug-resistant cancer Will be described in detail later. (Example 4)
樹高 3mの日本ィチイの針葉部 (生針葉部) からタキサン関連化合物 である化合物 (6) および化合物 ( 1 1 ) を取り出した。 即ち、 上記の 針葉部 42 28 gを n—へキサン 8. 7 5 L (X 2回) に 1週間浸漬す ることにより脱脂した後、 酢酸ェチル 8. 75 L (X 2回) に浸漬した。 1週間浸潰した後、針葉部を濾別し、濾液である抽出液を得た。次いで、 抽出液から酢酸ェチルを室温で減圧除去し、 該酢酸ェチルに可溶な成分 (粗酢酸ェチル抽出物) 74. 00 gを得た。  Compounds (6) and (11), which are taxane-related compounds, were extracted from needles (raw needles) of Nippon yew with a height of 3 m. That is, after immersing 4228 g of the above needle portion in 8.75 L (x 2 times) of n-hexane for 1 week, degrease it, and then immersing in 8.75 L (2 times) of ethyl acetate did. After immersion for one week, the needle portion was separated by filtration to obtain an extract as a filtrate. Next, ethyl acetate was removed from the extract under reduced pressure at room temperature to obtain 74.00 g of a component soluble in the ethyl acetate (crude ethyl acetate extract).
上記の成分を、 n—へキサンと酢酸ェチルとを容量比 1 : 1で混合し てなる混合溶液 2. 5 Lに投入し、 該混合溶液に不溶な成分 52. 5 g を得た。 次に、 該不溶成分を、 メチルアルコールと酢酸ェチルとを容量 比 1 : 3で混合してなる混合溶液 1 Lに溶解した。 次に、 該溶液を、 酸 性水溶液である 0. 5M硫酸 250m 1で 3回 (3 X 250m 1 ) 抽出 した。 得られた抽出液に 29 %アンモニア水を添加して pHを 9. 0と した後、 クロ口ホルム 30 Om 1で 5回 ( 5 X 300m l ) 抽出した。 さらに、 該抽出液を無水硫酸ナトリウムで乾燥させ、 クロ口ホルムを除 去した。 これにより、 粗アルカロイド分画 9. 26 g (針葉部の量を基 準として 0. 22 %) を得た。  The above-mentioned components were added to 2.5 L of a mixed solution obtained by mixing n-hexane and ethyl acetate at a volume ratio of 1: 1 to obtain 52.5 g of a component insoluble in the mixed solution. Next, the insoluble component was dissolved in 1 L of a mixed solution obtained by mixing methyl alcohol and ethyl acetate at a volume ratio of 1: 3. Next, the solution was extracted three times (3 × 250 ml) with 250 ml of 0.5 M sulfuric acid, which is an acidic aqueous solution. 29% ammonia water was added to the obtained extract to adjust the pH to 9.0, and the extract was extracted five times (5 × 300 ml) with a black-mouthed form 30 Om1. Further, the extract was dried over anhydrous sodium sulfate, and the form of cloper was removed. This gave 9.26 g of crude alkaloid fraction (0.22% based on the amount of needles).
次に、 上記の粗アルカロイド分画を、 活性アルミナ (中性, 活性度 I ) 920 gを用いたオープンカラムクロマトグラフィーを採用して、 溶媒 として n—へキサンと酢酸ェチルとメチルアルコールとを用いたグラジ ユエント溶出を行うことにより、 〜?17の 1 7個のフラクションに 分画 (荒分け) した。 Fi の溶出液は酢酸ェチル: n—へキサンが容量 比 3 : 7で混合してなる混合溶液であり、 F2 · F3 の溶出液は同容量比 1: 1で混合してなる混合溶液であり、 F4 · F5 の溶出液は同容量比 7 : 3で混合してなる混合溶液であり、 F6 - F7 の溶出液は酢酸ェチルであ り、 F8 の溶出液は酢酸ェチル: メチルアルコールが容量比 9 : 1で混 合してなる混合溶液であり、 F9 の溶出液は同容量比 4 : 1で混合して なる混合溶液であり、 F10の溶出液は同容量比 7 : 3で混合してなる混 合溶液であり、 Fuの溶出液は同容量比 1 : 1で混合してなる混合溶液 であり、 F12〜F17の溶出液はメチルアルコールであった。各フラクショ ンの収量は、 1番目 (低極性側) から 1 7番目 (高極性側) に向かって 順に、 Fi 82. 5mg, F2 1 03. 6mg, F3 1 08. 0 mg, F 4 1 55. 2mg, F5 183. 3mg, F6 1 95. Omg, F7 24 3. 7 mg, F8 220 1 mg, F9 2327 mg, F 1065 1. 5mg, F 1141 7. Omg, F i2483. Omg, Fi3774. 9 mg, F143 68. 9 mg, F15270. Omg, F 1635. 8 m g , F1714. 9m gであった。 Next, the crude alkaloid fraction was subjected to open column chromatography using 920 g of activated alumina (neutral, activity I), using n-hexane, ethyl acetate and methyl alcohol as solvents. By performing gradient gradient elution, It was fractionated (roughly divided) into 17 17 fractions. The eluate of Fi is a mixed solution of ethyl acetate: n-hexane mixed at a volume ratio of 3: 7, and the eluate of F 2 · F 3 is the same volume ratio A mixed solution obtained by mixing 1: 1. The eluate of F 4 · F 5 is a mixed solution obtained by mixing at the same volume ratio of 7: 3. The eluate of F 6 -F 7 is ethyl acetate. Ah is, eluate F 8 is acetate Echiru: methyl alcohol volume ratio of 9: 1 is a mixed solution composed engaged mixed in, eluant F 9 is the volume ratio 4: mixed solution prepared by mixing 1 , and the eluate F 10 is equal volume ratio 7: mixed-solution prepared by mixing 3, eluant F u is the volume ratio of 1: is a mixed solution obtained by mixing with 1, F 12 eluate to F 17 were methyl alcohol. The yield of each fraction was Fi 82.5 mg, F 2 103.6 mg, F 3 108.0 mg, F 4, in order from the first (low polarity side) to the 17th (high polarity side). 1 55.2 mg, F 5 183.3 mg, F 6 1 95.Omg, F 7 24 3.7 mg, F 8 220 1 mg, F 9 2327 mg, F 10 651.5 mg, F 1141 7.Omg, F i2483. Omg, it was Fi 3 774. 9 mg, F 14 3 68. 9 mg, F 15 270. Omg, F 16 35. 8 mg, F 17 14. 9m g.
そして、 上記 1〜 9番目のフラクション (Fi 〜F9 ) を、 ODSステ ンレスカラム (INERTSIL PREP ODS, GL Science, 250 X 20mm i.d., UV 254nm ) を用いた逆相高速液体クロマトグラフィーを採用して、 メ チルアルコールと 0. 05M酢酸アンモニゥム緩衝液 (pH4. 8) と ァセトニトリルとを容量比 1 : 1 : 2で混合してなる混合溶液 (キヤリ ァ, 流速 1 5 ml/min.) を用いて単離 ·精製した。 これにより、 アル力 ロイド分画の主成分であるタキシン 1189 Omg (保持時間 8. 9分、 針葉部の量を基準として 0. 02 1 1 %)、 および、 化合物 (6) 1 82 lmg (保持時間 6. 8分、 針葉部の量を基準として 0. 043 1 %) を取り出した。 Then, the 1-9-th fraction (Fi to F 9), employs ODS stearyl Nresukaramu (INERTSIL PREP ODS, GL Science, 250 X 20mm id, UV 254nm) reverse-phase high performance liquid chromatography using, A single solution (carrier, flow rate 15 ml / min.) Was prepared by mixing methyl alcohol, 0.05M ammonium acetate buffer (pH 4.8) and acetonitrile at a volume ratio of 1: 1: 2. Separated and purified. As a result, taxin 1189 Omg (retention time: 8.9 min, 0.021 1% based on the amount of needles), which is the main component of the alkyloid fraction, and compound (6) 182 lmg ( With a retention time of 6.8 minutes, 0.043 1%) was taken out based on the amount of needles.
次に、 上記主成分を取り出した後の (残余の) 低極性分画 460. 8 mg (保持時間 9. 0〜 2 2. 0分) を、 OD Sステンレスカラム (INERTSIL PREP-ODS, GL Science, 250 X 10mm i.d., UV 254誰 ) を用いた逆相高速液体クロマトグラフィーを採用して、 メチルアルコー ルと 0. 05M酢酸アンモニゥム緩衝液 (pH4. 8) とァセトニトリ ルとを容量比 1 : 1. 8 : 2で混合してなる混合溶液 (キャリア, 流速 5. 0 ml/min.) を用いて単離 ·精製した。 これにより、 化合物 (1 1) 16. 6mg (保持時間 47. 5分、 針葉部の量を基準として 0. 00 04%) を得た。 Next, 460.8 mg (remaining time: 9.0 to 22.0 minutes) of the (remaining) low-polarity fraction after removing the main component was transferred to an ODS stainless steel column. (INERTSIL PREP-ODS, GL Science, 250 x 10 mm id, UV 254) using reversed-phase high-performance liquid chromatography with methyl alcohol, 0.05 M ammonium acetate buffer (pH 4.8) and acetate nitrile. Was isolated and purified using a mixed solution (carrier, flow rate: 5.0 ml / min.) Prepared by mixing the mixture at a volume ratio of 1: 1.8: 2. Thus, 16.6 mg of the compound (11) (retention time: 47.5 minutes, 0.0004% based on the amount of needles) was obtained.
化合物 (1 1) の iH— NMRおよび i3C— NMRの測定結果を第 1 7表 ·第 1 8表にまとめて示す。 これにより、 化合物 (1 1) の構造を 解析した。 また、 該化合物 (1 1) の物性値等を測定した結果、  The measurements of iH-NMR and i3C-NMR of compound (11) are summarized in Tables 17 and 18. Thus, the structure of the compound (11) was analyzed. In addition, as a result of measuring the physical properties and the like of the compound (11),
分子式 : C37H5109 N Molecular formula: C 37 H 51 0 9 N
分子量 : 653. 3558 (測定値)、 653. 3564 (計算値) 融 点 : 1 86で〜 1 88t:  Molecular weight: 653.3558 (measured value), 653.3536 (calculated value) Melting point: 186 to 188t:
〔ひ〕 D 20 : + 80. 2。 (c = 0. 500, CHC 13 ) [H] D 20: +80.2. (c = 0.50, CHC 1 3 )
I R : 3328, 1 740 c m i (CHC 13 ) IR: 3328, 1 740 cmi ( CHC 1 3)
であった。 Met.
Figure imgf000099_0001
Figure imgf000099_0001
〔第 1 8表〕 (Table 18)
Figure imgf000100_0001
化合物(1 1)の多剤耐性癌克服作用については、後段にて詳述する, (多剤耐性癌克服作用の測定)
Figure imgf000100_0001
The overcoming effect of compound (11) on multidrug-resistant cancer will be described in detail later. (Measurement of action to overcome multidrug-resistant cancer)
化合物 (6) 〜 (1 9), (22) 〜 (26) の多剤耐性癌克服作用 (こ こでは、 P—糖蛋白質阻害剤としての機能) を、 上述した方法で以て測 定した。  The effect of compounds (6) to (19) and (22) to (26) on overcoming multidrug-resistant cancer (here, the function as a P-glycoprotein inhibitor) was measured by the method described above. .
尚、 化合物 (24) は、 針葉部の量を基準として 0. 0008 %の割 合で、 日本ィチイの針葉部 (生針葉部) から取り出すことができた。 化 合物(24) の iH— NMRおよび i3C— NMRの測定結果を第 1 9表 · 第 20表にまとめて示す。 これにより、 化合物 (24) の構造を解析し た。 また、 該化合物 (24) の物性値等を測定した結果、  The compound (24) could be taken out from the needle portion of the yew tree (raw needle portion) at a rate of 0.0008% based on the amount of the needle portion. The measurements of iH-NMR and i3C-NMR of compound (24) are summarized in Tables 19 and 20. Thus, the structure of compound (24) was analyzed. In addition, as a result of measuring physical properties and the like of the compound (24),
融 点 : 280で〜 282で  Melting point: from 280 to 282
「284で〜286で …文献①」  "From 284 to 286 ... Literature ①"
「240で (分解) …文献②」  "At 240 (disassembled)… literature ②"
[ α ] D 20: + 14. 6° (c = 0. 500, CHC 13 ) [Α] D 20: + 14. 6 ° (c = 0. 500, CHC 1 3)
「〔ひ〕 D 20 + 1 3 7. 1 ° (c = 0. 03, CHC 13 ) …文 献①」 "[Shed] D 20 + 1 3 7. 1 ° (c = 0. 03, CHC 1 3) ... literature ①"
「〔ひ〕 D 22 + 7 1. 7° (c = 0. 03, CHC 13 ) …文 献②」 "[H] D 22 + 71.7 ° (c = 0.03, CHC 13)… References"
I R : 2 9 64, 742 , 1 640, 1 246 c m—i (CHC 1 a )  I R: 2964, 742, 1640, 1246 cm—i (CHC 1 a)
「1 740, 1 7 1 0, 1 695, 1670, 1640, 1 250, 1 230 cm-i (KB r ) …文献①」  "1 740, 1710, 1695, 1670, 1640, 1250, 1230 cm-i (KBr)… Reference ①"
「3050, 1 746, 1 62 5, 1400, 1 2 50 cm-i (KB r ) …文献①」  "3050, 1746, 1625, 1400, 1250 cm-i (KBr)… Reference ①"
であった。 上記文献①は Liang, J.Y., et al. Phytochem., 47, 69-72, (1998)であり、 文献②は Shrestha, T.B., et al. J. Nat. Prod., 60, NMR S e c t r a l Da t a o f c omp. ( 24 ) i n CDC " Met. Reference, is Liang, JY, et al. Phytochem., 47, 69-72, (1998), and reference ② is Shrestha, TB, et al. J. Nat.Prod., 60, NMR Sectral Da taofc omp. (24) in CDC "
f ound r e f e r en c e *3 f ound refer en ce * 3
位置 し COn6Cl€Q Π. concCteQ n CO  Located COn6Cl € Q Π. ConcCteQ n CO
1  1
1 40.86 (d) 2.21 (ra) a 2.21 (m)  1 40.86 (d) 2.21 (ra) a 2.21 (m)
ti 25.72 (t) a) 1.81 (ra) c a) 1.81 (m)  ti 25.72 (t) a) 1.81 (ra) c a) 1.81 (m)
b) 2.04 (ra) b) 2.04 (m)  b) 2.04 (ra) b) 2.04 (m)
o 36.84 (d) 3.06 (br d. 4.9) g. Q) 3.04 (d. 4.9)  o 36.84 (d) 3.06 (br d. 4.9) g.Q) 3.04 (d. 4.9)
A Q  A Q
146.90 Cs) SJ  146.90 Cs) SJ
J 74.38 (d) 5.46 (t, 3.0) 7 j j 5. 3 (t. 3.2, 2.4)  J 74.38 (d) 5.46 (t, 3.0) 7 j j 5.3 (t. 3.2, 2.4)
U 34.38 (t) a) 2.04 (ra) o4. U l) a) 2.04 (m)  U 34.38 (t) a) 2.04 (ra) o4.U l) a) 2.04 (m)
b) 1.78 On) b) 1.77 (m)  b) 1.78 On) b) 1.77 (m)
7 69.82 (d) 5.54 (dd, 11.5, 5.2) 09. O 5.52 (dd, 11.5, 5.2)  7 69.82 (d) 5.54 (dd, 11.5, 5.2) 09.O 5.52 (dd, 11.5, 5.2)
0  0
0 46.54 (s) a a 0 46.54 (s) a a
S)  S)
Q 75.70 (d) 5.93 (d. 10.8) 7C 7 u 5.90 (d. 10.8)  Q 75.70 (d) 5.93 (d. 10.8) 7C 7 u 5.90 (d. 10.8)
1丄 Π u 72.93 (d) 6.31 (d. 10.8) 72· 9 (d) 6.29 (d, 10.8) 〇 1 丄 Π u 72.93 (d) 6.31 (d.10.8) 729 (d) 6.29 (d, 10.8) 〇
11 11
丄 i 152.24 (s) 152.2 (s)  丄 i 152.24 (s) 152.2 (s)
13821 (&) 138.2 (s)  13821 (&) 138.2 (s)
13 200.05 (s) 200.0 (s)  13 200.05 (s) 200.0 (s)
14 39.53 (t) a) 2.94 (dd, 20.0, 7.2) 39.5 (t) a) 2.92 (dd. 19.8, 7.3)  14 39.53 (t) a) 2.94 (dd, 20.0, 7.2) 39.5 (t) a) 2.92 (dd.19.8, 7.3)
b) 1.99 (m) b) 1.94 (br d, 19.8)  b) 1.99 (m) b) 1.94 (br d, 19.8)
15 39.79 (s) 39.8 (s)  15 39.79 (s) 39.8 (s)
16 25.56 (q) 1.64 (s) 25.6 (q) 1.61 (s)  16 25.56 (q) 1.64 (s) 25.6 (q) 1.61 (s)
17 37.07 (q) 1.13 (s) 37,1 (q) 1.11 (s)  17 37.07 (q) 1.13 (s) 37,1 (q) 1.11 (s)
18 14.11 (q) 2.39 (s) 14· 1 (q) 2.37 (s)  18 14.11 (q) 2.39 (s) 14.1 (q) 2.37 (s)
19 12.82 (q) 0.89 (s) 12.8 (q) 0.86 (s)  19 12.82 (q) 0.89 (s) 12.8 (q) 0.86 (s)
20 114.60 (t) a) 5.31 (d. 1.0) 114.6 (t) a) 5.29 (br s)  20 114.60 (t) a) 5.31 (d.1.0) 114.6 (t) a) 5.29 (br s)
b) 4.95 (d. 1.5) b) 4.93 (d, 1.4)  b) 4.95 (d. 1.5) b) 4.93 (d, 1.4)
)¾ 8〇822997 1e--《 〔第 20表〕 ) ¾ 8〇822997 1e-- 《 (Table 20)
QH I!p!;u¾ U 952s. d suo: QH I! P!; U¾ U 952s.d suo:
( (866£I λΓ1s 69u130B-: -, ..  ((866 £ I λΓ1s 69u130B-:-, ..
0  0
<0 *^ 00<0 * ^ 00
CM C  CM C
Figure imgf000103_0001
多剤耐性癌克服作用の測定結果を、 まとめて第 2 1表〜第 26表に示 す。 同表において、 「投与濃度」 とは、 反応溶液における上記化合物また はべラバミルの濃度 ( gZm l ) を示し、 「VCR蓄積量の平均値」 と は、 各ゥエルにおける、 2780 A D細胞中のピンクリスチン (VCR) 蓄積量の平均値 (dpm/well) を示す。 また、 「ベラパミル比」 とは、 ベラ パミルを対照薬剤とした比較実験との比較結果を示し、 「評価」の項にお ける (最大べラパミル比) および (濃度) とは、 ベラパミルと比較して 最も効果が高かったときの比および濃度を示すものとする。 尚、 多剤耐 性癌克服作用が高い化合物については、 再測定を行った (第 25表 ·第 26表)。
Figure imgf000103_0001
The results of measuring the effect of overcoming multidrug-resistant cancer are summarized in Tables 21 to 26. You. In the table, “dose concentration” indicates the concentration (gZml) of the above compound or veravamil in the reaction solution, and “average VCR accumulation amount” indicates the pin concentration in 2780 AD cells in each well. Shows the average value of the accumulated amount of Christin (VCR) (dpm / well). In addition, “verapamil ratio” indicates the results of comparison with a comparative experiment using verapamil as a control drug, and (maximum verapamil ratio) and (concentration) in the “Evaluation” section are compared with those of verapamil. And the ratio and concentration at which the effect was highest. Re-measurement was performed for compounds with high ability to overcome multidrug-resistant cancer (Tables 25 and 26).
濃度 VCR蓄積量 コント ベラパミル比 評 価 皲 化合物 の平均植 ロール比 判 定 (最大べラバミル比) IN3 Concentration VCR accumulation control Verapamil ratio Evaluation 皲 Average compound vegetation ratio Judgment (maximum veravamil ratio) IN3
(dpm/well) (%) (濃 度)  (dpm / well) (%) (concentration)
0. 1 557 1 07 N  0.1 557 1 07 N
化合物 Compound
62 1 20 + 一 %'  62 1 20 + 1% '
(6 )  (6)
1 0 873 1 67 + g/m 1  1 0 873 1 67 + g / m 1
0. 1 443 85 N  0.1 443 85 N
化合物 Compound
1 9 十 一 %  1 9 11%
(7)  (7)
1 0 1 336 255 十 U g/m 1  1 0 1 336 255 Ten U g / m 1
0. 1 44 1 84 N  0.144 1 84 N
化合物 Compound
62 1 20 十 一 %  62 1 20 11%
(8)  (8)
1 0 1 245 238 + fi g/ 1  1 0 1 245 238 + fi g / 1
0. 1 5 23 1 0 0 N  0.15 23 1 0 0 N
化合物 Compound
6 5 1 26 + 一 %  6 5 1 26 + 1%
(9)  (9)
1 0 1 0 57 20 2 + g/m 1  1 0 1 0 57 20 2 + g / m 1
0. 1 529 1 0 1 N  0.1 529 1 0 1 N
化合物 Compound
28 1 39 + - %  28 1 39 +-%
(10)  (Ten)
1 0 1 26 5 24 2 + g/m 1  1 0 1 26 5 24 2 + g / m 1
C 1M C 1M
o
Figure imgf000106_0001
o
Figure imgf000106_0001
^ 22 投与濃度 VCR蓄積量 っン卜 ベラパミル比 評 価 ^ 22 Administration concentration VCR accumulation amount Verapamil ratio Evaluation
化合物 の平均値 ロール比 判 定 (最大べラバミル比)  Average value of compounds Roll ratio judgment (maximum verabamil ratio)
(β g/ml) (dpm/well) (濃 度) (β g / ml) (dpm / well) (concentration)
0. 1 46 1 1 08 94 再測定 化合物 0.1 46 1 1 08 94 Re-measurement Compound
1 735 1 73 + 1 08 1 29 % 1 735 1 73 + 1 08 1 29%
(15) (15)
1 0 1 726 406 + + 1 29 1 0 iL g/m 1  1 0 1 726 406 + + 1 29 1 0 iL g / m 1
0. 1 46 1 1 08 94 再測定 化合物  0.1 46 1 1 08 94 Re-measurement Compound
5 1 78 + 1 1 1 1 1 1 %  5 1 78 + 1 1 1 1 1 1 1%
(16)  (16)
1 0 1 345 3 1 6 + + 1 0 1 1 g/m】  1 0 1 345 3 1 6 + + 1 0 1 1 g / m]
0. 1 468 I 1 0 96 再測定 化合物  0.1 468 I 1 0 96 Remeasured compound
900 2 1 2 + 1 33 1 39 %  900 2 1 2 + 1 33 1 39%
(17)  (17)
1 0 1 85 1 436 + + 1 39 1.0 g/m 1  1 0 1 85 1 436 + + 1 39 1.0 g / m 1
0. 1 1 3 9 N 化合物  0.1 1 3 9 N compound
406 96 + 一 %  406 96 + 1%
(18)  (18)
1 0 45 1 1 06 II g/m 1  1 0 45 1 1 06 II g / m 1
0. 1 1 1 97 N 化合物  0.1 1 1 97 N compound
475 1 1 2 + - %  475 1 1 2 +-%
(19)  (19)
1 0 82 1 84 + a g/m 1  1 0 82 1 84 + a g / m 1
^ 23 ^ 23
o
Figure imgf000108_0001
o
Figure imgf000108_0001
^^24 再 測 定 投与濃度 VCR蓄積量 3ン卜 ベラバミル比 評 価 ^^ 24 Re-measurement Dose concentration VCR accumulation 3 ton Berabamil ratio Evaluation
化合物 の平均値 ロール比 判 定 (最大べラバミル比)  Average value of compounds Roll ratio judgment (maximum verabamil ratio)
( / cr /ml) (dpm/well) (%) (%) (濃 度)  (/ cr / ml) (dpm / well) (%) (%) (concentration)
0. 1 25 9 1 1 4 + 97 P  0.125 9 1 1 4 + 97 P
化合物 Compound
1 726 3 1 8 + + 1 53 200 %  1 726 3 1 8 + + 1 53 200%
(11)  (11)
1 0 2421 1 0 62 + + + + 200 1 0 a g/m】  1 0 2 421 1 0 62 + + + + 200 1 0 a g / m]
0, 1 280 1 23 + 1 05 P  0, 1 280 1 23 + 1 05 P
化合物 Compound
1 6 87 30 1 + + 1 45 1 85 %  1 6 87 30 1 + + 1 45 1 85%
(15)  (15)
1 0 2235 98 0 + + + 1 85 1 0 fi g/m 1  1 0 2235 98 0 + + + 1 85 1 0 fi g / m 1
0. 1 276 1 2 1 + 1 04 P  0.1 276 1 2 1 + 104 P
化合物 Compound
1 673 295 十 1 42 1 42 %  1 673 295 tens 1 42 1 42%
(16)  (16)
1 0 1 26 8 5 5 6 十 + + 1 05 1 μ. g/m 1  1 0 1 26 8 5 5 6 10 + + 1 05 1 μ.g / m 1
0. 1 28 3 1 24 + 1 06 P  0.128 3 1 24 + 1 06 P
化合物 Compound
1 732 32 1 + + 1 55 1 9 1 %  1 732 32 1 + + 1 55 1 9 1%
(17)  (17)
1 0 23 1 8 1 0 1 7 + + + + 1 9 1 1 0 {i g/m 1  1 0 23 1 8 1 0 1 7 + + + + 1 9 1 1 0 (i g / m 1
08 1 〔第 26表〕 08 1 (Table 26)
Figure imgf000110_0001
Figure imgf000110_0001
第 2 1表〜第 26表より明らかなように、 前記化合物群のうち、 特に 再測定を行った化合物(1 1), (1 5)〜(1 7), (22), (24), (2 6) は、 何れも多剤耐性癌克服作用を有し、 しかも、 ベラパミルを上回 る多剤耐性癌克服作用を有する高活性化合物であることから、 それ単独 で特に有効な (新規な) 多剤耐性癌克服剤となり得ることが判る。 As is clear from Tables 21 to 26, among the compound groups, the compounds (11), (15) to (17), (22), (24), (2 6) is a highly active (new) compound, which is a highly active compound that has a multidrug-resistant cancer-overcoming action and a multidrug-resistant cancer-overcoming action that surpasses verapamil. It turns out that it can be a drug-resistant cancer overcoming agent.
〔第 27表〕  (Table 27)
Figure imgf000111_0001
前記化学式 ( 1 7), (22), ( 27 ), (28), (29) で表される 5 種類の夕キシニン化合物は、 新規物質である。
Figure imgf000111_0001
The chemical formula (17), (22), (27), (28), (29) Kind of xinine compounds are new substances.
〔実施例 5〕 (Example 5)
(カルスからの化合物 (3 0), (3 1), (3 3) 〜 (36) の取り出し) 日本イチィ (7¾ us cus fe a Sieb. et Zucc.) の若茎 (針葉部) を外 植体として用い、 カルスの誘導並びに組織培養を行った。 上記外植体と しての若茎は、冬季(採取前 1力月間の平均気温 5で以下) に採取した。 はじめに、 上記切片を一般的な方法で洗浄 ·滅菌処理した後、 ショ糖 (スクロース)、 寒天粉末、 およびオーキシンである NAAが、 順に 2 0 g/L, 1 0 gZL、 1. OmgZLの濃度となるよう添加された改変 ガンボーグ培地 (以下、 改変ガンボーグ固体培地と称する) に、 該切片 を置床した。すなわち、本実施例における改変ガンボーグ固体培地とは、 以下の第 28表に示す組成よりなる固体培地である。 (Extraction of compounds (30), (31), (33)-(36) from callus) Remove the young stem (needle) of Japanese yew (7¾ uscus fe a Sieb. Et Zucc.) Callus induction and tissue culture were performed as a plant. The young shoots as the above explants were collected in winter (at an average temperature of 5 months during the first month before collection). First, after washing and sterilizing the above sections by a general method, sucrose (sucrose), agar powder, and NAA, which is an auxin, were added in the order of 20 g / L, 10 gZL, and 1.OmgZL. The slices were placed on a modified Gamborg's medium (hereinafter, referred to as a modified Gamborg's solid medium) to which the addition was made. That is, the modified Gamborg's solid medium in this example is a solid medium having the composition shown in Table 28 below.
〔第 2 8表〕 (Table 28)
Figure imgf000113_0001
Figure imgf000113_0001
そして、 上記の固体培地を 2 5 :、 喑所下で静置した。 これにより、 培養細胞を静置培養して、 日本ィチイのカルスを得た。 次に、 約 4 0 5 0日毎に増殖性の良好なカルスを選抜し、 同一組成の培地を用いて継 代培養を行うことで、 系統を確立した。 次に、 寒天粉末、 NAA、 ジャ スモン酸メチル (J M)、 オリゴサッカライド (KTO S) が、 順に、 1 0 g/L, 1. Omg/L, 1 00 M、 0. 3mgZLの濃度となる よう添加された改変ガンボーグ培地 (以下、 生産培地 1と称する) にこ のカルスを置床し、 25 、 暗所下で 60日間静置培養を行った。 この 結果、 新鮮重量で 83 1. 9 gのカルス (新鮮カルスと称する) が得ら れた。 Then, the above-mentioned solid medium was allowed to stand under 25: 5 place. As a result, the cultured cells were allowed to stand and cultured to obtain calluses of Japanese yew. Next, a callus with good growth potential was selected about every 450 days, and the callus was passaged using a medium of the same composition. The strain was established by subculture. Next, the concentration of agar powder, NAA, methyl jasmonate (JM), and oligosaccharide (KTOS) were adjusted to 10 g / L, 1.0 mg / L, 100 M, and 0.3 mg ZL, respectively. The callus was placed on the added modified gamborg culture medium (hereinafter referred to as production medium 1), and cultured statically for 25 days in the dark for 25 days. As a result, a fresh weight of 831.9 g of callus (referred to as fresh callus) was obtained.
次に、 上記新鮮カルスを用いて上記化合物 (30), (3 1), (33) 〜 (35)、 及び新規なタキシニン化合物としての化合物 (36) の抽出 を行った。 先ず、 新鮮カルス 83 1. 9 gを採取し、 凍結乾燥させるこ とにより、 乾燥カルス 86. 97 gを得た。 続いて、 n—へキサン 1. 3 L中で該乾燥カルスを 1時間撹拌し、 抽出操作を行った。 この撹拌 · 抽出操作は 3回繰り返し行われた。  Next, the compounds (30), (31), (33) to (35), and the compound (36) as a novel taxinine compound were extracted using the fresh callus. First, 83.19 g of fresh callus was collected and freeze-dried to obtain 86.97 g of dry callus. Subsequently, the dried callus was stirred in 1.3 L of n-hexane for 1 hour to perform an extraction operation. This stirring / extraction operation was repeated three times.
そして、 n—へキサン層 (抽出液) から n—へキサンを留去して、 粗 n—へキサン抽出物 1, 847 mgを得た。 続いて、 シリカゲルステン レスカラムを用いた順相高速液体クロマトグラフィー(INERTSIL PREP-SIL; 25 X 1 c m; GL Science;溶媒 酢酸ェチル: n—へキ サン = 2 : 8 ;流速 5 m 1 /m i n) を用いて、 粗 n—へキサン抽出物 より各種化合物を分離した。  Then, n-hexane was distilled off from the n-hexane layer (extract) to obtain 1,847 mg of a crude n-hexane extract. Subsequently, normal-phase high-performance liquid chromatography using a silica gel stainless steel column (INERTSIL PREP-SIL; 25 X 1 cm; GL Science; solvent: ethyl acetate: n-hexane = 2: 8; flow rate: 5 m1 / min) , Various compounds were separated from the crude n-hexane extract.
その結果、 n—へキサン層から上記化合物 (30), (3 1), (33) 〜 (36) が単離された。 より具体的には、 化合物 (35) が 1 0. 0 mg (乾燥カルスに対して 0. 0 1 2重量%、 保持時間 ( t R) 2 1. 6分)、 化合物 (36) が 1 5. Omg (乾燥カルスに対して 0. 0 1 7 重量%、 t R 28. 1分) 単離された。 また、 化合物 (30) と、 その 14位ァシルォキシ同族体である化合物 (3 1), (33), (34) とが 順に、 37 7mg (乾燥カルスに対して 0. 433重量%、 t R 1 5. 2分)、 38mg (乾燥カルスに対して 0. 044重量%、 t R 1 1. 5 分)、 354mg (乾燥カルスに対して 0. 40 7重量%、 t R 8. 3分)、 3 7mg (乾燥カルスに対して 0. 043重量%、 t R 9. 4分) 単離 された。 すなわち、 n—へキサン層からの化合物 (3 0) と、 その 1 4 位ァシルォキシ同族体との合計単離収量は 8 0 6mg (乾燥カルスに対 して 9 2 7重量%) に達した。 As a result, the compounds (30), (31), (33) to (36) were isolated from the n-hexane layer. More specifically, compound (35) contained 10.0 mg (0.012% by weight based on dry callus, retention time (tR) 21.6 minutes) and compound (36) contained 15 Omg (0.017% by weight based on dry callus, tR 28.1 min) Isolated. In addition, compound (30) and its 14-hydroxyl homologue compounds (31), (33), and (34) were sequentially converted to 377 mg (0.433% by weight based on dry callus, t R 1 Five. 2 minutes), 38 mg (0.044% by weight based on dry callus, tR 11.5 minutes), 354 mg (0.47% by weight based on dry callus, tR 8.3 minutes), 37 mg (0.043% by weight based on dry callus, tR 9.4 min) Isolated. That is, the total isolated yield of the compound (30) from the n-hexane layer and its 14-position acyloxy homolog reached 806 mg (927% by weight based on dry callus).
n—へキサン層からは、その他に、タクスァビエタン A(taxusabietane A) が 8. Omg (乾燥カルスに対して 0. 00 9重量%、 t R 1 3. 6 分)、 タクスシン (taxusin) が 5 7mg (乾燥カルスに対して 0. 0 6 6 重量%、 t R 1 8. 3分)、 2 α—ァセトキシタクスシン(2 α— acetoxy taxusin)が 5. Omg (乾燥カルスに対して 0. 0 0 6重量%、 t R 4 1. 0分) 単離された。  From the n-hexane layer, in addition, 8. Omg (0.009% by weight based on dry callus, tR 13.6 minutes) of taxusabietane A, 57 mg of taxusin (0.066% by weight based on dry callus, t R 18.3 min), 2 α-acetoxitaxin (2 α- acetoxy taxusin) 5.Omg (0. 0.6% by weight, tR 4 1.0 min) Isolated.
単離された化合物の同定並びに構造決定は、 インバースプローブを装 着した 50 0MH zの NMRを用い、 P FG— COSY、 P FG— HM QC、 P F G— HMB C等の測定結果を解析することによって行った。 尚、 これらの測定結果のうち、 新規なタキシニン化合物としての化合物 (3 6) の iH— NMR及び 13C— NMRの測定結果を第 2 9表、 第 3 0表にまとめて示す。 これにより、 化合物 (3 6) の構造を解析した。 また、 該化合物 (3 6) の物性値等を測定した結果、  Identification and structure determination of the isolated compound were performed by analyzing the measurement results of PFG-COSY, PFG-HMQC, PFG-HMBC, etc. using 500 MHz NMR equipped with an inverse probe. went. Among these measurement results, the measurement results of iH-NMR and 13C-NMR of compound (36) as a novel taxinine compound are summarized in Tables 29 and 30. As a result, the structure of the compound (36) was analyzed. In addition, as a result of measuring physical properties of the compound (36),
分子式 : C33H4207 Molecular formula: C 33 H 42 0 7
分子量 : 5 5 0. 2 9 3 6 (測定値)、 55 0. 2 9 3 1 (計算値) 融 点 : 22 8で〜 2 3 1  Molecular weight: 55.29.36 (measured value), 55.29.31 (calculated value) Melting point: 228 to 23.1
〔ひ〕 D 20 : + 9 9. 8° (c = 0. 5 38, CHC 13 ) [Shed] D 20: + 9 9. 8 ° (c = 0. 5 38, CHC 1 3)
I R : 296 0, 1 7 32, 1 642, 1 2 5 2 c (CH であった。 Sp e c t r a l Da t a o f c omp o u n d (36) i n C 6 D ( IR: 2960, 1732, 1642, 1252c (CH2. Sp ectral Da taofc omp ound (36) in C 6 D (
13 * 1 f>n  13 * 1 f> n
し connected ri n rl し UovY * 3 H BC Μ U C V * 5  UovY * 3 H BC Μ U C V * 5
IN U C. I  IN U C. I
1 40.76 (d) 1.50 (m) H2, 14 β, 3. 14tt. β, H2 m). 14 s)  1 40.76 (d) 1.50 (m) H2, 14 β, 3.14tt. Β, H2 m). 14 s)
16, 17 16(m), 17(m)  16, 17 16 (m), 17 (m)
2 28.54 (t) <r)l.45(br dd. 15.5. 5.5) HI. 3 HI. 3, 14a, β. H2 s), 3(w),  2 28.54 (t) <r) l.45 (br dd. 15.5.5.5) HI. 3 HI. 3, 14a, β.H2 s), 3 (w),
20b(s)  20b (s)
j l.65(ddd, 15.5, 6.0.1.5) HI, 3 HI Cm), 2fl(s),  j l.65 (ddd, 15.5, 6.0.1.5) HI, 3 HI Cm), 2fl (s),
9(s)  9 (s)
3 38.41 (d) 3.10 (br d, 6.0) H2ff, jJ, 20a, b HI. 2a, β, 5. 7β, 9, H2c(w), 7 m).  3 38.41 (d) 3.10 (br d, 6.0) H2ff, jJ, 20a, b HI.2a, β, 5.7β, 9, H2c (w), 7 m).
19, 20a, ¾ 10( ), 14a(s)  19, 20a, ¾ 10 (), 14a (s)
4 149.61 (s) H2a. 3, 5. 6 , 20a. b  4 149.61 (s) H2a. 3, 5.6, 20a.b
5 76.30 (d) 5.53 (dd, 2.5, 2.5) H6«, j? H3, 6e, 7α. ' 20a, b H6a(m). 6i(m).  5 76.30 (d) 5.53 (dd, 2.5, 2.5) H6 «, j? H3, 6e, 7α. '20a, b H6a (m). 6i (m).
20a (DI)  20a (DI)
6 27.86 (t) a) 1.78 (m) H5, 6β, 7a, β H5, 7a, β H5(n0  6 27.86 (t) a) 1.78 (m) H5, 6β, 7a, β H5, 7a, β H5 (n0
β) 1.57 (m) E5, 6a. 7a, β H5(m), 7 m)  β) 1.57 (m) E5, 6a.7a, β H5 (m), 7 m)
7 27.86 (t) fl) 1.84 (m) H6i. β. H5, 6a. β, 9. 19 H3(m), 7 s).  7 27.86 (t) fl) 1.84 (m) H6i.β.H5, 6a.β, 9.19 H3 (m), 7 s).
Or Or
10 On) 10 On)
β) 1.92 (m) Η6ι, β, 7a H6 ra), 7a(s),  β) 1.92 (m) Η6ι, β, 7a H6 ra), 7a (s),
19 (in)  19 (in)
8 43.46 (S) H2ff, £. 3, 6α. 7α. β,  8 43.46 (S) H2ff, £. 3, 6α. 7α. Β,
9. 19  9.19
9 80.61 (d) 6.18 (d. 10.5) H10 H7a, 10, 19 H2.i(s). 17(s),  9 80.61 (d) 6.18 (d. 10.5) H10 H7a, 10, 19 H2.i (s). 17 (s),
19(m)  19 (m)
10 70.84 (d) 5.04 (d, 10.5) H9 H9 H3(w). 7fl(m).  10 70.84 (d) 5.04 (d, 10.5) H9 H9 H3 (w) .7fl (m).
18(s)  18 (s)
11 138.95 (s) HI, 10, 13, 16, 17, 18  11 138.95 (s) HI, 10, 13, 16, 17, 18
12 133.63 (s) H10. 13. 14 18  12 133.63 (s) H10.13.14 18
13 70.68 (d) 6.10(ddd. 10.0. 6.5, 1.2) H14c, β, 18 HI. 14ff. β, 18 H14a(m).14i(s)  13 70.68 (d) 6.10 (ddd. 10.0. 6.5, 1.2) H14c, β, 18 HI. 14ff.β, 18 H14a (m) .14i (s)
16(m), 18(m)  16 (m), 18 (m)
» 29 〔第 30表〕 »29 (Table 30)
Figure imgf000117_0002
Figure imgf000117_0002
Figure imgf000117_0001
尚、 上記化合物のうち化合物 (35) 及び、 化合物 (36) は、 上記 生産培地 1にかえて以下に示す組成の生産培地 2 6を用いて誘導 ·組 織培養されてなるカルスからも単離された。
Figure imgf000117_0001
The compound (35) and the compound (36) among the above compounds were induced using a production medium 26 having the following composition instead of the production medium 1. It was also isolated from calli that had been woven.
上記生産培地 2とは、寒天粉末、 NAA及び、 ジャスモン酸メチルが、 順に 1 0 gZL、 0. Smg/L, 1 0 0 mMとなるように添加された 改変ガンボーグ培地であり、該生産培地 2における化合物(3 5)及び、 化合物 (3 6) の単離収量は順に、 0mg、 1 9. 2mg (乾燥カルス に対して 0. 02 6重量%)であった。 上記生産培地 3とは、寒天粉末、 NAA、 ジャスモン酸メチル、 フエ二ルァラニン、 酢酸が、 順に 1 0 g ZL、 0. 5mgZL、 1 00mM、 0. 2 6 3 g/L, 1 m 1 /L, となるように添加された改変ガンボーグ培地であり、 該生産培地 3にお ける化合物 (3 5) 及び、 化合物 (36) の単離収率は順に、 0mg、 1 9. 2mg (乾燥カルスに対して 0. 0 26重量%) であった。  The production medium 2 is a modified gamborg medium to which agar powder, NAA, and methyl jasmonate are added in order of 10 gZL, 0.1 Smg / L, and 100 mM, respectively. The isolation yields of the compound (35) and the compound (36) were 0 mg and 19.2 mg (0.026% by weight based on dry callus), respectively. The production medium 3 is agar powder, NAA, methyl jasmonate, phenylalanine, and acetic acid, in that order: 10 g ZL, 0.5 mg ZL, 100 mM, 0.26 g / L, 1 m 1 / L The isolated yield of compound (35) and compound (36) in production medium 3 was 0 mg and 19.2 mg (in dry callus, respectively). 0.026% by weight).
また、 上記生産培地 4とは、 寒天粉末、 NAA、 フエ二ルァラニン、 酢^が、 順に 1 0 gZL、 0. 5mg/L, 0. 2 6 3 g/L, 1 m l /L, となるように添加された改変ガンボーグ培地であり、 該生産培地 4における化合物 (3 5) 及び、 化合物 (3 6) の単離収率は順に、 5 8. 8mg (乾燥カルスに対して 0. 1 0 5重量%)、 Omgであった。 上記生産培地 5とは、寒天粉末、 NAA、 キトサンが、 順に 1 0 gZL、 0. 5mg/L, 1. 2 5 gZL、 となるように添加された改変ガンボ ーグ培地であり、 該生産培地 5における化合物 (3 5) 及び、 化合物 (3 6) の単離収率は順に、 58. 8mg (乾燥カルスに対して 0. 1 0 5 重量%)、 Omgであった。 上記生産培地 6とは、 寒天粉末、 ΝΑΑ、 β —シクロデキストリンが、 順に 1 0 gZL、 0. 5mgZL、 1 0mM、 となるように添加された改変ガンボーグ培地であり、 該生産培地 6にお ける化合物 (3 5) 及び、 化合物 (3 6) の単離収率は順に、 7. 5m g (乾燥カルスに対して 0. 0 1 5重量%)、 Omgであった。 〔実施例 6〕 The production medium 4 is such that agar powder, NAA, phenylalanine, and vinegar become 10 gZL, 0.5 mg / L, 0.263 g / L, 1 ml / L, respectively. The isolated yield of the compound (35) and the compound (36) in the production medium 4 was 58.8 mg (0.15% with respect to the dry callus) in the production medium 4 in this order. Wt%), Omg. The production medium 5 is a modified gambog medium in which agar powder, NAA, and chitosan are added in such a manner as to give 10 gZL, 0.5 mg / L, and 1.25 gZL, respectively. The isolation yields of the compound (35) and the compound (36) in 5 were 58.8 mg (0.105% by weight based on dry callus) and Omg, respectively. The production medium 6 is a modified Gamborg's medium to which agar powder, ΝΑΑ, β-cyclodextrin is added in order of 10 gZL, 0.5 mgZL, and 10 mM, respectively. The isolation yields of the compound (35) and the compound (36) were 7.5 mg (0.015% by weight based on dry callus) and Omg, respectively. (Example 6)
(カルスからの化合物 (30) 〜 (3 5)、 並びにタキソ一ル関連化合 物の取り出し)  (Removal of compounds (30) to (35) and taxol-related compounds from callus)
日本イチィ ( ¾ATUS cuswWa a Sieb. et Zuc ) の若茎 (針葉部) を外 植体として用い、 上記実施例 1と同一の条件、 培地でカルスの誘導並び に組織培養を行った。 続いて、 寒天粉末、 NAA、 フエ二ルァラニン、 酢酸が、 順に、 1 0 gZL、 0. 5mg/L, 0. 2 6 3 g/L, 1. 0m l ZLの濃度となるよう添加された改変ガンボーグ培地に得られた カルスを置床し、 2 5で、 喑所下で 6 0日間静置培養を行った。 この結 果、 新鮮重量で 60 5. 5 gのカルス (新鮮カルスと称する) が得られ た。  The callus induction and tissue culture were carried out under the same conditions and in the same medium as in Example 1 above, using the young stems (needles) of the Japanese yew (@ATUS cuswWa a Sieb. Et Zuc) as explants. Subsequently, the agar powder, NAA, phenylalanine, and acetic acid were added in order to obtain a concentration of 10 gZL, 0.5 mg / L, 0.263 g / L, and 1.0 ml ZL. The calli obtained in a Gamborg's medium were placed on a bed, and static culture was performed at 25 under a place for 60 days. As a result, a fresh weight of 605.5 g of callus (referred to as fresh callus) was obtained.
次に、 上記新鮮カルスを用いて上記化合物 (3 0) 〜 (3 5)、 並びに タキソ一ル関連化合物の抽出を行った。 先ず、 新鮮カルス 6 0 5. 5 g を採取し、 凍結乾燥させることにより、 乾燥カルス 5 5. 9 gを得た。 続いて、 有機溶媒として n—へキサン、 酢酸ェチル、 メチルアルコール を用いて、 該乾燥カルスからの抽出操作を順に行った。 即ち、 n—へキ サン 1 Lを用いて一時間撹拌 ·抽出操作 (3回繰り返す) を行った後、 酢酸ェチル 1 Lを用いて一時間撹拌 ·抽出操作(3回繰り返す) を行い、 次いで、 メチルアルコール 1 Lを用いて一時間撹拌 ·抽出操作 (3回繰 り返す) を行った。  Next, the compounds (30) to (35) and taxol-related compounds were extracted using the fresh callus. First, 605.5 g of fresh callus was collected and freeze-dried to obtain 55.9 g of dry callus. Subsequently, extraction operations from the dried calli were sequentially performed using n-hexane, ethyl acetate, and methyl alcohol as organic solvents. That is, after performing 1 hour of stirring and extraction operation (repeated three times) using 1 L of n-hexane, stirring and 1 hour of extraction and extraction operation (repeated three times) using 1 L of ethyl acetate, Using 1 L of methyl alcohol, the mixture was stirred and extracted for 1 hour (repeated three times).
そして、 n—へキサン層 (抽出液) から n—へキサンを留去して、 粗 n—へキサン抽出物 1 0 6 1. 7mgを得た。 また、 酢酸ェチル層 (抽 出液) から酢酸ェチルを留去して、 粗酢酸ェチル抽出物 8 5 6. 2mg を得た。 この粗酢酸ェチル抽出物は、 メチルアルコール 酢酸ェチル混 合溶液 (混合量、 1 0m 1 : 40m 1 ) に溶解された後に、 a) 0. 5 M硫酸水溶液 2 Om 1で洗浄されてアルカロイド分画 (塩基性成分) の 除去が行われ、 ついで、 b) 1 M水酸化ナトリウム水溶液 20m 1で洗 浄されてフエノール分画 (酸性成分) の除去が行われた。 続いて、 溶媒 (酢酸ェチル) の除去が行われ、 粗酢酸ェチル中性分画 543. 6mg を得た。 Then, n-hexane was distilled off from the n-hexane layer (extract) to obtain 1061.7 mg of a crude n-hexane extract. Ethyl acetate was distilled off from the ethyl acetate layer (extracted liquid) to obtain 856.2 mg of a crude ethyl acetate extract. The crude ethyl acetate extract is dissolved in a mixed solution of methyl alcohol and ethyl acetate (mixture amount: 10 ml: 40 ml), and then a) washed with 0.5 M aqueous sulfuric acid solution (2 Om1) and subjected to alkaloid fractionation. (Basic component) Removal was carried out, and then b) washing with 20 ml of a 1 M aqueous sodium hydroxide solution to remove the phenol fraction (acidic component). Subsequently, the solvent (ethyl acetate) was removed to obtain 543.6 mg of a crude ethyl acetate neutral fraction.
一方、 メチルアルコール層 (抽出液) からメチルアルコールを除去し て、 粗メチルアルコール抽出物を得た。 この粗メチルアルコール抽出物 は、 メチルアルコール Z水混合溶液 (混合量、 50m l : 40m l ) に 溶解された後に、 クロ口ホルム 1 0 Om 1で 3回抽出操作が行われ、 続 いてクロ口ホルムが除去されて粗クロ口ホルム抽出物 9 1 8. 3mgが 得られた。 この粗クロ口ホルム抽出物は、 メチルアルコール 酢酸ェチ ル混合溶液 (混合量、 1 0m l : 40m l ) に溶解された後に、 a) 0. 5M硫酸水溶液で洗浄されてアルカロイド (塩基性成分) の除去が行わ れ、 ついで、 b) 1 M水酸化ナトリウム水溶液で洗浄されてフエノール 分画 (酸性成分) の除去が行われた。 続いて、 溶媒 (酢酸ェチル) の除 去が行われ、 粗クロ口ホルム中性分画 693. 2mgを得た。  On the other hand, methyl alcohol was removed from the methyl alcohol layer (extract) to obtain a crude methyl alcohol extract. This crude methyl alcohol extract is dissolved in a mixed solution of methyl alcohol Z water (mixing volume, 50 ml: 40 ml), and then extracted three times with a black form 10 Om1. The form was removed to obtain 98.3 mg of a crude black-mouthed form extract. This crude form-form extract is dissolved in a mixed solution of methyl alcohol and ethyl acetate (mixture volume: 10 ml: 40 ml), and then a) washed with a 0.5 M aqueous sulfuric acid solution to remove alkaloids (basic components). ) Was removed, followed by b) washing with a 1 M aqueous sodium hydroxide solution to remove the phenol fraction (acidic component). Subsequently, the solvent (ethyl acetate) was removed to obtain 693.2 mg of a crude fraction having a neutral form in crude form.
続いて、 シリカゲルステンレスカラムを用いた順相高速液体クロマト グラフィ一 (INERTSIL PREP-SIL; 25 X 2 c m; GL Science;溶媒 酢 酸ェチル: n—へキサン = 2 : 8 ;流速 1 0. Om l /m i n) を用い て、 上記の粗 n—へキサン抽出物 1 06 1. 7mgより各種化合物を分 離した。  Subsequently, normal-phase high-performance liquid chromatography using a silica gel stainless steel column (INERTSIL PREP-SIL; 25 × 2 cm; GL Science; solvent: ethyl acetate: n-hexane = 2: 8; flow rate: 10 Oml) / min) to isolate various compounds from the above crude n-hexane extract 1061.7 mg.
その結果、 上記化合物 (30), (3 1), (33) 〜 (35) が単離さ れた。 より具体的には、 化合物 (33) が 200. 3mg (保持時間 ( t R) 1 1. 0分)、 化合物 (34) が 1 7. Omg ( t R 1 2. 9分)、 化合物 (3 1) が 24. 6mg ( t R 16. 5分)、 化合物 (30) が 3 1 0. 7mg ( t R 22. 5分)、 化合物 (35) が 29. 5mg ( t R 3 1. 2分)単離された。 また、 夕クスシン (taxusin) が 1 9. 7mg ( t R 28. 9分) 単離された。 As a result, the above compounds (30), (31), (33) to (35) were isolated. More specifically, the compound (33) was 200.3 mg (retention time (tR) 11.0 minutes), the compound (34) was 17.0 mg (tR 12.9 minutes), the compound (3 1) at 24.6 mg (tR 16.5 min), compound (30) at 30.7 mg (tR 22.5 min), compound (35) at 29.5 mg (tR3 1.2 min) ) Isolated. In addition, 19.7 mg (t R 28.9 min) Isolated.
また、 上記の粗酢酸ェチル中性分画 543. 6 mgをシリカゲルフラ ッシユカラムを用いたシリカゲルカラムクロマトグラフィーにより分取 し、 以下に説明する 1 2フラクション ( f r l〜 f r l 2) に粗分けし た。 フラクション f r 1〜 f r 6は、 n—へキサンと酢酸ェチルとを容 量比 5 : 5で混合してなる溶離液を用いて得られた分画であり、 フラク シヨン f r 7〜 f r 9は、 n—へキサンと酢酸ェチルとを容量比 7 : 3 で混合してなる溶離液を用いて得られた分画であり、 フラクション f r 10, f r l lは、 酢酸ェチルを溶離液として得られた分画であり、 フ ラクシヨン f r 1 2は、 メチルアルコールを溶離液として得られた分画 である。  Also, 543.6 mg of the above crude ethyl acetate neutral fraction was separated by silica gel column chromatography using a silica gel flash column, and roughly separated into 12 fractions (frl to frl2) described below. Fractions fr 1 to fr 6 are fractions obtained using an eluent obtained by mixing n-hexane and ethyl acetate at a volume ratio of 5: 5, and fractions fr 7 to fr 9 are: This is a fraction obtained using an eluent obtained by mixing n-hexane and ethyl acetate in a volume ratio of 7: 3. Fractions fr 10 and frll are fractions obtained using ethyl ethyl acetate as an eluent. The fraction fr 12 is a fraction obtained using methyl alcohol as an eluent.
続いて、 低極性部のフラクション f r 1 ~ f r 3 (合計 2 1 2. 2m g) をシリカゲルステンレスカラムを用いた順相高速液体クロマトダラ フィ一 (INERTSILPREP-SIL; 25 X 1 c m; GL Science;溶媒 酢酸 ェチル: n—へキサン = 2 : 8 ;流速 5. 0m 1 /m i n) を用いて分 取した。 その結果、 化合物 (33) が 40. 8mg (保持時間 ( t R) 8. 6分)、 化合物 (34) が 2. 7mg ( t R 9. 5分)、 化合物 (3 1 ) が 3. 7mg ( t R 1 1. 8分)、 化合物 ( 30 ) が 59. 1 mg ( t R 16. 9分)、 化合物 (35) が 6. lmg ( t R 22. 1分) 単離さ れた。 また、 タクスシン (taxusin) が 2. 7mg ( t R 1 9. 3分) 単離 された。  Subsequently, the low-polarity fractions fr 1 to fr 3 (total 2 12.2 mg) were subjected to normal-phase high-performance liquid chromatography (INERTSILPREP-SIL; 25 × 1 cm; GL Science; solvent) using a silica gel stainless steel column. Separation was performed using ethyl acetate: n-hexane = 2: 8; flow rate: 5.0 m 1 / min). As a result, compound (33) was 40.8 mg (retention time (tR) 8.6 minutes), compound (34) was 2.7 mg (tR 9.5 minutes), and compound (31) was 3.7 mg (tR11.8 min), compound (30) was isolated at 59.1 mg (tR16.9 min), and compound (35) was isolated at 6.1 mg (tR22.1 min). In addition, 2.7 mg (tR19.3 min) of taxusin was isolated.
また、 フラクション f r 4、 及び f r 5 (合計 1 29. 5mg) をシ リカゲルステンレスカラムを用いた順相高速液体クロマトグラフィー (INERTSIL PREP-SIL; 25 X 1 c m; GL Science;溶媒 酢酸ェチル: n—へキサン = 3 : 7 ;流速 5. Om 1 /m i n) を用いて分取した。 その結果、 化合物 (32) が 5. 7mg (保持時間 ( t R) 29. 8分) 単離された。 また、 アビェ夕ン化合物であるタキサマイ リ ン C (taxamairin C)が 2. 0 m g ( t R 32. 6分) 単離された。 Fractions fr 4 and fr 5 (total 129.5 mg) were subjected to normal-phase high-performance liquid chromatography using a silica gel stainless steel column (INERTSIL PREP-SIL; 25 × 1 cm; GL Science; solvent ethyl acetate: n— Hexane = 3: 7; flow rate 5.Om 1 / min). As a result, 5.7 mg of compound (32) (retention time (tR) 29.8 minutes) Isolated. In addition, 2.0 mg (tR32.6 minutes) of taxamairin C, an abyssin compound, was isolated.
さらに、 フラクション f r 6〜: f r 9 (合計 67. 8mg) をシリカ ゲルステンレス力ラムを用いた順相高速液体ク口マトグラフィー (INERTSIL PREP-SIL; 25 X 1 c m; GL Science;溶媒 酢酸ェチル: n—へキサン = 5 : 5 ;流速 5. 0 m 1 / i n) を用いて分取した。 その結果、 7—ェピタキソール (7-epitaxol)が 0. 6mg (保持時間 ( t R) 1 0. 5分)、 タキソール C(taxol C) が 0. 2mg ( t R 20. 3 分)、 タキソ一ル (taxol) が 1. 6mg ( t R 29. 8分) 単離された。 上記の粗クロ口ホルム中性分画 693. 2mgは、 シリカゲルフラッ シュカラムを用いたシリカゲルカラムクロマトグラフィーにより分取さ れ、 以下に説明する 18フラクション (F r l〜F r l 8) に粗分けさ れた。 フラクション F r 1〜F r 1 3は、 n—へキサンと酢酸ェチルと を容量比 5 : 5で混合してなる溶離液を用いて得られた分画であり、 フ ラクシヨン F r l 4, F r 1 5は、 酢酸ェチルを溶離液として得られた 分画であり、 フラクション F r 1 6〜F r 1 8は、 メチルアルコールを 溶離液として得られた分画である。  Further, fractions fr 6 to: fr 9 (total 67.8 mg) were subjected to normal-phase high-performance liquid chromatography using silica gel stainless steel ram (INERTSIL PREP-SIL; 25 X 1 cm; GL Science; solvent ethyl acetate: n-hexane = 5: 5; flow rate was 5.0 m 1 / in). As a result, 7-epitaxol (7-epitaxol) was 0.6 mg (retention time (tR) 10.5 minutes), taxol C (taxol C) was 0.2 mg (tR 20.3 minutes), 1.6 mg (tR 29.8 min) was isolated. 693.2 mg of the above crude black-mouthed form neutral fraction was fractionated by silica gel column chromatography using a silica gel flash column, and roughly separated into 18 fractions (Frl to Frl8) described below. Was. Fractions Fr 1 to Fr 13 are fractions obtained using an eluent obtained by mixing n-hexane and ethyl acetate at a volume ratio of 5: 5, and the fractions F rl4, F r15 is the fraction obtained using ethyl acetate as the eluent, and fractions Fr16 to Fr18 are the fractions obtained using methyl alcohol as the eluent.
続いて、 低極性部のフラクション F r 1, F r 2 (合計 44. 3mg) をシリカゲルステンレスカラムを用いた順相高速液体クロマトグラフィ 一 (INERTSIL PREP-SIL; 2 5 X 1 c m; GL Science;溶媒 酢酸ェチ ル: n—へキサン = 2 : 8 ;流速 5. 0 m 1 /m i n) を用いて分取し た。 その結果、 化合物 (33) が 3. 8mg (保持時間 ( t R) 8. 2 分)、 化合物 (34) が 0. 5mg ( t R 9. 5分) 単離された。  Subsequently, the low-polar fraction Fr 1 and Fr 2 (total 44.3 mg) were subjected to normal phase high performance liquid chromatography using a silica gel stainless steel column (INERTSIL PREP-SIL; 25 X 1 cm; GL Science; solvent). The sample was collected using ethyl acetate: n-hexane = 2: 8; flow rate: 5.0 m 1 / min). As a result, 3.8 mg of the compound (33) was isolated (retention time (tR) 8.2 minutes), and 0.5 mg of the compound (34) was isolated (tR 9.5 minutes).
また、 フラクション F r 3〜F r 6 (合計 290. Omg) をシリカ ゲルステンレスカラムを用いた順相高速液体ク口マトグラフィー (INERTSIL PREP-SIL; 25 X 1 c m; GL Science;溶媒 酢酸ェチル: n—へキサン = 2 : 8 ;流速 5. 0m l /m i n) を用いて分取した。 その結果、 化合物 (33) が 27. 4mg (保持時間 ( t R) 8. 0分)、 化合物 (34) が 7. 5mg ( t R 8. 9分)、 化合物 (3 1 ) が 2. 6 mg ( t R 1 1. 0分)、 化合物 (30) が 80. 7mg ( t R 14. 5 分)、 化合物 (35) が 23. lmg ( t R 23. 1分) 単離された。 ま た、 タクシン (taxusin) が 7. 8 m g ( t R 17. 4分) 単離された。 さ らに、 フラクション F r 3〜F r 6の高極性部 (合計 58. 3mg) を シリカゲルステンレスカラムを用いた順相高速液体クロマトグラフィー (INERTSIL PREP-SIL; 2 5 X 1 c m; GL Science;溶媒 酢酸ェチル: n—へキサン = 3 : 7 ;流速 5. 0m 1 Zm i n) を用いて分取した。 その結果、 化合物 (32) が 1 6. 6mg ( t R 24. 6分) 単離され た。 また、 タキサマイリン C(taxamairin C)が 3. 2 m g ( t R 29. 9分) 単離された。 The fractions Fr3 to Fr6 (total 290.Omg) were subjected to normal-phase high-performance liquid chromatography using a silica gel stainless steel column (INERTSIL PREP-SIL; 25 X 1 cm; GL Science; solvent ethyl acetate). n-hexane = 2: 8; flow rate 5.0 ml / min). As a result, compound (33) was 27.4 mg (retention time (tR) 8.0 min), compound (34) was 7.5 mg (tR 8.9 min), and compound (31) was 2.6 mg (tR11.0 min), compound (30) was isolated at 80.7 mg (tR14.5 min), and compound (35) was isolated at 23.1 mg (tR23.1 min). In addition, 7.8 mg (tR17.4 minutes) of taxusin was isolated. In addition, the high-polarity fractions (total 58.3 mg) of fractions Fr3 to Fr6 were subjected to normal-phase high-performance liquid chromatography using silica gel stainless steel columns (INERTSIL PREP-SIL; 25 X 1 cm; GL Science; Solvent: Ethyl acetate: n-hexane = 3: 7; fractionated using a flow rate of 5.0 ml 1 Zmin). As a result, compound (32) was isolated in an amount of 16.6 mg (tR 24.6 minutes). In addition, 3.2 mg (tR 29.9 minutes) of taxamairin C was isolated.
さらに、 フラクション F r 7〜F r l 3 (合計 5 3. 9mg) を、 逆 相 OD Sカラムとしての OD Sステンレスカラムを用いた逆相高速液体 クロマトグラフィー(INERTSIL PREP ODS ; 2 5 X 1 c m ; GL Science;溶媒 メチルアルコール:酢酸アンモニゥムバッファ一 (pH 4. 8):ァセトニトリル = 1 : 2 : 2混合溶液;流速 5. Om 1 /m i n) を用いて分取した。その結果、 7—ェピタキソール (7-epitaxol)が 0. 5 mg (保持時間 ( t R) 8. 5分)、 タキソ一ル C(taxol C) が 0. 5 mg ( t R 8. 6分)、 ノ ッ力チン V baccatin VI) が 1. 5mg ( t R 6. 3分)、 タキソール (taxol) が 2. 2mg ( t R 5. 9分) 単離され た。  Further, the fractions Fr7 to Frl3 (total 53.9 mg) were subjected to reverse-phase high-performance liquid chromatography (INERTSIL PREP ODS; 25 X 1 cm; using an ODS stainless steel column as a reverse-phase ODS column). GL Science; Solvent Methyl alcohol: ammonium acetate buffer (pH 4.8): acetonitrile = 1: 2: 2 mixed solution; flow rate: 5.Om 1 / min). As a result, 7-epitaxol (7-epitaxol) was 0.5 mg (retention time (tR) 8.5 minutes), and taxol C (taxol C) was 0.5 mg (tR 8.6 minutes). 1.5 mg (tR 6.3 min) and 2.2 mg (tR 5.9 min) of taxol were isolated.
以上の結果をまとめると、 上記の乾燥カルス 55. 9 gより、 化合物 (30)が総計 450. 5mg (乾燥カルスに対して 0. 806重量%)、 化合物 (3 1) が総計 30. 9mg (乾燥カルスに対して 0. 055重 量%)、 化合物 (32) が総計 22. 3mg (乾燥カルスに対して 0. 0 40重量%)、 化合物 (33) が総計 272. 3mg (乾燥カルスに対し て 0. 487重量%)、 化合物 (34) が総計 27. 7 m g (乾燥カルス に対して 0. 0 50重量%) 得られた。 すなわち、 化合物 (30) と、 その 14位ァシルォキシ同族体である化合物 (3 1) 〜 (34) との合 計単離収量は 803. 7mg (乾燥カルスに対して 1. 438重量%) と極めて高い値となった。 また、 化合物 (3 5) は総計 58. 7mg得 られた。 〔実施例 7〕 Summarizing the above results, based on the above-mentioned dried callus (55.9 g), the compound (30) had a total of 455.5 mg (0.806% by weight based on the dry callus), and the compound (31) had a total of 30.9 mg ( 0.055 weight of dried callus %), Compound (32) totaling 22.3 mg (0.040% by weight based on dry callus), compound (33) totaling 272.3 mg (0.487% by weight based on dry callus), compound (34) was obtained in a total of 27.7 mg (0.050% by weight based on dry callus). That is, the total isolated yield of the compound (30) and the compounds (31) to (34), which are 14-hydroxyl homologues, was extremely high at 803.7 mg (1.438% by weight based on dry callus). It was a high value. Compound (35) was obtained in a total amount of 58.7 mg. (Example 7)
(化合物 (30) 〜 (34)、 及び(36) の多剤耐性癌克服作用の測定) 化合物 (30) 〜 (34)、 及び (36) の多剤耐性癌克服作用 (ここ では、 P—糖蛋白質阻害剤としての機能) を、 上記説明の方法にて測定 した。 多剤耐性癌克服作用の測定結果は以下の第 3 1表〜第 33表にま とめて示す。第 3 1表〜第 33表において、 「投与濃度」とは、化合物(3 0) 〜 (34)、 化合物 (36)、 または、 ベラパミルの反応溶液におけ る濃度を示し、 「VCR蓄積量の平均値」 とは、 各ゥエルにおける、 27 80AD細胞中のビンクリスチン (VCR) 蓄積量の平均値を示す。 ま た、 「ベラパミル比」 とは、 ベラパミルを対照薬剤とした比較実験との比 較結果を示し、 「評価」 の項における (最大べラパミル比)、 及び (濃度) とはそれぞれ、 ベラパミルと比較して最も効果が高かったときの比およ び濃度を示すものとする。 尚、 多剤耐性癌克服作用が高い化合物につい ては、 再測定を行った (第 33表)。 投与濃度 VCRf っン ベラパミル比 評 (Measurement of Compounds (30) to (34) and (36) for Overcoming Multidrug-Resistant Cancer) Compounds (30) to (34) and (36) for Overcoming Multidrug-Resistant Cancer (here, P— Function as a glycoprotein inhibitor) was measured by the method described above. The results of measuring the effect of overcoming multidrug-resistant cancer are summarized in Tables 31 to 33 below. In Tables 31 to 33, “dose concentration” refers to the concentration in a reaction solution of compound (30) to (34), compound (36), or verapamil, and “the concentration of VCR accumulation”. The “average value” indicates the average value of the amount of vincristine (VCR) accumulated in 2780AD cells in each well. In addition, “verapamil ratio” indicates the results of comparison with a comparative experiment using verapamil as a control drug, and (maximum verapamil ratio) and (concentration) in the “Evaluation” section are compared with verapamil, respectively. Then, the ratio and concentration when the effect is the highest is shown. Re-measurements were performed for compounds with a high effect of overcoming multidrug-resistant cancer (Table 33). Dosing concentration VCRf Verapamil ratio
化合物 霉養 卜 価  Compound nutrient value
ロール 判 定  Roll judgment
(u g/ral) (最 ラバ 00  (ug / ral) (most mule 00
(dpm/nelD (¾) ノレ比)  (dpm / nelD (¾) Nore ratio)
0. 1 4 4 8 1 0 5 ±  0.1 4 4 8 1 0 5 ±
化合物 Compound
1 6 5 8 1 5 5 +  1 6 5 8 1 5 5 +
(30)  (30)
1 0 1 0 3 9 2 4 4 +  1 0 1 0 3 9 2 4 4 +
0. 1 4 7 7 1 1 2 +  0.1 4 7 7 1 1 2 +
化合物 Compound
1 6 3 7 1 5 0 +  1 6 3 7 1 5 0 +
(32)  (32)
1 0 9 9 5 2 3 4 +  1 0 9 9 5 2 3 4 +
0. 1 4 9 2 1 1 6 + 1 0 1 再測定  0.1 4 9 2 1 1 6 + 1 0 1 Re-measurement
化合物 Compound
1 6 5 0 1 5 3 + 9 6 1 0 3 %  1 6 5 0 1 5 3 + 9 6 1 0 3%
(33) t  (33) t
1 0 1 3 7 8 3 2 4 1 0 3 1 0 u /m 1  1 0 1 3 7 8 3 2 4 1 0 3 1 0 u / m 1
0. 1 5 7 7 1 3 6 + 1 1 8 再測定  0.15 7 7 1 3 6 + 1 1 8 Re-measurement
化合物 Compound
1 9 8 5 2 3 2 + 1 5 1 4 5 %  1 9 8 5 2 3 2 + 1 5 1 4 5%
(3ο  (3ο
1 0 1 3 1 5 3 0 9 + + 9 8 1 n g/m 1  1 0 1 3 1 5 3 0 9 + + 9 8 1 n g / m 1
0 4 2 5 1 0 0  0 4 2 5 1 0 0
ベラノ 0. 1 4 9 0 1 1 5 + Verano 0.1 4 9 0 1 1 5 +
ミル 1 6 8 0 1 6 0 +  Mill 1 6 8 0 1 6 0 +
1 0 1 3 3 5 3 1 + + 1 0 1 3 3 5 3 1 + +
投与濃度 V iン卜 ベラパミル比 評 価 Dosage concentration V int Verapamil ratio Evaluation
化合物 ロール^ 判 定  Compound Roll ^ Judgment
{p. g/ral)
Figure imgf000126_0001
(%) (%) (最 ラバ レ比)
(p. g / ral)
Figure imgf000126_0001
(%) (%) (Maximum rubber ratio)
0. 1 4 5 3 1 2 1 + 1 1 1 再測定 化合物  0.1 4 5 3 1 2 1 + 1 1 1 Re-measurement Compound
1 8 8 5 2 3 6 十 1 29 1 4 9 % 1 8 8 5 2 3 6 10 1 29 1 4 9%
(3D (3D
1 0 1 6 9 2 4 5 1 + + 1 4 9 1 0 u g/m 1  1 0 1 6 9 2 4 5 1 + + 1 4 9 1 0 u g / m 1
0. 1 4 8 0 1 28 + 1 1 7 再測定 化合物  0.1 4 8 0 1 28 + 1 1 7 Re-measurement Compound
1 7 1 2 1 9 0 + 1 0 4 1 1 7 % (34)  1 7 1 2 1 9 0 + 1 0 4 1 1 7% (34)
1 0 1 1 6 9 3 1 2 + + 1 0 3 0. 1 g/m 1  1 0 1 1 6 9 3 1 2 + + 1 0 3 0.1 g / m 1
0 3 7 5 1 0 0  0 3 7 5 1 0 0
ベラノ 0. 1 4 0 9 1 0 9 土 Verano 0.1 4 0 9 1 0 9 Sat
ミル 1 6 8 8 1 8 3 +  Mill 1 6 8 8 1 8 3 +
1 0 1 1 3 3 3 0 2 + + 1 0 1 1 3 3 3 0 2 + +
^ 32 〔第 3 3表〕 ^ 32 (Table 33)
Figure imgf000127_0001
Figure imgf000127_0001
第 3 1表〜第 3 3表より明らかなように、 上記化合物 (3 0 ) 〜 (3 4)、 及び (3 6 ) はいずれも多剤耐性癌克服作用を有し、 それ単独で新 規な多剤耐性癌克服剤となりうることが明らかになった。 特に、 第 3 3 表に示すように、 化合物 (3 1), (33), (36) は、 ベラパミルを上 回る多剤耐性癌克服作用を有する高活性化合物であることが明らかとな り、 それ単独で特に有効な多剤耐性癌克服剤となりうることが明らかに なった。 As is clear from Tables 31 to 33, all of the above compounds (30) to (34) and (36) have a multidrug-resistant cancer overcoming action, and they are new compounds alone. It has been revealed that it can be an effective drug for overcoming multidrug-resistant cancer. In particular, the third three As shown in the table, Compounds (31), (33), and (36) were found to be highly active compounds having a multidrug-resistant cancer-surpassing action superior to verapamil. It has become clear that it can be an effective drug for overcoming multidrug-resistant cancer.
〔実施例 8〕 (Example 8)
(カルスからの化合物 (37), (38) の取り出し)  (Removal of compounds (37) and (38) from callus)
日本イチィ ( ¾ ci/s Sieb. et Zucc.) の若茎 (針葉部) を外 植体として用い、 カルスの誘導並びに組織培養を行った。 上記外植体と しての若茎は、冬季(採取前 1力月間の平均気温 5で以下) に採取した。 はじめに、 上記切片を一般的な方法で洗浄 ·滅菌処理した後、 ショ糖 (スクロース)、 寒天粉末、 およびオーキシンである NAAが、 順に 20 g/L, 1 0 g/L, 0. 5mgZLの濃度となるよう添加された改変 ガンボーグ培地 (以下、 改変ガンボーグ固体培地と称する) に、 該切片 を置床した。すなわち、本実施例における改変ガンボーグ固体培地とは、 以下の第 34表に示す組成よりなる固体培地である。 Calli induction and tissue culture were performed using young stems (conifers) of Japanese yew (チ ci / s Sieb. Et Zucc.) As explants. The young shoots as the above explants were collected in winter (at an average temperature of 5 months during the first month before collection). First, the above sections are washed and sterilized by a general method, and then sucrose (sucrose), agar powder, and NAA, which is an auxin, have a concentration of 20 g / L, 10 g / L, and 0.5 mg ZL, respectively. The slices were placed on a modified Gamborg's medium (hereinafter, referred to as a modified Gamborg's solid medium) added so that That is, the modified Gamborg's solid medium in this example is a solid medium having the composition shown in Table 34 below.
〔第 3 4表〕 (Table 34)
Figure imgf000129_0001
Figure imgf000129_0001
そして、 上記の固体培地を 2 5で、 暗所下で静置した。 これにより、 培養細胞を静置培養して、 日本ィチイのカルスを得た。 次に、 約 4 0 5 0日毎に増殖性の良好なカルスを選抜し、 同一組成の培地を用いて継 代培養を行うことで、 系統を確立した。 次に、 寒天粉末、 ΝΑΑ、 β— シクロデキストリンが、 順に、 l O gZL, 0. 5mgZL、 1 00m Mの濃度となるよう添加された改変ガンボーグ培地 (生産培地) にこの カルスを置床し、 2 5で、 喑所下で 6 0日間静置培養を行った。 この結 果、 新鮮重量で 47 1. 4 gのカルス (新鮮カルスと称する) が得られ た。 Then, the solid medium was allowed to stand at 25 in the dark. As a result, the cultured cells were allowed to stand and cultured to obtain calluses of Japanese yew. Then, about 40 Every 50 days, calli having good proliferation were selected, and subculture was performed using a medium having the same composition to establish a strain. Next, the callus was placed on a modified gamborg medium (production medium) to which agar powder, ΝΑΑ, and β-cyclodextrin were added in order to obtain a concentration of lOgZL, 0.5 mgZL, and 100 mM. In step 5, static culture was performed for 60 days under the same location. As a result, a fresh weight of 471.4 g of callus (referred to as fresh callus) was obtained.
次に、 上記新鮮カルスを用いて新規なアビェ夕ン化合物としての化合 物 ( 3 7), ( 38) の抽出を行った。 先ず、 新鮮カルス 4 7 1. 4 gを 採取し、 凍結乾燥させることにより、 乾燥カルス 47. 2 gを得た。 該 乾燥カルスを粉砕した後、 有機溶媒として n—へキサン、 酢酸ェチル、 メチルアルコールを用いて、 順に抽出操作を行った。 即ち、 乾燥カルス 1 g当たり 2 0m 1の n—へキサンを用いて一時間撹拌 ·抽出操作 (3 回繰り返す) を行った後、 乾燥カルス 1 g当たり 2 0m 1の酢酸ェチル を用いて一時間撹拌 ·抽出操作 (3回繰り返す) を行い、 次いで、 乾燥 カルス 1 g当たり 2 Om 1のメチルアルコールを用いて一時間撹拌 ·抽 出操作 (3回繰り返す) を行った。  Next, compounds (37) and (38) were extracted as novel abiene compounds using the above fresh calli. First, 471.4 g of fresh callus was collected and lyophilized to obtain 47.2 g of dry callus. After pulverizing the dried callus, an extraction operation was sequentially performed using n-hexane, ethyl acetate and methyl alcohol as organic solvents. That is, after stirring and extracting (repeated three times) for 1 hour using 20 ml of n-hexane per 1 g of dry callus, and then for 1 hour using 20 ml of ethyl acetate per 1 g of dry callus Stirring / extraction operation (repeated three times) was performed, and then stirring / extraction operation (repeated three times) for 1 hour using 2 Om1 of methyl alcohol per 1 g of dry callus.
そして、 n—へキサン層 (抽出液) から n—へキサンを留去して、 粗 n—へキサン抽出物 2 9 7. 5mgを得た。 また、 酢酸ェチル層 (抽出 液) から酢酸ェチルを留去して、 粗酢酸ェチル抽出物 5 0 5. 8mgを 得た。 この粗酢酸ェチル抽出物は、 メチルアルコール/酢酸ェチル混合 溶液 (混合量、 1 0m l : 40m l ) に溶解された後に、 a) 0. 5M 硫酸水溶液 1 5m 1で 3回洗浄されて塩基性成分の除去が行われ、 つい で、 b) 2 M水酸化ナトリウム水溶液 1 5m 1で 3回洗浄されて酸性成 分 (フエノール誘導体) の除去が行われた。 続いて、 得られた酢酸ェチ ル層を無水硫酸水素ナトリウムで乾燥後、 溶媒 (酢酸ェチル) を除去し て酢酸ェチル抽出物 373. 6 mg (乾燥カルスに対して 0. 79重量%) を得た。 Then, n-hexane was distilled off from the n-hexane layer (extract) to obtain 297.5 mg of a crude n-hexane extract. Ethyl acetate was distilled off from the ethyl acetate layer (extract) to obtain 505.8 mg of a crude ethyl acetate extract. The crude ethyl acetate extract is dissolved in a mixed solution of methyl alcohol / ethyl acetate (mixing volume: 10 ml: 40 ml), and then a) washed three times with a 0.5 M aqueous sulfuric acid solution 15 ml 1 The components were removed, and then b) washing was performed three times with 15 ml of a 2 M aqueous sodium hydroxide solution to remove acidic components (phenol derivatives). Subsequently, the obtained ethyl acetate layer was dried over anhydrous sodium hydrogen sulfate, and the solvent (ethyl acetate) was removed. Thus, 373.6 mg (0.79% by weight based on dry callus) of the ethyl acetate extract was obtained.
一方、 メチルアルコール層 (抽出液) からメチルアルコールを除去し て、 粗メチルアルコール抽出物を得た。 この粗メチルアルコール抽出物 は、 メチルアルコール 水混合溶液 (混合量、 50m l : 200m l ) に溶解された後に、 クロ口ホルム 100m lで 3回抽出操作が行われ、 続いてクロ口ホルムが除去されて粗クロ口ホルム抽出物 1, 85 1. 7 mgが得られた。 この粗クロ口ホルム抽出物は、 メチルアルコール Z酢 酸ェチル混合溶液 (混合量、 1 0m l : 40m l ) に溶解された後に、 a) 0. 5M硫酸水溶液 1 5m 1で 3回洗浄されて塩基性成分の除去が 行われ、 ついで、 b) 2 M水酸化ナトリウム水溶液 1 5m 1で 3回洗浄 されて酸性成分 (フエノール誘導体) の除去が行われた。 続いて、 得ら れた酢酸ェチル層を無水硫酸水素ナトリウムで乾燥後、 溶媒 (齚酸ェチ ル) を除去してクロ口ホルム抽出物 9 14. 9mg (乾燥カルスに対し て 1. 94重量%) を得た。  On the other hand, methyl alcohol was removed from the methyl alcohol layer (extract) to obtain a crude methyl alcohol extract. The crude methyl alcohol extract was dissolved in a mixed solution of methyl alcohol and water (mixing volume, 50 ml: 200 ml), and then extracted three times with 100 ml of black form, followed by removal of form. As a result, 1,851.7 mg of a crude black-mouthed form extract was obtained. The crude black-mouthed form extract was dissolved in a mixed solution of methyl alcohol Z and ethyl acetate (mixture amount: 10 ml: 40 ml), and then a) washed three times with a 0.5 M sulfuric acid aqueous solution 15 ml 1 times. The basic components were removed, and then b) the substrate was washed three times with 15 ml of a 2 M aqueous sodium hydroxide solution to remove acidic components (phenol derivatives). Subsequently, the obtained ethyl acetate layer was dried over anhydrous sodium hydrogen sulfate, and the solvent (ethyl acetate) was removed to remove 91.49 mg of a black-mouthed form extract (1.94% by weight of the dried callus). %).
続いて、 上記の酢酸ェチル抽出物 373. 6mgをシリカゲルフラッ シュカラムを用いたシリカゲルカラムクロマトグラフィー(Merck 製: Silica gel 60,230-240mesh, 18.7g) で以下に説明する 5フラクショ ン ( f 1〜 f 5) に粗分けした。 フラクション f 1〜 f 3は、 n—へキ サンと酢酸ェチルとを容量比 5 : 5で混合してなる溶離液を用いて得ら れた分画であり、 フラクション f 4は、 酢酸ェチルを溶離液として得ら れた分画であり、 フラクション f 5は、 メチルアルコールを溶離液とし て得られた分画である。  Then, 373.6 mg of the above ethyl acetate extract was subjected to silica gel column chromatography using a silica gel flash column (manufactured by Merck: Silica gel 60, 230-240mesh, 18.7 g) to obtain 5 fractions (f 1 to f 5 ). Fractions f1 to f3 are fractions obtained using an eluent obtained by mixing n-hexane and ethyl acetate at a volume ratio of 5: 5, and fraction f4 is a fraction obtained by mixing ethyl ethyl acetate. This is the fraction obtained as eluent, and fraction f5 is the fraction obtained using methyl alcohol as eluent.
そして、 シリカゲルステンレスカラムを用いた順相高速液体クロマト グラフィ一 (INERTSILPREP-SIL; 2 5 X 1 cm;GLScience;溶媒 酢 酸ェチル: n—へキサン =4 : 6 ;流速 5 m 1 Zm i n) を用い、 上記 フラクション f 2 (22. 6mg) より各種化合物を分離したところ、 化合物 (37) が 3. 9mg (乾燥カルスに対して 0. 008重量%、 保持時間 ( t R) 28. 9分)、 単離された。 Then, normal-phase high-performance liquid chromatography using a silica gel stainless steel column (INERTSILPREP-SIL; 25 X 1 cm; GLScience; solvent ethyl acetate: n-hexane = 4: 6; flow rate 5 m1 Zmin) was performed. Used above Isolation of various compounds from fraction f2 (22.6 mg) revealed that Compound (37) was isolated in an amount of 3.9 mg (0.008% by weight based on dry callus, retention time (tR) 28.9 minutes). Was done.
単離された化合物 (37) の同定並びに構造決定は、 インバースプロ —ブを装着した 50 OMH zの NMRを用い、 PFG— COSY、 P F G— HMQC、 P F G— HMB C等の測定結果を解析することによって 行った。 これらの測定結果は、 第 35表、 第 36表にまとめて示す。 こ れにより、 化合物 (37) の構造を解析した。 また、 該化合物 (37) の物性値等を測定した結果、  Identification and structure determination of the isolated compound (37) should be carried out by analyzing the measurement results of PFG-COSY, PFG-HMQC, PFG-HMBC, etc. using 50 MHZ NMR equipped with an inverse probe. I went by. The results of these measurements are summarized in Tables 35 and 36. Thus, the structure of compound (37) was analyzed. In addition, as a result of measuring physical properties and the like of the compound (37),
分子式 : C21H30O5 Molecular formula: C 21 H 30 O 5
分子量 : 362. 2095 (測定値)、 362. 2093 (計算値) 融 点 : 1 16 :〜 1 1 8 :  Molecular weight: 362. 2095 (measured value), 362. 2093 (calculated value) Melting point: 116: ~ 1 18:
溶解性 : CHC 13 に可溶 Solubility: soluble in CHC 1 3
〔ひ〕 !) 20 : + 2 16. 7° (c = 0. 04, CHC 13 ) [H]! ) 20: + 2 16. 7 ° (c = 0. 04, CHC 1 3)
I R : 3530, 2950, 1 620 c m i (CHC l 3 )IR: 3530, 2950, 1620 cmi (CHC l 3 )
U V : 3 1 0. 5 ( 2. 0 8 ), 2 6 7. 5 ( 1. 3 2 ) 238. 5 (1. 75) nm ( 1 o g ε ) (C2 H5 OH) UV: 3 10.5 (2.08), 26.7.5 (1.32) 238.5 (1.75) nm (1 og ε) (C 2 H 5 OH)
であった。 Met.
S e c t r a l Da t a o f c omp oun d (37) i n C D C 1 s S e c t r a l Da t a o f c omp oun d (37) i n C D C 1 s
位置 1 J c connected 1 H H— H. CO Y HMBC ·* NO E S Y 'Position 1 J c connected 1 HH— H. CO Y HMBC
1 29.83 (t) j?) 3.38 (ddd, 13.0, Hl , 2afi H2(t. 20ab Hla. 2/1. 20a 1 29.83 (t) j?) 3.38 (ddd, 13.0, Hl, 2afi H2 (t. 20ab Hla. 2 / 1.20a
12.0, 5.7)  12.0, 5.7)
tt) 1.50 (dddd, 13.0, Hlc. 2i 20b Hli, 2«, 5  tt) 1.50 (dddd, 13.0, Hlc.2i 20b Hli, 2 «, 5
12.0. 3.0. 2.8)  12.0.3.0.2.8)
2 29.14 (t) ) 2.28 Cddd, 13.0. Η2ί. Ιαί Hla, 2 18  2 29.14 (t)) 2.28 Cddd, 13.0. Η2ί. Ιαί Hla, 2 18
12.0. 5.7)  12.0.5.7)
JJ) 1.87 (ddd, 13.0. HIJJ, 2ί, 6i  JJ) 1.87 (ddd, 13.0.HIJJ, 2ί, 6i
12.0, 3.0)  12.0, 3.0)
3 98.46 (s) H2fl, 18. 19, 20a  3 98.46 (s) H2fl, 18.19, 20a
4 40.31 (s) H2i|3, 18. 19  4 40.31 (s) H2i | 3, 18.19
5 41.50 Cd) 2.19 (br d, 14.0) Η6αί. 20a HIJJ. 7. 18, 19, 20a Hlfl' 6i, 18, 7- OH 5 41.50 Cd) 2.19 (br d, 14.0) Η6αί. 20a HIJJ. 7. 18, 19, 20a Hlfl '6i, 18, 7-OH
6 27.74 (t) a) 1.95 (ddd. 14.0. H5, 6i. 7 H5. ββ, 7, 18 6 27.74 (t) a) 1.95 (ddd. 14.0. H5, 6i. 7 H5. Ββ, 7, 18
3.0, 3.0)  3.0, 3.0)
i) 1.81 (ddd. 14.0, H5. 6a. 7  i) 1.81 (ddd.14.0, H5.6.a.7
14.0, 3.0)  14.0, 3.0)
7 68.73 (d) 4.78 (br ddd. 3.0. Η6σί, 7-0H H14 H6ffi, 14  7 68.73 (d) 4.78 (br ddd.3.0.Η6σί, 7-0H H14 H6ffi, 14
3.0, 3.0)  3.0, 3.0)
8 135.67 (s)  8 135.67 (s)
9 122.08 (s) H14. 11 -OH  9 122.08 (s) H14.11 -OH
10 36.46 (s) HU, 2a. 20a  10 36.46 (s) HU, 2a.20a
11 147.95 (s) H14, 11 -OH  11 147.95 (s) H14, 11 -OH
12 144.52 (s) H14. 15. 12-OMe. 11 -OH  12 144.52 (s) H14. 15. 12-OMe. 11 -OH
13 140.03 (s) H15. 16. 17  13 140.03 (s) H15.16.17
14 119.17 Cd) 6.74 (s) H15 H7, 16, 17  14 119.17 Cd) 6.74 (s) H15 H7, 16, 17
15 26.52 (d) 3.20 (Sep, 7.0) HI 6. 17 H14. 16. 17 H16,17, 12-0Me 15 26.52 (d) 3.20 (Sep, 7.0) HI 6.17 H14. 16.17 H16, 17, 12-0Me
16 23.61 (q) 1.23 (d, 7.0) H15, 17 H15, 17 H14r15 , 12-0Me 16 23.61 (q) 1.23 (d, 7.0) H15, 17 H15, 17 H14 r 15, 12-0Me
誡^ 3 〔第 36表〕 Commandment ^ 3 (Table 36)
Figure imgf000134_0001
Figure imgf000134_0001
一方、 クロ口ホルム抽出物 9 14. 9mgをシリカゲルフラッシュ力 ラムを用いたシリカゲルカラムクロマトグラフィー (Merck製: Silica gel 60,230-240mesh, 18.7 g) で以下に説明する 5フラクション (F 1〜F 5) に粗分けした。 フラクション F 1〜F 3は、 n—へキサンと酢酸ェ チルとを容量比 5 : 5で混合してなる溶離液を用いて得られた分画であ り、 フラクション F 4は、 酢酸ェチルを溶離液として得られた分画であ り、 フラクション F 5は、 メチルアルコールを溶離液として得られた分 画である。 On the other hand, 91.4.9 mg of the black-mouthed form extract was subjected to silica gel column chromatography (manufactured by Merck: Silica gel 60, 230-240mesh, 18.7 g) using a silica gel flash column. It was roughly divided into 5). Fractions F1 to F3 are fractions obtained using an eluent obtained by mixing n-hexane and ethyl acetate at a volume ratio of 5: 5, and fraction F4 is a mixture of ethyl acetate and ethyl acetate. This is a fraction obtained as an eluent, and fraction F5 is a fraction obtained using methyl alcohol as an eluent.
そして、 シリカゲルステンレスカラムを用いた順相高速液体クロマト グラフィ一 (INERTSIL PREP-SIL; 25 X 1 c m; GL Science;溶媒 酢 酸ェチル: n—へキサン =4 : 6 ;流速 5 m 1 / i n) を用い、 上記 フラクション F 2 (95. 8mg) より各種化合物を分離したところ、 化合物 (38) が 10. 7mg (乾燥カルスに対して 0. 023重量%、 保持時間 ( t R) 12. 1分)、 単離された。  Then, normal-phase high-performance liquid chromatography using a silica gel stainless steel column (INERTSIL PREP-SIL; 25 X 1 cm; GL Science; solvent Ethyl acetate: n-hexane = 4: 6; flow rate 5 m1 / in) The compound (38) was separated into 10.7 mg (0.023% by weight based on dry callus, retention time (tR) of 12.1 minutes) ), Isolated.
単離された化合物 (38) の同定並びに構造決定は、 上記化合物 (3 7) の同定並びに構造決定と同様の方法により行った。 これらの測定結 果は、 第 37表、 第 38表にまとめて示す。 これにより、 化合物 (38) の構造を解析した。 また、 該化合物(38) の物性値等を測定した結果、 分子式 : C20H28O2 Identification and structure determination of the isolated compound (38) were performed in the same manner as in the identification and structure determination of the compound (37). The results of these measurements are summarized in Tables 37 and 38. Thus, the structure of the compound (38) was analyzed. In addition, as a result of measuring the physical properties of the compound (38), the molecular formula: C 20 H 28 O 2
分子量 : 300. 2093 (測定値)、 300. 2089 (計算値) 融 点 : 129t:〜 1 3 1で  Molecular weight: 300.2093 (measured value), 300.2089 (calculated value) Melting point: 129t: up to 131
溶解性 : CHC 13 に可溶 Solubility: soluble in CHC 1 3
C α ) D 20: - 5. 57° (c = 0. 269, CHC 13 ) C α) D 20: - 5. 57 ° (c = 0. 269, CHC 1 3)
I R : 3680, 3520, 1 500 c mi (CHC 13 )IR: 3680, 3520, 1 500 c mi (CHC 1 3)
U V : 275. 5 (1. 30), 240. 5 (1. 56) nm ( l o g ε ) (C2 H5 OH) UV: 275.5 (1.30), 240.5 (1.56) nm (log ε) (C 2 H 5 OH)
であった。 Sp e c a 1 Da t a o c omp o un d (38) i n CDC 1 Met. Sp eca 1 Da taoc omp o un d (38) in CDC 1
位置 13C " connected JH " H-H COSY*3 HMBC • 4 NOESY " Position 13 C "connected J H" HH COSY * 3 HMBC • 4 NOESY "
1 QJ T7 ftノ) ^ \uf , o« ノ HI lif a ph H2aii. ■> H2JS, 11. 18, 19, 20  1 QJ T7 ftno) ^ \ uf, o «no HI lif a ph H2aii. ■> H2JS, 11.18,19,20
ノ ■ Ί ί ¾λίΑΑΑ nip, H2c. 3ff, 5  ノ ■ Ί ί ¾λ¾ nip, H2c. 3ff, 5
丄 A 9 q'ノ  丄 A 9 q '
97 、 n  97, n
00 しノ t Hlfl, 3 Hie. 3, 5  00 Shino t Hlfl, 3 Hie. 3, 5
4.1, 3ノ  4.1, 3 ノ
p 1. QQQQ, ΪΔ* \), Πώβ, 1 u , Q O wn. 19, 20  p 1. QQQQ, ΪΔ * \), Πώβ, 1 u, Q O wn. 19, 20
11.4, 11. , Υ) 11.4, 11., Υ)
0. ί 1 Q> q Q^^hr* ΑΑ A \ \ Ηΐββ, 2a, 5, 18.19 Hla, 2a. 5, 18 0.ί 1 Q> q Q ^^ hr * ΑΑ A \ \ Ηΐββ, 2a, 5, 18.19 Hla, 2a. 5, 18
ο oc f  ο oc f
4 οο. ! Sソ H2e, 5, 6, 18, 19  4 οο.! S S H2e, 5, 6, 18, 19
c  c
0 DU. (1 0 no CAA Q f 0 no, ί Hla. 6. 7. 18. 19 Hla. 2a. 3, 18  0 DU. (1 0 no CAA Q f 0 no, ί Hla. 6. 7. 18.19 Hla. 2a. 3, 18
(i 1 ft 7  (i 1 ft 7
Ό 丄^0* 44cひ 、(1ノ 3, 0Ό UU, 1 · v» , *jj no* / H5 H7  Ό 丄 ^ 0 * 44ch, (1 no 3, 0Ό UU, 1 · v », * jj no * / H5 H7
7 f 1^1. 1 Uノ 丄 not ひ H5, 14 H6. 14  7 f 1 ^ 1. 1 U no 丄 not hi H5, 14 H6. 14
Q 0 l l R 丄丄 &ノ、 H6. 11  Q 0 l l R 丄 丄 & no, H6.11
9 146.49 (s) H7. 14, 20  9 146.49 (s) H7. 14, 20
10 37.47 (s) H7  10 37.47 (s) H7
11 109.50 (d) 6.56 (s) 12-OH Hli, 12-OH  11 109.50 (d) 6.56 (s) 12-OH Hli, 12-OH
12 152.27 (s) Hll. 14, 15. 12-OH  12 152.27 (s) Hll. 14, 15. 12-OH
13 131.17 (s) H11.15.16, 17, 12-OH  13 131.17 (s) H11.15.16, 17, 12-OH
14 124.60 (d) 6.90 (s) H15 H7, 16, 17  14 124.60 (d) 6.90 (s) H15 H7, 16, 17
15 26.66 (d) 3.13 (Sep, 7.0) HI 6, 17 H14, 16, 17 H16. 17  15 26.66 (d) 3.13 (Sep, 7.0) HI 6, 17 H14, 16, 17 H16. 17
16 22.75 Cq) 1.26 (d. 7.0) HI 5, 17 H15. 17 H14. 15  16 22.75 Cq) 1.26 (d.7.0) HI 5, 17 H15.17 H14.15
17 22.39 (q) 1.23 (d, 7.0) HI 5, 16 H15, 16 H14, 15  17 22.39 (q) 1.23 (d, 7.0) HI 5, 16 H15, 16 H14, 15
18 16.47 (q) 1.083 (s) H19 H19 Hla, 3' 5  18 16.47 (q) 1.083 (s) H19 H19 Hla, 3 '5
减^ 37 19 27.78 (q) 1.019 (s) H18 H18 2β 减 ^ 37 19 27.78 (q) 1.019 (s) H18 H18 2β
20 20.20 (q) 1.016 (s) Hla. 5 HI . 2β  20 20.20 (q) 1.016 (s) Hla.5 HI .2β
12 - OH 4.73 (s) HU  12-OH 4.73 (s) HU
*l) Multiplicities were determined by DEPT.  * l) Multiplicities were determined by DEPT.
*2) Connections were determined by HMQC and multiplicities and coupling constants in Hz are in  * 2) Connections were determined by HMQC and multiplicities and coupling constants in Hz are in
*3) Determined by PFG-COSY.  * 3) Determined by PFG-COSY.
*4) Correlations from C to the indicated protons.  * 4) Correlations from C to the indicated protons.
*5) NOESY cross peaks.  * 5) NOESY cross peaks.
CO CO
誡 w 3∞ 〔実施例 9〕 Commandment w 3∞ (Example 9)
(カルスからの化合物 (3 9) の取り出し)  (Removal of compound (39) from callus)
日本イチィ (7¾ΛΓί^ cu^wWa Sieb. et Zucc.) の若茎 (針葉部) を外 植体として用い、 カルスの誘導並びに組織培養を行った。 上記外植体と しての若茎は、冬季(採取前 1力月間の平均気温 5で以下) に採取した。 はじめに、 上記切片を一般的な方法で洗浄 ·滅菌処理した後、 ショ糖 (スクロース)、 寒天粉末、 およびオーキシンである NAAが、 順に 2 0 gZL、 1 0 g/L、 0. 5mg/Lの濃度となるよう添加された改変 ガンボーグ培地 (以下、 改変ガンボーグ固体培地と称する) に、 該切片 を置床した。すなわち、本実施例における改変ガンボーグ固体培地とは、 上記の第 34表に示す組成よりなる固体培地 (実施例 8における改変ガ ンボーグ固体培地と同一のもの) である。  Calli induction and tissue culture were performed using young shoots (conifers) of Nipponbari (7¾ΛΓί ^ cu ^ wWa Sieb. Et Zucc.) As explants. The young shoots as the above explants were collected in winter (at an average temperature of 5 months during the first month before collection). First, after washing and sterilizing the above sections by a general method, sucrose (sucrose), agar powder, and NAA, which is an auxin, were added in the order of 20 gZL, 10 g / L, and 0.5 mg / L. The slices were placed on a modified Gamborg's medium (hereinafter, referred to as a modified Gamborg's solid medium) added to have a concentration. That is, the modified gamborg solid medium in the present example is a solid medium having the composition shown in Table 34 above (the same as the modified gamborg solid medium in Example 8).
そして、 上記の固体培地を 2 5t:、 暗所下で静置した。 これにより、 培養細胞を静置培養して、 日本ィチイのカルスを得た。 次に、 約 40〜 5 0日毎に増殖性の良好なカルスを選抜し、 同一組成の培地を用いて継 代培養を行うことで、 系統を確立した。 次に、 寒天粉末、 およびォーキ シンである 4—クロ口インドール酢酸 (4-C1 IAA)が、 順に、 1 0 g/L、 0. 5mgZLの濃度となるよう添加された改変ガンボーグ培地 (生産 培地) にこのカルスを置床し、 2 5で、 喑所下で 6 0日間静置培養を行 つた。 尚、 60日間の静置培養期間中に植え継ぎは一度行われた。 すな わち、 2代継代培養が行われた。 この結果、 新鮮重量で 849. 9 gの カルス (新鮮カルスと称する) が得られた。  Then, the solid medium was allowed to stand for 25 t in a dark place. As a result, the cultured cells were allowed to stand and cultured to obtain calluses of Japanese yew. Next, a callus with good growth was selected about every 40 to 50 days, and subculture was performed using a medium of the same composition to establish a strain. Next, a modified Gamborg's medium (production medium) was added with agar powder and 4-auxin indoleacetic acid (4-C1 IAA), which were added in a concentration of 10 g / L and 0.5 mg ZL, respectively. This callus was placed on the ground, and static culture was performed at 25 under the place for 60 days. The subculture was performed once during the stationary culture period of 60 days. That is, two subcultures were performed. As a result, 849.9 g of fresh calli (referred to as fresh calli) were obtained.
次に、 上記新鮮カルスを用いて新規なアビェ夕ン化合物としての化合 物 (3 9) の抽出を行った。 先ず、 新鮮カルス 849. 9 gを採取し、 凍結乾燥させることにより、 乾燥カルス 7 7. 5 gを得た。 該乾燥カル スを粉砕した後、 有機溶媒として n—へキサン、 酢酸ェチルを用いて、 順に抽出操作を行った。即ち、 1 Lの n—へキサンを用いて一時間撹拌 · 抽出操作 (3回繰り返す) を行った後、 1 Lの酢酸ェチルを用いて一時 間撹拌 ·抽出操作 (3回繰り返す) を行った。 そして、 n—へキサン層 (抽出液)から n—へキサンを留去して、粗 n—へキサン抽出物 577. 3mgを得た。 また、 酢酸ェチル層 (抽出液) から酢酸ェチルを留去し て、 粗酢酸ェチル抽出物 77 5. 2mgを得た。 Next, a compound (39) as a novel abiene compound was extracted using the above fresh calli. First, 849.9 g of fresh callus was collected and freeze-dried to obtain 77.5 g of dry callus. After pulverizing the dried callus, using n-hexane and ethyl acetate as an organic solvent, The extraction operation was performed in order. That is, the mixture was stirred for 1 hour with 1 L of n-hexane and extracted (repeated three times), and then temporarily stirred and extracted with 1 L of ethyl acetate (repeated three times). . Then, n-hexane was distilled off from the n-hexane layer (extract) to obtain 577.3 mg of a crude n-hexane extract. Ethyl acetate was distilled off from the ethyl acetate layer (extract) to obtain 775.2 mg of crude ethyl acetate extract.
続いて、 上記の粗酢酸ェチル抽出物 775. 2mgをシリカゲルフラ ッシュカラムを用いたシリカゲルカラムク口マトグラフィ一(Merck 製: Silica gel 60,230"240mesh, 18.7g) で以下に説明する 9フラクショ ン (F (1) 〜F (9)) に粗分けした。 フラクション F (1) は、 n— へキサンと酢酸ェチルとを容量比 8 : 2で混合してなる溶離液を用いて 得られた分画であり、 フラクション F (2) 〜F (5) は、 n—へキサ ンと酢酸ェチルとを容量比 7 : 3で混合してなる溶離液を用いて得られ た分画であり、 フラクション F (6) 及び F (7) は、 n—へキサンと 酢酸ェチルとを容量比 4 : 6で混合してなる溶離液を用いて得られた分 画である。 また、 フラクション F (8) は、 酢酸ェチルを溶離液として 得られた分画であり、 F (9) は、 メチルアルコールを溶離液として得 られた分画である。  Subsequently, 775.2 mg of the above crude ethyl acetate extract was subjected to silica gel column chromatography using a silica gel flash column (Merck: Silica gel 60, 230 "240mesh, 18.7 g) to obtain 9 fractions (F ( 1) to F (9)) Fraction F (1) is a fraction obtained using an eluent obtained by mixing n-hexane and ethyl acetate in a volume ratio of 8: 2. The fractions F (2) to F (5) are fractions obtained using an eluent obtained by mixing n-hexane and ethyl acetate in a volume ratio of 7: 3, and the fraction F ( 6) and F (7) are fractions obtained using an eluent obtained by mixing n-hexane and ethyl acetate in a volume ratio of 4: 6. Fraction obtained with ethyl acetate as eluent, F (9) is the fraction obtained with methyl alcohol as eluent. It is.
そして、 OD Sステンレスカラムを用いた逆相高速液体クロマトダラ フィ一 (INERTSIL PREP ODS; 25 X 1 c m; GL Science;溶媒 メ チルアルコール: 0. 05M酢酸アンモニゥムバッファー(pH4. 8) : ァセトニトリル = 1 : 2 : 2混合溶液;流速 5m 1 Zm i n) を用い、 上記フラクション F (6) (1 20. 4mg) より各種化合物を分離した ところ、 化合物 (39) が 2. 3mg (乾燥カルスに対して 0. 003 重量%、 保持時間 ( t R) 14. 5分)、 単離された。  Then, reverse-phase high-performance liquid chromatography using an ODS stainless steel column (INERTSIL PREP ODS; 25 X 1 cm; GL Science; solvent: methyl alcohol: 0.05 M ammonium acetate buffer (pH 4.8): acetonitrile = Using a 1: 2: 2 mixed solution at a flow rate of 5 ml 1 Zmin), various compounds were separated from the above fraction F (6) (120.4 mg). As a result, 2.3 mg of compound (39) was obtained (based on dry callus). 0.0003% by weight, retention time (tR) of 14.5 minutes).
単離された化合物 (39) の同定並びに構造決定は、 上記説明の化合 物 (37)、 (38) の同定並びに構造決定と同様の方法により行った。 これらの測定結果は、 第 39表にまとめて示す。 これにより、 化合物(3 9) の構造を解析した。 また、 該化合物 (39) の物性値等を測定した 結果、 The identity and structure of the isolated compound (39) were determined by combining the compounds described above. Identification and structure determination of the products (37) and (38) were performed in the same manner. The results of these measurements are summarized in Table 39. As a result, the structure of compound (39) was analyzed. In addition, as a result of measuring the physical property values and the like of the compound (39),
分卞:! \ : し 20H20O4  Min Byon :! \: then 20H20O4
分子量 : 296. 141 5 (測定値)、 296. 141 7 (計算値) 融 点 : 282t〜 284  Molecular weight: 296.141 5 (measured value), 296.141 7 (calculated value) Melting point: 282t-284
溶解性 : CHC 13 に可溶 Solubility: soluble in CHC 1 3
[ α ) D 20 : - 1 1 8. 84° (c = 0. 1 38, CHC 13 ) [α) D 20 :-1 1 8.84 ° (c = 0.138, CHC 13)
I R : 3624, 2949, 1 728 c m-i (CHC " ) U V : 5 1 9. 0 ( 1. 9 2), 2 30 8. 5 (2. 2 7), 244. 0 (2. 67), 242. 0 ( 2. 67), 238. 5 (4. 6 8) nm ( l o g s) (C2 H5 OH) IR: 3624, 2949, 1728 cmi (CHC ") UV: 51.9 (1.92), 2308.5 (2.27), 244.0 (2.67), 242 0 (2.67), 238.5 (4.6.8) nm (logs) (C 2 H 5 OH)
であった。 Met.
尚、 上記の分子量としては、 分子イオン( M+) からカルボニル基 (C =0) が抜けた状態 (すなわち、 C19H20O3 ) のフラグメントピークの 測定値と計算値とを示している。 In addition, the above-mentioned molecular weight shows the measured value and the calculated value of the fragment peak in a state where the carbonyl group (C = 0) is removed from the molecular ion (M +) (that is, C 19 H 20 O 3 ).
〔第 3 9表〕 (Table 39)
Figure imgf000141_0002
Figure imgf000141_0002
Figure imgf000141_0001
Figure imgf000141_0001
〔実施例 1 0〕 日本イチィ (7¾ATi^ci5 W'i¾ Sieb. et Zucc.) の若茎 (針葉部) を外 植体として用いてカルスの誘導並びに組織培養を行い、 該カルスから夕 キサン関連化合物である化合物 (40) の取り出しを行った。 上記外植 体としての若茎は、 冬季 (採取前 1力月間の平均気温 5で以下) に採取 した。 (Example 10) Callus induction and tissue culture were performed using young shoots (needles) of Nipponbari (7¾ATi ^ ci5 W'i¾ Sieb. Et Zucc.) As explants. 40) was taken out. The young shoots as the explants were collected in winter (at an average temperature of 5 during the first month before collection).
はじめに、 上記切片を一般的な方法で洗浄 ·滅菌処理した後、 ショ糖 (スクロース)、 寒天粉末、 およびオーキシンである NAAが、 順に 2 0 g/L, 1 0 g/L, 1. OmgZLの濃度となるよう添加された改変 ガンボーグ培地 (以下、 改変ガンボーグ固体培地と称する) に、 該切片 を置床した。すなわち、本実施例における改変ガンボーグ固体培地とは、 以下の第 40表に示す組成よりなる固体培地である。 First, after washing and sterilizing the above sections by a general method, sucrose (sucrose), agar powder, and NAA, which is an auxin, are added in order of 20 g / L, 10 g / L, and 1.OmgZL. The slices were placed on a modified Gamborg's medium (hereinafter, referred to as a modified Gamborg's solid medium) added to have a concentration. That is, the modified Gamborg's solid medium in this example is a solid medium having the composition shown in Table 40 below.
〔第 4 0表〕 (Table 40)
Figure imgf000143_0001
Figure imgf000143_0001
そして、 上記の固体培地を 2 5で、 暗所下で静置した。 これにより、 培養細胞を静置培養して、 日本ィチイのカルスを得た。 次に、 約 4 0 50日毎に増殖性の良好なカルスを選抜し、 同一組成の培地を用いて継 代培養を行うことで、 系統を確立した。 続いて、 寒天粉末、 NAA、 フ ェニルァラニン、 酢酸が、 順に、 1 0 gZL、 0. 5mg/L, 0. 2Then, the solid medium was allowed to stand at 25 in the dark. As a result, the cultured cells were allowed to stand and cultured to obtain calluses of Japanese yew. Then, about 40 A callus with good growth was selected every 50 days, and subculture was performed using a medium of the same composition to establish a strain. Subsequently, agar powder, NAA, phenylalanine, and acetic acid were added in that order: 10 gZL, 0.5 mg / L, 0.2
63 g/L、 1. 0m 1 ZLの濃度となるよう添加された改変ガンポー グ培地に得られたカルスを置床し、 25で、 暗所下で 60日間静置培養 を行った。 この結果、 新鮮重量で 605. 5 gのカルス (新鮮カルスと 称する) が得られた。 The callus obtained was placed on a modified gump medium supplemented with a concentration of 63 g / L and 1.0 ml ZL, and the resulting calli were cultured statically at 25 in the dark for 60 days. As a result, 605.5 g of callus having a fresh weight (referred to as fresh callus) was obtained.
次に、 上記新鮮カルスを用いて上記化合物 (40) の抽出を行った。 先ず、 新鮮カルス 605. 5 gを採取し、 凍結乾燥させることにより、 乾燥カルス 55. 9 gを得た。 続いて、 有機溶媒として n—へキサン、 酢酸ェチル、 メチルアルコールを用いて、 該乾燥カルスからの抽出操作 を順に行った。 即ち、 n—へキサン 1 Lを用いて一時間撹拌 ·抽出操作 (3回繰り返す) を行った後、 酢酸ェチル 1 Lを用いて一時間撹拌 ·抽 出操作 (3回繰り返す) を行い、 次いで、 メチルアルコール 1 Lを用い て一時間撹拌 ·抽出操作 (3回繰り返す) を行った。  Next, the compound (40) was extracted using the fresh callus. First, 605.5 g of fresh callus was collected and freeze-dried to obtain 55.9 g of dry callus. Subsequently, extraction operations from the dried calli were sequentially performed using n-hexane, ethyl acetate, and methyl alcohol as organic solvents. That is, after 1 hour stirring and extraction operation (repeated 3 times) using 1 L of n-hexane, 1 hour stirring and extraction operation (repeated 3 times) using 1 L of ethyl acetate, and then Using 1 L of methyl alcohol, stirring and extraction operations (repeated three times) were performed for one hour.
そして、 n—へキサン層 (抽出液) から n—へキサンを留去して、 粗 n—へキサン抽出物 1 06 1. 7mgを得た。 また、 酢酸ェチル層 (抽 出液) から酢酸ェチルを留去して、 粗酢酸ェチル抽出物 856. 2mg を得た。 この粗酢酸ェチル抽出物は、 メチルアルコール 酢酸ェチル混 合溶液 (混合量、 1 Om 1 : 4 Om 1 ) に溶解された後に、 a) 0. 5 M硫酸水溶^ 2 Om 1で洗浄されてアルカロイド分画 (塩基性成分) の 除去が行われ、 ついで、 b) 1 M水酸化ナトリウム水溶液 2 Om 1で洗 浄されてフエノール分画 (酸性成分) の除去が行われた。 続いて、 溶媒 (酢酸ェチル) の除去が行われ、 粗酢酸ェチル中性分画 543. 6mg を得た。  Then, n-hexane was distilled off from the n-hexane layer (extract) to obtain 1061.7 mg of a crude n-hexane extract. Ethyl acetate was distilled off from the ethyl acetate layer (extracted liquid) to obtain 856.2 mg of a crude ethyl acetate extract. This crude ethyl acetate extract is dissolved in a mixed solution of methyl alcohol and ethyl acetate (mixing amount, 1 Om 1: 4 Om 1), and then a) washed with 0.5 M sulfuric acid aqueous solution ^ 2 Om 1 and washed with alkaloids. The fraction (basic component) was removed, followed by b) washing with 2M 1M aqueous sodium hydroxide solution to remove the phenol fraction (acid component). Subsequently, the solvent (ethyl acetate) was removed to obtain 543.6 mg of a crude ethyl acetate neutral fraction.
一方、 メチルアルコール層 (抽出液) からメチルアルコールを除去し て、 粗メチルアルコール抽出物を得た。 この粗メチルアルコール抽出物 は、 メチルアルコール 水混合溶液 (混合量、 50m l : 40m l ) に 溶解された後に、 クロ口ホルム 1 00m 1で 3回抽出操作が行われ、 続 いてクロ口ホルムが除去されて粗クロ口ホルム抽出物 9 18. 3mgが 得られた。 この粗クロ口ホルム抽出物は、 メチルアルコール Z酢酸ェチ ル混合溶液 (混合量、 1 Om 1 : 4 Om 1 ) に溶解された後に、 a) 0. 5M硫酸水溶液で洗浄されてアルカロイド (塩基性成分) の除去が行わ れ、 ついで、 b) 1 M水酸化ナトリウム水溶液で洗浄されてフエノール 分画 (酸性成分) の除去が行われた。 続いて、 溶媒 (酢酸ェチル) の除 去が行われ、 粗クロ口ホルム中性分画 693. 2mgを得た。 On the other hand, methyl alcohol is removed from the methyl alcohol layer (extract) Thus, a crude methyl alcohol extract was obtained. The crude methyl alcohol extract was dissolved in a mixed solution of methyl alcohol and water (mixing amount, 50 ml: 40 ml), and then extracted three times with 100 ml of black-mouthed form. Removal yielded 918.3 mg of crude black-mouthed form extract. This crude form-form extract is dissolved in a mixed solution of methyl alcohol Z and ethyl acetate (mixing amount: 1 Om 1: 4 Om 1), and then a) washed with a 0.5 M aqueous sulfuric acid solution and washed with an alkaloid (base). Then, b) washing with a 1 M aqueous sodium hydroxide solution was performed to remove the phenol fraction (acidic component). Subsequently, the solvent (ethyl acetate) was removed to obtain 693.2 mg of a crude fraction having a neutral form in crude form.
続いて、 シリカゲルステンレスカラムを用いた順相高速液体クロマト グラフィ一 (INERTSILPREP-SIL; 25 X 2 c m; GL Science;溶媒 酢 酸ェチル: n—へキサン = 2 : 8 ;流速 1 0. Om l Zm i n) を用い て、 上記の粗 n—へキサン抽出物 1 06 1. 7mgより各種化合物を分 離した。 その結果、 上記化合物 (40) が、 1 9. 7mg (保持時間 ( t R) : 28. 9分) 単離された。  Subsequently, normal-phase high-performance liquid chromatography using a silica gel stainless steel column (INERTSILPREP-SIL; 25 X 2 cm; GL Science; solvent ethyl acetate: n-hexane = 2: 8; flow rate 10 Om l Zm In), various compounds were separated from the above crude n-hexane extract 1061.7 mg. As a result, the above compound (40) was isolated in an amount of 19.7 mg (retention time (tR): 28.9 minutes).
上記化合物 (40) の iH— NMRおよび 13C— NMRの測定結果を 第 41表にまとめて示す。 これにより、 化合物 (40) の構造を解析し た。 また、 該化合物 (40) の融点は 1 27で〜 1 28でであった。 Table 41 summarizes the iH-NMR and 13C-NMR measurements of the above compound (40). Thus, the structure of compound (40) was analyzed. The melting point of the compound (40) was 127 to 128.
〔第 4 1表〕 (Table 41)
KMR spectral data of compound (1) in CDC1 KMR spectral data of compound (1) in CDC1
Compound (40) reference  Compound (40) reference
位置  Position
C connected 1 H connected 1 H C connected 1 H connected 1 H
1 40.31 (d) 1.84 (n)  1 40.31 (d) 1.84 (n)
2 28.29 (d) a) 1.77 (m)  2 28.29 (d) a) 1.77 (m)
b) 1.69 (m)  b) 1.69 (m)
3 37.97 (d) 3.00 (brd. 5.4) 3.00 (ii)  3 37.97 (d) 3.00 (brd.5.4) 3.00 (ii)
4 148.75 (s)  4 148.75 (s)
5 76.30 (d) 5.36 (dd, 2.7, 2.7) 5.36 (t, 3.0)  5 76.30 (d) 5.36 (dd, 2.7, 2.7) 5.36 (t, 3.0)
6 27.29 (t) a) 1.84 (m)  6 27.29 (t) a) 1.84 (m)
b) 1.69 (m)  b) 1.69 (m)
7 27.26 (t) 1.77 (n)  7 27.26 (t) 1.77 (n)
8 42.90 (s)  8 42.90 (s)
9 77.42 (d) 5.87 (d, 10.7) 5.85 (d, 10.8)  9 77.42 (d) 5.87 (d, 10.7) 5.85 (d, 10.8)
10 72.50 (d) 6.08 (d, 10.7) 6.05 (d, 10.8)  10 72.50 (d) 6.08 (d, 10.7) 6.05 (d, 10.8)
11 134.81 (s)  11 134.81 (s)
12 136.98 (s)  12 136.98 (s)
13 70.72 (d) 5.87(ddq. 8.6, 7.7, 1.5) 5.86 (t)  13 70.72 (d) 5.87 (ddq. 8.6, 7.7, 1.5) 5.86 (t)
14 31.89 (t) a) 2.69(ddd, 14.6,9.9, 1.5)  14 31.89 (t) a) 2.69 (ddd, 14.6,9.9, 1.5)
b) 1.06(brdd, 14.6, 7.7)  b) 1.06 (brdd, 14.6, 7.7)
15 39.84 (q)  15 39.84 (q)
16 31.10 (q) 1.62 (s) 1.62 (s)  16 31.10 (q) 1.62 (s) 1.62 (s)
17 27.32 (t) 1.11 (s) 1.11 (s)  17 27.32 (t) 1.11 (s) 1.11 (s)
18 14.84 (t) 2.11 (d, 1.5) 2.27 (s)  18 14.84 (t) 2.11 (d, 1.5) 2.27 (s)
19 17.72 (t) 0.75 (s) 0.75 (s)  19 17.72 (t) 0.75 (s) 0.75 (s)
20 114.06 (t) a) 5.21 (drd, 1.5) 5.21 (brs)  20 114.06 (t) a) 5.21 (drd, 1.5) 5.21 (brs)
b) 4.85 (brd, 1.5) 4.85 (brs)  b) 4.85 (brd, 1.5) 4.85 (brs)
5-OAc 169.92(s)  5-OAc 169.92 (s)
9-OAc I70.40(s)  9-OAc I70.40 (s)
10-OAc 169.95(s)  10-OAc 169.95 (s)
13-OAc 170.38(s)  13-OAc 170.38 (s)
5-OAc 20.84(q) 2.17 (s)  5-OAc 20.84 (q) 2.17 (s)
9-0 Ac 21.43(q) 2.07 (s)  9-0 Ac 21.43 (q) 2.07 (s)
10-OAc 20.04 (q) 2.05 (s)  10-OAc 20.04 (q) 2.05 (s)
13-OAc 21.79(q) 2.01 (s) なお、 第 4 1 表に は文献 ( S.K.Chattopadhyay,et.al.,J.Med. Aroma.Plant Sci., 19,17:1997) に報告のある、 タクスシン (taxusin) の NMRデータを参照 (reference) として併記している。 上記の化合物 (4 0) の NMRデータや融点の値は、 該文献の記載とほぼ一致することが 判明した。 該化合物 (40) の多剤耐性癌克服作用については、 実施例 1 2にて詳述する。 〔実施例 1 1〕 13-OAc 21.79 (q) 2.01 (s) In Table 41, taxin was reported in the literature (SK Chattopadhyay, et.al., J. Med. Aroma. Plant Sci., 19, 17: 1997). (taxusin) NMR data is also provided as a reference. The NMR data and melting point of the above compound (40) were found to be almost the same as those described in the literature. The effect of compound (40) on overcoming multidrug-resistant cancer was described in Examples. This will be described in detail in 12. (Example 11)
樹高 2mの日本ィチイから採取した新鮮葉および若茎よりなる針葉部 から、 タキサン関連化合物である化合物 (4 1) 〜 (44)、 化合物 (4 6)、 並びに、 化合物 (47) を取り出した。 即ち、 上記の針葉部 1 24 8 gを n—へキサン 8 Lに 1週間浸漬することにより脱脂した後、 酢酸 ェチル 8 Lに浸潰した。 1週間浸潰した後、 針葉部を濾別し、 濾液であ る抽出液を得た。 次いで、 抽出液から酢酸ェチルを室温で減圧除去し、 該酢酸ェチルに可溶な成分(粗酢酸ェチル抽出物) 1 9. 69 gを得た。 上記の成分を、 メチルアルコールと酢酸ェチルとを容量比 1 : 3で混 合してなる混合溶液 400m 1に溶解した。 次に、 該溶液を、 酸性水溶 液である 0. 5M硫酸 1 00m lで 3回 (3 X 1 0 0m l ) 洗浄し、 続 いて、 塩基性水溶液である 2 M水酸化ナトリゥム水溶液 1 0 Om 1で 2 回(2 X 1 0 Om 1 )洗浄することによりフエノール分画 1. 08 g (針 葉部の量を基準として 0. 087 %) を除去した。 さらに、 上記の溶液 (油層) を飽和食塩水で洗浄した後、 無水硫酸ナトリウムで乾燥させ、 メチルアルコールおよび酢酸ェチルを除去した。 これにより、 粗中性分 画 (テルペン分画) 7. 49 g (同 0. 600 %) を得た。  Taxane-related compounds (41) to (44), compounds (46), and compound (47) were extracted from the needle portion consisting of fresh leaves and young stems collected from a 2m-high tree yew. . That is, 1248 g of the needle portion was degreased by immersing it in 8 L of n-hexane for 1 week, and then immersed in 8 L of ethyl acetate. After immersion for one week, the needle portion was separated by filtration to obtain an extract as a filtrate. Then, ethyl acetate was removed from the extract under reduced pressure at room temperature to obtain 19.69 g of a component soluble in the ethyl acetate (crude ethyl acetate extract). The above components were dissolved in 400 ml of a mixed solution obtained by mixing methyl alcohol and ethyl acetate at a volume ratio of 1: 3. Next, the solution was washed three times (3 × 100 ml) with 100 ml of 0.5 M sulfuric acid as an acidic aqueous solution, and subsequently, 10 Om 2 aqueous sodium hydroxide solution as a basic aqueous solution was added. By washing twice with 1 (2 × 10 Om 1), 1.08 g (0.087% based on the amount of needles) of the phenol fraction was removed. Further, the above solution (oil layer) was washed with a saturated saline solution and dried over anhydrous sodium sulfate to remove methyl alcohol and ethyl acetate. As a result, 7.49 g (0.600%) of a crude neutral fraction (terpene fraction) was obtained.
一方、 洗浄後の上記 0. 5M硫酸に 29重量%アンモニア水を添加し て pHを 9. 0とした後、 クロ口ホルム 20 Om 1で 3回 (3 X 200 m 1 ) 抽出した。 これにより、 粗アル力ロイド分画 3. 65 g (針葉部 の量を基準として 0. 292 %) を得た。  On the other hand, the pH of the washed 0.5M sulfuric acid was adjusted to 9.0 by adding 29% by weight of aqueous ammonia to the above 0.5M sulfuric acid, and then extracted three times (3 × 200 m 1) with 20 μm 1 of form-form. As a result, 3.65 g (0.292% based on the amount of needles) of the crude alkyloid fraction was obtained.
次に、 上記の粗中性分画を、 シリカゲルを用いたオープンカラムクロ マトグラフィ一を採用して、 溶媒として n—へキサンと酢酸ェチルとメ チルアルコールとを用いたグラジュェント溶出を行うことにより、 Fi 〜F10の 1 0個のフラクションに分画 (荒分け) した。 Fi 〜F8 の溶出 液は酢酸ェチル: n—へキサンが容量比 2 : 3で混合してなる混合溶液 であり、 F9 の溶出液は酢酸ェチルであり、 F1()の溶出液はメチルアル コールであった。 各フラクションの収量は、 1番目 (低極性側) から 1 0番目(高極性側)に向かって順に、 Fi S S Omg. Fs S Z S Smg, F3 722 mg, F4 405mg, F5 3 1 lmg, FG 208mg, F 7 1 77 mg, F8 144mg, F9 237mg, Fi0205 1 mgであ つた。 Next, the crude neutral fraction was subjected to a gradient elution using n-hexane, ethyl acetate and methyl alcohol as a solvent by using open column chromatography using silica gel. Fi It was fractionated (roughly divided) into 10 fractions of F10. Fi eluate to F 8 are acetate Echiru: hexane n- capacity ratio 2: mixed solution prepared by mixing 3, eluant F 9 is acetic Echiru, eluent F 1 () is It was methyl alcohol. The yield of each fraction was Fi SS Omg. Fs SZS Smg, F 3 722 mg, F 4 405 mg, F 4 405 mg, F 5 3 1 lmg, in order from the 1st (low polarity side) to the 10th (high polarity side). F G 208mg, F 7 1 77 mg, F 8 144mg, F 9 237mg, Fi 0 205 1 mg der ivy.
そして、 前記 2番目のフラクション (F2 ) を、 シリカゲルステンレ スカラム (INERTSIL PREP-SIL ; 25 X 1 cm; GL Science) を用い た順相高速液体クロマトグラフィー (順相 HP L C) を採用して、 酢酸 ェチルと n—へキサンとを容量比 3 : 7で混合してなる混合溶液 (キヤ リア, 流速 5m 1 分) を用いて分離し、 保持時間 0分〜 1 0. 0分の 分画 F 432. Omg、 および保持時間 1 2. 0分〜 45. 2分の分 画 F2-2 962 mgを分取した。 Then, the second fraction (F 2 ) was subjected to normal-phase high-performance liquid chromatography (normal-phase HP LC) using a silica gel stainless steel column (INERTSIL PREP-SIL; 25 × 1 cm; GL Science), Separation was performed using a mixed solution (carrier, flow rate: 5 m / min) consisting of ethyl acetate and n-hexane mixed at a volume ratio of 3: 7. Fraction F with a retention time of 0 to 10.0 min 432. Omg, and retention time 1 2.0 minute to 45.2 minutes of fractions F 2 - was separated and 2 962 mg.
続いて、 上記分画 を上記カラムを用いて同一条件でさらに分離し、 保持時間 6. 4分の分画 Fan (ピーク) 32. 7mgを分取した。 続 いて、 分画 F2-H を、 上記カラムを用いた順相 HP L Cを採用して、 酢 酸ェチルと n—へキサンとを容量比 2 : 8で混合してなる混合溶液 (キ ャリア, 流速 5m l Z分) にて分離し、 これにより、 化合物 (44) 2 2. 5mg (保持時間 1 0. 8分) を取り出した。 該化合物 (44) の 含有量は、 新鮮針葉部の重量を基準として 0. 00 1 8 %であった。 Subsequently, the above fraction was further separated under the same conditions using the above column, and 32.7 mg of a fraction (Fan) peak (retention time: 6.4 minutes) was collected. Subsequently, the fraction F 2 -H was mixed with normal-phase HP LC using the above column to mix ethyl acetate and n-hexane at a volume ratio of 2: 8 (carrier). , And a flow rate of 5 ml Z min), whereby 22.5 mg of the compound (44) (retention time: 10.8 min) was obtained. The content of the compound (44) was 0.0018% based on the weight of the fresh needle portion.
上記化合物 (44) の iH— NMRおよび 13C— NMRの測定結果を 第 42表 ·第 43表にまとめて示す。 これにより、 化合物 (44) の構 造を解析した。 また、 該化合物 (44) の物性値等を測定した結果、  The results of iH-NMR and 13C-NMR measurements of the above compound (44) are summarized in Tables 42 and 43. Thus, the structure of compound (44) was analyzed. In addition, as a result of measuring the physical properties and the like of the compound (44),
融 点 : 1 78 〜 180で NMR spectral data of compound (44) in CDC I Melting point: 1 78 to 180 NMR spectral data of compound (44) in CDC I
Compound (44) reference 0 位置 CO  Compound (44) reference 0 position CO
C connected 1 H C connected 1 H o C connected 1 HC connected 1 Ho
40.15 (d) 1.82 (n) 40.23 (d) 40.15 (d) 1.82 (n) 40.23 (d)
27.37 (t) a) 1.80 (m) 27.41 (t)  27.37 (t) a) 1.80 (m) 27.41 (t)
b) 1.75 (m)  b) 1.75 (m)
3 37.91 (d) 3.11 (br d, 5.0) 37.97 (d) 3.03 (br d, 5.2)  3 37.91 (d) 3.11 (br d, 5.0) 37.97 (d) 3.03 (br d, 5.2)
4 148.52 (s) 148.61 (s) CO 5 76.29 (d) 5.54 (t, 2, 5) 76.31 (d) 5.58 (t, 3.3)  4 148.52 (s) 148.61 (s) CO 5 76.29 (d) 5.54 (t, 2, 5) 76.31 (d) 5.58 (t, 3.3)
6 28.21 (t) a) 1.88 (m) 28.25 (t)  6 28.21 (t) a) 1.88 (m) 28.25 (t)
b) 1.75 (m)  b) 1.75 (m)
27.71 (t) a) 1.91 (ra) 27.75 (t)  27.71 (t) a) 1.91 (ra) 27.75 (t)
b) 1.56 (m)  b) 1.56 (m)
8 43.02 (s) 43.06 (s) o 9 77.37 (d) 5.90 (d, 10.0) 77.45 (d) 5.9 (d, 10.0)  8 43.02 (s) 43.06 (s) o 9 77.37 (d) 5.90 (d, 10.0) 77.45 (d) 5.9 (d, 10.0)
10 72.53 (d) 6.11 (d, 10.0) 72.57 (d) 6.11 (d, 10.0)  10 72.53 (d) 6.11 (d, 10.0) 72.57 (d) 6.11 (d, 10.0)
II 136.91 (s) 136.95 (s)  II 136.91 (s) 136.95 (s)
12 135.28 (s) 135.35 (s)  12 135.28 (s) 135.35 (s)
13 70.63 (d) 5.76(ddd. 10.0, 9.0, L 0) 70.63 (d) 5.76 (dd, 7.3) CO 14 32.38 (t) a) 2.76(ddd, 15, 10.9.0) 32.29 (t) a)2.75(ddd, 15.0, 10.0,9.0)  13 70.63 (d) 5.76 (ddd. 10.0, 9.0, L 0) 70.63 (d) 5.76 (dd, 7.3) CO 14 32.38 (t) a) 2.76 (ddd, 15, 10.9.0) 32.29 (t) a) 2.75 (ddd, 15.0, 10.0,9.0)
b) 1.04 (dd, 15.0, 7.0)  b) 1.04 (dd, 15.0, 7.0)
15 39.17 (s) 39.20 (s)  15 39.17 (s) 39.20 (s)
16 31.28 (q) 1.09 (s) 31.29 (q) 1.09 (s)  16 31.28 (q) 1.09 (s) 31.29 (q) 1.09 (s)
17 26.99 (q) 1.63 (s) 27.02 (q) 1.63 (s)  17 26.99 (q) 1.63 (s) 27.02 (q) 1.63 (s)
18 17.80 (q) 2.35 (s) 17.82 (q) 25.2 (s)  18 17.80 (q) 2.35 (s) 17.82 (q) 25.2 (s)
19 15.29 (q) 0.78 (s) 15.26 (q) 0.78(s)  19 15.29 (q) 0.78 (s) 15.26 (q) 0.78 (s)
20 114.29 (t) a) 5.31 (d, 1.0) 114.29 (t) a>5.30 (d, 1.0)  20 114.29 (t) a) 5.31 (d, 1.0) 114.29 (t) a> 5.30 (d, 1.0)
b) 4.91 (d. 1.0) b)4.91 (d, 1.0)  b) 4.91 (d. 1.0) b) 4.91 (d, 1.0)
(〇 crCHal ,017. w C 06 CH〇 2 + H 〔第 43表〕 (〇 crCHal, 017.w C 06 CH〇 2 + H (Table 43)
σ* - -o σ *--o
r o  r o
O C < oo—  O C <oo—
。 — o o to oo σ  . — O o to oo σ
B B S B B S
o o o o
co o σ co o σ
o o  o o
< ■ <■
o o o  o o o
なお、 第 42表 '第 43表には文献 (M.K.Yeh,Phytochem.,271534: 1988)に報告のある、 2 デスァセトキシ タキシニン Eの NMRデ一夕 を参照 (reference) として併記している。 上記の化合物 (44) の NMR デ一夕は、 該文献の記載とほぼ一致することが判明した。 In Table 42-Table 43, the NMR data of 2desacetoxytaxinin E reported in the literature (MKYeh, Phytochem., 271534: 1988) is also shown as a reference. NMR of compound (44) above It was found that the night was almost in agreement with the description in the document.
また、 前記分画 F 2.2 を、 シリカゲルステンレスカラム (INERTSIL PREP-SIL; 25 X 1 c m; GL Science) を用いた順相 H P L Cを採用 して、 酢酸ェチルと n—へキサンとを容量比 3 : 7で混合してなる混合 溶液 (キャリア, 流速 5m l Z分) を用いて分離し、 保持時間 1 7. 0 分〜 2 1. 8分の分画 F 2.24 479. 2mg、 および保持時間 2 1. 8 分〜 48. 0分の分画 F2-2-2 1 29. 3mgを分取した。 Also, the fraction F 2 2, silica gel stainless steel column. The normal phase HPLC using a (INERTSIL PREP-SIL;; 25 X 1 cm GL Science) is adopted, the capacitance ratio and hexane acetate Echiru and to n- Separation using a mixed solution (carrier, flow rate 5 ml Z min) mixed with 3: 7, retention time 17.0 min to 21.8 min fraction F 2.24 479.2 mg and retention time 2 1.8 minutes to 48.0 minutes of fractions F 2 - was separated and 2 -2 1 29. 3 mg.
そして、得られた分画 Fan を、 ODSステンレスカラム(INERTSIL PREP ODS ; 25 X 1 c m; GL Science) を用いた逆相高速液体ク口 マトグラフィ一 (逆相 HPLC) を採用して、 メチルアルコールと 0. 05 M酢酸アンモニゥム緩衝液 (pH4. 8) とァセトニトリルとを容 量比 1 : 1 : 2で混合してなる混合溶液 (キャリア, 流速 5m l 分) を用いてさらに分離し、 保持時間 24. 7分の分画 F2-2U 25. Omg を分取した。次いで、該分画 F2-2+i にメチルアルコールを加えて不純物 を再結晶させ、ろ過により結晶部が取り除かれたろ液部 1 7. 5mgを、 上記 OD Sステンレスカラムを用いて同一条件でさらに分離し、 タキシ ニン NN— 3 ( 9 -deacethyl taxinine) と化合物 (4 1) との混合物 分画 (保持時間 27. 5分) 7. 9mgを分取した。 Then, the obtained fractionated Fan was converted to methyl alcohol by reversed-phase high-performance liquid chromatography using ODS stainless steel column (INERTSIL PREP ODS; 25 X 1 cm; GL Science) (reverse-phase HPLC). The mixture was further separated using a mixed solution (carrier, flow rate 5 ml) consisting of a 0.05 M ammonium acetate buffer (pH 4.8) and acetonitrile in a volume ratio of 1: 1: 2. 7 minutes fraction F2-2U 25. Omg was collected. Then, fractionation F 2 - 2 + i by adding methyl alcohol was recrystallized impurities in the filtrate section 1 7. 5 mg of crystals portion is removed by filtration, under the same conditions using the OD S stainless steel column Further separation, 7.9 mg of a mixture fraction (retention time 27.5 minutes) of taxinine NN-3 (9-deacethyl taxinine) and compound (41) was collected.
上記混合物分画を、 シリカゲルステンレスカラム ( INERTSIL PREP-SIL ; 2 5 X 0. 6 c m; GL Science) を用いた順相 HPLCを 採用して、 酢酸ェチルと n—へキサンとを容量比 2 : 8で混合してなる 混合溶液 (キャリア, 流速 2m l 分) を用いて分離し、 これにより、 化合物 (4 1) 1. 2 mg (保持時間 38. 1分) を取り出した。 該化 合物(41) の含有量は、 新鮮針葉部の重量を基準として 0. 000 1 % であった。  The above mixture fraction was subjected to normal-phase HPLC using a silica gel stainless steel column (INERTSIL PREP-SIL; 25 × 0.6 cm; GL Science), and the volume ratio of ethyl acetate to n-hexane was 2: Separation was carried out using the mixed solution (carrier, flow rate 2 ml min) obtained by mixing in step 8, and 1.2 mg of the compound (41) (retention time 38.1 min) was taken out. The content of the compound (41) was 0.0001% based on the weight of the fresh needle portion.
上記化合物 (41) の iH— NMRおよび i3C— NMRの測定結果を 第 44表 ·第 45表にまとめて示す。 これにより、 化合物 (41) の構 造を解析した。 また、 該化合物 (41) の物性値等を測定した結果、 融 点 : 1 2 1で〜 1 23で The measurement results of iH-NMR and i3C-NMR of the above compound (41) Table 44 · Table 45 summarizes this. Thus, the structure of the compound (41) was analyzed. In addition, as a result of measuring the physical property values and the like of the compound (41), the melting point was: from 121 to 123.
〔ひ〕 D 20 : + 48. 05° (c = 0. 077, CHC 13 ) であった。 [Shed] D 20: + 48. was 05 ° (c = 0. 077, CHC 1 3).
N M R S p e c t r a l D a t a o f c o mp o u nd (41) i n C D C 1 N M R S p e c t r a l D a t a o f c o mp o u nd (41) in C D C 1
位置 C connected 1 H H - H COSY H M B C N O E S Y Location C connected 1 HH-H COSY HMBCNOESY
1 48.74 (d) 2.22 (dd, 6.9, 2.0) H2, 140 H3, Ua 16, 17 H2, 14D, 16. 17  1 48.74 (d) 2.22 (dd, 6.9, 2.0) H2, 140 H3, Ua 16, 17 H2, 14D, 16.17
2 69.69 (d) 5.54 (dd, 6.1, 2.0) Hll, 3 HI, 3. 14eB HI, 9, 17, 19  2 69.69 (d) 5.54 (dd, 6.1, 2.0) Hll, 3 HI, 3.14eB HI, 9, 17, 19
3 43.18 (d) 3.41 (d. 6.1) H2, 20ab HI, 2. 5, 19, 20ab H7o, 14a, 18, 20a  3 43.18 (d) 3.41 (d.6.1) H2, 20ab HI, 2.5, 19, 20ab H7o, 14a, 18, 20a
4 142.09 (s) H3, 5. 20ab  4 142.09 (s) H3, 5.20ab
5 78.36 (d) 5.34 (br s) H6«il H3, 60 β, 20ab H6a0, 20b  5 78.36 (d) 5.34 (br s) H6 «il H3, 60 β, 20ab H6a0, 20b
6 28.43 (t) 0) 1.98 <m) H5. 6D, 7a& H7U H5, 6B. 7e  6 28.43 (t) 0) 1.98 <m) H5.6D, 7a & H7U H5, 6B. 7e
(1) 1.72 (m) H5, 60, 7αβ H5. 6a. 11  (1) 1.72 (m) H5, 60, 7αβ H5.6.a. 11
7 27.42 (t) a) 1.77 n) Η6αϋ H3, 5, 66. 9, 19 H6S, 9  7 27.42 (t) a) 1.77 n) Η6αϋ H3, 5, 66. 9, 19 H6S, 9
1) 1.65 On) Η6α|ί H3, 6a, 10  1) 1.65 On) Η6α | ί H3, 6a, 10
8 44.48 (s) H2, 3,6αί, 7»ί, 9.19  8 44.48 (s) H2, 3,6αί, 7 »ί, 9.19
9 79.20 (d) 5.76 (d. 10.0) H10 鼠 19 H2, 17, 19  9 79.20 (d) 5.76 (d. 10.0) H10 Rat 19 H2, 17, 19
10 72.25 (d) 5,03 (dd, 10.0. 6.4) H9 09 H7a, 18  10 72.25 (d) 5,03 (dd, 10.0.6.4) H9 09 H7a, 18
01 11 154.52 (s) HI, 10, 16, 17. 18 to 12 135.62 (s) 鳳 18  01 11 154.52 (s) HI, 10, 16, 17. 18 to 12 135.62 (s) Otori 18
13 199.70 (s) HI, ΙΑ ί, 18  13 199.70 (s) HI, ΙΑ ί, 18
14 36.13 (t) d)2.86(dd, 19.8, 6.9) H], 14a HI, 2 HI. 14a. 16  14 36.13 (t) d) 2.86 (dd, 19.8, 6.9) H], 14a HI, 2 HI. 14a. 16
a) 2.34 (d, 19.8) HI, 14i H3. 140  a) 2.34 (d, 19.8) HI, 14i H3. 140
15 37.84 (s) HI, 10, 14σ, 16, 17  15 37.84 (s) HI, 10, 14σ, 16, 17
16 37.62 (q) 1.25 (s) H17 HI, 17 HI. 14/). 17  16 37.62 (q) 1.25 (s) H17 HI, 17 HI. 14 /). 17
17 25.33 (q) 1.83 (s) HI6 HI, 16 HI, 2, 9, 16  17 25.33 (q) 1.83 (s) HI6 HI, 16 HI, 2, 9, 16
18 14.06 (q) 2.18 (s) H3, 10, 2'  18 14.06 (q) 2.18 (s) H3, 10, 2 '
19 Π.64 (q) 0.94 (s) H3, 7αβ. 9 H2, 7β, 9  19 Π.64 (q) 0.94 (s) H3, 7αβ. 9 H2, 7β, 9
20 117.17 (t) a) 5.35 (br s) H3, 20b H3, 5 H3. 20b  20 117.17 (t) a) 5.35 (br s) H3, 20b H3, 5 H3. 20b
b) 4.85 (br s) H3. 20a H5, 20a b) 4.85 (br s) H3.20a H5, 20a
〔第 45表〕 (Table 45)
=Π 3= Ο 3 = Π 3 = Ο 3
t a 6 ε t a 6 ε
ασ  ασ
o t- ■¾· ^  o t- ■ ¾
CM CO  CM CO
V5 υ) cr cr -ο -σ -σ -σ -σ
Figure imgf000154_0001
V5 υ) cr cr -ο -σ -σ -σ -σ
Figure imgf000154_0001
a,。 β a これらの結果から、 上記化合物 (41) は、 新規な夕キシニン化合物 としての 1 0—デァセチル タキシニン (夕キシニン NN— 14) で あることが判明した。 尚、 1 0—デァセチル 夕キシニンは、 文献 (T.Oritani,et.al.,Biosci.Biochem.,63,924:i999) にその名称が記載さ れた物質ではあるが、該物質と化合物(4 1) とは NMR分析の結果(特 に、 9位と 1 0位のプロトンシフト値) が明らかに異なっており、 該文 献に記載の物質は 9一 deacethyl 体 (タキシニン NN— 3) であった ことが明らかとなった。 なお、 参照のため、 化合物 (4 1) および上記 文献に記載の物質 (reference) の NM Rデータを第 46表に併記する。 a ,. β a From these results, the above compound (41) was converted to 10-deacetyltaxinine (Yu-Xinine NN-14) as a new Xinine compound. It turned out to be. Although 10-deacetyl xyxinine is a substance whose name is described in the literature (T. Oritani, et. Al., Biosci. Biochem., 63, 924: i999), the substance and compound (41 ) And NMR analysis results (particularly, the proton shift values at the 9-position and the 10-position), and the substance described in the literature was a 91-deacethyl form (taxinine NN-3). It became clear. For reference, the NMR data of compound (41) and the substance (reference) described in the above literature are also shown in Table 46.
〔第 46表〕 (Table 46)
N R spectral data of compound (41) in CDC1 N R spectral data of compound (41) in CDC1
Figure imgf000156_0001
Figure imgf000156_0001
一方、前記分画 F 2-2 より得られた他方の分画 F2.2-2 を、前記分画 から分画 F2-2 1 を分取した要領に従ってさらに分離し、保持時間 0分〜Meanwhile, the fraction F 2 other fractions obtained from -2 F 2. 2 -2, further separated according to the manner in which a sample was collected fractions F2-2 1 from the fraction, retention time 0 minutes to
1 5. 8分の分画 F2-2H 64. lmgを分取した。次いで、該分画 を、 上記要領に従ってさらに分離し、 粗製の化合物 (42) を含む分画 5. 6mg (保持時間 14. 3分) を分取した。 そして、 上記粗製の化 合物 (42) を含む分画を、 シリカゲルステンレスカラム (INERTSIL PREP-SIL ; 25 X 0. 6 cm ; GL Science) を用いた順相 H P L Cを 採用して、 酢酸ェチルと n—へキサンとを容量比 2 : 8で混合してなる 混合溶液 (キャリア, 流速 1. 7m l Z分) を用いて分離し、 これによ り、 精製された化合物 (42) 3. lmg (保持時間 53. 3分) を取 り出した。 該化合物 (42) の含有量は、 新鮮針葉部の重量を基準とし て 0. 00025 %であった。 15.8 min Fractionation F2-2H 64. lmg was collected. Then, the fraction Was further separated according to the above procedure, and 5.6 mg (retention time: 14.3 minutes) of a fraction containing the crude compound (42) was collected. The fraction containing the above crude compound (42) was separated with ethyl acetate by normal phase HPLC using a silica gel stainless steel column (INERTSIL PREP-SIL; 25 × 0.6 cm; GL Science). Separation was performed using a mixed solution (carrier, flow rate: 1.7 ml / min) mixed with n-hexane at a volume ratio of 2: 8, and the purified compound (42) 3. lmg (Retention time 53.3 minutes). The content of the compound (42) was 0.00025% based on the weight of the fresh needle portion.
上記化合物 (42) の iH— NMRおよび 13C— NMRの測定結果を 第 47表 ·第 48表にまとめて示す。 これにより、 化合物 (42) の構 造を解析した。 また、 該化合物 (42) の物性値等を測定した結果、 分子式 し 37H44リ 12  The results of iH-NMR and 13C-NMR measurements of the above compound (42) are summarized in Tables 47 and 48. Thus, the structure of compound (42) was analyzed. In addition, as a result of measuring the physical properties and the like of the compound (42), the molecular formula was 37H44
分子量 680. 2822 (測定値)、 680. 2833 (計算値) 融 点 232で〜 234で (プリズム)  Molecular weight 680. 2822 (measured value), 680. 2833 (calculated value) Melting point 232 to 234 (prism)
Ca) D 20 + 30. 49 ° (c = 0. 223, CHC 13 ) Ca) D 20 + 30. 49 ° (c = 0.223, CHC 13)
I R 3044, 1 750, 1 7 14, 1 638 cm'i (CH であった。 IR 3044, 1 750, 17 14, 1638 cm'i (CH.
cn cn
p N M o m c r o n C D〇
Figure imgf000158_0002
p NM omcron CD〇
Figure imgf000158_0002
Figure imgf000158_0001
Figure imgf000158_0001
i 裟〕 i
191 191
0170.0/10 ΟΛ\ 〔第 48表〕 0170.0 / 10 ΟΛ \ (Table 48)
εε
Figure imgf000159_0001
Figure imgf000159_0001
) n  ) n
o o
3= 3= 3 3 = 3 = 3
OO  OO
σ in  σ in
o O  o O
to o¾ o  to o¾ o
-< -< -< -< _ >c  -<-<-<-<_> c
これらの結果から、 上記化合物 (42) は、 新規な夕キシニン化合物 としての夕キシニン NN— 1 3であることが判明した。 From these results, the above compound (42) is a novel quinine compound It was found to be Xinin NN-13 as evening.
また、 前記 2番目のフラクション(F2 ) 2233 mgを別に用意し、 シリカゲルステンレスカラム (INERTSIL PREP-SIL ; 25 X 1 cm ;Separately, 2233 mg of the second fraction (F 2 ) was separately prepared, and a silica gel stainless column (INERTSIL PREP-SIL; 25 × 1 cm;
GL Science) を用いた順相 H P L Cを採用して、 酢酸ェチルと n—へキ サンとを容量比 2 : 8で混合してなる混合溶液 (キャリア, 流速 5m lUsing a normal phase HPLC with GL Science), a mixed solution consisting of ethyl acetate and n-hexane mixed at a volume ratio of 2: 8 (carrier, flow rate 5 ml)
Z分) を用いて分離し、 保持時間 12. 0分〜45. 0分の分画 F2.3 9Were separated using Z min), retention time 12.0 min to 45. 0 min fractions F 2. 3 9
6 1. 7mgを分取した。 6 1.7 mg was collected.
続いて、 上記分画 F2-3 を、 上記カラムを用いた順相 HPLCを採用し て、 酢酸ェチルと n—へキサンとを容量比 3 : 7で混合してなる混合溶 液 (キャリア,流速 5m l Z分) にて分離し、 保持時間 0分〜 14. 0分 の分画 Fan 269. Omgを分取した。 続いて、 分画 を、 上記 カラムを用いて同一条件で分離し、 保持時間 20. 4分の分画 (ピーク)Subsequently, the fraction F 2 - 3, employs a normal phase HPLC using the column, acetic Echiru a volume ratio and a hexane n-3: mixture solvent solution obtained by mixing 7 (carrier, (Flow rate 5 ml Z min), and fractionated Fan 269. Omg with a retention time of 0 min to 14.0 min. Subsequently, the fractions were separated under the same conditions using the above column, and the fraction (peak) with a retention time of 20.4 minutes
Fa-si ! 1 1 3. Omgを分取した。 Fa-si! 1 1 3. Omg was collected.
そして、 分画 を、 OD Sステンレスカラム (INERTSIL PREP ODS ; 25 X 1 c m; GL Science) を用いた逆相 HPLCを採 用して、 メチルアルコールと 0. 05M酢酸アンモニゥム緩衝液 (pH The fraction was subjected to reverse phase HPLC using ODS stainless steel column (INERTSIL PREP ODS; 25 × 1 cm; GL Science), and methyl alcohol and 0.05 M ammonium acetate buffer (pH
4. 8) とァセトニトリルとを容量比 1 : 1 : 2で混合してなる混合溶 液 (キャリア, 流速 5m lZ分) を用いてさらに分離し、 これにより、 化合物 (46) 30. 7mg (保持時間 38. 2分) を取り出した。 該 化合物 (46) の含有量は、 新鮮針葉部の重量を基準として 0. 0024.8) and acetonitrile were further separated using a mixed solution (carrier, flow rate of 5 ml / min) mixed at a volume ratio of 1: 1: 2, whereby 30.7 mg of compound (46) (retained) Time 38.2 minutes). The content of the compound (46) is 0.0002 based on the weight of the fresh needle portion.
5 %であった。 5%.
上記化合物 (46) の iH— NMRおよび 13C— NMRの測定結果を 第 49表にまとめて示す。 これにより、 化合物 (46) の構造を解析し た。 また、 該化合物 (46〗 の物性値等を測定した結果、  The results of iH-NMR and 13C-NMR measurements of the above compound (46) are summarized in Table 49. Thus, the structure of the compound (46) was analyzed. In addition, as a result of measuring the physical properties of the compound (46%),
融 点 : 1 6 It〜 1 63で  Melting point: 16 It ~ 1 63
[a] D 2o : + 66. 3° (c = 2. 32, CHC 13 ) I R 42, 1 640, 1 246 cm.i (CHC l 3 ) であった。 [a] D 2o: + 66. 3 ° (c = 2. 32, CHC 1 3) It was IR 42, 1 640, 1 246 cm.i (CHC l 3).
〔第 49表〕  (Table 49)
NMR spectral data of compound (46) in CDCI » NMR spectral data of compound (46) in CDCI »
Conpound (46) reference  Conpound (46) reference
位置  Position
C connected 1 H H C connected 1 HH
40.16 (d) 1.85 (m) 1.84 (l  40.16 (d) 1.85 (m) 1.84 (l
27.27 (t) a) 1.86 On) a) 2.02 (m)  27.27 (t) a) 1.86 On) a) 2.02 (m)
b) 1.77 (dd, 16, 5) b) I.95(ddd, 14.4, 5.0,2.5〉 b) 1.77 (dd, 16, 5) b) I.95 (ddd, 14.4, 5.0, 2.5>
37.45 (d) 3.03 (brd, 5.0) 3.03 (brd, 5.0) 37.45 (d) 3.03 (brd, 5.0) 3.03 (brd, 5.0)
146.30 (s)  146.30 (s)
74.79 (d) 5.58 (t, 3.0) 5.58 (t, 3.3)  74.79 (d) 5.58 (t, 3.0) 5.58 (t, 3.3)
34.58 (t) a) 1.95 (ddd, 15, 5, 3) a) 1.86 (in)  34.58 (t) a) 1.95 (ddd, 15, 5, 3) a) 1.86 (in)
b) 1.86 (m) b) 1.78 (dd, 16.6, 4.6) b) 1.86 (m) b) 1.78 (dd, 16.6, 4.6)
7 70.02 (d) 5.69 (dd, 12, 5) 5.69 (dd, 11.5, 5.0) 7 70.02 (d) 5.69 (dd, 12, 5) 5.69 (dd, 11.5, 5.0)
8 46.32 (s)  8 46.32 (s)
9 76.74 (d) 5.95 (d, 11.0) 5.95 (d, 11.1)  9 76.74 (d) 5.95 (d, 11.0) 5.95 (d, 11.1)
10 71.70 (d) 6.31 (d, 11.0) 6.57 (d, 11.1)  10 71.70 (d) 6.31 (d, 11.0) 6.57 (d, 11.1)
11 135.01 (s)  11 135.01 (s)
12 137.23 (s)  12 137.23 (s)
13 70.60 (d) 5.81 (t, 8.2) 5.81 (t, 7.3)  13 70.60 (d) 5.81 (t, 8.2) 5.81 (t, 7.3)
14 31.89 (t) a) 2.71 (ddd, 15, 10. a) 2.73 (ddd, 14.6,9.8.4.8)  14 31.89 (t) a) 2.71 (ddd, 15, 10.a) 2.73 (ddd, 14.6,9.8.4.8)
10)  Ten)
b) 0.99(brdd.15, 7) b) 0.99(dd, 14.6. 7.0)  b) 0.99 (brdd.15, 7) b) 0.99 (dd, 14.6. 7.0)
15 39.39 (s)  15 39.39 (s)
16 31.18 (q) 1.10 (s)  16 31.18 (q) 1.10 (s)
17 27.23 (q) 1.63 (s)  17 27.23 (q) 1.63 (s)
18 15.31 (q) 2.34 (d, 1.2)  18 15.31 (q) 2.34 (d, 1.2)
)9 13.19 (q) 0.88 (s)  ) 9 13.19 (q) 0.88 (s)
20 115.97 (q) a) 5.39 (brd. 1.2) a) 5.39 (d. 1.2)  20 115.97 (q) a) 5.39 (brd. 1.2) a) 5.39 (d. 1.2)
b) 5.02 (brd, 1.5) b) 5.02 (d, 1.5)  b) 5.02 (brd, 1.5) b) 5.02 (d, 1.5)
13-OAc 170.68 (s)  13-OAc 170.68 (s)
9-OAc 170.23 (s)  9-OAc 170.23 (s)
7-OAc 169.85 (s)  7-OAc 169.85 (s)
10-OAc 168.13 (s)  10-OAc 168.13 (s)
21.46 (q) 1.72 (s)  21.46 (q) 1.72 (s)
21.01 (q) 2.00 (s)  21.01 (q) 2.00 (s)
20.96 (q) 2.05 (s)  20.96 (q) 2.05 (s)
20.85 (q) 2.08 (s)  20.85 (q) 2.08 (s)
1" 166.13 (s)  1 "166.13 (s)
2" 118.38 (d) 6.58 (d, 16.2)  2 "118.38 (d) 6.58 (d, 16.2)
3, 145.72 (d) 7.78 (d, 16.2)  3, 145.72 (d) 7.78 (d, 16.2)
q-Ph 130.59 (s)  q-Ph 130.59 (s)
o- 128.09 (d) 7.49-7.52 (m)  o- 128.09 (d) 7.49-7.52 (m)
m- 128.99 (d) 7.42-7.39 (m)  m- 128.99 (d) 7.42-7.39 (m)
p- 130.59 (d) 7.42-7.39 (m) なお、 第 4 9表には文献 (J.Zang.Chiniese Chem丄 ett., 5,497:1994) に報告のある、 2—デスァセトキシ 夕キシニン Jの NMRデ一夕を参 照 (reference) として併記している。 上記の化合物 (46) の NMRデー 夕や 〔a〕 D 2oの値は、 該文献の記載とほぼ一致することが判明した。 一方、 上記 3番目のフラクション (F3 ) を、 シリカゲルステンレス カラム (INERTSIL PREP-SIL ; 25 X 2 cm; GL Science) を用いた 順相 HPLCを採用して、 酢酸ェチルと n—へキサンとを容量比 1 : 1 で混合してなる混合溶液 (キャリア, 流速 1 5m l Z分) を用いて分離 し、 保持時間 1 5. 8分の分画 FHI 47. 8mg、 および、 保持時間 18. 0分〜22. 0分の分画 F3-2 164. 7mgを分取した。 p-130.59 (d) 7.42-7.39 (m) Table 49 shows references (J. Zang.Chiniese Chem 丄 ett., 5,497: 1994). The NMR data of 2-desacetoxin xinine J reported in Ref. The NMR data and the value of [a] D 2 o of the above compound (46) were found to be almost the same as those described in the literature. On the other hand, the third fraction (F 3 ) was subjected to normal-phase HPLC using a silica gel stainless steel column (INERTSIL PREP-SIL; 25 × 2 cm; GL Science) to separate ethyl acetate and n-hexane. Separation was performed using a mixed solution (carrier, flow rate: 15 ml / min) mixed at a volume ratio of 1: 1. Fractionation of retention time 15.8 min FHI 47.8 mg and retention time 18.0 min It was aliquoted 2 164. 7 mg -. min to 22 0 min fractions F 3.
得られた分画 F 3.1 を、 O D Sステンレスカラム ( INERTSIL PREP ODS ; 2 5 X 1 c m; GL Science) を用いた逆相 HPLCを採 用して、 メチルアルコールと 0. 05M酢酸アンモニゥム緩衝液 (pH 4. 8) とァセトニトリルとを容量比 1 : 1 : 2で混合してなる混合溶 液 (キャリア, 流速 5m l Z分) を用いて単離 '精製した。 これにより、 化合物 (43) 2. 5mg (保持時間 1 7. 4分) を取り出した。 該化 合物(43) の含有量は、 新鮮針葉部の重量を基準として 0. 0002 % であった。  The obtained fraction F3.1 was subjected to reverse phase HPLC using an ODS stainless steel column (INERTSIL PREP ODS; 25 × 1 cm; GL Science), and methyl alcohol and 0.05 M ammonium acetate buffer (pH 4.8) and acetonitrile were isolated and purified using a mixed solution (carrier, flow rate 5 ml / min) with a volume ratio of 1: 1: 2. As a result, 2.5 mg of compound (43) (retention time: 17.4 minutes) was obtained. The content of the compound (43) was 0.0002% based on the weight of the fresh needle portion.
上記化合物 (43) の iH— NMRおよび 13C— NMRの測定結果を 第 50表 ·第 5 1表にまとめて示す。 これにより、 化合物 (43) の構 造を解析した。 また、 該化合物 (43) の物性値等を測定した結果、  The results of iH-NMR and 13C-NMR measurements of the above compound (43) are summarized in Tables 50 and 51. Thus, the structure of the compound (43) was analyzed. In addition, as a result of measuring physical properties and the like of the compound (43),
77ナ A : C35山 2リ 10  77na A: C35 mountain 2 re 10
分子量 : 622. 2783 (測定値)、 622. 2778 (計算値) 融 点 : 247で〜 249  Molecular weight: 622. 2783 (measured value), 622. 2778 (calculated value) Melting point: 247 to 249
〔ひ〕 D 20 : + 1 73. 37° (c = 0. 1 54, CHC 13 ) [H] D 20: + 1 73. 37 ° (c = 0.154, CHC 13)
I R : 3548, 1 780, 1 7 14, 1 640 c mi (CH C 13 ) であった。IR: 3548, 1780, 1714, 1640 cmi (CH C13) Met.
〔第 5 0表〕
Figure imgf000163_0001
〔第 5 1表〕
(Table 50)
Figure imgf000163_0001
(Table 51)
1 1
ex  ex
E  E
^ -c  ^ -c
1 ^ a- o . 1  1 ^ a- o. 1
. o. E  . o. E
αο α. oo . .  αο α. oo.
— o o  — O o
zn o as ^ ^  zn o as ^ ^
Figure imgf000164_0001
Figure imgf000164_0001
OO
Q B E Q B E
35 3 3  35 3 3
CM CM « O  CM CM «O
oo oo co oo oo co
• " ^ oo ao
Figure imgf000164_0002
• "^ oo ao
Figure imgf000164_0002
上記の結果から、 化合物 (43) は、 本願発明者により新たに見いだ された新規な夕キシニン化合物としてのタキシニン NN— 12である ことが判明した。 From the above results, compound (43) was newly found by the present inventors. Was found to be taxinine NN-12 as a new amino acid compound.
一方、前記分画 F3-2 を、前記フラクション F3 から分画 · F3-2 を 分取した要領に従ってさらに分離し、保持時間 1 7. 5分の分画 Fan 5 4. lmg、 および、 保持時間 1 8. 7分の分画 F3-2-2 1 00. 3 m g を分取した。 次いで、 これら分画 Fan · F3-2-2 それぞれを、 ODSス テンレスカラム (INERTSIL PREP-ODS ; 25 X 1 c m; GL Science) を用いた逆相 HP L Cを採用して、 メチルアルコールと 0. 05M酢酸 アンモニゥム緩衝液(pH4. 8) とァセトニトリルとを容量比 1 : 2 : 2で混合してなる混合溶液 (キャリア, 流速 5. Om l Z分) を用いて 分離した。 これにより、 分画 から保持時間 7. 3分の分画 Meanwhile, the fraction F 3 - 2, further separated from the fractions F 3 according to the procedure preparative fractionation · F3-2 min, retention time 1 7.5 minute fractions Fan 5 4. lmg and, retention time 1 8. 7 min fractions F 3 - was separated and 2 -2 1 00. 3 mg. Then, each of these fractions, Fan · F 3 -2-2, was subjected to reverse phase HP LC using an ODS stainless steel column (INERTSIL PREP-ODS; 25 × 1 cm; GL Science), and methyl alcohol and Separation was performed using a mixed solution (carrier, flow rate of 5. OmIZ) of a mixture of .05M ammonium acetate buffer (pH 4.8) and acetonitrile at a volume ratio of 1: 2: 2. This allows fractionation from retention to retention time of 7.3 minutes.
2. 5mgを、 分画 F 3-2-2 から保持時間 7. 3分の分画 8. 7 mgを分取した。 2. 5 mg, Fraction F 3 - 2 - 2 was collected fractions 8. 7 mg of retention time 7.3 minutes.
得られた上記分画 F3-2-2-i を、上記 OD Sステンレスカラムを用い、 上 記と同一条件で再分離して、 保持時間 20. 7分の分画 1. lmgを分 取し、 続いて、 該分画と分画 F3-2小 1 とをそれぞれ NMR分析して、 両分 画に共通の化合物が含まれていることを確認した。 The resulting said fraction F 3-2-2 -i, using the OD S stainless steel column, collected and separated again on Symbol the same conditions, the fractions 1. lmg of retention time 20.7 min min and, subsequently, fractionation and fractions F 3 - 2 1 small and the by NMR analysis, respectively, it was confirmed that contains common compounds both fractions.
次いで、 上記分画 F3-2-2-i から分取された分画と分画 F3-2+1 とをあわ せて、 上記 ODSステンレスカラムを用い、 上記と同一条件で単離 '精 製を行った。 これにより、 化合物 (47) 1. 5mg (保持時間 35. 2分) を取り出した。 該化合物 (47) の含有量は、 新鮮針葉部の重量 を基準として 0. 000 1 2 %であった。 Then, the fraction F 3 - to match the 2 from -2-i is fractionated fractions and fraction F3-2 + 1, using the above ODS stainless steel column, isolated 'rectification in the same conditions as described above Was manufactured. As a result, 1.5 mg of the compound (47) (retention time: 35.2 minutes) was obtained. The content of the compound (47) was 0.0012% based on the weight of the fresh needle portion.
上記化合物 (47) の iH— NMRおよび lac— NMRの測定結果を 第 52表 ·第 53表にまとめて示す。 これにより、 化合物 (47) の構 造を解析した。 また、 該化合物 (47) の物性値等を測定した結果、 融 点 : 20 Ot:〜 202で 隱 spectral data of compound (47) in CDCl: ri The results of iH-NMR and lac-NMR measurements of the above compound (47) are summarized in Tables 52 and 53. Thus, the structure of the compound (47) was analyzed. In addition, as a result of measuring the physical properties of the compound (47), melting point: 20 Ot: up to 202 Hidden spectral data of compound (47) in CDCl: ri
s  s
Compound (47) reference 0 Q 位置 CO  Compound (47) reference 0 Q position CO
o  o
C connected 1 H , 3C connected 1 H V C connected 1 H , 3 C connected 1 HV
to to
1 51.25 (d) 2.36 (dd, 6.8, 2.2) 51.3 (d) 2.36 (dd. 6.5, 2 5) 1 51.25 (d) 2.36 (dd, 6.8, 2.2) 51.3 (d) 2.36 (dd. 6.5, 25)
2 68.13 (d) 4.20 (m) 68.2 (d) 4.19 (dd, 6.5, 2 5)  2 68.13 (d) 4.20 (m) 68.2 (d) 4.19 (dd, 6.5, 25)
•OH 45.21 (d) t.94 (br s) 45.2 (d) 1.98 (br s)  OH 45.21 (d) t.94 (br s) 45.2 (d) 1.98 (br s)
3 3.27 (br d, 6.1) 3.25 (br d, 6.3)  3 3.27 (br d, 6.1) 3.25 (br d, 6.3)
4 144.18 (s) 144.2 (s)  4 144.18 (s) 144.2 (s)
5 78.20 (d) 5.35 (t, 2.8) 78.3 (d) 5.34 (t. 2.8〉  5 78.20 (d) 5.35 (t, 2.8) 78.3 (d) 5.34 (t.2.8>
6 29.02 (t) a) 2.11 (m) 29.0 (t)  6 29.02 (t) a) 2.11 (m) 29.0 (t)
b) 1.76 (m)  b) 1.76 (m)
7 26.39 (t) a) 1.86{dd, 14.0,5.3,2.0) 26.4 (t)  7 26.39 (t) a) 1.86 (dd, 14.0,5.3,2.0) 26.4 (t)
b) 1.55 (m)  b) 1.55 (m)
8 45.21 (s) 45.2 (s)  8 45.21 (s) 45.2 (s)
9 75.71 (d) 4.23 (d, 9.8) 75.7 (d) 4.22 (dd, 9.6, 4.0)  9 75.71 (d) 4.23 (d, 9.8) 75.7 (d) 4.22 (dd, 9.6, 4.0)
OH 2.30 (d, 4.0)  OH 2.30 (d, 4.0)
10 77.00 (d) 5.84 (d, 9.8) 77.0 (d) 5.83 (d, 9.6)  10 77.00 (d) 5.84 (d, 9.8) 77.0 (d) 5.83 (d, 9.6)
11 151.28 (s) 151.3 (s)  11 151.28 (s) 151.3 (s)
12 137.20 (s) 137.2 (s)  12 137.20 (s) 137.2 (s)
13 199.72 (s) 199.8 (s)  13 199.72 (s) 199.8 (s)
】4 35.71 (t) a) 2.84 (dd. 19.2, 6.8) 35.7 (t) a) 2.83 (dd, 19.2, 6.5)  4 35.71 (t) a) 2.84 (dd.19.2, 6.8) 35.7 (t) a) 2.83 (dd, 19.2, 6.5)
b) 2.25 (m) b) 2.24 (d, 19.2)  b) 2.25 (m) b) 2.24 (d, 19.2)
15 37.61 (s) 37.6 (s)  15 37.61 (s) 37.6 (s)
16 25.76 (q) 1.58 (s) 25.8 (q) 1.57 (s)  16 25.76 (q) 1.58 (s) 25.8 (q) 1.57 (s)
17 37.61 (q) 1.18 (s) 37.6 ω 1.17 (s)  17 37.61 (q) 1.18 (s) 37.6 ω 1.17 (s)
18 13.88 (q) 2.26 (s) 13.9 (Q) 2.26 (s)  18 13.88 (q) 2.26 (s) 13.9 (Q) 2.26 (s)
19 17.77 (q) 1.18 (s) 17.8 (q) 1.17 (s)  19 17.77 (q) 1.18 (s) 17.8 (q) 1.17 (s)
20 117.83 (t) a) 5.45 (br s) 117.9 (t) a) 5.43 (br s)  20 117.83 (t) a) 5.45 (br s) 117.9 (t) a) 5.43 (br s)
b) 5.38 (t, 1.3) b) 5.38 (t, 1.4)  b) 5.38 (t, 1.3) b) 5.38 (t, 1.4)
61 c + 0 CCH卩 〔第 5 3表〕 61 c + 0 CCH (Table 53)
-σ =5 O OO o -σ = 5 O OO o
CO CM C C  CO CM C C
oo oo oo oo
1 1  1 1
O  O
OO OO OS CO O CO ^4* OO OO OS CO O CO ^ 4 *
o e cx なお、 第 5 2表 · 第 5 3表には文献 (G.Appendino Phytochem.1,4253:1992)に報告のある、 5—シンナモイルー 1 0—ァセチル—タキ シン II の NMRデータを参照 (reference) として併記している。 上記の 化合物 (47) の NMRデータや物性値 (融点、 並びに 〔ひ〕 D 20) は、 該文献の記載とほぼ一致することが判明した。 oe cx Tables 52 and 53 show 5-cinnamoyl-10-acetyl-taki reported in the literature (G. Appendix Phytochem. 1,4253: 1992). The NMR data for Syn II are also provided as a reference. The NMR data and physical property values (melting point, and [g] D20) of the above compound (47) were found to substantially match the descriptions in the literature.
取り出された上記の化合物 (4 1) 〜 (44)、 化合物 (46)、 並び に、 化合物 (47) の多剤耐性癌克服作用の測定結果は、 以下の実施例 12にまとめて示すものとする。  The measurement results of the above-mentioned compounds (41) to (44), compound (46), and compound (47) taken out of the compound (47) taken together with those obtained in Example 12 are summarized below. I do.
〔実施例 1 2〕 (Example 12)
仙台産の日本ィチイから採取した新鮮葉および若茎よりなる針葉部か ら、 タキサン関連化合物である化合物 (45) を取り出した。 即ち、 上 記の針葉部 1 674 gを n—へキサン 6. 9 5 Lに 1週間浸漬し、 n— へキサン抽出物 2. 8 gを得、 残りをさらに酢酸ェチル 6. 95 Lに一 週間浸漬し、 酢酸ェチル抽出物 29. 3 gを得た。 この酢酸ェチル抽出 物を酢酸ェチル /n—へキサン (体積比 1 : 1) 混合溶液に溶かし、 不 溶部として 20. 8 gのクル一ドオイルを得た。  Compound (45), a taxane-related compound, was extracted from needles consisting of fresh leaves and young stems collected from Nippon yew from Sendai. That is, 1674 g of the needle portion described above was immersed in 6.95 L of n-hexane for 1 week to obtain 2.8 g of the n-hexane extract, and the remainder was further added to 6.95 L of ethyl acetate. After soaking for one week, 29.3 g of ethyl acetate extract was obtained. The ethyl acetate extract was dissolved in a mixed solution of ethyl acetate / n-hexane (volume ratio 1: 1) to obtain 20.8 g of a cloud oil as an insoluble portion.
次に、 該クルードオイルをメチルアルコール 1 00m 1に溶かし、 さ らに酢酸ェチル 30 Om 1を加えた後、 酸性水溶液である 0. 5M硫酸 1 00m lで 3回 (3 X 100m l ) 洗浄することにより、 塩基性物質 を水層 (硫酸) 中に抽出した。 そして、 洗浄後の上記 0. 5M硫酸 (水 層) に 29重量%アンモニア水を添加して pHを 9. 0とした後、 クロ 口ホルム 1 00m lで 3回 (3 X 200m l ) 抽出した。 これにより、 粗アルカロイド分画 2. 8 gを得た。  Next, the crude oil is dissolved in 100 ml of methyl alcohol, 30 Om 1 of ethyl acetate is added, and the resultant is washed three times (3 × 100 ml) with 100 ml of 0.5 M sulfuric acid as an acidic aqueous solution. As a result, the basic substance was extracted into the aqueous layer (sulfuric acid). Then, 29% by weight aqueous ammonia was added to the above 0.5M sulfuric acid (aqueous layer) after the washing to adjust the pH to 9.0, and then extracted three times (3 × 200 ml) with 100 ml of black form. . Thus, 2.8 g of a crude alkaloid fraction was obtained.
こうして得られた粗アル力ロイド分画を、 アルミナ 272 gを充填剤 として用いたカラムクロマトグラフィーを採用して、 展開溶媒として n 一へキサンと酢酸ェチルとメチルアルコールとを用いたグラジュェント 溶出を行うことにより、 f l〜 f 1 4の 14個のフラクションに分画(荒 分け) した。 The crude alloid extract thus obtained is subjected to column elution using 272 g of alumina as a packing material and gradient elution using n-hexane, ethyl acetate and methyl alcohol as a developing solvent. By this, fractionation into 14 fractions from fl to f14 (rough Divided)
次に、 これらのフラクションを、 OD Sカラムを用いた逆相 HP L C で分析することにより以下に示す計 8つのフラクション F 1〜F 8にま とめた。 これらフラクションとはすなわち、 F l ( f l〜 f 4 : 350 mg)、 F 2 ( f 5〜 f 6 : 350mg)、 F 3 ( f 7 : 200mg)、 F 4 ( f 8 : 54mg)、 F 5 ( f 9 : 1. 1 8 g)、 F 6 ( f 1 0 : 27 1 mg), F 7 ( f 1 1 : 2 1 9mg), F 8 ( f l 2〜 f l 4 : 1 1 5 m g )、 である。  Next, these fractions were analyzed by reversed-phase HPLC using an ODS column to collect the following eight fractions F1 to F8. These fractions are Fl (fl to f4: 350 mg), F2 (f5 to f6: 350 mg), F3 (f7: 200 mg), F4 (f8: 54 mg), F5 (f 9: 1.18 g), F 6 (f 10: 27 1 mg), F 7 (f 11: 2 19 mg), F 8 (fl 2-fl 4: 1 15 mg), It is.
これらフラクションのうち、 逆相 HP L Cを採用してフラクション F 2の分取を行い、 次の条件で化合物 (45) を単離した。 すなわち、 測 定機器として SHIMAZU LU-6A を、 カラムとして OD Sステンレス力 ラム (QINERTSILPREP-ODS;25cmX2cm)を採用し、 検出 UVの波長 (λ) = 254 nmの条件下で、 メチルアルコールと 0. 05M酢酸一 酢酸アンモニゥム緩衝液(PH4) とァセトニトリルとを容積比 1 : 1 : 2で混合してなる展開溶液(キャリア, 流速 9. Om l Z分) を用いて、 フラクション F 2を分離した。 これにより、 フラクション F 2から保持 時間 0分〜 1 5分の分画 F 1— 1 1 93 mgを分取した。  Among these fractions, fraction F2 was fractionated using reverse phase HPLC, and compound (45) was isolated under the following conditions. In other words, SHIMAZU LU-6A was used as the measuring instrument, ODS stainless steel column (QINERTSILPREP-ODS; 25 cm × 2 cm) was used as the column, and methyl alcohol and methyl alcohol were used under the conditions of detection UV wavelength (λ) = 254 nm. Fraction F2 was separated using a developing solution (carrier, flow rate of 9. OmIZ) consisting of a mixture of 05M ammonium acetate buffer (PH4) and acetonitrile at a volume ratio of 1: 1: 2. As a result, a fraction F1-119 mg of fraction F0-1-15 minutes was collected from fraction F2.
続いて、 酢酸ェチルと n—へキサンとを体積比 4 : 6で含む展開溶媒 を使用して、 上記分画 F 1一 1をシリカゲルカラムクロマトグラフィー で分離し、得られた高極性部 1 54 mgを逆相 H PLCで粗分けした後、 再度分取を行い、 化合物 (45) 37. 7 mgを取り出した。 該化合物 (45) の含有量は、 新鮮針葉部の重量を基準として 0. 0022 %で あった。  Subsequently, the above fraction F111 was separated by silica gel column chromatography using a developing solvent containing ethyl acetate and n-hexane at a volume ratio of 4: 6, and the resulting highly polar portion was obtained. mg was roughly separated by reversed-phase HPLC, and then fractionated again to obtain 37.7 mg of compound (45). The content of the compound (45) was 0.0022% based on the weight of the fresh needle portion.
上記化合物 (45) の iH— NMRおよび i3C— NMRの測定結果を 第 54表 ·第 55表にまとめて示す。 これにより、 化合物 (45) の構 造を解析した。 また、 該化合物 (45) の物性値等を測定した結果、 融 点 : 6 1 〜 65 The measurement results of iH-NMR and i3C-NMR of the compound (45) are summarized in Tables 54 and 55. Thus, the structure of the compound (45) was analyzed. In addition, as a result of measuring physical properties and the like of the compound (45), Melting point: 6 1 to 65
〔ひ〕 D 20 : +46. 6° (c = 1. 1 7, CHC 13 )[Shed] D 20:. +46 6 ° ( c = 1. 1 7, CHC 1 3)
I R : 36 1 6, 1 740 c mi (CHC 13 ) であった。 IR: 36 1 6, was 1 740 c mi (CHC 1 3 ).
NMR spectral data of compound (45) in CDCl NMR spectral data of compound (45) in CDCl
位置 C connected 1 H H - H COSY HMB C Location C connected 1 HH-H COSY HMB C
51.28 (d) 2.09 (m) H2, 3. 14a H3, Uab, Me 16, 17  51.28 (d) 2.09 (m) H2, 3.14a H3, Uab, Me 16, 17
69.98 (d) 4.17 (dd, 7.0, 2.0) HI, 3 HI, 3, 14ab  69.98 (d) 4.17 (dd, 7.0, 2.0) HI, 3 HI, 3, 14ab
45.98 (d) 2.96 (dr d, 7.0) H2, 20ab HI. 2, 5, 7ab, 20ab  45.98 (d) 2.96 (dr d, 7.0) H2, 20ab HI. 2, 5, 7ab, 20ab
143.30 (s) H3, 5, 20ab  143.30 (s) H3, 5, 20ab
77.80 (d) 5.22 (br t, 2.5) H6ab H3, 7a. 20ab  77.80 (d) 5.22 (br t, 2.5) H6ab H3, 7a. 20ab
28.61 (t) a) 1.45(ddd, 15.0, 4.0,4.0) H6b. 7a H5, 7ab  28.61 (t) a) 1.45 (ddd, 15.0, 4.0,4.0) H6b.7a H5, 7ab
b) 1.08(br d, 15.0) H6a. 7b  b) 1.08 (br d, 15.0) H6a.7b
27.32 (t) a) 1.57(br d, 15.0) H6a. 7b H3. 5, 6a. 9, Mel9  27.32 (t) a) 1.57 (br d, 15.0) H6a. 7b H3. 5, 6a. 9, Mel9
b) 1.38(br d, 15.4) H6b. 7a  b) 1.38 (br d, 15.4) H6b.7a
8 44.25 (s) H2.3.7ab.9, 10, Mel9  8 44.25 (s) H2.3.7ab.9, 10, Mel9
9 76.67 (d) 5.82 (d, 10.5) H10 H10. Mel9  9 76.67 (d) 5.82 (d, 10.5) H10 H10. Mel9
10 72.31 (d) 5.97 (d, 10.5) H9 H9  10 72.31 (d) 5.97 (d, 10.5) H9 H9
--3 11 132.87 (s) H9, 10.13,Mel6, 17, 18 O 12 136.67 (s) H10, 13, 14a, el8  --3 11 132.87 (s) H9, 10.13, Mel6, 17, 18 O 12 136.67 (s) H10, 13, 14a, el8
13 70.33 (d) 5.91 (ddd,9.0, 8.0, 1.2) H14ab, Mel8 HI, Uab, Mel8  13 70.33 (d) 5.91 (ddd, 9.0, 8.0, 1.2) H14ab, Mel8 HI, Uab, Mel8
14 27.98 (t) a) 2.56(ddd, 15.0, 9.5, 9.0) HI, 13, 14b HI, 2, 13  14 27.98 (t) a) 2.56 (ddd, 15.0, 9.5, 9.0) HI, 13, 14b HI, 2, 13
b) 1.31(ddd, 15.0, 8.0, 1.0) HI, 13, 14a  b) 1.31 (ddd, 15.0, 8.0, 1.0) HI, 13, 14a
15 37.23 (s) HI, 10, 14b, Me 16, 17  15 37.23 (s) HI, 10, 14b, Me 16, 17
16 27.04 (q) 1.15 (s) Mel7 Mel7  16 27.04 (q) 1.15 (s) Mel7 Mel7
17 31.57 (q) 1.67 (s) Mel6 HI, el6  17 31.57 (q) 1.67 (s) Mel6 HI, el6
18 15.25 (q) 2.12 (br d, 1.2) H13  18 15.25 (q) 2.12 (br d, 1.2) H13
19 17.91 (q) 0.84 (s) H3, 7b, 9  19 17.91 (q) 0.84 (s) H3, 7b, 9
20 119.49 (t) a) 5.43 (br t, 1.0) H3, 20b H3, 5  20 119.49 (t) a) 5.43 (br t, 1.0) H3, 20b H3, 5
b) 5.39 (br s) H3, 20a b) 5.39 (br s) H3, 20a
〔第 5 5表〕 (Table 55)
o. o.
1  1
o  o
6  6
Figure imgf000172_0001
Figure imgf000172_0001
<o <o
• « ■  • «■
o o  o o
* *  * *
s e e »a m *  s e e »a m *
1  1
♦ .  ♦.
o o
Figure imgf000172_0002
oo
Figure imgf000172_0002
N1 «0 * cJ O <£t LA <\3 M —  N1 «0 * cJ O <£ t LA <\ 3 M —
OO OO CvJ oo  OO OO CvJ oo
OO OO OO M  OO OO OO M
<o OJ CsJ CJ co »a
Figure imgf000172_0003
上記の化合物 (4 5) の NMRデ一夕や物性値 (融点、 並びに D 0) は、 文献 (J.KobayashiChem.Pharm.Bull.45,1205:1997) の記載と ほぼ一致し、 タキシン NA— 2 (タクスピン Z :taxuspineZ)であること が判明した。 該化合物 (45) の多剤耐性癌克服作用の測定については 以下に示す。
<o OJ CsJ CJ co »a
Figure imgf000172_0003
The NMR data and physical properties (melting point, and D 0) of the above compound (45) were almost identical to those described in the literature (J. Kobayashi Chem. Pharm. Bull. 45, 1205: 1997). 2 (Taxpin Z: taxuspineZ) There was found. The measurement of the compound (45) 's action to overcome multidrug-resistant cancer is described below.
(化合物 (40) 〜 (47) の多剤耐性癌克服作用の測定) (Measurement of compounds (40) to (47) for overcoming multidrug-resistant cancer)
化合物 (40) 〜 (47) の多剤耐性癌克服作用 (ここでは、 P—糖 蛋白質阻害剤としての機能) を、 上記説明の方法にて測定した。 多剤耐 性癌克服作用の測定結果は以下の第 56表〜第 6 1表にまとめて示す。 第 56表〜第 6 1表において、 「投与濃度」 とは、 化合物 (40) 〜 (4 7)、 または、 ベラパミルの反応溶液における濃度を示し、 「VCR蓄積 量の平均値」 とは、 各ゥエルにおける、 2780 AD細胞中のビンクリ スチン (VCR) 蓄積量の平均値を示す。 また、 「ベラパミル比」 とは、 ベラパミルを対照薬剤とした比較実験との比較結果を示し、 「評価」の項 における (最大べラパミル比)、 及び (濃度) とはそれぞれ、 ベラパミル と比較して最も効果が高かったときの比および濃度を示すものとする。 尚、 多剤耐性癌克服作用がベラパミルより明らかに高い 7種の化合物に ついては、 再測定を行った (第 59表〜第 6 1表参照)。 The multidrug-resistant cancer overcoming action (here, the function as a P-glycoprotein inhibitor) of the compounds (40) to (47) was measured by the method described above. The results of measuring the effect of overcoming multidrug-resistant cancer are summarized in Tables 56 to 61 below. In Tables 56 to 61, “dose concentration” indicates the concentration of the compound (40) to (47) or verapamil in the reaction solution, and “average VCR accumulation amount” indicates The average value of the amount of vincristine (VCR) accumulated in 2780 AD cells in the column is shown. In addition, “verapamil ratio” indicates the results of comparison with a comparison experiment using verapamil as a control drug, and (maximum verapamil ratio) and (concentration) in the “Evaluation” section are compared with verapamil, respectively. The ratio and concentration at which the effect was the highest are shown. The re-measurement was conducted for seven compounds that clearly had a higher effect of overcoming multidrug-resistant cancer than verapamil (see Tables 59 to 61).
投与濃度 V C R蓄 量 3 ン 卜 ペラパミル比 評 価 Dosing concentration VCR storage 3 ton Perapamil ratio Evaluation
化合物 の平均値 ロール比 判 定 (最大ペラパミ ル比)  Average value of compounds Roll ratio judgment (maximum perapamil ratio)
( ju g / m (dpm/irel t) (%) (%) (濃 度)  (ju g / m (dpm / irel t) (%) (%) (concentration)
0. 1 3 9 8 1 3 】 + 1 1 5 再測定  0.13 9 8 1 3] + 1 1 5 Re-measurement
化合物 Compound
1 7 5 5 2 4 9 + 1 4 6 1 6 %  1 7 5 5 2 4 9 + 1 4 6 16%
(40)  (40)
1 0 1 2 9 3 4 2 7 + + 1 2 2 1 s / τα \  1 0 1 2 9 3 4 2 7 + + 1 2 2 1 s / τα \
0 3 0 3 CO ベラノく 0. 1 3 4 5 1 I 4 +  0 3 0 3 CO Veranoku 0.1 3 4 5 1 I 4 +
ミル 1 5 1 7 I 7 1 +  Mill 1 5 1 7 I 7 1 +
1 0 1 0 5 9 3 5 0 十 + 1 0 1 0 5 9 3 5 0 10 +
^ 56 投与 S度 V C R蓄積量 つン ト ベラパミル比 評 価 ^ 56 Administration S-degree VCR accumulation amount Verapamil ratio Evaluation
化合物 の平均値 口一ル比 判 定 (最大ペラパミル比)  Average value of compounds Judgment ratio (maximum perapamil ratio)
n g /ml ) (dpm/wel 1) (%) (%) (漉 度)  n g / ml) (dpm / wel 1) (%) (%) (filtration)
0. 1 2 2 3 7 8 再測定 化合物  0.1 2 2 3 7 8 Re-measurement compound
4 7 7 1 6 7 + 1 2 1 1 2 1 %  4 7 7 1 6 7 + 1 2 1 1 2 1%
(41)  (41)
1 0 1 0 9 6 3 8 3 +十 1 1 4 1 μ g / I  1 0 1 0 9 6 3 8 3 +10 1 1 4 1 μg / I
0 . 1 2 6 1 9 1 N 化合物  0.12 6 1 9 1 N compound
3 8 7 1 3 5 + %  3 8 7 1 3 5 +%
(42)  (42)
1 0 6 3 9 2 2 3 + u g / \  1 0 6 3 9 2 2 3 + u g / \
0. 1 2 5 9 9 1 8 9 再測定 化合物  0.1 2 5 9 9 1 8 9 Re-measurement Compound
4 6 1 1 6 1 + 1 1 7 1 1 7 %  4 6 1 1 6 1 + 1 1 7 1 1 7%
(43)  (43)
1 0 1 0 4 4 3 6 5 1 0 9 1 /i g /m 1  1 0 1 0 4 4 3 6 5 1 0 9 1 / i g / m 1
0 . 1 3 2 3 1 1 3 + 1 1 1 再測定 化合物  0.13 2 3 1 1 3 + 1 1 1
4 7 7 1 6 7 + 1 2 1 1 2 1 %  4 7 7 1 6 7 + 1 2 1 1 2 1%
(44)  (44)
1 0 6 9 8 2 4 4 + 1 w g /m 1  1 0 6 9 8 2 4 4 + 1 wg / m 1
2 8 6  2 8 6
ベ 'パ 2 9 1 1 0 2 Vapor 2 9 1 1 0 2
ミル 3 9 5 1 3 8  Mill 3 9 5 1 3 8
1 0 9 5 9 3 3 5 + +  1 0 9 5 9 3 3 5 + +
铖 w 57 投与濃度 V C R蓄 量 ン 卜 ペラパミル比 評 価 铖 w 57 Dosing concentration VCR accumulation int Perapamil ratio Evaluation
化合物 の平均値 ロール比 判 定 (最大べラバミル比)  Average value of compounds Roll ratio judgment (maximum verabamil ratio)
( U g /ml ) (dpm/wel 1) (%) (%) (濃 度) (U g / ml) (dpm / wel 1) (%) (%) (concentration)
0. 1 2 5 5 8 9 ― 8 7 再測定 化合物 0.1 2 5 5 8 9 ― 8 7 Re-measurement Compound
1 3 4 1 1 1 9 + 8 6 1 0 6 %  1 3 4 1 1 1 9 + 8 6 1 0 6%
(45)  (45)
1 0 1 0 1 9 3 5 6 + + 1 0 6 1 0 u g /m 1  1 0 1 0 1 9 3 5 6 + + 1 0 6 1 0 u g / m 1
0. 1 2 5 3 8 8 一 8 6 再測定 化合物  0.1 2 5 3 8 8 1 8 6 Re-measurement Compound
1 6 0 1 2 1 0 + 1 5 2 1 5 2 %  1 6 0 1 2 1 0 + 1 5 2 1 5 2%
(46)  (46)
1 0 1 1 4 1 3 9 9 + + 1 1 9 1 μ g / m 1  1 0 1 1 4 1 3 9 9 + + 1 1 9 1 μg / m 1
0. 1 2 5 2 8 8 ― 8 6 再測定 化合物  0.1 2 5 2 8 8 ― 8 6 Re-measurement Compound
1 4 8 0 1 6 8 十 】 2 2 1 2 2 %  1 4 8 0 1 6 8 10] 2 2 1 2 2%
(47)  (47)
1 0 1 0 8 8 3 8 0 + + 1 1 3 1 ^ g / m 1  1 0 1 0 8 8 3 8 0 + + 1 1 3 1 ^ g / m 1
0 2 8 6  0 2 8 6
ベラパ 0. 1 2 9 1 】 0 2 + Verapa 0.1 2 9 1) 0 2 +
ミル 1 3 9 5 1 3 8 +  Mill 1 3 9 5 1 3 8 +
1 0 9 5 9 3 3 5 + +  1 0 9 5 9 3 3 5 + +
皲^ 58 再 測 定 皲 ^ 58 Re-measurement
投与濃度 V C R蓄撢量 3ン 卜 ベラパミル比 評 価  Dosing concentration VCR stored amount 3 nt Verapamil ratio Evaluation
化合物 の平 ϋ値 ロール比 判 定 (最大ペラパミ ル比)  Average value of compound Roll ratio judgment (maximum perapamil ratio)
( li g /ml ) (dpm/vel 1) (%) (%) (濃 度)  (li g / ml) (dpm / vel 1) (%) (%) (concentration)
0. 1 3 6 1 1 0 6 + 1 0 2 P  0.13 6 1 1 0 6 + 1 0 2 P
化合物 Compound
1 7 7 4 2 2 8 十 1 5 7 1 5 7 %  1 7 7 4 2 2 8 10 1 5 7 1 5 7%
(40)  (40)
1 0 1 5 6 2 A 6 1 十十 1 3 1 1 μ g /m 1  1 0 1 5 6 2 A 6 1 Tens 1 3 1 1 μg / m 1
0 3 3 9  0 3 3 9
-3 ぺラバ 05  -3 Peraba 05
0. 1 3 5 3 1 0 4  0.1 3 5 3 1 0 4
ミ ル 1 4 9 0 1 4 5 +  Mills 1 4 9 0 1 4 5 +
1 0 】 1 9 3 3 5 2 + +  1 0] 1 9 3 3 5 2 + +
狨^ 9 再 測 定 狨 ^ 9 Re-measurement
投与港度 V C R蓄積置 コント ベラパミル比 評 価 化合物 の平均値 ロール比 判 定 (鼓大ぺフパ 5·ル比〉  Port of administration VCR accumulation storage controller Verapamil ratio Evaluation Average value of compound Roll ratio Judgment
( g /ml ) (dpm/wel 1) (%) (%) (S 度) (g / ml) (dpm / wel 1) (%) (%) (S degree)
0. 1 3 3 6 1 2 9 + 1 3 1 P 0.1 3 3 6 1 2 9 + 1 3 1 P
化合物 Compound
1 6 1 6 2 3 7 + 1 5 8 1 5 8 % (41)  1 6 1 6 2 3 7 + 1 5 8 1 5 8% (41)
1 0 1 3 8 0 5 3 1 十 + + 1 5 7 1 ju g / m 1  1 0 1 3 8 0 5 3 1 ten + + 1 5 7 1 ju g / m 1
0. 1 2 9 3 1 1 3 + 1 1 8 P  0.12 9 3 1 1 3 + 1 18 P
化合物 Compound
1 5 8 7 2 2 6 + 1 5 1 1 5 1 % (43)  1 5 8 7 2 2 6 + 1 5 1 1 5 1% (43)
1 0 1 2 4 4 4 7 8 + + 1 1 1 ju g / m 1  1 0 1 2 4 4 4 7 8 + + 1 1 1 ju g / m 1
0 2 6 0  0 2 6 0
ペラノ < 0. I 2 4 9 9 6 士 Perano <0.I 2 4 9 9 6
ミル 1 3 9 1 1 5.0 +  Mill 1 3 9 1 1 5.0 +
1 0 8 8 0 3 3 8 + +  1 0 8 8 0 3 3 8 + +
w 60 〔第 61表〕 w 60 (Table 61)
Figure imgf000179_0001
Figure imgf000179_0001
第 56表〜第 61表より明らかなように、 上記化合物 (40) 〜 (4 7) はいずれも多剤耐性癌克服作用を有し、 それ単独で新規な多剤耐性 癌克服剤となりうることが明らかになった。 特に、 第 59表〜第 61表 に示すように、 化合物 (40), (4 1), (43), (44), (45), (4 6), (47) は、 ベラパミルを上回る多剤耐性癌克服作用を有する高活 性化合物であることが明らかとなり、 それ単独で特に有効な多剤耐性癌 克服剤となりうることが明らかになった。 As is clear from Tables 56 to 61, all of the above compounds (40) to (47) have a multidrug-resistant cancer overcoming action, and can be used alone as a novel multidrug-resistant cancer-overcoming agent. Was revealed. In particular, Table 59 to Table 61 As shown in the figure, compounds (40), (41), (43), (44), (45), (46) and (47) are highly active It has been revealed that the compound is a sex compound, and that it can be a particularly effective multidrug-resistant cancer-surviving agent by itself.
〔生物テスト〕 [Biological test]
本発明化合物の抗癌作用を測定した。  The anticancer effect of the compound of the present invention was measured.
本発明化合物の代表として化合物(2 6) と化合物(3 6) を選んだ。 ヒト培養癌細胞パネルによる抗癌剤スクリーニングは, 米国国立研究 所 (NC I ) の方法に順じてインビトロテストを行った。 すなわち, ヒ ト培養癌細胞パネルは、肺癌 7系、 胃癌 6系、 大腸癌 5系、 卵巣癌 5系、 脳腫瘍 6系、 乳癌 5系、 腎癌 2系、 前立腺癌 2系およびメラノーマ系 1 系の計 3 9系よりなる。 これらを 1つのパネルとして扱い、 in vitro 薬 剤感受性試験を行った。 癌細胞を 96ゥエルプレートにまき込み、 翌日 検体溶液( 5 doses, 10·4から 10·8Μまで 1 log 間隔、最高濃度を High Cone と呼ぶ) を添加、 2日間培養後、 細胞増殖をスルホローダミン B による比色定量で測定した。 Compound (26) and compound (36) were selected as representatives of the compounds of the present invention. In vitro screening of anticancer drugs using a panel of cultured human cancer cells was performed according to the method of the US National Laboratory (NCI). That is, the human cultured cancer cell panel consists of 7 lung cancers, 6 stomach cancers, 5 colon cancers, 5 ovarian cancers, 6 brain tumors, 5 breast cancers, 5 renal cancers, 2 prostate cancers, and 1 melanoma line. It consists of a total of 39 systems. These were treated as one panel, and an in vitro drug susceptibility test was performed. Entrainment cancer cells in 96 © El plate, the next day the sample solution added (5 doses, 1 log interval from 10.4 to 10 · 8 Micromax, maximum density is referred to as High Cone), after 2 days of culture, cell proliferation It was measured by colorimetry with sulforhodamine B.
その結果を下記するが、 データ解析のパラメータとして、 薬をかける 直前 (Time Zero) の細胞数を基準として  The results are shown below. The data analysis parameters are based on the number of cells immediately before the drug application (Time Zero).
G 1 5 0 : Controlに比べて増殖を 50 %抑制する濃度  G150: concentration that inhibits proliferation by 50% compared to Control
TG I : Time Zero と同じ細胞数に増殖を抑制する濃度 (見かけ上 細胞数の増減がない濃度)  TG I: Concentration that suppresses proliferation to the same number of cells as Time Zero (concentration that apparently does not increase or decrease the number of cells)
L C 5 0 : Time Zeroの 50 %に細胞数を減少させる濃度  LC 50: concentration that reduces cell number to 50% of Time Zero
の 3種類を求めた。
Figure imgf000181_0001
I asked for three types.
Figure imgf000181_0001
081 081
9C0S0/00df/I3d 0 0L0/T0 OAV 180- ] 9C0S0 / 00df / I3d 0 0L0 / T0 OAV 180-]
〔第 6 2表〕 続き [Table 62] continued
Figure imgf000182_0001
Figure imgf000182_0001
Figure imgf000183_0001
Figure imgf000183_0001
181 ] 181]
〔第 63表〕 続き [Table 63] continued
Figure imgf000184_0001
Figure imgf000184_0001
上記において、 G 1 50 Tg Iおよび LC 50はそれぞれモル濃 182 度を表し、 E— 0 4、 E— 0 5および E— 0 6はそれぞれ 1 0 _ 4、 1 0 _ 5および 1 0—6を表す。 産業上の利用可能性 In the above, G 150 Tg I and LC 50 are molar concentrations, respectively. Represents 182 °, it represents the E- 0 4, E- 0 5 and E- 0 6 each 1 0 _ 4, 1 0 _ 5 and 1 0 6. Industrial applicability
本発明の多剤耐性癌克服剤は、 以上のように、 上記化学式 (1 ) 〜 (4 7 ) で表される化合物から選ばれる少なくとも一種の化合物を含む構成 である。  As described above, the multidrug-resistant cancer-overcoming agent of the present invention is configured to include at least one compound selected from the compounds represented by the above chemical formulas (1) to (47).
上記化合物は何れも、 複数の抗癌剤に対する耐性を獲得した癌細胞、 特に多剤耐性癌細胞に対する多剤耐性癌克服作用を有しており、 該癌細 胞に好適に投与することができる多剤耐性癌克服剤を提供することがで きるという効果を奏する。  All of the above compounds have a multidrug-resistant cancer overcoming effect on cancer cells that have acquired resistance to a plurality of anticancer drugs, particularly multidrug-resistant cancer cells, and can be suitably administered to the cancer cells. This has the effect of providing a drug for overcoming resistant cancer.
上記化学式 (1 ) 〜 (4 7 ) の化合物または当該化合物と他の抗癌作 用物質 (抗癌剤) とを組み合わせて使用して優れた癌治療効果を奏する ことができる。  An excellent cancer therapeutic effect can be obtained by using the compounds of the above chemical formulas (1) to (47) or a combination of the compound and another anticancer substance (anticancer agent).
本発明の多剤耐性癌克服剤の製造方法は、 化学式 (6 ) 〜 (4 7 ) で 示される化合物を日本ィチイの組織または日本ィチイの組織より誘導さ れるカルスから取り出す工程を含む方法である。  The method for producing the multidrug-resistant cancer-overcoming agent of the present invention is a method comprising the step of removing the compound represented by any one of the chemical formulas (6) to (47) from the tissue of Nippon yew or callus derived from the tissue of Nippon yew. .
上記の製造方法によれば、 環境を保全しながら、 該有用化合物を効率 的に製造することができる。 これにより、 上記多剤耐性癌克服剤を、 環 境を保全しながら効率的に製造することができるという効果を奏する。  According to the above production method, the useful compound can be produced efficiently while preserving the environment. As a result, the above-mentioned agent for overcoming a multidrug-resistant cancer can be efficiently produced while preserving the environment.

Claims

183 請 求 の 範 囲 式 ( 1)  183 Scope of Claim (1)
Figure imgf000186_0001
Figure imgf000186_0001
(式中、  (Where
R 1は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基を、 R 1 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified,
R 2は水素原子を、 または R1と R 2とが一緒になつて =0を、 R 2 represents a hydrogen atom, or R 1 and R 2 together form = 0,
R 3は水素原子を、 R 3 represents a hydrogen atom,
R4は水素原子もしくは水酸基を、 または R3と R4とが一緒になつて、 単結合または— O—を形成してもよく、 または R3と R6が一緒になつて — O— CH2—を形成してもよく、 R 4 may be a hydrogen atom or a hydroxyl group, or R 3 and R 4 may together form a single bond or —O—, or R 3 and R 6 together — O— CH 2 — may be formed,
R 5は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基を、 R 5 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified,
R6はメチル基、 ヒドロキシメチル基、 またはエステル化もしくはエーテ ル化されていてもよい水酸基、 R 6 is a methyl group, a hydroxymethyl group, or a hydroxyl group which may be esterified or etherified,
R 7はエステル化またはエーテル化されていてもよい水酸基、 R 7 is a hydroxyl group which may be esterified or etherified,
R 8は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基、 R 8 is a hydrogen atom or a hydroxyl group which may be esterified or etherified,
R9は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基、 R 9 is a hydrogen atom or a hydroxyl group which may be esterified or etherified,
R1 Qは水素原子または R 4と一緒になつて単結合を表してもよい、 184 R 1 Q may be taken together with a hydrogen atom or R 4 to represent a single bond, 184
R11はメチル基またはエステル化もしくはエーテル化されていてもよい 水酸基を有するメチル基、 R 11 is a methyl group or a methyl group having a hydroxyl group which may be esterified or etherified,
R 12は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基を、 R 12 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified,
R 13は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基を、 R 13 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified,
R 14は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基を示す) で表される化合物、 R 14 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified)
式 (2)Equation (2)
Figure imgf000187_0001
Figure imgf000187_0001
(式中、 R15はエステル化もしくはエーテル化されていてもよい水酸基 を、 (Wherein, R 15 represents a hydroxyl group which may be esterified or etherified,
R 16は水素原子を、 R 16 represents a hydrogen atom,
R 17はメチル基を、 または R16と R17とが一緒になつて—O— CH2— を形成してもよく、 R 17 may be a methyl group, or R 16 and R 17 may be taken together to form —O—CH 2 —,
R 18は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基を、 R 18 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified,
R 19はエステル化もしくはエーテル化されていてもよい水酸基を、 は二重結合または単結合を示す)で表される化合物、 R 19 represents a hydroxyl group which may be esterified or etherified, and represents a double bond or a single bond),
式 (3) 185Equation (3) 185
Figure imgf000188_0001
Figure imgf000188_0001
(式中、 R 2 Qはエステル化またはエーテル化されていてもよい水酸基を、 R21は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基を、 R22は水素原子を、 または R21と R22とが一緒になつて =0 を形成してもよく、 (Wherein, R 2 Q represents a hydroxyl group which may be esterified or etherified, R 21 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified, R 22 represents a hydrogen atom, or R 21 And R 22 may together form = 0,
R 23は水素原子またはエステル化もしくはエーテル化されていてもよい 水酸基を、 R24は水素原子を、 または R23と R24とが一緒になつて =0 を形成してもよく、 R 25は水素原子またはエステル化もしくはエーテル 化されていてもよい水酸基を、 R26は水素原子を示す。 もっとも R25と R26とが一緒になつて ==〇を形成してもよい) で表される化合物、 式 (4) R 23 is a hydrogen atom or a hydroxyl group which may be esterified or etherified, R 24 may be a hydrogen atom, or R 23 and R 24 may together form = 0, and R 25 is R 26 represents a hydrogen atom or a hydroxyl group which may be esterified or etherified, and R 26 represents a hydrogen atom. R 25 and R 26 may be joined together to form == 〇), a compound represented by the formula (4)
Figure imgf000188_0002
Figure imgf000188_0002
(式中、 R27、 R28、 R29、 R3Q、 R31および R32はそれぞれ同一ま たは異なって水素原子、 またはエステル化もしくはエーテル化されてい てもよい水酸基を示す。 R33、 R34は水素原子を示すか R32と R33ま たは R28と R 34とが一緒になつて =0を形成してもよい) で表される化 合物、 または 186 式 (5) (Wherein, R 27, R 28, R 29, R 3Q, R 31 and R 32 are each independently hydrogen were identical or respectively or be esterified or etherified represents an hydroxy group. R 33,, R 34 represents a hydrogen atom, or R 32 and R 33 or R 28 and R 34 can be taken together to form = 0), or 186 Equation (5)
Figure imgf000189_0001
Figure imgf000189_0001
(式中、 R35、 R36、 R37、 R38および R39はそれぞれ同一または異 なって、 エステル化またはエーテル化されていてもよい水酸基を示す) で表される化合物またはその塩を含有することを特徴とする医薬。 (Wherein, R 35 , R 36 , R 37 , R 38 and R 39 are the same or different and each represents a hydroxyl group which may be esterified or etherified) or a salt thereof. A medicament characterized in that:
2. 化合物が式 2. The compound has the formula
Figure imgf000189_0002
Figure imgf000189_0002
Figure imgf000189_0003
Figure imgf000189_0004
οτ
Figure imgf000189_0003
Figure imgf000189_0004
οτ
Figure imgf000190_0001
Figure imgf000190_0001
Figure imgf000190_0002
Figure imgf000190_0003
Figure imgf000190_0002
Figure imgf000190_0003
Figure imgf000190_0004
Figure imgf000190_0005
Figure imgf000190_0004
Figure imgf000190_0005
LSI  LSI
9C0S0/00df/X3d OtO O/IO OAV o 9C0S0 / 00df / X3d OtO O / IO OAV o
Figure imgf000191_0001
Figure imgf000191_0001
01
Figure imgf000192_0001
01
Figure imgf000192_0001
Figure imgf000192_0002
Figure imgf000192_0002
Figure imgf000192_0003
Figure imgf000192_0004
Figure imgf000192_0005
Figure imgf000192_0003
Figure imgf000192_0004
Figure imgf000192_0005
68T  68T
9£0S0/00df/X3d 01 9 £ 0S0 / 00df / X3d 01
(8 4c ( 8 4c
Figure imgf000193_0001
Figure imgf000193_0002
Figure imgf000193_0001
Figure imgf000193_0002
Figure imgf000193_0003
Figure imgf000193_0004
Figure imgf000193_0005
Figure imgf000193_0003
Figure imgf000193_0004
Figure imgf000193_0005
061  061
9C0S0/00df/X3d OtO O/IO OAV
Figure imgf000194_0001
Figure imgf000194_0002
Figure imgf000194_0003
9C0S0 / 00df / X3d OtO O / IO OAV
Figure imgf000194_0001
Figure imgf000194_0002
Figure imgf000194_0003
Figure imgf000194_0004
Figure imgf000194_0004
Τ6ΐ  Τ6ΐ
9C0S0/00df/X3d 0170.0/Ϊ0 OAV CO 9C0S0 / 00df / X3d 0170.0 / Ϊ0 OAV CO
CO CO CO  CO CO CO
Figure imgf000195_0001
Figure imgf000195_0001
193 193
Figure imgf000196_0001
Figure imgf000196_0002
Figure imgf000196_0001
Figure imgf000196_0002
Figure imgf000196_0003
Figure imgf000196_0003
AcO OAc  AcO OAc
(40) (40)
'OAc 194
Figure imgf000197_0001
'OAc 194
Figure imgf000197_0001
Figure imgf000197_0002
Figure imgf000197_0003
Figure imgf000197_0002
Figure imgf000197_0003
Figure imgf000197_0004
Figure imgf000197_0005
195
Figure imgf000198_0001
または
Figure imgf000197_0004
Figure imgf000197_0005
195
Figure imgf000198_0001
Or
Figure imgf000198_0002
Figure imgf000198_0002
(式中、 Acはァセチル基を、 B zはベンゾィル基を、 Meはメチル基 を、 Phはフエ二ル基を示す) で表される化合物を含有することを特徴 とする請求の範囲第 1項記載の医薬。 (Wherein Ac represents an acetyl group, Bz represents a benzoyl group, Me represents a methyl group, and Ph represents a phenyl group). The medicament according to the item.
3. 癌克服剤である請求の範囲第 1項または第 2項に記載の医薬。 3. The medicament according to claim 1 or 2, which is an agent for overcoming cancer.
4. 多剤耐性癌克服剤である請求の範囲第 1項または第 2項に記載の 4. The drug according to claim 1 or 2 which is a drug for overcoming multidrug resistance cancer.
5. 抗癌作用物質蓄積増強剤である請求の範囲第 1項〜第 4項のいず れかに記載の医薬。 5. The medicament according to any one of claims 1 to 4, which is an anticancer substance accumulation enhancer.
6. さらに抗癌作用物質を含有する請求の範囲第 1項〜第 5項のいず れかに記載の医薬。  6. The medicament according to any one of claims 1 to 5, further comprising an anticancer substance.
7. 請求の範囲第 1項または第 2項に記載の化合物と抗癌作用物質と を組み合わせて癌患者に投与することを特徴とする癌の治療方法。  7. A method for treating cancer, which comprises administering a compound according to claim 1 or 2 in combination with an anticancer substance to a cancer patient.
8. 式 01
Figure imgf000199_0001
Figure imgf000199_0002
Figure imgf000199_0003
8. Expression 01
Figure imgf000199_0001
Figure imgf000199_0002
Figure imgf000199_0003
Figure imgf000199_0004
Figure imgf000199_0004
Figure imgf000199_0005
Figure imgf000199_0005
96T 96T
9C0S0/00df/X3d OtO O/IO OAV
Figure imgf000200_0001
9C0S0 / 00df / X3d OtO O / IO OAV
Figure imgf000200_0001
198 198
Figure imgf000201_0001
Figure imgf000201_0001
Figure imgf000201_0002
Figure imgf000201_0002
または Or
Figure imgf000201_0003
Figure imgf000201_0003
(式中、 Acはァセチル基を、 Meはメチル基を、 P hはフエ二ル基を 示す) で表される化合物。  (Wherein Ac represents an acetyl group, Me represents a methyl group, and Ph represents a phenyl group).
9. 式 9. Expression
Figure imgf000201_0004
Figure imgf000201_0004
または 199 Or 199
Figure imgf000202_0001
Figure imgf000202_0001
(式中、 Acはァセチル基を、 P hはフエ二ル基を示す) で表される化 合物を含有することを特徴とする制癌剤。  (Wherein Ac represents an acetyl group and Ph represents a phenyl group), a carcinostatic agent comprising:
1 0. 日本イチィ iTaxus cusp i data Sieb. et Zucc. ) の組織または その組織より誘導されるカルスから請求の範囲第 2項に記載の化合物を 採取することを特徴とする請求の範囲第 2項に記載された化合物の製造 方法。  10. The claim according to claim 2, wherein the compound according to claim 2 is collected from a tissue of Nipponbari iTaxus cuspi data Sieb. Et Zucc.) Or callus derived from the tissue. A method for producing the compound described in the above.
1 1. タキシン骨格、 タキシニン骨格またはアビエタン骨格を有し、 抗癌作用物質蓄積増強作用または抗癌作用物質排出抑制作用のある化合 物を含有する医薬。  1 1. A medicament containing a taxin skeleton, a taxinin skeleton or an abietane skeleton, and containing a compound having an anticancer substance accumulation enhancing action or an anticancer substance discharge inhibitory action.
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CN115097035B (en) * 2022-06-21 2024-06-04 山西振东制药股份有限公司 LLTS intermediate and detection method and application of related impurities thereof

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