CN104649901B - A kind of preparation method of the material medicine ingenol methyl butene acid esters for treating actinic keratosiss - Google Patents

A kind of preparation method of the material medicine ingenol methyl butene acid esters for treating actinic keratosiss Download PDF

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CN104649901B
CN104649901B CN201510066743.3A CN201510066743A CN104649901B CN 104649901 B CN104649901 B CN 104649901B CN 201510066743 A CN201510066743 A CN 201510066743A CN 104649901 B CN104649901 B CN 104649901B
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silica gel
obtains
methanol
petroleum ether
extractum
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CN104649901A (en
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邱明华
万落生
彭惺蓉
李忠荣
周琳
于慕湲
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Kunming Institute of Botany of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/48—Separation; Purification; Stabilisation; Use of additives
    • C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/48—Separation; Purification; Stabilisation; Use of additives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/52—Esters of acyclic unsaturated carboxylic acids having the esterified carboxyl group bound to an acyclic carbon atom
    • C07C69/533—Monocarboxylic acid esters having only one carbon-to-carbon double bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/56—Ring systems containing bridged rings
    • C07C2603/58—Ring systems containing bridged rings containing three rings
    • C07C2603/76—Ring systems containing bridged rings containing three rings containing at least one ring with more than six ring members
    • C07C2603/84—Ring systems containing bridged rings containing three rings containing at least one ring with more than six ring members containing rings with more than eight members

Abstract

A kind of preparation method of the material medicine ingenol methyl butene acid esters for treating actinic keratosiss, comprises the steps：Raw material extraction, petroleum ether extraction, macroporous resin D101 absorption dry method are mixed sample and draw section, silica gel separation, Sephadex gel-purifieds, DIOL silica gel purifications.The method separates material medicine ingenol methyl butene acid esters from southern Europe Radix Euphorbiae Pekinensis herb plant, possess isolate and purify it is quick and easy, it is efficiently high-purity, the advantages of cost is relatively low.

Description

A kind of material medicine ingenol methyl butene acid esters for treating actinic keratosiss Preparation method
Technical field：
The invention belongs to process for preparing medicine technical field, in particular it relates to one kind treats actinic keratosiss crude drug The preparation method of thing ingenol.
Background technology
The U.S. FDA of on January 23rd, 2012 have approved ingenol methyl butene acid esters (Ingenol Mebutate), as For treating actinic keratosiss (Actinic Keratosis, AK, to a kind of light activated dermatosiss of sun) medicine listing.Should Trade name Picato of medicine, is researched and developed successfully by LEO Pharma AS.
Ingenol methyl butene acid esters (Ingenol Mebutate), can be from euphorbia (Euphorbia) Extraction separation and purification, such as Euphorbia peplus, Euphorbia hirta, Euphorbia drummondi, and Chinese medicine The congeners such as Euphorbia lathyris (Semen Euphorbiae), Euphorbia kansui (Radix Kansui) all contain the material medicine. But the material medicine content is very low, and the parent nucleus framework compound Ingenol of its Ingenol Mebutate, in congener Distribution is very wide, is available for extraction separation and purification.Parent nucleus Ingenol is tetracyclic diterpene natural product, and wherein B rings, C rings are by ring strain Very big bicyclic [4,4,1] undecane is constituted, due to the inside-outside on its end of the bridge Stereochemistry, that is, constitute C-11 to the C-14 of C rings in end of the bridge heteropleural so as to which tension force increases.Synthesis chemist is special to this Different spatial chemistry phenomenon is especially interested, also so that the complete synthesis research of Ingenol becomes focus, Wood, Tanino/ The research teams such as Kuwajima, Winkler, Funk, Rigby are completed the complete synthesis research of Ingenol.Most easily Ingenol is complete synthesis only to need seven steps to react.So that material medicine ingenol methyl butene acid esters (Ingenol Mebutate) Can obtain from synthesis and semi-synthetic approach.
Southern Europe Radix Euphorbiae Pekinensis (Euphorbia peplus Linn), originates in Mediterranean (southern Europe is to north African), and naturalization is in Asia Continent, America and Australia；The Taiwan (in the Taibei, platform) of China, Guangdong, Hong Kong, Fujian (Xiamen, Foochow), Guangxi (Nanning) Naturalization plant or population are in succession found with Yunnan (Kunming), as euphorbiaceous one kind, southern Europe Radix Euphorbiae Pekinensis in Yunnan Province Widely distributed, the edge of a field, afforestation region are seen everywhere, because its milk has toxicity, it is impossible to used as herbage, resource pole Greatly, be ingenol methyl butene acid esters raw material resources basis.
So far, there are no in prior art and ingenol methyl butene acid esters industry medicine is extracted from the Radix Euphorbiae Pekinensis of southern Europe The report of the process of raw material.
The content of the invention
It is an object of the invention to provide a kind of from the adventitious plant southern Europe Radix Euphorbiae Pekinensis (Euphorbia for blazoning Yunnan Province Peplus the side of extraction separation and purification natural material medicine ingenol methyl butene acid esters (Ingenol Mebutate) in) Method.The process possess isolate and purify it is quick and easy, it is efficiently high-purity, the advantages of cost is relatively low.
In order to realize the above-mentioned purpose of the present invention, the invention provides following technical scheme：
The preparation method of ingenol methyl butene acid esters, comprises the steps：Raw material extraction, petroleum ether extraction, macropore Resin D101 absorption dry method is mixed sample and draws section, silica gel separation, Sephadex gel-purifieds, DIOL silica gel purifications；
Described raw material is extracted as：Southern Europe Radix Euphorbiae Pekinensis of collection various places or congener herb, the dry in the sun under natural sunlight are done It is dry, soaked with methanol after chopping, heating and refluxing extraction three times, be filtered to remove residue, after filtrate concentration, obtain total extractum；
Described petroleum ether extraction is：Total extractum water is suspended after dispersion, with volume ratio 1:1 petroleum ether extraction 3 times, point liquid are dense Contracting oil ether moiety, obtains the total extractum of petroleum ether；
It is, with the total extractum of petroleum ether, to be dissolved with appropriate chloroform that described macroporous resin D101 absorption dry method is mixed sample and draws section, with Macroporous resin D101 dry method mixes sample, then uses macroporous resin column crude separation, using methanol/water solution as mobile phase, by 40%, 60%th, 80%, 100% eluting, collects 80% methanol aqueous solution eluting eluting stream part, and concentrating under reduced pressure reclaims solution, obtains extractum For total diterpene position；
The silica gel is separated into：With silica gel mixed sample after the dissolving of appropriate chloroform, silicagel column crude separation is filled, with petroleum ether/acetic acid Ethyl ester carries out gradient elution as eluent, obtains 8 main portions Fr I-VIII, wherein Fr VII with C18 post separations, then Obtain 10 sub-portion position subFr1-10；
The Sephadex gel-purifieds are：To sub-portion position subFr8-9, then upper Sephadex gel column methanol-eluted fractions, Stream part concentration is collected, target compound monomer is obtained；
The DIOL silica gel purifications are：Monomeric compound is obtained, Jing DIOL silica gel is isolated and purified again, obtains purity 98% Above ingenol methyl butene acid esters.
The preparation method of ingenol methyl butene acid esters as mentioned, wherein described method committed step is macropore tree Fat D101 absorption is mixed sample stroke section and obtains total diterpene position；Sephadex gel filtration chromatography purification obtains compound monomer；DIOL silicon It is gel column chromatography eluting to obtain high-purity target material monomer.
The preparation method of ingenol methyl butene acid esters as mentioned, wherein described method is complete to take southern Europe Radix Euphorbiae Pekinensis Grass, is dried, and is soaked with methanol, heating and refluxing extraction three times after chopping, is filtered to remove residue, total extractum, leaching are obtained after filtrate concentration Cream is suspended with 20L water after dispersion, volume ratio 1:1 petroleum ether extraction 5 times, inspissated oil ether moiety obtain the total extractum of petroleum ether, with suitable Amount chloroform dissolving extractum, is mixed sample with macroporous resin D101 dry method, then uses macroporous resin column crude separation, made with methanol/water solution For mobile phase, by 40%, 60%, 80%, 100% eluting；Total diterpene position is obtained to 80% meoh eluate concentration and recovery, with suitable After amount chloroform dissolving, 200～300 mesh of silica gel mixes sample, fills silicagel column crude separation, with 20:1-0:1 petroleum ether:Ethyl acetate is entered Row gradient elution, obtains 8 main portions Fr I-VIII, and wherein Fr VII obtain 10 sub-portion position subFr1- with C18 post separations 10, subFr9 go up sephadex gel column methanol-eluted fractions again, obtain the target compound of purity more than 90%, again Jing DIOL Isolate and purify, obtain the ingenol methyl butene acid esters Ingenol Mebutate of purity more than 98%.
Specific embodiment
Below with embodiments of the invention further illustrating the essentiality content of the present invention, but not with this limiting this Invention.
Embodiment 1
The preparation of Ingenol Mebutate：
1. material benzenemethanol is extracted：Southern Europe Radix Euphorbiae Pekinensis (Euphorbia peplus Linn) of collection various places or congener are complete Grass, the dryness in the sun under natural sunlight are soaked with methanol after chopping, and heating and refluxing extraction three times is filtered to remove residue, and filtrate is dense Total extractum is obtained after contracting.
2. petroleum ether extraction：Total extractum water is suspended after dispersion, with volume ratio 1:1 petroleum ether extraction 3 times, point liquid inspissated oil Ether moiety, obtains the total extractum of petroleum ether.
3. macroporous resin D101 absorption dry method is mixed sample and draws section：The total extractum of petroleum ether is dissolved with appropriate chloroform, with macroporous resin D101 dry method mixes sample, then uses macroporous resin column crude separation, using methanol/water solution as mobile phase, by 40%, 60%, 80%, 100% eluting.80% methanol aqueous solution eluting eluting stream part is collected, concentrating under reduced pressure reclaims solution, extractum is obtained for total diterpene portion Position.
4. silica gel is separated：With silica gel mixed sample after the dissolving of appropriate chloroform, silicagel column crude separation is filled, petrol ether/ethyl acetate is used Gradient elution is carried out as eluent, 8 main portions (Fr I-VIII) are obtained, wherein Fr VII are with C18 (Fuji Silysia Chemical companies) post separation, then obtain 10 sub-portion position subFr1-10.Target compound is mainly in subFr8-9.
5.Sephadex gel-purifieds：To sub-portion position subFr8-9, then upper Sephadex gel column methanol-eluted fractions, collect Stream part is concentrated, and obtains target compound monomer.
6.DIOL silica gel purifications：Monomeric compound is obtained, Jing DIOL silica gel is isolated and purified again, obtains purity more than 98% Ingenol methyl butene acid esters (Ingenol Mebutate).
The key point of said method is：
1. macroporous resin D101 absorption is mixed sample stroke section and obtains total diterpene position；
2.Sephadex gel-purifieds obtain compound monomer；
3.DIOL silica gel purifications obtain high-purity target material monomer.
Embodiment 2
The preparation of ingenol methyl butene acid esters (Ingenol Mebutate)：
From southern Europe Radix Euphorbiae Pekinensis, herb plant separates material medicine ingenol methyl butene acid esters：
(Euphorbia peplus Linn) is collected in the Kunming north suburb for southern Europe Radix Euphorbiae Pekinensis, is dried herb 20kg, with first after chopping Alcohol soaks, and heating and refluxing extraction three times (3 × 100L) is filtered to remove residue, after filtrate concentration total extractum about 6kg, extractum with 20L water is suspended after dispersion, volume ratio 1:1 petroleum ether extraction 5 times, inspissated oil ether moiety obtain the total extractum 4kg of petroleum ether, with suitable Amount chloroform dissolving extractum, mixes sample with macroporous resin D101 (Tianjin sea light Chemical Co., Ltd.) dry method, then uses macroporous resin Post crude separation, using methanol/water solution as mobile phase, by 40%, 60%, 80%, 100% eluting.To 80% meoh eluate Concentration and recovery obtains total diterpene position 300g, mixes sample with silica gel (200～300 mesh) after the dissolving of appropriate chloroform, fills silicagel column crude separation, With petroleum ether:Ethyl acetate (20:1-0:1) gradient elution is carried out, obtains 8 main portions (Fr I-VIII), wherein Fr VII With C18 (Fuji Silysia Chemical companies) post separation, 10 sub-portion position subFr1-10 are obtained, subFr9 is gone up again Sephadex gel column methanol-eluted fractions, obtain target compound (106mg), purity more than 90%.Jing DIOL (Fuji again Silysia Chemical companies) isolate and purify, obtain ingenol methyl butene acid esters (Ingenol Mebutate) (95mg), purity more than 98%, yield 4.75%/1000000th.
The chemical constitution of Ingenol Mebutate is characterized：
Ingenol Mebutate, unformed powder (amorphous powder), [α]25 D+46.6(0.25,MeOH)。 Its spectral data is：
Ultraviolet spectra UV (MeOH) λmax(logε)：203(4.03)nm；
Infrared spectrum IR (KBr) υmax：3434,2928,1715,1457,1382,1231,1156,1040cm-1；
Hydrogen is composed1H NMR (600MHz, CDCl3)δ：5.99(1H,m,H-1),5.57(1H,s,H-3),4.01(1H,s,H- 5), 6.00 (1H, m, H-7), 4.14 (1H, m, H-8), 0.88 (1H, dd, J=11.7,8.5Hz, H-9), 0.65 (1H, dd, J= 15.1,8.6Hz,H-11),2.24(1H,m,H-12),1.70(1H,m,H-12),2.52(1H,m,H-13),1.75(3H, Brs, H-16), 4.09 (2H, m, H-17), 1.01 (3H, s, H-18), 1.04 (3H, s, H-19), 0.93 (3H, d, J=7.1Hz, H-20), 6.11 (1H, tt, J=7.3,3.6Hz, H-3 '), 1.97 (3H, dd, J=7.2,1.2Hz, H-4 '), 1.88 (3H, m, H-5’)；
Carbon is composed13C NMR (150MHz, CDCl3)δ：131.9(C-1),135.9(C-2),82.4(C-3),84.9(C-4), 76.1(C-5),139.5(C-6),128.1(C-7),43.4(C-8),22.8(C-9),24.0(C-10),23.2(C-11), 30.9(C-12),38.3(C-13),207.4(C-14),71.9(C-15),15.5(C-16),66.9(C-16),28.4(C- 18),15.5(C-19),17.1(C-20),168.6(C-1’),127.3(C-2’),139.4(C-3’),15.9(C-4’),20.8 (C-5’)。
Compared with prior art, process of the invention has following excellent benefit：1) macroporous resin D101 directly adsorbs Mix sample stroke section and obtain total diterpene position, it is different from macroporous resin D101 column chromatography samples in conventional solvent dissolving wet method；2) Sephadex gel filtration chromatography purification obtains compound monomer；3) DIOL silica gel column chromatographies purification obtains high-purity target material list The purification that DIOL silica gel column chromatographies efficiently purifying is used for industrial medicine material is had no report by body.The process possesses separation Purification is quick and easy, efficiently high-purity, the advantages of cost is relatively low.

Claims (1)

1. the preparation method of ingenol methyl butene acid esters, it is characterised in that the method comprises the steps：Raw material is extracted, stone Oily ether extraction, macroporous resin D101 absorption dry method are mixed sample and draw section, silica gel separation, Sephadex gel-purifieds, DIOL silica gel purifications,
Described raw material is extracted as：Southern Europe Radix Euphorbiae Pekinensis or congener herb are taken, is dried, soaked with methanol after chopping, be heated to reflux Extract three times, be filtered to remove residue, after filtrate concentration, obtain total extractum；
Described petroleum ether extraction is：Total extractum water is suspended after dispersion, with volume ratio 1:1 petroleum ether extraction 5 times, point liquid concentration stone Oily ether moiety, obtains the total extractum of petroleum ether；
It is, with the total extractum of petroleum ether, to be dissolved with chloroform, with macroporous resin that described macroporous resin D101 absorption dry method is mixed sample and draws section D101 dry method mixes sample, then uses macroporous resin column crude separation, using methanol/water solution as mobile phase, by 40%, 60%, 80%, 100% eluting, collects 80% methanol aqueous solution eluting stream part, and concentrating under reduced pressure reclaims solution, obtains extractum total diterpene position；
The silica gel is separated into：With silica gel mixed sample after chloroform dissolving, silicagel column crude separation is filled, petrol ether/ethyl acetate conduct is used Eluent carries out gradient elution, obtains 8 main portions Fr I-VIII, wherein Fr VII with C18 post separations, then 10 it is sub- Position subFr1-10；
The Sephadex gel-purifieds are：To sub-portion position subFr8-9, then upper Sephadex gel column methanol-eluted fractions, collect Stream part is concentrated, and obtains target compound monomer；
The DIOL silica gel purifications are：The monomeric compound that previous step is obtained, Jing DIOL silica gel are isolated and purified again, obtain pure The ingenol methyl butene acid esters of degree more than 98%；
Described method is dried to take southern Europe Radix Euphorbiae Pekinensis herb, is soaked with methanol, heating and refluxing extraction three times after chopping, is crossed and is filtered Residue is removed, total extractum after filtrate concentration, is obtained, extractum is suspended with 20L water after dispersion, volume ratio 1:1 petroleum ether extraction 5 times, concentrates stone Oily ether moiety, obtains the total extractum of petroleum ether, dissolves extractum with appropriate chloroform, mixes sample with macroporous resin D101 dry method, then use macropore Resin column crude separation, using methanol/water solution as mobile phase, by 40%, 60%, 80%, 100% eluting；80% methanol is washed De- liquid concentration and recovery obtains total diterpene position, mixes sample with 200～300 mesh of silica gel after the dissolving of appropriate chloroform, fills silicagel column crude separation, with 20:1‐0:1 petroleum ether:Ethyl acetate carries out gradient elution, obtains 8 main portions Fr I-VIII, wherein Fr VII with C18 post separations, obtain 10 sub-portion position subFr1-10, and subFr9 goes up sephadex gel column methanol-eluted fractions again, obtains purity More than 90% target compound, then Jing DIOL isolate and purify, and obtain the ingenol methyl butene acid esters of purity more than 98% Ingenol Mebutate。
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