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

Abstract

A method for preparing a raw material drug ingenol methacrylate for treating actinic keratosis, comprising the following steps: raw material extraction, petroleum ether extraction, macroporous resin D101 adsorption dry method mixing section, silica gel Separation, Sephadex gel purification, DIOL silica gel purification. The method separates the raw drug ingenol methacrylate from the whole plant of Euphorbia euphorbia, and has the advantages of fast and simple separation and purification, high efficiency and high purity, and low cost.

Description

A raw material drug ingenol methacrylate for treating actinic keratosis Preparation

Technical field:

The invention belongs to the technical field of medicine preparation methods, and in particular relates to a preparation method of ingenol, a raw material medicine for treating actinic keratosis.

Background technique

On January 23, 2012, the US FDA approved Ingenol Mebutate as a drug for the treatment of Actinic Keratosis (AK, a skin disease sensitive to sunlight) listed. The drug, trade name Picato, was successfully developed by LEO Pharma AS.

Ingenol Mebutate can be extracted, isolated and purified from Euphorbia plants, such as Euphorbia peplus, Euphorbia hirta, Euphorbia drummondi, and traditional Chinese medicine Euphorbia lathyris (daughter of gold), Euphorbia kansui ( Gansui) and other plants of the same genus all contain the raw material medicine. However, the content of the raw drug is very low, and its Ingenol Mebutate's mother core skeleton compound Ingenol is also widely distributed in the same genus of plants, which can be used for extraction, separation and purification. The mother core Ingenol is a tetracyclic diterpene natural product, in which the B ring and C ring are composed of bicyclic[4,4,1]undecane with very large ring tension, due to the inside-outsidestereochemistry on the bridgehead, that is, the C -11 to C-14 are on the opposite side of the bridgehead, greatly increasing its tension. Synthetic chemists are particularly interested in this special stereochemical phenomenon, which also makes the total synthesis of Ingenol a hot spot. Wood, Tanino/Kuwajima, Winkler, Funk, Rigby and other research teams have completed the total synthesis of Ingenol. The most convenient total synthesis of Ingenol requires only seven steps. The raw material drug Ingenol Mebutate can be obtained from synthetic and semi-synthetic routes.

Southern European euphorbia (Euphorbia peplus Linn), native to the Mediterranean coast (Southern Europe to North Africa), naturalized in Asia, America and Australia; my country's Taiwan (Taipei, Taichung), Guangdong, Hong Kong, Fujian (Xiamen, Fuzhou), Guangxi ( Nanning) and Yunnan (Kunming) successively found naturalized plants or populations. As a kind of Euphorbia plant, Southern European Euphorbia has been widely distributed in Yunnan Province. It can be seen everywhere in fields and landscaping areas, because its milk is toxic. , can not be used as pasture, the resources are huge, and it is the raw material resource base of ingenol methacrylate.

So far, there is no report on the process of extracting ingenol methacrylate industrial pharmaceutical raw material from Euphorbia euphorbia in the prior art.

Contents of the invention

The object of the present invention is to provide a method for extracting, separating and purifying natural raw material drug Ingenol Mebutate from the exotic plant Euphorbiapeplus widely distributed in Yunnan area. The process method has the advantages of fast and simple separation and purification, high efficiency and high purity, and low cost.

In order to realize the above-mentioned purpose of the present invention, the present invention provides following technical scheme:

The preparation method of ingenol methyl crotonate comprises the following steps: raw material extraction, petroleum ether extraction, macroporous resin D101 adsorption dry method sample mixing section, silica gel separation, Sephadex gel purification, DIOL silica gel purification;

The extraction of the raw materials is as follows: collect Euphorbia euphorbia or the whole plant of the same genus from various places, dry it in the sun under natural sunlight, soak it in methanol after chopping, heat and reflux for extraction three times, filter to remove the residue, and obtain the total extract after concentrating the filtrate;

The petroleum ether extraction is as follows: after the total extract is suspended and dispersed in water, it is extracted 3 times with petroleum ether at a volume ratio of 1:1, and the petroleum ether part is separated and concentrated to obtain the total extract of petroleum ether;

The section of the macroporous resin D101 adsorption dry sample mixing is to use the total extract of petroleum ether, dissolve it with an appropriate amount of chloroform, mix the sample with the macroporous resin D101 dry method, and then roughly separate it with a macroporous resin column, and use methanol/water solution As the mobile phase, elute at 40%, 60%, 80%, and 100%, collect 80% methanol aqueous solution to elute the eluted fraction, concentrate and recover the solution under reduced pressure, and obtain the extract as the total diterpene fraction;

The silica gel separation is as follows: the silica gel sample is mixed with an appropriate amount of chloroform, the silica gel column is installed for rough separation, and petroleum ether/ethyl acetate is used as the eluent for gradient elution to obtain 8 main parts Fr I-VIII, wherein Fr VII Separation with C18 column to obtain 10 sub-parts subFr1-10;

The Sephadex gel purification is as follows: for the sub-site subFr8-9, and then eluted with methanol on the Sephadex gel column, the collected fractions are concentrated, and the target compound monomer is obtained;

The DIOL silica gel purification is as follows: the monomer compound is obtained, which is separated and purified again by DIOL silica gel to obtain ingenol methacrylate with a purity of more than 98%.

As described in the preparation method of ingenol methyl crotonate, wherein the key step of the method is to obtain the total diterpene parts by adsorption and mixing of macroporous resin D101; Sephadex gel column chromatography purification to obtain the compound single body; purified by DIOL silica gel column chromatography to obtain high-purity target raw material monomer.

The preparation method of ingenol methyl crotonate as described above, wherein the method is to take the whole plant of Euphorbia euphorbia, dry it, chop it, soak it in methanol, extract it under reflux for three times, filter to remove the residue, and concentrate the filtrate Finally, the total extract was obtained. After the extract was suspended and dispersed in 20L of water, it was extracted 5 times with petroleum ether at a volume ratio of 1:1, and the petroleum ether part was concentrated to obtain the total extract of petroleum ether. D101 dry mixed sample, then roughly separated with a macroporous resin column, using methanol/water solution as mobile phase, and eluted at 40%, 60%, 80%, 100%; 80% methanol eluate was concentrated and recovered to obtain total two For the terpene part, after dissolving with an appropriate amount of chloroform, mix the sample with 200-300 mesh silica gel, install a silica gel column for rough separation, and perform gradient elution with 20:1-0:1 petroleum ether: ethyl acetate to obtain 8 main parts Fr I- VIII, wherein Fr VII was separated by C18 column to obtain 10 sub-parts subFr1-10, and subFr9 was eluted with methanol on a sephadex gel column to obtain the target compound with a purity of more than 90%, which was separated and purified by DIOL again to obtain a purity of 98%. Ingenol Mebutate above.

detailed description

The following examples of the present invention are used to further illustrate the substantive content of the present invention, but the present invention is not limited thereto.

Example 1

Preparation of Ingenol Mebutate:

1. Methanol extraction of raw materials: Collect Euphorbia peplus Linn or the whole plant of the same genus from various places, dry it in the sun, chop it, soak it in methanol, extract it under reflux for three times, filter to remove the residue, and concentrate the filtrate to obtain total extract.

2. Petroleum ether extraction: After the total extract was suspended and dispersed in water, it was extracted 3 times with petroleum ether at a volume ratio of 1:1, and the petroleum ether part was concentrated by liquid separation to obtain the total extract of petroleum ether.

3. Macroporous resin D101 adsorption dry sample mixing section: the petroleum ether total extract is dissolved in an appropriate amount of chloroform, mixed with macroporous resin D101 dry method, and then roughly separated by a macroporous resin column, using methanol/water solution as the mobile phase , according to 40%, 60%, 80%, 100% elution. Collect 80% methanol aqueous solution to elute the eluted fraction, concentrate and recover the solution under reduced pressure, and obtain the extract as the total diterpene fraction.

4. Silica gel separation: mix the sample with silica gel after dissolving an appropriate amount of chloroform, install a silica gel column for rough separation, and use petroleum ether/ethyl acetate as the eluent for gradient elution to obtain 8 main parts (Fr I-VIII), of which Fr VII was separated on a C18 (FujiSilysia Chemical Company) column to obtain 10 subparts subFr1-10. The target compounds are mainly in subFr8-9.

5. Sephadex gel purification: for the sub-site subFr8-9, and then eluted with methanol on a Sephadex gel column, the collected fractions were concentrated to obtain the target compound monomer.

6. Purification on DIOL silica gel: the monomer compound was obtained, and separated and purified again by DIOL silica gel to obtain Ingenol Mebutate with a purity of more than 98%.

The key points of the above method are:

1. Macroporous resin D101 absorbs and mixes samples to obtain total diterpene parts;

2. Sephadex gel purification to obtain the compound monomer;

3. DIOL silica gel purification to obtain high-purity target raw material monomer.

Example 2

Preparation of Ingenol Mebutate:

Isolation of raw material drug ingenol methacrylate from whole plant of Euphorbia euphorbia:

Euphorbia peplus Linn was collected in the northern suburbs of Kunming, dried whole herb 20kg, chopped, soaked in methanol, heated and refluxed for three times (3×100L), filtered to remove the residue, and the filtrate was concentrated to obtain a total extract of about 6kg. After the extract was suspended and dispersed in 20L of water, it was extracted 5 times with petroleum ether at a volume ratio of 1:1, and the petroleum ether part was concentrated to obtain 4kg of total petroleum ether extract. Chemical Co., Ltd.) dry-mixed sample, and then roughly separated with a macroporous resin column, using methanol/water solution as the mobile phase, and eluted by 40%, 60%, 80%, and 100%. Concentrate and recover the 80% methanol eluent to obtain 300 g of total diterpene parts, dissolve with an appropriate amount of chloroform, mix the sample with silica gel (200-300 mesh), install a silica gel column for rough separation, and use petroleum ether: ethyl acetate (20:1-0 : 1) Carry out gradient elution to obtain 8 main parts (Fr I-VIII), wherein Fr VII is separated by C18 (Fuji Silysia Chemical Company) column, and 10 sub-parts subFr1-10 are obtained, and subFr9 is put on a sephadex gel column Eluted with methanol, the target compound (106 mg) was obtained with a purity of over 90%. Separation and purification by DIOL (FujiSilysia Chemical Company) again gave Ingenol Mebutate (95 mg) with a purity of over 98% and a yield of 4.75 parts per million.

Chemical structure characterization of Ingenol Mebutate:

Ingenol Mebutate, amorphous powder, [α] ²⁵ _D +46.6 (0.25, MeOH). Its spectral data are:

Ultraviolet spectrum UV (MeOH) λ _max (log ε): 203 (4.03) nm;

Infrared spectrum IR (KBr) υ _max : 3434, 2928, 1715, 1457, 1382, 1231, 1156, 1040cm ^-1 ;

Hydrogen spectrum ¹ H NMR (600MHz, CDCl ₃ ) 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 Spectrum ¹³ C NMR (150MHz, CDCl ₃ ) 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 the prior art, the process method of the present invention has the following advantages: 1) The macroporous resin D101 directly absorbs and mixes the samples and divides to obtain the total diterpene parts, and the macroporous resin D101 column on the conventional solvent-dissolved wet method Chromatographic samples are different; 2) Sephadex gel column chromatography purification to obtain compound monomers; 3) DIOL silica gel column chromatography purification to obtain high-purity target raw material monomers, and DIOL silica gel column chromatography for efficient purification of industrial pharmaceutical raw materials None reported. The process method has the advantages of fast and simple separation and purification, high efficiency and high purity, and low cost.

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。