CN100535000C - Method of preparing cyclopamine from jervine - Google Patents
Method of preparing cyclopamine from jervine Download PDFInfo
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- CN100535000C CN100535000C CNB2007100377510A CN200710037751A CN100535000C CN 100535000 C CN100535000 C CN 100535000C CN B2007100377510 A CNB2007100377510 A CN B2007100377510A CN 200710037751 A CN200710037751 A CN 200710037751A CN 100535000 C CN100535000 C CN 100535000C
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Abstract
The invention discloses a making method of cyclopamine and application in the medical domain, which comprises the following steps: adopting jervine from Veratrum plant as raw material; reacting jervine and hydrazine hydrate to produce hydrazone in the high-boiling point solvent; denitrifying acted by potassium hydroxide and tert-butanol potassium to form jervine; using carbowax 400 and TEBA as phase transmitting catalyst; connecting tumour cell Hh and Smo protein; dying cancer cell due to non-gene toxicity without influencing normal cell.
Description
Technical field
The invention belongs to medical technical field, be specifically related to a kind of preparation method of cyclopamine, relating to a kind of particularly is the method for feedstock production cyclopamine with the jervine that derives from the Veratrum plant, and the application of this cyclopamine aspect the tumour of Hh signal path expression such as treatment carcinoma of the pancreas, medulloblastoma, small cell lung cancer.
Background technology
Hedgehog (Hh) signal path is found in fruit bat the earliest, is to cause the signal path that drosophila larvae is grown.Studies show that further the Hh signal path comprises the complex body of Patched (Ptc) albumen and Smoothened (Smo) albumen and Ci (Gli) albumen, Cos2 albumen, Fused kinases, Protein S uFu, goal gene.Hh albumen is a kind of secretory protein, and is active through the modification acquisition of self; Patched albumen is the acceptor of Hh signal protein; Smo albumen is the transmodulator of Hh signal.When Hh albumen existed, Ptch lost the restraining effect to Smo, and the Smo protein activation for complex body provides phosphoric acid, impels Ci (Gli) to change catalyst mode (155kDa) into, entered nucleus startup downstream gene and transcribed; On the contrary, when the Hh protein delation, Smo is subjected to Ptch to be suppressed, and Ci (Gli) is by protease hydrolysis, and its inhibitor form (75kDa) stops gene transcription in nucleus.
The Hh signal path is the signal path of numerous animal embryo growths and internal organs, tissue development.The disappearance of Hh signal path can cause developmental defect or deformity among the embryo.The Hh signal path that notes abnormalities in increasing tumour activates, and therefore, Chinese scholars is progressively recognized close the getting in touch of having of Hh signal path and tumour.And along with this area research deeply and the expansion of scope, the unconventionality expression of discovery Hh signal path is the reason that causes tumor diseases such as basal cell tumor, medulloblastoma, pancreatic cell cancer, small cell lung cancer, mammary cancer.Therefore, the unconventionality expression of inhibition Hh signal path is one of effective ways of treatment tumour.
Cyclopamine (cyclopamine) is a kind of isocholestane Alkaloid that is present in black false hellebore and the Fritillaria plant, be found in a mao leaf black false hellebore (Veratrum grandiflorum (Maxim.) Loes.f.) (Masamune at first in 1964, T., et al., Bull.Chem.Soc.Jpn.38 (8): 1374-1378 (1965)).The sixties to the eighties in 20th century, the research for cyclopamine only limited to its teratogenesis, find just that up to the mid-90 in 20th century cyclopamine is the inhibitor of Hh signal path, relevant (the Cooper of generation development with kinds of tumors, M.K., et al., Science 280,1603-1607 (1998)).
The Berman study group of Johns Hopkings university migrates to rat with medulloblastoma cell, handles through cyclopamine, finds that cyclopamine can cell growth inhibiting, and the intravital tumour of rat is dwindled.The Olson of Hutchinson DKFZ carries out experiment in vitro to the medulloblastoma sample, finds that cyclopamine can kill 99.9% tumour cell (etal., Science 297,1559-1561 (2002) for Bermn, D.M).Found that the cell strain of Smo gene high expression is compared proliferation rate decline 75%-80% with control group after cyclopamine is handled, apoptosis increases 2.5-3.5 doubly the time spent of doing of pancreas cancer cell strain at the research cyclopamine.And find that the gross tumor volume of treatment group 50%-60% dwindles during to the nude mice of the cell strain of cyclopamine sensitivity with cyclopamine treatment inoculation.Show cyclopamine in vivo with external can be by Hh approach cell death inducing and blocking-up increment (etal., Nature 425,851-855 (2003) for Thayer, S.p.).
Above result as can be seen, cyclopamine is only inhibited to the tumour cell that the Hh signal path is expressed, and shows extremely strong specificity.And early stage toxicological study shows that cyclopamine does not have toxicity to normal cell, and is also very little to the toxic side effect of laboratory animal.Many experts think that this has opened a new way that has prospect of treatment tumour.The authority in this field, the P.A. of U.S. JohnsHopkings university, Beachy claims: present experiment shows, the speed of cyclopamine kill tumor cell is faster than in the past any medicine, and laboratory animal is not seen any side effect (Beachy, P.A., et al., Nature, 411,349-354 (2001)).But the content of cyclopamine in plant is very low, only have ten thousand/, and the complete synthesis very difficulty of chemistry.It is that raw material adopts the reduction of Wolff-Kishner method to obtain cyclopamine with jervine that report was once arranged, but adopt the hydrazine of inflammable high-risk in the method, reaction conditions requires harsh, productive rate is low, about 20% (Masamune are only arranged, T., et al., Bull.Chem.Soc.Jpn.38 (8): 1374-1378 (1965)).After again the someone adopt the imperial method reduction of the yellow ring of improved Wolff-Kishner-preparation cyclopamine, but still there is the low problem of product yield (about 50%), and have isomer 3-table cyclopamine exist (all swordsmen, etc. Chinese pharmaceutical chemistry magazine, 16 (5): 303-305 (2006)).The source problem that comes of cyclopamine becomes the big obstacle that it is developed to medicine.
This seminar has found the precursor of cyclopamine---jervine under study for action, and its content in this kind black false hellebore is higher, and the aboundresources of this kind black false hellebore.The contriver has carried out surplus in the of 40 time the experiment repeatedly for preparing cyclopamine with jervine, has optimized reaction conditions, can prepare cyclopamine easily with high yield, and required reaction conditions reaches easily, is suitable for industrial production.Prepare cyclopamine by jervine and become a practicable approach.
Summary of the invention
The purpose of this invention is to provide a kind ofly react quick and convenient, productive rate is high is the method for feedstock production cyclopamine with the jervine.
What the present invention proposed prepares the method for cyclopamine by jervine, its step is as follows: with jervine raw material and hydrazine hydrate, highly basic under the catalysis of phase-transfer catalyst, in high boiling solvent, react, the mol ratio of jervine and hydrazine hydrate is 1.1: 1-3: 1, temperature of reaction is 160-200 ℃, and the reaction times is 2-7 hour.Reaction is every g jervine 0.2-0.6L solvent with the consumption of high boiling solvent.After reaction finishes, add entry, and, collect organic solvent layer, concentrate after removing moisture, separate out crystallization and be cyclopamine with the organic solvent extraction of middle polarity.
Highly basic is potassium hydroxide or potassium tert.-butoxide in the reaction.High boiling solvent is-diglycol ethylene, dimethyl sulfoxide (DMSO), 1, ammediol or ethylene glycol.
Phase-transfer catalyst can be 400 polyoxyethylene glycol with molecular weight, and consumption is 2～8% (ml/ml) of reaction solvent volume, is preferably 5%.Phase-transfer catalyst also can be benzyltriethylammoinium chloride (TEBA), and consumption is 1～3% (g/g) of jervine weight.
The organic solvent of the middle polarity of extraction was one or more in chloroform, methylene dichloride, ethyl acetate, the acetone after reaction finished.
Separate out in the mother liquor after the crystallization and contain the part cyclopamine, be further purified, obtain cyclopamine with column chromatography.The used sorbent material of column chromatography is silica gel, aluminum oxide or polymeric amide.The solvent that wash-out is used is one or more in sherwood oil, methylene dichloride, chloroform, ethyl acetate, acetone, the methyl alcohol.
As follows from the reaction process of the synthetic cyclopamine of jervine:
The fusing point of the cyclopamine for preparing among the present invention and nuclear magnetic data and document (Gaffield, W., et al., J.Nat.Prod.49 (2): 286-292 (1986)) unanimity.
Among the present invention, described raw material jervine can extract acquisition from the black false hellebore plant, and concrete extraction step is as follows:
The dry rhizome of medicinal material black false hellebore plant is pulverized the back carry or cold soaking, extracting solution or cooling bath are concentrated with ethanol heat; Through acidifying and the heavy processing of alkali, recycle silicon plastic column chromatography method separates obtaining jervine to spissated crude extract; Used eluent is methylene dichloride and methanol system or sherwood oil and ethyl acetate system.
Adopt 75% ethanol when wherein heat is carried, heat is carried 2-4 time, and the 3-5 that each ethanol consumption is medicinal material (black false hellebore rhizome) volume doubly.Cold soaking is with the ethanol of 75%-95%, preferred 95% ethanol, cold soaking 2-4 time, the 4-10 that each consumption of ethanol is the medicinal material volume doubly, preferred 5-7 times of volume.The acid of acidification is sulfuric acid or the hydrochloric acid of 0.5%-5% for mass concentration, the mass concentration of preferred 1%-2%.The heavy alkali of handling usefulness of alkali is the saturated solution of strong aqua or yellow soda ash, and the pH value of solution value of regulating acidification is 7-10, preferred 8-9.
The structural formula of jervine is as follows:
The structural formula of cyclopamine is as follows:
The present invention also proposes to contain the pharmaceutical composition of above-mentioned cyclopamine and one or more pharmaceutically acceptable carriers.Pharmaceutical composition of the present invention preferably contains the activeconstituents that weight ratio is 0.1%-99.5%, most preferably contains the activeconstituents that weight ratio is 0.5%-95%.
Pharmaceutically acceptable carrier mentioned above is meant the pharmaceutical carrier of pharmaceutical field routine, for example: thinner, vehicle such as water etc., weighting agent such as starch, sucrose etc.; Tackiness agent such as derivatived cellulose, alginate, gelatin and polyethylene pyrrole be alkane ketone slightly; Wetting agent such as glycerine; Disintegrating agent such as agar, lime carbonate and sodium bicarbonate; Absorption enhancer such as quaternary ammonium compound; Tensio-active agent such as cetyl alcohol; Absorption carrier such as kaolin and soap clay; Lubricant such as talcum powder, calcium stearate and magnesium and polyoxyethylene glycol etc.Can also in composition, add other assistant agent such as flavouring agent, sweeting agent etc. in addition.
The compounds of this invention can composition form by Transdermal absorption, oral, patient that mode intravenous administration is used for this treatment of needs.When being used for Transdermal absorption, can be made into emulsifiable paste, ointment or gel paster preparation; Be used for when oral, can be made into conventional solid preparation such as tablet, pulvis, granula, capsule etc., make liquid preparation such as water or oil-suspending agent or other liquid preparation such as syrup, elixir etc.; When being used for intravenous administration, can be made into solution, water or the oiliness suspension agent etc. of injection.Preferred form is emulsifiable paste paster, tablet, coated tablet, capsule, suppository and injection.
Cyclopamine shows specific inhibitory effect to the propagation of the cell strain of the carcinoma of the pancreas that has the Hh signal path to express, medulloblastoma, small cell lung cancer.Therefore, the cyclopamine and the aforementioned pharmaceutical compositions of the present invention's preparation can be used for these tumor treatment.
The following examples can make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.
Description of drawings
Fig. 1 is the inhibition activity of cyclopamine to the pancreatic tumour cell strain.
Fig. 2 is the inhibition activity of cyclopamine to the small cell lung cancer cell strain
Fig. 3 is the inhibition activity of cyclopamine to colon cancer cell line
Embodiment
Get the meal of 5kg black false hellebore rhizome, the ethanol cold soaking twice with 95%, each consumption are the ethanol of 30L.Cooling bath is concentrated into flowing soaking paste, admixes the diatomite of 0.5kg, dries in the air to the nearly back dress post of doing, with the sulfuric acid wash-out of mass concentration 2%.The sour water that elutes is transferred pH=9 with strong aqua, leaves standstill the 2h after-filtration, the 40g total alkaloids that obtains.Admix in the 100g 200-300 purpose silica gel, in addition with 1kg 200-300 order silica gel dress.Use sherwood oil: ethyl acetate system (5: 1-2: 1-1: 1-0: 1) wash-out, collect the 11-16 cut, each 180mL.Each cut is concentrated into behind the 60mL standing over night in refrigerator, separates out faint yellow needle-like jervine, gross weight 325mg.Reach 99.1% through the HPLC detection level.
Embodiment 2
Get the meal of 5kg black false hellebore rhizome, carry 3 times with 75% ethanol heat, each consumption is the ethanol of 15L.Extracting solution is concentrated into does not have the alcohol flavor, residuum with 2% sulfuric acid acidation to pH=1.Centrifugal, get supernatant liquor, transfer pH=9 with strong aqua, use chloroform extraction 3 times, each 2L.Chloroform layer is concentrated into 0.5L, admixes evaporate to dryness in the 200g 200-300 purpose silica gel, in addition with 2kg200-300 order silica gel dress.Use methylene dichloride: methanol system (20: 1-10: 1-5: 1-2: 1) wash-out, collect the 8-12 cut, each 180mL concentrates the back and goes up Sephadex-LH20, uses methanol-eluted fractions, removes a spot of flavones and pigment.Cut is concentrated into behind the 60mL standing over night in refrigerator, separates out faint yellow needle-like jervine, gross weight 289mg.Reach more than 98% through the HPLC detection level.
The 50mg jervine is dissolved in the 30mL tirethylene glycol, adds 10 μ L hydrazine hydrates, the poly(oxyethylene glycol) 400 of 5% volume ratio and the potassium hydroxide of 0.2g stir down at 160 ℃, are cooled to 120 ℃ behind the 2h, remove moisture content, are warming up to 190 ± 5 ℃ again, about reaction 2.5h.Reaction solution adds in the 50mL water, with 100mL * 2 ethyl acetate extractions.Organic phase is used anhydrous sodium sulfate drying after with the saturated common salt water washing.Be concentrated into 5mL, standing over night in refrigerator is separated out colourless needle crystal 28mg, silicagel column on the mother liquor, and the sherwood oil of usefulness gradient: ethyl acetate mixed solution wash-out, obtain white powder 6mg, amount to 34mg, yield 70.3%.TLC launches back spray KBiI
4Colour developing has only a spot.Fusing point 125-128 ℃,
1H-NMR,
13C-NMR data such as table 1 are with document (Gaffield, W., et al., J.Nat.Prod.49 (2): 286-292 (1986)) basically identical.
Embodiment 4
The 50mg jervine is dissolved in the 15mL tirethylene glycol, adds 7 μ L hydrazine hydrates, the poly(oxyethylene glycol) 400 of 2% volume ratio stirs down at 160 ℃, is cooled to 120 ℃ behind the 2h, removes moisture content, adds the potassium hydroxide of 0.2g, is warming up to 190 ± 5 ℃ again, about reaction 4h.Reaction solution adds in the 50mL water, with 100mL * 2 ethyl acetate extractions.Organic phase is used anhydrous sodium sulfate drying after with the saturated common salt water washing.Be concentrated into 5mL, standing over night in refrigerator is separated out colourless needle crystal 36mg, silicagel column on the mother liquor, and the methylene dichloride of usefulness gradient: the methyl alcohol mixed liquor wash-out, obtain white powder 5mg, amount to 41mg, yield 82%.TLC launches back spray KBiI
4Colour developing has only a spot.Fusing point 125-128 ℃, mix the back fusing point with crystal among the embodiment 3 and do not descend.ESI：[M+H]412。
The 50mg jervine is dissolved in the 15mL tirethylene glycol, adds 10 μ L hydrazine hydrates, the TEBA of 1mg and the potassium hydroxide of 0.3g stir down at 160 ℃, are cooled to 120 ℃ behind the 2h, remove moisture content, are warming up to 190 ± 5 ℃ again, about reaction 2.5h.Reaction solution adds in the 50mL water, with 100mL * 2 chloroform extractions.Organic phase is used anhydrous sodium sulfate drying after with the saturated common salt water washing.Be concentrated into 6mL, standing over night in refrigerator is separated out colourless needle crystal 28mg, silicagel column on the mother liquor, and the methylene dichloride of usefulness gradient: the methyl alcohol mixed liquor wash-out, obtain white powder 5mg, amount to 33mg, yield 66%.TLC launches back spray KBiI
4Colour developing has only a spot.Fusing point 125-128 ℃, mix the back fusing point with crystal among the embodiment 3 and do not descend.ESI：[M+H]412。
Embodiment 6
The 50mg jervine is dissolved in 25mL ethylene glycol, adds 20 μ L hydrazine hydrates, the poly(oxyethylene glycol) 400 of 5% volume ratio and the potassium hydroxide of 0.2g, add de-watering apparatus on reaction flask, at 160 ℃, 2h refluxes under the 0.06MPa vacuum tightness, be warming up to 195-200 ℃ after returning to normal pressure, about reaction 4h.Reaction solution adds in the 40mL water, with 40mL * 2 dichloromethane extractions.Organic phase is used anhydrous sodium sulfate drying after with the saturated common salt water washing.Be concentrated into 5mL, standing over night in refrigerator is separated out colourless needle crystal 33mg, alumina column on the mother liquor, and the sherwood oil of usefulness gradient: ethyl acetate mixed solution wash-out, obtain white powder 5mg, amount to 38mg, yield 76%.TLC launches back spray KBiI
4Colour developing has only a spot.Fusing point 125-128 ℃, mix the back fusing point with crystal among the embodiment 3 and do not descend.ESI：[M+H]412。
Embodiment 7
The 50mg jervine is dissolved among the 15mL DMSO, adds 10 μ L hydrazine hydrates, the poly(oxyethylene glycol) 400 of 8% volume ratio and the potassium hydroxide of 0.1g stir down at 160 ℃, are cooled to 120 ℃ behind the 2h, remove moisture content, are warming up to 190 ± 5 ℃ again, about reaction 4h.Reaction solution adds in the 100mL water, with 100mL * 2 acetone extracts.Organic phase is used anhydrous sodium sulfate drying after with the saturated common salt water washing.Be concentrated into 5mL, standing over night in refrigerator is separated out colourless needle crystal 26mg, silicagel column on the mother liquor, and the sherwood oil of usefulness gradient: ethyl acetate mixed solution wash-out, obtain white powder 10mg, amount to 36mg, yield 72%.TLC launches back spray KBiI
4Colour developing has only a spot.Fusing point 125-128 ℃, mix the back fusing point with crystal among the embodiment 3 and do not descend.ESI：[M+H]412。
Embodiment 8
The 50mg jervine is dissolved in the 15mL tirethylene glycol, adds 10 μ L hydrazine hydrates, the poly(oxyethylene glycol) 400 of 5% volume ratio, at 160 ℃, the 2.5h that refluxes under the 0.06MPa vacuum degree condition is cooled to 30 ℃, adds the potassium tert.-butoxide of 0.4g, about reaction 6h.Reaction solution adds in the 60mL water, with 100mL * 2 ethyl acetate extractions.Organic phase is used anhydrous sodium sulfate drying after with the saturated common salt water washing.Be concentrated into 6mL, standing over night in refrigerator is separated out colourless needle crystal 29mg, silicagel column on the mother liquor, and the ethyl acetate of usefulness gradient: acetone mixed solution wash-out, obtain white powder 8mg, amount to 37mg, yield 74%.TLC launches back spray KBiI
4Colour developing has only a spot.Fusing point 125-128 ℃, mix the back fusing point with crystal among the embodiment 3 and do not descend.ESI：[M+H]412。
Embodiment 9
The 50mg jervine is dissolved in 20mL ethanol, adds 10 μ L hydrazine hydrates, the hydrochloric acid of the poly(oxyethylene glycol) 400 of 5% volume ratio and 0.2mL 37%, backflow 12h is concentrated into 5mL, and the faint yellow needle crystal of 46mg, yield 89.1% are separated out in cooling.It is dissolved among the 15mL DMSO, adds the 0.4g potassium tert.-butoxide, in 30 ℃ of stirring 6h, reaction solution adds in the 50mL water, with 100mL * 2 ethyl acetate extractions.Organic phase is used anhydrous sodium sulfate drying after with the saturated common salt water washing.Be concentrated into 5mL, standing over night in refrigerator is separated out colourless needle crystal 32mg, silicagel column on the mother liquor, and the sherwood oil of usefulness gradient: chloroform mixed solution wash-out, obtain white powder 5mg, amount to 37mg, yield 74%.TLC launches back spray KBiI
4Colour developing has only a spot.Fusing point 125-128 ℃, mix the back fusing point with crystal among the embodiment 3 and do not descend.ESI：[M+H]412。
Embodiment 10
Cyclopamine suppresses the propagation of pancreas cancer cell strain.
Adopt tetrazolium bromide reduction method (mtt assay) to measure cyclopamine to human pancreas cancer cell strain BXPC3, CEPAC, SW1990 proliferation inhibition activity.
Method: the human cancer cell strain in the vegetative period of taking the logarithm is sub-packed in 96 well culture plates, the 0.25mL/ hole.DMSO control group, 3 different concns cyclopamine groups are established in experiment.Medicine and cell cultures 96h, 48h changes liquid, and the every hole of 4h adds 20 μ L MTT liquid (5 μ g/mL) before experiment finishes, place to go nutrient solution behind the 4h, add 100 μ L DMSO/ holes, the rearmounted enzyme of dissolving to be crystallized joins detector, surveys the absorbance (A) in each hole with the 540nm wavelength.
The results are shown in Figure 1.As can be seen, pancreas cancer cell strain CEPAC and SW1990 have tangible dose-effect relationship to cyclopamine.This shows that cyclopamine has an antitumor action external, also shows that pancreas cancer cell strain CEPAC and SW199 are effective Hh path transduction inhibitor simultaneously.
Embodiment 11
Cyclopamine suppresses the propagation of small cell lung cancer cell strain.
Adopt tetrazolium bromide reduction method (mtt assay) to measure cyclopamine to human small cell lung carcinoma cell strain LTEP-Sml and NCI-H446 proliferation inhibition activity.
Method: the human small cell lung carcinoma cell strain in the vegetative period of taking the logarithm is sub-packed in 96 well culture plates, the 0.25mL/ hole.DMSO control group, 3 different concns cyclopamine groups are established in experiment.Medicine and cell cultures 96h, 48h changes liquid, and the every hole of 4h adds 20 μ L MTT liquid (5 μ g/mL) before experiment finishes, place to go nutrient solution behind the 4h, add 100 μ L DMSO/ holes, the rearmounted enzyme of dissolving to be crystallized joins detector, surveys the absorbance (A) in each hole with the 540nm wavelength.
The results are shown in Figure 2.As can be seen, human small cell lung carcinoma cell strain LTEP-Sml and NCI-H446 have tangible dose-effect relationship to cyclopamine.This shows that cyclopamine has an antitumor action external, also shows that human small cell lung carcinoma cell strain LTEP-Sml and NCI-H446 are effective Hh path transduction inhibitor simultaneously.
Embodiment 12
Cyclopamine suppresses the propagation of colon cancer cell line.
Adopt tetrazolium bromide reduction method (mtt assay) to measure cyclopamine to human colon cancer cell strain LOVO and HCT-8 proliferation inhibition activity.
Method: the human colon cancer cell strain in the vegetative period of taking the logarithm is sub-packed in 96 well culture plates, the 0.25mL/ hole.DMSO control group, 3 different concns cyclopamine groups are established in experiment.Medicine and cell cultures 96h, 48h changes liquid, and the every hole of 4h adds 20 μ L MTT liquid (5 μ g/mL) before experiment finishes, place to go nutrient solution behind the 4h, add 100 μ L DMSO/ holes, the rearmounted enzyme of dissolving to be crystallized joins detector, surveys the absorbance (A) in each hole with the 540nm wavelength.
The results are shown in Figure 3.As can be seen, human colon cancer cell strain LOVO and HCT-8 have tangible dose-effect relationship to cyclopamine.This shows that cyclopamine has an antitumor action external, also shows that pancreas cancer cell strain LOVO and HCT-8 are effective Hh path transduction inhibitor simultaneously.
The 1H-NMR of table 1. cyclopamine and 13C-NMR data
Claims (5)
1. the preparation method of a cyclopamine is characterized in that comprising the steps:
, reacted in high boiling solvent under the catalysis of phase-transfer catalyst by jervine, hydrazine hydrate, highly basic, the mol ratio of jervine and hydrazine hydrate is 1.1 in the reaction: 1-3: 1, temperature of reaction is 160-200 ℃, reaction times is 2-7 hour, and the consumption of high boiling solvent is every g jervine 0.2-0.6L solvent, after reaction finishes, add entry, and, collect organic solvent layer with the organic solvent extraction of middle polarity, concentrate after removing moisture, separate out crystallization and be cyclopamine, wherein:
Described highly basic is potassium hydroxide or potassium tert.-butoxide, and described phase-transfer catalyst employing molecular weight is 400 polyoxyethylene glycol, and consumption is 2～8% of a reaction solvent volume, or adopts benzyltriethylammoinium chloride, and consumption is the 1-3% of jervine weight; Described high boiling solvent is glycol ether, dimethyl sulfoxide (DMSO), 1, ammediol or ethylene glycol;
The organic solvent of described middle polarity is one or more in chloroform, methylene dichloride, ethyl acetate, the acetone.
2, preparation method according to claim 1 is characterized in that the solid of separating out after the cyclopamine crystallization is carried out column chromatography purification, obtains cyclopamine, and wherein the used sorbent material of column chromatography purification is silica gel, aluminum oxide or polymeric amide; The solvent that wash-out is used is one or more in sherwood oil, methylene dichloride, chloroform, ethyl acetate, acetone, the methyl alcohol.
3, preparation method according to claim 1, it is characterized in that used jervine raw material extracts acquisition from the Veratrum plant, concrete steps are as follows: the dry rhizome of medicinal material black false hellebore plant is pulverized the back carry or cold soaking with ethanol heat, extracting solution or cooling bath are concentrated; Through acidifying and the heavy processing of alkali, recycle silicon plastic column chromatography method separates obtaining jervine to spissated crude extract; Used eluent is methylene dichloride and methanol system or sherwood oil and ethyl acetate system.
4, preparation method according to claim 3 is characterized in that described heat is carried to adopt 75% ethanol that heat is carried 2-4 time, each ethanol consumption is the medicinal material volume 3-5 times; The cold soaking ethanol of 75%-95%, cold soaking 2-4 time, the 4-10 that each ethanol consumption is the medicinal material volume is doubly.
5, preparation method according to claim 3 is characterized in that the acid that described acidifying is used is sulfuric acid or the hydrochloric acid of mass concentration 0.5-5%, and the alkali of the heavy usefulness of alkali is the saturated solution of strong aqua or yellow soda ash, and the pH value of regulating the solution of acidification is 7-10.
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| 异甾体生物碱－环巴胺的研究进展. 周剑侠等.中国天然药物，第4卷第6期. 2006 |
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