CN104447950A - Romidepsin separation and purification method - Google Patents
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Abstract
The invention discloses a romidepsin separation and purification method. The method comprises the following steps: 1, mixing a romidepsin-containing Chromobacterium violaceum broth with macroporous adsorption resin, and stirring until complete adsorption; 2, separating out the macroporous adsorption resin adsorbing romidepsin from the broth, adding the macroporous adsorption resin adsorbing romidepsin to a column, cleaning, eluting to obtain an eluate 1, and concentrating to obtain a concentrate 1; 3, adding the concentrate 1 into macroporous adsorption resin, loading to the column, washing, eluting, detecting by using HPLC in the eluting process, collecting a romidepsin-containing elute 2, and concentrating to obtain a concentrate 2; 4, extracting the concentrate 2 to obtain an organic layer, and concentrating to obtain a crude product; and 5, purifying the crude product to obtain romidepsin. The separation and purification method has the advantages of high extraction purity, high yield, simple purification process and low cost.
Description
Technical field
The present invention relates to a kind of separation purification method of romidepsin.
Background technology
Romidepsin, English name is Romidepsin, is to be separated the natural polypeptide of the one obtained from the fermentation culture of chromobacterium violaceum (Chromobacteriumviolaceum), and its molecular formula is C
24h
36n
4o
6s
2, molecular weight is 540.
Romidepsin is a kind of novel histon deacetylase (HDAC) (HDAC) inhibitor; HDAC is a Zn-like ions Ntn hydrolase; when catalysis histone deacetylase; zine ion forms transition state with the hydrate chelating of oxygen on ethanoyl; excessive or the Abnormal regulation of HDAC makes the excessive deacetylation of histone; chromatin condensation makes to transcribe and is suppressed, and causes corresponding gene to express and declines, cause neoplastic conditions to occur.Romidepsin antitumor machanism is after romidepsin enters cell, and be sulfydryl by reductive glutathione (GSH) by its disulfide bond reduction, sulfydryl contestable is in conjunction with the Zn on HDAC
2+, HDAC was lost efficacy, thus reaches antineoplastic object.
Romidepsin, a novel antitumor bicyclic depsipeptide produced byChromobacterium violaceum No.968.I.Taxonomy, fermentation, isolation, physico-chemical and biological properties, and antitumor activity discloses a kind of method of purifying romidepsin from chromobacterium violaceum (Chromobacterium violaceum) WB968 fermented liquid, the method is the fermentor tank sterilizing 30min at 120 DEG C that will have fermented, the nutrient solution obtained adds diatomite drainage, by the filtrate that obtains with isopyknic extraction into ethyl acetate twice.The extraction liquid concentrating under reduced pressure obtained is obtained oily resistates.By oily resistates and silica gel (E.merk produces for Kiesel Gel160,70 ~ 230 orders) mixing, mixture makes jelly in methyl alcohol, evaporative removal solvent, and the dry powder obtained is carried out column chromatography.The elutriant solvent evaporation obtained obtains dry powder, again carries out column chromatography.Twice column chromatography uses identical silicagel column, silica gel and normal hexane fill post (mass volume ratio of silica gel and normal hexane is 4:1), developping agent is mixing solutions and the ethyl acetate of normal hexane, normal hexane and ethyl acetate, and in described normal hexane and the mixing solutions of ethyl acetate, the volume ratio of normal hexane and ethyl acetate is respectively 3:1,1:1 and 1:2.Collect the elutriant containing object compound, pressurization is concentrated, obtains the object compound of pale yellow powder, is dissolved in methyl alcohol: in methylene dichloride (v/v=10:1) solution, add the acetonitrile of two volumes, room temperature preservation, obtain the romidepsin of purifying.The yield of the last romidepsin of this purification process only has 31%, and industrial applications is restricted.
EP2124990A1 discloses a kind of method being separated romidepsin from fermented liquid, and the method is that fermented liquid sulfuric acid is adjusted pH to 2.5, acidifying fermentation liquid is added Sepabeads SP850 resin.First clean with tap water and 25% acetone, then carry out wash-out with 65% acetone.The elutriant obtained, is diluted with water to water content to 75%.By solution upper prop to Diaion HP20SS.Clean with 25% acetone and 40% acetone, with 47% acetone wash-out.By analysed by reverse phase HPLC elutriant, active part is merged in interceptor(-ter).Elutriant is diluted with water to containing 80% water yield, and upper prop, to DiaionHP20, washs with 20% aqueous acetone solution, then carries out wash-out with acetone.Elutriant carries out vacuum concentration, and concentrated solution adds ethyl acetate, reconcentration.The concentrated solution finally obtained, be dissolved in ethyl acetate, add aluminum oxide resin column to, with the mixing solutions of ethyl acetate/acetone and ethyl acetate, (wherein the volume ratio of ethyl acetate/acetone is 2:8, the volume ratio of ethyl acetate/acetone and ethyl acetate is 0.5:2) carry out wash-out, analyze and collect activated elutriant.Elutriant adds acetone and dissolves, and then adds methanol dilution, and vacuum concentration obtains active result crude extract, collecting by filtration active result crystal.Crystal is used 85% acetone solution, slowly add water, make Precipitation.Precipitation is cleaned with 15% acetone, and-70 DEG C of vacuum-dryings obtain the crystallization of purifying.Above per-cent is the volume of organic solvent: the volume of water and organic solvent.
From above-mentioned two kinds of methods, in prior art, the separation purification method purification procedures of romidepsin is too much, complicated operation, reagent and the instrument of purge process use are too much, the cost of separation and purification is caused to increase, there is irreversible adsorption in adsorption process, yield is lower and can not be applicable to industrial large-scale production.
Summary of the invention
In order to solve the separation purification method of romidepsin in prior art, to there is purification procedures too much in the present invention, complicated operation, reagent and the instrument of purge process use are too much, the cost of separation and purification is caused to increase, there is irreversible adsorption in adsorption process, yield is lower and can not be applicable to the defect of industrial mass production and provide a kind of separation purification method of romidepsin.Separation purification method DNA purity of the present invention is higher, and yield is high, and purifying process is simple, and cost is lower.
The invention provides a kind of separation purification method of romidepsin, it comprises the following step:
A. the fermented liquid of the chromobacterium violaceum (Chromobacterium violaceum) containing romidepsin is mixed with macroporous adsorbent resin, be stirred to absorption completely; Described macroporous adsorbent resin is one or more in nonpolar, low-pole and middle polarity macroporous resin;
B. separated from fermented liquid by the macroporous adsorbent resin of absorption romidepsin, upper prop, wash-out after cleaning, obtains elutriant 1, concentrated, obtains concentrated solution 1; The solvent of described cleaning is water; The solvent of described wash-out is organic solvent, or the mixed solvent 1 of organic solvent and water; In described mixed solvent 1, described organic solvent and the volume ratio of water are 3.5:6.5 ~ 6:4; Described organic solvent is ketones solvent, alcoholic solvent or nitrile solvents;
C. by described concentrated solution 1 loading to macroporous adsorbent resin, upper prop, wash-out after washing, detects with HPLC in elution process, collects elutriant 2, concentrated, obtains concentrated solution 2; The solvent of described washing is the mixed solvent 2 of organic solvent and water, and in described mixed solvent 2, described organic solvent and the volume ratio of water are 1:9 ~ 5:5; The solvent of described wash-out is the mixed solvent 3 of organic solvent and water, and in described mixed solvent 3, described organic solvent and the volume ratio of water are 3.5:6.5 ~ 7:3; Described organic solvent is ketones solvent, alcoholic solvent or nitrile solvents;
D. described concentrated solution 2 is extracted, obtain organic layer, concentrated, obtain crude product;
E. by described crude product refining, to obtain final product.
In step a, the mass volume ratio of described macroporous adsorbent resin and described fermented liquid is preferably 0.01g/mL ~ 0.06g/mL.The time of described stirring can be the time of this area routine, with described fermented liquid and absorption with macroporous adsorbent resin completely till, the time of described stirring is preferably 20 ~ 60min, is more preferably 20 ~ 40min.Described absorption is completely preferably fermented liquid lighter, more preferably for fermented liquid color is from the brown oyster white that becomes, best, detects to absorption complete by HPLC.
In step a and/or c, described macroporous adsorbent resin independently be one or more in nonpolar, low-pole and middle polarity macroporous resin, be preferably one or more in X-5, XAD-2, XAD-7, ADS-17, SP825, Diaion HP50, D101, HPD-100, HPD-300, AB-8, DA-201 and HPD-400, being more preferably one or more in X-5, XAD-2, XAD-7, HPD-100, HPD-300, AB-8, DA-201 and HPD-400, is one or more in SP825, Diaion HP50 and D101 best.
In step b, the solvent of described cleaning is water.In described mixed solvent 1, described organic solvent and the volume ratio of water are preferably 4:6 ~ 5:5.Described ketones solvent is preferably acetone.Described alcoholic solvent is preferably for carbon chain lengths is C
1~ C
3alkyl alcohol.Described carbon chain lengths is C
1~ C
3alkyl alcohol be preferably one or more in methyl alcohol, ethanol and Virahol, be more preferably ethanol.Described nitrile solvents is preferably acetonitrile.
In step b, the described concentrated concentration method that can adopt this area routine is preferably concentrating under reduced pressure.The color of described concentrated solution 1 is preferably brownish black.
In step c, in described mixed solvent 2, described organic solvent and the volume ratio of water are preferably 2:8 ~ 3.5:6.5.In described mixed solvent 3, described organic solvent and the volume ratio of water are 4:6 ~ 5:5.Described ketones solvent is preferably acetone.Described alcoholic solvent is preferably for carbon chain lengths is C
1~ C
3alkyl alcohol.Described carbon chain lengths is C
1~ C
3alkyl alcohol be preferably one or more in methyl alcohol, ethanol and Virahol, be more preferably ethanol.Described nitrile solvents is preferably acetonitrile.The flow velocity sv of described washing is preferably for being less than or equal to 7, and the flow velocity sv of described wash-out is preferably for being less than or equal to 5.
In step c, the mass volume ratio of described macroporous adsorbent resin and described concentrated solution 2 is preferably 0.03g/mL ~ 0.1g/mL.The described concentrated concentration method that can adopt this area routine is preferably concentrating under reduced pressure.The color of described concentrated solution 2 is preferably light brown.
In step c, the method that described HPLC detects and condition are preferably for chromatographic instrument is Waters510 type high performance liquid chromatograph, chromatographic column is C18 post, internal diameter is 4.6mm, column length is 250mm(4.6 × 250mm), the particle diameter of filler is 5 μm, determined wavelength is 210nm, moving phase is acetonitrile: 0.3% ammonium dihydrogen phosphate (v/v=30:70) (0.3% ammonium dihydrogen phosphate refers to that massfraction is the ammonium dihydrogen phosphate of 0.3%), flow velocity is 1mL/min, sample size is 20 μ L, and column temperature is 30 DEG C.
In steps d, the solvent of described extraction can be the conventional solvent of this area extraction, is preferably esters solvent and/or halogenated hydrocarbon solvent.Described esters solvent is preferably ethyl acetate.Described halogenated hydrocarbon solvent is preferably methylene dichloride and/or trichloromethane, is more preferably methylene dichloride.The number of times of described extraction is preferably twice.
In step e, described refining method and condition can be this area and refine conventional method and condition, preferably, adopt crystallization, or recrystallization two kinds of methods.Described crystallization preferably comprises the following steps: crude product steps d obtained, adds ether solvent, separates out solid.Described recrystallization preferably comprises the following steps: crude product steps d obtained, be dissolved in the mixed solvent 6 of the mixed solvent 5 of the mixed solvent 4 of ketones solvent, ketones solvent and water, alcoholic solvent, alcoholic solvent and water, nitrile solvents or nitrile solvents and water, add water more slowly to crystallization.
Wherein, described ether solvent is preferably ether.The consumption of described ether is 20 ~ 50mL/g with the volume mass ratio of described crude product.In described mixed solvent 4, described ketones solvent and the volume ratio of water are preferably 6:4 ~ 9:1; Be more preferably 7:3 ~ 9:1.In described mixed solvent 5, described alcoholic solvent and the volume ratio of water are preferably 6:4 ~ 9:1; Be more preferably 7:3 ~ 9:1.In described mixed solvent 6, described nitrile solvents and the volume ratio of water are preferably 6:4 ~ 9:1; Be more preferably 7:3 ~ 9:1.Described ketones solvent is preferably acetone.Described alcoholic solvent is preferably for carbon chain lengths is C
1~ C
3alkyl alcohol.Described carbon chain lengths is C
1~ C
3alkyl alcohol be preferably methyl alcohol, ethanol or Virahol, be more preferably methyl alcohol or ethanol.Described nitrile solvents is preferably acetonitrile.The consumption of described recrystallization solvent and the volume mass of described crude product are 15 ~ 20mL/g than preferably.
The separation purification method of above-mentioned romidepsin is applicable to the fermented liquid of the chromobacterium violaceum (Chromobacterium violaceum) containing romidepsin of all routines.
In the present invention, described chromobacterium violaceum (Chromobacterium violaceum), commercially.Preferably, purchased from Fermentation Research Institute Agency of Industrial Science andTechnology, Japan, is numbered chromobacterium violaceum (Chromobacteriumviolaceum) WB968 of FERM BP-1968, see US7608280B2.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is:
Romidepsin can be separated by the inventive method effectively from chromobacterium violaceum (Chromobacteriumviolaceum) fermented liquid, and wherein romidepsin purity can reach more than 99%, yield about 90%.Separation purification method DNA purity of the present invention is higher, and yield is high, and purifying process is simple, and cost is lower, simultaneous adaptation suitability for industrialized production.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
In the present invention, described chromobacterium violaceum (Chromobacterium violaceum), commercially.Preferably, purchased from Fermentation Research Institute Agency of Industrial Science andTechnology, Japan, is numbered chromobacterium violaceum (Chromobacteriumviolaceum) WB968 of FERM BP-1968, see US7608280B2.
In the present embodiment, the acquisition of the fermented liquid of the chromobacterium violaceum (Chromobacteriumviolaceum) containing romidepsin is carried out as follows.
To verify through throughput, well-grown chromobacterium violaceum (Chromobacteriumviolaceum) WB968 using skimmed milk as protective material, preserve by the mode of vacuum freezing.During use, chromobacterium violaceum (Chromobacterium violaceum) WB968 is transferred to plate culture medium, cultivate 2 days for 28 DEG C, then separation and purification 1 time, qualified chromobacterium violaceum (Chromobacteriumviolaceum) WB968 is transferred to slant medium, cultivate 1 day for 28 DEG C, be stored in 4 DEG C of refrigerators, in 7 days, can be used as chromobacterium violaceum (Chromobacterium violaceum) WB968 of fermentation culture at any time.Fermentation culture conditions is 30 DEG C, 1 day shake-flask culture time, and rotating speed is 220r/min.Fermentation condition is 25 DEG C, and the parameters such as fermenting process regulation and control dissolved oxygen, mixing speed, air flow, control mixing speed and air flow controls dissolved oxygen 20%, and add glucose, amino acid and water.
When the romidepsin in chromobacterium violaceum (Chromobacterium violaceum) the WB968 fermented liquid containing romidepsin tire no longer include obvious ascendant trend, other fermentation parameters performance fermentation just can put tank close to during finishing steps, fermentation time is generally 2 days.
Used medium is filled a prescription, specific as follows: slant medium (g/L): yeast extract paste 5, sodium-chlor 5, fish meal protein peptone 10, glucose 5, pH7.0; Seed culture based formulas is (g/L): yeast extract paste 5, fish meal protein peptone 10, sodium-chlor 5, glucose 10; Fermentative medium formula is (g/L): corn steep liquor 10, fish meal protein peptone 15, analysis for soybean powder 10, sodium-chlor 3, N.F,USP MANNITOL 25, glucose 15, Rice oil 2g.
Embodiment 1
A. by chromobacterium violaceum (Chromobacterium violaceum) the WB968 fermented liquid 3L containing romidepsin, add 150g SP825 macroporous adsorbent resin, stir 20min, detect absorption completely to HPLC.
B. will be adsorbed with romidepsin macroporous adsorbent resin to separate from fermented liquid, will the SP825 upper prop of romidepsin be adsorbed with, with tap water cleaning to clarification.With 1.5L acetone, romidepsin is eluted, obtain elutriant 1.Concentrating under reduced pressure operation is carried out to elutriant 1, the concentrated solution 1 of the brownish black obtained.
C. by concentrated solution 1 loading to SP825 macroporous adsorbent resin (mass volume ratio of SP825 and concentrated solution 1 is 0.07g/mL), be 3:7 by the volume ratio of acetone and water mixed solvent 2(acetone and water) wash, flow velocity sv controls to be no more than 7, and then acetone and the mixed solvent 3(acetone of water and the volume ratio of water be 4.5:5.5) carry out wash-out, flow velocity sv is no more than 5.Elution process HPLC detects, and collects containing romidepsin elutriant 2, altogether 1L.Concentrating under reduced pressure is carried out to elutriant 2, obtains the concentrated solution 2 of 500mL light brown.
D. extract concentrated solution 2 by isopyknic ethyl acetate, altogether extracting twice, collect ethyl acetate layer, concentrating under reduced pressure obtains lurid powder.
E. add anhydrous diethyl ether 25mL, separate out pale yellow powder is romidepsin product, vacuum-drying, the pale yellow powder shape material obtained.
The romidepsin finally obtained detects through HPLC, and purity is 97%, and yield is 80%.
Embodiment 2
Other conditions are constant relative to embodiment 1, and churning time being changed becomes 35min, and the romidepsin finally obtained detects through HPLC, and purity is 97%, and yield is 85%.
Embodiment 3
Other conditions are constant relative to embodiment 1, and churning time being changed becomes 60min, and the romidepsin finally obtained detects through HPLC, and purity is 93%, and yield is 80%.
Embodiment 4
Other conditions are constant relative to embodiment 1, and by extraction solvent by ethyl acetate, change and become methylene dichloride, the romidepsin finally obtained detects through HPLC, and purity is 97%, and yield is 87%.
Embodiment 5
Other conditions are constant relative to embodiment 1, and being changed by the SP825 in embodiment 1 becomes XAD-7, and the romidepsin finally obtained detects through HPLC, and purity is 91%, and yield is 87%.
Embodiment 6
Other conditions are constant relative to embodiment 1, and being changed by the SP825 in embodiment 1 becomes DiaionHP50, and the romidepsin finally obtained detects through HPLC, and purity is 96%, and yield is 86%.
Embodiment 7
Other conditions are constant relative to embodiment 1, and being changed by the SP850 in embodiment 1 becomes DA-201, and the romidepsin finally obtained detects through HPLC, and purity is 91%, and yield is 85%.
Embodiment 8
Other conditions are constant relative to embodiment 1, and being changed by the SP850 in embodiment 1 becomes D101, and the romidepsin finally obtained detects through HPLC, and purity is 97%, and yield is 88%.
Embodiment 9
Be methyl alcohol by the solvent replacement of wash-out in embodiment 1 step b, be the mixed solvent 2(methyl alcohol of methyl alcohol and water and the volume ratio of water by the solvent replacement washed in step c be 4:6), the solvent replacement of wash-out is the mixed solvent 3(methyl alcohol of methyl alcohol and water and the volume ratio of water is 6.5:3.5), other conditions are constant relative to embodiment 1, the romidepsin finally obtained detects through HPLC, purity is 97%, and yield is 83%.
Embodiment 10
Be ethanol by the solvent replacement of wash-out in embodiment 1 step b, be the mixed solvent 2(ethanol of ethanol and water and the volume ratio of water by the solvent replacement washed in step c be 3.5:6.5), the solvent replacement of wash-out is the mixed solvent 3(ethanol of ethanol and water and the volume ratio of water is 5:5), other conditions are constant relative to embodiment 1, the romidepsin finally obtained detects through HPLC, purity is 97%, and yield is 84%.
Embodiment 11
Recrystallization solvent in the step e of embodiment 1 is replaced by the mixed solvent 4 of acetone and water, the crude powder obtained in steps d is dissolved in acetone and (every 10g crude powder add 150mL acetone and water mix broad dose) in the mixed solvent (acetone is 7:3 with the volume ratio of water) of water, then purified water is added slowly, until crystallization, other conditions are constant relative to embodiment 1, the romidepsin finally obtained detects through HPLC, and purity is 90%, and yield is 85%.
Embodiment 12
Recrystallization solvent in the step e of embodiment 1 is replaced by the mixed solvent 5 changed and become ethanol and water.The crude powder obtained in steps d is dissolved in the mixed solvent (volume ratio of ethanol and water is 9:1) of ethanol and water (every 10g crude powder adds the mixed solvent of 200mL ethanol and water), then purified water is added slowly, until crystallization, other conditions are constant relative to embodiment 1, the romidepsin finally obtained detects through HPLC, purity is 91%, and yield is 84%.
Embodiment 13
Other conditions are constant relative to embodiment 8, the volume ratio of the mixed solvent 4(acetone and water that the pale yellow powder of separating out with anhydrous diethyl ether are dissolved in acetone and water is 8:2) in (in every 10g powder, adding the mixed solvent of 180mL acetone and water), then purified water is added slowly, until crystallization, the romidepsin finally obtained detects through HPLC, purity is 99%, and yield is 80%.
Comparative example 1
Other conditions are constant relative to example 1, and churning time being changed becomes 5min, and the romidepsin finally obtained detects through HPLC, and purity is 97%, and yield is 65%.
Comparative example 2
Other conditions are constant relative to example 1, and being changed by wherein used SP825 macroporous adsorbent resin becomes ADS-107 macroporous adsorbent resin, and the romidepsin finally obtained detects through HPLC, and purity is 85%, and yield is 78%.
Comparative example 3
Other conditions are constant relative to embodiment 1, be the volume ratio that 3:7 replaces acetone and water are 0.5:9.5 by the volume ratio of the acetone in washing process in step c and water, and the romidepsin finally obtained detects through HPLC, and purity is 78%, and yield is 88%.
Comparative example 4
Other conditions are constant relative to embodiment 1, be the volume ratio that 3:7 replaces acetone and water are 5.5:4.5 by the volume ratio of the acetone in washing process in step c and water, and the romidepsin finally obtained detects through HPLC, and purity is 92%, and yield is 75%.
Comparative example 5
Other conditions are constant relative to embodiment 1, be the volume ratio that 4.5:5.5 replaces acetone and water are 3:7 by the volume ratio of the acetone in elution process in step c and water, and the romidepsin finally obtained detects through HPLC, and purity is 72%, and yield is 80%.
Comparative example 6
Other conditions are constant relative to embodiment 1, be the volume ratio that 4.5:5.5 replaces acetone and water are 7.5:3.5 by the volume ratio of the acetone in elution process in step c and water, and the romidepsin finally obtained detects through HPLC, and purity is 70%, and yield is 89%.
Claims (10)
1. a separation purification method for romidepsin, is characterized in that, it comprises the following step:
A. the fermented liquid of the chromobacterium violaceum (Chromobacterium violaceum) containing romidepsin is mixed with macroporous adsorbent resin, be stirred to absorption completely; Described macroporous adsorbent resin is one or more in nonpolar, low-pole and middle polarity macroporous resin;
B. the macroporous adsorbent resin of absorption romidepsin is separated from fermented liquid, upper prop, wash-out after cleaning, collect the elutriant 1 containing romidepsin component, concentrated, obtain concentrated solution 1; The solvent of described cleaning is water; The solvent of described wash-out is organic solvent, or the mixed solvent 1 of organic solvent and water; In described mixed solvent 1, described organic solvent and the volume ratio of water are 3.5:6.5 ~ 6:4; Described organic solvent is ketones solvent, alcoholic solvent or nitrile solvents;
C. by described concentrated solution 1 loading to macroporous adsorbent resin, upper prop, wash-out after washing, detects with HPLC in elution process, collects containing the elutriant 2 of romidepsin component, concentrated, obtains concentrated solution 2; The solvent of described washing is the mixed solvent 2 of organic solvent and water; In described mixed solvent 2, described organic solvent and the volume ratio of water are 1:9 ~ 5:5; The solvent of described wash-out is the mixed solvent 3 of organic solvent and water; In described mixed solvent 3, described organic solvent and the volume ratio of water are 3.5:6.5 ~ 7:3; Described organic solvent is ketones solvent, alcoholic solvent or nitrile solvents;
D. described concentrated solution 2 is extracted, obtain organic layer, concentrated, obtain crude product;
E. by described crude product refining, to obtain final product.
2. separation purification method as claimed in claim 1, it is characterized in that, in step a, the mass volume ratio of described macroporous adsorbent resin and described fermented liquid is 0.01g/mL ~ 0.06g/mL; The time of described stirring is 20 ~ 60min; Described absorption is entirely fermented liquid lighter.
3. separation purification method as claimed in claim 1, it is characterized in that, in step a and/or c, described macroporous adsorbent resin independently be one or more in X-5, XAD-2, XAD-7, ADS-17, SP825, Diaion HP50, D101, HPD-100, HPD-300, AB-8, DA-201 and HPD-400.
4. separation purification method as claimed in claim 1, it is characterized in that, in step b, in described mixed solvent 1, described organic solvent and the volume ratio of water are 4:6 ~ 5:5; The color of described concentrated solution 1 is brownish black.
5. separation purification method as claimed in claim 1, it is characterized in that, in step b and/or c, described ketones solvent is acetone; Described alcoholic solvent is carbon chain lengths is C
1~ C
3alkyl alcohol; Described nitrile solvents is acetonitrile.
6. separation purification method as claimed in claim 1, it is characterized in that, in step c, in described mixed solvent 2, described organic solvent and the volume ratio of water are 2:8 ~ 3.5:6.5; In described mixed solvent 3, described organic solvent and the volume ratio of water are 4:6 ~ 5:5; The mass volume ratio of described macroporous adsorbent resin and described concentrated solution 2 is 0.03g/mL ~ 0.1g/mL; The color of described concentrated solution 2 is light brown.
7. separation purification method as claimed in claim 1, it is characterized in that, in steps d, the solvent of described extraction is esters solvent and/or halogenated hydrocarbon solvent; The number of times of described extraction is twice.
8. separation purification method as claimed in claim 7, it is characterized in that, described esters solvent is ethyl acetate; Described halogenated hydrocarbon solvent is methylene dichloride and/or trichloromethane.
9. separation purification method as claimed in claim 1, is characterized in that, in step e, described refining method is crystallization or recrystallization; Described crystallization comprises the following steps: crude product steps d obtained, adds ether solvent, separates out solid; Described recrystallization comprises the following steps: crude product steps d obtained, be dissolved in the mixed solvent 6 of the mixed solvent 5 of the mixed solvent 4 of ketones solvent, ketones solvent and water, alcoholic solvent, alcoholic solvent and water, nitrile solvents or nitrile solvents and water, add water again to crystallization.
10. separation purification method as claimed in claim 9, it is characterized in that, described ether solvent is ether; In described mixed solvent 4, described ketones solvent and the volume ratio of water are 6:4 ~ 9:1; In described mixed solvent 5, described alcoholic solvent and the volume ratio of water are 6:4 ~ 9:1; In described mixed solvent 6, described nitrile solvents and the volume ratio of water are 6:4 ~ 9:1.
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| CN109796521A (en) * | 2017-11-17 | 2019-05-24 | 上海医药工业研究院 | A kind of romidepsin acetate crystal form and preparation method thereof |
| CN111187337A (en) * | 2018-11-15 | 2020-05-22 | 上海医药工业研究院 | Romidepsin-isopropanol solvate and crystal form, preparation method and application thereof |
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Cited By (5)
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| WO2016037530A1 (en) * | 2014-09-11 | 2016-03-17 | 浙江海正药业股份有限公司 | Novel polymorph of romidepsin, pharmaceutical composition of same, preparation method for same, and uses thereof |
| CN109796521A (en) * | 2017-11-17 | 2019-05-24 | 上海医药工业研究院 | A kind of romidepsin acetate crystal form and preparation method thereof |
| CN109796521B (en) * | 2017-11-17 | 2022-04-19 | 上海医药工业研究院 | Romidepsin acetate crystal form and preparation method thereof |
| CN111187337A (en) * | 2018-11-15 | 2020-05-22 | 上海医药工业研究院 | Romidepsin-isopropanol solvate and crystal form, preparation method and application thereof |
| CN111187337B (en) * | 2018-11-15 | 2023-01-24 | 上海医药工业研究院 | Romidepsin-isopropanol solvate and crystal form, preparation method and application thereof |
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