CN102392053A - Method for preparing paricalcitol - Google Patents
Method for preparing paricalcitol Download PDFInfo
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- CN102392053A CN102392053A CN2011102793112A CN201110279311A CN102392053A CN 102392053 A CN102392053 A CN 102392053A CN 2011102793112 A CN2011102793112 A CN 2011102793112A CN 201110279311 A CN201110279311 A CN 201110279311A CN 102392053 A CN102392053 A CN 102392053A
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Abstract
The invention discloses a method for preparing drug paricalcitol for treating osteoporosis, which comprises the following steps of: taking vitamin D2 as raw materials and obtaining a target object through the steps of chemical reaction and biological reaction; omitting the use of an expensive toxic dihydroxylation reagent and bond-breaking dihydroxylation adopting cheap effective reagent oxide potassium permanganate solution into ketone; isomerizing into olefin under an alkaline reagent, carrying out hydroboration reaction by applying a borohydride reagent 9-BBN, borane and other reagents; introducing 1-hydroxyl, ring-opening to obtain 19-demethyl vitamin D2, taking pseudonocardiaautotrophica SEJTUQL-3701021 (Autotrophic false Nocardia CGMCC 5098) as hydroxylation bacteria, carrying out 25-direct hydroxylation by using a biotransformation method and obtaining the paricalcitol through extraction and separation. Through a chemical-biological transformation combination method, the reaction steps of the paricalcitol are shortened from known 27 to 9, so that the total yield is improved, and the process route is shortened. The invention provides a new process route for solving the industrial preparation of the paricalcitol.
Description
Technical field
The present invention relates to chemistry and bio-pharmaceuticals, the medicine 1-Alpha-hydroxy-19-that especially treats osteoporosis removes the methylene radical vitamins D
2Technical field.
Background technology
Zemplar (Paricalcitol) is the novel active vitamin drug of Abbott company in exploitation in 1998, and commodity Zemplar by name has obtained FDA approval listing, is used for prevention and treatment SHPT animal clinically.The pharmacological action that experiment and clinical studies show paricalcitol have a relative selectivity can suppress secretion and the parathyroid hyperplasia of Rat parathyroid hormone 1-34 safely and effectively and seldom cause hypercalcemia and hyperphosphatemia, and alternative CALCITRIOL USP (calcitriol) is used for the treatment that uremia is specially adapted to CH patient SHPT.
1-Alpha-hydroxy-19-removes the methylene radical vitamins D
2(1 α-19-nor-VD
2), i.e. the known synthetic employing mostly of Zemplar (paricalcitol) is complete synthesis, mainly contains two types of synthetic routes, and the one, adopt vitamins D like the said method of patent US7645911
2Or 25 hydroxy-vitamine D
2As raw material, through overprotection, 1 hydroxylation, 10,19 two key perosmic anhydride/sodium periodate scission of links, reduction, sulfonylation, LiAlH
4Synthetic or the protection of the step of reduction etc. or 10,19 two key perosmic anhydride/sodium periodate scission of links, 1,10 pair of key become alkene, and hydroboration introduces 1 hydroxyl, protection, 22,23 ozone scission of links, it is synthetic to introduce 25 hydroxyl side chains etc.; Adopt 25 hydroxy-vitamine Ds like patent US5237110, the said method of US5246925
2As raw material, through overprotection, 1 hydroxylation, 10,19 two key perosmic anhydride/sodium periodate scission of links, reduction, sulfonylation, LiAlH
4The step of reduction etc. is synthetic.The 2nd, like said employing vitamins Ds of patent such as patent US5086191, US5281731/5391755, US5486636, US5525745
2As raw material, adopt disconnected 6,7 the two keys of ozone; Obtain A ring and CD ring respectively, perhaps, pass through various chemically modifieds more respectively with the synthetic respectively A cyclization CD ring of chiral synthon; The A ring of having removed 10 methylene radical is reacted and the CD cyclic condensation through wittig; Again through Wittig-Horner reaction, the Suzuki-Miyaura linked reaction, Julia-type becomes alkene reaction etc. multistep is synthetic to obtain.The second method step is various, repeatedly carries out chiral separation, and yield is extremely low, and it is very uneconomical to be used for synthetic Zemplar.The first method step is less relatively, but still will use the expensive perosmic anhydride of severe toxicity as alkene bishydroxy reagent, or uses 25 rare hydroxy-vitamine Ds
2Do raw material, perhaps use ozone 22, the 23 pairs of keys that break, introduce 25 hydroxyl side chains again, still operation is too many on producing, complicated operation, and yield is on the low side, total recovery only about 0.1%.
Summary of the invention
Above deficiency in view of prior art the purpose of this invention is to provide a kind of novel method for preparing Zemplar, makes it to have the production process simplification, yield is high and gets rid of the expensive perosmic anhydride of severe toxicity as alkene bishydroxy reagent advantages of application.
The objective of the invention is to realize through following means.
A kind of method for preparing Zemplar is with vitamins D
2Be raw material, adopt like Fig. 1 expressed chemical reaction and biological respinse step and obtain treatment osteoporosis agents Zemplar: 1-Alpha-hydroxy-19-removes the methylene radical vitamins D
2(ix):
The chemical reaction part:
Potassium permanganate solution is under-20 ℃, and with alcoholic solvent dissolving cyclisation thing (iii): dripping concentration is the potassium permanganate solution of 5-20%, and stirring reaction 1-10 hour best 3 hours, concentrate and remove water, resistates is used with the volume alcohol dilution; Under 0 ℃, add the sodium periodate aqueous solution of 10-20%, stirred 2-5 hour, best 3 hours, with the chloroparaffin of an equal-volume 1-5 carbon, the extractions such as carboxylicesters of 1-5 carbon were concentrated into driedly, obtain 10 carbonyl cyclisation vitamins Ds
2(iv); Under-78 ℃, drip the highly basic of non-polar solvent, add Trifluoromethanesulfonic anhydride, two (trifluoromethane sulphonyl) imines of N-phenyl, obtain enol base trifluoromethayl sulfonic acid ester (V); The cyclohexene derivative that obtains 1,10 pair of key with formic acid and palladium reduction (vi), uses hydroborating agents to obtain 1-hydroxy-19-nor vitamins D through the alkaline hydrogen peroxide oxidation
2(vii), adopt the low molecular organic acids open loop, obtain 1-hydroxyl-10 nor-vitamin D through 4% sodium hydrate methanol solution hydrolysis again after the washing
2(viii);
The biological respinse part
1-hydroxy-19-nor vitamins D
2(viii) as substrate with bacterial strain CGMCC 5098 under the state of aeration-agitation, in the substratum of carbon source, nitrogenous source, mineral salt, carry out bio-transformation, leavening temperature 22-30 ℃; Preferably 27 ℃; PH scope 6.5-7.5, preferably 7.0, air flow is at 0.5-1.0v/v/min; With 1-hydroxy-19-nor vitamins D
2(viii) join in the nutrient solution as substrate; Concentration of substrate is the 1-15g/ liter preferably; The time that substrate adds substratum normally adds substrate the best nearly when carbon source runs out of; The consumption of carbon source can be controlled through the concentration of measuring sugar, adds substrate after normally dropping to 0.05-0.002%;
In the process that continues above-mentioned cultivation, produce 25 hydroxylation bio-transformation, the time of conversion is depended on the composition and the bacterial strain of substratum, continues usually to cultivate preferably 4 days 1-7 days.After solid-liquid separation is used the organic solvent extracting to mycelia and filtrating respectively, through silica gel column chromatography, the acetone recrystallization obtains pure title product 1-hydroxyl-25-hydroxy-19-nor vitamins D
2(ix), i.e. Zemplar.
The method for preparing Zemplar that the present invention adopts chemistry and bio-transformation to combine adopts low temperature potassium permanganate solution bishydroxy reaction cheap and easy to get to replace the expensive perosmic anhydride of severe toxicity on 10,19 pairs of key scission of links; Adopt biotransformation method to carry out 25 direct hydroxylation, gentleer than chemical method, need not very low temperature and ozone scission of link; And have regioselectivity, and need not carry out radical protection, 25 hydroxylation bio-transformation yields are high; Environmental friendliness substitutes 25 hydroxylated committed steps of chemical method, simplifies technical process; Through chemosynthesis and microbial transformation is that bonded method, shortens operational path greatly, improves yield; Reduce manufacturing cost; Known synthetic Zemplar 27 step reaction is shortened to the reaction of 9 steps, and total recovery reaches 3%, has improved more than 10 times than about 0.1% yield of existing public reported.
Description of drawings
Fig. 1 is the process route chart of the inventive method.
Embodiment
Below in conjunction with embodiment enforcement of the present invention is done further to describe.
Technical scheme route of the present invention is seen Fig. 1
Chemical part: chemosynthesis
Starting raw material of the present invention is a vitamins D
2, the method for (US4195027) such as employing DeLuca changes into 3,5-cyclisation vitamins D
2(iii), two keys of following 10,19 carry out bishydroxy, and the present invention adopts potassium permanganate solution under-20 ℃; With ethanol, propyl alcohol, Virahol etc. be the above-mentioned cyclisation thing of dissolution with solvents (iii), dripping concentration is the potassium permanganate solution of 5-20%, stirring reaction 1-10 hour, best 3 hours; Concentrate to remove water, resistates is used with the volume alcohol dilution, under 0 ℃, adds the sodium periodate aqueous solution of 10-20%; Stirred 2-5 hour, best 3 hours, with the chloroparaffin of an equal-volume 1-5 carbon; The extractions such as carboxylicesters of 1-5 carbon are concentrated into driedly, obtain 10 carbonyl cyclisation vitamins Ds
2(iv).10 carbonyl cyclisation vitamins Ds
2Be dissolved in non-polar solvent, like hexanaphthene, THF, ether, methylene dichloride etc., under-78 ℃; Drip the highly basic of foregoing non-polar solvent; Like hexamethyl two amido lithiums, n-Butyl Lithium, tert-butyl lithium, sodium amide etc., add Trifluoromethanesulfonic anhydride, two (trifluoromethane sulphonyl) imines of N-phenyl, obtain enol base trifluoromethayl sulfonic acid ester.Obtain 1 with formic acid and palladium reduction; The cyclohexene derivative of 10 pairs of keys (vi); Use common hydroborating agents, such as 9-boron two ring [3.3.1] nonanes (9-BBN), borine, diisopinocampheylchloroborane base chloroborane; Diethylammonium methoxyl group borine or the like obtains 1-hydroxy-19-nor vitamins D through the alkaline hydrogen peroxide oxidation
2((US4195027, low molecular organic acids US4260549) such as acetate open loop obtain 1-hydroxyl-10 nor-vitamin D through 4% sodium hydrate methanol solution hydrolysis after the washing more vii), to adopt DeLuca etc.
2(viii).
Biological part:
The contriver separates from the soil sample that Sichuan two E Shan take and obtains a strain bacterium QL-3721001, has 1-hydroxy-19-nor vitamins D
2(viii) have conversion capability, obtain bacterial strain QL-3721021 through natural seed selection and ultraviolet mutagenesis and have higher 25 more single-minded hydroxylation abilities, called after SWJTUQL-3721021.Adopt synthetic No. 1 nutrient agar of Gao Shi to carry out morphological specificity and observe, the substratum that adopts international chain mould plan (ISP) to recommend carries out cultural characteristic to be observed and serial physiological and biochemical test, and cultural characteristic is seen table 1; This bacterium fibrillae of spores is directly and slightly gentle bent; Several no branches, bacterium colony is fine and close, smooth surface.Bacterial strain can not liquefy gelatin, and starch is hydrolysis slightly, and milk peptonizes.To glucose, inositol, the D-seminose, N.F,USP MANNITOL, L-arabinose, fructose, rhamnosyl, raffinose, sugar alcohols such as D-wood sugar all have the ability of utilizing in various degree.Nitrate salt does not reduce.Do not grow on the Mierocrystalline cellulose.Type of generation melanochrome and tyrosine oxidase do not produce hydrogen sulfide.This bacterium can utilize sodium hydrogencarbonate to be sole carbon source.But also heterotrophic growth.Bacterium colony is a white, and the bacterium colony quality of formation is tight, and the pigmentary colours that the back side produces are little tawny.Bacterial strain produces two kinds of mycelia, substrate mycelium and aerial hyphae; Can also see simultaneously the thicker fibrillae of spores of black, be grown on the long and straight branch, conidium Dan Sheng, wall thickness has diaphragm, is DNA releaxed circular DNA or hook-shaped, and the ellipticity spore that is scattered is arranged.
The morphological specificity of table 1 bacterial strain SWJTUQL-3721021 on each substratum
According to outstanding bacterium handbook of uncle and above mensuration result; Preliminary mirror SWJTUQL-3721021 bacterial strain belongs to false Nocardia bacteria actinomyces actinoides; And name into false Nocardia bacteria SWJTUQL-3721021 (Pseudonocardia autotrophica SWJTUQL-3721021), deposit number is CGMCC No.5098.
Bacterial strain CGMCC No.5098 bio-transformation 1-hydroxy-19-nor vitamins D
2(conversion process viii):
Technology of the present invention relate to bacterial strain CGMCC No.5098 in the substratum of ventilation, stirring, carbon source, nitrogenous source, mineral salt to 1-hydroxy-19-nor vitamins D
2(viii) carry out bio-transformation, obtain 1-hydroxyl-25-hydroxy-19-nor vitamins D
2(ix), i.e. Zemplar.
Transform and cultivate and to carry out shaking in bottle or the fermentor tank of ventilation; Nutritive substance comprises that inorganic salt, nitrogenous source, sugar or other soluble substance are as carbon source; Inorganic salt comprise muriate, nitrate salt, carbonate, vitriol or the phosphoric acid salt of basic metal, earth alkali metal such as magnesium, iron, zinc, manganese; MgSO4 preferably, KH
2PO4.Nitrogenous source can be an ammonium salt, comprises Citrate trianion, tartrate, malate, SUMATRIPTAN SUCCINATE, oxalate, acetate etc.; Amino acid and mixture, peptide or protein and hydrolyzate thereof, the water soluble extract of meat extract, cereal such as corn, wheat; Malted maize extract, corn impregnation liquid, bean cake powder, peanut powder, hawk bean powder, cottonseed meal.Carbon source can be glucose, sucrose, starch, dextrin, sorbose, seminose, lactose or the like.No matter solid culture still is that deep layer is cultivated and all under aeration condition, carried out, and leavening temperature 22-30 ℃, preferably 27 ℃, pH scope 6.5-7.5, preferably 7.0, air flow is at 0.5-1.0v/v/min.
Process of the present invention is used conventional microbial transformation process, can carry out as follows very easily, with 1-hydroxy-19-nor vitamins D
2(viii) join in the nutrient solution as substrate, concentration of substrate does not have particular requirement, preferably the 1-15g/ liter; In the process that continues above-mentioned cultivation, produce 25 hydroxylation bio-transformation; The time that transforms is depended on the composition and the bacterial strain of substratum, continues usually to cultivate preferably 4 days 1-7 days.
The time of substrate adding substratum is extremely important, normally when carbon source runs out of, adds substrate the best nearly, and the consumption of carbon source can be controlled through the concentration of measuring sugar, normally drops to 0.05-0.002%.Normally strain culturing added substrate after 2 days.
Seed culture medium: 1.5% glucose, 1.5% soybean cake powder, 0.5% steeping water, 0.5%NaCl, 0.2%CaCO3, pH nature.With 121 ℃ of substratum, sterilization 20min.
Fermention medium: 1.5% glucose, 2.0% soybean cake powder, 1.0% steeping water, 0.5%NaCl, 0.2%CaCO3, pH nature.With 121 ℃ of substratum, sterilization 20min.
The fermentation culture process: first order seed inserts fermention medium with the inoculum size of 1-10%, 28 ℃ of leavening temperatures, and air flow 0.5v/v/min cultivated 48 hours.
The adding form of substrate can be a substrate 1-hydroxy-19-nor vitamins D
2(viii) raw material solid or be dissolved in organic solvent such as ethanol, methyl alcohol, terepthaloyl moietie, polyoxyethylene glycol; Tensio-active agent or dispersion agent such as tween 20, tween-80; The interpolation of solubility promoter such as Schardinger dextrins etc. more helps the dissolving of substrate in nutrient solution, helps to improve transformation efficiency.The best ratio of substrate and solubility promoter, tensio-active agent is 1: 0.5: 2.
The converted product Zemplar adopts solid-liquid separation filtering technique commonly used in the chemical engineering; To mycelium with organic solvent such as ethanol, acetone extraction, concentrate the back with organic chloroparaffin, ester class, fragrant alkane like extractions such as methylene dichloride, ETHYLE ACETATE, toluene, concentrated; Filtrating with aforesaid organic solvent such as organic chloroparaffin, ester class, fragrant alkane like extractions such as methylene dichloride, ETHYLE ACETATE, toluene; Obtain the upper strata organic layer, merge organic layer, concentrate and obtain the Zemplar bullion; Again through silica gel column chromatography; Pure Zemplar component is collected as eluent in organic solvent-normal hexane, sherwood oil etc. and ethyl ester gradient or 2: 1, obtains pure Zemplar through the acetone recrystallization.
Embodiment one
1-hydroxy-19-nor vitamins D
2(preparation viii)
A) 10-oxo-3,5 cyclization vitamins Ds
2Synthesizing (iv)
3,5 cyclization vitamins Ds
2The method of (US4195027) such as synthetic employing DeLuca (iii) is with vitamins D
2Change into 3,5-cyclisation vitamins D
2(iii), in reaction flask, add 3.88g, 9.46mmol) 3,5 cyclization vitamins Ds
2, with ethanol 300ml dissolving, be chilled to-20 ℃, drip potassium permanganate solution (KMnO then
43.33g, H
2O 100ml), about 20 minutes.Under this temperature, stir 1h, 15min is stirred in the back under 40 ℃ of water-baths.Filter, filtrate decompression is concentrated into dried, gets light yellow oily mixture 4 (3.67g), and this bullion adds saturated sodium periodate 100ml, stirring at room 2.5h with ethanol 300ml dissolving.Reaction solution is with ETHYLE ACETATE (2 * 200ml) extractions, saturated nacl aqueous solution (1 * 200ml) washing.Organic layer is used anhydrous sodium sulfate dehydration, filters, and filtrate decompression is concentrated into dried, gets light yellow oil.Column chromatography [column chromatography silica gel, ETHYLE ACETATE: sherwood oil (1: 10)] separates purifies, and concentrating under reduced pressure gets light yellow oil 5 (1.42g, 36.5% liang of step of productive rate).
1H?NMR(CDCl
3，400)δ0.57(s，3H，18-CH
3)，0.76-0.91(m，9H)，3.21(s，3H，6-H)，4.54(d，1H，6-H)，4.71(d，1H，7-H)，5.17(m，2H，22，23-H)。
B) 3,5 cyclizations-1-alkene vitamins D
2(synthesizing vi)
In reaction flask, add hexamethyl two amido lithium (LiHMDS) 11ml, be chilled to-78 ℃.Add with THF dissolved 10-oxo-3 5 cyclization vitamins Ds
21.75g (4.18mmol) and two (trifluoromethane sulphonyl) imines (PhN (CF of N-phenyl
35O
2)
2) 3.6g.After stirring 12h, reaction solution concentrates, and adds ETHYLE ACETATE, and (2 * 250ml), (2 * 250ml), (1 * 250ml) washs salt solution saturated sodium bicarbonate to use 1% hydrochloric acid respectively.Organic layer is used anhydrous sodium sulfate dehydration, filters, and filtrate decompression is concentrated into dried, product (2.16g, productive rate 94.8%); With 40ml DMF dissolving, add triphenylphosphine 0.219g (0.837mmol), palladium 0.094g (0.418mmol), N; N-diisopropyl ethyl amine 2.3ml, formic acid 0.32ml, under 60 ℃, reaction 1h; (2 * 250ml) extractions, (2 * 500ml), (2 * 500ml), (1 * 250ml) washs salt solution saturated sodium bicarbonate reaction solution to use 1% hydrochloric acid respectively with ETHYLE ACETATE.Organic layer is used anhydrous sodium sulfate dehydration, filters, and filtrate decompression is concentrated into dried, gets light yellow oil.Column chromatography [column chromatography silica gel, ETHYLE ACETATE: sherwood oil (1: 70)] separate to be purified, concentrating under reduced pressure get light yellow oil (vi) 0.7g,, yield 45%.
1H?NMR(CDCl
3，400)δ0.57(s，3H，18-CH
3)，0.76-0.91(m，9H)，3.28(s，3H，6-H)，3.97(d，1H，6-H)，4.85(d，1H，7-H)，5.18(m，2H，22，23-H)，5.39(brd，1H，1-H)，5.93(td，1H，10-H)。
C) 1-hydroxy-19-nor vitamins D
2(synthesizing viii)
With compound 3,5 cyclizations-1-alkene vitamins D
2(vi) 0.5g dissolves with THF, in the time of 0 ℃, drips 0.5mol/L 9-BBN solution, stirring at room reaction 1.5h, and the back adds entry, 30%H at 0 ℃
2O
2With 3mol/L sodium hydroxide solution (1: 1), stirring at room 30min.Extract with ETHYLE ACETATE, organic layer is used 3% hydrochloric acid respectively, saturated sodium bicarbonate solution, saturated nacl aqueous solution washing; Anhydrous sodium sulfate dehydration filters, and filtrating is concentrated into dried, gets light yellow oil; Add acetic acid 3mL, in 60 ℃ of stirrings 20 minutes, reaction solution was with the dilution of saturated sodium bicarbonate liquid, with ETHYLE ACETATE 5mL extraction three times; Use the saturated common salt water washing, dehydrate, enriched material is dissolved in the 10%NaOH methanol solution, and 0 ℃ was stirred 1 hour; Water 10mL dilution, ETHYLE ACETATE (2 * 20ml) extractions, saturated nacl aqueous solution (1 * 20ml) washing.Organic layer is used anhydrous sodium sulfate dehydration, filters, and filtrate decompression is concentrated into dried, and column chromatography [column chromatography silica gel, ETHYLE ACETATE: sherwood oil (1: 5)] separates purifies, and gets 1-hydroxy-19-nor vitamins D
2(viii) solid 0.25g, yield 40%.
1H?NMR(400MHz，CD
3OD)δ6.21(d，1H，J＝11Hz，H6)，5.88(d，1H，J＝11Hz，H7)，5.35(dd，1H，J＝8?and?15Hz，H23)，5.27(dd，1H，J＝8?and?15Hz，H22)，4.04(m，1H，H1)，3.98(m，1H，H3)，2.84(brd，1H，J＝11Hz，H9a)，2.59(brd，1H，J＝13Hz，H4a)，2.41(brd，1H，J＝13Hz，H10a)，2.21(dd，1H，J＝8?and?13Hz，H4b)，2.16(dd，1H，J＝7?and?13Hz，H10b)，2.04(m，4H)，1.84(m，1H，H2a)，1.76(m，2H)，1.67(m，2H)，1.55(m，3H)，1.35(m，3H)，1.13(s，3H，H26)，1.09(s，3H，H27)，1.04(d，3H，J＝7Hz，H21)，0.99(d，3H，J＝7Hz，H28)，0.58(s，3H，H18)；m/z：400.33[M]
+。
Embodiment two
A) 10-oxo-3,5 cyclization vitamins Ds
2Synthesizing (iv)
3,5 cyclization vitamins Ds
2The method of (US4195027) such as synthetic employing DeLuca (iii) is with vitamins D
2Change into 3,5-cyclisation vitamins D
2(iii), 3,5-cyclization VD
25 grams are dissolved in 150 milliliters of ethanol, place-20 ℃ of low temperature to bathe, and drip 3%KMnO
4(3% aqueous solution) 320 milliliters drips off, and continues reaction 2 hours, and regularly TLC detects; 3 hours, raw material consumption finished, and 40 ℃ were stirred 15 minutes, solidified Manganse Dioxide; Diatomite filtration concentrates and removes alcohol, adds 100 milliliters in ETHYLE ACETATE, extracted twice; Be concentrated into driedly, this bullion adds saturated sodium periodate 100ml, stirring at room 2.5h with ethanol 300ml dissolving.Reaction solution is with ETHYLE ACETATE (2 * 200ml) extractions, saturated nacl aqueous solution (1 * 200ml) washing.Organic layer is used anhydrous sodium sulfate dehydration, filters, and filtrate decompression is concentrated into dried, gets light yellow oil.Column chromatography [column chromatography silica gel, ETHYLE ACETATE: sherwood oil (1: 10)] separates purifies, and concentrating under reduced pressure gets light yellow oil 5 (1.42g, 36.5% liang of step of productive rate).
B) 3,5 cyclizations-1-alkene vitamins D
2(synthesizing vi)
In reaction flask, add hexamethyl two amido lithium (LiHMDS) 15ml, be chilled to-78 ℃.Add with THF dissolved 10-oxo-3 5 cyclization vitamins Ds
22.35g (4.18mmol) and two (trifluoromethane sulphonyl) imines (PhN (CF of N-phenyl
3SO
2)
2) 3.6g.After stirring 12h, reaction solution concentrates, and adds ETHYLE ACETATE, and (2 * 250ml), (2 * 250ml), (1 * 250ml) washs salt solution saturated sodium bicarbonate to use 1% hydrochloric acid respectively.Organic layer is used anhydrous sodium sulfate dehydration, filters, and filtrate decompression is concentrated into dried, product (2.8g, productive rate 94.8%); With 40ml DMF dissolving, add triphenylphosphine 0.219g (0.837mmol), palladium 0.094g (0.418mmol), N; N-diisopropyl ethyl amine 2.3ml, formic acid 0.32ml, under 60 ℃, reaction 1h; (2 * 250ml) extractions, (2 * 500ml), (2 * 500ml), (1 * 250ml) washs salt solution saturated sodium bicarbonate reaction solution to use 1% hydrochloric acid respectively with ETHYLE ACETATE.Organic layer is used anhydrous sodium sulfate dehydration, filters, and filtrate decompression is concentrated into dried, gets light yellow oil.Column chromatography [column chromatography silica gel, ETHYLE ACETATE: sherwood oil (1: 70)] separate to be purified, concentrating under reduced pressure get light yellow oil (vi) 1.2g,, yield 40%.
C) 1-hydroxy-19-nor vitamins D
2(synthesizing viii)
With compound 3,5 cyclizations-1-alkene vitamins D
2(vi) 1.0g dissolves with THF, in the time of 0 ℃, drips 0.5mol/L 9-BBN solution 10mL, stirring at room reaction 1.5h, and the back adds entry, 30%H at 0 ℃
2O
2With 3mol/L sodium hydroxide solution (1: 1) 5mL, stirring at room 30min.Extract with ETHYLE ACETATE, organic layer is used 3% hydrochloric acid respectively, saturated sodium bicarbonate solution, saturated nacl aqueous solution washing; Anhydrous sodium sulfate dehydration filters, and filtrating is concentrated into dried, gets light yellow oil; Add acetic acid 3mL, in 60 ℃ of stirrings 20 minutes, reaction solution was with the dilution of saturated sodium bicarbonate liquid, with ETHYLE ACETATE 5mL extraction three times; Use the saturated common salt water washing, dehydrate, enriched material is dissolved in the 10%NaOH methanol solution, and 0 ℃ was stirred 1 hour; Water 10mL dilution, ETHYLE ACETATE (2 * 20ml) extractions, saturated nacl aqueous solution (1 * 20ml) washing.Organic layer is used anhydrous sodium sulfate dehydration, filters, and filtrate decompression is concentrated into dried, and column chromatography [column chromatography silica gel, ETHYLE ACETATE: sherwood oil (1: 5)] separates purifies, and gets 1-hydroxy-19-nor vitamins D
2(viii) solid 0.45g, yield 37%.
Embodiment three
500m shakes bottled 100 milliliters of nutrient solutions, 1.5% glucose, 1.5% soybean cake powder, 0.5% steeping water, 0.5%NaCl, 0.2%CaCO
3, pH 7.0.With 121 ℃ of substratum, sterilization 20min.2 milliliters of the spore suspensions of access bacterial strain CGMCC 5098,27 ℃ of rotations, rotating speed 240rpm cultivated 50mg1-hydroxy-19-nor vitamins D 4 days
2(viii) be dissolved in 2mL ethanol, this mixture is added in nutrient solution, continue to cultivate 3 days with 10 milligrams beta-cyclodextrin, 10mg tween-80; Filter, filtrating is with ETHYLE ACETATE 2 * 50mL extraction, and extracting solution concentrates; With silicagel column [column chromatography silica gel; ETHYLE ACETATE: sherwood oil (1: 2)] separate, obtain 25 hydroxylation product Zemplar 31mg, yield 60%.
1H?NMR(400MHz，CD
3OD)δ6.21(d，1H，J＝11Hz，H6)，5.88(d，1H，J＝11Hz，H7)，5.35(dd，1H，J＝8?and?15Hz，H23)，5.27(dd，1H，J＝8?and?15Hz，H22)，4.04(m，1H，H1)，3.98(m，1H，H3)，2.84(brd，1H，J＝11Hz，H9a)，2.59(brd，1H，J＝13Hz，H4a)，2.41(brd，1H，J＝13Hz，H10a)，2.21(dd，1H，J＝8?and?13Hz，H4b)，2.16(dd，1H，J＝7?and?13Hz，H10b)，2.04(m，4H)，1.84(m，1H，H2a)，1.76(m，2H)，1.67(m，2H)，1.55(m，3H)，1.35(m，3H)，1.13(s，3H，H26)，1.09(s，3H，H27)，1.04(d，3H，J＝7Hz，H21)，0.99(d，3H，J＝7Hz，H28)，0.58(s，3H，H18)；m/z：416.3[M]
+。
Embodiment four
With embodiment three identical conversion process, substrate does not add tween-80, and the converted product Zemplar obtains 25mg, yield 50%.
Embodiment five
3 500m shake bottled 100 milliliters of nutrient solutions, 1.5% glucose, 1.5% soybean cake powder, 0.5% steeping water, 0.5%NaCl, 0.2%CaCO
3, pH 7.0.With 121 ℃ of substratum, sterilization 20min.2 milliliters of the spore suspensions of access bacterial strain CGMCC 5098,27 ℃ of rotations, rotating speed 240rpm cultivated 2 days; Again these 3 nutrient solutions that shake bottle are inserted in the 7L fermentor tank of 3 liters of chargings fermention medium: 1.5% glucose, 2.0% soybean cake powder; 1.0% steeping water, 0.5%NaCl, 0.2%CaCO
3, 0.05% silicone, pH nature, ventilation 1.0V/V/min; Stir 400rpm, cultivated 3 days, add, cultivated 3 days by 1 gram substrate and 10 gram tween-80 and 40 milliliters of conversion products that ethanol is formed; Filter, filtrating is with 3L ethyl acetate extraction secondary, be concentrated into dried, the silicagel column separation; ETHYLE ACETATE: sherwood oil (1: 2) separates, and the acetone recrystallization obtains 25 hydroxylation product Zemplar 600mg, yield 60%.
Claims (5)
1. method for preparing Zemplar is with vitamins D
2Be raw material, adopt following chemical reaction and biological respinse step to obtain treatment osteoporosis agents Zemplar: 1-Alpha-hydroxy-19-removes the methylene radical vitamins D
2(ix):
The chemical reaction part:
Potassium permanganate solution is under-20 ℃, and with alcoholic solvent dissolving cyclisation thing (iii): dripping concentration is the potassium permanganate solution of 5-20%, and stirring reaction 1-10 hour best 3 hours, concentrate and remove water, resistates is used with the volume alcohol dilution; Under 0 ℃, add the sodium periodate aqueous solution of 10-20%, stirred 2-5 hour, best 3 hours, with the chloroparaffin of an equal-volume 1-5 carbon, the extractions such as carboxylicesters of 1-5 carbon were concentrated into driedly, obtain 10 carbonyl cyclisation vitamins Ds
2(iv); Under-78 ℃, drip the highly basic of non-polar solvent, add Trifluoromethanesulfonic anhydride, two (trifluoromethane sulphonyl) imines of N-phenyl, obtain enol base trifluoromethayl sulfonic acid ester (V); The cyclohexene derivative that obtains 1,10 pair of key with formic acid and palladium reduction (vi), uses hydroborating agents to obtain 1-hydroxy-19-nor vitamins D through the alkaline hydrogen peroxide oxidation
2(vii), adopt the low molecular organic acids open loop, obtain 1-hydroxyl-10 nor-vitamin D through 4% sodium hydrate methanol solution hydrolysis again after the washing
2(viii);
The biological respinse part
1-hydroxy-19-nor vitamins D
2(viii) as substrate with bacterial strain CGMCC 5098 under the state of aeration-agitation, in the substratum of carbon source, nitrogenous source, mineral salt, carry out bio-transformation, leavening temperature 22-30 ℃; Preferably 27 ℃; PH scope 6.5-7.5, preferably 7.0, air flow is at 0.5-1.0v/v/min; With 1-hydroxy-19-nor vitamins D
2(viii) join in the nutrient solution as substrate, concentration of substrate is the 1-15g/ liter preferably, and the time that substrate adds substratum normally adds substrate the best nearly when carbon source runs out of, normally often be that strain culturing added substrate after 2 days;
In the process that continues above-mentioned cultivation, produce 25 hydroxylation bio-transformation, the time of conversion is depended on the composition and the bacterial strain of substratum, continues usually to cultivate preferably 4 days 1-7 days; After solid-liquid separation is used the organic solvent extracting to mycelia and filtrating respectively, through silica gel column chromatography, the acetone recrystallization obtains the pure title product 1-hydroxyl-25-hydroxy-19-nor vitamins D that obtains
2(ix), i.e. Zemplar.
2. a kind of method for preparing Zemplar according to claim 1 is characterized in that, said dissolving cyclisation thing alcoholic solvent (iii) can be: ethanol, propyl alcohol, Virahol.
3. a kind of method for preparing Zemplar according to claim 1 is characterized in that, and is said by 10 carbonyl cyclisation vitamins Ds
2The highly basic that (iv) obtains the non-polar solvent that drips in enol base trifluoromethayl sulfonic acid ester (V) step can be: hexamethyl two amido lithiums, n-Butyl Lithium, tert-butyl lithium, sodium amide.
4. a kind of method for preparing Zemplar according to claim 1 is characterized in that said cyclohexene derivative by 1,10 pair of key (vi) obtains 1-hydroxy-19-nor vitamins D through the alkaline hydrogen peroxide oxidation
2(vii) used hydroborating agents can be in the step: 9-boron two ring [3.3.1] nonanes (9-BBN), borine, diisopinocampheylchloroborane base chloroborane, diethylammonium methoxyl group borine.
5. a kind of method for preparing Zemplar according to claim 1 is characterized in that, said substratum adopts:
Seed culture medium: 1.5% glucose, 1.5% soybean cake powder, 0.5% steeping water, 0.5%NaCl, 0.2%CaCO3, pH nature; 121 ℃ of sterilization 20min;
Fermention medium: 1.5% glucose, 2.0% soybean cake powder, 1.0% steeping water, 0.5%NaCl, 0.2%CaCO3, pH nature; 121 ℃ of sterilization 20min;
First order seed inserts fermention medium with the inoculum size of 1-10%, 28 ℃ of leavening temperatures, and air flow 0.5v/v/min cultivated 48 hours.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451238A (en) * | 2013-07-23 | 2013-12-18 | 西南交通大学 | Method for preparing paricalcitol |
| CN114773151A (en) * | 2021-12-30 | 2022-07-22 | 正大制药（青岛）有限公司 | Preparation method of paricalcitol 20S isomer impurity |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451238A (en) * | 2013-07-23 | 2013-12-18 | 西南交通大学 | Method for preparing paricalcitol |
| CN114773151A (en) * | 2021-12-30 | 2022-07-22 | 正大制药（青岛）有限公司 | Preparation method of paricalcitol 20S isomer impurity |
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