CN103451238A - Method for preparing paricalcitol - Google Patents
Method for preparing paricalcitol Download PDFInfo
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- CN103451238A CN103451238A CN2013103118477A CN201310311847A CN103451238A CN 103451238 A CN103451238 A CN 103451238A CN 2013103118477 A CN2013103118477 A CN 2013103118477A CN 201310311847 A CN201310311847 A CN 201310311847A CN 103451238 A CN103451238 A CN 103451238A
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Abstract
The invention discloses a method for preparing a medicine paricalcitol treating osteoporosis. According to the method, vitamin D2 is taken as a raw material, and a target object is obtained through steps of chemical reaction and biological reaction. Expensive toxic double hydroxylation reagent is not used, and the double-hydroxylation broken bond of a cheap and effective oxidized reagent potassium permanganate solution is adopted for preparing ketone. The method comprises the following steps of preparing olefin in an alkaline reagent through isomerism; carrying out hydroboration through a hydroboration reagent 9-BBN and borane; introducing 1-OH, thereby obtaining 19-demethylation vitamin D2 through ring opening; and by taking Pseudonocardia autotrophica SWJTUQL-3721021 (autotrophy Pseudonocardia CGMCC 5098) as hydroxylation bacterium, carrying out direct hydroxylation on 25-OH through a biological conversion method, thereby obtaining the paricalcitol through extraction and separation. According to the method, 27 public known reaction steps are reduced to 9 reaction steps through combining the chemical conversion and biological conversion, so that the total recovery is improved, the process route is shortened, and the method provides a new process route for 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 that Abbott company developed in 1998, and commodity Zemplar by name has obtained FDA approval listing, clinically for preventing and treat the secondary hyperparathyroidism animal.The pharmacological action that experiment and clinical studies show paricalcitol have a relative selectivity can suppress safely and effectively the secretion of Rat parathyroid hormone 1-34 and parathyroid hyperplasia and seldom cause hypercalcemia and hyperphosphatemia, and alternative calcitriol (calcitriol) is specially adapted to the treatment of Patients Treated with Long-term Hemodialysis secondary hyperparathyroidism for uremia.
1-Alpha-hydroxy-19-removes the methylene radical vitamins D
2(1 α-19-nor-VD
2), Zemplar (paricalcitol) known synthetic adopts complete synthesisly mostly, mainly contains two class synthetic routes, and the one, as method as described in patent US7645911 adopts vitamins D
2or 25(OH)VD
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.; As method as described in patent US5237110, US5246925 adopts 25(OH)VD
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, as employing vitamins D as described in the patents such as patent US5086191, US5281731/5391755, US5486636, US5525745
2as raw material, adopt ozone disconnected 6,7 two keys, obtain respectively A ring and CD ring, or synthesize respectively A cyclization CD ring with chiral synthon, then pass through respectively various chemically modifieds, to remove the A ring of 10 methylene radical through wittig reaction and CD cyclic condensation, again through Wittig – Horner reaction, 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, very uneconomical for the synthesis of Zemplar.The first method step is relatively less, but still will use hypertoxic expensive perosmic anhydride as alkene bishydroxy reagent, or uses rare 25(OH)VD
2do raw material, or use disconnected 22, the 23 pairs of keys of ozone, then introduce 25 hydroxyl side chains, on producing, still operation is too many, complicated operation, and yield is on the low side, and total recovery is 0.1% left and right only.
Summary of the invention
In view of the above deficiency of prior art, the purpose of this invention is to provide a kind of novel method for preparing Zemplar, make it to have advantages of that production process is simplified, yield is high and get rid of the perosmic anhydride that severe toxicity is expensive and apply as alkene bishydroxy reagent.
The objective of the invention is to realize by following means.
A kind of method for preparing Zemplar, with vitamins D
2for raw material, adopt chemical reaction as expressed as Fig. 1 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 the alcoholic solvent of take dissolves cyclisation thing (III): the potassium permanganate solution that dropping concentration is 5-20%, stirring reaction 1-10 hour best 3 hours, concentrated water, the same volume alcohol dilution of resistates removed; Under 0 ℃, add the sodium periodate aqueous solution of 10-20%, stir 2-5 hour, best 3 hours, with the chloroparaffin of 1-5 carbon of equal-volume, the extractions such as carboxylicesters of 1-5 carbon, be concentrated into dryly, 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); Obtain the cyclohexene derivative (VI) of 1,10 pair of key with formic acid and palladium reduction, use 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, after washing again the sodium hydrate methanol solution hydrolysis through 4% obtain 1-hydroxyl-10 nor-vitamin D
2(VIII);
The biological respinse part
1-hydroxy-19-nor vitamins D
2(VIII) uses bacterial strain CGMCC5098 under the state of aeration-agitation as substrate, carries out bio-transformation in the substratum of carbon source, nitrogenous source, mineral salt, 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; By 1-hydroxy-19-nor vitamins D
2(VIII) joins in nutrient solution as substrate, concentration of substrate is the 1-15g/ liter preferably, substrate adds the time of substratum normally when carbon source runs out of, to add substrate the best nearly, the consumption of carbon source can be controlled by measuring sugared concentration, adds substrate after normally dropping to 0.05-0.002%;
Produce the hydroxylation bio-transformation of 25 in the process that continues above-mentioned cultivation, the time of conversion is depended on composition and the bacterial strain of substratum, usually continues to cultivate 1-7 days, preferably 4 days.Finally by solid-liquid separation, respectively mycelia and filtrate are used to the organic solvent extracting, through silica gel column chromatography, the acetone/water recrystallization obtains pure target product 1-hydroxyl-25-hydroxy-19-nor vitamins D
2(IV), i.e. Zemplar.
The method for preparing Zemplar that the present invention adopts chemistry and bio-transformation to combine, 10, on 19 pairs of key scission of links, adopt low temperature potassium permanganate solution dihydroxylation cheap and easy to get to replace the perosmic anhydride that severe toxicity is expensive, adopt biotransformation method to carry out the direct hydroxylation of 25, than chemical method gentleness, need not very low temperature and ozone scission of link, and there is regioselectivity, do not need to carry out radical protection, 25 hydroxylation bio-transformation yields are high, environmental friendliness, 25 hydroxylated committed steps of chemical method are substituted, simplification of flowsheet, it by chemosynthesis and microbial transformation, is the method for that combination, greatly shorten operational path, improve yield, reduce manufacturing cost, the 27 step reactions of known synthetic Zemplar are shortened to 9 step reactions, total recovery reaches 3%, yield than 0.1% left and right of existing open report has improved more than 10 times.
The accompanying drawing explanation
The process route chart that Fig. 1 is the inventive method.
Embodiment
Below in conjunction with embodiment, enforcement of the present invention is further described.
Technical scheme route of the present invention is shown in Fig. 1
Chemical part: chemosynthesis
Starting raw material of the present invention is vitamins D
2, the method for (US4195027) such as employing DeLuca changes into 3,5-cyclisation vitamins D
2(III), then to 10, two keys of 19 carry out bishydroxy, the present invention adopts potassium permanganate solution under-20 ℃, with ethanol, propyl alcohol, Virahols etc. are the above-mentioned cyclisation thing of dissolution with solvents (III), the potassium permanganate solution that dropping concentration is 5-20%, stirring reaction 1-10 hour, best 3 hours, the concentrated water of removing, the same volume alcohol dilution of resistates, under 0 ℃, the sodium periodate aqueous solution that adds 10-20%, stir 2-5 hour, best 3 hours, chloroparaffin with an equal-volume 1-5 carbon, the extractions such as carboxylicesters of 1-5 carbon, be concentrated into dry, obtain 10 carbonyl cyclisation vitamins Ds
2(IV).10 carbonyl cyclisation vitamins Ds
2be dissolved in non-polar solvent, as hexanaphthene, tetrahydrofuran (THF), ether, methylene dichloride etc., under-78 ℃, drip the highly basic of foregoing non-polar solvent, as 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, diethyl methoxyl group borine etc., obtain 1-hydroxy-19-nor vitamins D through the alkaline hydrogen peroxide oxidation
2(VII), adopt the low molecular organic acids of DeLuca etc. (US4195027, US4260549) as the acetic acid open loop, after washing again the sodium hydrate methanol solution hydrolysis through 4% obtain 1-hydroxyl-10 nor-vitamin D
2(VIII).
Biological part:
Separate and obtain a strain bacterium QL-3721001 the soil sample that the contriver takes from Er E mountain, Sichuan, have 1-hydroxy-19-nor vitamins D
2(VIII) has conversion capability, obtains bacterial strain QL-3721021 through natural seed selection and ultraviolet mutagenesis and has higher 25 more single-minded hydroxylation abilities, called after SWJTUQL-3721021.Adopt synthetic No. 1 nutrient agar of Gao Shi to carry out the morphological specificity observation, 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 in Table 1, this bacterium fibrillae of spores is directly and slightly flexible, several without branch, bacterium colony densification, smooth surface.Bacterial strain can not liquefy gelatin, and starch slightly is hydrolyzed, and milk peptonizes.To glucose, inositol, D-MANNOSE, N.F,USP MANNITOL, L-arabinose, fructose, rhamnosyl, raffinose, the sugar alcohols such as D-wood sugar all have the ability of utilizing in various degree.Nitrate does not reduce.On Mierocrystalline cellulose, do not grow.Do not produce Melanoidins and tyrosine oxidase, do not produce hydrogen sulfide.This bacterium can utilize sodium bicarbonate for sole carbon source.But also heterotrophic growth.Bacterium colony is white, and the bacterium colony quality of formation is tight, and the pigmentary colours that the back side produces are micro-tawny.Bacterial strain produces two kinds of mycelia, substrate mycelium and aerial hyphae; Can also see the thicker fibrillae of spores of black, be grown in long and straight branch upper simultaneously, conidium Dan Sheng, wall thickness, have diaphragm, is DNA releaxed circular DNA or hook-shaped, and the ellipticity spore be scattered is arranged.
The morphological specificity of table 1 bacterial strain SWJTUQL-3721021 on each substratum
According to the outstanding bacterium handbook of uncle and above measurement result, preliminary mirror SWJTUQL-3721021 bacterial strain belongs to the Selective medium actinomyces actinoides, and name the SWJTUQL-3721021 into Selective medium SWJTUQL-3721021(Pseudonocardia autotrophica), deposit number is CGMCC No.5098.
Bacterial strain CGMCC No.5098 bio-transformation 1-hydroxy-19-nor vitamins D
2the conversion process of (VIII):
Technique 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) carries out bio-transformation, obtains 1-hydroxyl-25-hydroxy-19-nor vitamins D
2(IV), i.e. Zemplar.
Transform and cultivate and can carry out in the shaking flask of ventilating or fermentor tank, nutritive substance comprises that inorganic salt, nitrogenous source, sugar or other soluble substance are as carbon source, inorganic salt comprise basic metal, alkaline-earth metal muriate, nitrate, carbonate, vitriol or the phosphoric acid salt as magnesium, iron, zinc, manganese, MgSO4 preferably, KH
2pO4.Nitrogenous source can be ammonium salt, comprises Citrate trianion, tartrate, malate, succinate, oxalate, acetate etc.; Amino acid and mixture, peptide or protein and hydrolyzate thereof, meat extract, cereal are as the water soluble extract of 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 etc.No matter solid culture or deep layer are cultivated and are all carried out under aeration condition, leavening temperature 22-30 ℃, and 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, by 1-hydroxy-19-nor vitamins D
2(VIII) joins in nutrient solution as substrate, and concentration of substrate is without particular requirement, preferably the 1-15g/ liter, produce the hydroxylation bio-transformation of 25 in the process that continues above-mentioned cultivation, the time transformed is depended on composition and the bacterial strain of substratum, usually continues to cultivate 1-7 days, preferably 4 days.
Substrate adds the time of substratum extremely important, normally when carbon source runs out of, adds substrate the best nearly, and the consumption of carbon source can be controlled by measuring sugared concentration, 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 powders, 0.5 ﹪ corn steep liquor, 0.5 ﹪ NaCl, 0.2 ﹪ CaCO3, pH nature.By 121 ℃ of substratum, sterilizing 20min.
Fermention medium: 1.5 ﹪ glucose, 2.0 ﹪ soybean cake powders, 1.0 ﹪ corn steep liquors, 0.5 ﹪ NaCl, 0.2 ﹪ CaCO3, pH nature.By 121 ℃ of substratum, sterilizing 20min.
The fermentation culture process: first order seed accesses fermention medium with the inoculum size of 1-10%, 28 ℃ of leavening temperatures, and air flow 0.5v/v/min, cultivate 48 hours.
The form that adds of substrate can be substrate 1-hydroxy-19-nor vitamins D
2(VIII) raw material solid or be dissolved in organic solvent as ethanol, methyl alcohol, ethylene glycol, polyoxyethylene glycol, tensio-active agent or dispersion agent are as tween 20, tween-80, solubility promoter, as the interpolation of cyclodextrin etc. more contributes to the dissolving of substrate in nutrient solution, contributes 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 chemical engineering, to mycelium, use organic solvent as ethanol, acetone extraction, after concentrating, with organic chloroparaffin, ester class, fragrant alkane, like methylene dichloride, ethyl acetate, toluene etc., extract, concentrate; Filtrate with aforesaid organic solvent as organic chloroparaffin, ester class, fragrant alkane like extractions such as methylene dichloride, ethyl acetate, toluene, divide and get the upper strata organic layer, merge organic layer, the concentrated Zemplar crude product that obtains, again through silica gel column chromatography, organic solvent-normal hexane, sherwood oil etc. and ethyl ester gradient or 2:1, as eluent, collect pure Zemplar component, through the acetone/water recrystallization, obtain pure Zemplar.
Embodiment mono-
1-hydroxy-19-nor vitamins D
2the preparation of (VIII)
A) 10-oxo-3,5 cyclization vitamins Ds
2synthesizing of (IV)
3,5 cyclization vitamins Ds
2the method of (US4195027) such as the synthetic employing DeLuca of (III) is by vitamins D
2change into 3,5-cyclisation vitamins D
2(III) adds 3.88g, 9.46mmol in reaction flask) 3,5 cyclization vitamins Ds
2, with ethanol 300ml, dissolve, be chilled to-20 ℃, then drip potassium permanganate solution (KMnO
43.33g, H
2o100ml), about 20 minutes.Stir 1h at this temperature, after stir 15min under 40 ℃ of water-baths.Filter, filtrate decompression is concentrated into dry, obtains light yellow oily mixture 4(3.67g), this crude product dissolves with ethanol 300ml, adds saturated sodium periodate 100ml, stirring at room 2.5h.Ethyl acetate for reaction solution (2 * 200ml) extraction, saturated nacl aqueous solution (1 * 200ml) washing.The organic layer anhydrous sodium sulfate dehydration, filter, and filtrate decompression is concentrated into dry, obtains light yellow oil.Column chromatography [column chromatography silica gel, ethyl acetate: sherwood oil (1:10)] separating-purifying, concentrating under reduced pressure obtains 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
2synthesizing of (VI)
Add hexamethyl two amido lithium (LiHMDS) 11ml in reaction flask, be chilled to-78 ℃.Add the 10-oxo-3 dissolved with tetrahydrofuran (THF), 5 cyclization vitamins Ds
21.75g (4.18mmol) and two (trifluoromethane sulphonyl) imines (PhN (CF of N-phenyl
3sO
2)
2) 3.6g.After stirring 12h, reaction solution is concentrated, adds ethyl acetate, uses respectively 1% hydrochloric acid (2 * 250ml), saturated sodium bicarbonate (2 * 250ml), salt solution (1 * 250ml) washing.The organic layer anhydrous sodium sulfate dehydration, filter, and filtrate decompression is concentrated into dry, product (2.16g, productive rate 94.8%), dissolve with 40ml DMF, 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, ethyl acetate for reaction solution (2 * 250ml) extraction, use respectively 1% hydrochloric acid (2 * 500ml), saturated sodium bicarbonate (2 * 500ml), salt solution (1 * 250ml) washing.The organic layer anhydrous sodium sulfate dehydration, filter, and filtrate decompression is concentrated into dry, obtains light yellow oil.Column chromatography [column chromatography silica gel, ethyl acetate: sherwood oil (1:70)] separating-purifying, concentrating under reduced pressure obtains 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
2synthesizing of (VIII)
By compound 3,5 cyclizations-1-alkene vitamins D
2(VI) 0.5g dissolves with tetrahydrofuran (THF), in the time of 0 ℃, drips 0.5mol/L9-BBN solution, stirring at room reaction 1.5h, after add water, 30%H at 0 ℃
2o
2with 3mol/L sodium hydroxide solution (1:1), stirring at room 30min.By ethyl acetate, extracted, organic layer is used respectively 3% hydrochloric acid, saturated sodium bicarbonate solution, saturated nacl aqueous solution washing, anhydrous sodium sulfate dehydration, filter, filtrate is concentrated into dry, obtains light yellow oil, adds acetic acid 3mL, in 60 ℃, stir 20 minutes, saturated sodium bicarbonate liquid dilution for reaction solution, with ethyl 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 ℃ is stirred 1 hour, water 10mL dilution, ethyl acetate (2 * 20ml) extraction, saturated nacl aqueous solution (1 * 20ml) washing.The organic layer anhydrous sodium sulfate dehydration, filter, and filtrate decompression is concentrated into dry, and column chromatography [column chromatography silica gel, ethyl acetate: sherwood oil (1:5)] separating-purifying, obtain 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=8and15Hz,H23),5.27(dd,1H,J=8and15Hz,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=8and13Hz,H4b),2.16(dd,1H,J=7and13Hz,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 bis-
A) 10-oxo-3,5 cyclization vitamins Ds
2synthesizing of (IV)
3,5 cyclization vitamins Ds
2the method of (US4195027) such as the synthetic employing DeLuca of (III) is by vitamins D
2change into 3,5-cyclisation vitamins D
2(III), 3,5-cyclization VD
25 grams, be dissolved in 150 milliliters of ethanol, is placed in-20 ℃ of low temperature and bathes, and drips 3%KMnO
4(3% aqueous solution) 320 milliliters, drip off, and continues reaction 2 hours, regularly TLC detects, and 3 hours, raw material consumption was complete, 40 ℃ are stirred 15 minutes, solidify Manganse Dioxide, diatomite filtration, concentrate and remove alcohol, add 100 milliliters of ethyl acetate, extracting twice, be concentrated into dry, this crude product dissolves with ethanol 300ml, adds saturated sodium periodate 100ml, stirring at room 2.5h.Ethyl acetate for reaction solution (2 * 200ml) extraction, saturated nacl aqueous solution (1 * 200ml) washing.The organic layer anhydrous sodium sulfate dehydration, filter, and filtrate decompression is concentrated into dry, obtains light yellow oil.Column chromatography [column chromatography silica gel, ethyl acetate: sherwood oil (1:10)] separating-purifying, concentrating under reduced pressure obtains light yellow oil 5(1.42g, 36.5% liang of step of productive rate).
B) 3,5 cyclizations-1-alkene vitamins D
2synthesizing of (VI)
Add hexamethyl two amido lithium (LiHMDS) 15ml in reaction flask, be chilled to-78 ℃.Add the 10-oxo-3 dissolved with tetrahydrofuran (THF), 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 is concentrated, adds ethyl acetate, uses respectively 1% hydrochloric acid (2 * 250ml), saturated sodium bicarbonate (2 * 250ml), salt solution (1 * 250ml) washing.The organic layer anhydrous sodium sulfate dehydration, filter, and filtrate decompression is concentrated into dry, product (2.8g, productive rate 94.8%), dissolve with 40ml DMF, 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, ethyl acetate for reaction solution (2 * 250ml) extraction, use respectively 1% hydrochloric acid (2 * 500ml), saturated sodium bicarbonate (2 * 500ml), salt solution (1 * 250ml) washing.The organic layer anhydrous sodium sulfate dehydration, filter, and filtrate decompression is concentrated into dry, obtains light yellow oil.Column chromatography [column chromatography silica gel, ethyl acetate: sherwood oil (1:70)] separating-purifying, concentrating under reduced pressure obtains light yellow oil (VI) 1.2g,, yield 40%.
C) 1-hydroxy-19-nor vitamins D
2synthesizing of (VIII)
By compound 3,5 cyclizations-1-alkene vitamins D
2(VI) 1.0g dissolves with tetrahydrofuran (THF), in the time of 0 ℃, drips 0.5mol/L9-BBN solution 10mL, stirring at room reaction 1.5h, after add water, 30%H at 0 ℃
2o
2with 3mol/L sodium hydroxide solution (1:1) 5mL, stirring at room 30min.By ethyl acetate, extracted, organic layer is used respectively 3% hydrochloric acid, saturated sodium bicarbonate solution, saturated nacl aqueous solution washing, anhydrous sodium sulfate dehydration, filter, filtrate is concentrated into dry, obtains light yellow oil, adds acetic acid 3mL, in 60 ℃, stir 20 minutes, saturated sodium bicarbonate liquid dilution for reaction solution, with ethyl 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 ℃ is stirred 1 hour, water 10mL dilution, ethyl acetate (2 * 20ml) extraction, saturated nacl aqueous solution (1 * 20ml) washing.The organic layer anhydrous sodium sulfate dehydration, filter, and filtrate decompression is concentrated into dry, and column chromatography [column chromatography silica gel, ethyl acetate: sherwood oil (1:5)] separating-purifying, obtain 1-hydroxy-19-nor vitamins D
2(VIII) solid 0.45g, yield 37%.
Embodiment tri-
The 500m shaking flask fills 100 milliliters of nutrient solutions, 1.5 ﹪ glucose, 1.5 ﹪ soybean cake powders, 0.5 ﹪ corn steep liquor, 0.5 ﹪ NaCl, 0.2 ﹪ CaCO
3, pH7.0.By 121 ℃ of substratum, sterilizing 20min.2 milliliters of the spore suspensions of access bacterial strain CGMCC5098,27 ℃ of rotations, rotating speed 240rpm, cultivate 4 days, 50mg1-hydroxy-19-nor vitamins D
2beta-cyclodextrin, the 10mg tween-80 of (VIII) and 10 milligrams are dissolved in 2mL ethanol, this mixture is added in nutrient solution, continue to cultivate 3 days, filter, filtrate extracts with ethyl acetate 2 * 50mL, and extracting solution is concentrated, with silicagel column [column chromatography silica gel, ethyl 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=8and15Hz,H23),5.27(dd,1H,J=8and15Hz,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=8and13Hz,H4b),2.16(dd,1H,J=7and13Hz,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 tetra-
Conversion process identical with embodiment tri-, substrate does not add tween-80, and the converted product Zemplar obtains 25mg, yield 50%.
Embodiment five
3 500m shaking flasks fill 100 milliliters of nutrient solutions, 1.5 ﹪ glucose, 1.5 ﹪ soybean cake powders, 0.5 ﹪ corn steep liquor, 0.5 ﹪ NaCl, 0.2 ﹪ CaCO
3, pH7.0.By 121 ℃ of substratum, sterilizing 20min.2 milliliters of the spore suspensions of access bacterial strain CGMCC5098,27 ℃ of rotations, rotating speed 240rpm, cultivate 2 days, again the nutrient solution of these 3 shaking flasks is accessed in the 7L fermentor tank of 3 liters of chargings to fermention medium: 1.5 ﹪ glucose, 2.0 ﹪ soybean cake powders, 1.0 the ﹪ corn steep liquor, 0.5 ﹪ NaCl, 0.2 ﹪ CaCO
3, 0.05% silicone, pH nature, ventilation 1.0V/V/min, stir 400rpm, cultivates 3 days, add by 1 gram substrate and 10 gram tween-80s and 40 milliliters of conversion products that ethanol forms, cultivate 3 days, filter, filtrate is with 3L ethyl acetate extraction secondary, is concentrated into dryly, and silicagel column separates, ethyl acetate: sherwood oil (1:2) separates, the acetone/water recrystallization, obtain 25 hydroxylation product Zemplar 600mg, yield 60%.
Claims (4)
1. a method for preparing Zemplar, with vitamins D
2for 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 the alcoholic solvent of take dissolves cyclisation thing (III): the potassium permanganate solution that dropping concentration is 5-20%, stirring reaction 1-10 hour best 3 hours, concentrated water, the same volume alcohol dilution of resistates removed; Under 0 ℃, add the sodium periodate aqueous solution of 10-20%, stir 2-5 hour, best 3 hours, with the chloroparaffin of 1-5 carbon of equal-volume, the extractions such as carboxylicesters of 1-5 carbon, be concentrated into dryly, 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); Obtain the cyclohexene derivative (VI) of 1,10 pair of key with formic acid and palladium reduction, use 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, after washing again the sodium hydrate methanol solution hydrolysis through 4% obtain 1-hydroxyl-10 nor-vitamin D
2(VIII);
The biological respinse part
1-hydroxy-19-nor vitamins D
2(VIII) uses bacterial strain CGMCC5098 under the state of aeration-agitation as substrate, carries out bio-transformation in the substratum of carbon source, nitrogenous source, mineral salt, 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; By 1-hydroxy-19-nor vitamins D
2(VIII) joins in nutrient solution as substrate, and concentration of substrate is the 1-15g/ liter preferably, and substrate adds the time of substratum normally when carbon source runs out of, to add substrate the best nearly, is often normally that strain culturing added substrate after 2 days;
Produce the hydroxylation bio-transformation of 25 in the process that continues above-mentioned cultivation, the time of conversion is depended on composition and the bacterial strain of substratum, usually continues to cultivate 1-7 days, preferably 4 days; Finally by solid-liquid separation, respectively mycelia and filtrate are used to the organic solvent extracting, through silica gel column chromatography, the acetone/water recrystallization obtains the pure target product 1-hydroxyl-25-hydroxy-19-nor vitamins D that obtains
2(IV), i.e. Zemplar.
2. a kind of method for preparing Zemplar according to claim 1, is characterized in that, the alcoholic solvent of described dissolving cyclisation thing (III) can be: ethanol, propyl alcohol, Virahol.
3. a kind of method for preparing Zemplar according to claim 1, is characterized in that, described 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, the described cyclohexene derivative by 1,10 pair of key (VI) obtains 1-hydroxy-19-nor vitamins D through the alkaline hydrogen peroxide oxidation
2in (VII) step, hydroborating agents used can be: 9-boron two ring [3.3.1] nonanes (9-BBN), borine, diisopinocampheylchloroborane base chloroborane, diethyl methoxyl group borine.5, a kind of method for preparing Zemplar according to claim 1, is characterized in that, described substratum adopts:
Seed culture medium: 1.5 ﹪ glucose, 1.5 ﹪ soybean cake powders, 0.5 ﹪ corn steep liquor, 0.5 ﹪ NaCl, 0.2 ﹪ CaCO3, pH nature; 121 ℃ of sterilizing 20min;
Fermention medium: 1.5 ﹪ glucose, 2.0 ﹪ soybean cake powders, 1.0 ﹪ corn steep liquors, 0.5 ﹪ NaCl, 0.2 ﹪ CaCO3, pH nature; 121 ℃ of sterilizing 20min;
First order seed accesses fermention medium with the inoculum size of 1-10%, 28 ℃ of leavening temperatures, and air flow 0.5v/v/min, cultivate 48 hours.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104860858A (en) * | 2015-04-28 | 2015-08-26 | 南京理工大学 | Amino acid modification based vitamin D2 derivative, synthesis and applications |
| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102131773A (en) * | 2008-07-22 | 2011-07-20 | Azad药物成分股份公司 | Methods for producing paricalcitol |
| CN102392053A (en) * | 2011-09-20 | 2012-03-28 | 西南交通大学 | Method for preparing paricalcitol |
-
2013
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102131773A (en) * | 2008-07-22 | 2011-07-20 | Azad药物成分股份公司 | Methods for producing paricalcitol |
| CN102392053A (en) * | 2011-09-20 | 2012-03-28 | 西南交通大学 | Method for preparing paricalcitol |
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| Title |
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| 万阳: "化学与生物转化法合成帕立骨化醇", 《有机化学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104860858A (en) * | 2015-04-28 | 2015-08-26 | 南京理工大学 | Amino acid modification based vitamin D2 derivative, synthesis and applications |
| 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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