CN103421025A - Method for preparing 7alpha-methoxyl cephalosporin C - Google Patents
Method for preparing 7alpha-methoxyl cephalosporin C Download PDFInfo
- Publication number
- CN103421025A CN103421025A CN2012101627209A CN201210162720A CN103421025A CN 103421025 A CN103421025 A CN 103421025A CN 2012101627209 A CN2012101627209 A CN 2012101627209A CN 201210162720 A CN201210162720 A CN 201210162720A CN 103421025 A CN103421025 A CN 103421025A
- Authority
- CN
- China
- Prior art keywords
- cephalosporin
- ethanol
- alcohol
- acid
- purity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Cephalosporin Compounds (AREA)
Abstract
The invention discloses a method for preparing 7alpha-methoxyl cephalosporin C. The method is characterized in that the method comprises steps of conversion liquid pretreatment, adsorbent resin chromatography treatment, reverse phase silica gel column chromatography treatment and preparation of 7alpha-methoxyl cephalosporin C from the conversion liquid converting cephalosporin C into 7alpha-methoxyl cephalosporin C. The method has simple processes, and can remove impurities such as conversion substrates, intermediate products and the like which have similar structures with 7alpha-methoxyl cephalosporin C well. The yield and purity of obtained 7alpha-methoxyl cephalosporin C are both high.
Description
Technical field
The invention belongs to the pharmaceutical engineering field, be specifically related to a kind of method for preparing high purity 7 α-methoxy cephalosporin C (7 α-Methoxycephalosporin C).
Background technology
Cephalosporins is that in anti-infectives, development early, is applied a wider large class.7 α that wherein grow up in early 1970s-methoxy cephalosporin (its chemical structural formula is as shown in the formula I), because there is a trans methoxyl group C7 position on beta-lactam nucleus, stop approaching of lactam nucleus and enzyme molecule, and then improved the stability of medicine to β-lactamase.Research for this class medicine is latest developments Disciplinary Frontiers more rapidly.
The existing multiple product listing of methoxy cephalosporins, comprise cefminox, cefmetazole etc.The common semi-synthetic intermediate of this class medicine is methoxy cephalosporin.The synthetic of methoxy cephalosporin generally can be by chemical synthesis and microbe transformation method.Chemical method is the method generally adopted in the industry at present, main direction of studying is all to take 7-ACA to be synthesized as raw material, for example Chinese patent CN200910075806.6 has reported and take 7-ACA as raw material, with first mercapto tetrazole, reaction obtains 7-TMCA, 7-TMCA and methyl sulphur bromine carry out imidization, then obtain intermediate with the hexichol diazomethane reaction, intermediate and methylating reagent methylation reaction, obtaining can be for the synthesis of the intermediate 7-MAC of methoxy cephalosporins medicine.In addition, Chinese patent CN101210019A reported with C4, and the cepham compound of 7 protections, C3 position monochloromethyl is raw material, the synthesizing methoxy cephalosporin intermediate.But the problems such as the method ubiquity organic solvent usage quantity of these chemosynthesis is large, and energy consumption is serious.And can effectively address these problems by the method for bio-transformation, the cephamycin C that utilizes bio-transformation to obtain is as synthetic methoxy cephalosporins, and for cleaner production, there is significant advantage the aspects such as reduction energy consumption.
Document (The Conversion of Cephalosporins to 7 α-Methoxycephalosporins by Cell-Free Extract of Streptomyces clavuligerus.Biochem.J, 1980,186:613-616) report utilizes clavuligerus (Streptomyces clavuligerus, ATCC27064) crude enzyme liquid after cytoclasis, add cofactor and cephalosporin substrate, conversion obtains 7 α-methoxy cephalosporin C, but there is no at present, reports from transformation system the method for product 7 α-methoxy cephalosporin C separation and purification.Document (Non-radioactive assay and HPLC analysis of cephalosporin C 7 α-methocxylation by cell-free extracts of Streptomyces clavuligerus.Appl Microbiol Biotechnol is separately arranged, 1991, 35:793-797) the method for report chemosynthesis 7 α-methoxy cephalosporin C, at first α-diphenyl-methyl-N-t-butanone-own diester of D-alpha-amino group and diphenyl-methyl-7-aminocephalosporinic acid ester are coupled, obtain the CPC of N-t-butanone-bis-benzhydryl ester, then using the methyl alcohol lithium salts as methyl donor, added the reaction of methoxyl group, finally slough hydroxyl and amino blocking group, obtain 7 α-methoxy cephalosporin C.In the chemosynthesis process, used more chemical reagent, reaction is complicated.Although in the chemosynthesis process, by organic solvent extraction, revolve the steps such as steaming and can obtain 7 αs of purity more than 90%-methoxy cephalosporin C, but because system after bio-transformation and chemosynthesis system have very big-difference, thereby the method for the separation methoxy cephalosporin C of report be not suitable for purifying methoxy cephalosporin C from converted product.
Summary of the invention
For solving the deficiencies in the prior art, the present invention relates to a kind of method for preparing high purity 7 α-methoxy cephalosporin C, the method not only technique is simple, can be good at removing the impurity such as the very similar conversion of substrate of structure, intermediate product, and yield and the purity of acquisition methoxy cephalosporin C are all very high.
Therefore, the invention provides a kind of method for preparing 7 α-methoxy cephalosporin C, it is characterized in that described method comprises by conversion fluid pre-treatment, polymeric adsorbent Image processing and reversed-phase silica gel column chromatography processes, from by the cephalosporin substrate conversion being the step of preparation 7 α-methoxy cephalosporin C the conversion fluid of 7 α-methoxy cephalosporin C.
According to of the present invention one preferred embodiment, described method comprises the following steps:
A) in every milliliter of conversion fluid, add the Glacial acetic acid of 30-70 μ l to mix, make the most of protein denaturation precipitation in conversion fluid, suction filtration is got the filtrate of clarification, with sour adjust pH, is 3.0-5.0, is the upper prop mother liquor;
B) the upper prop mother liquor obtained is slowly passed through to the macroporous adsorption resin chromatography post; The acid rinsing pillar 2-6CV (column volume) that is 2.5-4.5 with pH; Then with containing 10%-40% (v: v), preferably 15-30% (v: the v) aqueous solution of low fat alcohol, 2-5CV wash-out pillar, substep is collected and HPLC detects effluent liquid; 7 α-methoxy cephalosporin C HPLC purity is greater than to 15% receiving flask merging, adjusts pH3.0-5.0, the reduced vacuum concentrated by rotary evaporation obtains concentrated solution.
C) concentrated solution obtained sour water pre-treatment octadecyl (C18) the bonded silica gel chromatography column that is 3.0-5.0 by the pH value, and by b) is slowly by above-mentioned C18 reverse phase silica gel post; Then the acid rinsing pillar 3-4CV that is 3.0-5.0 by the pH value; Again with add containing 1%-3% (v: the v) aqueous solution of methyl alcohol or ethanol, 2-4CV wash-out pillar, substep is collected and HPLC detects effluent liquid, then with the aqueous solution containing 4%-8% methyl alcohol or ethanol, 3-8CV wash-out pillar, substep is collected and HPLC detects; Purity is greater than to 85% receiving flask merging, freeze-drying after the reduced vacuum concentrated by rotary evaporation, make 7 α-unformed powder of methoxy cephalosporin C.
The inventive method a) in step acid used be hydrochloric acid or sulfuric acid, be preferably hydrochloric acid.
The inventive method is a) after the conversion fluid suction filtration is got filtrate in step, and preferably the pH value is adjusted to 3.0-4.5.
The inventive method a) in step low fat alcohol be methyl alcohol, ethanol, acetone or Virahol, be preferably methyl alcohol or ethanol.
The inventive method b) in step, macroporous adsorbent resin is commercially available unmodified polystyrene-divinylbenzene type macrolattice middle polarity resin, its specific surface area>=300m
2/ g, characteristic aperture is
Mediation particle diameter 250-840 μ m.The preferred HZ-816 of Shanghai Huazhen Science and Technology Co., Ltd., HZ-803, HZ-818 type resin, the XAD-16N of Rhom and Hass, XAD-16HP, XAD1600N, XAD1180, XAD-4 or XAD-18 type resin, most preferably be the XAD-18 resin.
In the present invention, term " mediation particle diameter " is defined as in composite grain, the mean diameter of all particles.
The inventive method b), c) sour water in step refers to contain a small amount of aqueous acid, this acid is sulfuric acid or hydrochloric acid, is preferably hydrochloric acid.
The inventive method b), c) sour water described in step is the aqueous solution that pH is 2.5-4.5, preferred 3.0-4.0.
The inventive method b) lower aliphatic of 10%-40% described in step alcohol solution for adding the alcohol that volume is 10%-40% to prepare in distilled water or deionized water; Described 15%-30% lower aliphatic alcohol solution for adding the alcohol that volume is 15%-30% to prepare in distilled water or deionized water.
The inventive method c) in step, octadecyl (C18) bonded silica gel filler is spherical hydrophilic, and characteristic aperture is
The particle diameter that is in harmonious proportion is 5-70 μ m.Preferably point scientific and technological filler Bio-C8 of company limited, HP-C18 are matched in Suzhou; There are greatly the filler DYB7130 of chromatographic technique Services Co., Ltd, DYB7219 in Shanghai.
The inventive method c) contain 1%-3% (v: the methyl alcohol that the ethanol that v) in the aqueous solution of methyl alcohol or ethanol, preferred alcohols concentration is 1%-2% or determining alcohol are 2%-3% described in step; The methyl alcohol that the ethanol that in the aqueous solution of described 4%-8% (a little more than concentration before) methyl alcohol or ethanol, preferred alcohols concentration is 4%-5% or determining alcohol are 4%-7%.
The inventive method b), chromatography column aspect ratio c) used in step is more than or equal to 3: 1.
Step b in the inventive method) Main Function is to remove the protein in conversion fluid, salt, pigment etc. some not with the impurity of resin absorption, this is for improving step c) separating effect helpful.
Step c of the present invention) in, with the low fat alcohol of lower concentration, carry out wash-out purpose product, can remove well other strong polar impurities, and can be good at methoxy cephalosporin C is separated with substrate, the intermediate product of conversion, the purpose product purity obtained is higher.
Method of the present invention is simple, but resin and reverse phase silica gel filler Reusability, regeneration is convenient, the few and recovery capable of circulation of the organic solvent amount of use, and the purity of the purpose product obtained is very high.
The accompanying drawing explanation
Fig. 1 is the process that the substrate cephalosporin is converted into 7 α-methoxy cephalosporin C;
High performance liquid chromatography (HPLC) collection of illustrative plates (7 α in figure-methoxy cephalosporin C appearance time is 17.4min) of 7 α that Fig. 2 is embodiment 1 acquisition-methoxy cephalosporin C (HPLC purity 97.6%);
The mass-spectrogram of 7 α that Fig. 3 is embodiment 1 acquisition-methoxy cephalosporin C (HPLC purity 97.6%);
The nuclear-magnetism H1 spectrum of 7 α that Fig. 4 is embodiment 1 acquisition-methoxy cephalosporin C (HPLC purity 97.6%).
Embodiment
The present invention 7 α-methoxy cephalosporin C HPLC testing conditions is as follows:
Chromatographic column: post Ultimate AQ-C18,5 μ m, 250 * 4.6mm;
Detect wavelength: 272nm;
Column temperature: 30 ℃;
Flow velocity: 0.8ml/min;
Moving phase: A phase: add 2.4ml formic acid in 1L water, the 0.77g ammonium acetate
B phase: add 2ml formic acid in the 1L acetonitrile
Thalli growth condition used in the present invention is as follows:
Bacterial classification: Streptomyces clavuligerus ATCC27064 (NRRL3585)
Slant culture
Substratum (g/L): malt extract 10, yeast extract 4, glucose 4, agar 25, pH7.0-7.4,121 ℃ of sterilizing 30min;
Culture condition: cultivate 7-10 days for 28 ℃.
Seed culture
Substratum (g/L): glucose 10, import peptone 10,5,121 ℃ of sterilizing 20min of import yeast extract;
Culture condition: 28 ℃, 250rpm, cultivate 2 days.
Conversion condition used in the present invention is as follows:
Clavuligerus Streptomyces clavuligerus ATCC27064 cultivates after 2 days suction filtration and collects thalline in seed culture medium, and with after deionized water wash, put-20 ℃ frozen.
The every gram of frozen thalline is added to 10ml phosphate buffered saline buffer (pH7.4), melt rear ultrasonication, as first enzyme liquid, add cofactor 2-oxoglutaric acid (final concentration 2mM), L-AA (final concentration 1mM), FeSO
4(final concentration 1mM), S-adenosylmethionine (SAM, final concentration 1mM) and substrate cephalosporin (final concentration 500 μ g/ml) mix, and the constant temperature oscillation conversion (28 ℃, 240r/min) 4 to 6h.
Get conversion fluid 500ml, wherein the content of 7 α-methoxy cephalosporin C is about 0.2mg/ml, and HPLC purity is about 7%, adds the 25ml Glacial acetic acid in conversion fluid, mixes rear filtration, gets filtrate, and recording the pH value is 3.5, without regulating the pH value again.
Fill a high about 20cm, the XAD-18 macroporous adsorption resin chromatography post of diameter 5cm, cross post by above-mentioned filtrate with the flow velocity of 7ml/min, the sour water 3CV washing pillar of the pH3.5 prepared with hydrochloric acid; With 20% ethanolic soln 3CV washing pillar, each 70ml collects one bottle again.With HPLC, detect, the receiving flask that methoxy cephalosporin C content is higher merges, and purity is 24%.
Fill a high about 10cm, the sour water that the Bio-C18 chromatography column of diameter 2cm is 3.5 with the hydrochloric acid secure ph is processed pillar.The part reduced vacuum concentrated by rotary evaporation that previous step is merged, after adjust pH 3.5, upper prop, flow velocity is 1ml/min.The sour water 3CV washing pillar that the pH value of preparing with hydrochloric acid is 3.5, remove impurity; Then wash with 2% methanol solution 3CV, every 15ml, collect a pipe, HPLC detects; With 4% methanol solution 4CV, wash, every 10ml collects a pipe again, and HPLC detects.
The part that purity is higher merges, and the purity of last amalgamation liquid is 90%, and yield is 65%.
Embodiment 2
Get conversion fluid 500ml, wherein the content of 7 α-methoxy cephalosporin C is about 0.2mg/ml, and HPLC purity is about 6%, adds the 25ml Glacial acetic acid in conversion fluid, mixes rear filtration, gets filtrate, and recording the pH value is 3.6, without regulating the pH value again.
Fill a high about 20cm, the XAD-4 macroporous adsorption resin chromatography post of diameter 5cm, cross post by above-mentioned filtrate with the flow velocity of 7ml/min, the sour water 3CV washing pillar of the pH3.5 prepared with hydrochloric acid; With 30% ethanolic soln 3CV washing pillar, each 70ml collects one bottle again.Detect with HPLC, will have the part of more methoxy cephalosporin C to merge, purity is 23%.
Fill a high about 10cm, the sour water that the HP-C18 chromatography column of diameter 2cm is 3.5 with the hydrochloric acid secure ph is processed pillar.The part reduced vacuum concentrated by rotary evaporation that previous step is merged, after adjust pH 3.5, upper prop, flow velocity is 1ml/min.The sour water 3CV washing pillar that the pH value of preparing with hydrochloric acid is 3.5, remove impurity; Then wash with 1% ethanolic soln 3CV, every 15ml, collect a pipe, HPLC detects; With 4% ethanolic soln 4CV, wash, every 10ml collects a pipe again, and HPLC detects.
The part that purity is higher merges, and the purity of last amalgamation liquid is 92%, and yield is 62%.
Embodiment 3
Get conversion fluid 500ml, wherein the content of 7 α-methoxy cephalosporin C is about 0.15mg/ml, and HPLC purity is about 5%, adds the 25ml Glacial acetic acid in conversion fluid, mixes rear filtration, gets filtrate, and recording the pH value is 3.6, without regulating the pH value again.
Fill a high about 20cm, the HZ-816 macroporous adsorption resin chromatography post of diameter 5cm, cross post by above-mentioned filtrate with the flow velocity of 6ml/min, the sour water 3CV washing pillar of the pH3.5 prepared with hydrochloric acid; With 15% ethanolic soln 3CV washing pillar, each 70ml collects one bottle again.Detect with HPLC, will have the part of more methoxy cephalosporin C to merge, purity is 20%.
Fill a high about 10cm, the DYB7130 of diameter 2cm (mediation particle diameter 5 μ m) chromatography column, the sour water that is 3.5 with the hydrochloric acid secure ph is processed pillar.The part reduced vacuum concentrated by rotary evaporation that previous step is merged, after adjust pH 3.5, upper prop, flow velocity is 0.8ml/min.The sour water 3CV washing pillar that the pH value of preparing with hydrochloric acid is 3.5, remove impurity; Then wash with 3% methanol solution 3CV, every 15ml, collect a pipe, HPLC detects; With 6% methanol solution 4CV, wash, every 10ml collects a pipe again, and HPLC detects.
The part that purity is higher merges, and the purity of last amalgamation liquid is 92%, and yield is 64%.
Embodiment 4
Get conversion fluid 500ml, wherein the content of 7 α-methoxy cephalosporin C is about 0.2mg/ml, and HPLC purity is about 6%, adds the 25ml Glacial acetic acid in conversion fluid, mixes rear filtration, gets filtrate, and recording the pH value is 3.5, without regulating the pH value again.
Fill a high about 20cm, the HZ-803 macroporous adsorption resin chromatography post of diameter 5cm, cross post by above-mentioned filtrate with the flow velocity of 7ml/min, the sour water 3CV washing pillar of the pH3.5 prepared with hydrochloric acid; With 20% ethanolic soln 3CV washing pillar, each 70ml collects one bottle again.Detect with HPLC, will have the part of more methoxy cephalosporin C to merge, purity is 22%.
Fill a high about 10cm, the DYB7130 of diameter 2cm (mediation particle diameter 40 μ m) chromatography column, the sour water that is 3.5 with the hydrochloric acid secure ph is processed pillar.The part reduced vacuum concentrated by rotary evaporation that previous step is merged, after adjust pH 3.5, upper prop, flow velocity is 0.8ml/min.The sour water 3CV washing pillar that the pH value of preparing with hydrochloric acid is 3.5, remove impurity; Then wash with 3% methanol solution 3CV, every 15ml, collect a pipe, HPLC detects; With 7% methanol solution 4CV, wash, each 10ml collects a pipe again, and HPLC detects.
The part that purity is higher merges, and the purity of last amalgamation liquid is 92%, and yield is 62%.
Get conversion fluid 500ml, wherein the content of 7 α-methoxy cephalosporin C is about 0.2mg/ml, and HPLC purity is about 5%, adds the 25ml Glacial acetic acid in conversion fluid, mixes rear filtration, gets filtrate, and recording the pH value is 3.6, without regulating the pH value again.
Fill a high about 20cm, the HZ-818 macroporous adsorption resin chromatography post of diameter 5cm, cross post by above-mentioned filtrate with the flow velocity of 7ml/min, the sour water 3CV washing pillar of the pH3.5 prepared with hydrochloric acid; With 35% ethanolic soln 3CV washing pillar, each 70ml collects one bottle again.Detect with HPLC, will have the part of more methoxy cephalosporin C to merge, purity is 20%.
Fill a high about 10cm, the DYB7130 of diameter 2cm (mediation particle diameter 20 μ m) chromatography column, the sour water that is 3.5 with the hydrochloric acid secure ph is processed pillar.The part reduced vacuum concentrated by rotary evaporation that previous step is merged, after adjust pH 3.5, upper prop, flow velocity is 1ml/min.The sour water 3CV washing pillar that the pH value of preparing with hydrochloric acid is 3.5, remove impurity; Then wash with 2% ethanolic soln 3CV, every 15ml, collect a pipe, HPLC detects; With 4% ethanolic soln 4CV, wash, every 10ml collects a pipe again, and HPLC detects.
The part that purity is higher merges, and the purity of last amalgamation liquid is 90%, and yield is 65%.
Embodiment 6
Get conversion fluid 500ml, wherein the content of 7 α-methoxy cephalosporin C is about 0.2mg/ml, and HPLC purity is about 5%, adds the 25ml Glacial acetic acid in conversion fluid, mixes rear filtration, gets filtrate, and recording the pH value is 3.4, without regulating the pH value again.
Fill a high about 20cm, the XAD-16N macroporous adsorption resin chromatography post of diameter 5cm, cross post by above-mentioned filtrate with the flow velocity of 7ml/min, the sour water 3CV washing pillar of the pH3.5 prepared with hydrochloric acid; With 30% ethanolic soln 3CV washing pillar, each 70ml collects one bottle again.Detect with HPLC, will have the part of more methoxy cephalosporin C to merge, purity is 19%.
Fill a high about 10cm, the DYB7219 of diameter 2cm (mediation particle diameter 5 μ m) chromatography column, the sour water that is 3.5 with the hydrochloric acid secure ph is processed pillar.The part reduced vacuum concentrated by rotary evaporation that previous step is merged, after adjust pH 3.5, upper prop, flow velocity is 1ml/min.The sour water 3CV washing pillar that the pH value of preparing with hydrochloric acid is 3.5, remove impurity; Then wash with 2% methanol solution 3CV, every 15ml, collect a pipe, HPLC detects; With 5% methanol solution 4CV, wash, every 10ml collects a pipe again, and HPLC detects.
The part that purity is higher merges, and the purity of last amalgamation liquid is 93%, and yield is 62%.
Embodiment 7
Get conversion fluid 500ml, wherein the content of 7 α-methoxy cephalosporin C is about 0.2mg/ml, and HPLC purity is about 4%, adds the 25ml Glacial acetic acid in conversion fluid, mixes rear filtration, gets filtrate, and recording the pH value is 3.5, without regulating the pH value again.
Fill a high about 20cm, the XAD-1600N macroporous adsorption resin chromatography post of diameter 5cm, cross post by above-mentioned filtrate with the flow velocity of 7ml/min, the sour water 3CV washing pillar of the pH3.5 prepared with hydrochloric acid; With 20% ethanolic soln 3CV washing pillar, each 70ml collects one bottle again.Detect with HPLC, will have the part of more methoxy cephalosporin C to merge, purity is 23%.
Fill a high about 10cm, the DYB7219 of diameter 2cm (mediation particle diameter 50 μ m) chromatography column, the sour water that is 3.5 with the hydrochloric acid secure ph is processed pillar.The part reduced vacuum concentrated by rotary evaporation that previous step is merged, after adjust pH 3.5, upper prop, flow velocity is 1ml/min.The sour water 3CV washing pillar that the pH value of preparing with hydrochloric acid is 3.5, remove impurity; Then wash with 2% ethanolic soln 3CV, every 15ml, collect a pipe, HPLC detects; With 4% ethanolic soln 4CV, wash, every 10ml collects a pipe again, and HPLC detects.
The part that purity is higher merges, and the purity of last amalgamation liquid is 91%, and yield is 64%.
Embodiment 8
Get conversion fluid 500ml, wherein the content of 7 α-methoxy cephalosporin C is about 0.2mg/ml, and HPLC purity is about 7%, adds the 25ml Glacial acetic acid in conversion fluid, mixes rear filtration, gets filtrate, and recording the pH value is 3.5, without regulating the pH value again.
Fill a high about 20cm, the XAD-1180N macroporous adsorption resin chromatography post of diameter 5cm, cross post by above-mentioned filtrate with the flow velocity of 7ml/min, the sour water 3CV washing pillar of the pH3.5 prepared with hydrochloric acid; With 25% ethanolic soln 3CV washing pillar, each 70ml collects one bottle again.Detect with HPLC, will have the part of more methoxy cephalosporin C to merge, purity is 20%.
Fill a high about 10cm, the DYB7219 of diameter 2cm (mediation particle diameter 20 μ m) chromatography column, the sour water that is 3.5 with the hydrochloric acid secure ph is processed pillar.The part reduced vacuum concentrated by rotary evaporation that previous step is merged, after adjust pH 3.5, upper prop, flow velocity is 1ml/min.The sour water 3CV washing pillar that the pH value of preparing with hydrochloric acid is 3.5, remove impurity; Then wash with 3% methanol solution 3CV, every 15ml, collect a pipe, HPLC detects; With 6% methanol solution 4CV, wash, every 10ml collects a pipe again, and HPLC detects.
The part that purity is higher merges, and the purity of last amalgamation liquid is 91%, and yield is 64%.
Claims (14)
1. the method for preparing 7 α-methoxy cephalosporin C, it is characterized in that described method comprises by conversion fluid pre-treatment, polymeric adsorbent Image processing and reversed-phase silica gel column chromatography processes, from by the cephalosporin substrate conversion being the step of preparation 7 α-methoxy cephalosporin C the conversion fluid of 7 α-methoxy cephalosporin C.
2. the method for claim 1, is characterized in that described conversion fluid pre-treatment comprises the steps:
In every milliliter of conversion fluid, add the Glacial acetic acid of 30-70 μ l to mix, suction filtration is got the filtrate of clarification, with sour adjust pH, is 3.0-5.0, obtains the upper prop mother liquor.
3. method as claimed in claim 1 or 2, is characterized in that described polymeric adsorbent Image processing comprises the steps:
Described upper prop mother liquor, by the macroporous adsorption resin chromatography post, is obtained to concentrated solution; The acid rinsing pillar 2-6CV that is 2.5-4.5 with pH; Then with the low fat alcohol aqueous solution containing 10%-40%, 2-5CV wash-out pillar, collect step by step and HPLC detects effluent liquid; 7 α-methoxy cephalosporin C HPLC purity is greater than to 15% receiving flask merging, adjusts pH3.0-5.0, the reduced vacuum concentrated by rotary evaporation obtains concentrated solution.
4. method as described as claim 1 or 3, is characterized in that described reversed-phase silica gel column chromatography processing comprises the steps:
The sour water pre-treatment octadecyl silane chromatography column that is 3.0-5.0 by the pH value, and described concentrated solution is slowly passed through to above-mentioned C18 reverse phase silica gel post; Then the acid rinsing pillar 3-4CV that is 3.0-5.0 by the pH value; With adding the aqueous solution containing 1%-3% methyl alcohol or ethanol, 2-4CV wash-out pillar, collect step by step and HPLC detects effluent liquid again, then with the aqueous solution that contains 4%-8% methyl alcohol or ethanol, and 3-8CV wash-out pillar, substep is collected and HPLC detects; Purity is greater than to 85% receiving flask merging, freeze-drying after the reduced vacuum concentrated by rotary evaporation, make 7 α-unformed powder of methoxy cephalosporin C.
5. method as claimed in claim 2, is characterized in that described acid is hydrochloric acid or sulfuric acid.
6. method as claimed in claim 2, is characterized in that described pH value is adjusted to 3.0-4.5.
7. method as claimed in claim 3, is characterized in that described low fat alcohol is methyl alcohol, ethanol, acetone or Virahol.
8. method as claimed in claim 3, is characterized in that described macroporous adsorbent resin is polystyrene-divinylbenzene type macrolattice middle polarity resin, its specific surface area>=300m
2/ g, characteristic aperture is
, mediation particle diameter 250-840 μ m.
9. method as described as claim 3 or 4, is characterized in that described sour water is the aqueous solution that pH is 2.5-4.5, and described acid is sulfuric acid or hydrochloric acid.
10. method as claimed in claim 9, the pH value that it is characterized in that described sour water is 3.0-4.0.
11. method as claimed in claim 3, is characterized in that described 10%-40% lower aliphatic alcohol solution for adding the alcohol that volume is 10%-40% to prepare in distilled water or deionized water.
12. method as claimed in claim 4, the filler that it is characterized in that described octadecyl silane is spherical hydrophilic, and characteristic aperture is
, the particle diameter that is in harmonious proportion is 5-70 μ m.
13. method as claimed in claim 4, is characterized in that in the described aqueous solution containing 1%-3% methyl alcohol or ethanol, determining alcohol is 1%-2% ethanol or 2%-3% methyl alcohol; In the described aqueous solution containing 4%-8% methyl alcohol or ethanol, determining alcohol is 4%-5% ethanol or 4%-7% methyl alcohol.
14. method as described as claim 3 or 4, is characterized in that described chromatography column aspect ratio is more than or equal to 3: 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210162720.9A CN103421025B (en) | 2012-05-21 | 2012-05-21 | Prepare the method for 7 α-methoxy cephalosporin C |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210162720.9A CN103421025B (en) | 2012-05-21 | 2012-05-21 | Prepare the method for 7 α-methoxy cephalosporin C |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103421025A true CN103421025A (en) | 2013-12-04 |
| CN103421025B CN103421025B (en) | 2016-05-11 |
Family
ID=49646445
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210162720.9A Expired - Fee Related CN103421025B (en) | 2012-05-21 | 2012-05-21 | Prepare the method for 7 α-methoxy cephalosporin C |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103421025B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3467654A (en) * | 1966-08-19 | 1969-09-16 | Lilly Co Eli | Process for the recovery and purification of cephalosporin c |
| CN1226250A (en) * | 1996-07-29 | 1999-08-18 | 布里斯托尔-迈尔斯斯奎布公司 | Solvent extraction of 3-hydroxymethylcephalosporins |
| CN1234036A (en) * | 1996-09-20 | 1999-11-03 | 明治制果株式会社 | Crystalline substance of cefditoren pivoxyl and prodn. of same |
| CN101288841A (en) * | 2008-06-11 | 2008-10-22 | 山东鲁抗立科药物化学有限公司 | Macroporous adsorption resin special for extracting cephalosporin C and its preparation method |
| CN101497620A (en) * | 2009-02-16 | 2009-08-05 | 浙江大学 | Purification method of penicillin and cephalosporin |
| CN101556262A (en) * | 2009-05-14 | 2009-10-14 | 海口市制药厂有限公司 | Method for detecting impurities in cefoxitin product |
| CN102134251A (en) * | 2011-03-24 | 2011-07-27 | 上海交通大学 | Method for extracting cephalosporin C by using water soluble organic solvent/salt/water two-phase system |
| CN102212075A (en) * | 2010-04-06 | 2011-10-12 | 上海安瀚特生物医药技术有限公司 | Preparation method for cefbuperazone |
| CN202028233U (en) * | 2011-04-22 | 2011-11-09 | 南昌立健药业有限公司 | Column separation device used in technical process of cephalosporin antibiotics production |
-
2012
- 2012-05-21 CN CN201210162720.9A patent/CN103421025B/en not_active Expired - Fee Related
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3467654A (en) * | 1966-08-19 | 1969-09-16 | Lilly Co Eli | Process for the recovery and purification of cephalosporin c |
| CN1226250A (en) * | 1996-07-29 | 1999-08-18 | 布里斯托尔-迈尔斯斯奎布公司 | Solvent extraction of 3-hydroxymethylcephalosporins |
| CN1234036A (en) * | 1996-09-20 | 1999-11-03 | 明治制果株式会社 | Crystalline substance of cefditoren pivoxyl and prodn. of same |
| CN101288841A (en) * | 2008-06-11 | 2008-10-22 | 山东鲁抗立科药物化学有限公司 | Macroporous adsorption resin special for extracting cephalosporin C and its preparation method |
| CN101497620A (en) * | 2009-02-16 | 2009-08-05 | 浙江大学 | Purification method of penicillin and cephalosporin |
| CN101556262A (en) * | 2009-05-14 | 2009-10-14 | 海口市制药厂有限公司 | Method for detecting impurities in cefoxitin product |
| CN102212075A (en) * | 2010-04-06 | 2011-10-12 | 上海安瀚特生物医药技术有限公司 | Preparation method for cefbuperazone |
| CN102134251A (en) * | 2011-03-24 | 2011-07-27 | 上海交通大学 | Method for extracting cephalosporin C by using water soluble organic solvent/salt/water two-phase system |
| CN202028233U (en) * | 2011-04-22 | 2011-11-09 | 南昌立健药业有限公司 | Column separation device used in technical process of cephalosporin antibiotics production |
Non-Patent Citations (4)
Also Published As
| Publication number | Publication date |
|---|---|
| CN103421025B (en) | 2016-05-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20080312447A1 (en) | Process for Isolation of Crystalline Tacrolimus | |
| US20180179244A1 (en) | Fidaxomicin purification method | |
| CN108250270A (en) | A kind of method of the enrichment extraction Daptomycin from zymotic fluid | |
| CN101348494A (en) | High-purity cefmenoxime hydrochloride and preparation thereof | |
| CN103421025B (en) | Prepare the method for 7 α-methoxy cephalosporin C | |
| CN102690333B (en) | Preparation method of high-purity teicoplanin | |
| EP3962929B1 (en) | Method for the purification of lipoglycopeptide antibiotics | |
| CN112961891B (en) | Method for preparing icariin by using biphasic enzymatic reaction | |
| CN101638401A (en) | Method for preparing high-purity danshinolic acid B | |
| CN103224547A (en) | Daptomycin separation and purification method | |
| CN114660187A (en) | Preparation method of beta-nicotinamide mononucleotide | |
| CN101314605A (en) | Method for extracting cefaclor from cefaclor naphthalenol complexes | |
| JP3067183B2 (en) | Method for producing FR900506 substance | |
| CN103130874B (en) | A kind of preparation method of high purity ramoplanin | |
| CN111187339B (en) | Method for extracting FR901379 from fermentation broth | |
| CN112390817B (en) | Method for salting out and extracting tacrolimus fermentation liquor | |
| NZ244774A (en) | Protein-associated chromophore from actinomadura, pharmaceutical compositions and production | |
| CN106632551A (en) | Method for preparing fidaxomicin by flash chromatography | |
| CN101831417A (en) | Preparation method of L-asparaginase | |
| JPH101496A (en) | Purification of avermectin | |
| CN1131236C (en) | Process for purification of 11 beta'-21-dihydroxy-2'-methyl-5 beta H-pregna-1,4-dieno [17,16-D] oxazole-3,20-dione | |
| CN1030839C (en) | Process for 1-deoxy macnogiley mycin | |
| JP4443708B2 (en) | Mizoribine production method | |
| EP0547484A1 (en) | Process for producing antibiotic R106-I | |
| JP2690779B2 (en) | L-ascorbic acid derivative and method for producing the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160511 Termination date: 20210521 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |