CN102605033B - Method for preparing cefazolin intermediate TDA - Google Patents
Method for preparing cefazolin intermediate TDA Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 57
- MLYYVTUWGNIJIB-BXKDBHETSA-N cefazolin Chemical compound S1C(C)=NN=C1SCC1=C(C(O)=O)N2C(=O)[C@@H](NC(=O)CN3N=NN=C3)[C@H]2SC1 MLYYVTUWGNIJIB-BXKDBHETSA-N 0.000 title abstract description 5
- 229960001139 cefazolin Drugs 0.000 title abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims abstract description 149
- IXUSDMGLUJZNFO-BXUZGUMPSA-N (7R)-7-(4-carboxybutanamido)cephalosporanic acid Chemical compound S1CC(COC(=O)C)=C(C(O)=O)N2C(=O)[C@@H](NC(=O)CCCC(O)=O)[C@@H]12 IXUSDMGLUJZNFO-BXUZGUMPSA-N 0.000 claims abstract description 46
- FPVUWZFFEGYCGB-UHFFFAOYSA-N 5-methyl-3h-1,3,4-thiadiazole-2-thione Chemical compound CC1=NN=C(S)S1 FPVUWZFFEGYCGB-UHFFFAOYSA-N 0.000 claims abstract description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 44
- 238000003756 stirring Methods 0.000 claims description 31
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- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 25
- 239000012043 crude product Substances 0.000 claims description 25
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 19
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 18
- 238000000746 purification Methods 0.000 claims description 17
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- 239000000706 filtrate Substances 0.000 claims description 14
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- 239000012065 filter cake Substances 0.000 claims description 12
- 239000007791 liquid phase Substances 0.000 claims description 12
- HZWLVUKHUSRPCG-BJIHTTGYSA-M sodium;(6r,7r)-3-(acetyloxymethyl)-7-[(5-amino-5-carboxypentanoyl)amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate Chemical compound [Na+].S1CC(COC(=O)C)=C(C([O-])=O)N2C(=O)[C@@H](NC(=O)CCCC(N)C(O)=O)[C@@H]12 HZWLVUKHUSRPCG-BJIHTTGYSA-M 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 10
- 238000001291 vacuum drying Methods 0.000 claims description 10
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- 239000000843 powder Substances 0.000 claims description 9
- 239000002994 raw material Substances 0.000 claims description 8
- JFPVXVDWJQMJEE-QMTHXVAHSA-N Cefuroxime Chemical compound N([C@@H]1C(N2C(=C(COC(N)=O)CS[C@@H]21)C(O)=O)=O)C(=O)C(=NOC)C1=CC=CO1 JFPVXVDWJQMJEE-QMTHXVAHSA-N 0.000 claims description 7
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- HOKIDJSKDBPKTQ-GLXFQSAKSA-N cephalosporin C Chemical compound S1CC(COC(=O)C)=C(C(O)=O)N2C(=O)[C@@H](NC(=O)CCC[C@@H](N)C(O)=O)[C@@H]12 HOKIDJSKDBPKTQ-GLXFQSAKSA-N 0.000 abstract description 8
- 239000003960 organic solvent Substances 0.000 abstract description 8
- 108010034416 glutarylamidocephalosporanic acid acylase Proteins 0.000 abstract description 6
- -1 5-methyl-1,3,4-thiadiazol-2yl Chemical group 0.000 abstract description 4
- 102000004674 D-amino-acid oxidase Human genes 0.000 abstract 1
- 108010003989 D-amino-acid oxidase Proteins 0.000 abstract 1
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- VLLMWSRANPNYQX-UHFFFAOYSA-N thiadiazole Chemical compound C1=CSN=N1.C1=CSN=N1 VLLMWSRANPNYQX-UHFFFAOYSA-N 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 87
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- HSHGZXNAXBPPDL-HZGVNTEJSA-N 7beta-aminocephalosporanic acid Chemical compound S1CC(COC(=O)C)=C(C([O-])=O)N2C(=O)[C@@H]([NH3+])[C@@H]12 HSHGZXNAXBPPDL-HZGVNTEJSA-N 0.000 description 15
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- HOKIDJSKDBPKTQ-GLXFQSAKSA-M cephalosporin C(1-) Chemical compound S1CC(COC(=O)C)=C(C([O-])=O)N2C(=O)[C@@H](NC(=O)CCC[C@@H]([NH3+])C([O-])=O)[C@@H]12 HOKIDJSKDBPKTQ-GLXFQSAKSA-M 0.000 description 6
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- KFCMZNUGNLCSJQ-NFBKMPQASA-N (4-methoxyphenyl)methyl (6r,7r)-3-(chloromethyl)-8-oxo-7-[(2-phenylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate Chemical compound C1=CC(OC)=CC=C1COC(=O)C1=C(CCl)CS[C@H]2N1C(=O)[C@H]2NC(=O)CC1=CC=CC=C1 KFCMZNUGNLCSJQ-NFBKMPQASA-N 0.000 description 5
- 229930186147 Cephalosporin Natural products 0.000 description 5
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- JJJPTTANZGDADF-UHFFFAOYSA-N thiadiazole-4-thiol Chemical class SC1=CSN=N1 JJJPTTANZGDADF-UHFFFAOYSA-N 0.000 description 4
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- 230000008901 benefit Effects 0.000 description 3
- KZMGYPLQYOPHEL-UHFFFAOYSA-N boron trifluoride etherate Substances FB(F)F.CCOCC KZMGYPLQYOPHEL-UHFFFAOYSA-N 0.000 description 3
- FLKYBGKDCCEQQM-WYUVZMMLSA-M cefazolin sodium Chemical compound [Na+].S1C(C)=NN=C1SCC1=C(C([O-])=O)N2C(=O)[C@@H](NC(=O)CN3N=NN=C3)[C@H]2SC1 FLKYBGKDCCEQQM-WYUVZMMLSA-M 0.000 description 3
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- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
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- AIDLAEPHWROGFI-UHFFFAOYSA-N 2-methylbenzene-1,3-dicarboxylic acid Chemical compound CC1=C(C(O)=O)C=CC=C1C(O)=O AIDLAEPHWROGFI-UHFFFAOYSA-N 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
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- 241000193738 Bacillus anthracis Species 0.000 description 1
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- RZMVCLIAMAGMFW-UHFFFAOYSA-N OC(O)=O.F.F.F Chemical compound OC(O)=O.F.F.F RZMVCLIAMAGMFW-UHFFFAOYSA-N 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 108010073038 Penicillin Amidase Proteins 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
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- LYGJENNIWJXYER-UHFFFAOYSA-N nitromethane Chemical compound C[N+]([O-])=O LYGJENNIWJXYER-UHFFFAOYSA-N 0.000 description 1
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- Cephalosporin Compounds (AREA)
Abstract
The invention provides a method for preparing intermediate cefazolin TDA (7-amino-3-[(5-methyl-1,3,4-thiadiazol-2yl)thiomethyl]-3-cephalosporanicacid). The method comprises the following steps of: oxidizing cephalosporin C to be GL-7-ACA (glutaryl-7-aminocephalosporanic acid) by using D-amino acid oxidase; reacting with 2-methyl-5-sulfydryl-1,3,4-thiadiazole; removing glutaryl by using GL-7-ACA acylase to finally obtain TDA. The method provided by the invention improves a TDA synthetic route. By using reaction and synthesis based on the biological enzyme method, the method is green and environment-friendly and has no pollution. The problems in chemical production, such as large organic solvent using amount and long synthetic route, can be solved. Furthermore, thiadiazole can be recycled, so that the product quality is improved, the using amount of side chain is reduced, and the method is applicable to the industrial production.
Description
Technical field
The present invention relates to the preparation field of Kefzol Intermediate TDA, especially, relate to a kind of preparation method of Kefzol Intermediate TDA.
Background technology
Cephazolin sodium has another name called cephalosporin, and this medicine has good anti-microbial activity to gram-positive cocci (as streptococcus pneumoniae, Hemolytic streptococcus, diphtheria corynebacterium, anthrax bacillus, listeria bacteria and clostridium).This medicine also has good anti-microbial activity to part escherichia coli, Proteus mirabilis and klebostiella pneumoniae simultaneously.Cephazolin sodium is as first generation cephalosporin, have the advantages such as relatively anti-enzyme, efficient, low toxicity, pharmacokinetics be more satisfactory, for organ sensitive organisms such as liver, kidney, the heart, spleen, lung, muscle, infect, clinical treatment respiratory system and urinary system infection especially for some severe infections have unusual effect, and are widely used in the rear preventing infection of prevention of surgical operation.
In prior art, TDA commonly used is that raw material connects the tetrazole acetyl group by chemical method and obtains cefazolin on 7 of TDA.The sodium salt of cefazolin is cephazolin sodium.
TDA is 7-amino-3-(2-methyl isophthalic acid, 3,4-thiadiazoles-5-yl) thiomethyl-3-cephem-4-carboxylic acid, structural formula is as follows:
Existing TDA production technique is mainly with the 7-ACA(7-amino-cephalo-alkanoic acid) be raw material, at 70 ℃ of lower 7-ACA and MMTD(2-methyl-5-sulfydryl-1,3,4-thiadiazole) react and make, but yield is only 60～65%, as US5, and 387,679.And while adopting the method to produce TDA, side reaction easily occurs in 3 acetoxyl groups of 7-ACA when introducing (2-methyl isophthalic acid, 3,4-thiadiazoles-5-yl) thio group.This side reaction energy very exothermic.And the poor heat stability of 7-ACA, under high temperature, products therefrom is degraded into by product more, thus product yield and purity not high.
A kind of method for preparing Kefzol is disclosed in CN1178220A; the method be take GCLE(7-phenylacetyl amino-3-monochloromethyl-3-cephem-4-carboxylicesters) be raw material; GCLE elder generation and 2-methyl-5-sulfydryl-1; 3; the reaction of 4-thiadiazoles; degreasing under the existence of phenol again, then adopt penicillin acylase to make the product after degreasing slough phenylacetyl and obtain TDA.Wherein GCLE used passes through the penicillin oxidation, ring expansion, and the polystep reactions such as halo obtain.The step of preparation process of GCLE is loaded down with trivial details, complicated operation, and productive rate is low, and environmental pollution is serious.And the TDA purity prepared by GCLE is low, yield is low, and the cost of reaction is high.
Technical scheme in US4317907 is solvent for take 7-ACA as raw material is used Nitromethane 99Min. or acetic acid, adds boron trifluoride or boron trifluoride diethyl etherate under anhydrous condition, and 7-ACA is reacted with MMTD, and yield can be increased to approximately 86%.WO9,302,085 have proposed, and in the presence of dialkyl carbonate trifluoride complex compound and lipid acid, the high energy of yield reaches 89%.Although aforementioned two kinds of method productive rates slightly are improved, but the two all needs to use boron trifluoride to carry out reduction catalysts, and boron trifluoride is poisonous and very expensive, and also need to process the refuse that contains boride after reaction, and it is very high to carry out innoxious cost to these materials.So method also is unsuitable for scale operation.
Summary of the invention
The technical problems such as the object of the invention is to provide a kind of preparation method of Kefzol Intermediate TDA, low with the productive rate that solves TDA in prior art, that the boron trifluoride environmental pollution is serious.
For achieving the above object, according to an aspect of the present invention, provide a kind of preparation method of Kefzol Intermediate TDA, it is characterized in that, comprised the following steps:
1) take cephalosporin C Sodium makes GL-7-ACA solution as raw material reaction;
2) press the grams of 2-methyl-5-sulfydryl-1,3,4-thiadiazole: NaCO
3Grams: the milliliter number of water is warming up to 60～70 ℃ for (27:11:100)～(40:16:100) after mixing, add GL-7-ACA solution, obtain the first reaction solution, the first reaction solution is after reacting 3～4h under 60～70 ℃, obtain the GL-TDA crude product, the GL-TDA crude product obtains GL-TDA solution through the first purification step;
The GL-7-ACA ACY of 5000～7000U is added in GL-TDA solution; obtain the second reaction solution; under 18～25 ℃, react; during reaction, keeping the second reacting liquid pH value with ammoniacal liquor is 7.8～8.2; when in reaction to the second reaction solution, GL-TDA content is less than 1.5%, stop; filter, obtain the TDA crude product, the TDA crude product obtains TDA through the second purification step.
Further, 2) in step, the first reaction solution is under 65 ℃, to react 4h; The add-on of GL-7-ACA ACY is 6000U, and the second reacting liquid pH value is 8.
Further, the first purification step comprises the following steps: the GL-TDA crude product is cooled to room temperature, the gac and the vat powder decolouring 0.5～1h that add GL-TDA crude product volume 3～5 ‰, after filtration, be cooled to 0～5 ℃, with the first hydrochloric acid conditioning solution pH value to 5.0～5.2, slowly stir 0.5～2h, filter, gained filtrate is GL-TDA solution.
Further, the first hydrochloric acid equivalent concentration is 6.0N.
Further, the second purification step comprises the following steps: the TDA crude product is cooled to 0～5 ℃, is 4.0 by the second hydrochloric acid conditioning solution pH value, slowly stir 1～2h, filter, obtain filter cake, use respectively deionized water and washing with acetone the vacuum-drying filter cake 2h of 0～5 ℃.
Further, the second hydrochloric acid equivalent concentration is 3.0N.
Further, the preparation method of GL-7-ACA solution comprises the following steps:
1) to after adding D-AAO 3000～5000U in the ammonia soln of cephalosporin C Sodium, obtain the 3rd reaction solution;
2) under 15～20 ℃, react, during reaction, keeping the pH value of the 3rd reaction solution with ammoniacal liquor is 7.25～7.50, to the content of cephalosporin C Sodium in the liquid phase of the 3rd reaction solution during lower than 0.5wt.%, and stopped reaction, filtration, obtain GL-7-ACA solution.
Further, the add-on of D-AAO is 3000U, and during reaction, the pH value of the 3rd reaction solution is 7.3.
Further, ammoniacal liquor equivalent concentration is 3.0N.
The present invention has following beneficial effect:
Method provided by the invention is by 2-methyl-5-sulfydryl-1,3,4-thiadiazoles elder generation and GL-7-ACA(Glularyl-7-amino-cephalo-alkanoic acid) react, make 7-ACA and TDA can not be in simultaneously in same reaction system side reaction occurs, make gained TDA moderate purity high, the purifies and separates that is conducive to TDA, improve conversion yields.
Method provided by the invention be take enzyme and is the synthetic TDA of catalyzer, and the reaction process environmental protection of preparation TDA is pollution-free, produces without the boracic refuse.Synthetic route is short, saves production cost and has improved production efficiency.In traditional method, first make 7-ACA, but 7-ACA easily decomposition under 60～70 ℃, therefore yield is lower, and at first method provided by the invention generates GL-7-ACA, and GL-7-ACA is stable under 60～70 ℃.In reaction, product degradation is few, therefore the yield of TDA can, up to 93%, be applicable to suitability for industrialized production.And the thiadiazoles produced in process of production can also be recycled.
The whole reaction process reaction of method provided by the invention is all carried out in water, make organic solvent residual in prepared TDA seldom even not have, and is conducive to reduce the detrimentally affect of organic solvent to cefazolin.And in reaction consumption of organic solvent less also without using poisonous and harmful organic solvent, this method environmental protection.
The problems such as method provided by the invention has also been avoided because TDA and 2-methyl-5-sulfydryl-1,3,4-thiadiazole iso-electric point approach, and the TDA purifying complex caused, and product colour is dark, and the yield of TDA can reach 93%.
Except purpose described above, feature and advantage, the present invention also has other purpose, feature and advantage.Below with reference to figure, the present invention is further detailed explanation.
The accompanying drawing explanation
The accompanying drawing that forms the application's a part is used to provide a further understanding of the present invention, and schematic description and description of the present invention the present invention does not form inappropriate limitation of the present invention for explaining.In the accompanying drawings:
Fig. 1 is the efficient liquid phase chromatographic analysis schematic diagram of the preferred embodiment of the present invention 1 obtained TDA.
Embodiment
Below in conjunction with accompanying drawing, embodiments of the invention are elaborated, but the multitude of different ways that the present invention can be defined by the claims and cover is implemented.
The equivalent concentration of each solution refers to the concentration of solution herein, is the gram-equivalent number of contained solute in 1 liter of solution.Gram equivalent refers to the molar mass of the base mole unit of material.Equivalent=nucleidic mass/valency, if when nucleidic mass adopted gram atomic weight, equivalent just became gram equivalent.Gram atomic weight is exactly nucleidic mass.Monovalent is exactly the meaning of an electronics of gain and loss.The hydrochloric acid of take describes as example, and hydrochloric acid is often emitted a hydrogen ion, and equivalent concentration is the same with volumetric molar concentration.The sulfuric acid of take describes as example, the volumetric molar concentration equivalent concentration that equals two times.
At first method provided by the invention reacts on 3 of the GL-7-ACA that makes of reaction according to a conventional method.GL-7-ACA is stable under 60～70 ℃, and then has improved the output of GL-TDA.The recycling enzyme process carries out acidylate to GL-TDA after obtaining GL-TDA, can improve the purity of products therefrom.Method provided by the invention is not only to have above-mentioned thinking just attainable, and the application's proposition relates to the much extremely hard and bitter work of the inventor.Be described as follows.
Method provided by the invention comprises the following steps:
The cephalosporin C Sodium of take makes GL-7-ACA solution as raw material reaction;
Press the grams of 2-methyl-5-sulfydryl-1,3,4-thiadiazole: NaCO
3Grams: the milliliter number of water is warming up to 60～70 ℃ for (27:11:100)～(40:16:100) after mixing, add GL-7-ACA solution, obtain the first reaction solution, the first reaction solution is after reacting 3～4h under 60～70 ℃, obtain the GL-TDA crude product, the GL-TDA crude product obtains GL-TDA solution through the first purification step;
The GL-7-ACA ACY of 5000～7000U is added in GL-TDA solution; obtain the second reaction solution; under 18～25 ℃, react; during reaction, keeping the second reacting liquid pH value with ammoniacal liquor is 7.8～8.2; when in reaction to the second reaction solution, GL-TDA content is less than 1.5%, stop; filter, obtain the TDA crude product, the TDA crude product obtains TDA through the second purification step.
When the obvious method that the invention provides prepares GL-7-ACA, can take the sodium salt of cephalosporin by method commonly used and be raw material, make GL-7-ACA.Preferred GL-7-ACA makes according to the following steps:
After adding D-AAO 3000～5000U in the ammonia soln of cephalosporin C Sodium, obtain the 3rd reaction solution;
Reaction to keep the pH value of the 3rd reaction solution with ammoniacal liquor be 7.25～7.50 under 15～20 ℃, lower than 0.5wt.%, stopped reaction, filter, and obtains GL-7-ACA solution to the content of cephalosporin C Sodium in the liquid phase of the 3rd reaction solution.
The present invention preferably makes GL-7-ACA with D-AAO catalysis.The sodium salt of cephalosporin is mixed with the catalysts D-AAO and makes the 3rd reaction solution.Use the high conversion rate of D-AAO catalytic reaction activity fast, avoided boron-containing compound to participate in the environmental pollution that reaction causes.While usually with enzyme, making catalyzer, more speed of response is faster for institute's enzyme concentration, but the excessive not only cost of institute's enzyme concentration can increase but also react too violent, and while reacting violent, by product can increase again.So locating the add-on of preferred enzyme is 3000～5000U, further, optimum addition is 3000U.With enzyme, during as catalyzer, also need, for enzyme reaction provides suitable reaction conditions, to find by great many of experiments, during this reaction of catalysis, need the pH value of this reaction solution is controlled at 7.25～7.50 times.Preferably, the pH value of reaction soln is 7.3 o'clock reaction effect optimums.Because ammoniacal liquor affects little and low price to enzyme, therefore adopt ammoniacal liquor to regulate the pH value.The optimum reaction condition of D-AAO is 18 ℃ of pH7.3 temperature, and oxydase drops into 3000U, and the effect that this reaction of now catalysis is carried out is best.
The content that proceeds to cephalosporin C Sodium in the 3rd reaction solution when reaction is during lower than 0.5wt.%, sufficient reacting.In reaction process, pass through repeatedly the wherein content of cephalosporin C Sodium of sample detecting, determine the terminal of reaction.Visible in an embodiment, reaction process only needs 60min, has effectively shortened the reaction times, has improved production efficiency.
After making GL-7-ACA solution, at first by 2-methyl-5-sulfydryl-1,3,4-thiadiazole, NaCO
3The quality of pressing the 2-methyl-5-sulfydryl-1,3,4-thiadiazole with water: NaCO
3Quality: the volume of water is that 27:11:100～40:16:100 mixes.After in this ratio, mixing, GL-7-ACA is transformed and fully be conducive to improve yield.After mixing, solution is warming up to 60～70 ℃ again, obtains the first reaction solution, and gained GL-7-ACA solution is added in the first reaction solution.At this temperature the reaction times short, yield is high.Preferred temperature of reaction is 65 ℃.Obvious, temperature descends in order to prevent from reacting, and can solution be heated to 70 ℃ in advance, reacts after it is cooled to 65 ℃ again.GL-7-ACA is more stable than 7-ACA under 60～70 ℃, therefore can reduce decomposition because of intermediate product to the detrimentally affect that yield causes, is conducive to improve yield.The productive rate of GL-TDA can have been brought up to present 73% from original 55-60%.After reaction 3～4h, finish.This is chemical reaction, and after reaction 3～4h, the effective constituent reaction in the first reaction solution mixed according to the above ratio is the most abundant.Originally the reaction times reach 8～12h now only 3～4h just can complete, improved greatly production efficiency.Reaction times shortens greatly.The preferred reaction times is 4h.This is optimum reaction condition.In reaction process, avoid the use of virose boron-containing compound, played the effect of protection of the environment.Along with the enhancing of people's environmental consciousness, this alternative meaning is even more far-reaching on the impact that social and the mankind bring more than only improving yield.
After reaction finished, gained was only the crude product of GL-TDA, GL-TDA and 2-methyl-5-sulfydryl-1, the iso-electric point of 3,4-thiadiazoles differs far away, color than in prior art due to TDA and 2-methyl-5-sulfydryl-1,3,4-thiadiazoles mixes and the color that forms is shallow, makes the GL-TDA decolouring easily.Impurity in the GL-TDA crude product also needs further removal.In method provided by the invention, preferably this GL-TDA crude product being carried out to first purifies.This purification step can be method commonly used.This first purification step comprises the following steps: the temperature of the first reaction solution is reduced to room temperature from 60～70 ℃, to the gac and the vat powder decolouring 0.5～1h that add its volume 3～5 ‰ in the GL-TDA crude product.In prior art, because the products therefrom color is darker, only add the bleaching agent bleaching effect limited, adopt chromatography repeatedly to remove more, but this method inefficiency is unsuitable for industrial application.Because the GL-TDA color made in the inventive method is more shallow and water-soluble, therefore adopt discoloring agent can realize the purpose of decolouring.Discoloring agent is water insoluble not to react with product yet, thereby after decolouring, only needs to filter and can remove.Decolour easy, production efficiency is high.After filtration, be cooled to 0～5 ℃, with the first hydrochloric acid conditioning solution pH value to 5.0～5.2, slowly stir 0.5～2h.Low temperature slowly stirs under acidic conditions, can make the residual concentration of the GL-TDA in the mother liquor of crystallization reduce, and makes crystallization abundant.Reduce the impurity in the GL-TDA crystal powder, and improve yield.Filter, gained filtrate is GL-TDA solution.The equivalent concentration of the first hydrochloric acid used herein is 6.0N.With the hydrochloric acid of this concentration, regulate, liquor capacity can be not excessive, can reduce loss material caused owing to filtering, and yield is improved.
Afterwards GL-7-ACA ACY is dropped in GL-TDA it is carried out to catalysis, obtain TDA.This GL-7-ACA ACY is the key enzyme that catalysis generates cephalosporin analog antibiotic parent nucleus 7-ACA.This enzyme is better at 30-42 ℃ of scope internal stability, and optimal reactive temperature is 37 ℃, and the stable range that exceeds this enzyme vigor of enzyme is lost very soon, and in the time of 65 ℃, the remaining vigor of enzyme is less than 12%.GL-7-ACA ACY is that 7.0, pH is higher than 9.0 or descend rapidly lower than 7.0 enzyme activities referring to the optimum pH of reaction.And in method provided by the invention, the reaction conditions of this enzyme is that 18～25 ℃ and the pH value during with the ammoniacal liquor conditioned reaction are 7.8～8.2.Under this condition reaction enzymes live the highest, the good stability of the highest and enzyme of reaction conversion ratio, reusable batch at most.Can effectively improve reaction efficiency, Reaction time shorten.Operation is easy, only needs GL-7-ACA ACY is mixed with gained GL-TDA solution, obtains the second reaction solution, and gets final product by above-mentioned conditioned response.The addition of GL-7-ACA ACY is 5000～7000U herein, is preferably 6000U.In this ratio, add and neither can waste material, can make again the reaction times unlikely long, and affect production efficiency.
When reaction stops when GL-TDA content in the second reaction solution is less than 1.5%.After filtering, the gained liquid phase is the TDA crude product.Obvious, now also need the TDA crude product is purified and just can be obtained purer product.This purification step can be method commonly used.Preferred the second purification step of the present invention comprises the following steps: at first the TDA crude product being cooled to 0～5 ℃, is 4.0 by the second hydrochloric acid conditioning solution pH value, slowly stirs 1～2h.Under low temperature, maintaining pH and be acidity can make in crystalline mother solution TDA residual low.Slowly stirring can increase the contact between each material in solution, and the efficiency of purification is provided.This is optimum growing the grain parameter.After growing the grain, TDA separates out with crystalline form, and after filtering, the gained filter cake is again through twice of the deionized water wash of 0～5 ℃.Use afterwards the acetone soln washing leaching cake twice of 0～5 ℃, vacuum-drying 2h, except desolventizing, obtains TDA afterwards again.
The yield of TDA prepared by method provided by the invention can reach 93%.And in whole process, use a small amount of micro-malicious organic solvent a large amount of uses of organic solvent while having avoided chemical reaction in the prior art fully during except last washing.And avoided the use of poisonous boride.Both obtain high yield, and reached again present environmental requirement.
In above-mentioned the second purification step, preferably when crystallization, adopting equivalent concentration is the pH value of the hydrochloric acid control solution of 3.0N.Adopt the hydrochloric acid of this concentration to control the degraded that can avoid local overacidification to produce.
The equivalent concentration of ammoniacal liquor used is 3.0N in method provided by the invention.Ammoniacal liquor under this concentration can make the moderate unlikely generation that too much affects reaction due to addition of add-on.
Embodiment
In following examples, agents useful for same and instrument are commercially available, and wherein enzyme classes used is all from Fu Laige Bioisystech Co., Ltd.High performance liquid chromatograph: LC-10A, Japanese Shimadzu.Moving phase: precision takes the 1.542g ammonium acetate and is dissolved in the 850mL ultrapure water, adjusts pH to 6.0 with glacial acetic acid, and 0.45 μ m water system membrane filtration, add the 150mL acetonitrile, degassed 20min.Pillar type: Diamosil C185 μ m4.6 * 250mm.Flow velocity: 1ml/min wavelength: 254nm.
Embodiment 1
1) enzyme process prepares GL7-ACA
Get after 48 gram cephalosporin C Sodiums (anhydrous) add appropriate deionized water and add ammonia solvent, vacuum filtration is removed insolubles, be settled to 1200ml, throw D-AAO 3000U, control 18 ℃ of temperature of reaction, ammoniacal liquor with 3.0N in reaction process keeps PH7.30, and a C remains in 0.5% following termination reaction in liquid phase, leaches the oxidation stop buffer.Obtain containing 37.7gGL7-ACA solution 1215ml, productive rate is 96%.
2) synthetic GL-TDA
Get the 54.0g2-methyl-5-sulfydryl-1,3,4-thiadiazole, 22.0g NaCO
3200ml water is put into stirring is housed, thermometer, in the 2000ml tetra-neck flasks of reflux condensate device, open to stir and be warmed up to 70 ℃, after solution becomes clarification, the enzyme reaction solution of previous step is joined in above-mentioned solution, after after 65 ℃ of reaction 3.0h, being down to normal temperature, decolour half an hour with activated carbon and the vat powder of reaction solution volume 3 ‰.Filter cooling, the temperature of question response is down to 5 ℃ of left and right, starts to regulate pH value to 5.0 with the salt slow acid of 3N, slowly stirs and filters half an hour, and filtrate volume is that 1435ml contains GL-TDA32.75g, and productive rate is 73%.
3) the synthetic TDA of enzyme process
In above-mentioned filtrate, add GL7-ACA acylase 6000U, under the condition of 20 ℃, keep the PH8.00 reaction with 3.0N ammoniacal liquor, the 1.5% following termination reaction that remains in as Liquid Detection GL-TDA, leach reaction solution.Get above-mentioned reaction terminating liquid and be cooled to 5 ℃, regulate slowly pH value to 4.0 with 6.0N hydrochloric acid, slowly stir filtration in 1 hour.Filter cake is with cleaning twice with acetone again after twice of washed with de-ionized water of ice, and dry 2 hours of vacuum drying oven, obtain dry TDA22.82g (yield of TDA is 93%).
1) enzyme process prepares GL-7-ACA
Get after 48 gram cephalosporin C Sodiums (anhydrous) add appropriate deionized water and add ammonia solvent, vacuum filtration is removed insolubles, be settled to 1200ml, throw D-AAO 5000U, control 15 ℃ of temperature of reaction, ammoniacal liquor with 3.0N in reaction process keeps PH7.50, when a liquid phase C remains in 0.5% following termination reaction, leaches the oxidation stop buffer.Obtain containing 36.5gGL7-ACA solution 1215ml, productive rate is 93%.
2) synthetic GL-TDA
Get the 54.0g2-methyl-5-sulfydryl-1,3,4-thiadiazole, 22.0g NaCO
3200ml water is put into stirring is housed, thermometer, in the 2000ml tetra-neck flasks of reflux condensate device, open to stir and be warmed up to 65 ℃, after solution becomes clarification, the enzyme reaction solution of previous step is joined in above-mentioned solution, after after 60 ℃ of reaction 4.0h, being down to normal temperature, decolour half an hour with activated carbon and the vat powder of reaction solution volume 3 ‰.Filter cooling, the temperature of question response is down to 5 ℃ of left and right, starts to regulate pH value to 5.0 with the salt slow acid of 3N, slowly stirs and filters half an hour, and filtrate volume is that 1435ml contains GL-TDA30.95g, and productive rate is 71%.
3) the synthetic TDA of enzyme process
In above-mentioned filtrate, add GL7-ACA acylase 5000U, under the condition of 25 ℃, keep the PH8.20 reaction with 3.0N ammoniacal liquor, the 1.5% following termination reaction that remains in as Liquid Detection GL-TDA, leach reaction solution.Get above-mentioned reaction terminating liquid and be cooled to 5 ℃, regulate slowly pH value to 4.0 with 6.0N hydrochloric acid, slowly stir filtration in 1 hour.Filter cake is with cleaning twice with acetone again after twice of washed with de-ionized water of ice, and dry 2 hours of vacuum drying oven, obtain dry TDA20.87g (yield of TDA is 90%).
1) enzyme process prepares GL-7-ACA
Get after 48 gram cephalosporin C Sodiums (anhydrous) add appropriate deionized water and add ammonia solvent, vacuum filtration is removed insolubles, be settled to 1200ml, throw D-AAO 3000U, control 20 ℃ of temperature of reaction, ammoniacal liquor with 3.0N in reaction process keeps PH7.25, when a liquid phase C remains in 0.5% following termination reaction, leaches the oxidation stop buffer.Obtain containing 36.5gGL7-ACA solution 1215ml, productive rate is 93%.
2) synthetic GL-TDA
Get the 27.0g2-methyl-5-sulfydryl-1,3,4-thiadiazole, 11.0g NaCO
3100ml water is put into stirring is housed, thermometer, in the 2000ml tetra-neck flasks of reflux condensate device, open to stir and be warmed up to 60 ℃, after solution becomes clarification, the enzyme reaction solution of previous step is joined in above-mentioned solution, after after 60 ℃ of reaction 4.0h, being down to normal temperature, decoloured 1 hour with activated carbon and the vat powder of reaction solution volume 5 ‰.Filter cooling, the temperature of question response is down to 0 ℃ of left and right, starts to regulate pH value to 5.20 with the salt slow acid of 3N, slowly stirs and filters in 2 hours, and filtrate volume is that 1435ml contains GL-TDA30.95g, and productive rate is 71%.
3) the synthetic TDA of enzyme process
In above-mentioned filtrate, add GL7-ACA acylase 7000U, under the condition of 18 ℃, keep the PH7.80 reaction with 3.0N ammoniacal liquor, the 1.5% following termination reaction that remains in as Liquid Detection GL-TDA, leach reaction solution.Get above-mentioned reaction terminating liquid and be cooled to 0 ℃, regulate slowly pH value to 4.0 with 6.0N hydrochloric acid, slowly stir filtration in 2 hours.Filter cake is with after twice of 0 ℃ of washed with de-ionized water, with acetone, cleaning twice again, and dry 2 hours of vacuum drying oven, obtain dry TDA20.64g (yield of TDA is 89%).
Embodiment 4
1) enzyme process prepares GL-7-ACA
Get after 48 gram cephalosporin C Sodiums (anhydrous) add appropriate deionized water and add ammonia solvent, vacuum filtration is removed insolubles, be settled to 1200ml, throw D-AAO 3000U, control 15 ℃ of temperature of reaction, ammoniacal liquor with 3.0N in reaction process keeps PH7.30, when a liquid phase C remains in 0.5% following termination reaction, leaches the oxidation stop buffer.Obtain containing 36.10gGL7-ACA solution 1215ml, productive rate is 92%.
2) synthetic GL-TDA
Get the 40.0g2-methyl-5-sulfydryl-1,3,4-thiadiazole, 16.0g NaCO
3100ml water is put into stirring is housed, thermometer, in the 2000ml tetra-neck flasks of reflux condensate device, open to stir and be warmed up to 65 ℃, after solution becomes clarification, the enzyme reaction solution of previous step is joined in above-mentioned solution, after after 60 ℃ of reaction 4.0h, being down to normal temperature, decolour half an hour with activated carbon and the vat powder of reaction solution volume 3 ‰.Filter cooling, the temperature of question response is down to 5 ℃ of left and right, starts to regulate pH value to 5.0 with the salt slow acid of 3N, slowly stirs and filters half an hour, and filtrate volume is that 1435ml contains GL-TDA29.62g, and productive rate is 69%.
3) the synthetic TDA of enzyme process
In above-mentioned filtrate, add GL7-ACA acylase 5000U, under the condition of 25 ℃, keep the PH8.20 reaction with 3.0N ammoniacal liquor, the 1.5% following termination reaction that remains in as Liquid Detection GL-TDA, leach reaction solution.Get above-mentioned reaction terminating liquid and be cooled to 5 ℃, regulate slowly pH value to 4.0 with 6.0N hydrochloric acid, slowly stir filtration in 1 hour.Filter cake is with cleaning twice with acetone again after twice of washed with de-ionized water of ice, and dry 2 hours of vacuum drying oven, obtain dry TDA19.53g (yield of TDA is 88%).
Comparative Examples 1
This Comparative Examples 1 only is 2 with the difference of embodiment 1), during synthetic TDA, carry out according to the following steps.
1) enzyme process prepares GL7-ACA
Get after 48 gram cephalosporin C Sodiums (anhydrous) add appropriate deionized water and add ammonia solvent, vacuum filtration is removed insolubles, be settled to 1200ml, throw D-AAO 3000U, control 18 ℃ of temperature of reaction, ammoniacal liquor with 3.0N in reaction process keeps PH7.30, and a C remains in 0.5% following termination reaction in liquid phase, leaches the oxidation stop buffer.Obtain containing 37.7gGL7-ACA solution 1215ml, productive rate is 96%.
2) the synthetic 7-ACA of enzyme process
In above-mentioned filtrate, add GL-7-ACA ACY 6000U, under the condition of 20 ℃, keep the PH8.00 reaction with 3.0N ammoniacal liquor, the 1.5% following termination reaction that remains in as Liquid Detection GL-7-ACA, leach reaction solution.Get above-mentioned reaction terminating liquid and be cooled to 5 ℃, regulate slowly pH value to 4.2 with 6.0N hydrochloric acid, slowly stir filtration in 1 hour.Filter cake is with cleaning twice with acetone again after twice of washed with de-ionized water of ice, and dry 2 hours of vacuum drying oven, obtain dry 7-ACA23.41g (yield of 7-ACA is 88%).
3) synthetic TDA
Get the 54.0g2-methyl-5-sulfydryl-1,3,4-thiadiazole, 22.0g NaCO
3, 23.41g7-ACA, 1400ml water is put into stirring is housed, thermometer, in the 2000ml tetra-neck flasks of reflux condensate device, open to stir and be warmed up to 70 ℃, after solution becomes clarification, the enzyme reaction solution of previous step is joined in above-mentioned solution, after after 70 ℃ of reaction 2.0h, being down to normal temperature, with the salt slow acid of 3N, regulate pH value to 4.2, with after the excessive first mercapto thiadiazoles of ethyl acetate extraction, separating emulsion layer, filter, filter cake is with after twice of washed with de-ionized water of ice, with acetone, cleaning twice again, dry 2 hours of vacuum drying oven, obtain dry TDA17.19g, productive rate is 58%.
Comparative Examples 2
Comparative Examples 2 only is 2 with the difference of embodiment 1) while synthesizing GL-TDA, temperature of reaction is 50 ℃, during synthetic TDA, carry out according to the following steps.
1) enzyme process prepares GL7-ACA
Get after 48 gram cephalosporin C Sodiums (anhydrous) add appropriate deionized water and add ammonia solvent, vacuum filtration is removed insolubles, be settled to 1200ml, throw D-AAO 3000U, control 18 ℃ of temperature of reaction, ammoniacal liquor with 3.0N in reaction process keeps PH7.30, and a C remains in 0.5% following termination reaction in liquid phase, leaches the oxidation stop buffer.Obtain containing 37.7gGL7-ACA solution 1215ml, productive rate is 96%.
2) synthetic GL-TDA
Get the 40.0g2-methyl-5-sulfydryl-1,3,4-thiadiazole, 16.0g NaCO
3100ml water is put into stirring is housed, thermometer, in the 2000ml tetra-neck flasks of reflux condensate device, open to stir and be warmed up to 50 ℃, after solution becomes clarification, the enzyme reaction solution of previous step is joined in above-mentioned solution, after after 50 ℃ of reaction 6.0h, being down to normal temperature, decolour half an hour with activated carbon and the vat powder of reaction solution volume 3 ‰.Filter cooling, the temperature of question response is down to 5 ℃ of left and right, starts to regulate pH value to 5.0 with the salt slow acid of 3N, slowly stirs and filters half an hour, and filtrate volume is that 1435ml contains GL-TDA24.90g, and productive rate is 58%.
3) the synthetic TDA of enzyme process
In above-mentioned filtrate, add GL7-ACA acylase 5000U, under the condition of 25 ℃, keep the PH8.20 reaction with 3.0N ammoniacal liquor, the 1.5% following termination reaction that remains in as Liquid Detection GL-TDA, leach reaction solution.Get above-mentioned reaction terminating liquid and be cooled to 5 ℃, regulate slowly pH value to 4.0 with 6.0N hydrochloric acid, slowly stir filtration in 1 hour.Filter cake is with cleaning twice with acetone again after twice of washed with de-ionized water of ice, and dry 2 hours of vacuum drying oven, obtain dry TDA16.42g (yield of TDA is 88%).
In embodiment 1, the single step yield of TDA can reach 93%, and comprehensive yield also can reach 65.17%, far above 58% single step yield in Comparative Examples 1 and 48.99% comprehensive yield.In Comparative Examples 1 7-ACA is than the poor heat stability of GL-7-ACA, and when 3 of 7-ACA connected first mercapto thiadiazoles and react, the degraded aggravation, be difficult to control, therefore yield is low.And the separation difficulty of the TDA in Comparative Examples 1 and first mercapto thiadiazoles, must extract with a large amount of organic solvents, and adopt in time the impurity first mercapto thiadiazoles content in the purification by liquid extraction product still very high, poor product quality.
Gained TDA in embodiment 1 is carried out to efficient liquid phase chromatographic analysis, and acquired results as shown in Figure 1.The purity of TDA is very high as seen from the figure, and impurity in products and substrate seldom residual.
The relative embodiment 1 of the productive rate that carries out TDA along with reaction in Comparative Examples 2 can reduce, and this is mainly because reduced temperature of reaction, and the activation energy of the group on 3 of GL7-ACA reduces, thereby low conversion rate.Thereby in Comparative Examples 2, the one-step reaction yield of TDA is only 88%.Illustrate that the react yield of the TDA that makes of the temperature range that adopts method provided by the invention is the highest, and purity is also best.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (7)
1. the preparation method of a Kefzol Intermediate TDA, is characterized in that, comprises the following steps:
1) take cephalosporin C Sodium makes GL-7-ACA solution as raw material reaction;
2) press the grams of 2-methyl-5-sulfydryl-1,3,4-thiadiazole: NaCO
3Grams: the milliliter number of water is warming up to 60～70 ℃ for (27:11:100)～(40:16:100) after mixing, add described GL-7-ACA solution, obtain the first reaction solution, described the first reaction solution is after reacting 3～4h under 60～70 ℃, obtain the GL-TDA crude product, described GL-TDA crude product obtains described GL-TDA solution through the first purification step; Described the first purification step comprises the following steps:
Described GL-TDA crude product is cooled to room temperature, the gac and the vat powder decolouring 0.5～1h that add described GL-TDA crude product volume 3～5 ‰, after filtration, be cooled to 0～5 ℃, with the first hydrochloric acid conditioning solution pH value to 5.0～5.2, slowly stir 0.5～2h, filter, gained filtrate is described GL-TDA solution;
3) GL-7-ACA ACY of 5000～7000U is added in described GL-TDA solution; obtain the second reaction solution; under 18～25 ℃, react; during reaction, keeping described the second reacting liquid pH value with ammoniacal liquor is 7.8～8.2; when reaction to GL-TDA content in described the second reaction solution is less than 1.5%, only, filter, obtain the TDA crude product; described TDA crude product obtains described TDA through the second purification step, and described the second purification step comprises the following steps:
Described TDA crude product is cooled to 0～5 ℃, is 4.0 by the second hydrochloric acid conditioning solution pH value, slowly stirs 1～2h, filters, and obtains filter cake, uses respectively deionized water and washing with acetone the described filter cake 2h of vacuum-drying of 0～5 ℃.
2. method according to claim 1, is characterized in that, described 2) the first reaction solution described in step is for reacting 4h under 65 ℃; The add-on of described GL-7-ACA ACY is 6000U, and described the second reacting liquid pH value is 8.
3. method according to claim 2, is characterized in that, described the first hydrochloric acid equivalent concentration is 6.0N.
4. method according to claim 3, is characterized in that, described the second hydrochloric acid equivalent concentration is 3.0N.
5. method according to claim 1, is characterized in that, the preparation method of described GL-7-ACA solution comprises the following steps:
1) to after adding D-AAO 3000～5000U in the ammonia soln of described cephalosporin C Sodium, obtain the 3rd reaction solution;
2) under 15～20 ℃, react, during reaction, keeping the pH value of described the 3rd reaction solution with ammoniacal liquor is 7.25～7.50, when extremely described in the liquid phase of described the 3rd reaction solution, the content of cephalosporin C Sodium is lower than 0.5wt.%, and stopped reaction, filter, obtain described GL-7-ACA solution.
6. method according to claim 5, is characterized in that, the add-on of described D-AAO is 3000U, and during reaction, the pH value of described the 3rd reaction solution is 7.3.
7. according to the described method of any one in claim 1～6, it is characterized in that, described ammoniacal liquor equivalent concentration is 3.0N.
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Denomination of invention: Preparation method of TDA intermediate of cefazolin Granted publication date: 20131127 Pledgee: Changsha Bank city branch of Limited by Share Ltd. Pledgor: HUNAN FLAG BIOTECHNOLOGY Co.,Ltd. Registration number: Y2024980012003 |