TW459048B - Catalytic method for the desacetylation of cephalosporin intermediates - Google Patents

Abstract

A method is disclosed for the preparation of desacetyl cephalosporin intermediates. It comprises the steps of: (a) preparing a reaction mixture containing a cephalosporin intermediate and a lipase or a organic solvent in a pH 4 to 8 of phosphate buffer; (b) reacting the reaction mixture at temperatures between 4 DEG and 60 DEG C; and (c) recovering the reaction product by the precipitation of the product via isoelectric point. The advantages of this method are high yield and short reaction time.

Description

A7 A7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 0571TWF, DOC / 002 V. Description of the invention (丨) The present invention relates to a deacetylation reaction of a cephalosporin intermediate and its derivatives, and in particular A technology using enzymes to catalyze the deacetylation of cephalosporin intermediates and their derivatives. This cephalosporin intermediate and its derivatives have the following general formula:

Among them, the COOR2 group at the 4th position is an acid group, an alkyl group, or an aromatic functional group; the NHR1 group at the 7th position is a monoamine group, an alkyl group, or an aromatic palace group. During the synthesis of such intermediates such as Cefuroxime or Cefixime, the acetamidine group at position 3 must be removed to become a methanol-based intermediate, and then other synthetic steps are performed. For this deacetylation reaction technique, French Patent No. 1,449,610 (1966) has disclosed that strains such as Streptomyces sp. Or Bacillus subtilis are used in the fermentation process at 27 ° C for 24 hours. In 7 hours, 7-aminocephalosporanic acid (abbreviated as 7-ACA) with a starting concentration of 5-10% can be deacetylated, and the reaction yield is 80-100%. Similarly, Swiss Patent No. 498,152 (1966) also discloses that 7-ACA deacetylation reaction was performed with Bacillus subtilis ATCC No. 6633. German Patent No. 2,423,058 (1974) discloses that (6R, 7R) -3-acetoxymethyl-7- (D-5- aminocarboxylpentanamido) -ceph-3-em-4-carboxylate is removed with Rhodotorula rubra strain at room temperature. Acetylation reaction, after 60 hours, 90% of the reaction yield can be obtained. _3 _ This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) ί — I n I n (please first Read the notes on the back and fill in this page) A7 0571TWF.D0C / 0C2 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (meaning) A tritiated product. This invention was also patented in the United States (No. 3,912,589, 1974 (Years). When the reaction is performed by live bacteria, in order to maintain the pure bacteria culture state, it is necessary to prevent contamination by miscellaneous bacteria, so the reaction equipment and operation are more complicated; and because the general cephalosporin intermediate is in the liquid Phases are relatively easy to lyse by themselves, and the deacetylation reaction with live bacteria culture usually requires 24-60 hours of reaction operation time, so it is easy to cause the reaction yield to decrease. Japanese Patent Laid-Open No. 61-70 " No. 9 (I % 6 years) revealed that using wheat The enzyme in (wheat bran) is used to carry out the deacetylation reaction of 7-ACA and similar compounds. Taking Cephalosporin C (Cephalosporin C) as an example, the reaction yield can be obtained after treatment at 30 ° C for 13 hours. It is a 95% deacetylated product. However, the enzyme activity of this method is low, and a lot of wheat bran must be added to shorten the reaction time, so it is easy to cause high impurities in the reaction solution, and the downstream recovery of the product is more complicated. European patent No. 204,517 (1986) revealed that dehydroacetylation of cephalosporin intermediate derivatives using sodium hydroxide at ultra-low temperature (-10-40 ° C). The disadvantage of this technology is that it must be used in The reaction at ultra-low temperature has a large energy consumption. If the reaction temperature is increased, it is easy to cause matrix cleavage, resulting in increased by-products, reduced reaction yield, and difficult to separate by-products. Therefore, in order to solve the above problems, the main purpose of the present invention is to solve the above problems. Provided is a method for catalyzing the deacetylation reaction of a cephalosporin intermediate. By using this method, a high reaction yield can be obtained, and the reaction can be dissolved at normal temperature and pressure and neutral solvent. It does not need to add nutritional sources, and does not need to operate in a sterile state.!;.. II | — I II :: Order (please read the precautions on the back before filling this page) This paper size applies Chinese national standards (CNS) A4 specification (2IO × 297 mm) 459048 D57) TWF.DOC / OQ2 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (") According to the main purpose of the present invention," a catalytic cephalosporin A method for deacetylating an intermediate of an adiponin, including the following steps: a_ preparing a reaction mixture including a cephalosporin intermediate and a lipolytic enzyme dissolved in a buffer solution; b. Performing the reaction under a temperature environment; and c. Recovering a product of the cephalosporin intermediate catalyzed by the lipolytic enzyme. In order to make the above and other objects, features, and advantages of the present invention more comprehensible, a preferred embodiment is given below in conjunction with the accompanying drawings for detailed description as follows: Brief description of the drawings: FIG. 1 It is a flowchart of a first intermediate deacetylation reaction using different lipolytic enzymes according to a preferred embodiment of the present invention. Fig. 2 is a flowchart of a second intermediate deacetylation reaction using different lipolytic enzymes according to a preferred embodiment of the present invention. FIG. 3 is a flowchart of a third intermediate deacetylation reaction using different lipolytic enzymes according to a preferred embodiment of the present invention. Table 1 shows the results of deacetylation of the first intermediate with different lipolytic enzymes. Table 2 shows the results of deacetylation of the second intermediate with different lipolytic enzymes. Table 3 shows the results of deacetylation of the third intermediate with different lipolytic enzymes in the presence of organic solvents. Detailed description of the invention 5 -9 (Please read the precautions on the back before filling in this page)

This paper size is applicable to the standard of Chinese family standard (CNS) A4 (210X297mm) 459048 A7 0571TWF.DOC / 002 D i 5. Description of the invention (child) The present invention is to select commercially produced lipolytic enzymes for cephalospores The bacteriocin intermediate undergoes a deacetylation reaction; this reaction can be expressed by the following reaction formula:

According to the present invention, Ez is a lipolytic enzyme. The lipolytic enzyme is selected from the group consisting of Aspergillus niger, Pseudomonas sp., Rhizopus sp., Candida cylindracea, and Candida lipolytica ), Penicillium roqurforti, Rhizopus nineus, wheat germ, porcine pancreas, Mucor meihei, Miicor javariCUS, etc. Lipolytic enzymes. More specifically, this kind of lipolytic enzyme refers to a lipolytic enzyme derived from black fungus, wine bacterium, wheat germ, pig pancreas, and penicillium. Among them, the NHR1 group at the seventh position is a monoamine group, an alkyl group, or an aromatic functional group. More specifically, R1 may be a hydrogen atom or any one of the following functional groups: I—II j 1 (1 n (Please read the notes on the back before filling out this page) OMHC IN c · —ο π 2

This paper size applies to China National Standards (CNS) Λ4 specifications (2) ο X 297 mm) A7 V. Description of the invention

OCH- OHC j H2 Hc—Nvy

OH

/ V

/ \ 0 卩 C-I Hc—N -2

o = c3h I o Hc—SV /

I ------- f, install-(Please read the notes on the back before filling out this page) Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs

h2n

! lN—OCH-

This paper size applies to Chinese National Standard (CNS) A4 specification (210X297 mm) 057iTWF.DOC / 002 A7 B7 V. Description of the invention (II) Η2Ν—c

HOOC

〇IIc-? H2? —S ~ c—c h2 Please read H2N 0 \ II / CH ~~ u—s ~ c 1 〇 HOOC H2 H2 CN Cl

N 0I! N——C——C—, H2

C2H5—N

1¾ of the notes • Please fill in this page. Printed on the winter page. Printed by c-ch2cooh, the COOR2 group at the 4th position is an acid, alkyl, or aromatic functional group. More specifically, R2 It may be a hydrogen atom or any of the following functional groups. Factory ch3 I H3C—Si—

CH 3 This paper size applies to Chinese national standards. (CNS) A4 size (210X297 mm) 4β9048 057LTWF.DOC / 002 Α7 Β7 V. Description of the invention (Ο

ecu-C-3 h2 According to a preferred embodiment of the present invention, the cephalosporin intermediate may be: 1_ the first intermediate, 7-aminocephalosporanic acid (referred to as 7-ACA), and its structural formula is as follows: h2n- , S '

c / ~ NY ^ CH2OCOCH3COOH2. The second intermediate, (7- [2- (2-furyl) -2- (methoxyimino) acetamido] cephalosporanic acid), its 9 H person 'C—CN—I ~ > I! IJ, N c / ~ NY ^ CH2OCOCH3〇CH3 COOH structural formula is as follows; and 3. The third intermediate, (4-nitrobenzyl 7b-phenylacetamido-3-[(acetyloxy) methyl] -3-cephem-4- —- HI n ^ Pack-Order II (Please read the notes on the back before filling out this page) Printed by the Consumers' Cooperation of the Central Standards Bureau of the Ministry of Economic Affairs

carboxylate)), its structural formula is as follows: 〇II C—C—N— h2 H oo = c This paper size is applicable to China National Standard (CNS) A4 specification (210X 297 mm) ch2ococh3 0—c- -N〇2 A7 057 1TWF.DOC / 002 V. Description of the Invention (?) The following is a detailed description of the present invention with examples, but it does not mean that the present invention is limited to the content disclosed by these examples. The first example is printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). First, please refer to Figure 1 and perform the first step. Prepare the reaction mixture. An intermediate is dissolved in 50 ml of phosphate buffer solution (0.2 M), the appropriate pH is 4-8, preferably pH 7, and 0.5 g of lipolytic enzyme is added. The lipolytic enzyme can be, for example, , German Serva product number 27930 is derived from lipolytic enzymes of Saccharomyces cerevisiae, Amano product number AP6 is derived from lipolytic enzymes of Nigella spp. The company product number L-3001 is derived from the lipolytic enzyme of wheat germ. Then, the second step is carried out to start the deacetylation reaction 12: The reaction is performed at an appropriate reaction temperature of 4-60 ° C, preferably at 30 ° C for 3 hours, and then acid is added to adjust the pH to below 2.0 to Stop the enzyme-catalyzed reaction. Next, the yield analysis was performed. The reaction yield analysis was measured by a high-efficiency liquid chromatography. The chromatographic column used was RP-18 column (Lichrospher products of Merck, Germany). It is a 0.01 M potassium phosphate solution with a flow rate of 0.7 ml / min and a sample detection wavelength of 254 nm. According to the analysis result of liquid chromatography, the reaction yield was 97.8%. Subsequently, the method of product recovery 14 can be performed by adding an equal volume of methyl isobutyl ketone to the reaction solution for extraction, and placing it in an environment of 0-5 ° C to stand for analysis. The layer was washed with distilled water and methylpentanone in sequence, and finally dried under vacuum at 30 ° C. The deacetylation reaction of the lipolytic enzyme derived from Saccharomyces cerevisiae with German Serva product number 27930 , Can get 0.45 grams of deacetylation first___________10 This paper size applies Chinese National Standard (CNS) A4 specifications (2 丨 0〆297 public 0571TWF.DOC / 002 A7 B7 V. Description of the invention (ί) Intermediate products In addition, please refer to Table 1. When the reaction is catalyzed by lipolytic enzymes derived from black fungus and wheat germ, the reaction yield of the deacetylated first intermediate product can reach 90 °. /. Above. Table 1 {Binding (please read the precautions on the back before filling out this page) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs Lipolytic Enzyme Source Reaction Yield (%) Serva 27930 (Rhizopus sp.) 97.8 Amano AP6 Aspergillus niger 91.4 Fluka 62301 Aspergillus niger 90.1 Sigma L-3001 Wheat germ (Wheat germ) 100 This paper applies the Chinese National Standard (CNS) Λ4 specification (210X297 mm) A7 B7 Economy Printed by the Consumer Standards Cooperative of the Ministry of Standards of the People's Republic of China. 5. Description of Invention (π) For the second embodiment, please refer to Figure 2, and perform the first step. The reaction mixture is prepared in a 0.2M phosphate buffer solution. The appropriate pH is 4-8, preferably PH7, the second intermediate is prepared with a solution concentration of 1 g / liter, and 1 g / liter of lipolytic enzyme is added. This lipolytic enzyme can be, for example, German Serva company product number 27930 derived from Lipolytic enzymes in Saccharomyces cerevisiae, Amano company product number AP6 derived from lipolytic enzymes of black fungus, Fluka product number 62301 derived from lipolytic enzymes of black fungus or Sigma product number L-3001 Lipolytic enzymes in wheat germ. Then, the second step is carried out to start the deacetylation reaction 22, which is at an appropriate reaction temperature of 4-60 ° C, preferably 30 ° C for 6 hours to react After stopping, proceed Yield analysis 23, here, the reaction yield is using a high-efficiency liquid chromatography with RP-18 column in the mobile phase as the methanol / 0.01 M potassium phosphate solution, the volume ratio is 40: 60, the flow rate is 0.6 Ml / min, measured at 254 nm. Next, the product was recovered 24. Please refer to Table 2 for the results. When the deacetylation reaction was performed with a lipolytic enzyme derived from Saccharomyces cerevisiae, the reaction yield was 97.4%. When the source was S. niger ( When Aspergillus niger) and wheat germ are used to catalyze the reaction, the reaction yield of the deacetylated second intermediate product can reach more than 90%. Γ Please read the precautions on the back before filling this page),-= & This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) 459CU8 0571TWF, DOC / 002 A7 B7 V. Description of the invention (I! ) Table 2 Lipolytic enzyme source reaction yield (%) Serva 27930 Rhizopus sp. 90.9 Amano AP6 Aspergillus niger 97.9 Fluka 62301 Aspergillus niger 97.4 Sigma L-3001 Wheat germ (Wheat germ) 100 -------- ί. 'Installation ------ Order- (' Please read the notes on the back before filling this page) Refer to Figure 3 for the third embodiment, because the third intermediate is insoluble in water. When preparing the reaction mixture 31, it is prior to 0.2 M phosphate buffer, and the appropriate pH is about 4-8, preferably At about pH 7.0, ethyl acetate or dimethylformamide of different concentrations are added, and a third intermediate is added to make a solution having a concentration of 1 g / liter, and finally, 1 g / liter of lipolysis is added. Enzymes, this lipolytic enzyme can be, for example, Japanese Amano company product number AP6 Lipolytic enzyme aspergillus black or Sigma (USA) Sigma Catalog No wheat germ lipolytic enzymes of the L-3001. Next, a deacetylation reaction 32 is performed: the reaction is performed at an appropriate reaction temperature of 4-60 ° C, preferably at 30 ° C for 3 hours. After that, the “yield analysis 33” uses the RP-18 tube. I? The paper size applies the Chinese National Standard (CNS) Α4 specification (210 × 297 mm). Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs? ^ · 5 9 C £ 8 Α7 -------_ Β7 ___ V. Description of the invention (丨 >) The mobile phase composition of the column is methanol / chlorochloric acid solution (pH 1.7), the volume ratio is 25:75, The flow rate was 0.3 ml / min. The measurement was performed at a wavelength of 254 nm. Finally, product recovery 34 is performed. The experimental results are listed in Table 3. From the experimental results in Table 3, it is clear that the use of lipolytic enzymes can effectively perform the deacetylation reaction of the third intermediate under the condition that an organic solvent is added. (Please read the precautions on the reverse side before filling out this page) Pack. Order Table 3. Organic solvents from lipolytic enzyme sources and their added concentration (volume percentage) Reaction yield (%) Amano ΑΡ6 Nigrobacterium ethyl acetate ( 5%) 93.4 (Aspergillus ethyl acetate (10%) 85.1 niger) ethyl acetate (50%) 91.3 dimethylformamide 98.7 (2%) dimethylformamide 95.7 (5%) Sigma L- wheat Germ ethyl acetate (5%) 62.6 3001 (Wheat germ) ethyl acetate (10%) 71.0 ethyl acetate (50%) 87.2 dimethylformamide 84.0 (2%) dimethylformamide 62.4 (5 %) ___ This paper scale is applicable to China's national standard (CNS> A4 specification (2) ϋΧ2ίΐ7mm) A7 B7 0571TWF.DOC / 002 5. Invention description (15) Those skilled in the art can understand that the application of the present invention has the following Features: 1. Catalyzed reaction using partially purified lipolytic enzymes that have been commercially produced in large quantities. 2. The reaction activity is high, so the catalyst, namely lipolytic enzyme, has a low addition amount (S lg enzyme / g substrate): And the reaction time is short; 3. Reaction at normal temperature, saving Source; 4. No need to add nutrient source, and do not need to operate in a sterile state; 5. Can react between pH5. 5 ~ 8, the matrix is less likely to lyse itself; 6. High product recovery (greater than 95%) 7. Less by-products and impurities, simple product separation steps; 8. Wastewater treatment is easy. Although the present invention has been disclosed as above with a preferred embodiment, it is not intended to limit the present invention. Anyone skilled in the art will not Without departing from the spirit and scope of the present invention, some modifications and retouching can be made, so the scope of protection of the present invention shall be determined by the scope of the attached patent application. (Please read the precautions on the back before filling this page )

I Printed by the Central Standards Bureau of the Ministry of Economic Affairs—industrial-consumption cooperation. The paper size is applicable to China National Standards (CNS) A4 specifications (210X297 mm)

Claims (1)

Case No. 85109784 VI. Patent Application Fanyuan Xie 09/09 Version:. '' '丨 .__________________________ HI II II 丨 Including 1, a method for catalyzing the deacetylation reaction of cephalosporins to interstitial cells The following steps: a. Preparation A reaction mixture including the cephalosporin fluorescein intermediate and a lipolytic enzyme selected from the group consisting of black fungus, wine fungus, or wheat germ or a mixture thereof, dissolved in a pH 4 ~ 8 phosphate buffer solution; b. 44 ~ 60 t temperature reaction: and! C. The product solution is adjusted to pH 1 ~ 3 and isoelectrically precipitated to recover the catalytic reaction Page 15 2001.08. 06.015 459048 __Case No. 85109 _ 84_ ___ | VI. A patent scope I I 3. The method described in item 2 of the scope of patent application, wherein the organic solvent is ethyl acetate. 4. The method as described in item 2 of the scope of patent application, wherein the organic solvent is dimethylamidoxamine. ί 5. The method according to item 2 of the scope of patent application, wherein the organic solvent is 曱 alcohol = I I 6. The method according to item 2 of the scope of patent application, wherein the organic solvent is ethanol. i. The method according to item 2 of the scope of patent application, wherein the organic solvent is tetrazolium. 8. The method as described in item 2 of the scope of patent application, wherein the organic solvent is i-propion. Page 丨 6 2001.08.06,016