KR20120078687A - Process for the preparation of cephalosporin intermediate using new enzyme - Google Patents

Abstract

PURPOSE: A method for preparing a cephalosporin-based antibiotic intermediate is provided to industrially synthesize crystalline hydrate of the intermediate and to develop the antibiotics. CONSTITUTION: A method for preparing a cephalosporin-based antibiotic intermediate of chemical formula 1 comprises: a step of adding cresol and phenol to a compound of chemical formula 2 and stirring for deprotecting protection groups of a carboxyl group; a step of adding syntha CLEC-PA(cross-linked enzyme crystal-penicillin g amidase/acylase) while adjusting pH concentration using alkaline; a step of adjusting pH concentration using inorganic acid. The alkaline is hydrocarbon alkali metal, alkali hydroxide metal, ammonia solution, or trialkyl amine. The hydrocarbon alkali metal is sodium carbonate or potassium carbonate.

Description

Process for the preparation of cephalosporin intermediate using new enzyme}

The present invention relates to an efficient mass production method of cephalosporin antibiotic intermediates using a new enzyme.

Cephalosporin antibiotics are widely used in the treatment of diseases caused by pathogenic bacteria in humans and animals. useful. In many cases, it is desirable to use antibiotics that are active against both Gram-positive and Gram-negative microorganisms, and thus, studies on the development of various types of cephalosporin antibiotics have been continued.

7-amino-3-cepem-4-carboxylic acid (hereinafter 7-ANCA), 7-amino-3- [propen-1-yl] -3- Cefem-4-carboxylic acid (7-amino-3- [propen-1-yl] -3-cephem-4-carboxylic acid; 7-APRA), 7-amino-3- [2- (4-methyl Thiazol-5-yl) vinyl] -3-cepem-4-carboxylic acid (7-amino-3- [2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid; 7-ATVA) is a key intermediate compound used in the preparation of Ceftizoxime, Ceftibutene and Cefprozil, which are representative third-generation cephalosporin-based antibiotics having a wide range of antibacterial activity.

The 7-ANCA, 7-APRA and 7-ATVA is mainly present as a hydrate, as shown in the following formula (1).

[Formula 1]

(In Formula 1, R is H, propenyl, (4-methylthiazol-5-yl) vinyl,

x is 3 to 6)

At this time, the Ceftizoxime, Ceftibutene and Cefprozil has excellent antimicrobial activity against a wide range of pathogens, and because of its excellent stability in vivo, as one of the third generation cephaic antibiotics, sore throat, acute bronchitis, tonsillitis, pneumonia It is a popular sepatic antibiotic because it shows excellent efficacy in otitis media.

Therefore, the preparation of 7-ANCA, 7-APRA and 7-ATVA, which are used as intermediates in the preparation of cephalosporin antibiotics, exhibits high stability because they affect the purity and yield of the cephalosporin antibiotics, and are easy to handle. It is very important to manufacture 7-ANCA, 7-APRA and 7-ATVA with high purity and high yield.

In general, the preparation of the cephalosporin-based antibiotic intermediates is shown in Scheme 1 below.

Removing the protecting group of the carboxyl group and the protecting group of the amino group in the compound of formula (2).

Scheme 1

(In Scheme 1, R is as defined in Formula 1)

In this case, phenyl or cresol is used to remove the carboxyl protecting group, and phosphorus pentachloride, pyridine or the like is used to remove the amino group protecting group. However, contaminants used to remove the amino group protecting group, pyridine and the like are harmful to the human body because they are toxic solvents of strong acids or strong bases, cause environmental pollution, and produce a lot of by-products, resulting in a decrease in yield.

Therefore, in order to solve the above problem, a method of introducing an enzyme capable of removing the amino protecting group has been devised, and penicillysiase or penicyriamidase has been used as the enzyme. The enzymes are environmentally friendly because they can react with water and can be reused. However, the mass production has a lot of problems in the field application is low economical compared to the conventional chemical method.

Therefore, there is a need for a method for producing a new cephalosporin-based antibiotic intermediate with high stability, reproducibility and in situ, eco-friendliness, and high yield and purity.

Therefore, the inventors of the present invention while studying how to efficiently mass-produce cephalosporin-based antibiotic intermediates, using the Syntha CLEC-PA (Cross-Linked Enzyme Crystal-Penicillin G Amidase / Acylase) enzyme The present invention was completed by finding out that it is possible to mass-produce cephalosporin-based antibiotic intermediates having high, reproducibility and in situ, and high yield and purity.

It is an object of the present invention to provide a method for producing a cephalosporin-based antibiotic intermediate with high stability, reproducibility and in situ, high yield and purity, and which is capable of mass production.

In order to achieve the above object,

Adding a cresol or a phenol to the compound of Formula 2 and stirring to deprotect the protecting group of the carboxyl group (step 1);

Deprotecting the protecting group of the amino group by adding and stirring the synta CLEC-PA enzyme while adjusting the pH with a base (step 2); And

It provides a method for producing a cephalosporin antibiotic intermediate comprising the step (step 3) of preparing a cephalosporin antibiotic intermediate of the formula (1) by adjusting the pH with the inorganic acid.

In addition, the present invention is dissolved in the cephalosporin-based antibiotic intermediate produced in step 3 and dissolved in an aqueous methanol solution and a base, and then added to the acid to produce a crystalline hydrate (step 4) further comprising cephalosporin Provided is a method for preparing an antibiotic intermediate.

According to the present invention, it is possible to industrially synthesize crystalline hydrates of high-purity, high-efficiency cephalosporin-based antibiotic intermediates by using the synta CLEC-PA enzyme, which is easy to handle and stable in the deprotection step of the amino group protecting group. It can be usefully used when developing Sporin antibiotics.

1 is a photograph showing the crystal form of the synta CLEC-PA enzyme used in the present invention.
2 is an XRD graph of 7-APRA according to an embodiment of the present invention.
3 is an XRD graph of crystalline 7-APRA hydrate according to one embodiment of the present invention.
4 is an XRD graph of 7-ANCA according to an embodiment of the present invention.
5 is an XRD graph of crystalline 7-ANCA hydrate according to one embodiment of the present invention.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The present invention comprises the steps of deprotecting the protecting group of the carboxyl group by adding a cresol or phenol to the compound of the formula (2);

Deprotecting the protecting group of the amino group by adding and stirring the synta CLEC-PA enzyme while adjusting the pH with a base (step 2); And

It provides a method for producing a cephalosporin-based antibiotic intermediate of Formula 1 comprising the step (step 3) of preparing a cephalosporin-based antibiotic intermediate of the formula (1) by adjusting the pH with an inorganic acid.

[Formula 2]

[Formula 1]

(In Formulas 1 and 2, R is H, propenyl, (4-methylthiazol-5-yl) vinyl, and x is 3 to 6).

First, step 1 is a step of deprotecting the protecting group of the carboxyl group by adding a cresol or phenol to the compound of formula (2) and stirring.

In the preparation method according to the present invention, the compound of Formula 2 may be prepared or commercially available by the method of the prior art, and the amount of cresol or phenol to be added may be used in an appropriate amount such that the protecting group of the carboxyl group may be deprotected. Can be.

Next, step 2 is a step of deprotecting the protecting group of the amino group by adding a Cinta CLEC-PA enzyme while stirring the pH to the base to the compound in which the protecting group of the carboxyl group is deprotected in step 1.

In the production method according to the present invention, the base used may be an alkali metal carbonate such as sodium carbonate or potassium carbonate; Alkali metal hydroxides such as sodium bicarbonate, sodium oxide and potassium hydroxide; ammonia; Trialkylamines, such as trimethylamine, etc. are mentioned.

In the production method according to the invention, the pH is preferably maintained to 7.5 ~ 8.0.

In the production method according to the present invention, since the Cinta CLEC-PA enzyme is in free or crystallized form as shown in FIG . 1 , it is easy to handle and has high reaction efficiency, and thus may be industrially useful. The synta CLEC-PA enzyme is derived from E. coli. The amount of the enzyme varies depending on the type of enzyme, substrate, reaction, temperature, equilibrium point, reaction concentration, etc., but in this reaction, it is preferable to use 0.01-20 g for 1 g of substrate. The syntha CLEC-PA enzyme can be reused in the same reaction after filtration.

Next, step 3 is a step of crystallizing the intermediate by adjusting the pH with an inorganic acid to the intermediate compound deprotected in the protecting group of the carboxyl group and the amino group in the step 2.

In the production method according to the present invention, any inorganic acid can be used without limitation as long as it does not interfere with crystallization of the intermediate, and examples thereof include hydrochloric acid, nitric acid and sulfuric acid.

In the production method according to the invention, the pH is preferably maintained at 1.5 ~ 2.5.

In addition, the present invention may further comprise a step (step 4) of preparing a crystalline hydrate by adding an aqueous solution of methanol and a base in the cephalosporin-based antibiotic intermediate produced in step 3 and dissolving it.

In the production method according to the present invention, the methanol aqueous solution is preferably mixed in a ratio of methanol: water = 5: 1 to 1: 2, and more preferably methanol: water = 4: 1.

In the production method according to the present invention, the concentration of the cephalosporin antibiotic intermediate in the aqueous methanol solution is preferably 5-20%, more preferably 10%.

In the production method according to the invention, the base may be used sodium carbonate, potassium carbonate, sodium hydrogen carbonate and the like.

In the production method according to the present invention, the acid may be hydrochloric acid, sulfuric acid, nitric acid and the like.

After acid treatment in step 4, crystalline intermediate hydrate is formed through filtration, washing and drying. Crystalline intermediate hydrate prepared by the above method showed an XRD peak as shown in Figures 2 to 5 , the yield was up to 93%, the purity was also 99%.

In addition, in the deprotection experiment of the amino group protecting group, the Cinta CLEC-PA enzyme shortened the reaction time by 1 to 4 hours and improved the yield by about 10% compared to the penicillin amidase used as a conventional deprotection enzyme (Table 1 ~). 3).

Therefore, the Cinta CLEC-PA enzyme has high reaction efficiency, is easy to handle, and does not require a toxic organic solvent, which is environmentally friendly, and thus may be industrially useful in the preparation of crystalline hydrates.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the invention.

< Example  1> 7-amino-3- [ Propene -1-yl] -3- Sepem -4- Of carboxylic acids (7-APRA)  Produce

p-methoxybenzyl-7-phenylacetamido-3- [propen-1-yl] -3-cefe-4-carboxylate (200 g, 0.42 mol) was added to 500 mL of cresol and 35-40 ° C. Stir overnight at. After confirming the reaction by TLC, 2 liters of ethyl acetate was added, and a sodium bicarbonate solution (a solution of 50 g of sodium bicarbonate dissolved in 1.5 L of water) was slowly added thereto, followed by stirring at room temperature for 30 minutes. Next, ethyl acetate (500) was used to remove the cresol from the aqueous solution layer. mL) twice. After separating the water layer, 20 g of Synta CLEC-PA enzyme was added thereto, and the mixture was stirred for 3 hours while maintaining the pH of 7.5-8.0 using an aqueous sodium bicarbonate solution at 25-30 ° C. After confirming the reaction by TLC, the enzyme was filtered and separated. Carbon complex call was added to the water layer, and the mixture was stirred for 1 hour and then filtered. 2 L of methanol was added and the pH was adjusted to 3 with 2N-HCl to produce a white solid. The solid was filtered, washed with distilled water and acetone and dried to obtain the target compound (93.8 g, 93%).

¹ H-NMR (D ₂ O + NaHCO ₃ , 300 MHz): δ 1.68, 1.87 (3H, each, d, J = 6.0 Hz) 3.40 (d, 1H, J = 17.3 Hz), 3.69 (d, 1H, J = 17.3 Hz), 5.14 (d, 1H, J = 4.8 Hz), 5.51 (d, 1H, J = 11.1 Hz), 5.77 (dd, 1H, J = 5.6, 4.5 Hz), 6.02 (d, 1H, J = 11.1).

< Example  2> Crystallinity 7- APRA  Carb Synthesis

200 mL of H ₂ O and 50 mL of MeOH were added to 10 g of 7-APRA prepared in Example 1, 40 mL of 2N-sodium carbonate was added dropwise thereto, and the mixture was stirred until it was dissolved completely. Thereafter, about 20-30 mL of 2N-HCl was slowly added dropwise to the reaction solution while maintaining it at room temperature to completely dissolve it, and then stirred again at room temperature for 30 minutes, filtered, washed with a small amount of acetone, and dried at 50 ° C. for 1-2 hours. Crystalline 7-APRA hydrate was prepared.

Moisture (Kar Fischer Method) 2.2%.

Preparation of <Example 3> 7-amino-3- [2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid (7-ATVA)

P-methoxybenzyl-7-phenylacetamido, instead of p-methoxybenzyl-7-phenylacetamido-3- [propen-1-yl] -3-cefe-4-carboxylate as starting material Except for using -3- [2- (4-methylthiazol-5yl) vinyl] -3-cepem-4-carboxylate, the reaction was carried out in the same manner as in Example 1, to obtain the target compound (91% )

¹ H-NMR (D ₂ O + NaHCO ₃ , 300 MHz): δ 2.50 (3H, s, CH 3), 3.48 (d, 1H, J = 17.8 Hz), 3.77 (d, 1H, J = 17.8 Hz), 5.14 (d, 1H, J = 4.9 Hz), 5.24 (d, 1H, J = 4.9 Hz), 6.71 (s, 2H), 9.62 (s, 1H).

< Example  4> Crystallinity 7- ATVA  Carb Synthesis

Using 7-ATVA prepared in Example 3 was carried out in the same manner as in Example 2 to prepare a crystalline 7-ATVA hydrate.

Moisture (Karl Fischer method) 1.9%.

< Example  5> 7-amino-3- Sepem -4- Of carboxylic acids (7-ANCA)  Produce

P-methoxybenzyl-7-phenylacetamido, instead of p-methoxybenzyl-7-phenylacetamido-3- [propen-1-yl] -3-cefe-4-carboxylate as starting material A target compound (94%) was obtained in the same manner as in Example 1 except for using -3-cef-4-carboxylate.

¹ H-NMR (D ₂ O + NaHCO ₃ , 300 MHz): δ 3.55 (dd, 1H, J = 18.2, 5.5 Hz), 3.71 (dd, 1H, J = 18.2, 3.8 Hz), 5.11 (d, 1H, J = 5.5 Hz), 5.17 (d, 1H, J = 5.5 Hz), 6.55 (m, 1H).

< Example  6> Crystallinity 7- ANCA  Carb Synthesis

Using 7-ATVA prepared in Example 5 was carried out in the same manner as in Example 2 to prepare a crystalline 7-ANCA hydrate.

Moisture (Karl Fischer method) 2.5%.

<Analysis>

Compounds and crystalline hydrates prepared in the examples were analyzed using XRD.

As a result of measurement by XRD, 7-APRA compound showed a peak as shown in FIG. 2, and 7-APRA hydrate showed a peak as shown in FIG. 3 , indicating that the 7-APRA hydrate was crystalline unlike the 7-APRA compound. have. In addition, the 7-ANCA compound showed a peak as shown in Figure 4 , the 7-ANCA hydrate shows a peak as shown in Figure 5 it can be seen that the 7-ANCA hydrate is prepared in a crystalline form.

< Comparative example  1> 7-amino-3- [ Propene -1-yl] -3- Sepem -4- Carboxylic acid  Produce

7-Amino-3- [propen-1-yl] -3-cefe-4-carboxylic acid was prepared using conventional chemical synthesis.

22.8 g of phosphorus pentachloride, 150 mL of methylene chloride, and 8.88 mL of pyridine were added to a 500 mL flask, which was stirred for 30 minutes at room temperature. 30 g (62.6 mmol) of p-methoxybenzyl-7-phenylacetamido-3- [propen-1-yl] -3-cef-4-carboxylate was added dropwise to this solution, followed by 2 hours. Stirred. After cooling the reaction mixture to -10 ℃, 30 mL of 1,2-propanediol was added and stirred for 2 hours, then 120 mL of cresol was added dropwise and stirred for 2 hours.

200 mL of distilled water was added dropwise to the reaction mixture, stirred for 1 hour, the layers were separated, the water layer was sent to a crystal bath, and the organic layer was extracted with 300 mL of 2N HCl and sent to a crystal bath. 200 ml of a 30% sodium hydroxide solution was added dropwise to the crystallization bath, and the mixture was cooled to 0 ° C., and the precipitated solid was filtered and then dried in vacuo to give 12 g (20 mmol, 80% yield, 80%, Z /) as a pale yellow solid. E = 10.1 / 1).

¹ H-NMR (δ, D ₂ O + NaHCO ₃ ): 1.69 and 1.88 (3H, each, d, J = 6.0 Hz, -CH = CH-CH 3), 3.38 and 3.72 (2H, Abq, J = 17.0 Hz , H-2), 5.18 (1H, d, J = 5.0 Hz, H-6), 5.51 (1H, d, H-7), 5.8 (1H, m, -CH = CH-CH ₃ ), 6.06 ( 1H, d, J = 11 Hz, -CH = CH-CH ₃ )

< Comparative example  2> 7-amino-3- Sepem -4- Carboxylic acid  Produce

p-methoxybenzyl-7-phenylacetamido-3-cepem-4-carboxylate (100 g) was added to 500 mL of phenol at 45-50 ° C. The mixture was stirred at 45-50 ° C. for 7 hours (experimental with HPLC) and then cooled to 35-40 ° C. 1000 mL of n-butyl acetate was added to the mixture at 35-40 ° C. and cooled to 10-15 ° C. Sodium bicarbonate (1500 mL, 1.5%) was added at 10-20 ° C and stirred for 10-15 minutes. After extracting the water layer, the organic layer was extracted again with sodium hydrogen carbonate (1500 mL, 1.5%) at 10-20 ℃. Washing with n-butyl acetate to remove phenol in the extracted water layer. 50 g of wet penicillin-G amidase was added to the water layer at 20-25 ° C., and hydrolysis was performed. While stirring the mixture, an aqueous sodium hydrogen carbonate solution (5%) was added at 20-30 ° C. to maintain pH 7.5-7.9 and observed by HPLC. The enzyme was then filtered off and the remaining residue was washed with deionized water (200 mL) at 8-10 ° C. The pH of the cooled solution is adjusted to 2.4-2.5 with 10% hydrochloric acid. The resulting solid was filtered, washed with deionized water and acetone and dried in vacuo at 40-45 ° C. to obtain the target compound (yield 41.5 g, 81%).

¹ H-NMR (D ₂ O + NaHCO ₃ , 300 MHz): δ 3.57 (dd, 1H, J = 18.2, 5.4 Hz), 3.71 (dd, 1H, J = 18.2, 3.8 Hz), 5.09 (d, 1H, J = 5.4 Hz), 5.15 (d, 1H, J = 5.4 Hz), 6.55 (m, 1H).

<Comparative Example 3> 7-amino-3- [2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid Produce

Weigh 10 g p-methoxybenzyl-7-phenylacetamido-3- [2- (4-methylthiazol-5yl) vinyl] -3-cef-4-carboxylate into a 500 mL four-necked flask. And 55 mL of phenol was stirred at 50-55 ° C. for 5 hours. 100 mL of ethyl acetate and 200 mL of 5% aqueous sodium bicarbonate solution were added to the reaction solution, and the mixture was cooled to 10 ° C or lower. The organic layer was removed, the water layer was taken, and washed three times with 150 mL of ethyl acetate to obtain an aqueous solution. 5 g of PGA-450 was added to this aqueous solution, and the reaction was carried out at 20-30 ° C. for 5 hours using a 5% aqueous sodium carbonate solution within a range of pH = 7.5 to 8.5. After completion of the reaction, the enzyme was filtered off to obtain an aqueous solution of the compound in the flask. After cooling this aqueous solution to 10 degrees C or less, 5 g of activated carbon were added, and it stirred for 1 hour. Activated charcoal was removed by filtration, pH was adjusted to 4.0 by adding 4N hydrochloric acid to the filtrate, and aged at a temperature of 10 ° C. or lower for 1 hour. The precipitated crystals were collected by filtration, and the crystals were washed with water and acetone and dried to obtain the target compound (3.82 g, yield 79.8%).

¹ H-NMR (0.2 mol / L-DCl / D ₂ O ppm from TSP): δ 2.53 (3H, s, CH ₃ ), 3.56 to 3.61 (1H, d, S-CH (H), 18.4 Hz), 3.75 to 3.80 (1H, d, S-CH (H), 18.4 Hz), 5.26 to 5.27 (1H, d, S-CH, 5.2 Hz), 6.79 (2H, s, HC = CH), 9.79 (1H, s, S-CH = N)

< Experimental Example > Enzymes Deprotection  Efficiency experiment

In order to determine the deprotection efficiency according to the enzyme used in the preparation of 7-ATVA, 7-APRA and ANCA hydrate according to the present invention, the following experiment was performed.

50 g of each of the Cinta CLEC-PA enzyme used in the Example (manufactured by Altus Biologics) or the penicillin amidase used in the Comparative Example (manufactured by Sigma) was added, and the starting material (p-methoxybenzyl-7-phenylacetamido- 3-cefe-4-carboxylate, p-methoxybenzyl-7-phenylacetamido-3- [propen-1-yl] -3-cefe-4-carboxylate or p-methoxybenzyl- 7-phenylacetamido-3- [2- (4-methylthiazol-5yl) vinyl] -3-cef-4-carboxylate) was changed to 100 g, 200 g and 400 g, followed by amino group. The deprotection reaction of the protecting group was carried out.

After the reaction time and the yield of the product was measured and shown in Tables 1-3.

7- ATVA  synthesis Amount of starting material (g) Syntha CLEC-PA Enzyme Penicillin amidase Response time (hours) yield(%) Response time (hours) yield(%) 100 2 94 4 88 200 4 91 9 83 400 10 85 18 82

7- APRA  synthesis Amount of starting material (g) Syntha CLEC-PA Enzyme Penicillin amidase Response time (hours) yield(%) Response time (hours) yield(%) 100 3 93 4 85 200 6 92 10 85 400 12 84 26 72

7- ANCA  synthesis Amount of starting material (g) Syntha CLEC-PA Enzyme Penicillin amidase Response time (hours) yield(%) Response time (hours) yield(%) 100 3 93 4 90 200 6 89 7 87 400 11 82 22 76

As shown in Tables 1 to 3, the synta CLEC-PA enzyme used in the present invention was shown to shorten the reaction time by 1 to 4 hours and improve the yield by about 10% compared to the penicillin amidase used as a conventional deprotection enzyme. .

Therefore, the Cinta CLEC-PA enzyme has high reaction efficiency, is easy to handle, and does not require toxic organic solvents and is environmentally friendly, thus industrially preparing crystalline 7-ATVA, 7-APRA and ANCA hydrates. It can be usefully used.

Claims (16)

Adding a cresol or a phenol to the compound of Formula 2 to stir to deprotect the protecting group of the carboxyl group (step 1);
Adding a Syntha CLEC-PA (Syntha Cross-Linked Enzyme Crystal-Penicillin G Amidase / Acylase) enzyme while adjusting the pH with a base to stir to deprotect the protecting group of the amino group (step 2); And
A method for preparing the cephalosporin-based antibiotic intermediate of formula 1 comprising the step (step 3) of preparing a cephalosporin-based antibiotic intermediate of formula 1 by adjusting the pH with an inorganic acid.
(2)

[Formula 1]

(In Formulas 1 and 2, R is (4-methylthiazol-5-yl) vinyl, and x is 3 to 6). The method of claim 1, wherein the base of step 2 is selected from the group consisting of alkali metal carbonate, alkali metal hydroxide, ammonia water and trialkylamine. 3. The cephalosporin system according to claim 2, wherein the alkali metal carbonate is sodium carbonate or potassium carbonate, the alkyl hydroxide metal is sodium bicarbonate, sodium oxide or potassium hydroxide, and the trialkylamine is trimethylamine. Process for the preparation of antibiotic intermediates. According to claim 1, wherein the pH of step 2 is a method for producing a cephalosporin antibiotic intermediate, characterized in that to maintain at 7.5 ~ 8.0. The method of claim 1, wherein the inorganic acid of step 3 is a method for producing cephalosporin antibiotic intermediate, characterized in that hydrochloric acid, nitric acid or sulfuric acid. According to claim 1, wherein the pH of step 3 is a method for producing cephalosporin-based antibiotic intermediates, characterized in that it is maintained at 1.5 ~ 2.5. Adding a cresol or a phenol to the compound of Formula 2 to stir to deprotect the protecting group of the carboxyl group (step 1);
Deprotecting the protecting group of the amino group by adding and stirring the synta CLEC-PA enzyme while adjusting the pH with a base (step 2);
Preparing a cephalosporin antibiotic intermediate of Formula 1 by adjusting pH with an inorganic acid (step 3); And
The cephalosporin-based antibiotic of Formula 1 comprising the step (4) of preparing a crystalline hydrate by dissolving an aqueous methanol solution and a base in the cephalosporin-based antibiotic intermediate produced in step 3 and dissolving it. Method for preparing crystalline hydrate of intermediate.
(2)

[Formula 1]

(In Formulas 1 and 2, R is (4-methylthiazol-5-yl) vinyl, and x is 3 to 6). The method of claim 7, wherein the base of step 2 is selected from the group consisting of alkali metal carbonate, alkali metal hydroxide, ammonia water and trialkylamine. 9. The cephalosporin system according to claim 8, wherein the alkali metal carbonate is sodium carbonate or potassium carbonate, the alkyl hydroxide metal is sodium bicarbonate, sodium oxide or potassium hydroxide, and the trialkylamine is trimethylamine. Process for the preparation of crystalline hydrates of antibiotic intermediates. 8. The method of claim 7, wherein the pH of step 2 is maintained at 7.5 to 8.0. 8. The method of claim 7, wherein the inorganic acid of step 3 is hydrochloric acid, nitric acid or sulfuric acid. 8. The method of claim 7, wherein the pH of step 3 is maintained at 1.5 to 2.5. 8. The method of claim 7, wherein the aqueous methanol solution of step 4 is mixed in a ratio of methanol: water = 5: 1 to 1: 2. The method of claim 7, wherein the concentration of the cephalosporin antibiotic intermediate in the aqueous methanol solution of step 4 is 5 to 20%, the method for producing a crystalline hydrate of the cephalosporin antibiotic intermediate. 8. The method according to claim 7, wherein the base of step 4 is selected from the group consisting of sodium carbonate, potassium carbonate and sodium hydrogen carbonate. 8. The method of claim 7, wherein the acid of step 4 is hydrochloric acid, sulfuric acid, or nitric acid.

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