WO2003031451A1

WO2003031451A1 - Method for crystallization of 7-aminocephalosporanic acid

Info

Publication number: WO2003031451A1
Application number: PCT/KR2002/001851
Authority: WO
Inventors: Jung Woo Kim; Sang Kyu Shin; Sang Yong Choi; Jong Chan Yun; In Kyun Yun
Original assignee: Ckd Bio Corp.
Priority date: 2001-10-06
Filing date: 2002-10-04
Publication date: 2003-04-17
Also published as: KR20030029500A; CN1697837A; KR100509737B1; EP1436299A4; EP1436299A1

Abstract

The present invention relates to a method for preparing crystals of 7-aminocephalosporanic acid from 7-aminocephalosporanic acid solution by using additive and acid. The crystals of 7-aminocephalosporanic acid prepared according to the present invention exhibits superior granularity, purity, and can be prepared in a high yield.

Description

Method for crystallization of 7-aminocephalosporanic acid

Technical Field

The present invention relates to a method for preparing 7- aminocephalosporanic acid (7-ACA) crystals from 7-aminocephalosporanic acid solution by using additive and acid.

Background Art

7-ACA is used as an intermediate for synthesis of various cephalosporins. Cephalosporin antibiotics refer to antibiotics prepared from cephalosporin C (CPC), and CPC is generally isolated from fermentation product of Acremonium chrysogenium. Methods of preparing 7-ACA from CPC as raw material are divided into chemical process and enzymatic process. Synthesis of 7-ACA based on the chemical process requires a multi-step reaction at ultralow temperature, and includes problems, remaining of toxic solvent within final product or environmental pollution, accordingly, lately, enzymatic process has been developed and used. Representative method of the enzymatic process consists of, first, inverting CPC into glutaryl-7- aminocephalosporanic acid (G1-7-ACA) by reacting with D-amino acid oxidase (DAOD) as shown in reaction formula 1, then reacting G1-7-ACA with G1-7-ACA acylase to obtain 7- AC A as shown in reaction formula 2. [Reaction formula 1]

CPC G1-7-ACA [Reaction formula 2]

G1-7-ACA 7-ACA

Adjusting of pH value of the 7- AC A solution prepared via chemical process or enzymatic process with acid to isoelectric point leads to formation of crystals, and by filtration, 7-ACA crystals can be isolated from medium. The shape of 7-ACA crystals influences purity, that is, the larger and harder the crystals are, the higher the purity is, due to ease of filtration and wash, while the smaller and softer the crystals are, the lower the purity is, due to difficulty of filtration and wash. In general, in case of enzymatic reaction, precipitation of 7-ACA after the reaction leads to formation of small and soft particles sticking together, causing difficulty of filtration and wash, resulting in lower purity.

To increase purity, crystallization may be carried out after purifying 7-ACA solution with ion exchange resin or adsorption resin, but in this case, despite of increase in purity, problems occur, that is, difficulty of filtration due to small size of the particles and resultant low yield. Further, Korean Patent Application No. 1999-7011246 discloses attempts to raise yield by adding organic solvent such as methanol or acetone. Though this method improved granularity to some degree, but the effect was not enough. On the other hand, Korean Patent Application No. 1999-7011246 discloses a method of using additives such as Cycep 2411R (Cytec), polyacryl such as P3-pherocryl 7262R, organic carboxylic acid ester such as ethylacetate or butylacetate, polyamine such as C 592R(Cytec), polymeric glycol such as PEG 300R (Fluka), amines such as triethylene tetramine or tris-(2-aminoethyl)amine and amino acids such as lysine. In addition, Korean Patent Application No. 1999-7011246 includes a disclosure on method of isolating 7-ACA by using organic solvent such as methanol or acetone along with the additive or by dropping a portion of solution to the rest thereof.

The present invention was conceived to resolve the previous problems as described above, and the object of the present invention lies in providing a method for preparing 7-ACA crystals with improved granularity and purity in a high yield through crystallizing 7-aminocephalosporanic acid by using additive and acid.

Disclosure of the Invention The present invention relates to a method for preparing 7- aminocephalosporanic acid (7-ACA) crystals from 7-aminocephalosporanic acid solution by using additive and acid.

As the additive, an alkylsulfate of the following formula 1, an amineoxide of formula 2, a quarternary ammonium of formula 3 and a glycerylester of formula 4 can be used alone or in a combination of two or more. [Formula 1]

1

[Formula 2]

R₂

2 [Formula 3] R

3

[Formula 4] R e

4

Wherein, Ri represents C_1-22 alkyl, phenyl, naphthyl or dialkylether; R₂ , R , R₄ and R₅ represent independently hydrogen, -₂₂ alkyl, phenyl or naphthyl; X represents CI, Br or I; and R^ , R₇ and R₈ represent independently hydrogen, C₁-₂₂ alkyl, phenyl, naphthyl, OH or R₂COO (said alkyl, phenyl or naphthyl is unsubstituted or substituted with a group without chemical reactivity toward 7-ACA). As the alkylsulfate of the formula 1, sodiumlaurylsulfate, sodiumlaurylethersulfate, ammoniumlaurylsulfate, triethanolaminelaurylsulfate etc. can be enumerated. As the amineoxide of the formula 2, lauryldimethylamineoxide, cocamidopropylamineoxide and laurylpolyethylenedimethylamineoxide etc. can be enumerated. As the quarternary ammonium of the formula 3, lauryltrimethylamrnoniumchloride, cetyltrimethylammoniumchloride, distearyldimethylammoniumchloride, stearyldimethylbenzylammoniumchloride, lauryldimethylbenzylamrnoniumchloride, stearylditrimethylammoniumchloride and behenyltrimethylammoniumchloride etc. can be used. As the glycerylester of the formula 4, glycerylmonolaurate, glycerylmonomyristate, glycerylmonostearte, glycerylmonoisostearte, glycerylmonooleate, glyceryltricaprylate, glyceryltrioleate and glyceryltriisostearate etc. can be enumerated. Among them, it is preferable to use sodiumlaurylsulfate as an alkylsulfate, cocamidopropylamineoxide as an amineoxide, lauryltrimethylammoniumchloride as a quartemary ammomum, glycerylmonostearte and glyceryltricaprylate as glycerylester.

As the acid, hydrochloric acid, sulfuric acid and acetic acid can be used alone or in a combination of two or more.

The crystallization method of the present invention can be explained in detail as follows. First, additive is added to 7-ACA solution. The 7-ACA solution is a solution converted from CPC by enzymatic process, and concentration of a range of 10 to 40g/L is preferred. The amount of additive is preferred to be 0.001 to 5%(w/v) to 7- ACA solution. Addition of acid leads to precipitation of 7-ACA, and for a high yield, it is advantageous to adjust pH value to isoelectric point. Temperature at the time of crystallization is 0 to 30°C, preferably 2 to 15°C. 7-ACA crystallized according to the present invention is filtered by centrifuging, washed with water and acetone, and vacuum dried at 25 to 45°C.

On the other hand, addition of organic solvent in addition to the additive improves granularity and purity of 7-ACA crystals, thus preferable. As the organic solvent, one or more solvent selected from the group consisting of ethylacetate, carbon tetrachloride, dichloroethane, trichloroethane, methylenechloride, chloroform and methylisobutylketone can be used. Organic solvent is added along with the additive, and the amount of organic solvent is 0.5 to 60%(v/v) to 7-ACA solution, preferably 0.5 to 10%(v/v). Brief Description of Drawings

Fig. 1 shows crystal form of 7-aminocephalosporanic acid obtained according to Comparative Example 1 of the present invention.

Fig. 2 represents crystal form of 7-aminocephalosporanic acid obtained according to Example 1 of the present invention.

Fig.3 shows crystal form of 7-aminocephalosporanic acid obtained according to Example 23 of the present invention.

Best Mode for Carrying Out the Invention In the below, the present invention is disclosed in detail through Examples.

The following Examples specifically explain the present invention, and the scope of the present invention is not limited by the Examples.

Examples 1 to 22: Crystallization of 7- AC A using additive and acid Additive is added to 7-ACA solution (about HOmM) l,000mL, stirred for

30min at 8°C, 15% hydrochloric acid was added to adjust pH to 3.1, and stirred for 1 hr. Precipitated 7-ACA solution was filtered, 7-ACA crystals thus obtained were washed with water 200mL, acetone lOOmL, and vacuum dried at about 40°C for 14 hr. The kind and amount of the additive used and the filtration time for 7-ACA are as represented in Tables 1 to 4. Table 1. Crystallization of 7-ACA using an alkylsulfate

Table 2. Crystallization of 7- AC A using an amineoxide

Table 3. Crystallization of 7- AC A using a quartemary ammonium

Table 4. Crystallization of 7-ACA using a glycerylester

Examples 23 to 44: Crystallization of 7- AC A using additive, organic solvent and acid

Ethylacetate lOOmL and additive were added to 7-ACA solution (about llOmM) l,000mL, and stirred at 8 °C for 30 min, and 15% HCl was added to adjust pH value to 3.1 and stirred for 1 hr. Precipitated 7- AC A solution was filtered, 7- AC A crystals obtained were washed with water 200mL, acetone lOOmL, and vacuum dried at 40°C for 14 hr. The kind and amount of the additive used and the filtration time are as shown in Tables 5 to 8. Table 5. Crystallization of 7-ACA using an alkylsulfate

Table 6. Crystallization of 7-ACA using an amineoxide

Table 7. Crystallization of 7-ACA using a quartemary ammonium

Table 8. Crystallization of 7- AC A using a glycerylester

Comparative Example 1: Crystallization of 7-ACA using acid

7-ACA was crystallized according to the same method as in Example 1 except the addition of additive. Filtration time was 11 min.

Experimental Example 1: Measurement of content and bulk density for 7-ACA Content and bulk density of 7- AC A prepared according to said Comparative

Example 1 , Example 1 and Example 23 were analyzed and the result is shown in Table 9.

The content of 7-ACA was determined with HPLC analysis (measurement at 254nm) by dissolving lOmg of 7-ACA prepared in accordance with said Comparative Example 1, Example 1 and Example 23 in pH 7.3 phosphate buffer lOOmL HPLC was carried out in accordance with Liquid Chromatography of General Test Methods, Korea Pharmacopoeia, and the content was calculated based on relative peak area. The content of sample means relative value against the analysis result for standard substance by the same method, and was calculated by the following equation 1.

[Equation 1]

Content of sample (%) = (peak area of sample/ peak area of standard substance) x content of standard substance (%)

7-ACA prepared according to said Comparative Example 1, Example 1 and Example 23 were, respectively, filled in lOOmL graduated cylinder to 50mL without compacting, and the weight was measured and bulk density was calculated.

Table 9.

As can be seen from the Table 9, the content of 7- AC A in case of crystallization using additive and acid according to the present invention (Example 1) was at least 96%>, higher than that in case of crystallization using acid (Comparative Example 1), 92.3%. In particular, in case of crystallization using organic solvent in addition to the additive (Example 23), the content of 7-ACA was at least 98%>, showing very good purity.

Experimental Example 2: Observation of crystal form of 7- AC A

Shape and size of 7-ACA particles prepared according to said Comparative Example 1, Example 1, Example 23 were photographed and shown in Figs. 1 to 3. First, lOmg of 7-ACA was smeared on a slide glass, and crystal shape was photographed to 100 magnifications with Axiolab Microscope (Karl zeiss).

The result revealed that crystallinity of 7-ACA in case of crystallization using additive and acid (Example 1) was superior to that in case of crystallization using acid (Comparative Example 1). In particular, in case of crystallization using organic solvent in addition to the additive (Example 23), 7-ACA crystals were large, hard and uniform, exhibiting superior crystallinity.

Industrial Applicability 7-ACA obtained according to the crystallization of the present invention exhibits improved granularity, content, transmittance, and thus purity is superior to that by conventional technologies.

Claims

1. A method for preparing 7-aminocephalosporanic acid crystals from 7- aminocephalosporanic acid solution by using at least one additive selected from the group consisting of compounds of the following formulae 1 to 4 and acid:

R e

R

1 2 3 4

wherein, Ri represents Cι-₂₂ alkyl, phenyl, naphthyl or dialkylether; R₂ , R₃ , 4 and R₅ represent independently hydrogen, C1-₂₂ alkyl, phenyl or naphthyl; X represents CI, Br or I; and R_ό , R and R₈ represent independently hydrogen, Cj.₂₂ alkyl, phenyl, naphthyl, OH or R₂COO (said alkyl, phenyl or naphthyl is unsubstituted or substituted with a group without chemical reactivity toward 7- AC A).

2. The method as in Claim 1, characterized in that at least one organic solvent selected from the group consisting of ethylacetate, carbon tetrachloride, dichloroethane, trichloroethane, methylenechloride, chloroform and methylisobutylketone is further used in addition to the additive.

3. The method as in Claim 1 or Claim 2, characterized in that the concentration of 7- aminocephalosporanic acid solution is 10 to 40g/L.

4. The method as in Claim 1 or Claim 2, wherein said additive is sodiumlaurylsulfate, sodiumlaurylethersulfate, ammoniumlaurylsulfate, triethanolaminelaurylsulfate, lauryldimethylamineoxide, cocamidopropylamineoxide, laurylpolyethylenedimethylamineoxide, lauryltrimethylammoniumchloride, cetyltrimethylammoniumchloride, distearyldimethylammoniumchloride, stearyldimethylbenzylammoniumchloride, lauryldimethylbenzylammoniumchloride, stearylditrimethylammoniumchloride, behenyltrimethylammoniumchloride, glycerylmonolaurate, glycerylmonomyristate, glycerylmonostearte, glycerylmonoisostearate, glycerylmonooleate, glyceryltricaprylate, glyceryltrioleate or glyceryltriisostearate.

5. The method as in Claim 1 or Claim 2, characterized in that the amount of said additive is 0.001 to 5%(w/v) to 7-aminocephalosporanic acid solution.

6. The method as in Claim 2, characterized in that the amount of said organic solvent is 0.5 to 60%o(v/v) to the 7-aminocephalosporanic acid solution.

7. The method as in Claim 1 or Claim 2, characterized in that acid is added to adjust pH value to isoelectric point.

8. The method as in Claim 1 or Claim 2, characterized in that crystallization temperature is 0 to 30°C.