JPS6170999A - Production of cephalosporin compound - Google Patents

Abstract

PURPOSE:To obtain a cephalosporin compound wherein acetoxymethyl at the 3-position is converted into hydroxymethyl group by eliminating advantageously acetyl group from the acetoxymethyl without causing side reactions, by treating a cephalosporin compound with wheat bran. CONSTITUTION:(A) A cephalosporin compound such as cephalosporin C, 3- acetoxymethyl-7beta-amino-3-cephem-4-carboxylic acid, cephaloglycin, etc. is processed into preferably 1-50mg/ml aqueous solution, (B) wheat bran such as barley bran is washed with water to remove impurities, the component A is blended with 4-40 times calculated as weight before water washing as much barley as the component A. The component A is treated with the barley bran at 6-8pH for 24hr, the barley bran is filtered off, the filtrate is subjected to ultrafiltration to remove colored substances and protein, the solution is evaporated to dryness, or an organic solvent such as an alcohol, etc. is added to the solution, and a substance is precipitated, to give the aimed substance.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing a cephacesporin compound.

Cephalosporin compounds useful as chemotherapeutic agents have a cephem structure, and exhibit unique antibacterial activity by introducing various substituents into the 8- and 7-positions.

The raw materials often used in the production of these compounds are Cephalosporin C (hereinafter referred to as CPC) and 3-acetoxymethyl-7β-amino-8-cephem-4-carboxylic acid (hereinafter referred to as 7-ACA). (abbreviated). However, since an inactive acetoxymethyl group exists at the 3-position of these compounds, when attempting to introduce a substituent at the 3-position, deacetylation is first performed to produce an active hydroxymethyl intermediate. things are essential.

[Conventional technology]

There are two methods for removing the acetyl group from the acetoxymethyl group at the 3-position of a cephalosporin compound and converting it into a hydroxymethyl group: chemical cleavage by adding an acid or alkaline aqueous solution, and mild hydrolysis with an enzyme. There is.

Methods using chemical hydrolyzing reagents such as caustic soda and hydrochloric acid tend to cause decomposition of the inherently unstable cefterosporin compound, which is difficult to achieve industrially at -20 to -40°C.
However, in the reaction with an acidic reagent, a lactone ring is formed between the hydroxymethyl group formed at the 8-position and the carboxyl group at the 4-position, and when an alkaline reagent Now, the transferred 6 of type 2 packing (Δ3)
Two bodies are likely to occur as side effects.

On the other hand, enzymatic deacetylation methods that allow the reaction to proceed under mild conditions include (a) a method using esterase derived from citrus peel (British Patent No. 996.222);
Method using wheat germ esterase (Special Publication Publication No. 42-15
53), (c) Methods of hydrolysis using microbial enzymes such as Bacillus subtilis (Japanese Patent Publication No. 49-85993) are known, but these still have various problems from an industrial perspective. There is. For example, in method (a), it is difficult to obtain uniform pericarp as an enzyme source, and in method (naka), although the enzyme source is relatively easy to obtain, the esterase must be extracted and purified before use. The operation is extremely complicated. On the other hand, with the microbial enzymes that are considered the most promising industrially, such as the method (c), although there are differences in degree depending on the bacterial species, contamination with an enzyme (β-lactamase) that cleaves the β-lactam ring is a problem. It inevitably has drawbacks that cause a decrease in yield and purity.

Therefore, the present inventors conducted various studies on an advantageous production method that does not involve side reactions when carrying out enzymatic deacetylation of the 3-position side chain, using an enzyme source that can be obtained in large quantities at low cost and is easy to purify. As a result, the present invention was completed.

[Structure of the invention]

The present invention is a method for producing a compound represented by the general formula by treating the compound represented by the general formula with wrinkles (bran). However, R
t means hydrogen or an acyl group, and R2 means a lower alkyl group. - stoichiometrically represents a carboxyl group or a salt thereof or a protected carboxyl group.

In addition to apa and 7-ACA, compound (I) also includes cephaloglycin (R1: C5HsCjH(N)Iz)0
0-) + cephalothin (R1' 0GHx00-
) rcephapirin (R1: NG5C1hCO-)
, There is a compound with se7 otaxime (R1 nylamino group), and this acyl group In addition to the above, as an example of an acyl group, the deacylation reaction of the 3-position acyloxymethyl group is not affected by the difference in 2-□. Almost none.・(2-aminothiazol-4-yl)-2
Examples include -((imidazol-4-yl)methoxyimino)acetyl group and those shown in Japanese Patent Publication No. 58-43979.

The concentration of compound (1) in the reaction solution is usually 1 to 50 μ/g.
Although the concentration is in the range of IIt, even higher concentrations are possible because the rate of the deacylation reaction is high.

Wheat drums, which are inexpensive and available in large quantities, are suitable for the wrinkles, and it is preferable to use them after removing impurities adhering to the surface by washing with water one to several times. The amount used is the weight before washing with water, and is usually 4 to 40 times the weight of the substrate (compound (I)).

To carry out the reaction, add water-washed wrinkles to an aqueous solution of compound (I), maintain the pH at 6-8, preferably pH 6,5-7.0, and heat at 20-40"C, preferably 25-30"C.
Process for 24 hours, 1 hour, and 18 hours a day. At this time, a normal buffer such as a phosphate buffer can be used, but it is also possible to carry out the reaction without using a buffer, while neutralizing the free acid generated by deacylation, such as acetic acid, with an alkali as appropriate. is also possible.

To isolate the target compound (I[) from the reaction-completed solution.

The wrinkles are removed by an operation such as filtration, colored substances, proteins, etc. are removed by ultrafiltration, and the resulting liquid is dried, or an organic solvent such as alcohol, acetone, or ether is added to this solution to obtain the desired solution. It is common to precipitate substances. Ultrafiltration.

Commercially available ultrafiltration membranes such as cellulose acetate, polyamide, polysulfone, and polyacrylonitrile may be used singly or in combination as appropriate.

Example 1 5 ooq of cephalosporin C sodium salt dihydrate was dissolved in 100-ml of 0.1 M phosphate buffer (pH 7, -0), 2 g of wheat gluten was added, and the mixture was stirred at 80°C for 13 hours.

After the drum was filtered off, one reaction solution was analyzed by liquid chromatography, and it was found that 3-hydroxymethyl-7β-(D-5-amino-5-carboxypentanamide)-8-cephem-
The production rate of 4-carboxylic acid was 95%. After ultrafiltration of this reaction solution, nonionic porous polymer resin HP
-20 (Mitsubishi Kasei) 504, and the effluent has a pH of 7:
After adjusting the concentration to
6.1%).

The purified crystal obtained by recrystallizing this dry product with water-ethanol has an NMR δ (Dis, 020) -4,4 (2H
, S, O spider OH), (α)”+107° (H, O.

O-2,0)tE:, :2603fi-185, which matched the standard product.

Example 2 Except for using 3-acetoxymethyl-7β-amino-8-conjugated 7emu-4-carboxylic acid (7-ACA) in place of cephalosporin C in Example 1 and using wheat wheat 1a-g. was treated in the same manner as in Example 1, and reacted for 5 hours. When the reaction solution was analyzed by liquid chromatography, 3-
Hydroxymethyl-7β-aminocef-3-em-4-
The production rate of carboxylic acid was 100%.

After filtering the drum, the filtrate was ultrafiltered and then HP-2050
Concentrate the effluent to approximately 5-50% by passing the liquid through the bar, and adjust the pH value with acetic acid.
:Adjusted to 4.2. This concentrated solution was cooled with ice, and the precipitate was collected by filtration and dried to obtain 308 ml of 3-hydroxymethyl compound. The NMR of this product is δ(DSS, DzO)-4,2(2H,
8, COH) and TLc (n-butanol:acetic acid:pyridine:water-15=3:10:12) are Rf-0,37
The result was consistent with the standard product.

Example 3 - 3-acetoxymethyl-7β-(2-(2-aminothiazol-4-yl)-2-((imidazol-1-yl)methoxyimino)acetamido-3-cephem-
4-Carboxylic acid dihydrochloride 5QO■ was dissolved in 100% water and adjusted to pH 7.0 with 0.05N sodium hydroxide solution.

To this solution was added 4 g of wheat gluten which had been washed 5 times with 20°C of cold water, and the mixture was reacted at 80°C for 13 hours while the system pH was maintained at 7.0 with 0.05N sodium hydroxide.

After removing the wrinkles by filtration, the nine reaction solutions were analyzed by liquid chromatography, and the results showed that 3-hydroxymethyl-7β-(2-(2-
The production rate of aminothiazol-4-yl)-2-((imidazol-4-yl)methoxyimino)acetamido-3-cephem-4-carboxylic acid was 95.2%.

After ultrafiltration, this reaction solution was passed through HP-205Qm/, and the effluent was freeze-dried to obtain 8-hydroxymethyl compound 36.
2■ (calculated as anhydride) was obtained.

The NMR of this product is δ(DSL, DzO)-4,35(2
H, s, 030H), I R is νmax(KBr
) -1760, 1650, 1600 and supported the structure.

Example 4 The same procedure as in Example 8 was carried out, except that ce7-otaxime sodium was used instead of the raw material compound in Example 8, and the wheat gluten was washed 5 times with 40 cm of cold water before use. I let it happen. Liquid chromatography analysis of the reaction solution revealed that 3-hydroxymethyl-7β-(2-(2-aminothiazole-4-
yl)-2-methoxyiminoacetamide]-3-77
! The production rate of sodium mu-4-carboxylate is 94.7%
Met.

The obtained reaction solution was post-treated in the same manner as in Example 8 to obtain 8-hydroxymethyl compound 401119 (in terms of anhydride). The NMR of this product was δ(DSS, D20) = 4.8
5 (2H,8,CjlzOH), X R is νmax
(KBr)-1760, which was consistent with the standard product.

Example 5 In Example 2? - Cephalothin sodium salt was used instead of AOA, and the reaction was carried out in the same manner as in Example 2 except for the above. Liquid chromatography analysis of the reaction solution revealed that the production rate of sodium 8-hydroxymethyl-7β-(thio7en-2-yla-7doamide)-3-cephem-4-carboxylate was 97.9%. After adding sodium chloride to the resulting reaction solution, the pH was adjusted to 3.5, extracted with ethyl acetate, and the ethyl acetate layer was concentrated to dryness. The residual liquid was recrystallized with methanol to obtain crystals of 3-hydroxymethyl compound. This product has NMRδ(D19S, C0)-4,4(
2H,s, 0yaOH) and UVλmax260am(
4-8050), it was identified as the standard sample. ◎ Example 6 In Example 2? - Treated as in Example 2, except using Cephapirin Sodium instead of ACA.
Made it react. Analysis of the reaction solution by liquid chromatography revealed that the production rate of sodium 8-hydroxydimethyl-[beta]-(pyrid-4-ylthio-7tamide)-3-cephem-4-carboxylate was 97.2%.

The obtained reaction solution was post-treated in the same manner as in Example 8 to obtain crystals of the 3-hydroxymethyl compound, and its N MRδ (Dss
, C0)=4.2 (2H,s, GHs,OH), it was identified as the standard specimen.

Example 7 The same procedure as in Example 2 was repeated except that ceftetetraglycine dihydrate was used in place of 7-ACA in Example 2, and 20 g of wheat bran was used, and the reaction was carried out for 2.5 hours. When the reaction solution was analyzed by liquid chromatography, 3-hydroxymethyl-7β-(D-α-aminophenyl acetamide)-
The production rate of 8-cephem-4-carboxylic acid was 95.0%.

3- isolated from the resulting reaction solution in the same manner as in Example 3.
Crystals of hydroxymethyl form are NMR (Dss, D2o)
δ-4,2 (2He Is, Cji20H) and UV
2max260s@(a-8400) It was identified as the standard product.

Claims (1)

[Claims] Manufacture of a compound represented by the general formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ characterized by treating the compound represented by the general formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Law. However, R_1 means hydrogen or an acyl group, and R_2 means a lower alkyl group. R_3 means a carboxyl group, a salt thereof, or a protected carboxyl group.