DK149125B - 6- (D - (-) ALFA-AMINO-ALFA- (P-ACETOXYPHENYL) ACETAMIDO) PENICILLANIC ACID FOR INTERMEDIATE USE - Google Patents

Description

149125

The present invention relates to the novel compound 6- [D - (-) α-amino-α- (p-acetoxyphenyl) acetamiao] penicillanic acid of the formula [alpha] - [CH 3 CH 3 -NH —If

NH2 + -N - * - COOH

for use as an intermediate in the preparation of 6- [D - (-) - α-amino-ot- (p-hydroxyphenyl) acetamido] penicillanic acid (also known as amoxycillin or p-hydroxyampicillin), hydrate or a pharmaceutically acceptable salt thereof.

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Amoxycillin, having the formula OH J '\ _CH-CO- NH -_fy ^^ S \ y'CE3 x ^ / I I ^ ch3

'-' NH2 J-N__COOH

cr is a known antibiotic active compound which has heretofore been prepared by treating 6-aminopenicillanic acid with an acylating agent in the form of a reactive functional derivative of the side chain acid.

It has now been found that this valuable penicillin can be prepared by treating the corresponding p-acetoxy compound with certain esterases, and the invention is based on this disclosure.

It is known to cleave various chemical bonding types by esterases, e.g. an imide bond in the 6-side chain of a penicillin, cf. Danish Patent Application No. 534/68, and an oxygen bond in an O-acetyl group in the 3-position of a cephalosporin, cf. Danish Patent Application No. 3338/71 and Danish Patent No. 108,006. However, these are bond types which are not chemically analogous to the bond of an O-acetyl group which is cleaved in the intermediate of the present invention.

The conversion of the intermediate according to the invention to amoxycillin, hydrate or a pharmaceutically acceptable salt thereof is effected by treating 6- [D - (-) α-amino-α- (p-acetoxyphenyl) acetamido] -penicillanic acid in aqueous solution with esterase from human serum or wheat bran or citrus esterase at a pH between about 5.0 and approx. 7.5, isolates the product in a manner known per se, and if desired in a manner known per se, converts the product in the form of the free acid or hydrate to the corresponding pharmaceutically acceptable salt thereof.

According to a preferred embodiment, commercial coarse wheat bran is used as esterase, wherein the treatment in aqueous solution is carried out at a pH between 5.5 and 6.0 or optionally in the presence of a buffer at a pH of 7.0 at concentration of approx. 10 mg / ml esterase per total volume of solution.

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The above pharmaceutically acceptable salts include the non-toxic carboxylic acid salts, e.g. non-toxic metal salts such as the sodium, potassium, calcium and aluminum salts, ammonium salts and salts with non-toxic amines, e.g. trialkylamines, procaine, dibenzylamine, N-ben zy1-β-phenethylaminoη, 1-ephenamine, Ν, Ν'-dibenzylethylenediamine, N-alkylpiperidine and other amines used to form salts of penicillins. Furthermore, the definition of pharmaceutically acceptable salts includes the non-toxic acid addition salts (amine salts), e.g. salts with mineral acids such as hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric and sulfuric acids as well as salts with organic acids such as maleic, vinegar, lemon, oxal, amber, benzoic, wine, fumaric, , almond, ascorbic and malic acids.

6- [D - (-) α-Amino-α- (p-acetoxyphenyl) acetamido] penicillanic acid can be prepared by reacting 6-D-aminopenicillanic acid or a cilyl ester or a salt thereof with an acylating agent of the formula Λ

CH3 - c - O-C y- CH - COOH

NHB

wherein B represents an amino protecting group and removing the amino protecting group. The silyl esters can be prepared by methods described in the literature, e.g. U.S. Patent No. 3,249,622. The silyl ester group can be removed after the acylation reaction by hydrolysis.

Examples of suitable amino protecting groups include t-butoxy-carbonyl, carbobenzyloxy, 2-ethoxycarbonyl-1-methylvinyl and 2-methoxy-carbonyl-1-methylvinyl. A particularly valuable blocking group is a proton which, in the compound of the formula CH3 - C - 0 J ~ \ CH - COC NH2, HCl.

Eg. this group can be easily removed after the acylation by neutralization.

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The particular reaction conditions, e.g. temperature, solvent, reaction time, etc., selected for the acylation are determined by the nature of the acylation method used and are well known to those skilled in the art. In general, it is valuable to add an organic tertiary amine, e.g. triethylamine, Ν, Ν-dimethylaniline, ethylpiperidine, 2,6-lutidine or quinoline, which serve as a proton acceptor or salt-forming agent.

The following examples illustrate the preparation of the intermediate of the invention and its conversion.

Starting materials I. Preparation of D (-) α-amino-α- (p-acetoxyphenyl) acetic acid Process A (in acetic acid as solvent) 203.5 g (1 mole) of D (-) p-hydroxyphenylglycine hydrochloride, 800 ml of acetic acid and 314 g (4 moles) of acetyl chloride was stirred for 48 hours at room temperature. The solid was collected, washed 3 times with acetone (3 x 250 ml) and 2 times with ethanol (2 x 250 ml) and dried at 40 °. Yield 210 g (85.4%). This hydrochloride was dissolved in 3.0 liters of water; the solution was cooled to +5 - 10 ° C and the pH was adjusted to 4.5 with 20% NH 2 OH. The suspension was stirred for 1 hour at 5 ° C and the solid collected, washed twice with water and 2 times with acetone and dried at 40 ° C. Yield 133 g (64% from D (-) p-hydroxyphenylglycine). aD (1% HG1 N / 10 = -104.5.

Process B (in methylene chloride) 4.07 g (0.02 mol) of D (-) p-hydroxyphenylglycine hydrochloride, 30 ml of methylene chloride and 6.28 g (0.08 mol) of acetyl chloride were stirred for 48 hours at room temperature. The solid was collected, washed twice with acetone and 2 times with ethanol. Yield 4.17 g (84.5%).

Analysis calculated Cl = 14.8 (calculated acid)

Process C (in trifluoroacetic acid) 1.67 g (0.01 mole) of D (-) p-hydroxyphenylglycine was added with stirring to 10 ml of trifluoroacetic acid at room temperature. After solution, 1.57 g (0.02 mole) of acetyl chloride was added. After a slight exothermic reaction, a solid came. The suspension was stirred for 1.5 hours at room temperature and the trifluoroacetic acid removed in vacuo. The residual solid was collected, washed with methylene chloride and with ethanol. The D (-) α-amino-α- (p-acetoxyphenylacetic acid) was identical to that prepared by Methods A and B.

Yield: 1.9 g (75%).

Preparation of D (-) α-amino-α- (p-acetoxyphenyl) acetyl-chloride hydrochloride 83.6 g (0.40 mol) of D (-) α-amino-α- (p-acetoxyphenyl) acetic acid and 1, 25 liters of anhydrous methylene chloride were cooled to -5 ° C with stirring. Next, 152 g of phosphorus pentachloride was slowly added followed by 4 ml of dime thyformamide. The mixture was stirred for 4 hours at 0 ° C. The solid was collected, washed with anhydrous methylene chloride and vacuum dried at room temperature.

Yield; 61 g (57.5%)

Analysis. Total chlorine = 27.2% (theoretical 26.9%) III-Preparation of 6- [D- (-) ot-amino-α- (p-acetoxyphenyl) acetamido] -penicilic acid

Procedure A. Anhydrous: process.

15.27 g (0.071 mol) of 6-APA were stirred in 500 ml of anhydrous methylene chloride, 120 ml of methylene chloride distilled off and 11.8 ml of hexamethyldisilane was added. The mixture: stirred and refluxed for 20 hours (after about 10 to about 15 hours all 6-APA had dissolved). The above solution was cooled to 0 ° C and 120 ml of methylene chloride was added followed by 9.5 ml of dimethylaniline and 7 ml of a solution of dimethylaniline hydrochloride in methylene chloride (30%). Next, 20 g (0.0756 mol) of D (-) α-amino-α- (p-acetoxyphenyl) acetyl chloride, hydrochloride was added in small portions (about 1 1/2 hours) at +20 ° C and allowed to stand. ca.

12 to approx. 18 hours at + 5 ° C. Next, 5 ml of methanol was added followed by 240 ml of water. The pH was adjusted to 2.5 with triethylamine and the mixture was filtered through a "Celite" pad; The pH was then checked and the aqueous phase separated, washed twice (2 x 150 ml) with methylene chloride and treated with charcoal. The solution was adjusted to pH 4.5 and concentrated to a volume of ca. 150 ml. The suspension was left for approx. 12 to approx. 18 hours at + 5 ° C and the solid was collected and washed with water and acetone and dried at 40 ° C to give the title compound, substantially without the L - (+) isomer.

Yield = approx. 30% (of an 85-90% pure material). aD (0.5% HCl N / 10) = +205.5 149125 6

Elemental analysis for one trihydrate

Theory Found C 46.85 47.17 H 5.89 5.72 N 9.10 9.02 S 6.93 7.27 H2 O 11.7 11.33 (KF) and 10.77 (TGA) NMR conformity to the presumed structure, lithometric test (against ampicin standard) = 738 µg / ml

Procedure B. Wet process.

10.8 g (0.05 mole) of 6-APA was dissolved in 45 ml of water and 11.7 ml of HCl 6Nj 300 ml of acetone was added and the mixture was cooled to -5 ° C. Next, 7.4 g (0.028 mol) of D (-) α-amino-α- (p-acetoxyphenyl) acetyl chloride hydrochloride was added in small portions and the pH was kept constant at 1.4 - 1.6 by the addition of triethylamine. .

The second fraction of chloride hydrochloride was added at pH 1/2 - 1.4. After 1 hour at -5 ° C, the acetone was removed in vacuo and the pH of the solution was adjusted to 4.3 - 4.5. The solid material was discarded · The mother liquors were seeded and allowed to crystallize approx. 12 to approx. 18 hours at + 5 ° C. RN 1395 was collected, washed thoroughly with a little water and with acetone and dried at 40 ° C. Yield: 2.0 g (9%). The product obtained was identical to that of process A.

Example 3 »

Solutions of 0.5 mg / ml of 6- [D - (-) α-amino-α- (p-acetoxyphenyl) acetamido] penicillanic acid (p-acetoxyampicillin) in normal saline solution and in human serum were prepared. Standard solutions of 0.5 mg / ml of 6- [D - (-) α-amino-α- (p-hydroxyphenyl) acetamido] penicillanic acid (p-hydroxyampicillin) were also prepared in both normal saline and human serum.

All of the above solutions were incubated at 37 ° C with shaking and samples were taken for chromatography after 0, 2, 4, 8 and 24 hours ·. The solutions in an amount of approx. 5 microliters per strip was placed in the form of spots on Whatman No. 1 strips (approximately 12.7 mm) which were dried and developed in a solvent system containing 80 parts of butyl acetate, 15 parts of n-butanol, 40 parts of acetic acid and 24 parts of water. Next, the strips were bioautographed on plates seeded with bacillus subtilus at a pH of 6.0.

The biochromatograms showed that β-acetoxyampicillin is rapidly hydrolyzed to the β-hydroxy form in human serum but appears to be stable in normal saline.

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Example 2 The following solutions were prepared: 0.5 mg / ml of 6- [D- (-) ct-amino-α- (p-acetoxyphenyl) acetamido] penicillanic acid (p-acetoxyampicillin) in brine; 0.5 mg / ml p-acetoxyampicillin in a solution of citrus esterase diluted 10 times with 0.1 M potassium phosphate buffer to maintain a pH of 7.0; and 0.5 mg / ml p-acetoxyampicillin in a 10 mg / ml solution of coarse wheat bran (Shiloh) containing 0.1 'potassium phosphate buffer.

All of the above solutions were incubated at 37 ° C with shaking and samples were taken for chromatography as described in Example 1.

These biochromatograms showed that p-acetoxyampicillin is stable in saline but is rapidly hydrolyzed to the p-hydroxy form with both citrus esterase and bran esterase.

Example 3 The following reaction mixtures were prepared, shaken at 28 ° C and samples were taken after 0, 1/2, 1, 2, 3, 4 and 6 hours as described in Example 1 · 1. 25 mg of degreased bran (wheat bran obtained from Shiloh, treated with acetone and dried), 4.5 ml 0.1 M, pH 6.0, potassium phosphate buffer and 0.5 ml 5 mg / ml 6- [D - (-) α-amino-α- (p-acetoxyphenyl) acetamido ] penicillanic acid (p-acetoxyampicillin) in the same buffer.

2. 25 mg of degreased bran, 4.5 ml of 0.1 Mi pH 7.0, potassium phosphate buffer and 0.5 ml of 5 mg / ml p-acetoxyampicillin in the same buffer.

3. 25 mg degreased bran, 4.5 ml 0.1 M, pH 7.5, potassium phosphate buffer and 0.5 ml 5 mg / ml p-acetoxyampicillin in the same buffer.

4. 50 mg degreased bran, 4.5 ml 0.1 M, pH 6.0, potassium phosphate buffer and 0.5 ml 5 mg / ml p-acetoxyampicillin in the same buffer.

5. 50 mg degreased bran, 4.5 ml 0.1 M, pH 7.0, potassium phosphate buffer and 0.5 ml 5 mg / ml p-acetoxyampicillin in the same buffer.

6. 50 mg degreased bran, 4.5 ml 0.1 M, pH 7.5, potassium phosphate buffer and 0.5 ml 5 mg / ml p-acetoxyampicillin in the same buffer.

The results of the biochromatograms are shown in Table I.

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Tabol I

% conversion to p-hydroxyampicillin Reaction time (hours)

Reaction number 0 1/2 12346 1 12 23 32 64 100 91 95% 2 11 34 45 68 120 75 77% 3 11 32 39 45 68 75 98% 4 9 32 55 59 80 80 77% 5 14 34 64 109 131 104 104 % 6 16 66 66 91 98 116 86%

Optimal results: thus obtained by using reaction mixture # 5, where the total conversion to p-hydroxyampicillin is completed in 2 hours at an enzyme concentration of 10 mg / ml and at a pH of 7.0.

Example 4 The following reaction mixture was prepared containing 50 µg of degreased bran (Shiloh) 2.5 mg of 6- [D - (-) o-amino-α- (p-acetoxyphenyl) acetamido] penicillanic acid (p-acetoxyampicillin) and 5. 0 ml of water. The mixture was shaken at ca. 28 ° C and chromatographically determined after 1, 2 and 3 hours as described in Example 1.

The results of the biochromatograms showed 100% conversion of p-acetoxyampicillin to p-hydroxyampicillin in 3 hours. The pH of the reaction mixture remained constant at ca. 5.7 despite the lack of buffer.

Example 5 The following materials were combined: 20 g of degreased bran (Shiloh) 1.0 g of 6- [D - (-) α-amino-α- (p-acetoxyphenyl) acetamido] penicillanic acid (p-acetoxycampicillin) and 2 liters of a 0.01 M aqueous solution of pH 7.0 potassium phosphate buffer. The resulting mixture was stirred at ca. 28 ° C and samples were taken every hour as described in Example 1.

The biochromatogram studies showed 100% conversion in 3 hours. The mixture was then centrifuged. The supernatant was collected, adjusted to pH 4.0 with hydrochloric acid and lyophilized. The lyophilate was re-examined as described above and found to contain ca. 900 mg of p-hydroxyampicillin.

6.0 g of the lyophilate was slurried in 20 ml of water. To the resulting mixture was added 6N hydrochloric acid to gradually lower the pH to 2.0. Stirring was continued for another 15 minutes, followed by