DK149124B - METHOD OF ANALOGUE FOR PREPARATION OF 6- (D - (-) - ALFA-AMINO-ALFA- (P-ACETOXYPHENYL) ACETAMIDO) PENICILLANIC ACID OR SALTS THEREOF - Google Patents

Description

149124

The present invention relates to an analogous process for the preparation of 6- [D- (-) -α-amino-α- (p-acetoxyphenyl) acetamido] penicillanic acid or a pharmaceutically acceptable salt thereof, substantially free of L- ( +) isomers.

U.S.A. U.S. Patent No. 2,985,648 relates to α-aminobenzylpenicillins. and discloses a general formula for such penicillins which include, among other things, lower alkanoyloxy substituents on the benzene ring. However, this document does not provide further details regarding the compounds thus substituted, nor are specific examples of their preparation given. The document suggests that the α-aminobenzylpenicillins exist in various optical forms, and concrete examples of both D and L forms have been given.

2 U9124 O.S.A. U.S. Patent No. 3,520,876 also refers to the same general formula for o-aminobenzylpenicillins as in U.S.A. No. 2,985,648 and discloses among a list of 21 compounds 6-α-amino-4-acetoxy-benzylpenicillanic acid (or 6-α-amino-α- (p-acetoxy-phenyl) acetamide penicillanic acid. U.S. Patent No. 3,520,876 to any indication that a particular optical isomer of this compound was prepared, nor are there details of its antibacterial activity.

It has now been found that the compound 6 ~ [D - (-) α-amino-α- (p-acetoxy-phenylDacetamido] penicillanic acid possesses particularly valuable antibacterial properties and, according to the invention, the compound 6- [D - (-) α -amino-α- (p-acetoxyphenyl) acetamido] penicillanic acid or a pharmaceutically acceptable salt thereof, substantially free of the L - (+) isomer.

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-benzyl-8-phenethylamine, 1-ephenamine, N, N'-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.

The process of the invention is characterized by reacting a compound of the formula S .CH, V -.-

i-N - L__COOH

Cr (I) or silyl ester or salt thereof with an acylating agent in the form of the D - (-) isomer substantially without the L - (+) isomer of an acid of formula 3 149124

CH3 - C - O-V y- CH - COOH

NHB (II) wherein B represents an amino protecting group and the acid group may be converted to an activated derivative thereof, thus removing the amino protecting group to prepare the desired compound or a pharmaceutically acceptable salt thereof, if desired either before or after removal of the group B in a manner known per se, converts the product in the form of the free acid or silyl ester or salt thereof to the corresponding free acid or pharmaceutically acceptable salt thereof.

In the preparation of the aforementioned novel penicillins, acyls are acylated. a corresponding 6-amino-penicillanic acid of formula (I) or a silyl ester or salt thereof in a manner known per se with the appropriate acylating agent of formula (II).

The compound (I) can, if desired, be converted into a silyl ester or acid addition salt thereof before the acylation reaction. The silyl esters can be prepared by methods described in the literature, e.g. U.S.A. U.S. Patent No. 3,249,622. The silyl ester group can be removed after the acylation reaction by hydrolysis.

Prior to the acylation reaction, the amino group of the acylating agent II can be protected by a conventional amino blocking group B which can be easily removed at the end of the reaction in a manner known per se. Examples of suitable amino protecting or blocking groups include t-butoxycarbonyl, carbobenzyloxy, 2-ethoxycarbonyl-1-methylvinyl and 2-methoxycarbonyl-1-methylvinyl. A particularly valuable blocking group is a proton, which in the compound of the formula s jT \.

ch3 - c - o y- ch - coci NH2, HCl Eg. it can be easily removed by neutralization after the acylation coupling reaction. Of course, other functionally equivalent blocking groups for an amino group can be used and the use of such groups is considered to fall within the scope of the invention.

Acylation of a 6-amino group of a penicillin is a well-known reaction, and any of the functional equivalents of the compound (II) usually used as acylating agents for primary amino groups can be used. Examples of suitable acylating derivatives of the acid of formula (II) include the corresponding acid anhydrides, mixed anhydrides, e.g. alkoxy formic anhydrides, acid halides, acid azides, active esters and active thioesters. The free acid of formula (II) may be coupled to the compound (I) after prior reaction of the free acid with Ν, Ν'-dimethyl chloroforminium chloride or by the use of enzymes or by a Ν, Ν'-carbonyldiimidazole or an N, N'- carbonylditriazole or a carbodiimide reagent, e.g. Ν, Ν-diisopropylcarbodiimide, N, N'-dicyclohexylcarbodiimide or N-cyclohexylcarbodiimide or N-cyclohexyl-N '- (2-morpholinoethyl) carbodiimide or an alkylamine reagent or an isoxazolium salt reagent. Another equivalent of the free acid is a corresponding azolid, i.e. an amide of the corresponding acid whose amide nitrogen is included in a quasi-aromatic five-membered ring containing at least two nitrogen atoms, i.e. imidazole, pyrazole, triazoles, benzimidazole, benzotriazole and substituted derivatives thereof. Another reactive derivative of the phenylglycolic acid of formula (II) is the N-carboxy anhydride (Leuch's anhydride). In this structure, the group activating the carboxyl group also serves to protect the amino group.

A particularly preferred acylating agent is the D - (-) isomer of acid chloride, the hydrochloride of formula CH3 - c - o -C 'V CH - COC1 NH2, HCl which also serves a dual function in carboxyl activation and amino protection. Also mentioned above is the use of enzymes for coupling the free acid with its blocked amino group with compound (I). These processes include the use of an ester, e.g. the methyl ester of the free acid in question with enzymes provided by various microorganisms, e.g. those of T. Takahashi et al. described in J. Amer. Chem. Soc., 94 (11), 4035-4037 (1972). and by T. Nara et al. in J. Antibiotics (Japan) 24 (5), 321-323 (1971) and in West German Patent No. 2,216,113.

The particular reaction conditions, e.g. temperature, solvent, reaction time, etc., selected for the coupling reaction 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 compounds of the invention can be isolated in any of the ways normally used to isolate similar penicillins. Thus, the product can be obtained as the neutral molecule, although this is probably more precisely the zwitterion, or it can be isolated as a salt. Formation of a desired pharmaceutically acceptable carboxylic acid salt or acid addition salt is carried out in a manner known per se, e.g. by reaction of the acid .med. one suitable base or acid.

At the end of the acylation reaction, the product obtained can be converted (before or after removal of the amino protecting group) in a manner known per se to the desired form of the novel compound. Eg. For example, the compound in the form of a silylester or salt thereof can be converted to the free acid or pharmaceutically acceptable salt thereof by removal of the silylester group, e.g. by hydrolysis.

The disclosed pharmaceutically active compounds have potent antibacterial activity which makes them valuable for the treatment of infectious diseases in poultry, animals and humans, caused by numerous Gram-positive and Gram-negative bacteria. The active compounds are also valuable as prophylactic nutritional supplements for animal feed and as agents for the treatment of mastitis in cattle. Furthermore, the preferred compounds have unexpectedly been found to be effectively absorbed by oral administration.

The novel drug-active compounds can be formulated into pharmaceutical compositions containing, in addition to the active ingredient, a pharmaceutically acceptable carrier or diluent. The compounds can be administered both orally and parenterally. The pharmaceutical compositions may be in solid form such as capsules, tablets or emulsions. For the treatment of bacterial infections in humans, the compounds of the invention can be administered parenterally in an amount of from ca. 5 to approx. 200 mg / kg / day in divided doses, e.g. 3-4 times daily. They are administered in dosage units containing e.g. 125, 250 or 500 mg of active ingredient with suitable physiologically acceptable carriers or excipients.

In the following, the preparation of starting materials and the process according to the invention are illustrated.

starting materials

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 mol) 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 14 912 Λ 6 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% HCl 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-acetoxyphenyl) acetic acid was identical to that prepared by processes 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 was cooled to -5 ° C with stirring. Next, 152 g of phosphorus pentachloride was slowly added followed by 4 ml of dimethylformamide. 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% (theoretically 26.9%) In the following example which illustrates the process of the invention, all temperatures are in ° C and 6-aminopenicilic acid is abbreviated as 6-APA.

Example 1 6- [D - (-) α-Amino-α- (p-acetoxyphenyl) acetamido] penicillanic acid or acetoxycampicillin - or RN 1395. __ U9124 7

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 was distilled off and 11.8 ml of hexamethyldisilane was added. The mixture was 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 "Cell" 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 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 consistent with the putative structure.

Iodometric test (versus ampi in Hin's tooth) = 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 6N; 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, 8 149124 and the pH of the solution was adjusted to 4.3 - 4.5. The solid material was collected and 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.

Biological data

Table I shows comparative MIK data for amoxycillin (BL-P1410: p-hydroxy analogue of ampicillin) and p-acetoxyampicillin (RM-1395). Minimum inhibitory concentrations were determined by the double-substrate dilution method using equimolar concentrations of each compound.

.Table II and the associated Figures I and II show comparative blood level data in rats and dogs by administration of amoxycillin (BL-P1410) and p-acetoxyampicillin (RN-1395). Table III summarizes the results of Figs. I and II.

As can be seen from the biological data, p-acetoxyampicillin and amoxycillin have similar MIK properties, but p-acetoxyampicillin has surprisingly higher blood levels.

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