GB2125035A - 6 beta -Halopenicillanic acid derivatives - Google Patents

Abstract

A method is disclosed of preparing in essentially pure form penicillanic acid derivatives of the formula I <IMAGE> in which X stands for chlorine, bromine or iodine, pharmaceutically acceptable, non-toxic salts of the compounds of formula I, and pharmaceutically acceptable, easily hydrolysable esters thereof, including salts of such esters. The 6 beta -halopenicillanic acids of formula I are potent inhibitors of beta - lactamases from a variety of gram- positive and gram-negative bacteria, making the 6 beta -halopenicillanic acids as well as their salts and easily hydrolysable esters valuable in human and veterinary medicine. The derivatives of formula I and the salts and esters thereof are prepared by separating by chromatography or fractional crystallisation 6 beta -halopenicillanic acid or salt or ester thereof from a mixture comprising the 6 beta -halo derivative and the corresponding 6 alpha -halo and/or 6,6-dihalo derivatives. The mixture may be prepared by epimerization of a 6 alpha -halo derivative or by selective reduction with a boranate of a 6,6-dihalo derivative.

Description

SPECIFICATION 6,B-Halopenicillanic acid derivatives This invention relates to a method of preparing in essentially pure form penicillanic acid derivatives of the formula I

in which X stands for chlorine, bromine or iodine, pharmacetically acceptable, non-toxic salts of the compounds of formula I, and pharmaceutically acceptable, easily hydrolyzable esters thereof, including salts of such esters.

In the treatment of bacterial infections it is a serious problem that fi-lactamase producing bacteria occur with increasing frequency. These enzymes inactivate a variety of p-lactarn antibiotics, and it is well recognized that ss-lactamases from both gram-positive and gramnegative bacteria contribute significantly to the resistance of bacteria to p-lactam antibiotics.

It has been found that 6ss-halopenicillanic acids of Formula I are potent inhibitors of ss- lactamases from a variety of gram-positive and gram-negative bacteria. This property makes the 6ss-halopenicillanic acids as well as their salts and easily hydrolyzable esters valuable in human and veterinary medicine because they can protect fi-lactam antibiotics against inactivation when co-administered with these.

In addition to the inhibitory activity against ss-lactamases, the 6-halopenicillanic acids have antibacterial properties (cf. Table I) and are particularly active against Neisseria species.

It has been reported (J. Org. Chem. Val. 43, pp. 3611-3613, 1978; Proc. Natl. Acad. Sci.

U.S.A., Vol. 75, pp. 4145-4149, 1978; U.S. Patent No. 4,180,506 (Dec 25, 1979); Biochem. J., Vol. 177, pp. 365-367, 1979) that mixtures of 6ss- and 6a-bromopenicillanic acids are obtained either on epimerization of the latter or by selective hydrogenation of 6,6dibromopenicillanic acid, the 6fi-brnmo epimer being present in the reaction mixtures in estimated amounts of from 5 to 15%. (In this specification percentages are by weight in respect of solid materials and by volume in respect of liquid materials.) The same literature has reported that such epimeric mixtures act as inhibitors of fi-lactamases, and since pure 6a-bromopenicil- lanic acid has no effect on these enzymes, the inhibitory activity has been attributed to the 6ss- bromo isomer. After the submission of the priority document corresponding to the present application, it has been reported (Tetrahedron Letters No. 48, pp. 4631-4634, Nov 1979) that selective reduction of trimethylsilyl 6,6-dibromopenicillanate with tri-rrbutyltin hydride followed by hydrolysis and salt formation afforded a 30 per cent yield of sodium 6fl-brnmopenicillanate containing less than 5 per cent of the 6a-bromo epimer, but that the major side reaction was overreduction to penicillanic acid.The same literature also describes a similar reduction of the corresponding 6-chloro-6-iodopenicillanate providing a 39 per cent yield of a mixture of 6ss- and 6a-chloropenicillanic acids containing about 25 per cent of the 6a-epimer.

Thus, it is one object of the present invention to provide a method for the preparation of 6ss- halopenicillanic acids of the formula I and salts and easily hydrolyzable esters thereof in an essentially pure, crystalline form, suitable for medical use, these compounds exhibiting strong Plactamase inhibitory activity increasing in the order Cl Br I and also showing antibacterial activity, in particular against Neisseria species.

The salts of the 6ss-halopenicillanic acids are salts with pharmaceuticaly acceptable, non-toxic bases, and among the suitable salts mention may be made of alkali metal salts and alkaline earth metal salts, such as lithium, sodium, potassium, magnesium, and calcium salts, as well as salts with ammonia and suitable non-toxic amines, such as lower alkylamines, e.g. triethylamine, lower alkanolamines, e.g. diethanolamine or triethanolamine, procaine, cycloalkylamines, e.g.

dicyclohexylamine, benzylamines, e.g. N-methylbenzylamine, N-ethylbenzylamine, N-benzyl-ss- phenethylamine, N,N'-dibenzylethylendiamine or dibenzylamine, and heterocyclic amines, e.g.

morpholine, N-ethylpiperidine or the like. Also the salts may be those with e.g. fi-lactam antibiotics or pro-drugs thereof containing a basic group such as pivampicillin, pivmecillinam, becampicillin, bacmecillinam, penethamate, ampicillin or amoxycillin. In some instances, it is preferred to prepare salts which are readily soluble in water, whereas for other purposes, it may be appropriate to prepare an only slightly soluble salt, e.g. in order to obtain a prolonged effect or for preparation of aqueous suspensions.

The esters of the 6ss-halopenicillanic acids which may be prepared by the present method are esters which are readily hydrolyzed in vivo or in vitro. Such esters include acyloxyalkyl, alkoxycarbonyloxyalkyl or aminoacyloxyalkyl esters of the Formula II

in which X has the same meaning as in Formula I, R, is hydrogen, methyl or ethyl, and R2 is a straight or branched alkyl or alkoxy with from 1 to 6 carbon atoms, or an aryl or aryloxy radical, or R2 is a straight or brached aminoalkyl radical with from 1 to 6 carbon atoms, the alkyl moiety optionally being substituted by one or more additional groups, such as hydroxy, mercapto, alkoxy, alkylthio, carbalkoxy, carboxamido, phenyl or hydroxyphenyl. The asterisk in the ester moeity indicates the possibility of an asymmeric carbon atom.

Among the above esters the following are preferred: alkanoyloxymethyl with from 3 to 8 carbon atoms, 1 -(alkanoyloxy)ethyl with from 4 to 9 carbon atoms, alkoxycarbonyloxymethyl with from 3 to 6 carbon atoms, 1 -(alkoxycarbonyloxy)ethyl with from 4 to 7 carbon atoms, and a-aminoalkanolyloxymethyl with from 2 to 6 carbon atoms.

Other preferred esters are lactonyl esters, e.g. 3-phthalidyl, 4-crotonolactonyl or y-butyrolacton-4- yl esters.

Also, methoxymethyl, cyanomethyl, or mono- or dialkyl substituted aminoalkyl esters, e.g. 2dimethylaminoethyl, 2-diethylaminoethyl, or 3-dimethylaminopropyl esters may be prepared.

In particular, such esters are preferred which are well-absorbed upon oral administration and during or after the absorption are hydrolysed to the free acids of Formula I.

Esters which contain an amino group in the ester moiety can be prepared and used in the form of their salts with pharmaceutically acceptable, non-toxic inorganic or organic acids.

Examples of suitable inorganic and organic acids include, but are not limited to, the following: hydrohalide acids, e.g. hydrochloric, hydrobromic and hydriodic acid, phosphoric acid, sulphuric acid, nitric acid, p-toluenesulphonic acid, methanesulphonic acid, formic acid, acetic acid, propionic acid, citric acid, tartaric acid, maleic acid, pamoic acid, p-(dipropylsulfamyl)benzoic acid (probenecid), and phenoxymethylpenicillin or other acidic fi-lactam antibiotics. As mentioned above, easily soluble salts may be preferred for different purposes.

The present invention relates to methods for the preparation of the above compounds and according to the present invention there is provided a method for the preparation of an essentially pure compound of the formula I

in which X stands for chlorine, bromine or iodine, and salts and easily hydrolyzable esters thereof and salts of such esters;; wherein a mixture comprising a 6ss-halopenicillanic acid, together with the corresponding 6ahalo and/or 6,6-dihalo derivatives, or salts or esters thereof, is prepared by epimerization of a 6a-halopenicillanic acid or a salt or ester thereof in aqueous or organic solution in the presence of a base, or by reducing a 6,6-dihalopenicillanic acid or a salt or ester thereof with an alkali metal boranate or a tetraalkyammonium boranate, or with sodium cyanoborohydride in an organic solvent, and wherein the 6ss-halo-penicillanic acid or salt or ester thereof is separated from the corresponding 6a-halo and/or 6,6-dihalo derivatives by chromatography or fractional crystallization.

In one embodiment of this method, a mixture of epimeric 6-halopenicillanic acids is produced by aqueous equilibration of a salt of a salt of a 6y-halopenicillanic acid at 30-40"C and a moderately basic pH-value (8-10) for a period of 6-48 hours preferably at 30-32"C and pH 9.0-9.1 for 20-24 hours, the pH in the reaction mixture being held constant by addition of dilute aqueous base via an automatic titrator. The amount of 6halo epimer present in the resulting mixtures decreases in the order I > Br > Cl, but the yields of the fi-epimers are at least twice as high as those described in the literature for the epimerization of 6a-bromopenicillanic acid or by using said method for the epimerization of the corresponding 6a-chloro and 6a-iodo acids.

The epimeric mixtures of 6-chloro, 6-bromo-, or 6-iodopenicillanic acids thus obtained can be separated by column chromatography on silica gel using as developing solvent an appropriate mixture of organic solvents, e.g. ether-petroleum ether, ethyl acetate-petroleum ether, chloroform-benzene or ethyl acetate-cyclohexane, these solvent mixtures preferably containing a low percentage (0.1 -0.5%) of a carboxylic acid, such as formic acid or acetic acid.A highly efficient developing solvent for the separation of the epimeric mixtures referred to above by dry column chromatography on silica gel is e.g. ether-petroleum ether-formic acid, 70:30:0.1 Hereby the more polar 6ss-halopenicillanic acids are completely separated from their less polar 6a-epimers and, following a usual work-up procedure of the eluates, obtained in a pure crystalline state, either in the form of the free acids or as the corresponding potassium or sodium salts. The purity of the crystalline 6ss-halopenicillanic acids thus obtained as well as the respective potassium and sodium salts is at least 99 per cent, as determined by thin-layer and gas-liquid chromatography.

In another embodiment of the method 6,6-dihalopenicillanic acids or salts thereof can be selectively reduced by treatment with alkali metal boranates or tetraalkylammonium boranates, e.g. sodium borohydride, potassium borohydride, sodium cyanoborohydride, tetrabutylammonium boranate, or cetyl trimethylammonium boranate to afford favourably high yields ( > 50% of the free 6ss-halopenicillanic acids. The reactions are performed in an appropriate organic solvent, e.g. dimethyl sulphoxide, dimethylformamide, ethyl acetate or methylene chloride, and at temperatures between 0 and 80"C, preferably at room temperature.The 6ss-halopenicillanic acids can be separated from the corresponding 6a-halo- and/or 6,6-dihalopenicillanic acids present in the crude reaction mixtures by column chromatography as referred to above or by fractional crystallization known to the man skilled in the art.

According to a further embodiment of the present method; the esters of the halo acids of formula I can be prepared by epimerization of the corresponding 6a-halopenicillanic acid salts or esters in an appropriate solvent. The esters are conveniently epimerized in an organic solvent, e.g. methylene chloride, chloroform or dimethylformamide, in the presence of an organic base, e.g. 1 ,5-diazabicyclo[4.3.0] non-5-ene or triethylamine, and at temperatures between 10 C and room temperature. The epimeric mixtures of the corresponding 6 -halopenicillanic acid esters thus obtained are separated by column chromatography under similar conditions as mentioned above to afford the pure GP-halo isomers.

In a further embodiment of the method, the esters can be obtained by selective reduction of 6,6-dihalopenicillanic acid esters with alkali metal boranates or tetraalkylammonium boranates, similar to the procedure referred to before. The halo esters thus obtained are separated from minor amounts of the corresponding 6a-epimers and/or unreacted starting materials by column chromatography as described above.

The 6ss-halopenicillanic acids of formula I or their salts can be converted into the corresponding esters by well known esterification processes, and vice versa such esters can be cleaved chemically or enzymatically to give the corresponding free acids of formula I or salts thereof under conditions which do not result in any appreciable destruction of the remaining part of the molecule.

As it has been previously stated, the 6ss-halopenicillanic acids are potentiators of fi-lactamase sensitive antibiotics and may by themselves be useful in combatting some specific bacterial infections.

More specifically, the antibacterial spectra of the pure 6ss-halopenicillanic acids will appear from Table I below.

Table I Antibacterial spectra a) of 6ss-bromopenicillanic acid (A), 6ss- chloropenicillanic acid (B) and 6ss-iodopenicillanic acid (C) C50 ( g/ml) Organism A B C Staph.aureus CJ9 32 40 63 Dipl.pneumoniae EA 1.6 5.0 5.0 Strep.pyogenes EC 6.3 6.3 5.0 Strep.faecalis El3 100 100 100 Coryneb.xerosis FF 5.0 5.0 16 Bacillus subt. KA2 5.0 7.9 13 Pseud. aeruginosa BA2 100 100 100 Alcaligenes faecalis GA 2.0 2.5 10 Escherichia coli HA2 50 50 100 Escherichia coli HA58 (RTEM) 100 100 100 Kleb. pneumoniae HE 63 63 100 Enterobact.aerogenes HC7A 63 63 100 Proteus vulg.HJ 40 40 100 Salm.typhimurium HL2 100 100 100 Shigella dysenteriae HR 50 40 100 Neisseria gonorrhoeae DA2 0.25 0.20 0.79 Neisseria meningitidis DB 0.79 0.63 1.6 Haemophilus influenzae IX3 32 20 100 a) Determined by serial dilution in fluid medium inoculum 106 CFUb) (gram-positive organism) or 104 CFU (gram-negative organism) b) CFU = colony forming units In Table II is shown the in vitro activity against fi-lactamase producing bacterial strains of selected fi-lactam antibiotics in 1:1 combinations with the fi-halopenicillanic acids. These data indicate that, in combination with 6ss-halopenicillanic acids, benzylpenicillin and ampicillin are highly active against otherwise resistant strains of Staphylococcus aureus.A similar synergistic effect against strains of Klebsiella pneumoniae, Proteus mirabilis, and Escherichia coli is shown by combinations of ampicillin as well as mecillinam with the halo acids.

Table II Susceptibility# of ss-lactamase producing bacterial strains to selected ss-lactam antibiotics in 1:1 combinations with 6ssbromopenicillanic acid (A), 6ss-chloropenicillanic acid (B). and 6ss-iodopenicillanic acid (C).

IC50 ( g/mL) Staphylococcus Staphylococcus Klebsiella Proteus Escherischia Antibiotic aureus aureus pneumoniae mirabilis coli CJ9 CJ145 HE7 HJ28 HA58 (RTEM) A 13 10 > 50 > 50 > 50 B 16 13 > 50 > 50 > 50 C 20 6.3 > 100 > 100 > 100 Benzylpenicillin > 100 10 > 100 > 100 > 100 Benzylpenicillin + A 0.5 + 0.5 0.2 + 0.2 NDb) ND ND Benzylpenicillin + B 1.3 + 1.3 0.4 + 0.4 ND ND ND Benzylpenicillin + C 0.79 + 0.79 0.08 + 0.08 7.9 + 7.9 2.5 + 2.5 7.9 + 7.9 Ampicillin 10 1.6 > 50 > 50 > 100 Ampicillin + A 0.4 + 0.4 0.16 + 0.16 1.3 + 1.3 5.0 + 5.0 4.0 + 4.0 Ampicillin + B 1.6 + 1.6 0.32 + 0.32 ND ND ND Ampicillin + C 0.79 + 0.79 0.16 + 0.16 2.5 + 2.5 2.5 + 2.5 3.2 + 3.2 Mecillinam > 50 > 50 > 25 6.3 1.3 Mecillinam + A 4.0 + 4.0 3.2 + 3.2 5.0 + 5.0 1.3 + 1.3 0.16 + 0.16 Mecillinam + B ND 4.0 + 40 ND ND 0.05 + 0.05 Mecillinam + C 10 + 10 3.2 + 3.2 0.79 + 0.79 0.63 + 0.63 0.13 + 0.13 a) Serial dilutions in agar medium, inoculum 105 CFU (gram-positive organisms) or 104 CFU (gram-negative organisms) b) ND = not determined c) Corresponding to an IC50 of 0.4 g/ml for mecillinam in this test The compounds of formula I prepared by the present method may be used to provide pharmaceutical compositions for use in the treatment of infectious diseases and adapted for enteral, parenteral, intramammary or topical use for the treatment of infections in mammals including humans.

The invention will be further described in the following Examples which are not to be construed as limiting the invention.

Example 1 Potassium 6ss-bromopenicillanate A solution of potassium 6a-bromopenicillanate (7.64 g, mmol) in 0.04 M aqueous disodium hydrogen phosphate (800 ml) was incubated for 72 hours at 30"C. According to an NMRspectrum (D20) of a freeze-dried 5 ml sample, the epimeric mixture contained 10-12% of the 6fl-brnmo compound.

After addition of sodium chloride (160 9), the mixture was stirred at 0 C under a layer of ether (250 ml), and the ph of the aqueous phase was adjusted to 3 with 4 N aqueous hydrochloride. The organic layer was separated, the aqueous phase was re-extracted with ether (100 ml), and the combined ethereal extracts were washed with saturated aqueous sodium chloride (10 ml), dried, and concentrated to about 40 ml at reduced pressure. The concentrated solution was subjected to dry column chromatography on silica gel (Silica Woelm TSC, Woelm Pharma, Eschwege, Western Germany).The column (f5.6 cm, length 46 cm) was developed with ether-petroleum ether-formic acid, 70:30:0.1 (1200 ml), fractions a 2 cm were scraped out, suspended in ethyl acetate (10 ml/fraction), and samples of the supernatants were examined by thin-layer chromatography using the above mentioned solvent system. Fractions containing the pure, more polar 6-brnmopenicillanic acid were combined and eluted with ethyl acetate. The resulting ethyl acetate eluate was concentrated to about 50 ml at reduced pressure and washed throughly with water (6 X 5 ml) to remove the major amount of formic acid. To the organic layer was added water; (40 ml), and the apparent pH of the mixture was adjusted to 7.2 by addition of 0.5 M aqueous potassium bicarbonate.The aqueous layer was separated and freeze-dried to afford 0.54 g of pure potassium GP-bromopenicillanate as a colourless amorphous powder which crystallized from n-butanol, [a]2D0+ 240 (c = 0.2, H20).

The detailed FT proton NMR-spectrum (Fig. 1) showed signals at 8 = 1.47 (s, 3H; CH3-2a), 1.59 (s, 3H; CH3-2ss), 4.27 (s, 1 H; CH-3), 5.52 and 5.58 (doublets, J = 4 Hz, 2H; CH-5a and CH-6a, confer Fig. 1a) ppm.

Instrument JEOL FX 100. Concentration 50 mg per ml. All data converted to tetramethylsilane as 0.00 ppm Sscale.

Example 2 Potassium 6,6-chloropenicillanate A solution of potasium 6a-chloropenicillanate (13.14 g, 48 mmol) in 0.04 M aqueous disodium hydrogen phosphate (1600 ml) was incubated for 96 hours at 30"C to yield, as revealed by an NMR-spectrum (D20) of a freeze-dried 5 ml sample of the reaction mixture, about 5-6% of 6ss-chloropenicillanic acid in admixture with the starting material.

To the reaction -mixture was added sodium chloride (320 g) and ether (400 ml), and the pH of the aqueous phase was adjusted to 3 by addition of 4 N aqueous hydrochloric acid at 0 C with stirring. The organic phase was separated, the aqueous layer was re-extracted with ether (200 ml), and the combined ethereal extracts were washed with saturated aqueous sodium chloride (20 ml), dried, and concentrated to about 50 ml at reduced pressure. The concentrate was subjected to dry column chromatography on silica gel (as described in Example 1 for the separation of the corresponding 6-epimeric bromopenicillanic acids).Fractions containing the pure 6ss-chloropenicillanic acid were eluted with ethyl acetate, and the resulting solution was worked up in a similar manner as described in Example 1 to afford 0.68 g of potassium 6Pchloropenicillanate as an amorphous powder which crystallized from n-butanol.

The NMR-spectrum (D20) showed signals at 8 = 1.48 (s, 3H; CH3-2a), 1.58 (s, 3H; CH3-2ss), 4.27 (s, 1H, CH-3), 5.43 and 5.63 (2d, J = 4 Hz, 2H; CH-5a and CH-6a) ppm.

Tetramethylsilane was used as external reference.

Example 3 Potassium 6ssiodopenicillanate By following the procedure of Example 1, but substituting potassium 6a-iodopenicillanate for the potassium 6a-bromopenicillanate, the desired compound was obtained as an amorphous product which crystallized from n-butanol.

Example 4 Potassium 6fi-bromopenicillanate A solution of potassium 6a-bromopenicillanate (15.28 g, 48 mmol) in water (320 ml) was adjusted to pH 9.0 with 1 N aqueous sodium hydroxide and stirred for 24 hours at 30 C During the reaction of pH of 9.0 was maintained in the solution by addition of 1 N aqueous sodium hydroxide via an automatic titrator. An NMR spectrum (D20) obtained from freeze-dried 1 ml sample of the solution indicated the presence of approximately 25% of the 6ss-bromo compound in the epimeric mixture formed.

The mixture was worked up and purified by column chromatography as described in Example 1 to yield crystalline potassium 6/?-bromopenicillanate identical with the product prepared in Example 1; [aTh0+ 253 (c = 0.5, 1 M phosphate buffer, pH 7).

Calculated for C8HgBrKNO3S: C, 30.19; H, 2.85; Br, 25.11; N, 4.40; S, 10.08%. Found: C, 30.16; H, 2.95; Br, 25.28; N, 4.33; S, 10.07%.

Example 5 Potassium 6fi-chlqropeniclllanate By following the procedure of Example 4, but substituting potassium 6a-chloroperlicillanate for the potassium 6a-bromopenicillanate, an epimeric mixture containing abqut 15% of the 6ss- chloro compound. was obtained, as revealed by an NMR spectrum D20) of a freeze-dried sample of the reaction mixture.

The crystalline title compound was obtained using a similar work-up and chromatography method as described in Example 1; [&alpha;]D20= 243e (c = 0.5, 1 M phosphate buffer pH 7).

Example 6 Potassium 6ss-iodopenicillanate A. Acetoxymethyl 6-diazopenicillanate To a stirred solution of acetoxymethyl 6ss-aminopenicillanate (5.77 g, 20 mmol) and sodium nitrate (2.76 g 40 mmol) in a mixture of dichloromethane (120 ml) and water (120 ml) was added dropwise at 0-3 C 4 N aqueous sulphuric acid (7 ml).

After stirring at the low temperature for a further 30 minutes, the organic phase was separated, dried (Na2SO4), and concentrated to approximately 30 ml at reduced pressure.

This concentrate was used immediately in the following step.

B. Acetoxymethyl 6&alpha;-iodopenicillanate The concentrated solution of acetoxymethyl 6-diazopenicillanate from step A above was diluted with ice-cold acetone (180 ml), and to the stirred mixture was added dropwise at 0-3 C a solution of sodium iodide (9.0 g, 60 mmol) and 57% hydroiodic acid (7.4 ml) in water (1 5 ml). After stirring at 0-3 C for a further 25 minutes, the mixture was treated with solid sodium bicarbonate (10 g) and filtered. The filtrate was diluted with ethyl acetate (150 ml), acetone was removed at reduced pressure, and the remaining organic layer was separated, washed with 0.5 M aqueous sodium thiosulphate (2 X 100 ml), dried (Na2504). and concentrated to about 10 ml at reduced pressure.

This concentrated solution was subjected to dry column chromatography on silica gel (etherpetroleum ether, 4:6) to yield pure acetoxymethyl 6a-iodopenicillanate as a slightly yellowish oil.

The NMR spectrum (CDCl3) showed signals at 8 = 1.48 (s, 3H; CH3-2a), 1.63 (s, 3H; CH3-2ss), 1.63 (s, H; CH3-2ss, 2.11 (s, 3H; COCH3), 4.56 (s, 1H; CH-3), 4.99 (d, J= 15 Hz, 1 H; CH-.6), 5.45 (d, 4 = 1.5 Hz, 1 H; CH-5), and 5.79 (ABq, J = 5.5 Hz, 2H; OCH2O) ppm.

Tetramethylsilane was used as internal reference.

C. Potassium 6a-iodopenicillanate To a solution of acetoxymethyl 6a-iodopenicillanate (2.0 g, 5 mmol) in 70% aqueous methanol (50 ml) was added 4 N aqueous hydrochloric acid (1.5 ml), and, after protection from light, the mixture was stirred at room temperature for 3 days. The mixture was poured into water (150 ml), extracted twice with ether (100 ml), and the combined ethereal extracts were washed with water (2 X 25 ml). To the organic layer was added fresh water (40 ml), and the pH in the aqueous phase was adjusted to 6.8 by addition of 1 M potassium bicarbonate with stirring. The aqueous phase was separated and freeze-dried to give potassium 6a-iodopenicillanate as an amorphous powder, which crystallized from acetone.

The NMR-spectrum (D20) showed signals at 6= 1.46 (s, 3H; CH3-2a), 1.57 (s, 3H; CH3-2ss), 4.30 (s, 1 H; CH-3), 5.24 (d, J = 1.5 Hz, 1 H; CH-6), and 5.46 (d, J = 1.5 Hz, 1 H; CH-5) ppm.

D. Potassium 6ss-iodopenicillanate A solution of potassium 6a-iodopenicillanate (3.65 g, 10 mmol) in water (200 ml) was stirred at 30"C for 20 hours, a constant pH of 9.0 being maintained in the reaction mixture by additions of 0.1 N sodium hydroxide via an automatic titrator. According to the NMR spectrum (D20) of a freeze-dried 1 ml-sample, the epimeric mixture of 6-iodopenicillanates thus formed contained approximately 30% of the 6ss-iodo compound.

To the mixture was added ether (150 ml), and the pH of the aqueous phase was adjusted to 3.0 by addition of 4 N hydrochloric acid with stirring. The organic phase was separated, the aqueous phase re-extracted with ether (50 ml), and the combined ethereal extracts were washed with saturated aqueous sodium chloride (2 X 20 ml), dried (MgSO4), and concentrated to about 6-8 ml at reduced pressure. The concentrate thus obtained was subjected to dry column chromatography on silica gel (ether-petroleum ether-formic acid, 70:30:0.1), and, analogously to the procedure described in Example 1 for the separation and isolation of the corresponding 6ss- and 6a-bromo compounds, potassium 6ss-iodopenicillanate was obtained in-a crystalline state; [ayD0+ 260 (c = 0.5, 1 M phosphate buffer pH 7).

The NMR spectrum (D20) showed signals at S = 1.49 (s, 3H; CH3-2a), 1.65 (s, 3H; CH3-2ss), 4.29 (s, 1 H; CH-3), 5.42 and 5.80 (2d, J = 3.5 Hz, 2H; CH-5, and CH-6) ppm.

Calculated for C8HgIKNO3S: C, 26.31; H, 2.48; I, 34.75; N, 3.84; S, 8.78%. Found C, 26.51; H, 2.58; I, 34.91; N, 3.75; S, 8.80%.

Example 7 Pivaloyloxymeth yl 6fi-brnmopen icillana te To a solution of potassium 6ss-bromopenicillanate (0.64 g, 2 mmol) in dimethylformamide (15 ml) was added chloromethyl pivalate (0.27 ml, 2.5 mmol), and the mixture was stirred for 16 hours at room temperature. After dilution with ethyl acetate (45 ml), the mixture was washed with water (4 X 10 ml), dried (MgSO4), and evaporated in vacuo. The residual oil crystallized from diisopropyl ether to give the title compound, melting point: 67-68 C.

The NMR spectrum (CDCl3) showed signals at 6 = 1.23 (s, 9H; C(CH3)3), 1.51 (s, 3H; CH3-2a), 1.68 (s, 3H; CH3-2ss), 4.54 (s, 1 H; CH-3), 5.32 and 5.57 (2 d, J = 4 Hz, 2H; CH-5 and CH-6), and 5.82 (ABq, J = 5.5 Hz, 2H; OCH2O) ppm.

Tetramethylsilane was used as internal reference.

Example 8 Pivaloyloxymethyl 6fi-chloropenicillanate By substituting potassium 6ss-chloropenicillanate for the potassium 6ss-bromopenicillanate in the procedure of Example 7, pivaloyloxymethyl 6ss-chloropenicillanate was obtained as colourless crystals, melting point: 68-69 C.

The NMR spectrum (CDCl3) showed signals at # = 1.22 (s, 9H; C(CH3)3), 1.51 (s, 3H; CH3-2a), 1.66e(s, 3H; CH3-2ss), 4.53 (s, 1 H; CH-3), 5.22 and 5.60 (2d, J = 4 Hz, 2H; CH-5 and CH-6), and 5.82 (ABq, J = 5.5 Hz, 2H; OCH20) ppm.

Tetramethylsilane was used as internal reference.

Example 9 Pivaloyloxymethyl 6ss-iodopenicillanate A. Pivaloyloxymethyl 6-diazopenicillanate A stirred mixture of pivaloyloxymethyl 6ss-aminopenicillanate (3.30 g, 10 mmol) and sodium nitrite (1.38 g, 20 mmol) in methylene chloride (120 ml) and water (120 ml) was cooled to 0 C and treated with 2 N aqueous sulphuric acid (7.5 ml) for 40 minutes. The organic phase was separated, dried (Na2504), and concentrated at reduced pressure to about 30 ml. The concentrate was used immediately for the subsequent transformation.

B. Pivaloyloxymethyl 6&alpha;-iodopenicillanate The concentrate of pivaloyloxymethyl 6-diazopenicillanate from step A above was diluted with acetone (120 ml), cooled to 0 C, and to the stirred mixture was added a cold solution 67% aqueous hydroiodic acid (3.5 ml) and sodium iodide (4.5 g) in water (20 ml). After stirring for a further 20 minutes at the low temperature, the mixture was treated with solid sodium bicarbonate, filtered, and evaporated. The residue obtained was taken up in ethyl acetate (100 ml) and washed with 5% aqueous sodium thiosulphate (2 x 75 ml). The organic phase was separated, dried (MgSO4), and evaporated in vacuo.The residual oil was purified by column chromatography on silica gel using ether-petroleum ether, 30:70, as the eluant to yield pure pivaloyloxymethyl 6a-iodopenicillanate as colourless crystals from diisopropyl ether, melting point : 63-64 C.

C. Pivaloyloxymethyl 6ss-iodopenicillanate To a stirred solution of pivaloyloxymethyl 6a-iodopenicillanate (0.88 g, 2 mmol) in dry mothylene chloride (20 ml) added at - 5 C a 1 M solution of 1,5-diazabicyclo[4.3.0]non5-ene (DBN) in dry methylene chloride (2 ml). The mixture was stirred at 0 C for 20 minutes, sliken with 1 N aqueous acetic acid (2 mi), diluted with methylene chloride (20 ml), washed with water (2 x 10 ml), dried (Na2SO4), and evaporated in vacuo to a dark oil.The residue was purified by column chromatography on silica gel using ether-petroleum ether, 30:70, as the eiuant to afford pivaloyloxymethyl 6ss-iodopenicillanate as a slightly yellowish oil which ctystallized from diisopropyl ether, melting point: 78-79 C.

The NMR spectrum (CDCl3) showed signals at # = 1.23 (s, 9H; C(CH3)3), 1.49 (s, 3H; CH3-2a, 1.70 (s, 3H; CH3-2ss), 4.55 (s, 1 H; CH-3), 5.38 and 5.62 (2d, J = 4 Hz, 2H; CH-5 and CH-6). and 5.81 (ABq, J = 5.5 Hz, 2H; OCH20) ppm. Tetramethylsilane was used as internal reference.

Example 10 Acetoxymethyl 6/3-bromopenicillanate Chloromethyl acetate (0.11 ml, 1.2 mmol) was added to a solution of potassium 6ss- cromopenicillanate (0.32 g, 1 mmol) in dimethylformamide (5 ml), and -ihs mixture was stirred for 1 6 hours at room temperature in a dark room. After dilution with ether (20 ml), the mixture was washed with water (4 X 5 ml), dried (MgSO4), and evaporated in vacuo to yield the title compound as a yellowish oil.

The NMR spectrum (CDCl3) showed signals at S = 1.49 (s, 3H; CH3-2&alpha;), 1.68 (s, 3H ; CH3-2ss), 2.11 (s, 3H; COCH3), 4.54 (s, 1 H; CH-3), 5.33 and 5.59 (2d, J = 4 Hz, 2H; CH-5a and CH-6&alpha;). and 5.82 (ABq, J = 5.5 Hz, 2H; OCH20) ppm. Tetramethylsilane was used as internal reference.

Example 11 Acetoxymethyl 6ss-iodopenicillanate Following the procedure of Example 10, but substituting potassium 6ss-iodopenicillanate for the potassium 6ss-iodopenicillanate the title compound was obtained as a yellowish oil.

The NMR spectrum (CDCI3) showed signals at 8 = 1.50 (s, 3H; CH3-2a), 1.70 (s, 3H; CH3-2ss), 2.12 (s, 3H; COCH3), 4.55 (s, 1H;, CH-3), 5.39 and 5.63 (2d, J = 3.5 Hz, 2H; OH-S and CH-6a), and 5.83 (ABq, J = 5.5 Hz 2H; OCH2O) ppm. Tetramethylsilane was used as intomal reference.

Examples 12-14 6ss-Halopenicillanic acids The he crystalline 6/?-halopenicillanic acids listed in Table Ill below could be obtained as follows: (a) By concentrations at reduced pressure of the ethyl acetate solutions containing the pure /?-h3lo compounds obtained after separation from the corresponding 6a-epimers by dry column chromatography on silica gel (as described in Example 1).

(b) By liberation from aqueous solutions of the corresponding potassium salts under a layer of ether or ethyl acetate at pH 3 followed by separation of the organic phase, drying, and crystallization from ether-diisopropylether or ethyl acetate-hexane.

Table 111

[a]2D0 1H-NMR data (8/ppm; CD3CN) Example X (e = 0.5, CHCl3) CH5 and CH6 12 Br + 272 5.48 and 5.54, 2d, J = 4.0 Hz 13 Cl + 264 5.38 and 5.58, 2d, J = 4.0 Hz 14 1 +276' 5.35 and 5.74, 2d, J =4.0 Hz The above acids decompose at about 80-100"C, therefore a welldefined melting point cannot be determined.

Example 15 Sodium 6fi-bromopenicillanate A. Tetrabutylammonium 6, 6-dibromopenicillanate To a solution of tetrabutylammonium hydrogen sulphate (34 g, 0.1 mol) in water (50 ml), methylene chloride (100 ml) was added, followed by 2N sodium hydroxide (50 ml). To the stirred mixture was added 6,6-dibromopenicillanic acid (36 g, 0.1 mol) and the pH adjusted to 7.0 with 2N sodium hydroxide. The organic layer was separated, and the aqueous pahse was re-extracted with methylene chloride (50 ml). After drying of the combined organic phases, ethyl acetate (500 ml) was added, and the solution was concentrated to about 300 ml in vacuo.

B. Sodium 6fi-bromopenicillanate To the above solution of tetrabutylammonium 6,6-dibromopenicillanate, tetrabutylammonium boranate (24.9 g, 0.1 mol) was added in one portion with stirring. After about 30 minutes, the temperature in the mixture had raised to 45-50"C, whereafter it slowly decreased. After standing for 1 hour, the solution was diluted with ether (300 ml), water (300 ml) was added, and the pH was adjusted to 3 with hydrochloric acid. The organic phase was separated and washed with water. Fresh water (50 ml) was added to the organic phase, and the pH was adjusted to 6.8 with aqueous potassium bicarbonate.The aqueous layer was separated, and water removed azeotropically with n-butanol in vacuo to yield a crystalline mixture of the potassium salts of 6ss-bromo-, 6a-bromo-, and 6,6-dibromopeniciilanic acid in an approximate ratio of 65:25:10.

From the aqueous solution of the above salts, the free acids were liberated at pH 3 (dilute hydrochloric acid) under a layer of ether, and the resulting mixture was separated by column chromatography in a similar way as described in Example 1 to yield, after salt formation with 0.5 M sodium bicarbonate and removal of water by azeotropic distillation with nbutanol, crystalline sodium 6/?-bromopenicillanate; [a]2D0 + 266 (c = 0.5, 1 M phosphate buffer pH 7).

Calculated for C8HgBrNNaO3S: C, 31.80; H, 3.00; Br, 26.45; N, 4.64; S, 10.61%. Found: C, 31.85; H, 3.04; Br, 26.53; N, 4.56; S, 10.72%.

Example 16 6ssBromopenicillanic acid To a stirred suspension of potassium 6,6-dibromopenicillanate (11.91 g, 30 mmol) in dimethylformamide (30 ml) was added sodium borohydride (1.14 9, 30 mmol). In the course of approximately 30 minutes, the temperature in the reaction mixture rose to 50"C, whereafter it slowly decreased to normal. After stirring at room temperature for 20 hours, water (100 ml) and ether (100 ml) were added, and the pH of the mixture was adjusted to 3 with dilute hydrochloric acid. The organic layer was separated, the aqueous layer was extracted with ether (25 ml), and the combined organic extracts were washed with water (25 ml). To the organic phase was added fresh water (25 ml), and the pH of the aqueous phase was adjusted to 7 by addition of 1 M potassium bicarbonate with stirring.The aqueous layer was separated, and the water removed azeotropically by distillation with nbutanol in vacuo to give a crystalline mixture of the potassium salts of 6ss- and 6a-bromopenicillanic acid in an approximate ratio of 55:45, as indicated by NMR spectroscopy.

The above potassium salts were dissolved in water (5 ml/g salt), and the pH of the aqueous phase was adjusted to 3 with 4 N hydrochloric acid under a layer of ethyl acetate 5 ml/g salt).

The organic phase was separated , washed with water, dried, and diluted with an equal volume of hexane. Seeding of the resulting solution followed by concentration at reduced pressure to about half the volume afforded crystalline 6ss-bromopenicillanic acid which was filtered off, washed with ethyl acetate-hexane (1:1), and dried. Recystallization from ether-diisopropyl ether gave the analytical sample, [a]2D0 + 268 (c = 0.5, CHCI3) Calculated for C8H,OBrNO3S: C, 34.30; H, 3.60; Br, 28.53; N, 5.00; S 11.45%. Found C, 34.47; H, 3.81; Br. 28.66; N, 4.99; S, 11.43%.

Example 17 6ssBromopenicillanic acid A. Dicyclohexylammoniurn 6,8-bromopenicillanate To a solution of 6,6-dibromopenicillanic acid (10.8 g, 30 mmol) in dimethylsulphoxide (75 ml) was added sodium cyanoborohydride (2.1 g; 90% pure), and the mixture was stirred until a clear solution was obtained (about 30 minutes). After standing for 72 hours, the mixture was diluted with water (75 ml) to precipitate unreacted starting material as dimethylsulphoxide solvate (C8H9Br2NOaS, C2H ;OS). The crystals were filtered off, washed with water and dried.The filtrate was extracted with methylene chloride (4 X 25 ml), and the combined extracts were washed with water (50 ml), dried (Na2504), and concentrated at reduced pressure to about half the volume. After addition of dicyclohexylamine (2.5 ml) and acetone (50 ml), the mixture was further concentrated to about 25 ml. Crystallization was induced by scratching, and, after standing for 1 hour at room temperature, the pure dicyclohexylammonium 6ss-bromopenicilla- nate was filtered off, washed with acetone, and dried. The compound exhibited no well-defined melting point, after darkening at about 170"C, it decomposed at 280-290"C.

B. 6ss-Bromopenicillanic acid A stirred suspension of dicyclohexylammonium 6ss-beomopenicillanate in ethylaetate-water (1:1) (20 ml/g salt) was adjusted to pH 3 with 4 N hydrochloric acid. Precipitated dicyclohexylammonium chloride was filtered off, and the organic layer was separated, washed twice with water, and dried. Addition of an equal volume of hexane followed by concentration of the solution at reduced pressure yielded pure, crystalline 6ss-b;omopenicillanic acid, identical with the compound described in Examples 12 and 16.

Example 18 Sodium 6ssiodopenicillanate A. 6, 6-DiXodopenicillanic acid morpholine salt To a stirred solution of 6-aminopenicillanic acid (110 g, ""0.5 mol) in a mixture of 5 N sulphuric acid (400 ml) and methylene chloride (1.5 litre) were added dropwise and simultaneously at 0 C 2.5 M aqueous sodium nitrite (340 ml) and 0.5 M methanolic iodine (1 litre). After the addition was finished, the cooling bath was removed, and stirring of the mixture was continued for 1 hour. The organic layer was separated, and the aqueous phase was extracted with methylene chloride (200 ml). The combined organic extracts were washed with 1 M aqueous sodium thiosulphate (600 ml) and dried (Na2504). After addition of morpholine (32.6 ml, 0.375 mol), the resulting solution was concentrated at reduced pressure to about 200-250 ml to afford, after cooling, the title compound as colourless crystals which were collected, washed with acetone, and dried. Yield: 162.6g; melting points: 152-154"C (decomposition).

Calculated for C,2H1812N204S: C, 26.68; H, 3.36; I, 46.99; N, 5.19; S, 5.94%. Found: C, 27.01; H, 3.44; 1, 46.70; N, 5.18; S, 5.64%.

B. Sodium 6ssiodopenicillanate A stirred solution of 6,6-diiodopenicillanic acid morpholine salt (54 g, 0.1 mol) in methylene chloride (500 ml) was protected from light, and cetyl trimethylammonium boranate (36 g, 0.12 mol) was added. After stirring for 15 minutes at room temperature, the mixture was evaporated in vacuo. The residue was triturated with acetone (250 ml), insoluble salt was removed by filtration and the filtrate evaporated to dryness. The residual oil was dissolved in ethyl acetate (200 ml), water (200 ml), water (200 ml) was added, and the pH in the aqueous phase was adjusted to 7 by addition of 2 N sodium hydroxide with stirring. The aqueous layer was separated, the organic phase washed with water (50 ml), and the pH of the combined aqueous phases was adjusted to 3 with dilute hydrochloric acid under a layer of ether (200 ml).The organic phase was separated, the aqueous phase was re-extracted with ether, and combined ethereal extracts were dried and concentrated at reduced pressure to about 80-100 ml. The concentrate contained a mixture of 6ss-and 6a-iodopenicillanic acids as well as minor amounts of penicillanic acid (approximate ratio 50:40:10) which were separated by dry column chromatography using a similar procedure as described in Example 1. The pure 6ss-iodo acid thus obtained gave, after salt formation with 0.5 M aqueous sodium bicarbonate and removal of water by azeotropic distillation with n-butanol, crystalline sodium 6ss-iodopenicillanate; [aJ2D0 + 274 (c = 0.5, 1 M phosphate buffer pH 7).

Calculated for C8HgINNaO3S: C, 27.52; H, 2.60; I, 36.35; N, 4.01; S, 9.18%. Found C, 27.31; H, 2.64; I, 36.12; N, 3.92; S, 9.34%.

Example 19 6ss-lodopenicillanic acid A. 6,.6-Diiodopenicillanic acid dimethylsulphoxide solvate To a cooled solution of 6,6-diiodopenicillanic acid morpholine salt (10.8 g, 20 mmol) in dimethylsulphoxide (20 ml) was added 1 N hydrochloric acid (20 ml), and crystallization was induced by scratching. After further addition of water (20 ml), the crystals were filtered off, washed with water, and dried to give an almost quantitative yield of the title compound which showed an ill-defined melting point with slow decomposition above 120-115 C.

Calculated for C8Hgl2NO3S, C2H60S: C, 22.61; H, 2.85; 1, 47.78; N, 2.64; S, 12.07%.

Found: C, 22.96; H, 2.81; 1, 47.64; N, 2.74; S, 12.14%.

B. Dicyclohexylammonium GP-iodopenicillanate To a solution of 6,6-diiodopenicillanic acid dimethylsulphoxide solvate (5.31 g, 10 mmol) in dimethylsulphoxide (25 ml) was added sodium cyanoborohydride (0.7 g; 90% pure), and the mixture was stirred until a clear solution was obtained (about 30 minutes). After standing for 40 hours at room temperature, water (50 ml) was added, and the mixture was cooled to 5"C to preciptate unreacted starting material which was collected, washed with water, and dried. The filtrate was extracted with methylene chloride (3 x 25 ml), and the combined extracts were washed with water (2 x 10 ml), dried (Na2SO4), and carefully evaporated in vacuo. The residual oil was dissolved in acetone (25 ml), an equivalent amount of dicyclohexylamine was added, and crystallization was induced by scratching.After standing for 1 hour, the pure dicyclohexylammonium 6ss-iodopenicillanate was filtered off, washed with acetone, and dried. The compound showed no well-defined melting point, after darkening at about 150"C, it decomposed slowly above this temperature.

C. 6ss-Iodopenicillanic acid By substituting dicyclohexylammonium 6ss-iodopenicillanate for the corresponding 6ss-bromopenicillanate in the procedure of Example 12 B, 6ss-iodopenicillanic acid was obtained as colourles crystals. Recrystallization from ether-diisopropyl ether afforded the analytical sample [a]2D0 + 278 (c = 0.5 CHC13).

Calculated for C8H10lN035: C, 29.37; H, 3.08; I, 38.79; N, 4.28; S, 9.80%. Found: C, 29.46; H, 3.13; 1, 38.96; N, 4.27; S, 9.81% Example 20 Pivaloyloxym ethyl GP-bromopenicillanate A. PivaloyloxmethVl 6, 6-dibromopenicillanate To a solution of potassium 6,6-dibromopenicillanate (5.96 g, 15 mmol) in dimethylformamide (30 ml) was added chloromethyl pivalate (2.22 ml), 15 mmol), and the mixture was stirred for 16 hours at room temperature. After dilution with ethyl acetate (120 ml), the mixture was washed with water (4 x 30 ml), dried, decolourized by stirring with charcoal (0.5 g ; 1 hour), and evaporated to dryness to give the desired compound as a yellow oil which crystallized from ether-hexane; melting point: 101-102"C.

B. Pivaloyloxymethyl 6ssromopenicillanate To a solution of pivaloyloxymethyl 6,6-dibromopenicillanate (5.68 g, 12 mmol) in dimethylsulphoxide (25ml) was added sodium cyanoborohydride (0.84 g; pure), and the mixture was stirred for 24 hours at room temperature in a dark room. After addition of water (75ml), the mixture was extracted with ether (3 X 25 ml). and the combined ethereal extracts were washed with water (3 X 1 Oml), dried, and concentrated at reduced pressure to about 20 ml, dried, and concentrated at reduced pressure to about 20ml. The cqncentrate was subjected to column chromatography on silica gel similar to the procedure described in Example 9 C. Hereby, the 6/?- bromo compound was separated from unreacted starting material.Fractions containing the more polar 6ss-bromo ester were combined and evaporated in vacuo. The residual oil was crystallized from ether-diisopropyl ether to give pivaloyloxmethyl 6/?-bromopenicillanate, melting point: 66-68 C, identical with the compound described in Example 7.

Example 21 6ssBromopenicillanic acid pivampicillin salt To a stirred sotution of pivampicillin hydrochloride (2.50 g, 5 mmol) in water (100 ml) was added dropwise 0.1 M aqueous potassium 6/?-bromopenicillanate (50 ml). The colorless precipitate thus obtained was filtered off, washed with water (3 X 1 0 ml), and dried in vacuo to give the pure title compound as colourless crystals which began to decompose at 120-130 C without melting.

The IR-spectrum (KBr) showed bands at 3030, 2970, 2935, 2870, 1790, 1770, 1680, 1600, and 627 cm.

The NMR-spectrum (CDCl3) showed signals at 8 = 1.20 (s, 9H; C(CH3)3), 1.36 (s, 3H; CH3-2), 1.44 (s, 3H; CH3-2), 1.52 (s, 3H; CH3-2), 1.54 (s, 3H; CH3-2), 4.26 (s, 1 H; CH-3), 4.40 (s, 1H; CH-3), 5.11 (s, 1H, CHCO), 5.23, 5.36, 5.43, and ""5.76 (4 doublets, J = 3.8-4.2 Hz, 4H; CH-5 and CH-6), 5.79 (ABq, J = 5.5 Hz, 2H; OCH2O), 7.40 (s, 5H; arom. CH), and 7.82 (d, J = 8.2 Hz, 1 H; CONH) ppm. Tetramethylsilane was used as internal reference.

Example 22 6ss-lodopenicillanic acid bacampicillin salt A solution of potassium 6ss-iodopenicillanate (0.73 g, 2 mmol) in water (20 ml) was added dropwise to a stirred solution of bacampicillin hydrochloride (1.0 g, 2 mmol) in water (40 ml).

The resulting crystalline precipitate was filtered off, washed with water, and dried to afford the pure title compound which decomposed at 110-120 C without melting.

The IR-spectrum (KBr) showed bands at 3030, 2980, 2870, 1780, 1765, 1695, 1625, and 618 cm-1.

The NMR-spectrum (CDC13) showed signals at S = 1.31 (t, J = 7 Hz, 3H; OCH2CH3), 1.38 (s, 3H; CH3-2), 1.49 (s, 3H; CH3-2), 1.54 (s, 3H; CH3-2), 1.59 (s, 3H; CH3-2), 4.25 (m, 4H; OCH2CH3 and CH-3), 4.99 (s, 1H; CHCO), 5.12 (bs, NH+), 5.21, 5.45, 5.54, and 5.4 (4 doublets, J = 3.8-4.2 HZ, 4H; CH-5 and CH-6), 6.76 (ABq, J = 5.5 Hz, 1H; CHCH3), 7.39 (s, 5H; arom. CH), and 7.72 (d, J = 8.5 Hz, 1 H; CONH) ppm. Tetramethylsilane was used as internal reference.

Examples 23 to 25 Further salts of 6ss-halopenicillanic acids with inorganic bases Treatment of an ethereal solution of the 6/?-halopenicillanic acid with an equivalent amount of aqueous base followed by separation of the aqueous phase and freeze-drying afforded the salts listed in Table IV as colourless powders.

Table IV

Example X n Z 23a Cl 1 Na+ 23b CI 2 Ca++ 24a Br 1 Li+ 24b Br 2 Ca++ 25a I 1 Li+ 25b 1 2 Ca++ Examples 26-28 Further salts of 6fi-halopenicillanic acids with organic bases By treatment of a solution of the 6/?-halopenicillanic acid in a suitable organic solvent, e.g.

acetone, ethyl acetate or ether, with an equivalent amount of the organic base (preferably dissolved in the same solvent), the desired salt was obtained as a crystalline precipitate which was filtered off and dried in vacuo. The salts obtained by this method are listed in Table V below.

Table V

Example X n Z' 26a CI 1 dicyclohexylamine 26b Cl 2 N,N'-dibenzylethylenediamine 27a Br 1 morpholine 27b Br 2 N,N'-dibenzylethylenediamine 27c Br 1 N-ethylpiperidine 27d Br 1 procaine 28a I 1 dibenzylamine 28b 1 2 N,N'-dibenzylethylenediamine 28c I 1 N-methylbenzylamine 28d 1 1 procaine Examples 29 to 30 Salts of 6fi-halopenicillanic acids with ss-lactan antibiotics and pro-drugs thereof containing a basic group The salts listed in Table VI below were prepared by procedures similar to those described in the preceding Examples 21-22 (A), 23-25 (B), or 26-28 (C).

Table VI

Table VI Example x Z" Procedure 1R-date (cm-1) 29a Br Bacampicillin A 3010, 2980, 2940, 1785, 1765, 1690, 1620, 628 29b Br Pivmecillinam C 2970, 2935, 2860, 1770, 1685, 1630, 1600, 625 29c Br Bacmecillinam C 2970, 2935, 2860, 1770, 1685, 1630, 1600, 628 29d Br Penethamate C 3455, 3020, 2940, 2860, 2660, 1800, 1780, 1745 29e Br Ampicillin B 1675, 625 29f Br Amoxycillin B 30a | Pivampicillin A 3040, 2975, 2935, 2870, 1782, 1770, 1685, 1600, 618 30b | Pivmecillinam C 2970, 2935, 2865, 1780, 1770, 1685, 1630, 1600, 616 30c | Bacmecillinam C 2970, 2930, 2860, 1775, 1760, 1685, 1630, 1600, 616 30d | Penethamate C 3460, 3018, 2940, 2855, 2665, 1795, 1775, 1745 30e | Ampicillin B 1670, 615 30f | Amoxycillin B

Claims (9)

1. A method for the preparation of an essentially pure compound of the formula I:

in which X stands for chlorine, bromine or iodine, and salts and easily hydrolysable esters thereof and salts of such esters; wherein a mixture comprising a 6/?-halopenicillanic acid, together with corresponding 6a-halo and/or 6,6-dihalo derivatives, or salts or esters thereof, is prepared by epimerization of a 6ahalopenicillanic acid or salt or ester thereof in aqueous or organic solution in the presence of a base, or by reducing a 6,6-dihalopenicillanic acid or a salt or ester thereof with an alkali metal boranate or a tetraalkylammonium boranate, or with sodium cyanoborohydride in an organic solvent, and wherein the 6/?-halopenicillanic acid or salt or ester thereof is separated from the corresponding 6a-halo and/or 6,6-dihalo derivatives by chromatography or fractional crystallization.

2. A method as claimed in Claim 1, wherein the separation is performed by chromatography using as a developing solvent a mixture of organic solvents containing a low percentage of a carboxylic acid.

3. A method as claimed in Claim 2, wherein the developing solvent comprises a mixture of ether-petroleum ether-formic acid in the proportions 70:30:0. 1.

4. A method as claimed in any one of Claims 1 to 3, wherein the aqueous equilibration is carried out at 30 to 32"C at a pH of 9.0 to 9.1 and for a period of 20 to 24 hours, the pH being held substantially constant by addition of dilute aqueous base via an automatic titrator.

5. A method as claimed in any one of Claims 1 to 4, wherein the compound of formula I is obtained in the form of the free acid or salt and is converted into an ester by esterification process.

6. A method as claimed in any one of Claims 1 to 4, wherein the compound of formula I is obtained in the form of an ester and the ester is cleaved chemically or enzymatically to provide the free acid or salt thereof.

7. A method as claimed in Claim 1, wherein the separation is performed by fractional crystallization.

8. A method as claimed in Claim 7, wherein the separation is performed using the dicylohexylammonium salt.

9. A method for the preparation of an essentially pure compound of the formula I defined in Claim 1 substantially as hereinbefore described in any one of the foregoing Examples 1 to 6, 9 and 12 to 20.

9. A method for the preparation of an essentially pure compound of the formula I defined in Claim 1 substantially as hereinbefore described in any one of the foregoing Examples.

10. An essentially pure compound of the formula I

in which X stands for chlorine, bromine or iodine, or salt or easily hydrolysable ester thereof, or salt of such ester, whenever prepared by the method claimed in any one of Claims 1 to 8.

CLAIMS (5 Oct 1983)

1. A method for the preparation of an essentially pure compound of the formula I:

in which X stands for chlorine, bromine or iodine, and salts and easily hydrolysable esters thereof and salts of such esters; wherein a mixture comprising a 6/?-halopenicillanic acid, together with the corresponding Sa- halo and/or 6,6-dihalo derivatives, or salts or esters thereof, is prepared by epimerization of a 6a-halopenicillanic acid or salt or ester thereof in aqueous or organic solution in the presence of a base, or by reducing a 6,6-dihalopenicillanic acid or a salt or ester thereof with an alkali metal boranate or a tetraakylammonium boranate, or with sodium cyanoborohydride in an organic solvent, and the resulting 6/?-halopenicillanic acid or salt or ester thereof being separated from the corresponding 6a-halo and/or 6,6-dihalo derivatives by chromatography or fractional crystallization and wherein in the case where the compound of formula I is obtained in one of the free acid, salt or ester forms, it is optionally converted into another of said forms.