IE45559B1 - Lactam antibiotics - Google Patents

Abstract

Salts of the compounds of the formula II are obtained by esterifying a salt of a corresponding 2-carboxylic acid. The products are antibacterially active and can increase the activity of penicillins and cephalosporins against ss-lactamase-producing strains of various Gram-positive and Gram-negative bacteria.

Description

The present invention relates to novel compounds, to their preparation and to pharmaceutical compositions containing them.

The compounds of the formula (I): and salts thereof wherein X is a trans -SO.CH=CH-, trans -S.CH=CH- or -S-CHj-CHj- group are disclosed in Belgian Patents Nos. 827331, 827332 and 839324 where they are designated MM 4550, MM 13902 and MM 17880 respectively.

It has now been found that the esters of the compounds of the formula (I) are antibacterial agents. In addition the esters of the compounds Of the formula (I) are also able to enhance the effectiveness of penicillins and cephalosporins against g-lactamase producing strains of various gram-positive and gram-negative bacteria such as Staphylococcus aureus, Klebsiella aerogenes, Escherichia coli, Proteus mirabilis and other similar organisms. - 2 4S559 Accordingly the present invention provides the salts of the compounds of the formula (II)s (III wherein R is an organic group of up to 16 carbon atoms and X is as defined in relation to formula (I).

Suitably X in the compounds of the formula (II) is a trans -SO-CH=CH- group. Suitably X in the compounds of the formula (II) is a trans -S-CH=CH- group. Suitably X in the compounds of the formula (II) is a -S-CH^-CH^group. - 3 Suitably the group R is selected from aryl, alkyl, aralkyl and alkenyl groups whioh may be substituted by halogen, or by hydroxy, alkoxy, acyloxy, aryloxy, aralkoxy, alkylthio, arylthio, aralkylthio, nitro, cyano, carboxamido, acetamido, sulphonamido or phenylsulphonyl groups. Alkyl and alkenyl groups are suitably straight or branched chained but straight chained groups are generally more convenient.

More suitably R will contain up to 12 carbon atoms.

Thus R may be a methyl, ethyl, propyl, butyl or benzyl group or a benzyl group substituted by one or two halogen atoms or C1-4 alkoxy or nitro groups. The benzhydryl group and the benzhydryl group substituted by one or two halogen atoms C1-4 alkoxy or nitro groups are also suitable.

Particularly suitable esters include those wherein the ester group COjR is of the formula COjR1 wherein the group COjR1 is readily hydrolysed in the human body, for example in tissues or blood, to yield the parent acid or its salt, or in equivalent mammalian tissues. other particularly suitable esters include those wherein the ester group CO2R is as in an Example herein. - 4 4 5 5 5 9 Suitably R1 ia a group of the sub-formula (a) - (o): I 3 - CH - 0 - CO - RJ (a) 3 wherein R is a hydrogen atom or a methyl group? R is a A C1^4 alkyl, Cj_4 alkoxy, phenyl or benzyl group? and R is a hydrogen atom or a methoxy group.

Suitably R is a hydrogen atom.

Suitably R is a methyl, t-butyl, phenyl or ethoxy group.

Most suitably R1 is a group of the sub-formula (a) or (b) and is especially suitably of the sub-formula (b).

Preferably R1 is a phthalidyl group as the resulting compounds have particularly good activity when administered by injection.

Thus preferred compounds of this invention include 5 the pharmaceutically acceptable salts of the compounds of the formula (IHis -S-CH2-CH2- group.

A particularly suitable value for X1 is the trans -S-CH=CH- group.

A further favoured value for X1. is the. .-S-CHg-CHggroup.

Those compounds of the invention wherein R contains a chiral centre may be presented as pure optical isomers (for example the R- isomer or the S- isomer) or a mixture thereof (for example the R,S- form). Thus for example the R- and S- phthalidyl esters are included within this invention as well as mixtures thereof such as the R,S- phthalidyl esters. - 6 45559 Suitable salts of the compounds of the formula (li) . include the sodium, potassium, calcium, magnesium, ammonium or substituted ammonium, for example trimethylammonium, trimethylamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)-amine, tri-(2-hydroxyethyl)-amine, benzathine, procaine, bicyclohexylamine, dibenzylamine, Ν,Ν-dibenzylethylenediamine, 1-ephenamine, N-ethylpiperidine, N-benzyl-g-phenethylamine, pyridine, collidine, quinoline, or a quaternary ammonium salt such as tetramethylammonium.

Most suitably the salt will be a sodium, potassium or quaternary ammonium, for example tetra-C1_g alkyl ammonium such as tetra-n-butyl ammonium and tetra-n-hexyl ammonium. .

Preferably the salt will be a sodium or potassium salt and most preferably the sodium salt. - 7 •lSS'5 The present invention also provides a process for the preparation of the compounds of the formula (II) by esterification of the corresponding compound of the formula (I) or salt thereof.

Suitable methods of ester formation are those conventionally used to prepare esters of β-lactam containing compounds and include: (a) the reaction of a di-salfof a compound of the formula (I) with a compound of the formula (IV): R - Q (IV) wherein R is as defined in relation to formula (II) and Q is a readily displaceable group; and (b) the reaction of a mono-salt of a compound of the formula (I) with a diazoalkane.

Suitable di-salts of the compounds of the formula (I) which may be reacted with compounds R - Q include alkali metal salts such as the sodium or potassium salts or other conventional salts such as the quaternary ammonium salts.

Suitable groups Q include those atoms or groups known to be displaceable by carboxylate anions and include chlorine, bromine and iodine atoms, sulphonic acid esters such as OSOgCHg or OSOgCgH^CHg and other conventional groups displaceable by nuoelophlles.

A particularly suitable compound of the formula (IV) is phthalidyl bromide. Other phthalidyl derivatives are also favoured. 4B3S9 This displacement reaction is normally carried out in an aprotic organic solvent such as dimethylformamide, dimethylsulphoxide, hexamethylphosphoric triamide, acetone, dioxane or acetonitrile optionally in the presence of a crown ether and the like solvents and at a non-extreme temperature such as -5°c to 100°C. We have found that the reaction may suitably be carried out in anhydrous dimethylformamide at ambient temperature.

Those compounds in which the group R contains a chiral centre are generally initially formed as the R,S- form. Separation into the R- and S- isomers may be achieved by conventional methods such as chromatography, seeding out and crystallisation.

The reaction of a mono-salt of a compound of the formula (I) with a diazocompound is a mild method of making alkyl, aralkyl or substituted alkyl or aralkyl esters. The diazotization reaction may be performed under conventional reaction conditions, for example at a nonextreme temperature and in a conventional solvent. Such reactions are normally carried out at between -5°C and 1QO°C and conveniently at ambient temperature. Suitable solvents for this reaction include Cj__^ alkanols such as methanol and ethanol and solvents such as tetrahydrofuran and dioxane. These solvents will normally be present with a small amount of water which aids the solubility of the compounds of the formula (I) in the solvent. - 9 45553 Aqueous ethanol has proved a particularly useful solvent for this reaction.

The mono ester mono quaternary ammonium salts of the compounds of the formula (II) may also conveniently be prepared by the dissolution of a water soluble salt of a compound of the formula (II) in water followed by the mixing of this solution with a solution of a quaternary ammonium halide in a water immiscible inert organic solvent. The organic solvent is then separated and evaporated to yield the quaternary ammonium salt. Suitably the organic solvent will be a halogen substituted hydrocarbon, such as methylene dichloride or chloroform, and the mixing of the two solutions will take place between 1°C and 10Q°C, for example between 1°C and 30°C. The two solutions will normally be mixed by shaking or stirring.

Salts of the compounds of the formula (IIJ may be interconverted in conventional manner, for example by using ion exchange resins. Metal ion salts useful for this purpose include lithium, sodium and potassium salts.

Salts within this invention, e.g. of the compounds of the formula (III) may be obtained crystalline, for example by crystallisation from inert organic solvents such as ethyl acetate, cyclohexane or their mixtures. ‘1S S 5' 9 In a further aspect this invention provides a pharmaceutical composition which comprises a salt of a compound of the formula (III as hereinbefore defined together with a pharmaceutically acceptable carrier.

Such compositions may be in a form suitable for oral, topical or parenteral use. For example, tablets, capsules, syrup, reconstitutable powders and more suitably sterile forms suitable for injection or infusion may be used. Such compositions may contain conventional pharmaceutically acceptable materials such as diluents, binders, colours, flavours, preservatives and disintegrants in accordance with conventional pharmaceutical practice in a manner well known to those skilled in the formulation of antibiotics such as penicillins and cephalsporins. Preferred compositions are adapted for injection, for example as a sterile solution in water.

The salt of the compound of the formula (IIJ may be present in the composition of the invention as sole therapeutic agent or it may be present together with a β-lactam antibiotic. Suitable β-lactam antibiotics include those known to be susceptible to β-lactamases and also those having some intrinsic resistance to β-lactamases. Such β-lactam antibiotics include ampicillin, amoxycillin, benzylpenicillin, phenoxymethylpenicillin, propicillin, cephaloridine, cefoxitin, cephalothin, cephalexin, carbenicillin, ticarcillin, and in-vivo hydrolysable esters of such compounds such as the phenyl, - 11 ,StS5'j® tolyl or indanyl esters of carbenicillin or tioarcillin or the acetoxymethyl, pivaioyloxymethyl or phthalidyl esters of ampioillin, benzylpenicillin, amoxycillin, cephaloridine and cephaloglycin.

The ratio of the salt of the compound of the formula (II). to β-lactam antibiotic is normally between 2Q:1 and 1:20 and more usually between 10:1 and 1:10, for example between 3:1 and 1:3.

The total quantity of compound of this invention 10 present in any unit dosage form will normally be between and 1500 mgs and will usually be between 100 and 1000 mgs. Preferred unit dosage compositions according to this invention may be administered one or more times a day, for example, 2 to 4 times a day, in the treatment of diseases of the urinary tract, respiratory tract and soft tissues. Thus the compositions may be used in the treatment of such diseases as bronchitis, gonorrhea, otitus media and mastitis. •a S 5 a J The following Examples illustrate the invention: Example 1 The disodium ss.lt of MM 13902 (0.60 g, 1.37 mmol) was treated with methyl iodide (0.53 g, 4.17 mmol) in dimethylformamide (10 ml) at room temperature for 2h.

The solvent was removed in vacuo and the product chromatographed on silica gel using chloroform-ethanol mixtures as eluant.

The mono-methyl ester mono-sodium salt of MM 13902 was obtained as a white solid (0.36 g, 61%); Xmax (H20) 323 and 228 nm vmax (KBr) 1760, 1685, Ϊ620 and 1220-1280 br cm-1 n.m.r. (DMSO-de) 1.33 (3H, d, J, 6 Hz, CH3.CH), 1.92 (3H, s, CH3), 2.92 (IH, dd, J 19, J’ 10 Hz, Ha of ABX system), ca. 3.5 (IH, m, Hb of ABX system), 3.75 (4H, s + m, CH3O2C and CH.CH.CH), 4.20 (IH, m, Hx of ABX system), 4.38 (IH, dq, J 10, J' 6 Hz, CH3CH.CH), 5.82 (IH, d, J 14 Hz, CHsCHNH), 7.07 (IH, dd, J 14, J' 11 Hz, CH:CHNH), and 10.45 6 br (IH, d, J 11 Hz, tJH.CH;).

The compound was also prepared as follows: The disodium salt of MM 13902 (1,1 g, 2,52 mmol), was stirred at room temperature with methyl iodide (1,5 g, 10,56 mmol) in NN-dimethylformamide (20 ml) for 2 hours. The solvent was removed in vacuo and the residue chromatographed on silica gel (30 g) employing a gradient elution from chloroform to chloroform:ethanol (1:1). Fractions containing the product were combined and evaporated in vacuo. Ethyl acetate* was added to the residue and then removed in vacuo to afford 3 59 the monomethyl ester monosodium salt of MM 13902 as a white solid (0.68 g, 63%).

* Other solvents Which will azeotropically remove ethanol (e.g. CH^CN, toluene) may be employed at this stage.

Example 2 The disodium salt of MM 13902 (0.50 g, 1.15 mmol) was treated with benzyl bromide (1.00 g, 5.85 mmol) in dimethylformamide (10 ml) for 2 hours at room temperature. Work-up and chromatography as in Example 1 afforded the mono-benzyl ester mono-sodium salt of MM 13902 (0.29 g, 50%) λ max (H2O) 325 and 226 nm. v max (KBr) 1765, 1685, 1620 and 1210 - 1280 br cm Example 3 The di-sodium salt of MM 13902 (0.970 g, 2.22 mmol) was treated with £-bromobenzyl bromide (1.7 g, 6.80 mmol) in DMF (17 ml) for 2.5 hours at room temperature. Work-up and chromatography as described in Example 1 afforded the mono £-bromobenzyl ester mono-sodium salt of MM 13902 as a white solid (0.73 g, 56%) t λ max (H2O) 325 and 226 nm. υ max (KBr) 1760, 1675, 1620 and 1210 - 1280 br cm1. - 14 •1S559 Example 4 The di-sodium salt of MM 13902 (1.67 g, 3.83 mmol) was stirred at room temperature with p-nitrobenzyl bromide (2.48 g, 11.6 mmol) in dimethylformamide (30 ml). Work-up and chromatography after 2 hours as described in Example 1 gave the p-nitrobenzyl ester mono-sodium salt of MM 13902 as a pale yellow solid (1.27 g, 60%); λ max (EtOH) 325, 266, 225 sh and 220 nm. v max (KBr) 1760, 1680, 1620, 1210 - 1280 br cm1; (DMSO - d,) 1.36 (3H, d, J 6 Hz, CH,CH), 1.92 (3H, s, o — — CHgCO), 2.96 (IH, dd, J 19 and 10 Hz, CH^ of ABX), 3.52 (IH, dd, J 19 and 9 Hz, CHg of ABX), 3.67 (IH, dd, J 10.5 and 6 Hz, CH.CH.CH), ca. 4.20 (IH, m, 0Ηχ of ABX), 4.42 (IH, dq, J 10.5 and 6 Hz, CH.CHg), 5.22 and 5.42 (each IH, d, J 14 Hz, CHgAr), 5.80 (IH, d, J 14 Hz, CHsCH.S), 7.04 (IH, dd, J 14 and 10.5 Hz, CHsCH.NH), 7.63 and 8.17 (each 2H, d, J 8 Hz, aromatic protons) and 10.38 (IH, d, J 10.5 Hz, NH).

Pounds C, 42.94; H, 3.77; N, 7.21; S, 11.54; Na, 4.41%, C20H20N3S2°10Na ^H2° recIuires C' 43.01; H, 3.76; N, 7.54; S, 11.47; Na, 4.12%. 4SSl>a Example 5 The di-sodium salt of MM 13902 (0.30g, 0.69 mmol) was treated with p-iodobenzyl bromide (0.90g, 3.03 mmol) in dimethylformamide (9 ml) at room temperature. Work-up and chromatography after h as described in Example 1 afforded the mono p-iodobenzyl ester mono-sodium salt of MM 13902 as a white solid (0.15g, 34%), λ max (H20) 325 and 232 nm V max (KBr) 1765, 1675, 1620 and 1210-1280 br cm1.

Example 6 The di-sodium salt of MM 13902 (0.18g, 0.41 mmol) was treated with o-iodobenzyl bromide (0.55g, 1.85 mmol) in DMF (5ml) for 6 h at room temperature. Work-up and chromatography as described in Example 1 afforded the mono o-iodobenzyl ester monosodium salt of MM 13902 as a buff-coloured solid (0.07a, 26%) v max (KBr) 1760, 1680, 1620 and 1210-1280 br cm1.

Example 7 The di-sodium salt of MM 13902 (0.25g, 0.57 mmol) was stirred at room temperature with p-bromophenacyl bromide (0.80g, 2.88 mmol) in dimethylformamide (5 ml). After 1.5h, work-up and chromatography 20 as described in Example 1 afforded the mono p-bromophenacyl ester mono-sodium salt of MM 13902 as a cream-coloured solid (0.21g, 60%) λ max (EtOH) 327 and 257 nm υ max (KBr) 1765, 1690, 1620 and 1210-1280 cm1 5 Found: C, 39.90? H, 3.64; N, 4.09; S, 10.10; Br, 12.55% C21H20N2BrO9S2Na,H2° re9uires cr 40.06; H, 3.50: N, 4.45; S, 10.17? Br, 12.72%.

Example 6 The di-sodium salt of MM 13902 (0.30g, 0.69 mmol) was treated with bromophthalide (0.50g, 2.35 mmol) in DMF (5ml). Work-up and chromatography after 1.5h as described in Example 1 afforded the phthalide ester mono-sodium salt of MM 13902 as a buff-coloured solid (0.08g, 21%). λ max (HgO) 333 and 233 nm v max (KBr) 1775, 1680 br., 1620, 1210 and 1275 cm1 Example 9 The di-sodium salt of MM 13902 (0.30g, 0.69 mmol) was treated with N-chloromethylbenzoxazolone (0.50g, 2.72 mmol) in DMF (6 ml) at room temperature for 2 h. Work-up and chromatography as described in Example 1 afforded the mono N-benzoxazolonylmethyl ester mono-sodium salt of MM 13902 (0.155g, 40%) λ max (h20) 330, 267 and 225 nm \> max (KBr) 1775 br., 1690, 1620 and 1220-12180 br cm1 - 17 45559 Example 10 The di-sodium salt of MM 17880 (0.3 g, 0.68 mmol) in Ν,Ν-dimethylformamide (4 ml) was treated with p-bromobenzyl bromide (0.75 g, 3 mmol) and the mixture was stirred for 2¾ hours. The solvent was removed on a rotary evaporator and the residue chromatographed on silica gel, eluting with chloroform/ethanol mixtures (up to 34 2) . Evaporation of the fractions containing the ester (followed by evaporation of the residue from toluene) gave the mono £-bromobenzyl ester mono-sodium salt of MM 17880 (0.195 g, 48%); V max (KBr) 1765, 1690, 1635, 1220 cm-1; λ max (H20) 320 nm; δ (DMSO - dg) 1.37 (3H, d, J 6 Hz, CH3CH), 1.81 (3H, s, CH3CO), 2.8 - 3.6 (6H, m, (¾,C.SCH2CH2N), 3.69 (IH, dd, J 11 and 6 Hz, CH.CH.CH), 4.05 - 4.6 (2H, m, CH.CH.CH), 5.08 and 5.26 (each IH, d, J 14 Hz, CHjAr), 7.36 and 7.57 (each 2H, d, J 8.5 Hz, aromatic protons) and 8.10 (IH, broad, NH).

Example 11 The di-sodium salt of MM 4550 (816 mg) in W,N-dimethylformamide (10 ml) was treated with iodomethane (0.9 ml).

The mixture was stirred for 2 hours. The solvent was then removed on a rotary evaporator and the residue chromatographed on silica gel (40 g), eluting initially with chloroform, and then with chloroform/ethanol (3:2).

The monomethyl ester mono-sodium salt of MM 4550 was isolated as a solid (260 mg) after combination and evaporation in vacuo of the fractions containing the ester followed by evaporation in vacuo of the residue from toluene. v max (KBr) 1775, 1710, 1620, 1260 cm1; λ max 297 and 245 nm; The n.m.r. spectrum in (CD3)2SO showed inter alia signals at δ 1.36 (3H, d, J » 6 Hz), 2.0 (3H, s), 3.77 (3H, s), 6.22 (IH, d, J = 15 Hz), 7.38 (IH, dd, = 15 Hz, J2 = 10 HZ) .

Example 12 The di-eodium salt of MM 4550 (500 mg) in dimethylformamide (5 ml) was treated with p-bromobenzyl bromide (1.5 g) and the mixture was stirred for 3 hours, work-up as in Example 11 gave the mono p-bromobenzyl ester mono-sodium salt of MM 4550 (350 mg). v max (KBr) 1780, 1710, 1620, 1260 - 1220 cm-1; λ max (H2O) 305, 244 (sh), 228 nm. The n.m.r. spectrum in D20 showed signals inter alia at δ 1.43 (3H, d, 0 » 6 Hz), 2.00 (3H, S), 6.23 (IH, d, J = 15 Hz), 6.96 (2H, d, J = 8 Hz), 7.14 (2H, d, J = 8 Hz), 7.32 (IH, d, J » 15 Hz). - 19 45 553 Example 13 The di-sodium salt of MM 4550 (800 mg) in .Ν,Ν-dimethylformamide (8 ml) was treated with benzyl bromide (1.0 ml).

The mixture was stirred for 3 hours, and then worked up as in Example 11. The monobenzyl ester mono-sodium salt of MM 4550 was obtained as a solid (420 mg). v max (KBr) 1780, 1705, 1620, 1220 - 1260 cm1; λ max (H2O) 306, 246 nm; C(CD3)2SO] inter alia 1.36 (3H, d, J 6 Hz, CH3CH), 2.01 (3H, s, CH3CO), 5.26 (2H, broad s, OCff2Ph), 6.24 (IH, d, J 14 Hz, CH=CH).

Example 14 The di-sodium salt of MM 4550 (400 mg) in Ν,Ν-dimethylformamide (4 ml) was treated with p-nitrobenzyl bromide (1.2 g). The mixture was stirred for 2.25 hours and then worked up as in Example 11 to give the mono p-nitrobenzyl ester monosodium salt of MM 4550 (234 mg). v max (KBr) 1780, 1710, 1620, 1260 - 1220 cm-1; λ max 250, 296 (sh) nm. The n.m.r. spectrum in D20 showed inter alia signals at about 6 1.53 (3H, d, J = 6 Hz), 2.1 (3H, s), 6.4 (IH, d, J == 15 Hz), 7.33 (IH, d, J = 15 Hz), 7.38 (2H, d, 1 = 8 Hz), 8.03 ,(2H, d, J = 8 Hz).

Example 15 The di-sodium salt of MM 4550 (670 mg) and p-methoxybenzyl bromide (1.58 g) were stirred together in N,N-dimethyl formamide (10 ml) for 3 hours. Work-up as in Example 11 gave the mono p-methoxyben2yl ester mono-sodium salt of MM 4550.

S 5 5 9 Example 16 The di-sodium salt of MM 4550 (50 mg) and o-iodobenzvl bromide (190 mg) were stirred together in Ν,Ν-dimethyIformamide (2 ml) for 6 hr. The solvent was evaporated off and the residue was triturated under toluene. After centrifuging the residue (92 mg) was collected and then chromatographed on cellulose, eluting with isopropanol/water (9;1). The requisite fractions were combined and evaporated down -in vacuo to give .the mono o-iodobenzyl ester mono-sodium salt of MM 4550. v max (CHClg) 1785, 1710, 1620, 1260-1220 cm1.

Example 17 The di-sodium salt of MM 4550 (185 mg) in N,N-dimethylformamide (2 ml) was treated with pivaioyloxymethyl bromide (80 mg) in Ν,Ν-dimethyIformamide (1 ml). The mixture was stirred at room temperature for 44 hr., and then in a refrigerator for 2 days.

The solvent was evaporated off, and then the residue placed on a very short silicagel chromatography column and eluted off with ethanol/chloroform (3:2). The mono pivaioyloxymethyl ester mono-sodium salt of MM 4550 was obtained from the eluate as a solid, v max (KBr) 1780, 1705, 1610, 1260 cm1. The u.v. spectrum showed a maximum at 296 nm (in HgO).

Example 18 The di-sodium salt of MM 4550 (300 mg) and N-chloromethylbenzoxazolone (360 mg) were stirred together in DMF (5 ml) for 3 hr.

The solvent was then evaporated off and the mixture was worked up as in Example 11 to give the mono-N-benzoxazolonylmethyl ester 4S5S9 v mono-sodium salt of MM 4550,vmax (KBr) 1780, 1720-1710, 1620, 1260 cm-*, λ max (HgO) 306, 247, and 226 nm. 5(DgO, HOD internal standard at 64.6) inter alia. 1.40 (3H, d, J 6Hz, CHICHI, .2,00 (3H, s, CH^CO) 5.90 (2H, broad s, OCHgN), 6.25 (1H, d, J 14Hz, CH = CH), 7-0 - 7.25 (4H, m, ArH), 7.36 (1H,d, J 14 Hz, CH=CH).

Example 19 The di-sodium salt of MM 13902 (60mg) was suspended in ethanol (5ml) and water was added, until dissolution was complete. The solution was cooled to 0° (ice bath) and treated with a cooled (0°) solution of p-toluenesulphonic acid (90 mgs) in ethanol (6 mis). The resulting solution was stirred for ca. 5 seconds and treated with excess ethereal diazomethane. After 20 minutes at room temperature the solvents were removed in vacuo. The residual semi-solid was triturated with ether, filtered and washed with ether.

Chromatography of the solid on silica gel, eluting with butanolethanol -water (16:4:7), gave the monomethyl ester mono-sodium salt as an amorphous solid.

Example 20 The di-sodium salt of MM 4550 (500 mg) was dissolved in water and passed down a column of DOWEX 50W-X8 ion exchange resin which had been converted to its calcium form by treatment with an aqueous slurry of calcium carbonate (DGWEX is a„ '.Trade Mark). Evaporation in vacuo of the relevant fractions gave the calcium salt of MM 4550,V max (KBr) 1755, 1690, 1020, 1200-1220cm1 The calcium salt so obtained was taken up in Ν,Ν-dimethylformamide (9 ml) and p-bromobenzyl bromide (1.4g) was added. 5 5 9 The mixture was stirred for 3 hr., and the solvent was then evaporated. The residue was chromatographed on silica gel eluting with chloroform/ethanol mixtures to give the mono-pbromobenzyl ester of MM 4550 calcium salt (180 mg) v max (CHCl3/Me2NCHO) 1790, 1720, 1620, 1260 cm-1.

Example 21 The mono-sodium salt mono-p-bromobenzyl ester of MM 13902 (191 mg) in water (6 ml) was shaken with a solution of tetrahexylammonium bromide (138 mg; 0.95 equivalent) in methylene chloride (10 ml).

The organic la^er was separated, filtered through cotton and evaporated to give the product as a foam in almost quantitative yield; V (CH2Cl2) 3200-3400 (broad), 1775 (β-lactam C=O), 1695 (broad, ester and amide carbonyls), 1620 cm-1 (C=C); n.m.r.

(CDClj) showed inter alia 6.20 (IH, d, J 14Hz, -S-CH=CH-NH), 7.55 (5H, m, -S-CH=CH-NH- and aromatic protons), 10.06 δ (IH, d, J 10Hz, SCHCHNH).

Example 22 The monomethyl ester, mono-sodium shit of MM 13902 (0.05g) was dissolved in water, and the solution stirred, vigorously with a solution of tetrabutylammonium hydrogen sulphate (0.04g) in CH2C12 (5 ml). After 5 min. the organic layer was separated and dried (MgSO^).

Evaporation of the solvent gave the crude tetrabutylammonium salt (II, R=(C4Hg)4N, R^CHg) (O.Slg). Rapid chromatography on silica (CHClg-EtOH as eluant) afforded a pure sample of the mono tetrabutylammonium salt monomethyl ester of MM 13902 <0.04 g); v max (CHClj) 1775 (β lactam C=0), 1695 (unsaturated ester and enamide C=0), 1625 and 1200-1280 br cm-! (sulphate)., Example 23 The mono-p-nitrobenzyl ester mono sodium salt of MM 13902 -(0.05g) 5 was treated with tetrabutylammonium hydrogen sulphate as described in Example 22. The mono tetrabutylammonium salt mono-p-hitrobenzyl ester Of MM 13902 was obtained as a gum (0.05 g) υ max (CHClj) 1775 (8-laotam C=0), 1690 (unsat. ester and enamide C=0), 1625 and 1200-1280 br cm-}-(sulphate) .

Example 24 The mono-p-iodobenzyl ester mono sodium salt of MM 13902 (0.22g) was dissolved in water (2 ml) and the solution treated with an aqueous solution of S-benzyl-iso-thiouronium chloride (0.100 g) at 5°. An immediate white precipitate was formed. Chloroform was added and the product was extracted into the organic layer, which was dried (MgSOj) and evaporated to afford the mono S-benzylisothiouronium salt mono p-iodobenzyl ester of MM 13902 (0.15 g); υ max (CHCl3) 1780, 1690, 1670 and 1625 cm-1.

Example 25 The mono-p-bromophenacyl ester mono sodium salt of MM 13902 (0.055 g) was treated with S-benzyliso-thiouronium chloride (0.04 g) in the manner described in Example 24. The mono S-benzylisothiouronium salt, mono p-bromophenaeyl ester of MM 13902 was obtained as a gum (0.047 g); v max (CHClg) 1780, 1695, 1670 and 1625 cm-1.

Example 26 The mono p-nitrobenzyl ester mono sodium salt of MK 13902 (0.11g) was treated with' S-henzylisothiouronium chloride (0.075g) as described in Example 24. The S-benzylisothipu'ronium salt of the 5 p-nitrobenzyl ester of MM 13902 was. obtained as a gum C0.080 gi; \> max (CHClg) 1775, 1690 br, and 1620 cm1.

Example 27 MM 4550 monobenzyl ester mono-sodium salt was dissolved in water and a chloroform solution containing Aliquat 336 (methyl trioctyl10 ammonium chloride from General Mills Chemical Inc., Kanakee, Illinois, U.S.A.) was added, the mixture was shaken thoroughly, the layers separated and the chloroform layer was evaporated to give MM 4550 monobenzyl ester monomethyl-trioctylammoniura salt, v max (CHgClg) 1785, 1720, 1620, 1240-1220 cm-1.

Example 28 Antibacterial and Synergistic Activity of the Mono-Pivaloyloxymethyl Ester Mono-Sodium Salt of MM 4550 (el) and the Mono-Phthalide Ester Mono-Sodium Salt of MM 13902 (e2) The above two esters (el) and (e2j were tested in parallel with 20 MM 13902 and MM 4550 against 5 β-laotamase producing organisms both alone and in combination with ampioillin/ against<'Vieof these organisms. The results are shown in Tables 1 and 2. 4S559 Table 1 Antibacterial Activity of (el) and (e2) expressed as minimum inhibitory concentration (MIC) in ug/ml β-lactamase Compound Staphylococcus aureus Russell Klebsiella aerogenes E70 Proteus mirabilis C889 E.coli JT39 E.coli στιο Di-Sodium MM 4550 12.5 6.25 25 12.5 25 Di-Sodium MM 13902 0.2 0.8 0.2 0.8 0.4 el 125 125 125 8.0 250 e2 31 15.6 15.6 31 62 Table 2 Synergistic Activity of (el) and (e2) with ampicillin and cephaloridine expressed as minimum inhibitory concentration (MIC) in ug/ml Compound Cone, of compound in ug/ml Staphylococcus aureus Russell Proteus mirabilis C889 Klebsiella aerogenes E70 Ampici- llin 250 >2000 1000 el 1.0 250 > 500 >500 10 4 15.6 500 e2 1.0 4 15.6 >500 5 < 0.5 1.0 250 45S59 Example 29 In-vivo Activity of the mono-phthalide ester mono-sodium salt of MM 13902 (e2) and the mono-phthalide ester of the mono-sodium salt of MM 4550 (e3).

The above two esters (e2) and (e3) were tested in parallel with MM 13902 and MM 4550 by administration by sub-cutaneous injection in aqueous pH 6.6 phosphate buffer solution.

In this test four doses were applied at 1, 2, 3 and 4 hour post intra -peritoneal infection by E-coli 8.

The results were as follows; Test compound MM 4550 Di-sodium (03) MM 13902 Di-sodium (e2) 3.9 mg/kg x 4 22.0 mg/kg x 4 10.5 mg/kg x 4 4.4 mg/kg x 4 In a similar test against Staphylococcus aureus Smith the following results were obtained Test Compound MM 13902 Di-Sodium (e2) 23.5 mg/kg x 4 7.8 mg/kg x 4 • 4S569 Example 30 MM 4550 di-sodium salt (l.05g) and bromophthalide (625mg) were stirred together in Ν,Ν-dimethylformamide (19ml) for 2 hours at ambient temperature. The solvent was then removed on a rotary evaporator and the residue chromatographed on silica gel (50g) eluting with a 1:1 mixture of chloroform and ethanol. The fractions containing the ester were combined and evaporated on a rotary evaporator, toluene was added to the residue and evaporated off in the evaporator to give the mono-phthalide ester mono sodium salt of MM 4550 as a solid λ max (H20) 506 and 230-235 nm. v max (KBr) 1785, 1720-1700 (br.), 1620, 1260, and 980 cm"\ 6 [(cd3)2so] (inter alia) 1.33 (3H, d, J •ca 6Hz), 1.98 (s) and 2.01 (s) (3H, C0CH3 of the two epimers), 6.20 (d, J = 14Hz) and 6.26 (d, J = 14Hz) (1H, S-CH = C, of the two epimers!.

Example 31 The di-sodium salt of MM 4550 (200mg) in water (2ml) was shaken with cetylbenzyl dimethyl ammonium chloride ' (350mg, 2 eq.) in dichloromethane (2ml). The dichloromethane layer was dried (MgSO^) and evaporated down to leave the di-quatemary ammonium salt of MM 4550 as a ' gum (397mg). v max (CH2C12) 1765, 1690, 1620 cm-·1.

Methyl chloroformate (33mg) in dichloromethane (5ml) was cooled to -30°C and tho dlquaternary ammonium salt of MM 4550 in dichloromethane (8ml) containing one drop of 4S559 dimethylbenzylamine was added drop-wise. The mixture was stirred at -30° C to -10°C for 45 minutes after addition was complete, when the infra red spectrum indicated that the mixed anhydride had formed [t max (CHgClg) 1820, 1790, 1620 cm’1]. With the temperature at -3o° ethanol (1 ml) was added and the mixture was then stirred at -10° for 15 minutes, then at 0°C for ca 10 minutes, then at 10°C for 45 minutes and finally at room temperature for 1 hour.

The mixture was then evaporated in vacuo and the 10 residue was taken up in a mixture of water (pH 7.0) and ethanol and passed down an Amberlite 120 (Na) ion exchange resin* (Amberlite is a Registered Trade Mark). Evaporation of the eluate, and chromatography of the residue on silica gel (10 g) eluting with chloroform/ethanol (3s2) gave the mono ethyl ester mono sodium salt of MM 4550.

(DgO) (inter alia) (HOD internal standard δ 4.6 ppm) 1.26 (3H, t, J 8 Hz, CHgCHg), 1.44 (3H, d, J 6 Hz, CHgCH), 2.04 (3H, s, CHgCO), 6.34 (IH, d, J 14 Hz, CH=CH), 7.62 (IH, d, J 14 Hz, CH»CH).

* An alternative method for conversion of a mono ester mono d1 quaternary ammonium salt to the mono ester mono sodium salt is to shake a dichloromethane solution of the ester with water containing a sodium salt such as sodium tetrafluoroborate or sodium iodide. Separation of the layers and evaporation of the aqueous layers gives the mono ester 45553 mono sodium salt of MM 455Q, This works provided the quaternary ammonium iodide or tetrafluoroborate is relatively insoluble in water, Example 32 The mono methyl ester mono sodium salt of MM 4550 (30mg) in water (2ml) was treated with chloroform (3ml) contain.ing cetylbenzyl-dimethylammonium chloride (27mg). After shaking the chloroform layer was separated, dried (Mgso4) and evaporated in vacuo to give the mono methyl ester mono cetylbenzyldimethyiammonium salt of MM 4550 (ca 80$ yield), υ max (CHC13) 1785, 1705, (broad), 1620 cm .

Example 35 The di-sodium salt of MM 4550 (105mg) in water (2ml) was treated with cetylbenzyldimethyiammonium chloride (184mg) in diehloromethane (2ml). After shaking the dichlorometh ane layer was separated, dried (Mgso4J and evaporated, in vacuo to leave the di (Cetylbenzyldimethyiammonium) salt of MM 4550,υ max 1775, 1695, 1625 cm1.

The diquaternary ammonium salt of MM 4550 was redissolved in diehloromethane (2ml) and. methyl iodide(0.5inl) was added. After stirring the mixture for ca 2 hr. the solvent and excess methyl iodide were removed in vacuo. Chromatography of the residue on silica gel (10g) eluting 4555 with chloroform/ethanol mixtures (gradient elution from CHClj to CHClj/EtOH 3:2) gave the mono methyl ester mono cetylbenzyldimethylammonium salt of MM 4550. (oa 30# yield from MM 4550).

Alternatively the esterification of the di (cetylbenzyldimethylammonium) salt of MM 4550 can be accomplished in solution in dry 1,2-dimethoxyethane; after evaporation to a low volume cetylbenzyldimethylammonium iodide crystallises out, leaving the mono methyl ester mono cetyl10 benzyldimethylammonium salt of MM 4550 in the mother liquors Confirmation of the structure of the ester can be obtained by conversion to the mono sodium salt by two methods; a) sodium iodide (1 equivalent) is added to a concentrated solution of the mono methyl ester mono cetylbenzyldimethyl ' ammonium salt of MM 4550 in 1,2-dimethoxyethane. Cetylbenzyldimethyl ammonium iodide crystallises out leaving the sodium salt in the mother liquors. After chromatographic purification the mono methyl ester mono sodium salt of MM 4550, identical to that described in Example 11 is obtained. b) A solution of the mono methyl ester mono cetylbenzyldimethylammonium salt of MM 4550 in dichloromethane is shaken with water containing sodium tetrafluoroborate SBS9 (1 equivalent), separation and evaporation of the aqueous layer, followed by chromatography of the residue on silica gel gives the mono methyl ester mono sodium salt of MM 4550.

Example 34.

The mono p-nitrobenzyl ester mono sodium salt of MM 4550 (lOOmg) and cetylbenzyldimethylammonium chloride (70mg) were dissolved in water (5ml) and chloroform (5ml). After shaking the layers were separated and the chloroform layer dried (MgSO^) and evaporated in vacuo to give the mono p-nitrobenzyl ester mono cetylbenzyldimethylammonium salt of MM 4550 (ca 80% yield) Umax (CHC13) 1785, 1710 (broad), 1620 cm1.

Example 35 j5 The mono methyl ester mono sodium salt of MM 4550 (52mg) and benzyltriphenylphosphonium chloride (44 mg) were shaken together in water (5ml) and diehloromethane (5ml). •Qn separation the diehloromethane layer was dried (MgSO^) and evaporated in vacuo to leave the mono methyl ester . mono benzyltriphenylphosphonium salt of MM 4550 as a semi-solid foam (76mg) Umax (CHgClg) 1790, 1710'(broad) 1610 cm1.

Example 36 The di-sodium salt of MM 17880 (492 mg) in DMF (5 ml) -15 S 5 8 was treated with p-nitrobenzylbromide (1.0g) and the mixture was stirred for 1i hr. Work-up as in Example 1 gave the mono p-nitrobenzyl ester mono sodium salt of MM 17880 (200 mg).

Example 57 The di-sodium salt of MM 13902 (0.2g, O.46mmol) was treated with methyl tosylate (0.25g, 1.47mmol) in dimethylformamide (5ml) for 3 h at room temperature. Work-up and chromatography as described in Example 1 afforded the mono methyl ester, mono sodium salt of MM 13902 as a white solid (0.06g, 30%).

Example 38 To a suspension of the disodium salt of MM 13902 (0.3g, 0.69 mmol) in acetonitrile (10ml) was added 15-crown-5 (3 drops) and benzyl bromide (0.35g, 2.05 mmol). After stirring at room temperature for 3 h, the mixture was treated as in Example 1. The mono-benzyl ester, mono sodium salt of MM 13902 was obtained as an off white solid (0.145g,42%).

Example 39 A sample of crude (a& 60% pure) disodium salt of MM 17880 (lg) was treated with methyl iodide (1ml) in IMF (15ml). After 2 h the solvent was removed in vacuo, and the residue chromatographed on silica-gel (30g) using a gradient elution from chloroform to chloroform/ethanol (2.3). The fractions containing the product were combined and evaporated in vacuo. Ethyl acetate was added to the residue and then removed in vacuo to afford the monomethyl ester mono-sodium salt of MM 17880 as a light-yellow solid (0.25g); λ max. (HgO) 3l4nm. v max. (KBr) 1765, 1700, 1.660br and 1220-1270br cm1. s(DMS0-dJ 1.35 a (3H, d, J 6Hz, CH3CH), 1.80 (3H, s, CHgCO), 2.8-3.8 (7H, m,CH.CH.CH.CH2 C.SCH2CH2N), 3.65 (3H,s,C02CH3) 3.95 -4.6 (2H,m,CH.CH.CH and 8.0 (1H, br NH).

Example 40 The mono p-bromobenzyl, mono sodium salt of MM 13902 (0.05g, 0.086mmol) was dissolved in water (5ml) and the solution shaken with one of cetylbenzyldimethylammonium chloride (0.032g, 0.08mmol) in dichloromethane (5ml).

The organic layer was separated, dried (MgSO^) and evaporated in vacuo to afford the mono p-bromobenzyl ester, mono cetylbenzyldimethylammonium salt of MM 13902 as a foam (0.074g,93%); vmax. (CH2C12) 1780, 1700, 1685sh and 1625cm~1.

Example 41 A solution of the mono methyl ester, mono sodium salt of 3 3 59 MM 13902 (0.10 g, 0.23 mmol) in water (5ml) was treated with a solution of cetylbenzyldimethylammonium chloride (0.09 g, 0.23 mmol) in CH2C12 (5 ml) as described in Example 40. The mono methyl ester, mono-cetylbenzyldimethyl5 ammonium salt of MM 13902 was obtained as a gum (0.156 g, 87%) υ max (CHC13) 1780, 1695 and 1630 cm-1.

Example 42 The di-sodium salt of MM 13902 (1.0 g, 2.29 mmol) was treated with bromophthalide (0.60 g, 2.82 mmol) in dimethylformamide (15 ml) at room temperature. After 1.5 hours the dimethylformamide was removed in vacuo, and the residue chromatographed on silica gel (30 g) using a gradient elution (CHC13 to CHCl3:EtOH, 1:1).

The first few fractions which contained the mono15 phthalide ester of MM 13902 (by thin layer chromatography) slowly deposited white crystals. These were obtained by filtration (0.03 g). The mother liquors and the remaining fractions were combined and evaporated in vacuo. Ethyl acetate was added and the solvents again removed in vacuo to afford the mono-phthalide ester, mono sodium salt of MM 13902 as a cream-coloured amorphous solid (0.62 g, 50%). λ max (H2O) 333 and 233 nm; υ max (KBr) 1775, 1680 br, 1620, 1210 and 1275 cm1; - 35 4SS59 The white crystals also consisted of the required mono-phthalide ester of-MM 13902, S(DMSO-dg) 1.32 (3H, d, J 6 Hz, CH3CH), 1.92 (3H, s, CHjCO), 2.98 (lH, dd, J 20 and 10 Hz, HA of ABX), 3.45 - 3.75 (2H, m, Ηβ of ABX and CHCHCH), 4.05 - 4.55 (2H, m, CHCHCH), 5.80 (IH, d, J 14 Hz, CH=CH.S), 7.05 (IH, dd, J 14 and 11 Hz, NH.CH=CH) 7.49 (IH, s, COjCHO), 7.55 - 8.0 (4H, m, aromatic protons) Example 43 Using the determination method of Belgian Patent No. 827926, the sodium salts of esters of this invention have been found to have the following values: MM 13902 Esters Staph. aureus Russell 0.563 TT CJ • O 2.2 23 0.2 0.22 0.2 1.6 0.68 rt Dl 43 CO ta 0 03 CJ © to Cl © s τ! iri O ta Γ- •cr to fQ © ri fQ o o © © rri to taw cu s ta • © © • © © © © © o O GH rt rt V rt Q •ri in fQ ta *4* Tf fQ' Cl in 0 Ί* 0 64 © o © ri 40 O © © © © © o • fa Λ Λ ω 0} Φ in in Kleb. roger E 70 O ri1 o rri ta © o > 40 0.4 © © 0.02 0.02 0.23 rt rt » tn *ri o oo CJ 10 Cl CJ CJ to 3 η © © o © ri iri O 0 »ri oo © © © © © O o o w ΑΑ» o rt © • © O o © © © o o MMU V rt 03 to to Cl in rri fi in © CJ •ri © o o © w in S'*3 Cl ri ta O fQ © © © O Pu a ri 14 Λ rt rt 14 Ol rtOl 44 □ CU rt 4e O) * «fi Φ N © CM Ό* fQ © © Tf 0 rt CJ cf O ri © © © © to u © © © O © © © © © Φ rt 44 O • o • © © © © t © © © © fi H V ω υ iri >< >1 iri to a ri Sh Φ 0 rri ϋ 44 ri X Sh iri iri rt ri ω 0 fi X >4 >1 fi >i a N Φ a N X Φ rt © rt a a 43 •ri rri X ri CQ Φ Φ rri ί>Η 0 N >» I CQ CQ 1 rt N N o X{ to to 43 fi fi a 4-1 a M H £Q 44 Φ Φ 1 Φ 1 Ol rfi CQ a hl s cu cu 0 CU β ο to ω rt N fi Φ 0) rfi -P to Φ A3 •ri Φ W •ri υ •ri £ •ri 44 fi >1 rt n S fi 0) I to rt φ tn M »ri rt S H rt o 44 Ο Φ rt rt rri o I rt ca 0 tn 0 •5. 01 0 3 b ϋ Ό rt rt Λ C 0 •H 1-1 « rt +) 4-1 Z3 G Ο H * 37 MM 17880 Esters Staph. aureus | Russell I 1.4 Ps. aeruginosa Dalgleish 2.32 0.057 Ή r-I 0 rp □ • « 19.5 0.4 Kleb. aerogenes E 70 0.6 Proteus mirabilis C 889 0.004 1 0.007 Ps. aeruginosa A 0.6 1 > 40 1 M 0\ Φ cn P U Cm rtf X» Φ 0 « Μ ϋ φ d +) o G H ω □ 0.116 0.022 Ester Methyl -1 N fl Staph. aureus Russell 0.82 23.0 2.8 (0 Μ Λ 0 w 00 G *H © in •cp • *rl Φ © 00 H r-l M tni-l • • • • ft fl O' © © © © M «Μ V (0 Q •M r-ί tn 00 0 * n ϋ fH • • EH o © o • A ω ta • c ni σ\ neo • • •tf r-l fH f-l W M W 0) rd Λ Λ us lis 9 CM 0) *h co rd © •UflS o •cP © © 0 ni • • • « MMO & Ή o © © o s (ϋ ω •cj* 0 00 fl « • «Μ fH o o M Cnfli ft fl Λ ΈΡ rp M Λ A 0 Λ A rtf M o\ Φ cn +> 0 ft oo tn . fd © f-l Λ © © 0 n> • • © O W 0 o o •cp rp Φ fl +J 0 V A A fl i-t H 0 H >1 x3 4J H 0) ε Λ H 4J 5>t M fl g ¢) 0 bn «Μ H hi ta 0 0 ω M H rd >i X 0 H H 0 H >1 N ftf Χί N G +1 fl •M J} 0) ffl ft E cq

Claims (37)

1. The salts of the compounds.of the formula (II): wherein R is an organic group of up to 16 carbon atoms 5 and X is a trans so.ch=ch, trans S.CH=CH or S.CH 2 CH 2 group.

2. The salts as claimed in claim 1 of the compound of the formula (II) wherein X is a trans SO.CHCH group.

3. The salts as claimed in claim 1 of the compound of the formula (II) wherein X is a trans S.CH=CH group. 10

4. The salts as claimed in claim 1 of the compound of the formula (IX) wherein X is a S.CH 2 CH 2 group.

5. A salt as claimed in any of claims 1-4 wherein R is a methyl, ethyl, propyl, butyl or benzyl group or a benzyl group substituted by one or two halogen atoms or C 1-4 alkoxy or nitro groups or is a benzhydryl group or a benzhydryl group substituted by one or two halogen atoms or C 1-4 alkoxy or nitro groups.

6. A salt as claimed in any of claims 1-4 wherein the ester group C0 2 R is of the formula CO^ 1 wherein the group COjR 1 is readily hydrolysed in the human body.

7. A salt as claimed in claim 6 wherein R 1 is a group of the sub-formulae (a) - (c): R 2 / 3 - CH - 0 - CO - R J (a) 2 3 wherein R is a hydrogen atom or a methyl group; R is a C lu , 4 alkyl, alkoxy, phenyl or benzyl group; and R 4 is a hydrogen atom or a methoxy group. 40 4SS59

8. A salt as claimed in claim 7 wherein R is hydrogen.

9. A salt as claimed in claims 7 or 8 wherein R is methyl, t-butyl, phenyl or ethoxy.

10. A salt as claimed in claim 7 wherein the group R 1 is S of the sub-formula (b).

11. A salt as claimed in claim 1 of a compound of the 10 wherein X 1 is a trans -SO-CH=CH-, trans -S-CH=CH- or -S-CH 2 ~CH 2 - group.

12. A salt as claimed in claim 11 wherein X 1 is a trans -S-CH«CH- group.

13. A salt as claimed in claim 11 wherein X 1 is a 15 -S-CH 2 -CH 2 - group.

14. A salt as claimed in any of claims 1 to 13 which is the sodium or potassium salt.

15. A salt as claimed in any of claims 1 to 13 which is the sodium salt.

16. A process for the preparation of a compound as claimed in claim 1 which process comprises the esterification of the corresponding compound of the formula (I) wherein X is as defined in claim 1 or a salt thereof. 10

17. A process as claimed in claim 16 which comprises the esterification of a salt of a compound of the formula (I).

18. A process as claimed in claim 17 which comprises the reaction of a di-salt of a compound of the formula (I) 15 with a compound of the formula (IV): R - Q (IV) wherein R is as defined in claim 1 and Q is a readily displaceable group. 45553

19. A process as claimed in claim 17 which comprises the reaction of a mono-salt of the compound of the formula (I) with a diazoalkane.

20. A process as claimed in claim 18 wherein Q is a 5 chlorine, bromine or iodine atom or a O.SOgCHg or O.SO 2 CgH 4 CH 3 group.

21. A process as claimed in claim 18 or 20 wherein R is a phthalidyl group.

22. A process as claimed in claim 18 wherein R - Q is phthalidyl bromide.

23. A process as claimed in any of claims 17 to 22 wherein X is a trans SO.CH»CH group.

24. A process as claimed in any of claims 17 to 22 wherein X is a trans S.CH=CH group. 15

25. A process as claimed in any of claims 17 to 22 wherein X is a S.CH 2 CH 2 group.

26. A pharmaceutical composition which comprises a compound as claimed in any of claims 1 to 15 and a pharmaceutically acceptable carrier. 43 4S3S9

27. A composition, as claimed in claim 26 adapted for administration by injection.

28. A composition as claimed in claims 26 or 27 which also comprises a penicillin or cephalosporin. 5

29. A composition as claimed in claim 28 wherein the ratio of compound of the invention to penicillin or cephalosporin is from 10:1 to 1:10.

30. A composition as claimed in claim 29 wherein the ratio is 3:1 to 1:3. 10

31. A composition as claimed in any of claims 26 to 30 which comprises a compound as claimed in claim 11.

32. A composition as claimed in any of claims 26 to 30 which contains a compound as claimed in claim 12.

33. A process as claimed in claim 16 substantially as 15 described in any Example herein.

34. A compound as claimed in claim 1 prepared in any Example herein.

35. A compound as claimed in claim 1 whenever prepared substantially as described in any Example herein. - 44 JSS59

36. The sodium salt of the compound as claimed in claim 12.

37. The sodium salt as claimed in claim 36 in crystalline form.