GB2049660A - 2-Spirocyclopropanbisnor- penicillanic acid derivatives - Google Patents

Abstract

The compounds have the 3S, 5R, 6R configuration and are represented by formula (IV): <IMAGE> wherein R represents an optionally substituted amino group. (including acylamine). They have antibiotic activity.

Description

SPECIFICATION Penicillin Derivatives The present invention relates to penicillin derivatives, to a process for their preparation, to intermediates useful in that process and to pharmaceutical compositions containing the penicillin derivatives.

Belgian Patent No. 854,946 discloses 6-amino-spiro-cycloalkane-penicillanic acid derivatives of formula (1):

wherein n represents an integer from 3 to 6 and Belgian Patent No. 854,947 discloses 6-acylamino derivatives of compound (I).

Although the process described for the preparation of compound (I) gives the correct relative stereo-chemistry at the three chiral centres, the products are racemic mixtures. By analogy with established properties of penicillins, it is generally believed that all the antibacterial activity resides in a single isomer, the 3S, 5R, 6R, or "natural" isomer.

Co-pending British Patent Application No. 6722/76 (Serial No. 1,546,622) discloses (3S, 5R, 6R) bisnor penicillanic acid compounds derived from the 6-amino compound (ill):

which may be prepared from the optically active mercaptoazetidinone of formula (III) disclosed in copending British Patent Application No. 4342/76 (Serial No. 1,543,046), and which is itself derived from natural Denicillin V.

It has now been found that the mercaptoazetidinone (III) may be used to prepare 2 spirocyclopropanbisnor-penicillanic acid derivatives having the 3S, SR, 6R configuration. Belgian Patent Nos. 854,946 and 854,947 do not disclose spiro-cyclopropane derivatives and it is unlikely that such compounds could be prepared by the process described therein. In addition, whereas the Belgian patents produce a racemic mixture the present process is capable of leading directly to a single desired stereoisomer.

The present invention provides a compound of formula (IV) or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof:

wherein R represents an organic acylamino group, a group of formula: our a group of formula:

wherein R1 and R2 each represent a C13 alkyl group of R1 and R2 taken together with the nitrogen atom to which they are attached form a monocyclic ring.

The compounds of the present invention include the pharmaceutically acceptable esters of compound (IV) which hydrolyse readily in the human body to produce the parent acid, for example acyloxyalkyl groups such as acetoxymethyl, pivaloyloxymethyl, a-acetoxyethyl, ct-acetoxybenzyl and a- pivaloyloxyethyl groups; alkoxy-carbonyloxyalkyl groups, such as ethoxycarbonyloxymethyl and aethoxycarbonyloxyethyl; dialkylaminoalkyl groups such as dimethylaminomethyl, dimethylaminoethyl, diethylaminomethyl or diethylaminoethyl; and lactone groups such as phthalidyl.

Suitable salts of the compound of formula (IV) include metal salts, e.g. aluminium, alkali metal salts such as sodium or potassium, alkaline earth metal salts such as calcium or magnesium, and ammonium or substituted ammonium salts, for example those with lower alkylamino such as triethylamine, hydroxy-lower alkylamines such as 2-hydroxyethylamine, bis-(2-hydroxyethyl)-amine or tri-(2-hydroxyethyl)-amine, cycloalkylamines such as bicyclohexylamine, or with procaine, dibenzylamine, N,N-dibenzylethylenediamine, 1-ephenamine, N-ethylpiperidine, N-benzyl-P- phenethyalamine, dehydroabietylamine, N,N"-bis-dehydroabietyl-ethyienediamine, or bases of the pyridine type such as pyridine, collidine or quinoline, or other amines which have been used for form salts with benzyl-penicillin.

When the compound of formula (IV) contains a free amino group, pharmaceutically acceptable acid addition salts of such a compound are also included within this invention. Suitable acid addition salts of the compounds of formula (IV) include, for example, inorganic salts such as the sulphate, nitrate, phosphate and borate; hydrohalides e.g. hydrochloride, hydrobromide and hydroiodide; and organic acid addition salts such as acetate, oxalate, tartrate, maleate, citrate, succinate, benzoate, ascorbate, methanesulphonate and p-toluenesulphonate, trifluoroacetate.

When R is an organic acylamino group, specific examples include any of the acylamino side chains found in known antibacterially active penicillins. It has been found over the years that by varying the identity of the 6-acylamino group of penams, the spectrum and/or level or antibacterial activity of any given penam can be modified. Similarly, in the present case, a very large number of 6-acylamino groups can be introduced, producing a range of compounds of widely differing spectra and levels of activity. In general, however, whatever the identity of the acylamino group R, the compounds of formula (IV) possess some activity and those who are familiar with the penicillin art will be aware of the range of acylamino groups R which may be introduced.

In general, therefore, R in formula (IV) may be any of the organic acylamino groups which are present in the reported natural and semi-synthetic penicillins. The acyl portion may be, for example, one of the following groups of formulae (i)-(iv):

wherein RU represents hydrogen, alkyl, cycloalkyl (especially C3 to Ce cycloalkyl) alkenyl, cycloalkenyl, aryl (especially phenyl or substituted phenyl) or heterocyclic; X represents hydrogen, halogen, carboxy, esterified carboxy, hydroxy, azide, amino, substituted amino (including ureido, substituted ureido, for example acylureido, guanidino and substituted guanidino groups), a triazolyl group, a tetrazolyl group, a cyano group, an acyloxy group (e.g. formyloxy or lower alkanoyloxy group) or an esterified hydroxy group; and p and q each separately represent 0, 1, 2 or 3.

wherein X1 is -CH20CH2-, -CH2SCH2- or (CH2)r wherein r is an integer from 1 to 4 and X is as defined in (i) above.

wherein R5 is an alkyl, aralkyl, aryl (especially phenyl or substituted phenyl group), cycloalkyl (especially a C3 to C6 cycloalkyl or substituted cycloalkyl group), cycloalkenyl (especially a cyclohexenyl or cyclohexadienyl group) or a heterocyclic group (especially a thienyl or pyridyl group); R6 and R7 are each hydrogen, lower alkyl, phenyl, benzyl or phenylethyl groups; and Z is oxygen or sulphur.

wherein R6 is a lower alkyl group and RU is as defined above.

R9--COO- (v) wherein R9 is a bulky aromatic group such as a 2,6-dimethoxyphenyl, 2-alkoxy-1 -naphthyl, 3 arylisoxoazolyl or 3-aryl-5-methylisoxazolyl group.

Specific examples of organic acylamino groups R which may be present in the compounds of this invention include 2-thienylacetamido, 3-thienylacetamido, phenylacetamido, 2 hydroxyphenylaceta m ido, 2-a m inophenylacetam ido, 4-pyridylacetam ido, 2-a m ino-2-(4 hydroxyphenyl)acetamido, 2-methoxyimino-2-fur-2'-ylacetamido, 2-carboxy-2-thien-3-ylacetamido, 2-carboxy-2-phenylacetam ido, 2-carboxy-2-(4-hydroxyphenyl)a ceta mido, 1 -tetrazolylaceta mido and phenoxyacetamido.

One class of compounds of formula (I) are those wherein the acyl portion of the group R is of formula (i) wherein p and q are zero; RU represents a furyl, thienyl, phenoxy, cycloalkyl, cycloalkenyl or phenyl group, or a phenyl group substituted by hydroxy, halogen, nitro, lower alkyl, lower alkoxy, amino, or carboxy; and X represents hydrogen, hydroxy, amino, carboxy, salted carboxy, esterified carboxy, ureido, or acylureido.

Within this class, suitable groups RU include 2- and 3-furyl, 2- and 3-thienyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexa-1, 4-dienyl, phenyl, 4-hydroxyphenyl, 3-chloro-4-hydroxyphenyl, 3,4-dihydroxyphenyl, 4-methoxyphenyl.

Preferably RU is a 2- or 3-thienyl, phenyl or 4-hydroxyphenyl group.

Specific examples of groups of compounds of formula (IV) are those of formulae (V), (VI) and (VII):

wherein R10 is phenyl or p-hydroxyphenyl;

wherein R1' is phenyl, 2-thienyl or 3-thienyl and R12 is hydrogen or a C110 hydrocarbon radical;

Compounds of formula (IV), salts and esters thereof, may be prepared by reacting a compound of formula (VII I):

wherein RX is hydrogen or a carboxyl-blocking group, with an N-acylating derivative of an acid of formula (IX):: Rq OH OH (IX) wherein Rq is an organic acyl group such that din formula (IV) represents RqNH and wherein any reactive groups (such as amino, carboxy and hydroxy groups) may be blocked, and thereafter, if necessary, carrying out one or more of the following steps: i) removing any blocking group from the group RX; ii) removing any blocking group from the acyl side chain; iii) converting the product to a salt or ester thereof.

Suitable carboxyl-blocking derivatives for the group CO2RX in formula (VIII) include salts, ester, and anhydride derivatives of the carboxylic acid. The derivative is preferably one which may readily be cleaved at a later stage of the reaction. Suitable salts include tertiary amine salts, such as those with tri-!ower-alkylamines, N-ethylpiperidine, 2,6-lutidine, pyridine, N-methylpyrrolidine, dimethylpiperazine. A preferred salt is with triethylamine.

Suitable ester groups of formula CO2RX include the following: i) COOCRcRdRe wherein at least one of Rc, Re and Re is an electron-donor e.g. p-methoxyphenyl, 2,4,6-trimethyiphenyl, 9-anthryl, methoxy, acetoxy, orfur-2-yl. The remaining Rc, Rd and R, groups may be hydrogen or organic substituting groups. Suitable ester groups of this type include pmethoxybenzyloxycarbonyl, 2,4,6-trimethylbenzyloxy carbonyl, bis-(pmethoxyphenyl)methoxycarbonyl, and 2,5- di-t-butyl-4-hydroxybenzyloxycarbonyl.

ii) COOCRCRdRe wherein at least one of Rc, Rd and Re is an electron-attracting group e.g.

benzoyl, p-nitrophenyl, 4-pyridyl, trichloromethyl, tribromomethyl, iodomethyl, cyanomethyl, ethoxycarbonylmethyl, arylsulphonylmethyl, 2-dimethylsulphonylmethyl, 2-dimethylsulphoniumethyl, o-nitrophenyl or cyano. The remaining Rc, Rd and Re groups may be hydrogen or organic substituting groups. Suitable esters of this type include benzoylmethoxycarbonyl, p-nitrobenzyloxycarbonyl, 4pyridylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl and 2,2,2-tribromoethoxycarbonyl.

iii) COOCRoRdRe wherein at least two of Rc, Rd and Re are hydrocarbon such as alkyl e.g. methyl or ethyl, or aryl e.g. phenyl and the remaining Rc, Rd and Re group, if there is one, is hydrogen. Suitable esters of this type include t-butyloxycarbonyl, t-amyloxycarbonyl, diphenylmethoxycarbonyl and triphenylmethoxycarbonyl.

iv) COORf wherein Rf is adamantyl, 2-benzyloxyphenyl, 4-methylthiophenyl, tetrahydrofur-2-yl, tetrahydropyran-2-yl, pentachlorophenyl.

v) Silyloxycarbonyl groups obtained by reaction of a silylating agent, such as trimethylsilylchloride with the carboxylic acid group.

vi) Trialkyltin esters.

vii) Oxime esters of formula CO2N=CH Rg where Rg is aryl or heterocyclic.

The carboxyl group may be regenerated from any of the above esters by usual methods appropriate to the particular RX group, for example, acid- and base-catalysed hydrolysis, or by enzymically-catalysed hydrolysis, provided that the method selected is sufficiently mild as not to cause substantial degradation of the sensitive fused ring system (IV).

Alternative methods of cleavage include: reduction with agents such as zinc/aqueous acetic acid, zinc/formic acid, zinc/lower alcohol, zinc/pyridine, or hydrogen and palladised-charcoal or other supported hydrogenation catalysts; attack by nucleophiles, such as those containing a nucleophilic oxygen or sulphur atom for example alcohols, mercaptans and water; oxidative methods, for example, those which involve the use of hydrogen peroxide and acetic acid; and irradiation with light or u.v.

A reactive N-acylating derivative of the acid (IX) is employed in the above process. The choice of reactive derivative will of course be influenced by the chemical nature of the substituents of the acid.

Suitable N-acylating derivatives include an acid halide preferably the acid chloride or bromide.

Acylation with an acid halide may be effected in the presence of an acid binding agent for example tertiary amine (such as triethylamine or dimethylaniline), an inorganic base (such as calcium carbonate or sodium bicarbonate) or an oxirane, which binds hydrogen halide liberated in the acylation reaction.

The oxirane is preferably a (C16)-1 ,2-alkylene oxide - such as ethylene oxide or propylene oxide. The acylation reaction using an acid halide may be carried out at a temperature in the range -500C to +500 C, preferably20 C to +200 C, in an aqueous or non-aqueous media such as aqueous acetone, ethyl acetate, dimethylacetamide, dimethylformamide, acetonitrile, dichloromethane, 1,2 dichloroethane, or mixtures thereof. Alternatively, the reaction may be carried out in an unstable emulsion of water-immiscible solvent, especially an aliphatic ester or ketone, such as methyl isobutyl ketone or butyl acetate.

The acid halide may be prepared by reacting the acid (IX) or a salt thereof with a halogenating (e.g. chlorinating or brominating) agent such as phosphorus pentachloride, thionyl chloride or oxalyl chloride.

Alternatively, the N-acylating derivative of the acid (IX) may be a symmetrical or mixed anhydride.

Suitable mixed anhydrides are alkoxyformic anhydrides, or anhydrides with, for example carbonic acid monoesters, trimethyl acetic acid, thioacetic acid, diphenylacetic acid, benzoic acid, phosphorus acids (such as phosphoric or phosphorous acids), sulphuric acid or aliphatic or aromatic sulphonic acids (such as p-toluenesulphonic acid). The mixed or symmetrical anhydrides may be generated in situ. For example, a mixed an hydroxide may be generated using N-ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline.

When a symmetrical anhydride is employed, the reaction may be carried out in the presence of 2,4lutidine as catalyst.

Alternative N-acylating derivatives of acid (IX) are the acid azide, or activated esters such as esters with 2-mercaptopyridine, cyanomethanol, p-nitrophenol, 2,4-dinitrophenol, thiophenol, halophenol, including pentachlorophenol, monomethoxyphenol or 8-hydroxyquinoline; or amides such as N-acylsaccharins or N-acyl-phthalimides; or an alkylidene iminoester prepared by reaction of the acid (IX) with an oxime.

Some activated esters, for example the ester formed with 1 -hydroxybenztriazole or Nhydroxysuccinimide, may be prepared in situ by the reaction of the acid with the appropriate hydroxy compound in the presence of a carbodiimide, preferably dicyclohexylcarbodiimide.

Other reactive N-acylating derivatives of the acid (IX) include the reactive intermediate formed by reaction in situ with a condensing agent such as a carbodiimide, for example N,N-diethyl-, dipropylor diisopropylcarbodiimide, N,N'-di-cyclohexylcarbodiimide, or N-ethyl-N'-ydimethylaminopropylcarbodiimide; a suitable carbonyl compound, for example N,N'carbonyldiimidazole or N,N'-carbonylditriazole; an isoxazolinium salt, for example N-ethyl-5phenylisoxazolinium-3-sulphonate or N-t-butyl-5-methylisoxazolinium perchlorate; or an Nalkoxycarbonyl-2-alkoxy- 1 ,2-dihydroquinoline, such as N-ethoxycarbonyl-2-ethoxy-1 ,2dihydroquinoline. Other condensing agents include Lewis acids (for example BBr3-C6H6); or a phosphoric acid condensing agent such as diethylphosphorylcyanide.The condensation reaction is preferably carried out in an organic reaction medium, for example methylene chloride, dimethylformamide, acetonitrile, alcohol, benzene, dioxan, or tetrahydrofuran.

With the above route it is preferable to protect any reactive groups in the acyl side chain prior to the acylation reaction. When the group to be protected is an amino group, any of the amino protecting groups known from the literature on the synthesis of a-aminobenzyl penicillin are suitable.

Examples of protected amino groups include the protonated amino group (NH+3) which after the acylation reaction can be converted to free amino group by simple neutralisation; the benzyloxycarbonylamino group or substituted benzyloxy-carbonylamino groups which are subsequently converted to NH2 by catalytic hydrogenation; and various groups which after the acylation reaction regenerate the amino group on mild acid hydrolysis, such as the 2-hydroxy-1-naphthylidene amino group which may be removed by treatment with very dilute aqueous acid.

Another example of a protected amino group which may subsequently be converted to NH2 by mild acid hydrolysis includes groups of formula (X):

wherein RA is an alkyl, aralkyl or aryl group, R6 is an alkyl, aralkyl, aryl, alkoxy, aralkoxy or aryloxy group, and Rc is a hydrogen atom or an alkyl, aralkyl, or aryl group, or Rc together with either RA or R6 completes a carbocylic ring.

An example of a "protected amino" which can be converted to NH2 after the N-acylation reaction is the azido group. In this case, the final conversion into NH2 may be brought about by either catalytic hydrogenation or electrolytic reduction. Alternatively the amino group may be blocked as the nitro group which is later converted to the amino group by reduction.

When the group R in formula (IV) represents a group of formula:

then the compounds of the invention may be prepared by reacting a compound of formula (VIII) with a reactive derivative of an amide of formula (XI):

wherein R1 and R2 are as defined above with respect to formula (IV) and optionally thereafter carrying out one or both of the following steps: i) removing any blocking groups from the group RX; ii) converting the product to a salt of ester thereof.

The compounds of formula (VIII) are valuable intermediates and as such form a further aspect of this invention.

Compound (VIII) may be prepared by: a) treating a compound of formula (XII):

wherein RV is a carboxyl blocking group with an agent forming an imino halide; b) treating the imino halide with a compound to introduce a group QRf on the imino carbon atom, wherein Q is oxygen, sulphur or nitrogen and Rf is an alkyl group of from 1 to 12 carbon atoms or an aralkyl group of from 5 to 1 4 carbon atoms, to form an iminoether, iminothioether, or amidine (when O is 0, S, or N respectively); c) treating with water; and d) optionally removing the carboxyl-blocking group RY.

A suitable agent for preparing an imino halide is an acid halide in the presence of an acid binding agent such as a tertiary amine, e.g. pyridine, triethylamine, or N,N-dimethylaniline. Examples of suitable acid halides are phosphorus pentachloride, phosgene, phosphorus pentabromide, phosphorus oxychloride, oxalyl chloride and p-toluene sulphonic acid chloride. Phosphorus pentachloride and phosphorus oxychloride are preferred. The reaction may be conducted under cooling, preferably at temperatures from OOC to -300C when phosphorus pentachloride is employed. The amount of the tertiary amine is preferably 3-5 mols per mol of phosphorus pentachloride. It is also preferable to use the phosphorus halide in an amount slightly in excess of that of the starting material.

The resulting imino compounds are then treated to introduce a QRf grouping onto the imino carbon atom. This is preferably effected by reacting the imino halide with a corresponding alcohol.

Examples of suitable alcohols for reaction with the imino halide are aliphatic alcohols containing from 1 to 12 carbon atoms, preferably 1 to 5 carbon atoms, such as methanol, ethanol, propanol, isopropyl alcohol, amyl alcohol and butyl alcohol, and aralkyl alcohols such as benzyl alcohol and 2phenylethanol.

The reaction of the alcohol with the imino halide is preferably effected in the presence of an acid binding agent, such as a tertiary amine, preferably pyridine, and the reaction is usually carried out without isolating the imino halide from the reaction mixture.

Finally, the product is treated with water. The water treatment may be conducted together with the isolation of the desired material. That is the reaction mixture may be added to water or a saturated aqueous solution of sodium chloride and then the aqueous layer formed is separated from the organic solvent layer.

The compound of formula (XII) may be prepared from the mercaptoazetidinone (III) by the following reaction sequence:

The antibiotic compounds according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other antibiotics, and the invention therefore includes within its scope a pharmaceutical composition comprising a compound of formula (IV) above together with a pharmaceutical carrier or excipient.

The compositions may be formulated for administration by any route, such as oral, topical or parenteral. The compositions may be in the form of tablets, capsules, powders, granules, lozenges, creams or liquid preparations, such as oral or sterile parenteral solutions or suspensions.

Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan mono-oleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl phydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.

Suppositories will contain conventional suppository bases, e.g. cocoa-butter or other glyceride.

For parenteral administration, fluid unit dosage forms are prepared utilizing the compound and a sterile vehicle, water being preferred. The compound, depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions the compound can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampoule and sealing. Advantageously, agents such as a local anesthetic, preservative and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use.

Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and sterilization cannot be accomplished by filtration. The compound can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.

The compositions may contain from 0.1% by weight, preferably from 1060% by weight, of the active material, depending on the method of administration. Where the compositions comprise dosage units, each unit will preferably contain from 50-500 mg of the active ingredient. The dosage as employed for adult human treatment will preferably range from 100--3000 mg per day, for instance 1 500 mg per day depending on the route and frequency of administration.

The compound of formula (IV) may be the sole therepeutic agent in the compositions of the invention or a combination with other antibiotics may be employed. Advantageously the compositions also comprise a compound of formula (XIII) or a pharmaceutically acceptable salt or ester thereof:

wherein A is hydroxyl, substituted hydroxyl, thiol, substituted thiol, amino, mono- or di-hydrocarbylsubstituted amino, or mono or di-acylamino.

The following Examples illustrate the preparation of some of the compounds of this invention.

Example 1 (3S 5R, 6R)-Benzyl 6-amino-2-spirocyclopropanbisnor-penicillanate a) Carbobenzyloxybromomethylene triphenylphosphorane

Carbobenzyloxybromamethylene triphenylphosphorane (1) was prepared by the method described for the preparation of carbomethoxybromomethylene triphenylphosphorane (G. Märkl, Chem.

Ber., 94, 2996 (1961)).

Thus treatment of carbobenzyloxymethyl triphenylphosphonium bromide (prepared from benzyl bromoacetate and triphenylphosphine in refluxing benzene) with a solution of bromine in water followed by sodium hydroxide solution gave the desired ylid (1) (67%) as an off white amorphous solid.

mp 122-1250C Vmex (CHCl3) 1630, (shoulder) cm-1.

b) Benzyl 2-bromocyclopropylideneacetate

A solution of carbobenzyloxybromomethylene triphenylphosphorane (1) (62.8 g) in dry benzene (600 ml) was added dropwise to a slowly distilling mixture of 1-ethoxycyclopropanol (2) (13.1 g) (J.

Salaün, J. Org. Chem. 41, 1237, (1976)), benzoic acid (3.14 g), and dry benzene (500 ml). The rate of addition was adjusted to maintain a constant volume in the distillation flask. After the addition was complete the reaction mixture was cooled and evaporated to give a crude oil. The residual oil was extracted with a mixture of ethyl acetate/petrol (1/9) (4x300 ml). The combined extracts were concentrated and chromatographed on silica gel eluting with ethyl acetate/petroleum spirit 60-800C mixtures to give an oil which partially crystallised (10.0 g). The NMR revealed the presence of benzyl bromoacetate (10--20%) but this material proved to be of sufficient purity for further synthetic work.

However, rapid distillation of the mixture gave rise to pure benzyl 2-bromocyclopropylideneacetate (3) bp. 115-1 200C (0.01 mm). Vmax (CHCl3) 1720 cm-. a ppm (CCl4) 1.10-1.92 (4 H, m), 5.29 (2 H, s), 7.40 (5 H, s).

c) (3S SR, 6R)-BenzyI 2-spirocyclopropan-6-phenoxy-acetamidobisnorpenicillanate

(i) With Isolation of the Intermediate Bromide (5) Finely powdered anhydrous potassium carbonate (275 mg) was added portionwise in 5 minutes to a stirred mixture of the 4-mercaptoazetidinone (4) (2.0 g) (UK Patent Application No. 04342/76) (Serial No. 1,543,046) and benzyl 2-bromocyclopropylideneacetate (3) (2.16 g, containing 1020% benzylbromoacetate) in hexamethylphosphoramide (30 ml) at room temperature. The mixture was stirred at room temperature for a further 30 minutes, diluted with ethyl acetate (1 50 ml) and washed with brine. The dried (MgSO4) organic layer was evaporated to give a crude gum (4.37 g).

Chromatography of the crude product on silica gel eluting with ethyl acetate/petroleum spirit 60-- 800C mixtures gave the bromoester (5) (1.76 g), a mixture of stereoisomers, as an amorphous solid.

Vsx (CHCl3) 3430, 1780, 1740, 1690 cm-1. # ppm (CDCl3) 1.04-1.30 (4 H, m), 4.19 (+ H, s), 4.24 ( H, s), 4.44(2 H, s), 4.87 ( H, d, J 5 Hz), 5.08-5.19 (26 H, m). 5.30-5.55 (1 H, m), 6.48 br (1 H, s, exch. D2O), 6.78-7.36 (11 H, m).Also obtained was a more polar product, the benzyloxycarbonylmethylthioazetidinone (8) derived from the impurity in the starting material, as a solid mp 119-1 200C. #max (CHCl3) 3400, 1780, 1735, 1690 cm-1. a ppm (CDCl3) 3.30 (2 H, s), 4.60 (2 H, s), 5.10-5.24 (3 H, m), 5.61(1 H, dd, J 4 and 9 Hz), 6.70-7.70 (12 H, m). (Found C, 59.7; H, 5.0; N, 6.9; S, 8.1%. C20H20N205S requires C, 60.0; N, 5.0; N, 7.0; S,8.0%).

A mixture of the bromide stereoisomers (5) (2.0 g) and finely powdered anhydrous potassium carbonate (2.56 g) were stirred in hexamethylphosphoramide (30 ml) at room temperature for 8 hours.

The mixture was diluted with ethyl acetate (150 ml) and washed with brine (3x40 ml). The dried (MgS04) organic layer was evaporated to give a gum (3.54 g). Chromatography of the crude gum on silica gel eluting with ethyl acetate/petroleum spirit 60-800C mixtures gave two products. The less polar product, the desired 3S-epimer (6) (427 mg), was obtained as a gum. [&alpha;]D22+744 (c 1% in CHCl3) vial (CHCl3) 3420, 1795, 1745, 1690 cm-'. a ppm (CDCl3) 0.55-1.30 (4 H, m) 4.28 (1 H, s), 4.50(2H,s),5.13(2 H,s), 5.52(1 H, d, J 4Hz), 5.70(1 H, dd, J 4 and 8Hz), 6.76-7.36(11 H, m).

(Found M+,438.1 252. C23H22N2O5S requires M+, 438.1250). The more polar product, the 3R-epimer (7) (58 mg), was also obtained as a gum [&alpha;]D22+147.5 (c, 1% in CHCI3). Vmax (CHCI3) 3420, 1795, 1745, 1690 cm-'. a ppm (CDCI3) 0.85-1.30 (4 H, m), 3.58 (1 H, d, J approx 1 Hz), 4.47 (2 H, s), 5.1 5 (2 H, s), 5.33 (1 H, d, J 5 Hz), 5.59 (1 H, ddd, J 4,9 and approx 1 Hz), 6.76-7.35 (11 H, m). (Found M+, 438.1233. C23H22N2O5S requires M+, 438.1250).

(ii) Without Isolation of the Intermediate Bromide (5) Finely powdered anhydrous potassium carbonate (11.2 g) was added portionwise in 30 minutes to a stirred, ice bath cooled, mixture of the 4-mercaptoazetidinone (4) (4.10 g) and benzyl 2bromocyclopropylideneacetate (3) (4.35 g, containing 1020% benzyl bromoacetate) in hexamethylphosphoramide (80 ml). The mixture was stirred at room temperature for a further 40 hours. Work up as in (i) gave, after chromatography, the 3S-epimer (6) (1.07 g) as a gum and an impure sample of the 3R-epimer (7) (258 mg). No attempt was made to isolate the benzyloxycarbonylmethylthioazetidinone (8) by-product in this reaction.

d) (3S, SR, 6R)-Benzyl 6-amino-2-spirocyclopropanbisnorpenicillanate

A solution of phosphorous pentachloride (1 25 mg) in dry methylene chloride (2 ml) was added dropwise in 3 minutes to a stirred solution of the penam ester (6) (219 mg) and N-methylmorpholine (101 mg) in dry methylene chloride (5 ml) at -250C. The mixture was stirred for a further 30 minutes during which the temperature was allowed to attain OOC. The mixture was re-cooled to -250C and treated with N-methylmorpholine (101 mg) followed by dropwise addition of dry methanol (2 ml).After stirring at 05 C OC for 1 2 hours the mixture was poured into ice water (10 ml) and stirred at pH 2 for 10 minutes with cooling at 0--5 OC. The pH of the vigorously stirred mixture was adjusted to 6 using dilute ammonium hydroxide solution. The organic layer was separated and the aqueous layer reextracted with methylene chloride (2x3 ml). The combined organic layers were washed with saturated sodium bicarbonate solution (3 ml) and brine (3x5 ml). The dried (MgSO4) organic layer was evaporated to give a crude gum. The crude product was immediately dissolved in acetone (1 ml) and treated with a solution of p-toluene sulphonic acid monohydrate (95 mg) in acetone (0.5 ml). The mixture was diluted with ether and allowed to crystallise with cooling.The product was filtered off, washed with a little acetone, and dried under vacuum to give the p-toluene sulphonic acid salt (10) (33 mg) as a solid [a]22+47.40 (c, 0.5% in methanol). Vmex (nujol mull) 1785, 1730 cm-'. a ppm (CD3OD) 0.85-1.30 (4 H, m), 2.32 (3 H, s), 4.54 (1 H, s), 4.73 (s, -N+H3 hydrated), 5.04 (1 H, d, J 4 Hz), 5.17 (2 H, s), 5.58(1 H, d,J 4Hz), 7.15(2 H,d,J 8Hz), 7.30(5 H, s), 7.64(2 H, d,J 8Hz).

The p-toluene sulphonic acid salt (10) (15 mg) was shaken with ethyl acetate (2 ml) and saturated sodium bicarbonate solution (0.5 mi). The organic layer was separated and the aqueous layer re-extracted with ethyl acetate (2x 1 ml). The combined organic layers were washed with brine, dried (MgSO4), and evaporated to give the 6-amino-penam (11) (9 mg) as a gum. Vmex (CHCl3) 3400 (br), 1780, 1740 cm-. a ppm (CDCl3) 0.75-1.36 (cyclopropane m), 1.94 br (2 H, s), 4.27 (1 H, s), 4.56 (1 H,d,J4Hz),5.13(2H,s), 5.49(1 H, d,J4Hz), 7.28 (5 H, s).

Example 2 (3S, SR, 6R)-6-Phenoxyacetamido-2-spirocyclopropanbisnorpenicillanic Acid

The penam ester (6) (200 mg) was dissolved in a mixture of tetrahydrofuran (16 ml) and water 4 ml) and was hydrogenated over 10% palladium on charcoal catalyst (200 mg) at NTP for 15 minutes.

The mixture was filtered through Kieselghur and the residue washed with a little tetrahydrofuran. The combined filtrates were evaporated to low volume and diluted with ethyl acetate (10 ml). The vigorously stirred mixture was cooled in an ice-bath and the pH was adjusted to 7 using saturated sodium bicarbonate solution. The aqueous layer was separated and the organic layer was re-extracted with water (5 ml). The combined aqueous extracts were washed with ethyl acetate (5 ml). The vigorously stirred aqueous layer was covered with ethyl acetate (10 ml), cooled in an ice-bath, and the pH adjusted to 3 using dilute hydrochloric acid. The organic layer was separated and the aqueous layer was re-extracted with ethyl acetate (2x5 ml). The combined organic layers were washed with brine (3x5 ml), dried (MgSO4), and evaporated to give the free acid (9) (50 mg) as an amorphous solid.

[(D22+68.60 (c, 1% in CHCl3). Vmex (CHCl3) 3660-2300 (br), 1795, 1735, 1690 cm-. ô ppm (CDCl3) 0.50-1.50 (m, cyclopropyl CH2), 4.29 (1 H, s), 4.52 (2 H, s), 5.55 (1 H, d, J4 Hz), 5.71 (1 H, dd, J 4 and 8 Hz), 6.31 br (2 H, s, exch. D2O), 6.75-7.40 (aromatic m+NH).

The minimum inhibitory concentrations (MIC) of this compound required to inhibit the growth of various bacteria on nutrient agar are tabulated below.

Organism MIC {yg/ml) B. Subtilis 0.4 Staph. aureus Oxford 0.4 Staph. aureus Russeíl* > 100 Staph. aureus 1517* > 100 Strep. faecalis 1 6.2 /3-Haemolytic Strep. CN10 0.1 * Penicillin-resistant strains.

Example 3 (3S SR, 6R)-6-Da-a minophenylaceta mido-2-spirocyclo-propanbisnorpenicillanate a) (3S, SR, 6R)-Benzyl 6(N-p-nitrobenzyloxywarbonyl-Da-aminophenylacetamido)-2- spirocyclopropanbisnorpenicillanate

A solution containing N-p-nitrobenzyloxycarbonyl-Da-phenylglycine (48 mg), dry triethylamine (15 mg), and N,N-dimethylbenzylamine (1 drop) in dry tetrahydrofuran (1 ml) was added dropwise in 2 minutes to a stirred solution of methyl chloroformate (15 mg) in dry tetrahydrofuran (1 ml) at -100C.

After 25 minutes stirring at -1 00C a solution of the 6-amino penam ester (11) (40 mg) in dry tetrahydrofuran (0.5 ml) was added dropwise in 1 minute. The mixture was stirred at -1 00C for further 45 minutes, filtered and evaporated. The residual gum was dissolved in ethyl acetate (10 ml), washed with sodium bicarbonate solution and brine, dried (MgS04), and evaporated to give an amorphous solid (90 mg).Chromatography of the crude product on silica gel eluting with ethyl acetate/petroleum spirit 60-800C mixtures gave the desired penam ester (12) (52 mg) as an amorphous solid. [c}]D2+53.2 (c, 1% in CHCl3). Vmax (CHCl3) 3430, 3340, 1785, 1730, 1685 cam~1. ô ppm (CDCl3) 0.75-1.37 (4 H, m), 4.21 (1 H,s), 5.10 (4 H, s), 5.23 (1 H, d,J 6 Hz, collapses to s on exch. D2O), 5.42 (1 H, d,J 4Hz), 5.55 (1 H, dd, J 4 and 9 Hz, collapses to d, J 4 Hz, on exch. D20), 6.18 (1 H, d, J 6 Hz, exch. D2O), 6.38 (1 H, d, J 9 Hz, each. D2O), 7.18-7.34(12 H,m), 8.08 (2 H, d, J 9 Hz).

b) (3S,5R,6R)-6-D-aminophenylacetamido-2-spirocyclopropanbisnorpenicillanate Acid-ptoluidine Salt

The penam ester (12) (100 mg) was dissolved in a mixture of tetrahydrofuran (9 ml) and water (1 ml) and hydrogenated overpre-hydrogenated 10% palladium on charcoal catalyst (100 mg) at NTP for 1 hour. The mixture was filtered through Kieselghur and evaporated. The residual gum was reevaporated from dry benzene (2x2 ml) and triturated with dry ether to give the p-toluidine salt (13) (65 mg) as a yellow amorphous solid Vmex (nujol mull) 3330 br, 1770, 1670, 1615 cm-1.

Example 4 (38 SR, 6R)-6-Da-aminophenylacetamido-2-spirocyclopropanbisnorpenicillanic Acid a) (3S, SR, 6R)-Benzyl 6-Da-aminophenylacetamido-2-spirocyclopropanbisnorpenicillanate

D-Phenylglycyl chloride hydrochloride (63 mg) was added portionwise in 5 minutes to a stirred ice-bath cooled mixture of the 6-amino penam ester (11) (78 mg) and N,N-dimethyianiline (37 mg) in dry methylene chloride (2 ml). The mixture was stirred at ice-bath temperature for 20 minutes and then for a further 20 minutes whilst it attained room temperature. The mixture was diluted with ethyl acetate (5 ml), washed with sodium bicarbonate solution and brine, dried (MgSO4), and evaporated to give a gum (102 mg).Chromatography of the crude product on silica gel eluting with ethyl acetate/petroleum spirit 60-800C mixtures gave the penam ester (14) (53 mg) as an amorphous solid [ & 20+57.30 (c, 1% in CHCl3). Vmex (CHCl3) 3400,3350, 1790, 1740, 1680 cm-'. a ppm (CDCl3) 0.72-1.30 (4 H, m), 1.98 br (2 H, s, exch. D2O), 4.42 (1 H, s), 4.62 (1 H, s), 5.25 (2 H, s), 5.53-5.90 (2 H, m, collapsing to 5.57 and 5.90, 2d, J 4 Hz on exch.D2O) 7.44 (10 H, s), 8.07 (1 H, d, J 8 Hz exch. D2O).

b) (3S,5R,6R)-6-Da-aminophenylacetamido-2-spirocyclopropanbisnorpenicillanic Acid

The penam ester (14) (1 30 mg) was dissolved in a mixture of tetrahydrofuran (8 ml) and water (2 ml) and hydrogenated over prehydrogenated 10% palladium on charcoal catalyst (130 mg) at NTP for 40 minutes. The mixture was evaporated to low volume, diluted with water (5 ml), cooled in an icebath, and acidified to pH 2 with dilute hydrochloric acid. The cooled mixture was stirred at pH 2 for 5 minutes and filtered through Kieselghur. The pH of the ice-bath cooled filtrate was adjusted to 5 with dilute sodium bicarbonate solution and the aqueous mixture was washed with a little ethyl acetate.

Evaporation of the aqueous layer gave the amino-acid (1 5) (53 mg), contaminated with sodium chloride, as an amorphous solid. z'mex (KBr) 3650-2500 br, 1 780, 1 760 slight shoulder, 1675 cm-'.

The minimum inhibitor concentrations (MIC) of this compound required to inhibit the growth of various bacteria on nutrient aga are tabulated overleaf.

Organism MIC (yg/mlJ E. coli JT1 10 E. coli NCTC 10418 25 P. mirabilis C977 5.0 B. Subtilis 0.1 Staph. aureus Oxford 0.1 Staph. aureus Russell 25 Strep. faecalis I 1.0 ,B-Haemolytic Strep. CN10 0.02

Claims (14)

Claims

1. A compound of formula (IV) or a pharmaceutically acceptable salt or in vivo hydrolysabie ester thereof:

wherein R represents an organic acylamino group, a group of formula:

our a group of formula:

wherein R' and R2 each represent a C13 alkyl group or R1 and R2 taken together with the nitrogen atom to which they are attached form an optionally substituted monocyclic ring.

2. A compound as claimed in claim 1, wherein the organic acylamino group R in formula (IV) is a group of formula:

wherein RU represents a furyl, thienyl, phenoxy, cycloalkyl, cycloalkenyl, or phenyl group, or a phenyl group substituted by hydroxy, halogen, nitro, lower alkyl, lower alkoxy, amino, or carboxy; and X represents hydrogen, hydroxy, amino, carboxy, salted carboxy, esterified carboxy, ureido, or acylureido.

3. A compound as claimed in either claim 1 or 2, wherein the group RU represents a 3-furyl, 2- or 3-thienyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexa-1 ,4-dienyl, phenyl, 4-hydroxyphenyl, 3-chloro-4-hydroxyphenyl, 3,4-dihydroxyphenyl, 4- methoxyphenyl or phenoxy.

4. A compound as claimed in any of claims 1 to 3, wherein RU is phenyl or p-hydroxyphenyl and X represents amino.

5. A compound as claimed in any of claims 1 to 3, wherein RU is phenyl, 2-thienyl or 3-thienyl and X is -CO2R12 wherein R12 is hydrogen or C,~,0 alkyl.

6. A compound as claimed in any of claims 1 to 3, wherein RU is phenyl and X is hydrogen.

7. (3S,5R,6R)-6-Phenoxyacetamido-2-spirocyclopropanbisnorpenicillanic acid or a carboxylblocked derivative thereof.

8. (3S,5R,6R)-6-DcE-aminophenylacetamido-2-spirocyclopropanbisnorpenicillanic acid or a pharmaceutically acceptable salt thereof.

9. A compound of formula (VIII), or an acid addition salt thereof:

wherein RX is hydrogen or a carboxyl-blocking group.

10. A compound as claimed in claim 9, wherein RX is benzyl.

11. A compound as claimed in claim 9, wherein RX is triethylammonium.

12. A process for the preparation of a compound as claimed in claim 1, which process comprises reacting a compound of formula (VIII):

wherein RX is hydrogen or a carboxyl-blocking group, with an N-acylating derivative of an acid of formula (IX): Rq- OH (IX) wherein Rq is an organic acyl group such that R in formula (IV) represents RqNHand wherein any reactive groups may be blocked, and thereafter, if necessary, carrying out one or more of the following steps: i) removing any blocking group from the group RX; ii) removing any blocking group from the acyl side-chain; iii) converting the product to a salt or ester thereof.

13. A process as claimed in claim 12, substantially as described with reference to Example 3 herein.

14. A process as claimed in claim 12, substantially as described with reference to Example 4 herein.

1 5. A process for the preparation of a compound as claimed in claim 9, which process comprises a) treating a compound of formula (XII):

wherein RV is a carboxyl blocking group with an agent forming an imino halide; b) treating the imino halide with a compound to introduce a group QRf on the imino carbon atom, wherein Q is oxygen, sulphur or nitrogen and Rf is an alkyl group of from 1 to 12 carbon atoms or an aralkyl group of from 5 to 14 carbon atoms, to form an iminoether, iminothioether, or amindine (when Q is 0, S or N respectiveiy); c) treating with water; and d) optionally removing the carboxyl-blocking group Rv.

1 6. A process as claimed in claim 1 5 substantially as described with reference to Example 1 herein.

1 7. A pharmaceutical composition comprising at least one compound as claimed in any one of claims 1 to 9 together with a pharmaceutically acceptable carrier.