WO1988007534A1 - Beta-lactam epoxides - Google Patents

Beta-lactam epoxides Download PDF

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Publication number
WO1988007534A1
WO1988007534A1 PCT/GB1988/000202 GB8800202W WO8807534A1 WO 1988007534 A1 WO1988007534 A1 WO 1988007534A1 GB 8800202 W GB8800202 W GB 8800202W WO 8807534 A1 WO8807534 A1 WO 8807534A1
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Prior art keywords
beta
lactam
group
isomer
ethyl acetate
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PCT/GB1988/000202
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French (fr)
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Barrie Walsham Bycroft
Richard Eden Shute
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National Research Development Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D499/00—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring

Definitions

  • This invention relates to derivatives of penicillins and cephalosporins having an epoxy group in the 6-posltion of the penicillin and 7-position of the cephalosporin fused beta-lactam ring system, their preparation and pharmaceutical compositions and preparations containing them.
  • the products comprise spiroepoxides and spiro(cyclic imines) (4) and the 6-methyloxalyl-6-chloropenicillanic ester (5). It was suggested that the 6-epoxides and cyclic imines (4) are unstable, undergoing cleavage of the beta-lactam ring and re-formation of the ring in an enlarged form having 5 or 7 ring members and loss of the 6-epoxy or cyclic imino group.
  • E is as defined above and J is Ph, p-NO 2 Ph or p-CH 3 OPh, but chiefly thiazolooxazinones of formula (7):
  • R represents the residue of a penicillin or cephalosporin ring
  • X represents a hydrogen or halogen atom
  • A represents a hydroxy, alkoxy, aryloxy, aralkyloxy, or alkyl group or an amino group, and their pharmaceutically acceptable salts, have useful pharmacological properties.
  • beta-lactamase-producing bacteria have developed a marked resistance to treatment by a variety of beta-lactam antibiotic compounds. Patients suffering from infection from such bacteria are often best treated by beta-lactam combination therapy. In this treatment the patient is treated with a compound which undergoes an irreversible reaction with the beta-lactamase, inhibiting its activity. The irreversibly inhibited beta-lactamase is thereby made ineffective against a subsequently administered beta-lactam antibiotic, of the kind which otherwise would be destroyed by the beta-lactamase enzyme (by ring-opening of the beta-lactam ring).
  • Beta-lactam epoxides as defined above are useful as Irreversible Inhibitors of beta-lactamases and some of them also have antibacterial activity against certain Gram-positive bacteria, viz. Staphylococcus aureus and epidermidis and Streptococcus faecalis, pyogenes and pneumoniae.
  • the compounds of formula (8) above comprise those of the penicillin and cephalosporin series, conveniently termed penams and cephems. They can have any of the conventional side-chains present in the five-membered ring of a penam or six-membered ring of a cephem.
  • the preferred penams are compounds of formula (9):
  • n 0, 1 or 2;
  • X and A are defined above in connection with formula (8) and their pharmaceutically acceptable salts and esters.
  • the invention lies primarily in the concept of fixing the bond angles of the 6'-penam or 7'-cephem side-chain by rigidifying the side-chain as an epoxide, and secondarily by attaching a carbonyl group on the 3-carbon atom of the epoxy ring, whereby any desired group A is attachable via the carbonyl group and the rigid epoxy linkage.
  • A is not of the essence of the invention.
  • Virtually all the exemplified compounds having an enormous variety of A groups show activity as beta-lactamase irreversible inhibitors. Description of the preferred embodiments
  • X is preferably bromine or chlorine and A is preferably alkoxy or alkyl of 1 to 4 carbon atoms, phenoxy, 2-phenylethoxy, or benzyloxy in each of which the benzene ring is unsubstituted or substituted by one or more substituents selected from methyl, methoxy, halogen, carboxy, cyano, nitro and amino, or A is an amino group of formula (11):
  • each of R 1 and R 2 independently represents a hydrogen atom, a hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from halogen, carboxy, cyano, nitro and amino or a heterocyclic group or R 1 , R 2 and N shown in formula (11) together represent a nitrogen-containing heterocyclic group.
  • R 1 is more peferably an aryl, especially phenyl, or aralkyl, especially benzyl or 2-phenethyl group, the alpha-carbon of which is optionally substituted.
  • R 1 , R 2 and N can together represent the residue of an aromatic amino acid, for example alpha- phenyl- glycine or phenyl alanine, in which event the alpha-carbon atom of R 1 is substituted by carboxy.
  • the benzene ring of the phenyl, benzyl or phenethyl group can be substituted by, for example, any of hydroxy, methoxy, halogen, methyl, cyano, nitro, amino and carboxy.
  • R 1 groups include alkyl and alkoxy of 1 to 4 carbon atoms, especially 1 or 2 carbon atoms, unsubstituted or substituted by halogen, carboxy, cyano, alkoxy, phenoxy or amino.
  • Heterocyclic R 1 groups include 2-thiazolyl, 2-oxazolyl, 2-imidazolyl, their benzo analogues, pyridyl, furyl, pyrrolyl and thienyl.
  • R 2 is preferably a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, preferably methyl or ethyl, which can be substituted, especially by carboxy.
  • R 1 is phenyl and R 2 is carboxymethyl
  • R 1 , R 2 and N together represent a residue of N-phenylglycine (Ph-NH-CH 2 COOH).
  • R 1 , R 2 and N together represent a heterocyclic group they preferably represent a saturated heterocyclic group such as piperidino, 4-methylpiperdino or morpholino.
  • the substituents can be any which are not clearly incompatible with formation of a covalent acyl-enzyme complex.
  • B.W. Bycroft and R.E. Shute Pharmaceutical Research 1, 3-14 (1985): "The molecular basis for the mode of action of beta-lactam antibiotics and mechanisms of resistance".
  • Sub-families of penams and cephems include those of the partial formulae (12 to 14):
  • R 3 being, for example, -O-CO-CH 3 or a group of formula (16):
  • the beta-lactam epoxides of the invention include salts and esters.
  • they include, for example, alkyl and benzyl esters of the 2'-carboxyl group.
  • Salts and esters of the 2'-carboxyl group and the carboxyl group or groups mentioned above as part of R 1 and/or R 2 form part of the invention.
  • Salts by internal compensation can be formed when, for example, the A group contains an -NH 2 substituent, between a nitrogen cation and a 2'-carboxyl ate anion.
  • Alkyl and aryl esters are preferred, especially alkyl esters having 1 to 4 carbon atoms in the alkyl group and phenyl esters.
  • the beta-lactam epoxides of the invention are capable of existing as geometric isomers at the 3-position.
  • the 1,3-bond is shown as a solid line and when depicting individual isomers as a broken line.
  • the invention includes these isomers individually and as a mixture in any proportions.
  • the compounds of the invention are optically active, e.g. those in which A is R 1 R 2 N-, where R 1 is benzyl alpha-substituted by carboxy.
  • the invention includes each optical isomer and mixtures thereof in any proportion including racemic mixtures.
  • the invention includes also a pharmaceutical composition comprising a beta-lactam epoxide as defined above in association with a pharmaceutically acceptable carrier.
  • a composition is useful in antibacterial therapy, either by itself or as part of a combination treatment with another beta-lactam.
  • the invention includes a combination-preparation or two-component pack, comprising (1) a beta-lactam antibiotic susceptible to attack, by a beta-lactamase, e.g. penicillin, ampicillin or amoxycillin, in association with (2) a beta-lactam epoxide of the invention (as an irreversible inhibitor or synergist).
  • a beta-lactam antibiotic susceptible to attack by a beta-lactamase, e.g. penicillin, ampicillin or amoxycillin
  • beta-lactam epoxide of the invention as an irreversible inhibitor or synergist.
  • Methods of formulation known in beta-lactam therapy can be used for the compositions and combination-preparations of
  • R being as defined above in connection with formula (8) provided that any group within R, especially a 2'-carboxy group, requiring to be protected is protected, under substantially anhydrous conditions, with a compound of formula (19):
  • V represents a halogen atom or an A group, A being as defined above in connection with formula (8) provided that any group within A, especially a carboxy group, requiring to be protected is protected, and X represents a halogen atom, and, when V represents a halogen atom, reacting the resultant acid halide of formula (20):
  • Y being a halogen atom, with a nucleophile A-L, L representing a leaving group reactive therewith to replace the Y halogen atom by the A group, and A being as defined above, and, if desired, removing any protecting group within R or A;
  • the beta-lactam epoxide to be prepared is a salt or ester, (other than a protective ester already present in R or A) converting the compound into its salt or esterifying it, respectively; and when the beta-lactam epoxide to be prepared is one in which n is 1 or 2 and the 6-diazo compound of formula (18) is one in which n is 0, oxidising a compound obtained above with an oxidising agent effective to generate a sulphoxide (4'-oxo for penams or 5'-oxo for cephems) or sulphone (4" ,4'-dioxo or 5',5'-dioxo) group.
  • Y and X are the same and are both chlorine or bromine.
  • one method is to react the starting diazo compound with oxalyl chloride or bromide to form the acid halide of formula (20) and then react this with an appropriate nucleophile such as an amine of formula NR 1 R 2 H or water.
  • an alkyl group A it is especially convenient to use a reactant of formula (19) wherein Y is alkyl, preferably pyruvyl chloride or bromide.
  • Preferred protecting groups are benzyl, t-butyl and 2,2,2-trichloroethyl. They are useful for protecting 2'-carboxyl groups and carboxyl groups present in A, especially when R 1 , R 2 and N together represent the residue of an amino acid. They can be removed in well known ways.
  • the oxidation of the 4'- or 5'- S-atom can take place in any way well known in beta-lactam chemistry.
  • the invention also includes the acid halide intermediates of formula (20) as new compounds, which through similarity of their structural formula can conveniently be grouped together with the compounds of formula (8).
  • Examples 1-47 relate to penams and
  • Examples 1 and 2 and 48 and 49 illustrate the preparation of the respective 6-diazo compounds which may be used as the starting point in the preparation of compounds in accordance with the invention.
  • the remaining Examples illustrate the preparation of compounds in accordance with the invention.
  • Benzyl 6-aminopenicillanate, tosylate salt (1.0 g, 2.1 x 10 -3 mole) was dissolved in 75% aqueous acetone (20 ml) and 2N hydrochloric acid (3 ml) was added. The solution was cooled to 0°C and a solution of sodium nitrite (152 mg, 2.2 x 10 -3 mole) in 1-2 ml water was added, with stirring, followed immediately by 10 ml ether. The yellow biphasic reaction was vigorously stirred at 0°C for 5-10 minutes, at the end of which time ether (10 ml) and water (20 ml) were added.
  • reaction was allowed to warm to room temperature and stood for % hour or until effervescence ceased.
  • the reaction mixture was washed with dilute HCO 3 - (x3), water, dried (MgSO 4 ) and evaporated. The residue was rapidly chromatographed on silica.
  • Benzyl 3-chloro-3-methoxycarbonylspiro-oxirane-2,6'-penicillanate (0.43 g, 1.05 x 10 -3 mole), prepared in Example 4, was dissolved in 80% aqueous acetic acid (10 ml). To this solution was added slowly a solution of potassium permanganate (0.5 g, 3.12 x 10 -3 mole) in water (5 ml). The solution was stirred at room temperature for 1 hour. The colour was quenched by addition of solid sodium metabisulphite and the clear solution was diluted with water (30 ml) and stored at -15°C for 11 ⁇ 2-2 hours to complete the precipitation.
  • the catalyst was filtered off through a tight Celite pad and washed with ethyl acetate. The filtrate and washings were evaporated to dryness at room temperature and the residue was taken up in ethanol (2 ml) and water (5 ml) and lyophilised to yield the product (84 mg) as a fluffy white solid homogeneous on TLC.
  • the catalyst was filtered off through a tight Celite pad and washed with ethyl acetate. The filtrate and washings were evaporated at room temperature and the residue was taken up again in ethyl acetate (3 ml). To this solution was added a solution of potassium 2-ethylhexanoate (200 mg) in ethyl acetate; a fine off-white precipitate formed immediately. The mixture was stored at -15°C for 1 hour to effect complete precipitation. at the end of which time the precipitate was collected, washed with ethyl acetate, taken up in water and lyophilised to yield the product (47 mg) as a yellow microcrystalllne powder.
  • Example 12 Following the procedure in Example 12, the isomeric mixture from Example 26 containing a 1:2 ratio of the 3R:3S-isomers of benzyl 3-bromo-3-(N-methyl-N-phenylcarboxamido)spiro-oxirane-2,6'-penicillanate (55 mg) and 10% palladium on charcoal (250 mg) yielded after lyophilisation, the product (35 mg) as a fluffywhite solid, homogeneous on TLC.
  • the yellow oil was taken up in anhydrous ether (10 ml) and anhydrous acetonitrile (10 ml) and added to a solution of 2,6-dichloroaniline (0.70 g) in anhydrous acetonitrile (20 ml) at room temperature. The solution was left at room temperature for 15 minutes, then dry pyridine (0.16 ml) was added. After a further 5 minutes at room temperature the reaction was diluted with water (50 ml) and ether (30 ml). The ethereal layer was separated and washed with water, dil. HCl (x2), dil. HCO 3 - (x2), water, dried (MgSO 4 ) and evaporated.
  • 2,6-Dimethoxyaniline was prepared as follows. 2-Nitroresorcinol (3.1 g) was dissolved in a solution of sodium hydroxide (2.0 g) in water (50 ml) and ethanol (10 ml). To this was added dimethyl sulphate (5 ml) and the reaction mixture was heated to 70°C. After 30 minutes a further portion (50 ml) of dimethyl sulphate and sodium hydroxide (1.0 g) were added. After 1 hour the reaction was cooled to room temperature and poured into a solution of 2 M NaOH (20 ml) and water (30 ml). The product was collected, washed with copious amounts of water, sucked as dry as possible and recrystallised from methanol. The yield of 2,6-dimethoxynitrobenzene was 2.86 g (78%), 129.5-130.5°C (lit. 130°C).
  • Benzyl 3-bromo-3-methoxycarbonylspiro-oxirane-2,6'-penicillanate (220 mg), prepared in Example 13, was dissolved in chloroform (5 ml) and the solution cooled to 0°C. A solution of 80-90% m-chloroperoxybenzoic acid (100 mg) in chloroform (5 ml) was added slowly with stirring to the above solution and the reaction mixture was stirred at 0°C for 1 hour. The reaction mixture was then washed with dil. HCO 3 - (x4), water, dried (MgSO 4 ), and evaporated.
  • Dibenzyl 3-chloro-3-(D-alpha-phenylglycinamido)spirooxirane-2,6'-penicillanate, 3S-isomer (190 mg), prepared in Example 17, was dissolved in ethyl acetate (10 ml) and a solution of potassium barcarbonate (61 mg, two equivalents) in water (15 ml) was added followed by 10% palladium on charcoal (700 mg). The mixture was hydrogenated with stirring for 1 hour. The catalyst was filtered off through a tight Celite pad and washed with water and ethyl acetate.
  • Example 45 the 1:13R:3S isomeric mixture of di benzyl 3-chloro-3-(D-alpha-phenylglycinamido)spirooxirane-2,6'-pencillanate (220 mg), prepared in Example 17, in ethyl acetate (10 ml), potassium bicarbonate (70 mg) in water (15 ml) and 10% palladium on charcoal (800 mg) yielded, after lyophilisation, the product (15 mg) as an amorphous solid, homogeneous on TLC, Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.86.
  • the reaction vessel was cooled to below 0 C, opened carefully and the contents poured into an ice-cold solution of sodium bicarbonate (75 g) in water (100 ml).
  • the solution was extracted with chloroform (3 x 300 ml) and the combined organic extracts were washed with water (2 x 300 ml), brine, dried (MgSO 4 ) and evaporated to leave a brown crystalline residue.
  • the crude product was dissolved in dichloromethane (100 ml) and extracted into ice-cold 0.5 M HCl (ca. 300 ml). The organic layer was discarded and the aqueous layer re-extracted with dichloromethane (2 x 100 ml), the organic layers being, once again, discarded. The aqueous layer was partitioned with dichloromethane and basified with saturated sodium bicarbonate solution. The organic layer was separated and the aqueous layer re-extracted with dichloromethane (2 x 100 ml). The combined organic extracts were washed once with water, dried (MgSO 4 ) and evaporated. The crystalline residue was dissolved in the minimum volume of warm dichloromethane (ca.
  • Oxanilic acid chloride (N-phenyl chloroglyoxamide) was prepared as follows. To a stirred suspension of aniline hydrochloride (3.0 g) in dry ether (20 ml) was added oxalyl chloride (5 ml). The reaction was stirred at room temperature for 2 days, after which time it was filtered and the solids washed with dry ether. The combined filtrate and washings were evaporated and the .residue was crystallised from dry ether/petrol at -15°C to yield the product (3.8 g, 89%) as white needles, m.p. 82-83°C (lit. 82.5°C).
  • Crystalline oxanilic acid chloride prepared as above was added to a solution of tert.-butyl 7-diazocephalosporanate (0.38 g) in anhydrous ether (10 ml) at 0°C.
  • the reaction was swirled to dissolve the solids and the solution was allowed to warm to room temperature as effervescence commenced. The golden colour faded slowly and the reaction was left at room temperature for 20 minutes.
  • the reaction was diluted with dil. HCO 3 - and the organic layer was separated and washed with water, dil. HCO 3 - (x2), water, dried (MgSO 4 ) and evaporated.
  • Example 50 fractional crystallisation of the residue yielded the crystalline 3S-isomer, identical to that of Example 50 (m.p., mixed m.p., NMR and IR). Investigation of the mother liquors revealed the presence of the minor, 3R-isomer, also identical to that of Example 50.
  • the two-phase reaction mixture with precipitated solids was filtered through Celite and the precipitate- was washed with cold ether. The filtrate and washings were combined, the organic layer was separated and washed with dil. HCl (x3), water, dil. HCO 3 - (x2), water, dried (MgSO 4 ) and evaporated.

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Abstract

Certain beta-lactam epoxides have been found to be irreversible inhibitors of beta-lactamase enzymes, thus making them alternatives to clavulanic acid and sulbactam, and others are useful intermediates in the preparation of such compounds. These epoxides have general formula (21), wherein R represents the residue of a penicillin or cephalo sporin ring; X represents a hydrogen or halogen atom; V represents a halogen atom (intermediates) or a group A defined below (final products); and A represents a hydroxy, alkoxy, aryloxy, aralkyloxy, or alkyl group or an amino group, and their pharmaceutically acceptable salts.

Description

BETA-LACTAM EPOXIDES Background of the Invention
1. Field of the invention
This invention relates to derivatives of penicillins and cephalosporins having an epoxy group in the 6-posltion of the penicillin and 7-position of the cephalosporin fused beta-lactam ring system, their preparation and pharmaceutical compositions and preparations containing them.
2. Description of the prior art
J.C. Sheehan, K. Nakajima and E. Chacko, Heterocycles 13, 227-234 (1979) have described reactions of certain 3-esters of 6-diazopenicillanic acid with an aldehyde, Schiff's base or oxalyl chloride according to the reaction scheme
Figure imgf000003_0001
wherein E = -CO2CH2CCI3 and J, X and L have the following meanings:
Figure imgf000004_0003
The products comprise spiroepoxides and spiro(cyclic imines) (4) and the 6-methyloxalyl-6-chloropenicillanic ester (5). It was suggested that the 6-epoxides and cyclic imines (4) are unstable, undergoing cleavage of the beta-lactam ring and re-formation of the ring in an enlarged form having 5 or 7 ring members and loss of the 6-epoxy or cyclic imino group.
V. Jephcote, D. John P.D. Edwards and Kong Luk, Tetrahedron Letters 25, 2915-2918 (1984) described reactions of 6-diazopenicillanates with aromatic aldehydes catalysed by boron trifluoride etherate giving various products including a proportion of the epoxide (6):
Figure imgf000004_0001
wherein E is as defined above and J is Ph, p-NO2Ph or p-CH3OPh, but chiefly thiazolooxazinones of formula (7):
Figure imgf000004_0002
It is known that the beta-lactamase TEM 1 is inactivated by 6-acetylmethylenepenicillanic add. See M. Arisawa et al., J. Antibiotics 35, 1578-1589 (1982), Biochem J. 209, 609-615 (1983) and ib. id. 211, 447-454 (1983). 6-Acylmethylene- penicillanic acids are the subject of European Patent Application Publication No. 50805A (Hoffman-La Roche). Summary of the invention
It has now been found that 6-spiroepoxy beta-lactams of the general formula (8):
Figure imgf000005_0001
wherein:
R represents the residue of a penicillin or cephalosporin ring;
X represents a hydrogen or halogen atom; and A represents a hydroxy, alkoxy, aryloxy, aralkyloxy, or alkyl group or an amino group, and their pharmaceutically acceptable salts, have useful pharmacological properties.
Many beta-lactamase-producing bacteria have developed a marked resistance to treatment by a variety of beta-lactam antibiotic compounds. Patients suffering from infection from such bacteria are often best treated by beta-lactam combination therapy. In this treatment the patient is treated with a compound which undergoes an irreversible reaction with the beta-lactamase, inhibiting its activity. The irreversibly inhibited beta-lactamase is thereby made ineffective against a subsequently administered beta-lactam antibiotic, of the kind which otherwise would be destroyed by the beta-lactamase enzyme (by ring-opening of the beta-lactam ring). Useful irreversible inhibitors are clavulanic acid (a component of "Augmentin") and CP - 45, 899 ("Sulbactam"). It has now been found that beta-lactam epoxides as defined above are useful as Irreversible Inhibitors of beta-lactamases and some of them also have antibacterial activity against certain Gram-positive bacteria, viz. Staphylococcus aureus and epidermidis and Streptococcus faecalis, pyogenes and pneumoniae.
Description of the preferred embodiments
The compounds of formula (8) above comprise those of the penicillin and cephalosporin series, conveniently termed penams and cephems. They can have any of the conventional side-chains present in the five-membered ring of a penam or six-membered ring of a cephem. The preferred penams are compounds of formula (9):
Figure imgf000006_0001
and the preferred cephems are compounds of formula (10):
Figure imgf000006_0002
wherein: n is 0, 1 or 2; and
X and A are defined above in connection with formula (8) and their pharmaceutically acceptable salts and esters.
The invention lies primarily in the concept of fixing the bond angles of the 6'-penam or 7'-cephem side-chain by rigidifying the side-chain as an epoxide, and secondarily by attaching a carbonyl group on the 3-carbon atom of the epoxy ring, whereby any desired group A is attachable via the carbonyl group and the rigid epoxy linkage. Thus, the precise structure of A is not of the essence of the invention. Virtually all the exemplified compounds having an enormous variety of A groups show activity as beta-lactamase irreversible inhibitors. Description of the preferred embodiments
In formulae (8), (9) and (10), X is preferably bromine or chlorine and A is preferably alkoxy or alkyl of 1 to 4 carbon atoms, phenoxy, 2-phenylethoxy, or benzyloxy in each of which the benzene ring is unsubstituted or substituted by one or more substituents selected from methyl, methoxy, halogen, carboxy, cyano, nitro and amino, or A is an amino group of formula (11):
Figure imgf000007_0001
wherein each of R1 and R2 independently represents a hydrogen atom, a hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from halogen, carboxy, cyano, nitro and amino or a heterocyclic group or R1, R2 and N shown in formula (11) together represent a nitrogen-containing heterocyclic group.
Referring in more detail to formula (11), more preferred meanings of R1 and R2 are as follows. R1 is more peferably an aryl, especially phenyl, or aralkyl, especially benzyl or 2-phenethyl group, the alpha-carbon of which is optionally substituted. In particular R1 , R2 and N can together represent the residue of an aromatic amino acid, for example alpha- phenyl- glycine or phenyl alanine, in which event the alpha-carbon atom of R1 is substituted by carboxy. The benzene ring of the phenyl, benzyl or phenethyl group can be substituted by, for example, any of hydroxy, methoxy, halogen, methyl, cyano, nitro, amino and carboxy. Other preferred R1 groups include alkyl and alkoxy of 1 to 4 carbon atoms, especially 1 or 2 carbon atoms, unsubstituted or substituted by halogen, carboxy, cyano, alkoxy, phenoxy or amino. In particular, R1 , R2 and N can together represent the residue of an alphatic amino acid, or an aliphatic diamine; R1 = cyanomethyl is also particularly preferred. Heterocyclic R1 groups include 2-thiazolyl, 2-oxazolyl, 2-imidazolyl, their benzo analogues, pyridyl, furyl, pyrrolyl and thienyl.
R2 is preferably a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, preferably methyl or ethyl, which can be substituted, especially by carboxy. Thus, when R1 is phenyl and R2 is carboxymethyl, R1, R2 and N together represent a residue of N-phenylglycine (Ph-NH-CH2COOH).
When R1, R2 and N together represent a heterocyclic group they preferably represent a saturated heterocyclic group such as piperidino, 4-methylpiperdino or morpholino.
As to the remainder of the penam or cephem ring system, i.e. substitution in the 21-, 31- and, for cephems, 4'-position, the substituents can be any which are not clearly incompatible with formation of a covalent acyl-enzyme complex. For a recent review of this subject, see B.W. Bycroft and R.E. Shute, Pharmaceutical Research 1, 3-14 (1985): "The molecular basis for the mode of action of beta-lactam antibiotics and mechanisms of resistance". Sub-families of penams and cephems include those of the partial formulae (12 to 14):
Figure imgf000008_0001
Figure imgf000008_0002
Figure imgf000008_0003
wherein the 3'-position is occupied by, for example, -CH3, -S-CH2CH3, -S-CH2CH2NH2 and derivatives thereof, and (15):
Figure imgf000009_0001
R3 being, for example, -O-CO-CH3 or a group of formula (16):
Figure imgf000009_0002
-OCONH2 or pyrido.
The beta-lactam epoxides of the invention include salts and esters. Thus they include, for example, alkyl and benzyl esters of the 2'-carboxyl group. Salts and esters of the 2'-carboxyl group and the carboxyl group or groups mentioned above as part of R1 and/or R2 form part of the invention. Salts and esters of the 3-carboxy group (A = OH) are also within the invention. Salts by internal compensation can be formed when, for example, the A group contains an -NH2 substituent, between a nitrogen cation and a 2'-carboxyl ate anion. Alkyl and aryl esters are preferred, especially alkyl esters having 1 to 4 carbon atoms in the alkyl group and phenyl esters.
The beta-lactam epoxides of the invention are capable of existing as geometric isomers at the 3-position. In formulae of the epoxides generically, the 1,3-bond is shown as a solid line and when depicting individual isomers as a broken line. The invention includes these isomers individually and as a mixture in any proportions. The isomers having the 3-position configuration of partial formula (17):
Figure imgf000009_0003
are preferred because in general they appear to show higher activity as beta-lactamase inhibitors than the alternative 3-position isomers. Only the formula (17) isomers are active against the Gram-positive bacteria mentioned above. Nomenclature of the formula (17) isomers is "3S" when X = hydrogen and "3R" when X = halogen.
Also some of the compounds of the invention are optically active, e.g. those in which A is R1R2N-, where R1 is benzyl alpha-substituted by carboxy. The invention includes each optical isomer and mixtures thereof in any proportion including racemic mixtures.
Beta-lactam epoxides of the invention found to be particularly valuable as beta-lactamase inhibitors are those of formula (9) or (10) in which n is O, X is chlorine or bromine and A is anilino or N-methylanilino (R1 = phenyl; R2 = hydrogen or methyl) and their pharmaceutically acceptable salts and esters.
The invention includes also a pharmaceutical composition comprising a beta-lactam epoxide as defined above in association with a pharmaceutically acceptable carrier. Such a composition is useful in antibacterial therapy, either by itself or as part of a combination treatment with another beta-lactam. For combination therapy, the invention includes a combination-preparation or two-component pack, comprising (1) a beta-lactam antibiotic susceptible to attack, by a beta-lactamase, e.g. penicillin, ampicillin or amoxycillin, in association with (2) a beta-lactam epoxide of the invention (as an irreversible inhibitor or synergist). Methods of formulation known in beta-lactam therapy can be used for the compositions and combination-preparations of the invention. The beta-lactam epoxides of the invention can be prepared by a method which comprises reacting' a 6-diazo compound of formula
(18):
Figure imgf000010_0001
R being as defined above in connection with formula (8) provided that any group within R, especially a 2'-carboxy group, requiring to be protected is protected, under substantially anhydrous conditions, with a compound of formula (19):
Figure imgf000011_0001
wherein V represents a halogen atom or an A group, A being as defined above in connection with formula (8) provided that any group within A, especially a carboxy group, requiring to be protected is protected, and X represents a halogen atom, and, when V represents a halogen atom, reacting the resultant acid halide of formula (20):
Figure imgf000011_0002
Y being a halogen atom, with a nucleophile A-L, L representing a leaving group reactive therewith to replace the Y halogen atom by the A group, and A being as defined above, and, if desired, removing any protecting group within R or A; further, when the beta-lactam epoxide to be prepared is a salt or ester, (other than a protective ester already present in R or A) converting the compound into its salt or esterifying it, respectively; and when the beta-lactam epoxide to be prepared is one in which n is 1 or 2 and the 6-diazo compound of formula (18) is one in which n is 0, oxidising a compound obtained above with an oxidising agent effective to generate a sulphoxide (4'-oxo for penams or 5'-oxo for cephems) or sulphone (4" ,4'-dioxo or 5',5'-dioxo) group. In the above method of preparation preferably Y and X are the same and are both chlorine or bromine. Thus one method is to react the starting diazo compound with oxalyl chloride or bromide to form the acid halide of formula (20) and then react this with an appropriate nucleophile such as an amine of formula NR1R2H or water. Where it is proposed to introduce an alkyl group A, it is especially convenient to use a reactant of formula (19) wherein Y is alkyl, preferably pyruvyl chloride or bromide.
Preferred protecting groups are benzyl, t-butyl and 2,2,2-trichloroethyl. They are useful for protecting 2'-carboxyl groups and carboxyl groups present in A, especially when R1 , R2 and N together represent the residue of an amino acid. They can be removed in well known ways.
The oxidation of the 4'- or 5'- S-atom can take place in any way well known in beta-lactam chemistry.
The invention also includes the acid halide intermediates of formula (20) as new compounds, which through similarity of their structural formula can conveniently be grouped together with the compounds of formula (8). In the following Examples, Examples 1-47 relate to penams and
48 onwards to cephems. Examples 1 and 2 and 48 and 49 illustrate the preparation of the respective 6-diazo compounds which may be used as the starting point in the preparation of compounds in accordance with the invention. The remaining Examples illustrate the preparation of compounds in accordance with the invention.
These compounds are summarised in a table which follows. Isomer ratios in RS mixtures are molar.
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
"Celite" and "Sulbactam" are trade marks. All ratios of elution solvents are volume:volume. All ratios of isomers are molar ratios. "Ether" means diethyl ether. "Petrol" means petroleum ether, b.p. 40-60°C. NMRs are 1H unless otherwise stated. Example 1
2,2,2-Trichloroethyl (2S,5R)-6-diazo-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylate(trichlorbethyl-6-diazopenicillanate). (a) A solution of trichloroethyl benzylpenicillanate (10.0 g, 2.1 x 10-2 mole) in dry dichloromethane (100 ml) was added in % hour with stirring at -10° to -15°C to a mixture of anhydrous sodium acetate (22 g), dinitrogen tetroxide (150 ml of a solution of dinitrogen tetroxide, 25 g, in 250 ml dry dichloromethane) and dry dichloromethane (100 ml). The mixture was stirred below 0°C for 1 hour. Additional portions of the dinitrogen tetroxide solution (20 ml and 80 ml) were added immediately after, and 30 minutes after, the addition of the penicillin derivative.
After 1 hour excess dinitrogen tetroxide was consumed by adding saturated aqueous sodium bicarbonate (about 500 ml). The organic phase was separated and the aqueous phase re-extracted with dichloromethane. The combined organic extracts were washed with water, dried and evaporated to yield trichloroethyl-6-beta-(N-nitrosophenylacetamido)pencillanate (10.6 g) as a yellow syrup which was not purified further and which was used in the next stage immediately.
(b) To a solution of trichloroethyl 6-beta-(N-nitrosophenylacetamido)penicillanate (10.6 g, 2.1 x 10-2 mole) in dichloromethane (300 ml), pyridine (3 ml) was added and the solution was refluxed for 3 hours.
The orange-brown solution was washed with water (x2), saturated HCO3- (x2), water (x1), rapidly once with dilute HCl and finally with water, dried and evaporated. The resultant trichloroethyl 6-diazopenicillanate product was recrystallised from carbon tetrachloride/petrol as yellow needles (5.3 g, 71%) m.p. 100-105°C. Example 2
Benzyl (2S,5R)-6-diazo-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo- [3.2.0]heptane-2-carboxylate (benzyl 6-diazopenicillanate)
Benzyl 6-aminopenicillanate, tosylate salt (1.0 g, 2.1 x 10-3 mole) was dissolved in 75% aqueous acetone (20 ml) and 2N hydrochloric acid (3 ml) was added. The solution was cooled to 0°C and a solution of sodium nitrite (152 mg, 2.2 x 10-3 mole) in 1-2 ml water was added, with stirring, followed immediately by 10 ml ether. The yellow biphasic reaction was vigorously stirred at 0°C for 5-10 minutes, at the end of which time ether (10 ml) and water (20 ml) were added. The ethereal layer was separated and the aqueous layer was re-extracted with ether (20 ml). The combined organic layers were washed with water repeatedly, dil. HCO3- (x1), water (x1), dried (MgSO4) and evaporated to yield the product (0.56 g, 85%) as a yellow oil, homogeneous on TLC. Rf (silica)-petrol/ethyl acetate (4:1):0.60; IR (film) 2080, 1740-1760 cm-1;
NMR delta, ppm (CDCl3) 1.32 (3H,s), 1.54 (3H,s), 4.34 (1H,s), 5.08 (2H,s), 6.07 (1H.s), 7.25 (5H,s). Example 3
2,2,2-Trichloroethyl (2S,3RS,2'S,5'R)-3-chloro-3-methoxycarbonyl-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia- (1)-azabicyclo[3.2.0]heptane]-2'-carboxylate.
Figure imgf000019_0001
To a solution of trichloroethyl 6-diazopenici 11 anate (200 mg, 5.6 x 10-4 mole), prepared in Example 1, in anhydrous ether (10 ml) at 0°C was added oxalyl chloride (0.06 ml). The solution was allowed to warm to room temperature and stood for 15 minutes or until effervescence ceased. The reaction was then cooled to 0°C and quenched with water (20 ml). The ethereal layer was washed repeatedly with water, dried (MgSO4) and evaporated to about 5 ml. The above ethereal solution was esterified with an ethereal solution of diazomethane. Excess diazomethane and solvent were removed by evaporation and the residue was chromato- graphed rapidly on silica. Elution with petrol/ethyl acetate (4:1) yielded the product (0.21 g, 83%) as a pale yellow oil. IR (film) 1800, 1760 cm-1;
NMR delta, ppm (CDCl3) 1.58 (3H,s), 1.64 (3H,s), 3.89 (3H,s,CO2CH3), 4.64 (1H,s,C2'-H) 4.79 (2H,s,-CH2CCl3) 5.58 (1H,s,C5'-H);
Found M+ 450.9200, C13H13 35Cl4NO6S requires 450.9217. Example 4
Benzyl (2S,3RS,2'S,5'R)-3-chloro-3-methoxycarbonyl-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo-[3.2.0]heptane]-2'-carboxylate (benzyl 3-chIoro-3-methoxycarbonyl spiro-oxirane-2,6'-penicillanate) To a solution of benzyl 6-diazopencillanate (560 mg, 1.77 x 10-3 mole), prepared in Example 2, in anhydrous ether (20 ml) at 0°C was added oxalyl chloride (0.16 ml). The solution was allowed to warm to room temperature and stood for 15 minutes or until effervescence ceased. The reaction was then cooled to 0°C and quenched with water (30 ml). The ethereal layer was worked up as described in Example 3, yielding the product (0.48 g, 67%) as a pale yellow oil,
Rf (silica)-petrol/ethyl acetate (4:1):0.55; IR (film) 1792, 1740-1760 cm-1; NMR delta, ppm (CDCl3) 1.40 (3H,s), 1.53 (3H,s), 3.83 (3H,s,CO2CH3), 4.51 (1H,s,C2'-H), 5.16 (2H,s,CO2CH2Ph), 5.52 (1H,s,C5'-H), 7.34 (5H,s,CH2Ph);
Found M+ 411.0537, C18H18 35ClNO6S requires 411.0539. Example 5 (2S,3RS,2'S.5'R)-3-chloro-3-methoxycarbonyl-3',3'-dimethyl- 7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxvlic acid Benzyl 3-chloro-3-methoxycarbonylspiro-oxirane-2,6'-penicillanate (0.2 g, 4.9 x 10-4 mole), prepared in Example 4, was dissolved in ethanol (15 ml). 10% Palladium on charcoal (700 mg) was added. The mixture was stirred for 1 hour and then hydrogenated with stirring for a further 1 hour. The catalyst was filtered off through a tight Celite pad and washed with ethanol. The ethanolic solution was diluted with water (30 ml) and the resultant solution lyophilised to yield the product (0.15 g) as an off-white fluffy solid homogeneous on TLC. Rf (silica)-n-butanol/acetic acid/water (4:1 :1):0.84. Example 6
Benzyl (2S,3S,2'S,5'R)-3-chloro-3-methoxycarbonyl-3'-3'- dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo- [3.2.0]heptane]-2'-carboxylate-4'-oxide Benzyl 3-chloro-3-methoxycarbonylspiro-oxirane-2,6'-penicillanate (450 mg, 1.1 x 10-3 mole), prepared in Example 5, was dissolved in chloroform (10 ml) and the solution cooled to 0°C. A solution of 80-90% m-chloroperoxybenzoic acid (225 mg) in chloroform (10 ml) was added slowly with stirring to the above solution and the reaction was stirred at 0°C for 1 hour. The reaction mixture was washed with dil. HCO3- (x4), water, dried (MgSO4) and evaporated to yield a colourless oil which crystallised on addition of a few drops of ether to yield the product (240 mg, 51%) as a white crystalline solid which was recrystallised from ethyl acetate/hexane, m.p. 159-161°C. IR (KBr) 1810, 1760, 1745, 1048 cm-1;
NMR delta, ppm (CDCl3) 1.12 (3H,s), 1.68 (3H,s) 3.88 (3H,s,CO2CH3), 4.62 (1H,s,C2'-H), 5.03-5.45 (2H,ABq,CO2CH2Ph) 5.11 (1H,s,C5'-H), 7.34 (5H,s,CH2Ph); Found C.50.77; H.4.32; N,3.23%, C18H18ClNO7S requires C,50.53; H.4.21; N,3.27%. Example 7
Benzyl (2S,3RS,2'S,5'R)-3-chloro-3-phenoxycarbonyl-3',3'-dimethyl-7-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo-[3.2.0]heptane]-2'-carboxylate
To a solution of benzyl 6-diazopenicil lanate (0.53 g, 1.7 x 10-3 mole) in anhydrous ether (10 ml) at 0°C was added a solution of phenyl chloroglyoxalate (0.36 g, 2.2 x 10-3 mole).
The reaction was allowed to warm to room temperature and stood for % hour or until effervescence ceased. The reaction mixture was washed with dilute HCO3- (x3), water, dried (MgSO4) and evaporated. The residue was rapidly chromatographed on silica.
Elution with petrol/ethyl acetate (4:1) yielded the product (0.52 g, 66%) as a pale yellow oil.
IR (film) 1790; 1740-1750 cm-1;
NMR delta, ppm (CDCl3) 1.37 (3H,s), 1.51 (3H,s), 4.57 (1H,s,C2'-H), 5.15 (2H,s,-CH2Ph), 5.60 (1H,s,C5'-H), 7.1-7.5
(10H,m, aromatics);
Found M+ 473.0687, C23H20 35ClNO6S requires 473.0697.
Example 8
Benzyl (2S,3S and 3R,2'S,5'R)-3-chloro-3-(N-phenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo- [3.2.0]heptane]-2'-carboxylate (benzyl 3-chloro-3-(N-phenylcarboxamido)spiro-oxirane-2,6'-penicillinate)
To a solution of benzyl 6-diazopenicillanate (0.55 g, 1.74 x
10-3 mole) in anhydrous ether (20 ml) at 0°C was added oxalyl chloride (0.16 ml). The solution was allowed to warm to room temperature and stood for 15 minutes or until effervescence ceased. The reaction was evaporated at room temperature and the resultant yellow oil was taken up again in anhydrous ether and evaporated a second time. The above oil was taken up in anhydrous ether (20 ml) and aniline, redistilled from zinc dust, (0.36 ml) was added.
Reaction, as indicated by precipitation of aniline hydrochloride, was instantaneous. Water (20 ml) and ethyl acetate (20 ml) were added and the organic layer was separated and washed with water (x2), dil. HCl (x2), dil. HCO3- (x2), water, dried (MgSO4) and evaporated. The residue was rapidly chromatographed on silica.
Elution with hexane/ethyl acetate (4:1) yielded the 3S-isomer as the major product [0.51 g, 62%, Rf (silica)-hexane/ethyl acetate
(3:1):0.463 and the 3R-isomer as the minor product [0.07 g, 9%, Rf (silica)-hexane/ethyl acetate (3:1):0.36], both as unstable pale yellow oils. Major product - 3S-isomer
IR (film) 3330, 1792, 1740, 1700 cm-1
NMR delta, ppm (CDCl3) 1.43 (3H,s), 1.57 (3H,s), 4.52 (1H,s,C2'-H), 5.18 (2H,s), 5.58 (1H,s,C5'-H), 7.10-7.60 (10H.m), 8.00-8.20 (1H,br,s,-NHPh, exchangeable).
Found M+ 472.0843, C23H21 35ClN2O5S requires 472.0860. Minor product - 3R-isomer
IR (film) 3330, 1792, 1740, 1700 cm-1
NMR delta, ppm (CDCl3) 1.37 (3H,s), 1.55 (3H,s), 4.62 (1H,s,C2'-H), 5.16 (2H,s), 5.65 (1H,s,C5'-H), 7.10-7.60 (10H,m), 8.15-8.30 (1H,br, s,-NHPh, exchangeable).
Found M+ 472.0861, C23H21 35ClN2O5S requires 472.0860. Example 9 Benzyl (2S,3S,2'S,5'R)-3-chloro-3-methoxycarbonyl-3',3'- dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabieyclo- [3.2.0]heptane]-2'-carboxylate-4',4'-dioxide
Benzyl 3-chloro-3-methoxycarbonylspiro-oxirane-2,6'-penicillanate (0.43 g, 1.05 x 10-3 mole), prepared in Example 4, was dissolved in 80% aqueous acetic acid (10 ml). To this solution was added slowly a solution of potassium permanganate (0.5 g, 3.12 x 10-3 mole) in water (5 ml). The solution was stirred at room temperature for 1 hour. The colour was quenched by addition of solid sodium metabisulphite and the clear solution was diluted with water (30 ml) and stored at -15°C for 1½-2 hours to complete the precipitation. The white precipitate was filtered off, washed thoroughly with water and dried overnight over P2O5 in vacuo to yield the product (0.21 g, 45%) as a white powder which was recrystallised from ethyl acetate/petrol, Rf (silica)-petrol/ethyl acetate (1:1):0.78; m.p. 160-161 °C. IR (KBr) 1818, 1760, 1745, 1332, 1175 cm-1; NMR delta, ppm (CDCI3) 1.32 (3H,s), 1.59 (3H,s), 3.91 (3H,s,-CO2CH3), 4.53 (1H,s,C2'-H), 4.98 (1H,s,C5'-H), 5.02-5.50 (2H,ABq,-CH2Ph), 7.39 (5H,s,-CH2Ph); Found C.48.89; H.4.42; N,3.24%, C18H18ClNO8S requires C.48.70; H.4.06; N,3.16%. Example 10
(2S,3S,2'S,5'R)-3-chloro-3-methoxycarbonyl-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]-heptane]-2'-carboxylic acid-4',4'-dioxide Benzyl 3-chloro-3-methoxycarbonylspiro-oxirane-2,6'-pencillanate-4',4'-dioxide, 3S-isomer (150 mg, 3.4 x 10-4 mole), prepared in Example 9, was dissolved in 2 ml ethyl acetate and 10 ml ethanol. 10% palladium on charcoal (250 mg) was added and the reaction was hydrogenated, with stirring for 1 hour. The catalyst was filtered off through a tight Celite pad and washed with ethanol. The ethanolic filtrate and washings were diluted with water (20 ml) and lyophilised overnight to yield the product (100 mg) as a white fluffy solid homogeneous on TLC.
Rf (silica)-n-butanol/acetic acid/water (4:1 :1):0.66. Example 11
(2S,3S,2'S,5'R)-3-chloro-3-(N-phenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylic acid
Benzyl 3-chloro-3-(N-phenylcarboxamido)spiro-oxirane-2,6'-penicillanate, 3S-isomer (220 mg), prepared in Example 8, was dissolved in ethyl acetate (25 ml). 10% palladium on charcoal (1.1 g) was added and the reaction was hydrogenated with stirring for 1 hour. The catalyst was filtered off through a tight Celite pad and washed with ethyl acetate. The filtrate and washings were evaporated to dryness at room temperature to yield the product (120 mg) as an off-white crystalline solid recrystalli sable from ethyl acetate/hexane.
Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.86 -toluene/acetone/acetic acid (12:7:1):0.64; IR (KBr) 3260, 1800, 1740, 1675 cm-1. Example 12
(2S,3R,2'S,5'R)-3-chloro-3-(N-phenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylic acid Benzyl 3-chloro-3-(N-phenylcarboxamido)spiro-oxirane-2,6'-penicillanate, 3R-isomer (140 mg), prepared in Example 9, sas dissolved in ethyl acetate (20 ml). 10% palladium on charcoal (700 mg) was added and the reaction hydrogenated with stirring for 1 hour. The catalyst was filtered off through a tight Celite pad and washed with ethyl acetate. The filtrate and washings were evaporated to dryness at room temperature and the residue was taken up in ethanol (2 ml) and water (5 ml) and lyophilised to yield the product (84 mg) as a fluffy white solid homogeneous on TLC.
Rf (silica)-n-butanol/acetic acid/water (4:1 : 1 ) :0.84 -toluene/acetone/acetic acid (12:7:1):0.60;
IR (KBr) 3200-3250 (sh), 1790, 1690-1740 (br) cm-1. Example 13
Benzyl (2S,3RS,2'S,5'R)-3-bromo-3-methoxycarbonyl-3',3'-dimethyl- 7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane] 2'-carboxylate
Following the procedure in Example 4, benzyl 6-diazopenicil lanate (0.62 g) and oxalyl bromide (0.2 ml) yielded, after rapid chromatography on silica, the above product (0.51 g, 51%) as a yeHow oil . Rf (silica)-hexane/ethyl acetate (3:1):0.47; IR (film) 1797, 1750 cm-1;
NMR delta, ppm (CDCl3) 1.43 (3H,s), 1.56 (3H,S), 3.83 (3H,s), 4.53 (1H,S,C2'-H), 5.17 (2H,s), 5.51 (1H,s,C5'-H), 7.31 (5H,s); Found M+ 454.9981, C18H18 79BrNO6S requires 455.0035; F.A.B.S. +ve ion mode 456, 458. Example 14
(2S,3RS,2'S,5'R)-3-bromo-3-methoxycarbonyl-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]-heptane]-2'-carboxylic acid Following the procedure in Example 12, hydrogenation of benzyl 3-bromo-3-methoxycarbonylspiro-oxirane-2,6'-penicillanate (300 mg), prepared in Example 13, over 10% palladium on charcoal (1.3 g) yielded, after lyophilisation, the product (180 mg) as a fluffy off-white solid, homogeneous on TLC. Rf (silica)-n-butanol/acetic acid/water (4:1 :1 ):0.85. Example 15
Benzyl (2S,3RS,2'S,5'R)-3-chloro-3-benzyloxycarbonyl-3',3'-dimethyl-7-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylate To a solution of benzyl 6-diazopenicillanate (0.23 g) in anhydrous ether (10 ml) at 0°C was added oxalyl chloride (0.08 ml). The solution was allowed to warm to room temperature and stood for 15 minutes or until effervescence ceased. The reaction was then cooled to 0°C and quenched with water (20 ml). The ethereal layer was washed repeatedly with water, dried (MgSO4) and evaporated to about 5 ml.
The above ethereal solution was esterified with an ethereal solution of phenyldiazomethane. Excess phenyldiazomethane was quenched by pouring the reaction into water (25 ml) containing citric acid (2.0 g). The ethereal layer was separated and washed with water (x2), dil. HCO3- (x2), water (x1), dried (MgSO4) and evaporated. The residue was chromatographed rapidly on silica. Elution with hexane/ethyl acetate (4:1) yielded the product (0.19 g, 54%) as a pale yellow oil. Rf (silica)-hexane/ethyl acetate (3:1):0.47; IR (film) 1795, 1745-1755 cm-1;
NMR delta, ppm (CDCI3) 1.38 (3H,s), 1.43 (3H,s), 4.51 (1H,s,C2'-H), 5.00-5.40 (2H,ABq, PhCH2OCO-), 5.15 (2H,s), 5.52 (1H,s,C5'-H), 7.30 (10H,s); Found M+ 487.0820, C24H22 35ClNO6S requires 486.0854. Example 16
Dipotassium (2S,3RS,2'S,5,R)-3-chloro-3',3'-dimethyl-7'-oxo-spiro[oxirane-2',6'-(4)-thia-(1)-azabicyclo[3.2.0]-heptane]-2',3-dicarboxylate Benzyl 3-chloro-3-benzyloxycarbonylspiro-oxirane-2,6'-penicillanate (96 mg), prepared in Example 15, was dissolved in ethyl acetate (15 ml). 10% palladium on charcoal (500 mg) was added and the reaction was hydrogenated with stirring for 1 hour. The catalyst was filtered off through a tight Celite pad and washed with ethyl acetate. The filtrate and washings were evaporated at room temperature and the residue was taken up again in ethyl acetate (3 ml). To this solution was added a solution of potassium 2-ethylhexanoate (200 mg) in ethyl acetate; a fine off-white precipitate formed immediately. The mixture was stored at -15°C for 1 hour to effect complete precipitation. at the end of which time the precipitate was collected, washed with ethyl acetate, taken up in water and lyophilised to yield the product (47 mg) as a yellow microcrystalllne powder.
Rf (silica)-n-butanol/aceti c acid/water (4:1:1):0.51. Example 17
Benzyl (R)-2-{(2S,3RS and 3S,2'S,5'R)-2'-benzyloxycarbonyl- 3-chloro-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)- azabicyclo[3.2.0]heptane]-3-carboxamido}-2-phenylacetate
To a solution of benzyl 6-diazopenicillanate (0.62 g) in anhydrous ether (20 ml) at 0°C was added oxalyl chloride (0.18 ml). The solution was allowed to warm to room temperature and stood for 15 minutes or until effervescence ceased. The reaction mixture was evaporated at room temperature and the resultant yellow oil was taken up again in anhydrous ether and evaporated a second time.
The above oil was taken up in anhydrous ether (20 ml), cooled to 0°C and added to a solution of D-alpha-phenylglycine, benzyl ester (1.60 g) in anhydrous ether (20 ml) also at 0°C. Reaction, as indicated by precipitation of the amino acid ester hydrochloride, was instantaneous. The mixture was diluted with water (20 ml) and the ethereal layer was separated and washed with water, dil. HCl (x2), dil. HCO3- (x2), water, dried (MgSO4) and evaporated. The residue was rapidly chromatographed on silica by elution with hexane/ethyl acetate (3:1) to yield the product mixture of isomers (0.63 g, 52%) as a pale yellow oil.
Repeated chromatography as before, yielded, in the early fractions, pure 3S-isomer and, in the later fractions, a 1:1 mixture (NMR) of 3R and 3S-isomers.
Rf (silica)-hexane/ethyl acetate (3:1):0.35-0.43 (isomeric mixture);
Major product - 3S-isomer IR (film) 3390, 1797, 1740-1750, 1705 cm-1;
NMR delta, ppm (CDCl3) 1.37 (3H,s), 1.46 (3H,s), 4.43 (1H,S,C2'-H), 5.10 (4H,s,2 x PhCH2O-), 5.43-5.55 (2H,m,C5'-H + alpha-H), 7.15 (5H,s), 7.27 (10H,s), 7.59 (1H,br,d,-NH, exchangeable);
Found, F.A.B.S. +ve ion mode 621, 623. Example 18 Benzyl (S)-2-{(2S,3RS and 3S,2'S,5'R)-2'-benzyloxycarbonyl-3-chloro-3',3'-dimethyl-7'-oxo-spiro[oxirane-2-6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-3-carboxamido}-2-phenyl acetate
Following the procedure in Example 17, benzyl 6-diazopenicil lanate (0.62 g), oxalyl chloride (0.18 ml) and L-alpha-phenylglycine, benzyl ester (1.70 g) yielded, after rapid chromatography, the product mixture of isomers (0.64 g, 53%) as a pale yellow oil. Repeated chromatography as before yielded, in the early fractions, pure crystalline 3S-isomer and, in the later fractions, a slightly greater than 1:1 mixture (NMR) of 3R and 3S isomers. The pure 3S-isomer was recrystallised from ethyl acetate/petrol, m.p. 137-138ºC.
Rf (silica)-hexane/ethyl acetate (3:1):0.35-0.43 (isomer mixture) and 0.40 (pure 3S-isomer); Major product - 3S-isomer IR (KBr) 3400, 1790, 1740 (sh), 1730, 1710 cm-1;
NMR delta, ppm (CDCl3) 1.42 (3H,s), 1.55 (3H,s), 4.55 (1H,S,C2'-H), 5.10-5.22 (2H,ABq, L-Phg PhCH2O-), 5.22 (2H,s, penam PhCH2-), 5.53-5.56 (1H,d,J=6.7 Hz, L-Phg alpha-H), 5.59 (1H,S,C5'-H), 7.13-7.40 (15H,m), 7.58-7.60 (1H,br,d,J=6.6 Hz,-NH-);
Found C,61.80; H,4.97; N,4.52%, C32H29 ClN2O7S requires C,61.89; H,4.67; N.4.51%. Example 19 Benzyl (2S,3S and 3R,2'S,5'R)-3-chloro-3-(N-(2-thiazolyl)carboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane3-2'-carboxylate
Following the procedure in Example 17, benzyl 6-diazopencillanate (0.62 g) oxalyl chloride (0.18 ml) and a solution of 2-aminothiazole (500 mg) in anhydrous THF (10 ml) and anhydrous ether (10 ml) yielded, after rapid chromatography on silica, eluting with hexane/ethyl acetate (2:1), the 3S-isomer (90 mg 10%), Rf (silica)-hexane/ethyl acetate (2:1):0.37 and the 3R-isomer (140 mg, 15%, Rf (silica)-hexane/ethyl acetate (2:1):0.31, both as highly unstable yellow oils. 3S-isomer
IR (film) 3080-3120 (br), 1790, 1735, 1690 cm-1; NMR delta, ppm (CDCl3) 1.43 (3H,s), 1.60 (3H,s), 4.55 (1H,s,C2'-H), 5.19 (2H,s), 5.62 (1H,s,C5'-H), 7.1-7.7 (3H,br,m, thiazole + -NH-), 7.33 (5H,s). 3R-isomer
IR (film) 3060-3120 (br), 1795, 1740, 1695 cm-1; NMR delta, ppm (CDCl3) 1.33 (3H,s), 1.51 (3H,s), 4.62 (1H,s,C2'-H), 5.12 (2H,s), 5.61 (1H,s,C5'-H), 7.00-7.06 (1H,d,J=3.5Hz), 7.50-7.56 (1H,d,J=3.5 Hz,1H), 7.30 (5H,s), 9.6-10.2 (1H,br,s,-NH-, exchangeable). Example 20 Benzyl (2S,3RS and 3S,2'S,5'R)-3-chloro-3-(N-methyl-N-phenylcarboxamido)-3',3'-dimethyl-7'oxo-spiro[oxirane-2,6'-(4)- thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylate
Following the procedure in Example 8, benzyl 6-diazopenicillanate (0.62 g) oxalyl chloride (0.18 ml) and N-methylaniline (0.42 ml) yielded, after rapid chromatography, the product mixture of isomers (0.60 g, 63%) as a pale yellow oil. Repeated chromatography yielded in the early fractions pure 3S-isomer and in the later fractions, a greater than 1:1 mixture (NMR) of the
3R and 3S-isomers. Rf (silica)-hexane/ethyl acetate (3:1):0.32 (isomeric mixture)
IR (film) 1792, 1742, 1670 cm-1;
NMR delta, ppm (CDCl3) (3S-isomer) 1.37 (3H,s), 1.59 (3H,s) 3.28 (3H,s), 4.42 (1H,s ,C2'-H), 5.10 (2H,s), 5.29 (1H,s,C5'-H), 7.30 (10H.s); Found M+ 486.0987, C24H23 35ClN2O5S requires 486.1013. Example 21
(2S,3RS,2'S,5'R)-3-chloro-3-(N-methyl-N-phenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylic acid Following the procedure in Example 12, the isomeric mixture from Example 20 containing an excess of the 3R-isomer of benzyl 3-chloro-3-(N-methyl-N-phenylcarboxamido)spiro-oxirane-2,6'-peni cillanate (110 mg) and 10% palladium on charcoal (500 mg) yielded, after lyophilisation, the product (61 mg) as a fluffy white solid homogenous on TLC.
Rf (silica)-n-butanol/acetic acid/water (4:1 : 1 ) :0.85 -toluene/acetone/acetic acid (12:7:1):0.72; IR (KBr) 1790, 1730, 1665 cm-1. Example 22
Benzyl (2S,3RS and 3S,2'S,5'R)-3-chloro-3-(N-benzyloxycarbonylmethyl-N-phenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicycIo[3.2.0]heptane]-2'-carboxylate
Following the procedure in Example 17, benzyl 6-diazopenicillanate (0.62 g), oxalyl chloride (0.18 ml); and a solution of N-phenylglycine, benzyl ester (0.50 g) and dry pyridine (0.16 g) in anhydrous ether (20 ml) yielded, after rapid chromatography, the product mixture of isomers (0.80 g, 65%) as a pale yellow oil. Repeated chromatography yielded, in the early fractions, predominantly the 3S-isomer and, in the later fractions, a 5:3 mixture (NMR) of 3R and 3S-isomers.
Rf (silica)-hexane/ethyl acetate (3:1):0.38; IR (film) 1795, 1745-1755, 1675 cm-1 (isomeric mixture);
NMR delta, ppm (CDCl3) (3S-isomer) 1.39 (3H,s), 1.62 (4H,s), 4.32 (1H,S,C2'-H), 4.32-4.83 (2H,ABq), 5.16 (4H,s), 5.37 (1H,s,C5'-H), 7.34 (10H,s), 7.40 (5H,s,Ph- );
Figure imgf000030_0001
Found F.A.B.S. +ve ion mode 621, 623. Example 23
(2S,3RS,2'S,5'R)-3-chloro-3-(N-carboxymethyl-N-phenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiroCoxirane-2,6'-(4)-thia-(1)-azabicyclo-[3.2.0]heptane]-2'-carboxylic acid
Following the procedure in Example 12, the isomeric mixture from Example 22 containing an excess of the 3R-isomer of benzyl 3-chIoro-3-(N-benzyloxycarbonylmethyl-N-phenylcarboxamido)spirooxirane-2,6'-penicillanate (100 mg) and 10% palladium on charcoal (400 mg) yielded, after lyophilisation, the product (56 mg) as a white fluffy solid, which on TLC could be shown to be a mixture of two products.
Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.85 -toluene/acetone/acetic acid (12:7:1):0.46 (3S-isomer) and 0.42 (3R-isomer);
IR (KBr) 1790, 1725-1735, 1670 cm-1. Example 24
Benzyl (2S,3S and 3R,2'S,5'R)-3-bromo-3-(N-phenylcarboxamido)- 3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabieyclo- [3.2.0]heptane]-2'-carboxylate
Following the procedure in Example 8, benzyl 6-diazopenicillanate (0.55 g), oxalyl bromide (0.18 ml) and aniline (0.4 ml) yielded, after rapid chromatography, pure 3S-isomer and pure 3R-1somer in a combined yield of 270 mg (31%, ratio about 9:1, 3S:3R) both as unstable yellow oils. Major product - 3S-isomer
Rf (silica)-hexane/ethyl acetate (3:1):0.45; IR (film) 3300-3330 (br), 1790, 1740, 1690 cm-1; NMR delta, ppm (CDCl3) 1.40 (3H,s) 1.53 (3H,s), 4.53 (1H,s,C2'-H), 5.17 (2H,s), 5.55 (1H,s,C5'-H), 7.10-7.60 (5H,m, PhNH-), 7.30 (5H,s,PhCH2O), 8.05-8.20 (1H,br,s, exchangeable). Minor product - 3R-isomer
Rf (silica)-hexane/ethyl acetate (3:1):0.37; IR (film) 3300-3340 (br), 1795, 1740, 1690 cm-1;
NMR delta, ppm (CDCl3) 1.37 (3H,s), 1.55 (3H,s), 4.62 (1H,S,C2'-H), 5.16 (2H,s) 5.67 (1H,s,C5'-H), 7.10-7.60 (5H,m, PhNH-), 7.30 (5H,s,PhCH2O), 8.15-8.30 (1H,br,s,-NH- exchangeable). Example 25 (2S,3R,2'S,5'R)-3-bromo-3-(N-phenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2-carboxylic acid
Following the procedure in Example 12, benzyl 3-bromo-3-(N-phenylcarboxamido)spiro-oxirane-2,6'-penicillanate, 3R-isomer (60 mg), prepared in Example 24, and 10% palladium on charcoal (250 mg) yielded, after lyophilisation, the product (19 mg) as a fluffy off-white solid, homogeneous on TLC.
Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.90 -toluene/acetone/acetic acid (12:7:1):0.58; IR (KBr) 1785, 1720 (sh), 1685 cm-1. Example 26
Benzyl (2S,3RS,2'S,5'R)-3-bromo-3-(N-methyl-N-phenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia(1)-azabicyclo-[3.2.0]heptane]-2'-carboxylate Following the procedure in Example 8, benzyl 6-diazopenicillanate (0.56 g), oxalyl bromide (0.20 ml) and N-methylaniline (0.5 ml) yielded, after rapid chromatography, the product mixture of isomers (0.35 g, 37%) as a yellow oil. Repeated chromatography yielded, in the early fractions, pure 3S-isomer and in the later fractions a 1:2 mixture (NMR) of 3R:3S-isomers.
Rf (silica)-hexane/ethyl acetate (3:1):0.33 (isomeric mixture) IR (film) 1790, 1740, 1665 cm-1;
NMR delta, ppm (CDCl3) (3S-isomer) 1.40 (3H,s), 1.62 (3H,s), 3.30 (3H,s), 4.50 (1H,S,C2'-H), 5.16 (2H,s), 5.32 (1H,s,C5'-H), 7.30 (10H,s);
Found M+ 530.0456, C24H23 79BrN2O5S requires 530.0507. Example 27
(2S,3RS,2'S,5'R)-3-bromo-3-(N-methyl-N-phenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylic acid
Following the procedure in Example 12, the isomeric mixture from Example 26 containing a 1:2 ratio of the 3R:3S-isomers of benzyl 3-bromo-3-(N-methyl-N-phenylcarboxamido)spiro-oxirane-2,6'-penicillanate (55 mg) and 10% palladium on charcoal (250 mg) yielded after lyophilisation, the product (35 mg) as a fluffywhite solid, homogeneous on TLC.
Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.87
-toluene/acetone/acetic acid (12:1 :1):0.69; IR (KBr) 1790, 1735, 1660 cm-1. Example 28
Benzyl (2S,3RS and 3S,2'S,5'R)-3-acetyl-3-chloro-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylate
Following the procedure in Example 7, benzyl 6-diazopenicillanate (0.62 g) and pyruvyl chloride (0.30 ml) yielded, after rapid chromatography, the product mixture of isomers (200 mg,
26%) as a yellow highly unstable oil. Repeated chromatography yielded, in the early fractions, pure 3S-isomer and, in the later fractions a mixture of the 3R and 3S-isomers.
Rf (silica)-hexane/ethyl acetate (3:1):0.60 (isomeric mixture) IR (film) 1790, 1740 cm-1; NMR delta, ppm (CDCI3) (3S-isomer) 1.46 (3H,s), 1.57 (3H,s), 2.50 (3H,s), 4.58 (1H,s,C2'-H), 5.28 (2H,s), 5.62 (1H,s,C5'-H), 7.40 (5H,s);
Found M+ 395.0605, C18H18 35ClNO5S requires 395.0592. Example 29 Benzyl (2S,3S and 3R,2'S,5'R)-3-chloro-3-(N-4-hydroxyphenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia- (1)-azabicyclo[3.2,0]heptane]-2'-carboxylate
Following the procedure in Example 17, benzyl 6-diazopenicillanate (0.61 g) and oxalyl chloride (0.18 ml) gave a yellow oil. The oil was taken up in anhydrous ether (20 ml) and added to a cold solution of 4-aminophenol (0.42 g) and dry pyridine (0.16 g) in anhydrous THF (20 ml). Reaction, as indicated by precipitation, was instantaneous. After standing at room temperature for 5 minutes the reaction was diluted with water (50 ml) and ether (50 ml). The ethereal layer was separated and washed with water, dil. HCl (x2), dil. HCO3- (x2), water, dried (MgSO4) and evaporated. The residue was rapidly chromatographed on silica, followed by elution with hexane/ethyl acetate (3:2) to yield the 3S-isomer as the major product (0.38 g, 41%), Rf (silica)-hexane/ethyl acetate (2:1):0.21; hexane/ethyl acetate (1:1):0.56 and the 3R-isomer as the minor product (0.10 g, 10%), Rf (silica)-hexane/ethyl acetate (2:1):0.16; hexane/ethyl acetate (1:1):0.50, both as unstable pale yellow oils. Major product - 3S-isomer IR (film) 3320-3360 (br), 3060, 3030, 2960, 2930, 1795, 1740, 1685, 1270 (sh), 700 cm-1;
NMR delta, ppm (CDCI3) 1.43 (3H,s), 1.56 (3H,s), 4.57 (1H.s), 5.28 (2H,s), 5.64 (1H,s), 6.70-6.80 and 7.30-7.43 (10H,m, AA' BB' + aromatics + phenolic proton), 8.15 (lH,Br,s); Found F.A.B.S +ve ion mode 489, 491 (MH+). Minor product - 3R-isomer
IR (film) 3310-3350 (br), 3030, 3060, 2960, 2920, 1795, 1745, 1690, 1270-1290, 700 cm-1;
NMR delta, ppm (CDCl3) 1.40 (3H,s), 1.56 (3H,s), 4.66 (1H.s), 5.23 (2H,s), 5.70 (1H,s), 6.78-6.88 and 7.30-7.43 (10H,m, AA' BB' + aromatics + phenolic proton), 8.29 (1H,br,s);
Found F.A.B.S +ve ion mode 489, 491 (MH+). Example 30
(2S,3R,2'S,5'R)-3-chloro-3-(N-4-hydroxyphenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylic acid
Following the procedure in Example 12, benzyl 3-chloro-3-(N-4-hydroxyphenylcarboxamido)spiro-oxirane-2,6'-penicillanate, 3R-isomer (100 mg), prepared in Example 29, in ethyl acetate (15 ml) and 10% palladium on charcoal (500 mg) yielded, after lyophilisation, the product (34 mg) as a white fluffy solid homogeneous on TLC.
Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.82 -toluene/acetone/acetic acid (12:7:1):0.41; IR (KBr) 3300 (br), 2980, 2925, 1785, 1725, 1695, 1515, 1275 (br), 1240 (br) cm-1;
Found F.A.B.S +ve ion mode 399, 401 (MH+). Example 31
(2S,3S,2'S,5'R)-3-chloro-3-(N-4-hydroxyphenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylic acid
Following the procedure in Example 12, benzyl 3-chloro-3-(N-4-hydroxyphenylcarboxamido)spiro-oxirane-2,6'-penicillanate, 3R-isomer (360 mg), prepared in Example 29, in ethyl acetate (30 ml) and 10% palladium on charcoal (1.8 g) yielded, after lyophilisation, the product (105 mg) as a white fluffy solid homogeneous on TLC.
Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.89 -toluene/acetone/acetic acid (12:7:1):0.44; IR (KBr) 3300 (br), 2980, 2920, 1785, 1725, 1685, 1515, 1245 (sh), 1225 (br) cm-1; Example 32
Benzyl (2S,3S and 3R,2'S,5'R)-3-chloro-3-(N-2,6-dichlorophenylcarboxamido)-3',3'-dlmethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia- (1)-azabicyclo[3.2.0]heptane]-2'-carboxylate Following the procedure in Example 17, benzyl 6-diazopenicillanate (0.59 g) and oxalyl chloride (0.18 ml) gave a yellow oil. The yellow oil was taken up in anhydrous ether (10 ml) and anhydrous acetonitrile (10 ml) and added to a solution of 2,6-dichloroaniline (0.70 g) in anhydrous acetonitrile (20 ml) at room temperature. The solution was left at room temperature for 15 minutes, then dry pyridine (0.16 ml) was added. After a further 5 minutes at room temperature the reaction was diluted with water (50 ml) and ether (30 ml). The ethereal layer was separated and washed with water, dil. HCl (x2), dil. HCO3- (x2), water, dried (MgSO4) and evaporated. The residue was rapdily chromatographed on silica by elution with hexane/ethyl acetate (3:1) to yield the product mixture of isomers (0.53 g, 53%) as a pale yellow oil. Repeated chromatography yielded the 3S-isomer as the major product, Rf (silica)-hexane/ethyl acetate (3:1):0.35 and the 3R-isomer as the minor product, Rf (silica)-hexane/ethyl acetate (3:1):0.30. Minor product - 3R-isomer
IR (film) 3295 (br), 3060, 3030, 2960, 2920, 1795, 1740, 1720, 1290, 700 cm-1; NMR delta, ppm (CDCl3) 1.41 (3H,s), 1.61 (3H,s), 4.67 (1H,s), 5.25 (2H,s), 5.71 (1H,s), 7.10-7.53 (8H,m), 8.20 (1H,br,s).
Found F.A.B.S +ve ion mode 541, 543, 545 (MH+); 513, 515, 517 (MH+ -CO); 507, 509 (MH+ -HCl). Example 33 (2S,3R,2'S,5'R)-3-chloro-3-(N-2,6-dichlorophenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylic acid
Following the procedure in Example 12, benzyl 3-chloro-3- (N-2,6-dichlorophenylcarboxamido)spiro-oxirane-2,6'-penicillanate, 3R-isomer (50 mg), prepared in Example 32, in ethyl acetate (10 ml) and 10% palladium on charcoal (200 mg) yielded, after lyophilsation, the product (23 mg) as a white fluffy solid homogeneous on TLC.
Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.84
-toluene/acetone/acetic acid (12:7:1) :0-52; IR (KBr) 3300 (br), 2955, 2920, 2860, 1790, 1720, 1710 (sh), 1500, 1285 cm-1;
Found F.A.B.S +ve ion mode 451, 453, 455 (MH+). Example 34
Benzyl (2S,3S and 3R,2'S,5'R)-3-chloro-3-(N-2,6-dichlorophenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylate
2,6-Dimethoxyaniline was prepared as follows. 2-Nitroresorcinol (3.1 g) was dissolved in a solution of sodium hydroxide (2.0 g) in water (50 ml) and ethanol (10 ml). To this was added dimethyl sulphate (5 ml) and the reaction mixture was heated to 70°C. After 30 minutes a further portion (50 ml) of dimethyl sulphate and sodium hydroxide (1.0 g) were added. After 1 hour the reaction was cooled to room temperature and poured into a solution of 2 M NaOH (20 ml) and water (30 ml). The product was collected, washed with copious amounts of water, sucked as dry as possible and recrystallised from methanol. The yield of 2,6-dimethoxynitrobenzene was 2.86 g (78%), 129.5-130.5°C (lit. 130°C).
To a stirred suspension of 2,6-dimethoxynitrobenzene (1.83 g) in 95% aqueous ethanol (20 ml), was added platinum oxide catalyst (35 mg). The reaction was hydrogenated at slightly higher than atmospheric pressure until 3 molar equivalents of hydrogen had been taken up (ca. 1.5-2 hours) and the solution went clear. The catalyst was filtered off through a tight Celite pad and washed with ethanol. The combined filtrate and washings were evaporated to dryness and the oily residue taken up in hot petrol, filtered and allowed to cool. The product crystallised as pale yellow plates. The yield of 2,6-dimethoxyaniline was 1.30 g (85%), m.p. 74°C (lit. 75°C).
Following the procedure in Example 29, benzyl 6-diazopenicillanate (0.51 g) and oxalyl chloride (0.17 ml); and a solution of 2,6-dimethoxyaniline (0.28 g) and dry pyridine (0.15 g) in anhydrous ether (20 ml) yielded, after rapid chromatography on silica, using as eluting solvent hexane/ethyl acetate (3:1), the product mixture of isomers (0.50 g, 50%) as a pale yellow oil. Repeated chromatography yielded the 3S-isomer as the major product, Rf (silica)-hexane/ethyl acetate (2:1):0.31; hexane/ ethyl acetate (3:2):0.38 and the 3R-isomer as the minor product, Rf (silica)-hexane/ethyl acetate (2:1):0.27; hexane/ethyl acetate (3:2):0.35. Minor product - 3R-isomer IR (film) 3330-3370, 3030. 3010 (sh), 2960, 2930, 1795, 1740, 1715, 1290, 700 cm-1;
NMR delta, ppm (CDCI3) 1.42 (3H,s), 1.59 (3H,s), 3.85 (6H,s), 4.68 (1H.s), 5.25 (2H,s), 5.72 (1H,s) 6.56-6.66 (2H,dd, J=1Hz and 10Hz), 7.15-7.35 (1H,m), 7.43 (5H,s), 7.77 (1H,br,s); Found M+ 532.1071, C25H25 35ClN2O7S requires 532.1067. Major product - 3S-isomer
IR (film) 3330-3370, 3030, 3010 (sh), 2960, 2930, 1795, 1740, 1715. 1290, 700 cm-1;
NMR delta, ppm (CDCI3) 1.46 (3H,s), 1.62 (3H,s), 3.85 (6H,s), 4.63 (1H,s), 5.25 (2H,s), 5.63 (1H,s) 6.54-6.64 (2H,dd, J=1Hz and 10Hz), 7.10-7.30 (1H,t+t.j=1Hz and 10Hz), 7.67 (1H.br, s); Example 35
(2S,3R,2'S,5'R)-3-chloro-3-(N-2,6-dimethoxyphenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2,0]heptane]-2'-carboxylic acid
Following the procedure in Example 12, benzyl 3-chloro-3- (N-2,6-dimethoxyphenylcarboxamido)spiro-oxirane-2,6'-penicillanate
(3R:3S-isomer ratio 3:1; 50 mg) in ethyl acetate (10 ml) and 10% palladium on charcoal (200 mg) yielded, after lyophilisation, the product (14 mg) as a white fluffy solid homogeneous on TLC.
Rf (silica)-n-butanol/acetic acid/water (4:1 : 1 ) :0.85
-toluene/acetone/acetic acid (12:7:1):0.54; IR (KBr) 3400 (br), 2960, 2930, 1790, 1725, 1710 (sh), 1600, 1260, 1115 cm-1; Found F.A.B.S +ve ion mode 443, 445 (MH+). Example 36
Benzyl (2S,3S and 3R,2'S,5'R)-3-chloro-3-(N-cyanomethylcarboxamido-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia- (1)-azabicvclo[3.2.0]heptane]-2'-carboxylate Following the procedure in Example 29, benzyl 6-diazopenicillanate (0.62 g), oxalyl chloride (0.18 ml) and a solution of aminoacetonitrile (0.13 g) and dry pyridine (0.15 g) in anhydrous ether (20 ml) yielded, after rapid chromatography on silica, using as eluting solvent hexane/ethyl acetate (2:1), the 3S-isomer as the major product, Rf (silica)-hexane/ethyl acetate
(2:1):0.31 and the 3R-isomer as the minor product, Rf (silica)-hexane/ethyl acetate (2:1):0.24, in a combined yield of 0.37 g
(44%), both as unstable pale yellow oils.
Major product - 3S-isomer IR (film) 3340 (br), 3060, 3030, 2960, 2930, 2250 (weak)
1795, 1740, 1710, 1295, 1270, 700 cm-1;
NMR delta, ppm (CDCI3) 1.44 (3H.s), 1.57 (3H,s), 4.22
(2H,d,J=6Hz), 4.59 (1H,s), 5.26 (2H,s), 5.61 (1H,s), 7.27-7.40
(1H.br, t,J=6Hz), 7.40 (5H,s); Found F.A.B.S +ve ion mode 436, 438 (MH+); 408, 410 (MH+
-CO); 402 (MH+ -HCl).
Minor product - 3R-isomer
IR (film) 3340 (br), 3060, 3030, 2960, 2930, 2250 (weak)
1795, 1740, 1700, 1285 (br), 700 cm-1; NMR delta, ppm (CDCI3) 1.41 '(3H,s), 1.54 (3H,s), 4.26
(2H,d,J=6Hz), 4.66 (1H,s), 5.26 (2H,s), 5.69 (1H,s), 7.40 (6H,m); Found F.A.B.S +ve ion mode 436, 438 (MH+); 408, 410 (MH+
-CO); 402 (MH+ -HCl).
Example 37 (2S,3R,2'S,5'R)-3-chloro-3-[N-ethylammoniurn]carboxamido)- 3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylate
Following the procedure in Example 5, benzyl 3-chloro-3-(N-cyanomethylcarboxamido)spiro-oxirane-2,6'-penicillanate, 3R-isomer (80 mg) in ethanol (10 ml) and 10% palladium on charcoal
(400 mg) yielded, after lyophilsation, the product (48 mg) as a water-soluble, white fluffy solid, homogeneous on TLC. Rf (silica)-n-butanol/acetic acid/water (4: 1 : 1 ) :0.81
-toluene/acetone/acetic acid (12:7:1):0.46; IR (KBr) 3410 (br), 2960, 2930, 1785, 1695, 1610, 1520 cm-1; Found F.A.B.S. +ve ion mode 350, 352 (MH+). Example 38
Benzyl (2S,3R,2'S,5'R)-3-chloro-3-(N-phenylcarboxamido)- 3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)- azabicyclo[3.2.0]heptane]-2'-carboxylate-4',4'-dioxide
To a solution of benzyl 3-chloro-3-(N-phenylcarboxamido)- spiro-oxirane-2,6'-penicillanate, 3R-isomer (100 mg), prepared in Example 8, in dichloromethane (15 ml) was added 80-90% m-chloroperoxybenzoic acid 150 mg) and the solution was refluxed for 4 hours. The solvent was evaporated and the residue taken up in ether (30 ml) and washed repeatedly with saturated HCO3-, then with water (x1), dried (MgSO4) and evaporated to yield the sulphone as a colourless oil in quantitative yield. Rf (silica)-hexane/ethyl acetate (3:1):0.24 -hexane/ethyl acetate (2:1):0.38; IR (film) 3340 (br), 3060, 3030, 2950, 2920, 1815, 1755, 1700, 1330, 1280, 1120, 690 cm-1;
NMR delta, ppm (CDCl3) 1.31 (3H,s), 1.53 (3H,s), 4.53 (1H.s), 5.10-5.43 (2H,ABq,J=12Hz), 5.27 (1H,S), 7.15-7.70 (5H,m) 7.43 (5H,s), 8.61 (1H,br,s).
Found F.A.B.S +ve ion mode 505, 507 (MH+). Example 39
(2S,3R,2'S,5'R)-3-chloro-3-(N-phenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylic acid-4',4'-dioxide
Following the procedure in Example 12, benzyl 3-chloro-3- (N-phenylcarboxamido)spiro-oxirane-2,6'-penicillanate-4',4'-dioxide, 3R-isomer (120 mg) in ethyl acetate (5 ml) and ethanol (5 ml) with palladium 10% on charcoal (300 mg) yielded, after lyophilisation, the product (75 mg) as a highly unstable white fluffy solid essentially homogeneous on TLC, but which had undergone slight degradation on hydrogenolysis.
Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.65 -toluene/acetone/acetic acid (12:7:1):0.25; IR (KBr) 3400 (br), 2930, 1815, 1700 (br), 1600, 1450, 1310, 1120, 690 cm-1;
Found F.A.B.S +ve ion mode 415, 417 (MH+). Example 40 Benzyl (2S,3S,2'S,5'R)-3-bromo-3-methoxycarbonyl-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylate-4'-oxide
Benzyl 3-bromo-3-methoxycarbonylspiro-oxirane-2,6'-penicillanate (220 mg), prepared in Example 13, was dissolved in chloroform (5 ml) and the solution cooled to 0°C. A solution of 80-90% m-chloroperoxybenzoic acid (100 mg) in chloroform (5 ml) was added slowly with stirring to the above solution and the reaction mixture was stirred at 0°C for 1 hour. The reaction mixture was then washed with dil. HCO3- (x4), water, dried (MgSO4), and evaporated. The resultant oily residue crystallised on addition of a drop of ether and the product (125 mg, 55%) was recrystallised from ethyl acetate/hexane, m.p. 130-133°C (dec). Rf (silica)-hexane/ethyl acetate (1:1):0.63 IR (KBr) 2970, 1820, 1755, 1740, 1265, 1048, 765, 705 cm-1; NMR delta, ppm (CDCl3) 1.12 (3H,s), 1.68 (3H,s), 3.91 (3H,s), 4.66 (1H,s), 5-10-5.43 (2H,Abq,J=12Hz), 5.13 (1H,s), 7.43 (5H,s); Found F.A.B.S +ve ion mode 472, 474 (MH+). Example 41
Benzyl (2S,3R,2'S,5'R)-3-methoxycarbonyl-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylate-4'-oxide
(This is the hydrodehalogenated analogue of the 3-bromo compound of Example 40 having the same 3-position configuration but now designated "3R".) A highly active zinc-copper couple was prepared following a procedure described by E. LeGoff, J. Org. Chem. 29, 2048 (1964), as follows. To a rapidly stirred hot solution of cupric acetate, monohydrate (1 g) in glacial acetic acid (25 ml) was added zinc dust (15 g). A vigorous exothermic reaction occurred and the reddish-grey silt-like couple precipitated. The hot reaction was swirled for 1-2 minutes and then cooled to room temperature. A further quantity of acetic acid (25 ml) was added and the couple swirled. The supernatant was decanted and the moist couple was washed repeatedly with ether to remove all traces of acetic acid. The couple was stored under ether in a tightly stoppered flask until required. To a solution of benzyl 3-bromo-3-methoxycarbonylspirooxirane-2,6'-penicillanate-4'-oxide, 3S-isomer (50 mg), prepared in Example 40, in tetrahydrofuran (2 ml) was added pH 4 buffer (1 ml) and excess of the zinc-copper couple. The suspension was stirred at room temperature for 30 minutes. The solids were filtered off through a tight Celite pad and washed with ether. The combined filtrate and washings were diluted with water (30 ml), the organic layer was separated and washed repeatedly with water, dried (MgSO4) and evaporated. The residue was chromatographed on silica plates, using as eluting solvent acetate/toluene (1:1) to yield the product (30 mg, 72%), Rf (silica)-ethyl acetate/toluene (1:1):0.24 which was recrystall ised from dichloromethane/ether (1 :1):hexane, m.p. 149-150°C.
Trace amounts of a second product (Rf (silica)-ethyl acetate/toluene (1:1):0.51), which could have been the 3S-isomeric product, were also isolated from the reaction, but in too small a quantity to allow full characterisation.
IR (KBr) 3050, 3005, 2910, 1785, 1750, 1735, 1285, 1050 700 cm-1; NMR delta, ppm (CDCl3) 1.13 (3H,s), 1.67 (3H,s), 3.86 (3H,s), 3.98 (1H,s) 4.66 (1H,s), 5.10-5.43 (2H,Abq,J=12Hz), 5.21 (1H,s), 7.42 (5H,S);
Found F.A.B.S +ve ion mode 394 (MH+); no halogen observed in any fragments; Found M+ 393.0878, C18N19NO7S requires 393.0879. Example 42
Benzyl(2S,3S and 3R,2'S,5'R)-3-(N-phenylcarboxamido)-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylate Following the procedure in Example 41, benzyl 3-bromo-3-(N-phenylcarboxamido)spiro-oxirane-2,6'-penicil lanate, mixture of 3S- and 3R-isomers (0.56 g), prepared in Example 24, in tetra hydrofuran (30 ml), pH 4 buffer (6 ml) and excess zinc-copper couple yielded, after chromatography on silica, using as eluting solvent hexane/ethyl acetate (3:1), in the early fractions pure crystalline 3R-isomer (290 mg, 61%), Rf (silica)-hexane/ethyl acetate (3:1):0.32, m.p. 154-156°C and in later fractions pure 3S-isomer (58 mg, 12%), Rf (si lica)-hexane/ethyl acetate (3:1):0.22.
A similar procedure was used, starting from the pure 3S- and 3R-isomers of the 3-bromo compound, prepared as in Example 24, to prepare the pure 3S- and 3R-isomers of the title compound, the properties of which were as follows. 3S-isomer
IR (KBr) 3370 (br), 3025, 2960, 2930, 1785, 1745, 1695, 1600, 1535, 1445, 1315, 1300, 695 cm-1; NMR delta, ppm (CDCI3) 1.43 (3H,s), 1.52 (3H,s), 3.97 (1H,s), 4.61 (1H,s) 5.22 (2H,s), 5.61 (1H,s) 6.98-7.67 (5H,m) 7.41 (5H,s), 7.96 (1H.br, s).
13C NMR delta, ppm 24.88 (-CH3), 34.08 (-CH3), 58.24 (>CH<),
63.70 (>C<), 67.58 (-CH2-), 69.93 (-CH<), 70.19 (-CH<), 75.63 (>C<), 120.62, 125.10, 128.64, 128.71, 128.77, 128.98 (six aromatic -CH=), 134.65, 136.36 (two aromatic >C=) 161.71, 166.79,
169.59 (three >C=0).
Found C,62.59; H.5.06; N,6.41%, C23H22N2O5S requires C.63.01; H,5.02; N,6.39%. 3R-isomer
IR (film) 3300 (br), 3060, 3030, 2960, 2920, 2860, 1790, 1740, 1680, 1290, 695 cm-1;
NMR delta, ppm (CDCl3) 1.37 (3H,s), 1.56 (3H,s), 4.01 (1H,s), 4.67 (1H,s), 5.22 (2H,s), 5.68 (1H,s), 7.15-7.60 (5H,m), 7.42 (5H,s), 7.77 (1H,br, s);
Found M+ 438.127, C23H22N2O5S requires 438.1246. Example 43
(2S,3R,2'S,5'R)-3-(N-phenylcarboxamido)-3',3'-dimethyl-7'-oxospiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylic acid
Following the procedure in Example 12, crystalline benzyl 3-(N-phenylcarboxamido)spiro-oxirane-2,6'-penicillanate, 3R-isomer (80 mg) in ethyl acetate (10 ml) and ethanol (5 ml) with 10% palladium on charcoal (450 mg) yielded, after lyophilisation, the product (36 mg) as a white fluffy solid homogeneous on TLC. Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.91
-toluene/acetone/acetic acid (12:7:1) :0.52; IR (KBr) 3420 (br), 2960, 2920, 1770, 1680, 1600, 1540, 1445, 1315, 1250, 695 cm-1;
Found F.A.B.S +ve ion mode 349 (MH+). Example 44
(2S,3S,2'S,5'R)-3-(N-phenylcarboxamido)-3',3'-dimethyl-7'-oxospiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'- carboxylic acid
Following the procedure in Example 12, benzyl 3-(N- phenylcarboxamido)spiro-oxirane-2,6'-penicillanate, 3S-isomer (58 mg) in ethyl acetate (10 ml) and ethanol (3 ml) with 10% palladium on charcoal (250 mg) yielded, after lyophilisation, the product (27 mg) as a white fluffy solid homogeneous on TLC. Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.73 -toluene/acetone/acetic acid (12:7:1) :0.38;
IR (KBr) 3430 (br), 2960, 2920, 1780, 1730, 1680, 1600, 1550, 1445, 1295, 695 cm-1;
Found M+ (low resolution), 348; F.A.B.S +ve ion mode 349 (MH+). Example 45
Dipotassiurn (R)-2-{(2S,3S,2'S,5'R)-2'-carboxy-3-chloro-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-3-carboxamido}-2-phenylacetate
Dibenzyl 3-chloro-3-(D-alpha-phenylglycinamido)spirooxirane-2,6'-penicillanate, 3S-isomer (190 mg), prepared in Example 17, was dissolved in ethyl acetate (10 ml) and a solution of potassium barcarbonate (61 mg, two equivalents) in water (15 ml) was added followed by 10% palladium on charcoal (700 mg). The mixture was hydrogenated with stirring for 1 hour. The catalyst was filtered off through a tight Celite pad and washed with water and ethyl acetate. The aqueous layer of the combined filtrate and washings was separated, washed with ether (x1) and lyophilised to yield the product (160 mg) as an amorphous solid homogeneous on TLC, Rf (silica)-butanol/acetic acid/water (4:1:1):0.84. Example 46 Dipotassium (R)-2-{(2S,3RS,2'S,5'R)-2'-carboxy-3-chioro-3'.3,-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-3-carboxamido}-2-phenylacetate
Following the procedure in Example 45, the 1:13R:3S isomeric mixture of di benzyl 3-chloro-3-(D-alpha-phenylglycinamido)spirooxirane-2,6'-pencillanate (220 mg), prepared in Example 17, in ethyl acetate (10 ml), potassium bicarbonate (70 mg) in water (15 ml) and 10% palladium on charcoal (800 mg) yielded, after lyophilisation, the product (15 mg) as an amorphous solid, homogeneous on TLC, Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.86.
IR (KBr) 3400 (br), 2965, 2925, 1780, 1685 (sh), 1610 (br), 1385, 1125 cm-1. Example 47 Dipotassium (S)-2-{(2S,3RS,2'S,5'R)-2'-carboxy-3-chloro-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-3-carboxamido}-2-phenylacetate
Following the procedure in Example 12, the 3R:3S isomeric mixture of dibenzyl 3-chloro-3-(L-alpha-phenylglycinamido)spirooxirane-2,6'-penicillanate (110 mg), prepared in Example 18, in ethyl acetate (15 ml) and 10% palladium on charcoal (550 mg) yielded, after lyophilisation, the product (30 mg) as a buffcoloured fluffy solid, Rf (silica)-n-butanol/acetic acid/water (4:1:1), 0.92 and 0.83 (i.e. separation of the two diastereoisomers). Example 48
(2S,3R,2'S,5'R)-3-chloro-3-CN-(2-thiazolyl)carboxamido]-3',3'-dimethyl-7'-oxo-spiro[oxirane-2,6'-(4)-thia-(1)-azabicyclo[3.2.0]heptane]-2'-carboxylic acid
Benzyl 3-chloro-3-[N-(2-thiazolyl)carboxamido]spiro-oxirane-2,6'-penicillanate, 3R-isomer (140 mg) prepared in Example 19, was dissolved in ethyl acetate (15 ml) and 10% palladium on charcoal (600 mg) was added. The reaction was hydrogenated with stirring for 30 minutes, an extra portion of catalyst (200 mg) was added and the reaction was re-hydrogenated for a further 30 minutes. TLC indicated that the deprotection was still not complete, so the catalyst was filtered off through a tight Celite pad, washed with ethyl acetate and the combined filtrate and washings were evaporated to approximately 15 ml. Fresh catalyst (300 mg) was added and the mixture was further hydrogenated with stirring for 30 minutes. Work-up above and lyophilisation from aqueous ethanol yielded the product (15 mg) as a yellow, poorly stable amorphous solid.
Rf (silica)-n-butanol/acetic acid/water (4:1 :1 ) :0.88; -toluene/acetone/acetic acid (12:7:1):0.57. Example 49
1,1-Dimethylethyl (6R)-3-acetoxymethyl-7-diazo-8-oxo-5- thia-1-azabicyclo[3.2.0]oct-2-ene-2-carboxylate (tert.-butyl 7-diazocephalosporanate)
(a) Concentrated sulphuric acid (15 ml) was added to tetrahydrofuran (300 ml) in an autoclave/pressure bottle and the resultant solution was cooled to -10°C. To this stirred solution was added liquid isobutylene (100 ml) followed by 7-aminocephalosporanic acid (15 g). The reaction vessel was tightly sealed and stirred at room temperature for 20 hours.
The reaction vessel was cooled to below 0 C, opened carefully and the contents poured into an ice-cold solution of sodium bicarbonate (75 g) in water (100 ml). The solution was extracted with chloroform (3 x 300 ml) and the combined organic extracts were washed with water (2 x 300 ml), brine, dried (MgSO4) and evaporated to leave a brown crystalline residue.
The crude product was dissolved in dichloromethane (100 ml) and extracted into ice-cold 0.5 M HCl (ca. 300 ml). The organic layer was discarded and the aqueous layer re-extracted with dichloromethane (2 x 100 ml), the organic layers being, once again, discarded. The aqueous layer was partitioned with dichloromethane and basified with saturated sodium bicarbonate solution. The organic layer was separated and the aqueous layer re-extracted with dichloromethane (2 x 100 ml). The combined organic extracts were washed once with water, dried (MgSO4) and evaporated. The crystalline residue was dissolved in the minimum volume of warm dichloromethane (ca. 10 ml), diluted with ether (ca. 20 ml) and crystallised by gradual addition of hexane and storage at -15°C overnight. The yield of t-butyl 7-aminocephalosporanate was 5.88 g (33%), m.p. 115ºC. (b) tert.-Butyl 7-aminocephalosporanate (0.68 g, 2.1 mmol), prepared above was dissolved in 75% aqueous acetone (20 ml) and 2 M hydrochloric acid (3 ml) was added. The solution was cooled to 0°C and a solution of sodium nitrite (160 mg) in water (1-2 ml) was added, with stirring, followed immediately by ether (10 ml). The yellow biphasic reaction liquid was vigorously stirred at 0°C for 10 minutes, at the end of which time ether (20 ml) and water (20 ml) were added. The ethereal layer was separated and the aqueous layer re-extracted with ether (20 ml). The combined organic layers were washed with water repeatedly, dil. HCO3- (x1), water, dried (MgSO4) and evaporated to yield the product (0.63 g, 90%) as a yellow oil of sufficient purity for all subsequent manipulations.
Rf (silica)-hexane/ethyl acetate (3:1):0.30;
IR (film) 2975, 2930, 2090, 1775, 1735 (br), 1635 cm-1;
NMR delta, ppm (CDCl3) 1.60 (9H,s), 2.11 (3H,s), 3.24-3.70 (2H,ABq,J=18Hz), 4.71-5.18 (2H,ABq,J=13Hz), 5.63 (1H,s). Example 50
1,1-Dimethylethyl (2S,3S and 3R,6'R)-3-chloro-3-(N-phenylcarboxamido)-3'-acetoxymethyl-8'-oxo-spiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'-ene]-2'-carboxylate (tert.-butyl 3-chloro-[N-phenylcarboxamido]spiro-oxirane-2,7'-cephalosporanate)
Figure imgf000046_0001
Following the procedure in Example 8, tert.-butyl 7-diazocephalosporanate (0.61 g) prepared in Example 49, oxalyl chloride (0.16 ml) and aniline (0.4 ml) yielded a yellow oil. The major product, 3S-isomer, was fractionally crystallised from the oil by dissolving the residue in warm chloroform (3 ml), adding ether (3 ml), diluting to faint turbidity with petrol and storing overnight at below 0 C.
The crystals, 280 mg, m.p. 138-143°C (dec), were collected, washed with ether and recrystallised from chloroform/ether (1:1) and petrol. The combined filtrate and washings, containing the 3R-isomer, were evaporated and chromatographed on silica. Elution with hexane/ethyl acetate (2:1) yielded, in the early fractions further quantities of the 3S-isomer, Rf (silica)-hexane/ethyl acetate (2:1):0.37 and, in later fractions, a 5:1 mixture (NMR) of the 3R- and 3S-isomer, Rf (silica)-hexane/ethyl acetate (2 :1):0.34. The combined yield of both isomers was 0.43 g (48%). Major product - 3S-isomer
IR (KBr) 3380, 3000, 2980, 2960, 1785, 1735, 1695, 1220, 690 cm-1; NMR delta, ppm (CDCl3) 1.60 (9H,s), 2.13 (3H,s), 3.27-3.78 (2H,ABq,J=18Hz), 4.74-5.17 (2H,ABq,J=13Hz), 5.38 (1H,s), 7.15-7.70 (5H,m), 8.46 (1H.br, s);
13C NMR delta, ppm* 20.62 (-CH3), 26.78 (-CH2-), 27.97 (-CH3), 58.51 (CH<), 62.92 (-CH2-). 76.78 (>C<) 84.66 (>C<), 120.85 (-CH=), 123.91 (>C=), 125.66 (-CH=), 127.91 (>C=), 129.15 (-CH=), 136.53 (>C=), 157.68 (>C=O), 158.47 (>C=O), 159.74 (>C=O), 170.44 (>C=O), one quaternary carbon (>C<) obscured by solvent peak;
* J modulated spin echo experiment -CH3 = Methyl carbon, appears +ve phase
-CH2- = Methylene carbon, appears -ve phase -CH< = Methine carbon, appears +ve phase >C< = Quaternary saturated carbon, appears -ve phase -CH= = Aromatic methine carbon, appears +ve phase >C= = Quaternary aromatic or unsaturated carbon, appears
-ve phase >C=O = Quaternary carbonyl carbon, appears -ve phase. Found F.A.B.S. +ve ion mode 495, 497 (MH+); 439, 441 (MH+ -t.Bu; 379, 381 (MH+ -t.Bu - CH3CO2H);
Found C,53.30; H.4.71; N, 5.40%, C22H23CIN2O7S requires C.53.39; H.4.65; N,5.66%. Minor product - 3R-isomer
IR (film) 3320 (br), 3050, 3010, 2980, 2930, 1795, 1740 (sh), 1720 (br), 1645, 1240 (sh), 1225, 690 cm-1;
NMR delta, ppm (CDCI3) 1.64 (9H,s), 2.09 (3H,s), 3.16-3.67 (2H,ABq,J=18Hz), 4.71-5.13 (2H,ABq,3=13Hz), 5.23 (1H,s), 7.22-7.66 (5H,m), 8.33 (1H.br, s);
Found F.A.B.S. +ve ion mode 495 (very weak MH+); 459 (MH+ -HCl); 435, 437 (MH+ -C4H8); 379, 381 (MH+ -C4H8 -CH3CO2H); 343 (MH+ -C4H8 -CH3CO2H -HCl); Also, characteristic of these spiro-oxirane cephalosporin 3R-isomers, MH+ -2 (493) and MH+ -2 -C4H8 (437, 439). Example 51
(2S,3S,6'R)-3-chloro-3-(N-phenylcarboxamido)-3'-acetoxymethyl-8'-oxo-spiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'ene-]-2'-carboxylic acid Tert.-butyl 3-chloro-3-(N-phenylcarboxamido)spiro-oxirane-2,7'-cephalosporanate, 3S-isomer (100 mg), prepared in Example 50, was taken up in freshly distilled ice-cold trifluoracetic acid, TFA, (10 ml). The solution was stored at below 5°C for 1½ hours and the solvent was evaporated at below 15°C. The residue was taken up in ethyl acetate and the solvent again evaporated at low temperature. This evaporation procedure was repeated to remove the least traces of TFA. The residue was taken up in acetone (1 ml) and water (5 ml) and lyophilised overnight to yield the product quantitatively as a pale yellow fluffy solid. Rf (silica)-toluene/acetone/acetic acid (12:7:1):0.40;
IR (KBr) 3420 (br), 1790, 1725 (sh), 1700, 1635, 1600, 1540, 1445, 1235 (br), 695 cm-1;
NMR delta, ppm (CDCI3) 2.12 (3H,s) 3.27-3.75(2H,ABq,J=18Hz), 4.83-5.20 (H2O, from lyophilisation, + 2H,ABq,-CH2-OAc), 5.36 (1H,s), 7.19-7.67 (5H,m), 8.25 (1H.br, s);
Found F.A.B.S +ve ion mode 439, 441 (MH+); 379, 381 (MH+ -CH3CO2H). Example 52
(2S,3R,6'R)-3-chloro-3-(N-phenylcarboxamido)-3'-acetoxymethyl-8'- oxo-spiroCoxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'ene-]- 2'-carboxylic acid Following the procedure in Example 51, the isomeric mixture of that Example containing a 5:1 excess of the 3R-isomer of tert.-butyl 3-chloro-3-(N-phenylcarboxamido)spiro-oxirane-2,7'- cephalosporanate (70 mg) and ice-cold TFA (10 ml) yielded, after lyophilisation, the product quantitatively as an unstable buff- coloured fluffy solid, which showed, on TLC, slight traces of degradation.
Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.74
-toluene/acetone/acetic acid (12:7:1):0.45; IR (KBr) 3420 (br), 2920, 1790, 1730, 1710 (sh), 1640, 1600, 1535, 1445, 1235 (br), 695 cm-1;
Found F.A.B.S +ve ion mode 439, 441 (MH+); 379, 381 (MH+ -CH3CO2H). Example 53
1,1-Dimethylethyl (2S,3S and 3R,6'R)-3-chloro-3-(N-phenylcarboxamido)-3'-acetoxymethyl-8'-oxo-spiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'-ene]-2'-carboxylate (the same compound as prepared in Example 50)
Oxanilic acid chloride (N-phenyl chloroglyoxamide) was prepared as follows. To a stirred suspension of aniline hydrochloride (3.0 g) in dry ether (20 ml) was added oxalyl chloride (5 ml). The reaction was stirred at room temperature for 2 days, after which time it was filtered and the solids washed with dry ether. The combined filtrate and washings were evaporated and the .residue was crystallised from dry ether/petrol at -15°C to yield the product (3.8 g, 89%) as white needles, m.p. 82-83°C (lit. 82.5°C).
Crystalline oxanilic acid chloride prepared as above was added to a solution of tert.-butyl 7-diazocephalosporanate (0.38 g) in anhydrous ether (10 ml) at 0°C. The reaction was swirled to dissolve the solids and the solution was allowed to warm to room temperature as effervescence commenced. The golden colour faded slowly and the reaction was left at room temperature for 20 minutes. The reaction was diluted with dil. HCO3- and the organic layer was separated and washed with water, dil. HCO3- (x2), water, dried (MgSO4) and evaporated. As in Example 50, fractional crystallisation of the residue yielded the crystalline 3S-isomer, identical to that of Example 50 (m.p., mixed m.p., NMR and IR). Investigation of the mother liquors revealed the presence of the minor, 3R-isomer, also identical to that of Example 50. Example 54 1,1-Dimethylethyl (S)-{(2S,3S and 3R,6'R)-3-chloro-3-acetoxymethyl-2'-(1,1-dimethyl)ethyloxycarbonyl-8'-oxospiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'-ene]-3-carboxamido}-2-phenylacetate
Following the procedure in Example 8, tert.-butyl 7-diazocephalosporanate (0.67 g) and oxalyl chloride (0.18 ml) gave a yellow oil. The oil was taken up in anhydrous ether (20 ml) and added to a solution of L-alpha-phenyl glycine, tert.-butyl ester (0.88 g, from 1.6 g of the tosylate salt) in anhydrous ether (20 ml) at 0°C. Reaction, as indicated by precipitation of, the amino acid, ester hydrochloride was almost instantaneous. The reaction was left at room temperature for 5 minutes and then diluted with water (50 ml). The organic layer was separated and washed with water, dil. HCO3- (x2), dil. HCl (x2), water, dried (MgSO4) and evaporated. The residue was rapidly chromatographed on silica, using as eluting solvent hexane/ethyl acetate (2:1), and all the fractions containing the desired product as a mixture of isomers (0.57 g, 47%), Rf (silica)-hexane/ethyl acetate (2:1):0.38-0.46 were bulked and evaporated. From this residue the major product, 3S-isomer, was fractionally crystallised by dissolving in ether, diluting to faint turbidity with petrol and storing at -15°C overnight.
The crystals (240 mg) m.p. 130-132°C (dec), Rf (silica)-hexane/ethyl acetate (2:1):0.42, were collected, washed with ice-cold petrol /ether (2:1) and recrystallised from ether/hexane. The combined filtrate and washings containing the minor product, 3R-isomer, were evaporated and re-chromatographed on silica. Elution with hexane/ethyl acetate (2:1) yielded, in the early fractions, further quantities of the 3S-isomer and in the later fractions a 5:1 mixture (NMR) of the 3R-and 3S-isomers, Rf (silica)-hexane/ethyl acetate (2:1):0.40.
IR (KBr), 3410, 3000, 2975, 2920, 1785, 1740, 1730, 1715, 1220, 695 cm-1;
NMR delta, ppm (CDCI3) 1.47 (9H,s) 1.63 (9H,s) 2.13 (3H,s), 3.24-3.74 (2H,ABq,J=18Hz), 4.74-5.16 (2H,ABq,J=14Hz), 5.31 (1H,s), 5.43 (1H,d,J=6Hz), 7.42 (5H,s), 7.63 (1H,br,d,J=6Hz);
Found C,55.42; H,5.56; N,4.56%, C28H33ClN2O9S requires C,55.22; H,5.42; N,4.60%. Minor product - 3R-isomer
IR (film) 3380 (br), 3060, 3030, 3000 (sh), 2975. 2920. 1795, 1730, 1700 (sh), 1220, 695 cm-1;
NMR delta, ppm (CDCI3) 1.45 (9H,s), 1.61 (9H,s), 2.10 (3H,s), 3.12-3.62 (2H,ABq,J=18Hz), 4.72-5.11 (2H,ABq,J=13Hz), 5.14 (1H,s), 5.44 (1H,d,J=7Hz), 7.41 (5H,s), 7.82 (1H,br,s,J=7Hz);
Found F.A.B.S +ve ion mode 553, 555 (MH+ -C4H8); 549, 551 (MH+ -CH3CO2H); 497, 499 (MH+ -2 x C4H8 -CO2; 437, 439 (MH+ -2 x C4H8 -CH3CO2H). No MH+ observed. Example 55
(S)-2-{(2S,3S,6'R)-3-chloro-3'-aectoxymethyl-2'-carboxy-8'-oxospiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'-ene]-3-carboxamido}-2-phenylacetic acid
Following the procedure in Example 51, di-tert.-butyl 3-chloro-3-(L-alpha-phenylglycinylcarbonyl)spiro-oxirane-2,7'-ceph alo- sporanate, 3S-isomer (65 mg) prepared in Example 54, and ice-cold TFA (10 ml) yielded, after lyophilisation, the product (47 mg) as a buff-coloured fluffy solid homogeneous on TLC. Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.46 -toluene/acetone/acetic acid (12:7:1):0.14-0.28;
IR (KBr) 3390 (br), 3060, 3030, 2920, 1790, 1735, 1705 (sh), 1635, 1515, 1380, 1230 (br) 700 cm-1;
Found F.A.B.S +ve ion mode 497, 499 (MH+ -CH3CO2H). Example 56 (S)-2-{(2S,3R,6'R)-3-chloro-3'-aectoxymethyl-2'-carboxy-8'-oxospiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'-ene]-3-carboxamido}-2-phenylacetic acid Following the procedure in Example 51, the 3R, 3S (5:1) isomeric mixture of di-tert.-butyl 3-chloro-3-(L-alpha-phenylglycinylcarbonyl)spiro-oxirane-2-7'-cephalosporanate (63 mg) prepared in Example 54 and ice-cold TFA (10 ml) yielded after lyophilisation, the product (51 mg) as a buff-coloured fluffy solid which, on TLC (silica; n-butanol /acetic acid water) could be seen to contain, as the major product, the less polar 3R-isomer (Rf:0.52) with traces of the more polar 3S-isomer (Rf:0.46). IR (KBr) 3400 (br), 3060, 3030, 2960, 2930, 1795, 1735, 1705 (sh), 1635, 1515, 1380, 1235 (br) 700 cm-1;
Found F.A.B.S +ve ion mode 497, 499 (MH+) 437, 439 (MH+ -CH3CO2H). Example 57 1,1-Dimethylethyl (2S,3S,6'R)-3-chloro-3-(N-1,1-dimethylethyloxycarbonylmethyl-N-phenylcarboxamido)-3'-acetoxymethyl-8'-oxospiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'-ene]-2'-carboxylate
Following the procedure in Example 8, tert.-butyl 7-diazocephalosporanate (0.55 g) and oxalyl chloride (0.15 ml) gave a yellow oil. Following the procedure of Example 54, the oil, N-phenylglycine, tert.-butyl ester (0.35 g) and dry pyridine (0.15 g), yielded after rapid chromatography on silica, using as eluting solvent hexane/ethyl acetate (2:1), the product (290 mg, 30%) as a pale yellow oil.
Rf (silica)-hexane/ethyl acetate (2:1):0.43; IR (film) 3055, 3000 (sh), 2970, 2920, 1790, 1730 (br), 1680, 1220, 700 cm-1;
NMR delta, ppm (CDCl3) 1.51 (9H,s), 1.61 (9H,s), 2.12 (3H,s), 3.02-3.56 (2H,ABq,J=18Hz), 4.09-4.48 (2H,ABq,J=16Hz), 4.68-5.05 (2H,ABq,J=14Hz), 5.08 (1H,s), 7.46 (5H,s);
Found F.A.B.S +ve ion mode 609, 611 (very weak MH+); 573 (MH+ -HCl); 497, 499 (MH+ -2 x C4H8); 493, 495 (MH+ -C4H8 -CH3CO2H); 437, 439 (MH+ -2 x C4H8 -CH3CO2H); 401 (MH+ -2 x C4H8 -CH3CO2H -HCl). Example 58
(2S,3S,6'R)-3-chlpro-3-(N-carboxymethyl-N-phenylcarboxamido)- 3'-acetoxymethy1-8'-oxo-spiro[oxirane-2,7'-(5)-thia-(1)- azabicyclo[4.2.0]oct-2'-ene-]-2'-carboxylic acid Following the procedure in Example 51, di-tert. -butyl 3-chloro-3-(N-phenylglycinylcarbonyl)spiro-oxirane-2,7'-cephalosporanate, 3S-isomer (160 mg) and ice-cold TFA (10 ml) yielded, after lyophilisation, the product (125 mg) as a pale yellow fluffy solid homogeneous on TLC. Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.63
-toluene/acetone/acetic acid (12:7:1):0.10-0.22; IR (KBr) 3440 (br), 2950 (sh), 2920, 1785, 1730 (br), 1675, 1595, 1490, 1385, 1230, 700 cm-1;
Found F.A.B.S +ve ion mode 497, 499 (very weak MH+); 437, 439 (MH+ -CH3CO2H). Example 59
1,1-Dimethylethyl (2S,3S and 3R,6'R)-3-chloro-3-(N-4-hydroxyphenylcarboxamido)-3'-acetoxymethyl-8'-oxo-spiro[oxirane-2,7'-(5)- thia-(1)-azabicyclo[4.2.0]oct-2'-ene-]-2'-carboxylate Following the procedure in Example 57, tert.-butyl 7-diazocephalosporanate (0.53 g), oxalyl chloride (0.15 ml) and a solution of p-aminophenol (0.34 g) and dry pyridine (0.13 g) in anhydrous tetrahydrofuran (20 ml) yielded, after rapid chromatography on silica, using as eluting solvent hexane/ethyl acetate (1:1), the 3S-isomer (0.39 g, 49%), Rf (silica)-hexane/ethyl acetate (1:1):0.41, as the major product and the 3R-isomer (0.08 g, 10%), Rf (silica)-hexane/ethyl acetate (1:1):0.31, both as yellow oils.
Major product - 3S-isomer IR (film) 3350 (br), 3010 (sh), 2970, 2920, 1790, 1715 (br), 1510, 1245 (sh), 1200 cm-1;
NMR delta, ppm (CDCl3) 1.59 (9H,s), 2.12 (3H,s), 3.22-3.72 (2H,ABq,J=18Hz), 4.69-5.12 (2H,ABq,J=14Hz), 5.28 (1H,s), 6.72-6.81 and 7.29-7.39(4H,AA'BB'), 7.31 (1H,s), 8.36 (1H,s). Found F.A.B.S +ve ion mode 511, 513 (MH+); 510, 512 (M+); 475 (MH+ -HCl); 455, 457 (MH+ -C4H8); 395, 397 (MH+ -C4H8 -CH3CO2H); 359 (MH+ -C4H8 -CH3CO2H -HCl). Minor product - 3R-isomer
IR (film) 3300 (br), 3050, 2970, 2920, 1790, 1720, 1700 (sh), 1515, 1245 (sh), 1220 cm-1;
NMR delta, ppm (CDCl3) 1.61 (9H,s), 2.07 (3H,s), 3.23-3.73 (2H,ABq,J=18Hz), 4.70-5.12 (2H,ABq,J=13Hz), 5.31 (1H,s), 6.80-6.90 and 7.42-7.52 (4H,AA'BB'), 7.58 (1H,s), 9.10 (1H,br,s);
Found F.A.B.S +ve ion mode 475 (MH+ -HCl); 451, 453 (MH+ -CH3CO2H); 395, 397 (MH+ -C4H8 -CH3CO2H); 359 (MH+ -C4H8 -CH3CO2H -HCl). Also observed 509, 511 (MH+ -2 -C4H8); 453,455 (MH+ -2-C4H8): characteristic of 3R-isomers. Example 60
(2S,3S,6'R)-3-chloro-3-(N-4-hydroxy-phenylcarboxamido)-3'-acetoxymethyl-8'-oxo-spiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'-ene-]-2'-carboxylic acid Following the procedure in Example 51, tert.-butyl 3-chloro-3-(N-4-hydroxyphenylcarboxamido)spiro-oxirane-2,7'-cephalosporanate, 3S-isomer (150 mg) from Example 59 and ice-cold TFA (10 ml) yielded, after lyophilisation, the product (122 mg) as a pale yellow fluffy solid, homogeneous on TLC. Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.78
-toluene/acetone/acetic acid (12:7:1):0.29; IR (KBr) 3400 (br), 2950, 2920, 1785, 1720, 1695 (sh) 1635 (sh), 1515, 1240 (br) cm-1;
Found F.A.B.S +ve ion mode 455, 457 (MH+); 454, 456 (MH+); 395, 397 (MH+ -CH3CO2H); 359 (MH+ -CH3CO2H -HCl). Example 61
1,1-Dimethylethyl (2S,3S and 3R,6'R)-3-chloro-3-(N-cyanomethylcarboxamido)-3'-acetoxymethyl-8'-oxo-spiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'-ene-]-2'-carboxylate Following the procedure in Example 57, tert.-butyl 7-diazocephalosporanate (0.63 g), oxalyl chloride (0.18 ml) and a solution of aminoacetonitrile (0.20 g) and dry pyridine (0.1 ml) in anhydrous ether (20 ml) yielded, after rapid chromatography on silica, using as eluting solvent hexane/ethyl acetate (1:1), the 3S-isomer (0.39 g, 46%), Rf (silica)-hexane/ethyl acetate (1:1):0.41 as the major product and the 3R-isomer (0.14 g, 17%), Rf (silica)-hexane/ethyl acetate (1:1):0.18, both as pale yellow oils. Major product - 3S-isomer
IR (film) 3320 (br), 2980, 2930, 2250, 1795, 1740 (sh) 1720 (br), 1640, 1230 (br) cm-1;
NMR delta, ppm (CDCI3) 1.60 (9H,s), 2.11 (3H,s), 3.23-3.74 (2H,ABq,J=18Hz), 3.98-4.46 (2H,ABq, + ABq,J=16Hz and 6Hz), 4.68-5.08 (2H,ABq,J=13Hz), 5.32 (1H.s), 7.50 (1H,br, tr,J=6Hz and 6Hz);
Found F.A.B.S +ve ion mode 458, 460 (MH+); 422 (MH+ -HCl); 402, 404 (MH+ -C4H8); 342, 344 (MH+ -C4H8 -CH3CO2H); 456 (MH+ -2).
Minor product - 3R-isomer
IR (film) 3320 (br), 2985, 2930, 2250 (weak), 1795, 1740 (sh), 1715 (br), 1645, 1240 (sh), 1220 cm-1;
NMR delta, ppm (CDCI3) 1.60 (9H,s), 2.08 (3H,s), 3.20-3.70 (2H,ABq,J=18Hz), 4.28 (2H,d,J=6Hz), 4.69-5.11 (2H,ABq,J=13Hz). 5.22 (1H.s), 7.63 (1H.br, tr,J=6Hz);
Found F.A.B.S +ve ion mode 456, 458 (MH+ -2; no MH+ observed); 400, 402 (MH+ -2 -C4H8); 398, 400 (MH+ -CH3CO2H); 342, 344 (MH+ -C4H8 -CH3CO2H); 306 (MH+ -C4H8 -CH3CO2H -HCl). Example 62
(2S,3R,6'R)-3-chloro-3-(N-cyanomethyl-carboxamido)-3'-acetoxymethyl-8'-oxo-spiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2,0]oct-2'-ene-3-2'-carboxylic acid
Following the procedure in Example 51, tert.-butyl 3-chloro-3-(N-cyanomethylcarboxamido)spiro-oxirane-2,7'-cephalosporanate, prepared in Example 61, 3S-isomer (95 mg) and ice-cold, redistilled TFA (10 ml) yielded, after lyophilisation the product (79 mg) as a buff-coloured fluffy solid homogeneous on TLC. Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.57 -toluene/acetone/acetic acid (12:7:1):0.19;
IR (KBr) 3400 (br), 2950, 2920, 2250 (weak), 1790, 1725 (sh), 1710 (br), 1640, 1520, 1235 (br) cm-1;
Found F.A.B.S +ve ion mode 402, 404 (MH+); 342, 344 (MH+ -CH3CO2H). Example 63
1,1-Dimethylethyl (2S,3S and 3R,6'R)-3-bromo-3-(N-phenylcarboxamido)-3'-acetoxymethyl-8'-oxo-spiro[oxirane-2,7'-(5)- thia-(1)-azabicvclo[4.2.0]oct-2'-ene-]-2'-carboxylate
To a solution of tert.-butyl 7-diazocephalosporanate (0.61 g) in anhydrous ether (20 ml) at -15°C was added freshly distilled oxalyl bromide (0.18 ml). The reaction mixture effervesced vigorously and the colour faded to a pale straw. After 5 minutes, or when effervescence ceased, the reaction mixture, still at -15°C, was quenched by addition of redistilled aniline (0.7 ml). Reaction, as indicated by precipitation, was immediate. The reaction product was stored for 5 minutes at room temperature and then diluted with water (30 ml). The two-phase reaction mixture with precipitated solids (oxanilide) was filtered through Celite and the precipitate- was washed with cold ether. The filtrate and washings were combined, the organic layer was separated and washed with dil. HCl (x3), water, dil. HCO3- (x2), water, dried (MgSO4) and evaporated. The residue was rapidly chromatographed on silica, using as eluting solvent hexane/ethyl acetate (2:1), and all the fractions containing the desired product as a mixture of isomers (0.32 g, 33%), Rf (silica)-hexane/ethyl acetate (2:1 ):0.29-0.37, in the ratio, 3S:3R, 5:2, bulked and evaporated. From this residue the major product 3S-isomer was fractionally crystallised by dissolving in dichloromethane (1 ml) and ether (1 ml), diluting to faint turbidity with petrol and storing at 15°C overnight.
The crystals [132 mg, m.p. 140-150°C (dec)], Rf (silica)-hexane/ethyl acetate (2:1):0.35, were collected, washed with cold petrol /ether (1:1) and recrystallised from dichloromethane/ether (1:1) and petrol. The combined filtrate and washings were evaporated to yield predominantly the 3R-isomer, Rf (silica)-hexane/ethyl acetate (2:1):0.33, as a yellow oil. Major product - 3S-isomer
IR (KBr) 3380, 3060, 3010, 2970, 2950, 2930, 1790, 1740, 1710, 1695, 1635, 1600, 1220, 695 cm-1;
NMR delta, ppm (CDCl3) 1.56 (9H,s), 2.12 (3H,s), 3.23-3.75 (2H,ABq,J=19Hz), 4.71-5.12 (2H,ABq,J=13Hz), 5.32 (1H,s), 7.10-7.65 (5H,m), 8.26 (1H,br,s);
Found C,49.21; H,4.46; N,5.29%, C22H23BrN2O7S requires C,48.98; H,4.27; N,5.19%. Minor product - 3R-isomer
IR (film) 3310 (br), 3050, 2970, 2920, 1795, 1745 (sh), 1725, 1705 (sh), 1245 (sh), 1230, 695 cm-1;
NMR delta, ppm (CDCI3) 1.57 (9H,s), 2.07 (3H,s), 3.22-3.73 (2H,ABq,J=18Hz), 4.69-5.10 (2H,Abq,J=13Hz), 5.21 (1H,s), 7.07-7.72 (5H,m), 8.50 (1H,br,s);
Found F.A.B.S +ve ion mode 483, 485 (MH+ -C4H8); 481, 483 (MH+ -2 -C4H8); 423, 425 (MH+ -C4H8 -CH3CO2H). No MH+ observed. Example 64 1 ,1-Dimethylethyl (2S,3S,6'R)-3-(N-phenylcarboxamido)-3'- acetoxymethyl-8'-oxo-spiro[oxirane-2,7'-(5)-thia-(1)- azabicyclo[4.2,0]oct-2'-ene-3-2'-carboxylate
Following the procedure in Example 41, tert.-butyl 3-bromo- 3-(N-phenylcarboxamido)spiro-oxirane-2,7'-cephalosporanate, 3R-isomer (150 mg), prepared in tetrahydrofuran (10 ml), pH 4 buffer (2 ml) and excess zinc-copper couple yielded after chromatography on silica, using as eluting solvent hexane/ethyl acetate (2:1), in the early fractions the product (50 mg, 42%), Rf (silica)-hexane/ethyl acetate (2:1) :0.28; hexane/ethyl acetate (1:1):0.66, as a colourless oil and in later fractions, trace amounts of a second product, Rf (silica)-hexane/ethyl acetate (2:1):0.28; hexane/ethyl acetate (1:1) -.0.48. subsequently identified as the 3R-isomer (see Example 66).
IR (film) 3310 (br), 3050, 2970 (sh), 2950, 2925, 1790, 1740 (sh), 1725, 1690, 1245, 1225, 690 cm-1;
NMR delta, ppm (CDCl3) 1.60 (9H,s), 2.06 (3H,s), 3.11-3.61 (2H,ABq,J=18Hz), 4.05 (1H,s), 4.67-5.10 (2H,ABq,d=13Hz), 5.19 (1H,s), 7.15-7.70 (5H,m), 8.04 (1H,br,s);
Found F.A.B.S +ve ion mode 459 (MH+ -2; no MH+ observed); 403 (MH+ -2 -C4H8); 401 (MH+ -CH3CO2H); 345 (MH+ -C4H8 -CH3CO2H). Example 65
(2S,3S,6'R)-3-(N-phenylcarboxamido)-3'-acetoxymethy!-8'-oxospiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'-ene-]-2'-carboxylate
Following the procedure in Example 51, tert.-butyl 3-(N-phenylcarboxamido)spiro-oxirane-2,7'-cephalosporanate, 3R-isomer (33 mg), prepared in Example 64, and ice-cold TFA (5 ml) yielded, after lyophilisation, the product (29 mg) as a buff-coloured fluffy solid homogeneous on TLC.
Rf (si!ica)-n-butanol/acetic acid/water (4:1:1):0.56 -toluene/acetone/acetic acid (12:7:1):0.14;
IR (KBr) 3420 (br), 3040, 2920, 1785, 1730, 1685, 1635, 1600, 1540, 1500, 1445, 1240 (br), 695 cm-1;
Found F.A.B.S +ve ion mode 405 (MH+); 345 (MH+ -CH3CO2H). Example 66 1,1-Dimethylethyl (2S,3R,6'R)-3-(N-phenylcarboxamido)-3-acetoxymethyl-8'-oxo-spiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'-ene-]-2'-carboxylate
Following the procedure in Example 41, crystalline tert.-butyl 3-bromo-3-(N-phenylcarboxamido)spiro-oxirane-2,7'-cephalosporanate, 3S-isomer (90 mg), prepared in Example 63, in tetrahydrofuran (5 ml), pH 4 buffer (1 ml) and excess zinc-copper couple yielded, after chromatography on silica, using as eluting solvent hexane/ethyl acetate (2:1), in the early fractions, trace amounts of the 3S-isomer, Rf (silica)-hexane/ethyl acetate (1:1):0.66 - see Example 64, and in the later fractions the crystalline product (54 mg, 73%), Rf (silica)-hexane/ethyl acetate (1:1):0.48, which was recrystallised from dichloromethane/ether (1:1) and petrol, m.p. 163.5-164°C.
IR (KBr) 3375, 3035, 3005, 2985, 2970 (sh), 2925, 1795, 1740, 1720, 1685, 1645, 1220, 700 cm-1;
NMR delta, ppm (CDCl3) 1.59 (9H,s), 2.09 (3H,s), 3.21-3.71 (2H,ABq,J=18Hz), 4.03 (1H,s), 4.72-5.13 (2H,ABq,J=13Hz), 5.06 (1H,s), 7.05-7.60 (5H,m), 8.10 (1H,br, s);
Found F.A.B.S +ve ion mode 461 (MH+); 405 (MH+ -C4H8); 401 (MH+ -CH3CO2H); 345 (MH+ -C4H8 -CH3CO2H).
Found C,57.43; H,5.37; N.6.01%, C22H24N2O9S requires C,57.39; H,5.22; N,6.09%. Example 67
(2S,3R,6'R)-3-(N-phenylcarboxamido)-3'-acetoxymethyl-8'-oxo- spiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'-ene-]-2'-carboxylic acid
Following the procedure in Example 51, tert.-butyl 3-(N-phenylcarboxamido)spiro-oxirane-2,7'-cephalosporanate, 3R-isomer (45 mg), and ice-cold TFA (10 ml) yielded, after lyophilisation, the product (38 mg) as a buff-coloured fluffy solid homogeneous on TLC. Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.50
-toluene/acetone/acetic acid (12:7:1):0.30; IR (KBr) 3360 (br), 3040, 2950, (sh), 2920, 1790, 1735, 1685, 1650 (sh), 1600, 1545, 1445, 1235 (br) 695 cm-1;
Found F.A.B.S +ve ion mode 405 (MH+); 345 (MH+ -CH3CO2H). Example 68
1,1-Dimethylethyl (2S,3S and 3R,6'R)-3-bromo-3-(N-2,6-dimethoxyphenylcarboxamido)-3'-acetoxymethyl-8'-oxo-spiro[oxirane-2,7'- (5)-thia-(1)-azabicyclo[4.2.0]oct-2'-ene-3-2'-carboxylate
Following the procedure in Example 63, tert.-butyl 7-diazocephalosporanate (0.59 g) and a solution of 2,6-dimethoxyaniline (0.40 g) and dry pyridine (0.20 g) in anhydrous ether (10 ml) also at -15°C, yielded after rapid chromatography on silica, using as eluting solvent hexane/ethyl acetate (3:2), in the early fractions, the major product 3S-isomer (140 mg, 13%), Rf (silica)- hexane/ethyl acetate (1:1):0.49 and, in the later fractions, the 3R-isomer (60 mg, 6%), Rf (silica)-hexane/ethyl acetate (1:1):0.36, both as pale yellow oils. Major product - 3S-isomer
IR (film) 3300 (br), 3000 (sh), 2975, 2930, 1795, 1735 (sh), 1720, 1710 (sh), 1260, 1225 cm-1;
NMR delta, ppm (CDCl3) 1.59 (9H,s), 2.12 (3H,s), 3.22-3.72 (2H,ABq,J=18Hz), 3.85 (6H,s), 4.69-5.11 (2H,ABq,J=13Hz), 5.30 (1H,s), 6.56 (2H,d,J=9Hz), 7.14 (1H,d,J=9Hz), 7.65 (1H,br, s);
Found F.A.B.S +ve ion mode 599, 601 (MH+); 543, 545 (MH+ -C4H8).
Minor product - 3R-isomer
IR (film) 3320 (br), 3000 (sh), 2970, 2925, 1790, 1735 (sh), 1720 (br), 1260, 1225 cm-1;
NMR delta, ppm (CDCl3) 1.60 (9H,s), 2.08 (3H,s), 3.13-3.63 (2H,ABq,J=18Hz), 3.87 (6H,s), 4.71-5.13 (2H,Abq,J=13Hz), 5.26 (1H,s), 6.60 (2H,d,J=8Hz), 7.19 (1H,d,J=8Hz), 7.91 (1H,br,s);
Found F.A.B.S +ve ion mode 599, 601 (MH+) 543, 545 (MH+ -C4H8); 539, 511 (MH+ -CH3CO2H); 483, 485 (MH+ -C4H8 -CH3CO2H). Example 69
(2S,3R,6'R)-3-bromo-3-(N-2,6-dimethoxyphenylcarboxamido)-3'-acetoxymethyl-8'-oxo-spiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'-ene-]-2'-carboxylic acid
Following the procedure in Example 51, tert.-butyl 3-bromo-3-(N-2,6-dimethoxyphenylcarboxamido)spiro-oxirane-2,7'-cephalosporanate, 3R-isomer (41- mg), prepared in Example 68, and ice-cold TFA (10 ml) yielded, after lyophilisation, the product (37 mg) as a pale yellow fluffy solid which, on TLC , showed slight traces of degradation.
Rf (silica)-n-butanol/acetic acid/water (4:1:1):0.55
-toluene/acetone/acetic acid (12:7:1):0.25; IR (KBr) 3400 (br), 3000 (sh), 2935, 2840, 1790, 1730 (br), 1635, 1600, 1515, 1480, 1260, 1230, 1110 cm-1;
Found F.A.B.S +ve ion mode 543, 545 (MH+); 483, 485 (MH+ -CH3CO2H); 481, 483 (MH+ -2 -CH3CO2H). Example 70
1,1-Dimethylethyl (2S,3R,6'R)-3-(N-2,6-dimethoxyphenylcarboxamido)-3'-acetoxymethyl-8'-oxo-spiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'-ene-]-2'-carboxylate
Following the procedure in Example 41, tert.-butyl 3-bromo-3-(N-2,6-dimethoxyphenylcarboxamido)spiro-oxirane-2,7'-cephalosporanate, 3S-isomer (140 mg), prepared in Example 68, in tetrahydrofuran (10 ml), pH 4 buffer (2 ml) and excess zinccopper couple yielded, after chromatography on silica, using as eluting solvent hexane/ethyl acetate (2:3), the product 884 mg, (69%), Rf (silica)-hexane/ethyl acetate (1:1):0.18, as a colourless oil. IR (film) 3310 (br), 3000 (sh), 2970, 2930, 2835, 1795, 1740, 1720, 1700 (sh), 1260, 1225 cm-1;
NMR delta, ppm (CDCI3) 1.60 (9H,s), 2.11 (3H,s), 3.21-3.71 (2H,ABq,J=18Hz), 3.83 (6H,s), 4.03 (1H,s), 4.71-5.03 (2H,ABq,J=13Hz), 5.03 (1H,s), 6.58 (2H,d,J=9Hz), 7.15 (1H,d,J=9Hz), 7.51 (1H,br,s);
Found F.A.B.S +ve ion mode 521 (MH+); 519 (MH+ -2); 465 (MH+-C4H8); 461 (MH+ -CH3CO2H); 405 (MH+ -CH3CO2H -C4H8); 403 (MH+ -2 -CH3CO2H -C4H8); Example 7!
1,1-Dimethylethyl (2S,3S,6'R)-3-bromo-3-(N-2,6-cyanomethylcarboxamido)-3'-acetoxymethyl-8'-oxo-spiro[oxirane-2,7'- (5)-thia-(1)-azabicydo[4.2,0]oct-2'-ene-]-2'-carboxy!ate Following the procedure in Example 68, tert.-butyl 7-diazocephalosporanate (0.58 g), oxalyl bromide (0.17 ml) and a solution of aminoacetonitrile (0.40 g) in anhydrous ether (20 ml) yielded, after chromatography on silica, using as eluting solvent hexane/ethyl acetate (1:1), in the early fractions, the major product 3S-isomer (270 mg, 31%), Rf (silica)-hexane/ethyl acetate (1:1):0.55, and in the later fractions, the 3R-isomer (70 mg, 8%), Rf (silica)-hexane/ethyl acetate (1:1):0.25, both as highly unstable pale yellow oils. Major product - 3S-isomer IR (film) 3320 (br), 2970, 2920, 2245, 1790, 1735 (sh), 1720 (br), 1245 (sh), 1220 cm-1;
NMR delta, ppm (CDCI3) 1.61 (9H,s), 2.11 (3H,s), 3.23-3.75 (2H,ABq,J=18Hz), 4.05-4.43 (2H,ABq + ABq,J=12Hz and 6Hz), 4.69-5.09 (2H,ABq,J=13Hz), 5.31 (1H,s), 7.41 (1H,br, tr,J=6Hz); Found F.A.B.S +ve ion mode 386, 388 (MH+ -CH3CO2H -C4H8). No MH+ observed. Minor product - 3R-isomer
IR (film) 3320 (br), 2960, 2950, 2920, 2245 (weak), 1790, 1735 (sh), 1720 (br), 1235 (sh), 1225 cm-1; NMR delta, ppm (CDCl3) 1.61 (9H,s), 2.10 (3H,s), 3.21-3.71 (2H,ABq,J=18Hz), 4.30 (2H,d,J=6Hz), 4.71-5.14 (2H,ABq,J=13Hz), 5.23 (1H.s), 7.36 (1H,br,tr,J=6Hz);
Found F.A.B.S +ve ion mode 386, 388 (MH+ -C4H8 -CH3CO2H). No MH+ observed . Exampl e 72
1,1-Dimethylethyl (2S,3S,6'R)-3-(N-cyanomethylcarboxamido)-3'-acetoxymethyl-8'-oxo-spiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'-ene-]-2'-carboxylate
Following the procedure in Example 41, tert.-butyl 3-bromo-3-(N-cyanomethylcarboxamido)spiro-oxirane-2,7'-cephalosporanate, 3R-isomer (70 mg) in tetrahydrofuran (5 ml), pH 4 buffer (1 ml) and excess zinc-copper couple yielded, after chromatography on silica plates, using as eluting solvent hexane/ethyl acetate (2:3), the product (27 mg, 46%), Rf (silica)- hexane/ethyl acetate (1:1):0.26, as a pale yellow oil. Trace amounts of a second product, Rf (silica)-hexane/ethyl acetate (1:1):0.34, probably the 3R-isomer, were also Isolated.
IR (film) 3320 (br), 2980, 2935, 2250 (weak), 1790, 1735 (sh), 1720, 1700 (sh), 1240 (sh), 1225 cm-1;
NMR delta, ppm (CDCl3) 1.61 (9H,s), 2.11 (3H,s), 3.19-3.70 (2H,ABq,J=18Hz), 4.02 (1H,s), 4.24 (2H,d,J=6Hz), 4.69-5.12 (2H,ABq,J=13Hz), 5.21 (1H,s), 7.21 (1H,br,tr,J=6Hz);
Found F.A.B.S +ve ion mode 422 (MH+ -2); 368 (MH+ -C4H8); 366 (MH+ -2 -C4H8); 308 (MH+ -C4H8 -CH3CO2H); 306 (MH+ -2 -C4H8 -CH3CO2H). No MH+ observed. Example 73 1,1-Dimethylethyl (2S,3S and 3R,6'R)-3-chloro-3-benzy!oxycarbonyl-3'-acetoxymethyl-8'-oxo-spiro[oxirane-2,7'-(5)-thia-(1)-azabicyclo[4.2.0]oct-2'-ene-]-2'-carboxylate
To a solution of tert.-butyl 7-diazocephalosporanate (0.57 g) in anhydrous ether (20 ml) at 0°C was added oxalyl chloride (0.17 ml). The reaction was allowed to warm to room temperature and stood for 15 minutes, or until effervescence ceased. The reaction mixture was cooled to 0 C and diluted with water (30 ml). The organic layer was separated and washed repeatedly with water, dried (MgSO4) and evaporated. The yellow oily residue was taken up in dry ether (10 ml) and to it was added dropwise an ethereal solution of phenyldiazomethane in sufficient quantity until effervescence ceased and a red-orange colour persisted. The solution was stored at room temperature for 10 minutes, diluted with ether (20 ml) and poured into a solution of citric acid (2 g) in water (25 ml). The two-phase system was shaken to quench excess phenyl diazomethane, the organic layer was separated, washed with water, dil HCO3- (x3), dried and evaporated. The residue was rapidly chromatographed on silica, using as eluting solvent hexane/ethyl acetate (3:1), to yield, in the early fractions the major product, 3S-isomer (0.18 g, 21%), Rf (silica)-hexane/ethyl acetate (2:1):0.52, as a pale yellow oil and in the later fractions a mixture of the 3S- and 3R-isomers (0.12 g, 14%). Major product - 3S-isomer
IR (film) 3060, 3030, 2980, 2930, 1795, 1745, 1730 (sh), 1640, 1455, 1370, 1270, 1240 (sh), 695 cm-1; NMR delta, ppm (CDCl3) 1.58 (9H,s), 2.08 (3H,s), 3.19-3.69 (2H,ABq,J=18Hz), 4.68-5.11 (2H,ABq,J=13Hz), 5.25 (1H,s), 5.32 (2H,s), 7.41 (5H,s);
Found F.A.B.S +ve ion mode 510, 512 (very weak MH+); 454, 456 (MH+ -C4H8); 452,454 (MH+ -2 -C4H8); 394, 396 (MH+ -C4H8 -CH3CO2H); 304, 306 (MH+ -C4H8 -CH3CO2H -PhCH).

Claims

1. Beta-lactam epoxides which are compounds of the general formula (21):
Figure imgf000064_0001
wherein: R represents the residue of a penicillin or cephalosporin ring; X represents a hydrogen or halogen atom;
V represents a halogen atom or a group A defined below; and A represents a hydroxy, alkoxy, aryloxy, aralkyloxy, or alkyl group or an amino group, and their pharmaceutically acceptable salts.
2. Beta-lactam epoxides which are compounds of the general formula (22):
Figure imgf000064_0002
wherein: n is 0, 1, or 2; and
V vand X are as defined in Claim 1; and their pharmaceutically acceptable salts and esters.
3. Beta-lactam epoxides which are compounds of the general formula (23):
Figure imgf000065_0001
wherein: n is 0, 1 or 2; and V and X are as defined in Claim 1; and their pharmaceutically acceptable salts and esters.
4. Beta-lactam epoxides according to Claim 1 , 2 or 3 wherein V represents an A group which is alkoxy or alkyl of 1 to 4 carbon atoms, phenoxy, 2-pheny1ethoxy or benzyloxy in each of which the benzene ring is unsubstituted or is substituted by one or more substituents selected from methyl, methoxy, halogen, carboxy, cyano, nitro and amino or A is an amino group of formula (11):
Figure imgf000065_0002
wherein each of R1 and R2 independently represents a hydrogen atom, a hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from halogen, carboxy, alkoxy of 1 to 4 carbon atoms, cyano, nitro and amino or a heterocyclic group or R1, R2 and N shown in formula (11) together represent a nitrogen-containing heterocyclic group.
5. Beta-lactam epoxides according to Claim 4 wherein n is 0, and A represents a said amino group in which R1 represents phenyl and R2 hydrogen or methyl.
6. Beta-lactam epoxides according to Claim 1, 2, 3, 4 or 5 in the form of their isomers having the 3-position configuration of partial formula (17):
Figure imgf000066_0001
7. Each of the beta-lactam epoxides according to Claim 1 specifically hereinbefore mentioned.
8. A method of preparing a beta-lactam epoxide defined in Claim 1, which method comprises reacting a 6-diazo compound of formula (18):
Figure imgf000066_0002
R being as defined in Claim 1, provided that any group within R requiring to be protected is protected, under substantially anhydrous conditions, with a compound of formula (19):
Figure imgf000066_0003
wherein V represents a halogen atom or an A group, A being as defined in Claim 1, provided that any group within A requiring to be protected is protected, and X represents a halogen atom, and when Y represents a halogen atom, reacting the resultant acid halide of formula (20):
Figure imgf000067_0001
with a nucleophile A-L, L representing a leaving group reactive therewith to replace the Y halogen atom by the A group, and A being as defined above, and, if desired removing any protecting group; further, when the beta-lactam epoxide to be prepared is a salt or ester, (other than a protective ester already present in R or A), converting the carboxylic acid into its salt or esterifying it, respectively; and when the beta-lactam epoxide to be prepared is one in which n is 1 or 2 and the 6-diazo compound of formula (18) is one in which n is 0, oxidising a compound obtained above with an oxidising agent effective to generate a sulphoxide or sulphone group.
9. A pharmaceutical composition comprising a beta-lactam epoxide claimed in any one of Claims 1 to 7 or prepared by a method claimed in Claim 8, in association with a pharmaceutically acceptable carrier.
10. A combination-preparation or two-component pack for combatting a bacterial infection resistant to therapy by beta-lactams, comprising a beta-lactam antibiotic susceptible to attack by a beta-lactamase enzyme, in association with an irreversible inhibitor of the beta-lactamase, characterised in that the irreversible inhibitor is a beta-lactam epoxide claimed in any one of Claims 1 to 7 or prepared by a method claimed in Claim 8.
PCT/GB1988/000202 1987-03-23 1988-03-15 Beta-lactam epoxides WO1988007534A1 (en)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF THE CHEMICAL SOCIETY, PERKIN TRANSACTIONS I, Vol. 12, 1986, V.J. JEPHCOTE et al., "Lewis Acid-Catalysed Reactions of 2,2,2,-Trichloroethyl 6-Diazopenicillanate and Imines: Rearrangements of Spiro-6-Aziridine- and Spiro-6-Oxirane-Penicillanates. X-Ray Crystal Structures of (3S,6'S -2,2,2-Trichloroethyl3-(4-Nitrophenyl)-1-Phenylspiro (Aziridine-2,6'-Penicillanate) and (3S,7aR)-2,2,2-Trichloroethyl 2,3,5,7a-Tetrahydro-7-Methoxy-2,2-Dimethyl-6-(4-Nitrophenyl)-5-Oxopyrrolo (2,1-b) Thiazole-3-Carboxylate", pages 2195-2201. *
THE JOURNAL OF ANTIBIOTICS, Vol. 37, No. 11, November 1984, JAPAN ANTIBIOTICS RESEARCH ASSOCIATION (Tokyo, JP), J.C. SHEEHAN et al., "In Vitro Biological Activities of 6-Isosteric Penicillins and 7-Isosteric Cephalosporins", pages 1441-1448. *

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