Imide and isatin derivatives as γ -lactam mimics of ß-lactam antibiotics

Activated γ -lactams, which are derivatives of succinimide, phthalimide and isatin with suitable elements of molecular recognition, have been synthesised as mimics of the ß-lactam antibiotics and their chemical and biological reactivity determined


Introduction
The traditional antibiotics which interact with the bacterial enzymes, the DD-peptidases and the ß-lactamases, are ß-lactams, for example, benzyl penicillin, 1.The strain energy inherent in the four-membered ring and the non-planarity of the ß-lactam were, for many years, thought to be vital for this activity which involves the acylation of an active site serine residue. 1 However, it has been demonstrated that the chemical reactivity of ß-lactams is not unusual and that molecular recognition is as equally important as acylating power. 2 Furthermore non-ß-lactam derivatives such as γ-lactams may show chemical reactivity similar to that of the classical penicillins and cephalosporins 3 and consequently offer the potential to act as inhibitors of the bacterial enzymes.We report here the synthesis of activated γ-lactams, e.g.For example, we have shown that ring closure can occur by the intramolecular aminolysis of acylenzymes to form γ-lactams but not ß-lactams. 11This paper describes some attempts to discourage the recyclisation whilst retaining features required for good antibiotic activity.Activated structures based on succinimide, phthalimide and isatin which have a potential leaving group (Lg) α-substituted to the incipient amine may not only reduce the probability of reclosure of the ring but also generate a 'second trap' for enzyme inactivation (Scheme 2).Nucleophilic attack at the γ-lactam carbonyl followed by ring opening and concomitant elimination of the leaving group generates an electrophilic imine.As a result of expulsion of the leaving group the nucleophilicity of the nitrogen is reduced, so the ease of the 'back-reaction' and ring closure with regeneration of the free enzyme is discouraged.In addition, the electrophilic imine could also covalently react with any nucleophilic species in the active site, increasing the likelihood of enhanced inactivation, potentially resulting in autolysis and death of the bacterium.
The introduction of a second carbonyl group into a γ-lactam ring either adjacent to the nitrogen, as in phthalimide and succinimide, 3 or adjacent to the carbonyl as in isatin, 12 activates the amide bond sufficiently to bring its reactivity close to that of the ß-lactam of ß-lactam antibiotics.To be an effective acylating agent it appears that the second order rate constant for hydrolysis, k OH , should be in the range exhibited by the biologically active penicillins and cephalosporins, 2 ie 0.01 to 1 M -1 s -1 .The γ-lactams of imides and isatins, which have alkaline hydrolysis rates that fall within this range, 2 are activated enough to be considered as starting points for potential antibiotics and/or inactivators of the ß-lactamase enzymes.The synthesis of

Results and Discussion
The structure-activity relationships of ß-lactam antibacterial agents are not very well defined although it is clear that the minimum requirements involve good acylating power combined with the correct geometrical relationship between the acylating centre and the carboxylate anion. 3,13 e compounds synthesised here represent a variety of different molecular shapes but with reactivities falling into the required range.
The N-α-hydroxyacetic acid adducts of succinimide 4 (Scheme 3), phthalimide 5 (Scheme 4) and isatin 6 (Scheme 5) were synthesised by condensation with glyoxylic acid. 14The chemical shifts of the methine hydrogens in the 1 H NMR spectra of these structures are particularly diagnostic being at 5.67δ (4), 5.88δ (5) and 6.01δ (6).These adducts are unlikely to be able to expel the hydroxyl group to generate the imine trap, nonetheless they were tested for antibacterial activity, but none was found.

Scheme 3
Reagents and conditions: i) Glyoxylic acid (50 wt%), THF, reflux; ii) 7; SOCl 2 , THF, 0°C, H 2 O; 9; acetyl chloride, pyridine, 0°C; 11; phenylacetyl chloride, pyridine, 0°C These adducts were found to be unstable in water and quickly decomposed to their corresponding imide and glyoxylic acid, the half-life of the isatin derivative 6 was only 2-3 minutes at pH 7 and 20°C.The hydroxyl function was consequently converted to a number of more convenient leaving groups.The first leaving groups to be considered were sulphonates but attempts at sulphonation of the hydroxyl group using various sulphonyl halides met with little success.For example, the reaction of 4 with either methanesulphonyl or p-toluenesulphonyl chloride resulted in sulphonate derivatives which were very labile and immediate decomposition occurred on attempted isolation.

Scheme 4
Reagents and conditions: i) Glyoxylic acid (50 wt%), THF, reflux; ii) 8; SOCl 2 , THF, 0 °C, H 2 O; 10; acetyl chloride, pyridine, 0 °C; 12; phenylacetyl chloride, pyridine, 0 °C; iii) methyl glyoxylate, THF, reflux; iv) 2,6-dichlorobenzoyl chloride, pyridine, 0 °C; v) 13; LiI.H 2 O, pyridine, reflux Compounds 4, 5 and 6 were reacted with 2.5 equivalents of thionyl chloride to give the corresponding halides as potential leaving groups -7, 8 and 19, respectively.The free carboxylic acid groups present in these derivatives also reacted to give rise to the formation of the corresponding acyl chlorides and their subsequent reaction with free hydroxy residues formed polymeric esters as side products.Hydrolysis of the acyl chlorides with ice water for 3 h.followed by a work-up and trituration using dichloromethane was the best method found for purifying the products.The characteristic methine protons moved downfield to 6.42δ (7), 6.70δ (8) and 7.18δ (19), respectively.Reaction of the glycolic acid derivatives 4 and 5 with acid chlorides gave esters 9-12 which, in the active site of the enzymes, could in principle react by expelling their corresponding carboxylate anions.Pyridine was used as both solvent and base in the synthesis but numerous purification procedures using silica gel chromatography were required to obtain pure products in relatively low yield.
In the active sites of the enzymes which recognise ß-lactam antibiotics [15][16][17][18][19] there are a number of different functional groups close enough to the proposed incipient imine intermediate formed by ring opening to be able to react with this 'second trap' electrophilic centre.However, such nucleophiles could also attack the carbonyl carbon of the ester "leaving group" in 9-12 and so compete with that at the γ-lactam centre.In order to protect this site from nucleophilic attack a more sterically hindered ester was used.Subramanyam 20 has reported the use of 2,6-disubstituted benzoates as leaving groups in compounds used to acylate the serine protease Human Leukocyte Elastase (HLE).The inhibition activity was dependent on the nature of the leaving group and the 2,6-disubstituents sterically prevented undesirable nucleophilic attack on the benzoyl carbonyl group.We therefore attempted to prepare the corresponding phthalimide derivative, 13.The acylation of the free hydroxy acid 5 with 2,6-dichlorobenzoyl chloride proved ineffective probably because of competing anhydride formation.The carboxylic acid residue was consequently protected as its methyl ester before acylation.
The first stage was the reaction of phthalimide with methyl glyoxylate to give Nphthalimido-α-hydroxy-acetic acid methyl ester 14. 1 H NMR showed a characteristic methyl ester singlet at 3.77δ and the methine proton of these glyoxylates at 5.93δ.Reaction of 14 with 2,6-dichlorobenzoyl chloride in pyridine to give N-phthalimido-α-2,6-dichlrobenzoyl-acetic acid methyl ester 15, the methine proton of the product now appearing at 7.24δ.When Nphthalimido-α-2,6-dichlorobenzoyl-acetic acid methyl ester 15 was reacted with 1 equivalent of lithium iodide monohydrate in refluxing pyridine to give 13 in high yield.Having established a three step synthesis involving facile purifications and good overall yield, it was decided to try this method to make the phenylacetyl derivative 12, previously synthesised by the much lower yielding, but only two step synthesis, described earlier.The reaction of N-phthalimido-αhydroxy-acetic acid methyl ester 14 with phenylacetyl chloride gave the diester 16, but in low yield.The phenylacetylation of isatin-N-α-hydroxy-acetic acid methyl ester also gave poor yields compared with that obtained for the 2,6-dichloro derivative.The methine shifted from 5.93δ in the starting material 14 to 7.02δ in the product 16.However, when the lithium iodide demethylation of the methyl ester was attempted degradation occurred with the formation of phenylacetic acid as well as some unidentifiable products.The suggestion that lithium iodide is a specific reagent for ethyl and methyl esters only 21 does not appear universal.Iodide attack on Nphthalimido-α-phenylacetyl-acetic acid methyl ester 16 apparently occurs at the more electron deficient methine carbon (Scheme 6), even though this site is more sterically hindered.Presumably the 2,6-dichloro substituted compound 15 has this methine position shielded by the two bulky chloride groups preventing attack at this site.Attempts to selectively remove the methyl ester using pig liver esterase also failed as did other attempts to remove the carboxyl protecting ester group.Using a 4-nitrobenzyl ester protecting group for the carboxylic acid group, 17, followed by acylation of the hydroxyl group led to products 20 and 21 which also could not be selectively deprotected.
It was felt that more enzyme recognition needed to be incorporated into the structures such as an acylamino side chain commonly found in active penicillins.As well as improving the affinity of the substrate for the target enzymes, the reactivity of the amide bond would also be increased.(R/S)-Aspartic acid was N-acylated using phenylacetyl chloride and then converted to the substituted succinic anhydride by the method of Mardle 4 using acetic anhydride.When this anhydride was heated with ammonium carbonate only an 18% conversion to the imide was observed.A series of attempts to heat N-acylated succinic acid with various nitrogen sources gave differing yields of the desired product, ie ammonia (8%), ammonium carbonate (6%) and finally urea (51%).Now that an effective route was available to the imide stage, optically pure (S)-aspartic acid was acylated to give the diacid 30 followed by cyclisation to the imide 31 using urea (Scheme 7).The next step was the previously established reaction of 31 with glyoxylic acid which resulted in formation of 31 with a 50/50 mixture of disastereoisomers.Conversion of the hydroxy acid 32 to the chloro acid 33 by use of thionyl chloride gave a near equal mixture of the required diastereoisomeric product (Scheme 7).The α-carbonyl group of isatin was converted to O-benzyloxime derivatives 27-29 to act also as a potential mimic of the N-phenylacetamido side chain of benzylpenicillin, 1. Reagents and conditions: i) Phenylacetyl chloride, NaOH, 0°C; ii) Urea, 170°C; iii) Glyoxylic acid.H 2 O, THF, reflux; iv) SOCl 2 , THF, 0°C, H 2 O Chemical Reactivity -The second-order rate constants for the hydroxide-ion catalysed hydrolysis of the γ-lactams are given in Table 1.For comparison the second-order rate constant for the alkaline hydrolysis of benzyl penicillin is 0.15 M -1 s -1 and so the γ-lactams have a similar or even greater reactivity indicating their suitability, on chemical grounds, as potential acylating agents of ß-lactamases and DD-peptidases. 2 It has been suggested that the second order rate constant, k OH , is a good indicator of enzyme acylating power and should have a value of 0.01 to 1.0 M -1 s -1 to maximise inactivation with competing hydrolysis.Inhibition Studies -The γ-lactams were tested for inhibition 23 of the ß-lactamase enzymes from Bacillus cereus 569/H class A and class B and the class C enzyme from Enterobacter cloacae P99 but no significant activity was found.The γ-lactams were also screened for antibacterial activity against a wide range of micro-organisms but they showed no significant activity up to a concentration of 128 µg/cm 3 .

Experimental Section
General Procedures.Melting points were determined on a Gallenkamp melting point apparatus and are uncorrected.270 MHz 1 H and 67 MHz 13 C NMR were determined on a Bruker AC-270 spectrometer with tetramethylsilane as internal standard.All J values are given in Hz.IR spectra were recorded on a Perkin Elmer 1600 Series FTIR and FAB MS were performed by Swansea Mass Spectrometry Service and Zeneca Pharmaceuticals.All elemental analyses were performed by MEDAC Ltd., Brunel University.Fluka silica gel 60 was used for all chromatographic separations and thin layer chromatographic separations and thin layer chromatographic techniques used Merck silica gel 60 F 254 TLC plates.Ether refers to diethyl ether.Tetrahydrofuran was dried by distilling over lithium aluminium hydride under dry nitrogen.
Dichloromethane was dried by passing it through a column of Grade I activated alumina into the reaction flask under argon.
Method A. N-Succinimido-α-hydroxyacetic acid (4) 14 Succinimide (50.0 g, 0.51 mol) was dissolved in THF (300 cm 3 ) at room temperature with stirring.To this was added glyoxylic acid (50 wt%) (74.80 g, 0.51 mol) and the reaction mixture was heated to reflux for 3 h.Evaporation to dryness under reduced pressure produced a yellow oil which eventually crystallised.This was recrystallised from ethyl acetate yielding 4 (52.95 g, 60%) as white crystals.M. p. 140-141°C (lit.m.p. 140-141°C) 14  Method B. N-Succinimido-α-chloroacetic acid (7).N-Succinimido-α-hydroxy-acetic acid 7 (2.00 g, 12.0 mmol) was dissolved in dry THF (50 cm 3 ) with stirring at room temperature under nitrogen.The solution was cooled to 0°C before thionyl chloride (3.44 g, 29.0 mmol) was added dropwise.Evolution of an acidic gas was observed.The reaction mixture was allowed to warm to room temperature overnight.Ice (10 g) was added and the mixture stirred for a further 3 h.The solution was extracted using ethyl acetate (3 x 50 cm 3 ), the organics dried (MgSO 4 ), filtered and evaporated to give a viscous brown oil.Isatin-N-α-chloroacetic acid (19).Method B was used in the synthesis of 19 using isatin-N-αhydroxy-acetic acid 6 (10.0 g, 45.0 mmol), thionyl chloride (16.15 g, 0.136 mol), a catalytic amount of DMF (0.5 cm 3 ) and dry CH 2 Cl 2 (150 cm 3 ).The solution was evaporated to dryness and re-dissolved in THF (150 cm 3 ).The mixture was stirred and cooled to 0°C before the addition of ice (50g).The evolution of gases was observed and this stopped after 2 h.The homogenous reaction mixture was again evaporated under reduced pressure until all THF had been removed and the aqueous layer extracted with ethyl acetate (3 x 75 cm 3 ).The combined organics were dried using MgSO 4 , filtered and reduced to leave an orange oil.This was dissolved in a small amount of dichloromethane and hexane was added until the product crystallised and was collected by filtration.Attempted de-esterification of N-phthalimide-α-phenylacetoxyacetic acid methyl ester (15).N-Phthalimido-α-phenylacetyl-acetic acid methyl ester 16 (0.75 g, 2.1 mmol) and lithium iodide monohydrate (0.355 g, 2.3 mmol) were dissolved in pyridine (10 cm 3 ) under nitrogen and the solution heated to reflux for 24 h.The ixture was evaporated to dryness and taken up into ethyl acetate (50 cm 3 ) then washed with saturated sodium hydrogen carbonate solution (50 cm 3 ).The aqueous was extrcted using ethyl acetate (3 x 50 cm 3 ) then taken to pH2 with 2M hydrochloric acid.The aqueous was re-extracted with ethyl cetate (3 x 50 cm 3 ) and the combined organics dried using MgSO 4 , filtered and evaporated to dryness to give a brown solid.Analysis of the crude reaction material revealed by-product phenylacetic acid and further degradation products of N-Phthalimido-α-iodo-acetic acid methyl ester.(R/S)-N-Phenylacetylaspartic anhydride (R/S)-N-Phenylacetylaspartic acid (50.0 g, 0.20 mol) and acetic anhydride (150 cm 3 ) were used as described. 4.The product (30.62 g, 66%) was a white crystalline solid.M.p. 160-162°C (lit.m.p. 162°C) 4  (R/S)-N-Phenylacetylaspartimide Method 1 (R/S)-N-Phenylacetylaspartic anhydride (24.83g, 0.107 mol) was added to finely ground ammonium carbonate (5.63g, 59.0 mmol), mixed thoroughly and heated to 180°C for 1 h.During this time, the mixture turned partially molten and more ammonium carbonate (5.63g, 59.0 mmol) was added until no further gases evolded.The mixture turned from colourless to brown and water droplets were observed condensing on the apparatus.The reaction was allowed to cool and then dissolved in a 50/50 ethyl acetate/THF mix (250 cm 3 ) and water (250 cm 3 ).The aqueous layer was extracted using ethyl acetate (3 x 150 cm 3 ), the combined organics were then washed with saturated sodium hydrogen carbonate solution (150 cm 3 ), brine (150 cm 3 ) and dried with Na 2 SO 4 .Filtration and evaporation gave a brown solid which when washed with cold methanol afforded the product (4.59g,18%) as a white powder.M. ).The reaction mixture was heated to reflux and the vapour allowed to escape from the reaction vessel.All solids eventually went into solution.When all the water had been evaporated, the molten residue was allowed to reach 180°C and was maintained at this temperature for a further 2.5 h.The mixture was cooled and dissolved in ehtyl acetate (250 cm 3 ) before the residue hardened.The organic layer was washed with saturated sodium hydrogen carbonate solution (150 cm 3 ), brine (150 cm 3 ) and dried with Na 2 SO 4 .Filtration and evaporation gave an impure brown solid product.Recrystallisation from ethyl acetate yielded the product (2.70g,8%) as a white powder.M.p. 195-196°C.All other analyses were identical to those in Method 1. Method 3 (R/S)-N-Phenylacetylaspartic acid (50.5g, 0.20 mol) was heated with finely ground ammonium carbonate (38.28g, 0.40 mol) as of Method 1.The product, after cooling the reaction mixture, was recrystallised from ethyl acetate yielding the product 21 (3.00g, 6%) as a white solid.M.p. 196-198°C.All other analyses were identical to those in Method 1. Method 4 (R/S)-N-Phenylacetylaspartic acid (100.0g,0.40 mol) was added to finely ground urea (47.9g, 0.80 mol) and the mixture heated with stirring to 170°C for 2 h.During this time, all solids dissolved and water vapur was observed condensing on the apparatus.The mixture was cooled and dissolved in a 50/50 water/ethyl acetate mix (500 cm 3 ), separated, and the aqueous layer extracted with ethyl acetate (3 x 250 cm 3 ).The combined organics were dried (MgSO 4 ), filtered and evaporated to give a light brown solid.Trituration using ether yielded the product (46.80g,51%) as a white solid.M.p. 197-198°C.All other analyses were identical to those in Method 1.

Scheme 2 .
Scheme 2. The potential mode of action with serine enzymes of γ-lactams with a leaving.