Novel insights into the mode of inhibition of class A SHV-1 beta-lactamases revealed by boronic acid transition state inhibitors.

Boronic acid transition state inhibitors (BATSIs) are potent class A and C ß-lactamase inactivators and are of particular interest due to their reversible nature mimicking the transition state. Here, we present structural and kinetic data describing the inhibition of the SHV-1 ß-lactamase, a clinically important enzyme found in Klebsiella pneumoniae, by BATSI compounds possessing the R1 side chains of ceftazidime and cefoperazone and designed variants of the latter, compounds 1 and 2. The ceftazidime and cefoperazone BATSI compounds inhibit the SHV-1 ß-lactamase with micromolar affinity that is considerably weaker than their inhibition of other ß-lactamases. The solved crystal structures of these two BATSIs in complex with SHV-1 reveal a possible reason for SHV-1's relative resistance to inhibition, as the BATSIs adopt a deacylation transition state conformation compared to the usual acylation transition state conformation when complexed to other ß-lactamases. Active-site comparison suggests that these conformational differences might be attributed to a subtle shift of residue A237 in SHV-1. The ceftazidime BATSI structure revealed that the carboxyl-dimethyl moiety is positioned in SHV-1's carboxyl binding pocket. In contrast, the cefoperazone BATSI has its R1 group pointing away from the active site such that its phenol moiety moves residue Y105 from the active site via end-on stacking interactions. To work toward improving the affinity of the cefoperazone BATSI, we synthesized two variants in which either one or two extra carbons were added to the phenol linker. Both variants yielded improved affinity against SHV-1, possibly as a consequence of releasing the strain of its interaction with the unusual Y105 conformation.

Inhibition of the class C beta-lactamase from Acinetobacter spp.: insights into effective inhibitor design.

The need to develop beta-lactamase inhibitors against class C cephalosporinases of Gram-negative pathogens represents an urgent clinical priority. To respond to this challenge, five boronic acid derivatives, including a new cefoperazone analogue, were synthesized and tested against the class C cephalosporinase of Acinetobacter baumannii [Acinetobacter-derived cephalosporinase (ADC)]. The commercially available carbapenem antibiotics were also assayed. In the boronic acid series, a chiral cephalothin analogue with a meta-carboxyphenyl moiety corresponding to the C(3)/C(4) carboxylate of beta-lactams showed the lowest K(i) (11 +/- 1 nM). In antimicrobial susceptibility tests, this cephalothin analogue lowered the ceftazidime and cefotaxime minimum inhibitory concentrations (MICs) of Escherichia coli DH10B cells carrying bla(ADC) from 16 to 4 microg/mL and from 8 to 1 microg/mL, respectively. On the other hand, each carbapenem exhibited a K(i) of <20 microM, and timed electrospray ionization mass spectrometry (ESI-MS) demonstrated the formation of adducts corresponding to acyl-enzyme intermediates with both intact carbapenem and carbapenem lacking the C(6) hydroxyethyl group. To improve our understanding of the interactions between the beta-lactamase and the inhibitors, we constructed models of ADC as an acyl-enzyme intermediate with (i) the meta-carboxyphenyl cephalothin analogue and (ii) the carbapenems, imipenem and meropenem. Our first model suggests that this chiral cephalothin analogue adopts a novel conformation in the beta-lactamase active site. Further, the addition of the substituent mimicking the cephalosporin dihydrothiazine ring may significantly improve affinity for the ADC beta-lactamase. In contrast, the ADC-carbapenem models offer a novel role for the R(2) side group and also suggest that elimination of the C(6) hydroxyethyl group by retroaldolic reaction leads to a significant conformational change in the acyl-enzyme intermediate. Lessons from the diverse mechanisms and structures of the boronic acid derivatives and carbapenems provide insights for the development of new beta-lactamase inhibitors against these critical drug resistance targets.

One-Pot Synthesis of Imidazole-4-Carboxylates by Microwave-Assisted 1,5-Electrocyclization of Azavinyl Azomethine Ylides.

Diversely functionalized imidazole-4-carboxylates were synthesized by microwave-assisted 1,5-eletrocyclization of 1,2-diaza-1,3-diene-derived azavinyl azomethine ylides. 1,2-Diaza-1,3-dienes were treated with primary aliphatic or aromatic amines and subjected to microwave irradiation in the presence of aldehydes. 3-Alkyl- and 3-arylimidazole-4-carboxylates were prepared in good yields through a one-pot multicomponent procedure. Modulation of the substituents at C-2, N-3 and C-5 was possible, and 2-unsubstituted imidazoles were obtained when paraformaldehyde was used.

Alpha-aminoester-derived imidazoles by 1,5-electrocyclization of azavinyl azomethine ylides.

An efficient and practical method for the preparation of alpha-imidazol-1-yl esters from 1,2-diaza-1,3-dienes (DDs), alpha-amino esters, and aldehydes is described. The overall sequence features a Michael-type conjugate addition between the alpha-amino ester and the DD, followed by iminium ion formation via condensation with the aldehyde and 1,5-electrocyclization of the resulting thermally generated azavinyl azomethine ylide to afford eventually alpha-imidazol-1-yl esters. Such a protocol allows access to enantiomerically pure imidazoles from optically pure alpha-amino esters.

Isolation, structure elucidation and total synthesis of a cytotoxic dienone from Echinacea pallida.

The isolation and structure characterization of a dienone from the roots of Echinacea pallida, namely (8Z,11Z)-pentadeca-8,11-dien-2-one, are described here. To assess the configuration of this secondary metabolite, the stereoselective total synthesis of the two isomeric forms, (8Z,11Z)- and (8Z,11E)-pentadeca-8,11-dien-2-one, was undertaken and the structure elucidation of the natural compound was unambiguously carried out. The cytotoxic activity of both isomers was also evaluated on a human T cell leukaemia cancer line (Jurkat cells). The results indicated that these compounds exert a dose-dependent cytotoxicity with a medium-level potency on the tested cell line.