5,5,6-Fused tricycles bearing imidazole and pyrazole 6-methylidene penems as broad-spectrum inhibitors of beta-lactamases.

Beta-lactamases are serine- and metal-dependent hydrolases, produced by the bacteria as defense against beta-lactam antibiotics. Commercially available inhibitors such as clavulanic acid, sulbactam, and tazobactam, which are currently used in the hospital settings, have reduced activity against newly emerging beta-lactamases. Bacterial production of diverse beta-lactamases including class-A, class-C, and ESBLs has motivated several research groups to search for inhibitors with a broader spectrum of activity. Previously, several novel 6-methylidene penems bearing, [5,5] [5,6] and [5,5,5] heterocycles have been synthesized in our laboratory and were shown to be potent and broad-spectrum beta-lactamase inhibitors. As a continuation of our previous work and in order to extend the structure-activity relationships, in this paper, we describe herein the synthesis and in vitro, in vivo activities of several novel 5,5,6-fused tricyclic heterocycles attached to the 6-methylidene penem core. The compounds presented in the current paper are potent and broad-spectrum inhibitors of the TEM-1 and AmpC beta-lactamases. In combination with piperacillin, their in vitro activities showed enhanced susceptibility to class A- and C-resistant strains studied in various bacteria. Some of the newly synthesized compounds such as 12a-c were shown to have in vivo activity in the acute lethal infection model against TEM-1 producing organisms. The 5,5,6-fused heterocyclic ring cores such as 21, 25, and 35 reported here are hitherto unknown in the literature.

Discovery of Novel Pyrazole-Based Selective Aldosterone Synthase (CYP11B2) Inhibitors: A New Template to Coordinate the Heme-Iron Motif of CYP11B2.

It is necessary for aldosterone synthase (CYP11B2) inhibitors to have both high potency and high selectivity over 11ß-hydroxylase (CYP11B1), a critical enzyme for cortisol synthesis. Previous studies have reported a number of CYP11B2 inhibitors, most of which have an imidazole or pyridine ring to coordinate the heme-iron motif of CYP11B2; however, highly selective inhibitors of human CYP11B2 are still needed. To expand the selectivity in humans, we explored alternative templates and found that pyrazoles were suitable templates for CYP11B2 inhibitors. Investigation of pyrazoles, especially N-alkyl pyrazoles, as a new template to coordinate the heme-iron motif led to a potent and highly selective CYP11B2 inhibitor 28 with an aldosterone-lowering effect at 1 mg/kg dosing in cynomolgus monkeys.

Structure-activity relationship of 6-methylidene penems bearing 6,5 bicyclic heterocycles as broad-spectrum beta-lactamase inhibitors: evidence for 1,4-thiazepine intermediates with C7 R stereochemistry by computational methods.

The design and synthesis of a series of 6-methylidene penems containing [6,5]-fused bicycles (thiophene, imidazole, or pyrazle-fused system) as novel class A, B, and C beta-lactamase inhibitors is described. These penems proved to be potent inhibitors of the TEM-1 (class A) and AmpC (class C) beta-lactamases and less so against the class B metallo-beta-lactamase CcrA. Their in vitro and in vivo activities in combination with piperacillin are discussed. On the basis of the crystallographic structures of a serine-bound reaction intermediate of 2 with SHV-1 (class A) and GC1 (class C) enzymes, compounds 14a-l were designed and synthesized. Penems are proposed to form a seven-membered 1,4 thiazepine ring in both class A and C beta-lactamases. The interaction energy calculation for the enzyme-bound intermediates favor the formation of the C7 R enantiomer over the S enantiomer of the 1,4-thiazepine in both beta-lactamases, which is consistent with those obtained from the crystal structure of 2 with SHV-1 and GC1.

Syntheses and pharmacokinetic studies of prodrug esters for the development of oral carbapenem, L-084.

We discovered an orally active carbapenem, L-084, through pharmacokinetic studies on various prodrug esters of (1R,5S,6S)-6-[(R)-1-hydroxyethyl]-l-methyl-2-[1-(1,3-thiazolin-2-yl)azetidin-3-yl]thio-1-carbapen-2-em-3-carboxylic acid (LJC11,036). L-084 showed a strong antimicrobial activity against Gram-positive and Gram-negative bacteria and exhibited the highest intestinal absorption among synthesized prodrugs of LJC11,036.

Novel imidazole substituted 6-methylidene-penems as broad-spectrum beta-lactamase inhibitors.

Beta-lactamases are serine and metallo-dependent enzymes produced by the bacteria in defense against beta-lactam antibiotics. Production of class-A, class-B, and class-C enzymes by the bacteria make the use of beta-lactam antibiotics ineffective in certain cases. To overcome resistance to beta-lactam antibiotics, several beta-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam are widely used in the clinic in combination with beta-lactam antibiotics. However, single point mutations within these enzymes have allowed bacteria to overcome the inhibitory effect of the commercially approved beta-lactamase inhibitors. Although the commercially available beta-lactamase inhibitor/beta-lactam antibiotic combinations are effective against class-A producing bacteria and many extended spectrum beta-lactamase (ESBL's) producing bacteria they are less effective against class-C enzymes expressing bacteria. To circumvent this problem, based on modeling studies several novel imidazole substituted 6-methylidene-penem derivatives were synthesized and tested against various beta-lactamase producing isolates. The present paper deals with the synthesis and structure-activity relationships (SAR) of these compounds.

A novel, mild, and facile method to prepare 6-methylidene penem derivatives.

A novel and mild method was established to synthesize 6-methylidene penem compounds. This method entails a MgBr(2)/Et(3)N-promoted aldol-type condensation on 6-bromopenem 12 with an appropriately substituted aldehyde to yield the intermediate acetylated bromohydrin, which was smoothly converted to the final product with simultaneous deprotection of C3 carboxylic acid ester using activated zinc dust and phosphate buffer at pH 6.5. This process provides a useful variation of C-C bond formation method for penem derivatives and also serves as a practical synthetic method to prepare 6-exomethylenepenem derivatives without racemization at the C5 position.

In vitro and in vivo activities of novel 6-methylidene penems as beta-lactamase inhibitors.

Novel penem molecules with heterocycle substitutions at the 6 position via a methylidene linkage were investigated for their activities and efficacy as beta-lactamase inhibitors. The concentrations of these molecules that resulted in 50% inhibition of enzyme activity were 0.4 to 3.1 nM for the TEM-1 enzyme, 7.8 to 72 nM for Imi-1, 1.5 to 4.8 nM for AmpC, and 14 to 260 nM for a CcrA metalloenzyme. All the inhibitors were more stable than imipenem against hydrolysis by hog and human dehydropeptidases. Piperacillin was combined with a constant 4-microg/ml concentration of each inhibitor for MIC determinations. The combinations reduced piperacillin MICs by 2- to 32-fold for extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae strains. The MICs for piperacillin-resistant (MIC of piperacillin, >64 microg/ml) strains of Enterobacter spp., Citrobacter spp., and Serratia spp. were reduced to the level of susceptibility (MIC of piperacillin, < or =16 microg/ml) when the drug was combined with 4, 2, or 1 microg of these penem inhibitors/ml. Protection against acute lethal bacterial infections with class A and C beta-lactamase- and ESBL-producing organisms in mice was also demonstrated with piperacillin plus inhibitor. Median effective doses were reduced by approximately two- to eightfold compared to those of piperacillin alone when the drug was combined with the various inhibitors at a 4:1 ratio. Pharmacokinetic analysis after intravenous administration of the various inhibitors showed mean residence times of 0.1 to 0.5 h, clearance rates of 15 to 81 ml/min/kg, and volumes of distribution between 0.4 and 2.5 liters/kg. The novel methylidene penem molecules inhibit both class A and class C enzymes and warrant further investigation for potential as therapeutic agents when used in combination with a beta-lactam antibiotic.