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.     

Mechanism of inactivation of beta-lactamases by novel 6-methylidene penems elucidated using electrospray ionization mass spectrometry

The reactions of 6-methylidene penems 4-7 with beta-lactamases (TEM-1, SHV-1, Amp-C) were characterized by electrospray ionization mass spectrometry (ESI-MS). The kinetics of the reactions were monitored, demonstrating that only one penem molecule reacts to form an acyl-enzyme complex. For penem 5, the ESI-MS/MS spectrum of the hydrolysis product produced in the reaction was identical to the spectrum generated from a synthesized dihydro[1,4]thiazepine 10, confirming the rearrangement of the penem ring system to a seven-membered dihydro[1,4]thiazepine structure. Gas-phase ESI-MS/MS fragmentation data were rationalized due to tautomerization between imine and enamine substructures. ESI-MS/MS analysis of the T-6 trypsin-digested fragments of TEM-1 and SHV-1 demonstrated that the penems were only attached to Ser-70 of these class A beta-lactamases and that the penem ring structures were rearranged to seven-membered dihydro[1,4]thiazepines.     

In vitro comparative assessment of beta-lactamase inhibitors and their penicillin combinations against selected enterobacteria

A comparative in vitro assessment of the inhibitory activity of clavulanate, sulbactam and tazobactam against the most frequent beta-lactamases found in enterobacteria isolated in Greek hospitals, has been performed. Tazobactam and clavulanate were potent inhibitors of TEM-1, SHV-1, SHV-2 and SHV-5 while sulbactam was less effective. In addition, tazobactam exerted a moderate inhibitory activity against AmpC-type beta-lactamases. In contrast with clavulanate, tazobactam could not cause significant induction of chromosomal cephalosporinases. Potentiation of the activity of penicillins (amoxycillin, ampicillin, ticarcillin and piperacillin), when combined with the inhibitors, has also been evaluated using a sample of recently isolated enterobacteria with beta-lactamase-mediated resistance. The results showed that the most effective among the commercially available combinations, is piperacillin/tazobactam followed by ticarcillin/clavulanate.     

RU 29 246, the active compound of the cephalosporin-prodrug-ester HR 916. II. Stability to beta-lactamases and affinity for penicillin-binding proteins.

The aminothiazolyl-cephalosporin RU 29 246, the active metabolite of the prodrug-ester HR 916, is active against strains producing the widespread plasmid-encoded TEM-1, TEM-2 and SHV-1 beta-lactamases. Except for TEM-7 the activity of RU 29 246 against strains producing extended broad spectrum beta-lactamases (TEM-3, TEM-5, TEM-6, SHV-2, SHV-4, SHV-5, CMY-1, CTX-M), however, is low. Relative hydrolysis rates of RU 29 246 are comparable with those of cefpodoxime, the active metabolite of CS-807, and are extremely low for the TEM-1 and SHV-1 beta-lactamases. The compound demonstrates remarkable inhibitory activity against the chromosomal beta-lactamase of Enterobacter cloacae P99. In the presence of 1.7 microM this enzyme loses 50% of its activity. At concentrations of 0.43, 0.003 and 0.01 micrograms/ml the compound binds preferentially to the penicillin-binding protein (PBP) 3 of Escherichia coli K12, to the PBPs 2x and 3 of Streptococcus pneumoniae R6 and to PBP 1 of Staphylococcus aureus SG 511, respectively.     

Interaction of clavulanic acid, sulbactam and cephamycin antibiotics with beta-lactamases

The inhibitory effects of clavulanic acid, sulbactam and cephamycin antibiotics on chromosomally-mediated or plasmid-mediated beta-lactamases were investigated. The inhibition constants were determined by a non-linear regression analysis. Clavulanic acid and sulbactam had high affinities for the purified plasmid-mediated beta-lactamases such as SHV-1, TEM-1 and PSE-4, and were potent inhibitors as shown by their low Ki values. Except for Bacteroides beta-lactamase, which is sensitive to inhibition by cephamycin antibiotics, clavulanic acid and sulbactam were found not to be as effective against chromosomally-mediated beta-lactamases. The cephamycin antibiotics were better inhibitors of chromosomally-mediated beta-lactamases than those that are plasmid mediated. Except for P99 beta-lactamase, against which sulbactam and clavulanic acid were inactive, the cephamycin antibiotics were less effective inhibitors than sulbactam and clavulanic acid.     

The kinetics of SHV-2 plasmid-mediated beta-lactamase compared to those of the parent enzyme from which it is derived

Kinetic constants were determined for two closely related plasmid-mediated beta-lactamases: SHV-2 and PIT-2 (also known as SHV-1). These enzymes were synthesized constitutively. They were highly sensitive to the action of the inhibitors clavulanic acid and sulbactam, and they lacked activity against cephamycins and also imipenem. Both enzymes were significantly active against penicillins and first-generation cephalosporins, and the main difference concerned the third-generation cephalosporins: SHV-2 was highly active against these compounds whereas PIT-2 was not.     

Impact of beta-lactamases on the clinical use of beta-lactam antibiotics

beta-Lactamase production is a very common mechanism of antibiotic resistance, occurring in a wide variety of important pathogens involved in both community-acquired and nosocomial infections. The beta-lactamases can be divided into four classes (A, B, C, and D), each of which contains both chromosomal and plasmid-encoded enzymes. The impact of beta-lactamases on the clinical use of beta-lactam antibiotics depends on the spectrum of activity of the enzymes, the prevalence of beta-lactamase production in a given species, the frequency of involvement of the pathogen in infections, and whether the infection is treated in hospital or in the community. The class A and class C beta-lactamases generally have the most impact on clinical practice. Plasmid-mediated class A enzymes are produced by a wide range of common pathogens; these enzymes are primarily penicillinases such as TEM(1), SHV(1) and ROB(1), but in addition include extended-spectrum beta-lactamases which can also hydrolyze cephalosporins. The vast majority of the class A enzymes can be inhibited by currently-available beta-lactamase inhibitors. The class C enzymes or cephalosporinases, production of which is related to the presence of beta-lactam inducers, are present in nosocomial pathogens such as members of the Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp. Constitutive hyperproduction of class C enzymes, resulting from mutations in regulatory genes, leads to third-generation cephalosporin resistance in these organisms. To provide the best antimicrobial treatment, and to ensure that beta-lactam antibiotics are used to their optimum effect, clinicians need to remain aware of the prevalence of particular beta-lactamases in their areas or institutions, and to make use of appropriate counter measures such as beta- lactam beta- lactamase inhibitor combinations.     

Emergence and prevalence of beta-lactamase-producing Klebsiella pneumoniae resistant to cephems in Japan

Forty-six cephem-resistant Klebsiella pneumoniae strains with minimum inhibitory concentrations>8 microg/mL for cefpodoxime and cefmetazole were selected from clinical isolates obtained between 2000 and 2002 from eight hospitals on Northern Kyushu Island, Japan. We investigated the mechanisms of resistance to cephems in these 46 K. pneumoniae isolates. The results of isoelectric focusing of beta-lactamases produced by these isolates, polymerase chain reaction for detection of various Class A, Class B and Class C beta-lactamases, and determination of the sequence of the beta-lactamase structural gene showed that most of these isolates had various types of broad-spectrum beta-lactamases. Of the 46 isolates, 2 were CMY-2 beta-lactamase producers and 41 were DHA-1 beta-lactamase producers. Forty of the 41 DHA-1 beta-lactamase producers simultaneously produced SHV-12 extended-spectrum beta-lactamase (ESBL), and the remaining isolate simultaneously produced SHV-27. Furthermore, one DHA-1 and SHV-12 beta-lactamase producer also produced IMP-1 beta-lactamase. The only broad-spectrum beta-lactamase with another isolate was IMP-1. Chromosomal DNA restriction fragment analysis using XbaI suggested that nosocomial infection due to DHA-1 and SHV-12 beta-lactamase producers had occurred at two centres. This is the first report of nosocomial infection due to DHA-1 beta-lactamase-producing K. pneumoniae including other plasmid-encoded AmpC beta-lactamases in Japan. The mechanisms of resistance of 44 of the 46 isolates to cephalosporins and cephamycins were ESBL production and/or plasmid-encoded AmpC beta-lactamase and/or IMP-1 beta-lactamase production. For two isolates, the mechanism of resistance to could not be identified. These results show that it is necessary to minimise the prevalence of these resistant strains as it will be a very serious problem if organisms producing these broad-spectrum beta-lactamases increase in clinical situations. It is important to detect these strains sooner and to perform rigorous infection control earlier.     

Synthesis and antiinflammatory/analgesic activities of 11H-dibenzo[b, e,][1,4]dioxepinacetic acids

A new class of tricyclic arylacetic acids was synthesized and evaluated as antiinflammatory/analgesic agents as well as inhibitors of prostaglandin synthetase. 11H-Dibenzo[b,e][1,4]dioxepin-2-, -3, -7, and -8-acetic and alpha-methylacetic acids and their derivatives were prepared by cyclization of diaryl ether precursors or by condensation of catechol and an aryl dihalide. The most potent compound in the carrageenan foot edema assay was alpha-methyl-11H-dibenzo[b,e][1,4]dioxepin-8-acetic acid (1 mg/kg = 43% inhibition). The most potent enzyme inhibitors were the 2-acetic acid and the alpha-methyl-7-acetic acid (IC50 = 0.1 microM). Some of these compounds were also found to be highly ulcerogenic.     

Design, synthesis, and evaluation of 3,4-dihydro-2H-[1,4]diazepino[6,7,1-hi]indol-1-ones as inhibitors of poly(ADP-ribose) polymerase

The design, synthesis, and biological evaluation of potent inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) are reported. A novel series of 3,4-dihydro-2H-[1,4]diazepino[6,7,1-hi]indol-1-ones were designed using a combination of protein structure-based drug design, molecular modeling, and structure-activity relationships (SAR). These novel submicromolar inhibitors possess a tricyclic ring system conformationally restricting the benzamide in the preferred cis orientation. The compounds were designed to optimize space-filling and atomic interactions within the NAD+ binding site of PARP-1. Previously described and newly adapted methods were applied to syntheses of these tricyclic inhibitors. Various modifications were made to the diazepinoindolones at the 6- and 7-positions in order to study this region of the active site and optimize noncovalent interactions. The electron density of derivative 28 bound to chicken PARP-1 revealed that the oxime makes a tight hydrogen bond with the catalytic gamma-carboxylate of glutamic acid (Glu) 988 in accordance with our original designs and models. Most of the compounds have been evaluated for inhibition of human PARP-1. Selected inhibitors were also tested for the ability to potentiate the cytotoxic effect of the DNA-damaging agent Topotecan.     

Molecular and crystal structures of MDL27,467A hydrochloride and quinapril hydrochloride, two ester derivatives of potent angiotensin converting enzyme inhibitors

The molecular structures of MDL27,467A hydrochloride, [4 alpha,7 alpha(R*),12b beta]-7-[[1-(ethoxycarbonyl)-3-phenyl-propyl] amino]-1,2,3,4,6,7,12a,12b-octahydro-6-oxopyrido[2,1- a][2]benzazepine-4-carboxylic acid diphenylmethyl ester hydrochloride, and quinapril hydrochloride, [3S-[2[R*(R*)],3R]]-2-[2[[1- (ethoxycarbonyl)-3-phenylpropyl]-amino]-1-oxopropyl]-1,2,3,4- tetrahydro-3-isoquinolinecarboxylic acid hydrochloride, were determined by X-ray diffraction methods. The modified, C-terminal dipeptide portions and the phenylpropyl fragments in both crystal structures adopt similar conformations. The binding positions for several pharmacophores are defined by the constraint of the tricyclic system in the crystallographic structure of MDL27,467A hydrochloride. Conformational energy calculations show that the phenyl ring of the tetrahydro-3-isoquinoline system of quinapril does not fit into the S2 hydrophobic pocket of angiotensin converting enzyme.     

The kinetics of CAZ-5, a novel SHV-related plasmid-mediated beta-lactamase with enhanced hydrolytic activity against ceftazidime

The kinetic constants for "CAZ-5", a novel plasmid-mediated beta-lactamase with noticeable activity against third-generation cephalosporins and particularly ceftazidime have been determined. Two closely-related plasmid-mediated beta-lactamases have also been studied: SHV-2 and PIT-2 (also known as SHV-1). These enzymes were synthesized constitutively; they were highly sensitive to the action of the inhibitors clavulanic acid and sulbactam and they lacked activity against the cephamycins and imipenem. PIT-2/SHV-1 had poor hydrolytic activity against the third-generation cephalosporins, SHV-2 was markedly active against cefotaxime and related compounds, whereas the new enzyme, which was also active against these cephalosporins, had a noticeably greater activity against ceftazidime. Aztreonam was slowly hydrolysed by CAZ-5 beta-lactamase, but demonstrated an unusually high affinity for this enzyme.     

Synthesis and biological activity of AM-112 and related oxapenem analogues

Thirty five oxapenem analogues substituted with a range of tertiary groups at C-2 have been synthesised and evaluated as broad-spectrum beta-lactamase inhibitors. All analogues enhanced the activity of ceftazidime against bacterial isolates producing Class A and Class C beta-lactamases. Compounds with cyclic substituents at C-1' (attached to C-6) were associated with enhanced antibacterial activity against Staphylococcus aureus. (R) Stereochemistry at C-1' led to synergistic activity against beta-lactamase negative enterococci. (S) Stereochemistry at C-1' was associated with enhanced inhibition of Class A beta-lactamases and lack of synergistic activity against enterococci. AM-113 was unstable in serum and not detectable following subcutaneous or oral dosing in mice. AM-112 and AM-115 achieved good serum levels following subcutaneous dosing. AM-114 exhibited 30% bioavailability following oral dosing. AM-112 [(1'R,5R,6R)-2-(4-ammonio-1,1-dimethylbutyl)-6-(1'-hydroxyethyl)oxapenem-3-carboxylate] achieved the greatest protection of ceftazidime against Gram-negatives producing Class A or C beta-lactamases.     

Ceftibuten stability to active-site serine and metallo-beta-lactamases

Ceftibuten is an oral third-generation cephalosporin active against a wide range of bacteria and shows an improved stability to hydrolysis by several beta-lactamases because of the carboxyethilidine moiety at position 7 of the ss-acyl side chain. The kinetic interactions between ceftibuten and active-site serine and metallo-ss-lactamases were investigated. The activity of several TEM-derived extended spectrum beta-lactamases (ESbetaLs) against ceftibuten, cefotaxime and ceftazidime was compared using K(m), K(cat) and K(cat)/K(m). Ceftibuten behaved as a poor substrate for class A and B beta-lactamases compared with cefotaxime. The chromosomal class C beta-lactamase from Enterobacter cloacae 908R gave a high K(cat) value (21 s(-1)), whereas there was poor activity with enzymes from Acinetobacter baumannii and Morganella morganii and ceftibuten. Ceftibuten resists hydrolysis in the presence of typical respiratory or urogenital-tract pathogens producing beta-lactamases.     

Beta-lactamase inhibitors: evolving compounds for evolving resistance targets

The many and diverse beta-lactamases produced by bacteria, particularly by Gram-negative pathogens, are increasingly posing a serious threat to the clinical utility of beta-lactams. First-generation inhibitors (clavulanic acid, sulbactam, tazobactam) focus on Ambler class A enzymes. However, recent structural upgrades of class A beta-lactamases (e.g. TEM, SHV) have extended their spectrum (extended-spectrum beta-lactamases and carbapenemases [Sme, NMC-A, IMI-1]) and have brought about the possibility of beta-lactamase-inhibitor resistance. Furthermore, the mobilisation and spread of originally chromosomal class C enzymes (CMY, MIR), the growing clinical importance of class B enzymes (IMP, VIM), the emergence of inhibitor-resistant, broad spectrum class D (OXA) enzymes and the co-existence of different classes of beta-lactamases in the same pathogen have spurred research toward universal inhibitors. A complicating issue is target accessibility in Gram-negative bacteria, particularly in Enterobacter, Acinetobacter, Pseudomonas, Stenotrophomonas and other organisms, which is necessary in order for the inhibitor to synergise with vulnerable beta-lactam antibiotics. Several new, broad-spectrum inhibitors have emerged: cephem sulfones and oxapenems are upgrades of penam sulfones and oxapenams, respectively, with cephem sulfones possibly extending their inhibition to class B metallo-enzymes; and boronates and phosphonates are designed de novo, based on common structural and mechanistic features of serine beta-lactamases.     

Beta-lactamase-inhibitor combinations in the 21st century: current agents and new developments.

Combinations of beta-lactams and beta-lactamase inhibitors have become one of the most successful antibacterial strategies in our global battle against bacterial infections. The success of these agents is particularly emphasized by the continued efficacy of Augmenting (amoxicillin and clavulanate) after nearly 20 years of clinical use. The clinical situation now dictates that second-generation beta-lactamase inhibitors capable of encompassing both class A and class C beta-lactamases would combat emerging resistance and provide a vital addition to our armory of hospital antibiotics. This realization has generated a renewed interest in beta-lactamase inhibitors and improved the prospects for the delivery of such agents in the future.     

4-Substituted trinems as broad spectrum beta-lactamase inhibitors: structure-based design, synthesis, and biological activity

A wide variety of pathogens have acquired antimicrobial resistance as an inevitable evolutionary response to the extensive use of antibacterial agents. In particular, one of the most widely used antibiotic structural classes is the beta-lactams, in which the most common and the most efficient mechanism of bacterial resistance is the synthesis of beta-lactamases. Class C beta-lactamase enzymes are primarily cephalosporinases, mostly chromosomally encoded, and are inducible by exposure to some beta-lactam agents and resistant to inhibition by marketed beta-lactamase inhibitors. In an ongoing effort to alleviate this problem a series of novel 4-substituted trinems was designed and synthesized. Significant in vitro inhibitory activity was measured against the bacterial beta-lactamases of class C and additionally against class A. The lead compound LK-157 was shown to be a potent mechanism-based inactivator. Acylation of the active site Ser 64 of the class C enzyme beta-lactamase was observed in the solved crystal structures of two inhibitors complexes to AmpC enzyme from E. cloacae. Structure-activity relationships in the series reveal the importance of the trinem scaffold for inhibitory activity and the interesting potential of the series for further development.     

Quinazolines and 1,4-benzodiazepines. 92. Conformational recognition of the receptor by 1,4-benzodiazepines

We propose that the conformation of 1,4-benzodiazepines that is recognized by the binding site on the benzodiazepine receptor complex is one in which the planes formed by the fused benzene ring and the methylene group (and the two adjoining atoms) of the diazepine ring are in the R configuration. The derivation of this conformation was based on comparisons of computer-generated 3-dimensional structures obtained from single-crystal X-ray data for diazepam, (R)- and (S)-1,3-dimethyl-5-(2-fluorophenyl)-7-nitro-1,3-dihydro-2H-1,4-benzodiazepin-2- one, and the structurally rigid ethyl (S)-7-chloro-11,12,13,13 alpha-tetrahydro-9-oxo-9H-imidazo[1,5-alpha] pyrrolo[2,1-d][1,4]benzodiazepine-1-carboxylate. The affinity of ligands for the benzodiazepine binding site was determined using the [3H]-diazepam binding assay.     

Axially chiral 1,7-naphthyridine-6-carboxamide derivatives as orally active tachykinin NK(1) receptor antagonists: synthesis, antagonistic activity, and effects on bladder functions.

Cyclic analogues of N-[3,5-bis(trifluoromethyl)benzyl]-7,8-dihydro-N, 7-dimethyl-5-(4-methylphenyl)-8-oxo-1,7-naphthyridine-6-carboxamide (1) having a 6-9-membered ring (6-9) were synthesized and evaluated for NK(1) antagonistic activities. The 8-membered ring compound with a beta-methyl group at the C((9))-position, (aR,9R)-7-[3, 5-bis(trifluoromethyl)benzyl]-8,9,10, 11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g] [1, 7]naphthyridine-6,13-dione [(aR,9R)-8b], was atropodiastereoselectively synthesized by cyclization of a chiral intermediate, 10g. On the other hand, the 7-membered ring compound with a beta-methyl group at the C((9))-position [(9S)-7b] was obtained as an equilibrium mixture of atropisomers with a ratio of ca. 3:2 in solution at room temperature (measured by NMR in CDCl(3)). Compounds (9S)-7b and (aR,9R)-8b exhibited excellent antagonistic activities both in vitro [IC(50) (inhibition of [(125)I]BH-SP binding in human IM-9 cells) = 0.28 and 0.45 nM, respectively] and in vivo (iv and po). Significantly, the in vitro activity of (aR, 9R)-8b was ca. 750-fold higher than that of its enantiomer (aS, 9S)-8b, ca. 40-fold higher than its atropisomer (aS,9R)-8b, and ca. 20-fold higher than its diastereomer (aR,9S)-8b. The structure-activity relationships in this series, along with the X-ray analysis of (aR,9R)-8b, indicated that the stereochemistry around the -C((6))(=O)-N((7))-CH(2)Ar moiety is important for NK(1) receptor recognition. The NK(1) antagonists showed effects on bladder functions in guinea pigs upon intravenous injection: i.e., the antagonists increased the shutdown time of distension-induced rhythmic bladder contractions and the bladder volume threshold, and the effects on the shutdown time were found to correlate well with the NK(1) antagonistic activities. Compound (aR,9R)-8b has been identified as a potential clinical candidate for the treatment of bladder function disorders.     

Crystal structures of monoamine oxidase B in complex with four inhibitors of the N-propargylaminoindan class

Monoamine oxidase B (MAO B) is an outer mitochondrial membrane enzyme that catalyzes the oxidation of arylalkylamine neurotransmitters. The crystal structures of MAO B in complex with four of the N-propargylaminoindan class of MAO covalent inhibitors (rasagiline, N-propargyl-1(S)-aminoindan, 6-hydroxy-N-propargyl-1(R)-aminoindan, and N-methyl-N-propargyl-1(R)-aminoindan) have been determined at a resolution of better than 2.1 A. Rasagiline, 6-hydroxy-N-propargyl-1(R)-aminoindan, and N-methyl-N-propargyl-1(R)-aminoindan adopt essentially the same conformation with the extended propargyl chain covalently bound to the flavin and the indan ring located in the rear of the substrate cavity. N-Propargyl-1(S)-aminoindan binds with the indan ring in a flipped conformation with respect to the other inhibitors, which causes a slight movement of the Tyr326 side chain. Four ordered water molecules are an integral part of the active site and establish H-bond interactions to the inhibitor atoms. These structural studies may guide future drug design to improve selectivity and efficacy by introducing appropriate substituents on the rasagiline molecular scaffold.