Pyrrolopyrimidine inhibitors of DNA gyrase B (GyrB) and topoisomerase IV (ParE), Part II: Development of inhibitors with broad spectrum,Gram-negative antibacterial activity

The structurally related bacterial topoisomerases DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as prime candidates for the development of broad spectrum antibacterial agents. However, GyrB/ParE targeting antibacterials with spectrum that encompasses robust Gram-negative pathogens have not yet been reported. Using structure-based inhibitor design, we optimized a novel pyrrolopyrimidine inhibitor series with potent, dual targeting activity against GyrB and ParE. Compounds were discovered with broad antibacterial spectrum, including activity against Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli. Herein we describe the SAR of the pyrrolopyrimidine series as it relates to key structural and electronic features necessary for Gram-negative antibacterial activity.     

Synthesis of novel dihydrotriazine derivatives bearing 1,3-diaryl pyrazole moieties as potential antibacterial agents

Three novel series of dihydrotriazine derivatives bearing 1,3-diaryl pyrazole moieties were designed, synthesized and evaluated in terms of their antibacterial and antifungal activities. Most of the synthesized compounds showed potent inhibition of several Gram-positive bacterial strains (including multidrug-resistant clinical isolates) and Gram-negative bacterial strains with minimum inhibitory concentration values in the range of 1–64?µg/mL. Compounds 4b and 4c presented the most potent inhibitory activity against Gram-positive bacteria (S. aureus 4220, MRSA 3167, QRSA 3519) and Gram-negative bacteria (E. coli 1924), with minimum inhibitory concentration values of 1 or 2?µg/mL. Compared with previous studies, these compounds exhibited a broad spectrum of inhibitory activity. The cytotoxic activity of the compounds 4a, 4b, 4c and 11n were assessed in L02 cells. In vitro enzyme study implied that compound 4c exerted its antibacterial activity through DHFR inhibition.     

Proline-based hydroxamates targeting the zinc-dependent deacetylase LpxC: Synthesis,antibacterial properties,and docking studies

The Zn²⁺-dependent deacetylase LpxC is an essential enzyme in Gram-negative bacteria, which has been validated as antibacterial drug target. Herein we report the chiral-pool synthesis of novel d- and l-proline-derived 3,4-dihydroxypyrrolidine hydroxamates and compare their antibacterial and LpxC inhibitory activities with the ones of 4-monosubstituted and 3,4-unsubstituted proline derivatives. With potent antibacterial activities against several Gram-negative pathogens, the l-proline-based tertiary amine 41g ((S)-N-hydroxy-1-(4-{[4-(morpholinomethyl)phenyl]ethynyl}benzyl)pyrrolidine-2-carboxamide) was found to be the most active antibacterial compound within the investigated series, also showing some selectivity toward EcLpxC (K_i?=?1.4?μM) over several human MMPs.     

Design,synthesis and antibacterial activity studies of thiazole derivatives as potent ecKAS III inhibitors

Two series of thiazole derivatives containing amide skeleton were synthesized and developed as potent Escherichia coli β-ketoacyl-(acyl-carrier-protein) synthase III (ecKAS III) inhibitors. All the 24 new synthesized compounds were assayed for antibacterial activity against the respective Gram-negative and Gram-positive bacterial strains, including E. coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus. In which, 10 compounds with broad-spectrum antibacterial activities were further tested for their ecKAS III inhibitory activity. Last, we have successfully found that compound 4e showed both the promising broad antibacterial activity with MIC of 1.56–6.25 μg/mL against the representative bacterial stains, and also processed the most potent ecKAS III inhibitory activity with IC₅₀ of 5.3 μM. In addition, docking simulation also carried out in this study to give a potent prediction binding mode between the small molecule and ecKAS III (PDB code: 1hnj) protein.     

Synthesis and antibacterial activity of benzyl-[3-(benzylamino-methyl)-cyclohexylmethyl]-amine derivatives

A series of benzyl-[3-(benzylamino-methyl)-cyclohexylmethyl]-amine derivatives with different substitution pattern on the aromatic ring have been prepared and evaluated for their antibacterial activity against Gram-positive and Gram-negative bacterial strains. Most of the compounds exhibit potent activity against Pseudomonas aeruginosa and Staphylococcus epidermidis while compounds 6l and 6m showed antibacterial activity against all the four bacterial strains with MIC values ranging from 0.002 to 0.016 μg/mL and no hemolytic activity up to 512 μg/mL in mammalian erythrocytes was observed.     

Towards Gram-negative antivirulence drugs: New inhibitors of HldE kinase

Gram-negative bacteria lacking heptoses in their lipopolysaccharide (LPS) display attenuated virulence and increased sensitivity to human serum and to some antibiotics. Thus inhibition of bacterial heptose synthesis represents an attractive target for the development of new antibacterial agents. HldE is a bifunctional enzyme involved in the synthesis of bacterial heptoses. Development of a biochemical assay suitable for high-throughput screening allowed the discovery of inhibitors 1 and 2 of HldE kinase. Study of the structure–activity relationship of this series of inhibitors led to highly potent compounds.     

Synthesis and antibacterial activities of new piperidine substituted (5R)-[1,2,3]triazolylmethyl and (5R)-[(4-F-[1,2,3]triazolyl)methyl] oxazolidinones

A novel series of 5(R)-[1,2,3]triazolylmethyl and (5R)-[(4-F-[1,2,3]triazolyl)methyl]oxazolidinones having various piperidine group were synthesized and evaluated antibacterial activity against clinically isolated resistant strains of Gram-positive and Gram-negative bacteria. The compound 12a having exo-cyanoethylidene group in the 4-position of piperidine ring was found to be two to threefold more potent than the linezolid against penicillin-resistant Staphylococcus pneumonia and Staphylococcus agalactiae, and also exhibited reduced MAO-B inhibitory activity.     

Design and synthesis of potent Gram-negative specific LpxC inhibitors

Antibiotic resistant hospital acquired infections are on the rise, creating an urgent need for novel bactericidal drugs. Enzymes involved in lipopolysaccharide (LPS) biosynthesis are attractive antibacterial targets since LPS is the major structural component of the outer membrane of Gram-negative bacteria. Lipid A is an essential hydrophobic anchor of LPS and the first committed step in lipid A biosynthesis is catalyzed by a unique zinc dependent metalloamidase, UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC). LpxC is an attractive Gram-negative only target that has been chemically validated by potent bactericidal hydroxamate inhibitors that work by coordination of the enzyme’s catalytic zinc ion. An exploratory chemistry effort focused on expanding the SAR around hydroxamic acid zinc-binding ‘warheads’ lead to the identification of novel compounds with enzyme potency and antibacterial activity similar to CHIR-090.     

6-Bromoindolglyoxylamido derivatives as antimicrobial agents and antibiotic enhancers

The combination of increased incidence of drug-resistant strains of bacteria and a lack of novel drugs in development creates an urgency for the search for new antimicrobials. Initial screening of compounds from an in-house library identified two 6-bromoindolglyoxylamide polyamine derivatives (3 and 4) that exhibited intrinsic antimicrobial activity towards Gram-positive bacteria, Staphylococcus aureus and S. intermedius with polyamine 3 also displaying in vitro antibiotic enhancing properties against the resistant Gram-negative bacterium Pseudomonas aeruginosa. A series of 6-bromo derivatives (5–15) were prepared and biologically evaluated, identifying analogues with enhanced antibacterial activity towards Escherichia coli and with moderate to excellent antifungal properties. Polyamine 3, which includes a spermine chain, was the most potent of the series – its mechanism of action was attributed to rapid membrane permeabilization and depolarization in both Gram-positive and Gram-negative bacteria.     

Synthesis and antibacterial activities of novel oxazolidinones having spiro[2,4]heptane moieties

The synthesis of a new series of oxazolidinones having spiro[2,4]heptane moieties is described. Their in vitro antibacterial activities against both Gram-positive and Gram-negative bacteria were tested and the effect of substituents on the oxazolidinone ring was investigated. A particular compound Ih having fluoro group showed the most potent antibacterial activity.     

Synthesis, characterization and antimicrobial activity of a series of substituted coumarin-3-carboxylatosilver(I) complexes

A series of new coumarin-derived carboxylate ligands and their silver(I) complexes have been synthesized, characterized and screened for their in vitro antibacterial activity against a range of Gram-positive and Gram-negative bacteria as well as for their antifungal activity against a clinical isolate of Candida albicans. The ligands were synthesised by either acid or base hydrolysis of their corresponding esters, which in turn were synthesised via the Knoevenegal reaction. The reaction of silver(I) nitrate with the coumarin carboxylate ligands in either aqueous or aqueous/ethanol solutions allowed the isolation of a series of novel Ag(I) carboxylate complexes. Whilst none of the ligands showed any antimicrobial activity, a number of the Ag(I) complexes exhibited potent activity. In particular, Ag(I) complexes of hydroxy-substituted coumarin carboxylates demonstrated potent activity against the clinically important methicillin-resistant Staphylococcus aureus (MRSA) bacterium (MIC₈₀ = 0.63 μM).     

Novel 3-arylfuran-2(5H)-one-fluoroquinolone hybrid: Design,synthesis and evaluation as antibacterial agent

3-Arylfuran-2(5H)-one, a novel antibacterial pharmacophore targeting tyrosyl-tRNA synthetase (TyrRS), was hybridized with the clinically used fluoroquinolones to give a series of novel multi-target antimicrobial agents. Thus, twenty seven 3-arylfuran-2(5H)-one-fluoroquinolone hybrids were synthesized and evaluated for their antimicrobial activities. Some of the hybrids exhibited merits from both parents, displaying a broad spectrum of activity against resistant strains including both Gram-negative and Gram-positive bacteria. The most potent compound (11) in antibacterial assay shows MIC₅₀ of 0.11 μg/mL against Multiple drug resistant Escherichia coli, being about 51-fold more potent than ciprofloxacin. The enzyme assays reveal that 11 is a potent multi-target inhibitor with IC₅₀ of 1.15 ± 0.07 μM against DNA gyrase and 0.12 ± 0.04 μM against TyrRS, respectively. Its excellent inhibitory activities against isolated enzymes and intact cells strongly suggest that 11 deserves to further research as a novel antibiotic.     

Design,synthesis and antibacterial evaluation of novel AHL analogues

Two series of novel AHL analogues were designed, synthesized and evaluated for antibacterial activity under cell membrane conditions in vitro. Analogues 4a–c and 4g–m presented potent activity against Gram-positive bacteria. Especially the analogue 4l exerted the most potent inhibition against Bacillus subtilis with MIC₅₀ value of 1.443 μg/ml. To our surprise, analogues 6a–c and 6g showed weak inhibition against Gram-negative bacteria with MIC₅₀ values ranging from 17.589 to 67.840 μg/ml. This was the first report about synthesis and antibacterial evaluation in vitro of AHL analogues containing dithioester linkage.     

Synthesis and evaluation of the antibacterial activities of aryl substituted dihydrotriazine derivatives

Five series of dihydrotriazine derivatives containing chalcone (13a–i), phenoxy acetophenone (14a–b), benzyl benzene (15a–c), naphthoxyl acetophenone (16a–b) and benzyl naphthalene (17a–h) moieties were designed and synthesized. The antibacterial and antifungal activities of these compounds were evaluated against several strains of Gram-positive and Gram-negative bacteria, as well as a single fungus. Compound 17h was found to be the most potent of all of the compounds tested, with an MIC value of 0.5?μg/mL against several Gram-positive (Staphylococcus aureus 4220 and QRSA CCARM 3505) and Gram-negative (Escherichia coli 1924) strains of bacteria. However, this compound was inactive against Pseudomonas aeruginosa 2742 and Salmonella typhimurium 2421, indicating that its antibacterial spectrum is similar to those of the positive controls gatifloxacin and moxifloxacin. The cytotoxic activity of the compound 13i, 16b and 17h was assessed in Human normal liver cells.     

Design,synthesis and antibacterial activity evaluation of moxifloxacin-amide-1,2,3-triazole-isatin hybrids

In this work, a series of novel moxifloxacin-amide-1,2,3-triazole-isatin hybrids 7a-l were designed and synthesized. The in vitro antibacterial activity against a panel of clinically important Gram-positive and Gram-negative bacteria including drug-resistant pathogens was also evaluated. All hybrids showed considerable activity against the tested pathogens with MIC values of ≤0.03 to 128 μg/mL, and some of them such as hybrids 7e, 7g and 7j were comparable to or better than the parent moxifloxacin (MIC: ≤0.03–8 μg/mL). Moreover, hybrids 7e, 7g and 7j also demonstrated low cytotoxicity towards CHO cells. However, the in vivo pharmacokinetic profiles of these three hybrids were generally far inferior to the parent moxifloxacin. The structure-activity relationship and structure-cytotoxicity relationship were also studied and discussed which may help with the identification of new chemical entities as potent antibacterial agents.     

Inhibitors for the bacterial ectonucleotidase Lp1NTPDase from Legionella pneumophila

Legionella pneumophila is an aerobic, Gram-negative bacterium of the genus Legionella, which constitutes the major causative agent of Legionnaires’ disease. Recently a nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila was identified and termed Lp1NTPDase; it was found to be a structural and functional homolog of mammalian NTPDases catalyzing the hydrolysis of ATP to ADP and ADP to AMP. Its activity is believed to contribute to the virulence of Legionella pneumophila. Therefore Lp1NTPDase inhibitors are considered as novel antibacterial drugs. However, only weakly potent compounds are available so far. In the present study, a capillary electrophoresis (CE)-based enzyme assay for monitoring the Lp1NTPDase activity was established. The enzymatic reaction was performed in a test tube followed by separation of substrate and products by CE and subsequent quantification by UV analysis. After kinetic characterization of the enzyme, a series of 1-amino-4-ar(alk)ylamino-2-sulfoanthraquinone derivatives structurally related to the anthraquinone dye Reactive Blue 2, a non-selective ecto-NTPDase inhibitor, was investigated for inhibitory activity on Lp1NTPDase using the CE-based enzyme assay. Derivatives bearing a large lipophilic substituent (e.g., fused aromatic rings) in the 4-position of the 1-amino-2-sulfoanthraquinone showed the highest inhibitory activity. Compounds with IC₅₀ values in the low micromolar range were identified. The most potent inhibitor was 1-amino-4-[phenanthrene-9-yl-amino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (28, PSB-16131), with an IC₅₀-value of 4.24 μM. It represents the most potent Lp1NTPDase inhibitor described to date. These findings may serve as a starting point for further optimization. Lp1NTPDase inhibition provides a novel approach for the (immuno)therapy of Legionella infections.     

Synthesis,antibacterial activities and molecular docking studies of peptide and Schiff bases as targeted antibiotics

A series of peptide and Schiff bases (PSB) were synthesized by reacting salicylic acid, primary diamines with salicylaldehyde or its derivatives, and 40 of which were newly reported. The inhibitory activities against Escherichia coli β-ketoacyl-acyl carrier protein synthase III (ecKAS III) were investigated in vitro and molecular docking simulation also surveyed. Top 10 PSB compounds which posses both good inhibitory activity and well binding affinities were picked out, and their antibacterial activities against Gram-negative and Gram-positive bacterial strains were tested, expecting to exploit potent antibacterial agent with broad-spectrum antibiotics activity. The results demonstrate compound N-(3-(5-bromo-2-hydroxybenzylideneamino)propyl)-2-hydroxybenzamide (2d) can be as a potential antibiotics agent, displaying minimal inhibitory concentration values in the range of 0.39–3.13 μg/mL against various bacteria.     

Expression of 4kD scorpion defensin and its in vitro synergistic activity with conventional antibiotics

The 4kD scorpion defensin (SD) is a potent disulfide-linked peptide. In this study, we expressed it in methylotrophic yeast Pichia pastoris and purified it using Ni–NTA His Bind Resin. We investigated its in vitro antibacterial activity and effect in combination with several conventional antibiotics. We first examined its antibacterial activity towards several Gram-positive and Gram-negative bacteria. Then we used the broth microdilution method to test drugs alone and in combination and used the fractional inhibitory concentration (FIC index) to classify the drug interactions. Our study showed the expressed SD peptide has antibacterial activity against Salmonella typhimurium, E. coli and S. aureus etc. Synergy or additive interaction was observed between SD and Norfloxacin, Polymyxin B and Ampicillin. Cell growth tests showed that combination of SD and Norfloxacin can improve their activity against bacteria. This result maybe permit lower using of the conventional antibiotic agents more effectively and safely.     

Design,synthesis and biological activity evaluation of novel 2,6-difluorobenzamide derivatives through FtsZ inhibition

Novel series of 3-substituted 2,6-difluorobenzamide derivatives as FtsZ inhibitors were designed, synthesized and evaluated for their in vitro antibacterial activity against various phenotype of Gram-positive and Gram-negative bacteria, and their cell division inhibitory activity against three representative strains. As a result, 3-chloroalkoxy derivative 7, 3-bromoalkoxy derivative 12 and 3-alkyloxy derivative 17 were found to exhibit the best antibacterial activity against Bacillus subtilis with MICs of 0.25–1 μg/mL, and good activity (MIC < 10 μg/mL) against both susceptible and resistant Staphylococcus aureus. Additionally, all the three compounds displayed potent cell division inhibitory activity with MIC values of below 1 μg/mL against Bacillus subtilis and Staphylococcus aureus.     

Isolation of the antibiotic pseudopyronine B and SAR evaluation of C3/C6 alkyl analogs

Natural products are an abundant source of structurally diverse compounds with antibacterial activity that can be used to develop new and potent antibiotics. One such class of natural products is the pseudopyronines. Here we present the isolation of pseudopyronine B (2) from a Pseudomonas species found in garden soil in Western North Carolina, and SAR evaluation of C3 and C6 alkyl analogs of the natural product for antibacterial activity against Gram-positive and Gram-negative bacteria. We found a direct relationship between antibacterial activity and C3/C6 alkyl chain length. For inhibition of Gram-positive bacteria, alkyl chain lengths between 6 and 7 carbons were found to be the most active (IC₅₀ = 0.04–3.8 µg/mL) whereas short alkyl chain analogs showed modest activity against Gram-negative bacteria (IC₅₀ = 223–304 µg/mL). This demonstrates the potential for this class of natural products to be optimized for selective activity against either Gram-positive or Gram-negative bacteria.