ATP-competitive DNA gyrase and topoisomerase IV inhibitors as antibacterial agents

Introduction: The bacterial topoisomerases DNA gyrase and topoisomerase IV are validated targets for development of novel antibacterial agents. Fluoroquinolones inhibit the catalytic GyrA and/or ParC(GrlA) subunit and have been commonly used, although these have toxicity liabilities that restrict their use. The ATPase GyrB and ParE(GrlB) subunits have been much less explored and after withdrawal of novobiocin, there are no further marketed inhibitors . ATP-competitive inhibitors of GyrB and/or ParE(GrlB) are of special interest, as this target has been validated, and it is expected that many of the problems associated with fluoroquinolones can be avoided.

Areas covered: This review summarises the development of ATP-competitive inhibitors of GyrB and/or ParE(GrlB) as novel antibacterial agents over the last 10 years. Structural features of the new inhibitors and their optimisation strategies are highlighted.

Expert opinion: The development of novel ATP-competitive inhibitors of GyrB and/or ParE(GrlB) is ongoing in industrial and academical research. Development of resistance is one of the most problematic issues, but GyrB/ParE(GrlB) inhibitors do not show cross-resistance with fluoroquinolones. Other common issues, such as low solubility, high protein binding, development of off-target resistance, are related to the structures of the inhibitors themselves, which is thus a main focus of design strategies. With some now in early clinical development, there is reasonable expectation that novel ATP-competitive inhibitors of GyrB/ParE(GrlB) will reach the market in the near future.     

Synthesis and antibacterial activity of a novel series of potent DNA gyrase inhibitors. Pyrazole derivatives

We have previously found that a pyrazole derivative 1 possesses antibacterial activity and inhibitory activity against DNA gyrase and topoisomerase IV. Here, we synthesized new pyrazole derivatives and found that 5-[(E)-2-(5-chloroindol-3-yl)vinyl]pyrazole 16 possesses potent antibacterial activity and selective inhibitory activity against bacterial topoisomerases. Many of the synthesized pyrazole derivatives were potent against clinically isolated quinolone- or coumarin-resistant Gram-positive strains and had minimal inhibitory concentration values against these strains equivalent to those against susceptible strains.     

Quercetin diacylglycoside analogues showing dual inhibition of DNA gyrase and topoisomerase IV as novel antibacterial agents

A structure-guided molecular design approach was used to optimize quercetin diacylglycoside analogues that inhibit bacterial DNA gyrase and topoisomerase IV and show potent antibacterial activity against a wide spectrum of relevant pathogens responsible for hospital- and community-acquired infections. In this paper, such novel 3,7-diacylquercetin, quercetin 6'-acylgalactoside, and quercetin 2',6'-diacylgalactoside analogues of lead compound 1 were prepared to assess their target specificities and preferences in bacteria. The significant enzymatic inhibition of both Escherichia coli DNA gyrase and Staphylococcus aureus topoIV suggest that these compounds are dual inhibitors. Most of the investigated compounds exhibited pronounced inhibition with MIC values ranging from 0.13 to 128 μg/mL toward the growth of multidrug-resistant Gram-positive methicillin-resistant S. aureus, methicillin sensitive S. aureus, vancomycin-resistant enterococci (VRE), vancomycin intermediate S. aureus, and Streptococcus pneumoniae bacterial strains. Structure-activity relationship studies revealed that the acyl moiety was absolutely essential for activity against Gram-positive organisms. The most active compound 5i was 512-fold more potent than vancomycin and 16-32-fold more potent than 1 against VRE strains. It also has realistic in situ intestinal absorption in rats and showed very low acute toxicity in mice. So far, this compound can be regarded as a leading antibacterial agent.     

Increased antibacterial activity of DW286, a novel fluoronaphthyridone antibiotic, against Staphylococcus aureus strains with defined mutations in DNA gyrase and topoisomerase IV

To investigate the activity of DW286, a new fluoronaphthyridone, the quinolone resistance determining regions (QRDRs) of gyrA, gyrB, grlA and grlB genes in 64 Staphylococcus aureus clinical isolates were analyzed and the MICs of DW286 and comparator quinolones determined. Double and triple mutants in gyrA and grlA were resistant to ciprofloxacin, sparfloxacin, trovafloxacin and gemifloxacin but susceptible to DW286 (MIC 0.25-0.5 mg/l). The fourth alteration, Ser85Pro of GyrA was required to make a strain resistant to DW286 (MIC 4-32 mg/l). For a strain with the mutations at GyrA Ser84Leu and GrlA Ser80Phe, the MBC of DW286 was two-fold higher than its corresponding MIC, in contrast to ciprofloxacin which was not bactericidal.     

Novel N-linked aminopiperidine inhibitors of bacterial topoisomerase type II: broad-spectrum antibacterial agents with reduced hERG activity

Novel non-fluoroquinolone inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. Aminopiperidines that have a bicyclic aromatic moiety linked through a carbon to an ethyl bridge, such as 1, generally show potent broad-spectrum antibacterial activity, including quinolone-resistant isolates, but suffer from potent hERG inhibition (IC(50)= 3 μM for 1). We now disclose the finding that new analogues of 1 with an N-linked cyclic amide moiety attached to the ethyl bridge, such as 24m, retain the broad-spectrum antibacterial activity of 1 but show significantly less hERG inhibition (IC(50)= 31 μM for 24m) and higher free fraction than 1. One optimized analogue, compound 24l, showed moderate clearance in the dog and promising efficacy against Staphylococcus aureus in a mouse thigh infection model.     

Azaindenoisoquinolines as topoisomerase I inhibitors and potential anticancer agents: a systematic study of structure-activity relationships

A comprehensive study of a series of azaindenoisoquinoline topoisomerase I (Top1) inhibitors is reported. The synthetic pathways have been developed to prepare 7-, 8-, 9-, and 10-azaindenoisoquinolines. The present study shows that 7-azaindenoisoquinolines possess the greatest Top1 inhibitory activity and cytotoxicity. Additionally, the introduction of a methoxy group into the D-ring of 7-azaindenoisoquinolines improved their biological activities, leading to new lead molecules for further development. A series of QM calculations were performed on the model "sandwich" complexes of azaindenoisoquinolines with flanking DNA base pairs from the Drug-Top1-DNA ternary complex. The results of these calculations demonstrate how changes in two forces contributing to the π-π stacking (dispersion and charge-transfer interactions) affect the binding of the drug to the Top1-DNA cleavage complex and thus modulate the drug's Top1 inhibitory activity.     

Discovery and structure-activity relationships of piperidinone- and piperidine-constrained phenethylamines as novel, potent, and selective dipeptidyl peptidase IV inhibitors

Dipeptidyl peptidase IV (DPP4) inhibitors are emerging as a new class of therapeutic agents for the treatment of type 2 diabetes. They exert their beneficial effects by increasing the levels of active glucagon-like peptide-1 and glucose-dependent insulinotropic peptide, which are two important incretins for glucose homeostasis. Starting from a high-throughput screening hit, we were able to identify a series of piperidinone- and piperidine-constrained phenethylamines as novel DPP4 inhibitors. Optimized compounds are potent, selective, and have good pharmacokinetic profiles.     

Syntheses, resolution, and structure-activity relationships of potent acetylcholinesterase inhibitors: 8-carbaphysostigmine analogues.

The synthesis of a series of 1,2,3,3a,8,8a-hexahydroindeno[2,1-b]pyrrole 5-alkylcarbamates and their resolution are reported. These compounds are structurally related to physostigmine with substitution of a methylene group in place of the NMe group at position 8 of physostigmine. Many of these 8-carbaphysostigmine analogues are more potent acetylcholinesterase inhibitors in vitro and less toxic in vivo than physostigmine. The (-)-enantiomer (e.g., 1d and 1g) possessing the same absolute configuration at C3a and C8a as that of physostigmine, is about 6 to 12-fold more potent at inhibiting acetylcholinesterase than the corresponding (+)-enantiomer (e.g., 1e and 1h).     

Novel potent orally active multitargeted receptor tyrosine kinase inhibitors: synthesis, structure-activity relationships, and antitumor activities of 2-indolinone derivatives

The inhibition of receptor tyrosine kinases (RTKs) has become a successful approach in the development of anticancer agents. Many potent small-molecule kinase inhibitors have been discovered. We report herein a series of pyrrolo-fused-heterocycle-2-indolinone analogues as inhibitors of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and c-Kit. Among them, some pyrrolo-fused six- and seven-membered-heterocycle derivatives such as 9, 15, 23, and 25 are potent inhibitors of VEGFR, PDGFR, and c-Kit both enzymatically (<50 nM) and cellularly (<50 nM). Furthermore, compounds 9 and 25 possess favorable pharmacokinetic profiles and demonstrate good efficacies against human HT-29 cell colon tumor xenografts in nude mice. Further evaluations are in progress.     

Investigation of 5-nitrofuran derivatives: synthesis, antibacterial activity, and quantitative structure-activity relationships

Three sets of antibacterial nitrofuran derivatives [set I, 5-R-substituted (Z)-2-(5-nitrofuran-2-ylmethylene)-3(2H)-benzofuranones (R = OCH(3), H, CH(3), C(2)H(5), nC(3)H(7), Cl, Br, CN, and NO(2)) and their 2-hydroxyphenyl and 2-acetoxyphenyl analogues; set II, 5-R-substituted (E)-1-(2-hydroxyphenyl)-3-(5-nitrofuryl)-2-propen-1-ones (R = H, CH(3), C(2)H(5), Cl, and NO(2)); and set III, 5-R-substituted (E)-1-(2-acetoxyphenyl)-3-(5-nitrofuryl)-2-propen-1-ones (R = H, CH(3); C(2)H(5), Cl, and NO(2))] were prepared and tested against a Gram-positive (Staphylococcus aureus, strain ATCC-25923) and a Gram-negative bacterium (Caulobacter crescentus, strain NA 1000). QSAR equations derived for the IC(50) values against both bacteria show negative contributions of two terms: an electronic one, expressed either by sigma, the Hammett substituent constant, or by E, the cyclic voltametric reduction potential. Another term described by an indicator variable, I(abs), is assigned the value of 0 for set I compounds and the value of 1 for sets II and III. No important contribution of the hydrophobic factor was found. For the three sets, the QSAR regressions suggest that the same structural features describe the activities for both bacteria and that, although reduction is a necessary step, it should not be the determining one. These results agree with those found for the QSAR of 5-nitroimidazole analogues.     

Development and radiosterilization of new hydrazono-quinoline hybrids as DNA gyrase and topoisomerase IV inhibitors: Antimicrobial and hemolytic activities against uropathogenic isolates with molecular docking study

This study aimed to synthesize new potent quinoline derivatives based on hydrazone moieties and evaluate their antimicrobial activity. The newly synthesized hydrazono-quinoline derivatives 2 , 5a , 9 , and 10b showed the highest antimicrobial activity with MIC values ≤1.0 μg/ml against bacteria and ≤8.0 μg/ml against the fungi. Further, these derivatives exhibited bactericidal and fungicidal effects with MBC/MIC and MFC/MIC ratio ≤4. Surprisingly, the most active compounds displayed good inhibition to biofilm formation with MBEC values ranging between (40.0 ± 10.0 – 230.0 ± 31.0) and (67.0 ± 24.0 – 347.0 ± 15.0) μg/ml against Staphylococcus aureus and Pseudomonas aeruginosa, respectively. The hemolytic assays confirmed that the hydrazono-quinoline derivatives are non-toxic with low % lysis values ranging from 4.62% to 14.4% at a 1.0 mg/ml concentration. Besides, compound 5a exhibited the lowest hemolytic activity value of ~4.62%. Furthermore, the study suggests that the hydrazono-quinoline analogs exert their antibacterial activity as dual inhibitors for DNA gyrase and DNA topoisomerase IV enzymes with IC₅₀ values ranging between (4.56 ± 0.3 – 21.67 ± 0.45) and (6.77 ± 0.4 – 20.41 ± 0.32) μM, respectively. Additionally, the recent work advocated that compound 5a showed the reference SAL at the ɣ-radiation dose of 10.0 kGy in the sterilization process without affecting its chemical structure. Finally, the in silico drug-likeness, toxicity properties, and molecular docking simulation were performed. Besides, the result exhibited good oral-bioavailability, lower toxicity prediction, and lower binding energy with good binding mode rather than the positive control.     

Synthesis and structure-activity relationships of ring-opened 17-hydroxywortmannins: potent phosphoinositide 3-kinase inhibitors with improved properties and anticancer efficacy

The phosphoinositide 3-kinase (PI3K) signaling pathway is frequently up-regulated in human cancer and is a promising target for the treatment of cancer. Wortmannin and its analogues are potent inhibitors of PI3K but suffer from inherent defects such as instability, insolubility, and toxicity. Opening of the reactive furan ring of 17-hydroxywortmannin with amines gives compounds with improved properties such as greater stability and aqueous solubility and a larger therapeutic index. Ring-opened analogues such as compound 13 containing basic amine groups have significantly increased PI3K inhibitory potency and greater efficacy in nude mouse xenograft assays.     

SM-216601, a novel parenteral 1beta-methylcarbapenem: structure-activity relationships of antibacterial activity and neurotoxicity in mice

It has been reported that 2-(4-substituted thiazol-2-ylthio)-1beta-methyl-carbapenems exhibit potent activity against methicillin-resistant staphylococci (MRS) and vancomycin-resistant enterococci (VRE). In order to develop a novel broad-spectrum carbapenem, the structure-activity relationships of a series of 2-(4-tetrahydropyridinylthiazol-2-ylthio)-1beta-methylcarbapenems and 4-dihydropyrrolyl thiazole analogs were investigated with regard to their activity against Gram-positive and especially Gram-negative bacteria and also their convulsant activity, which is a major side effect concern of carbapenems. The introduction of substituent(s) on the dihydropyrrole moiety did not cause remarkable changes in anti-MRS and VRE activities, but tended to lower the anti-Gram-negative bacterial activity except in some cases of methyl group introduction. These substitutions did however cause a reduction of the convulsant activity, which was affected by the size and also the configuration of the substituent. In the case of SM-216601 (6), introduction of a methyl group brought about significant reduction in neurotoxicity while maintaining favorable anti-Gram-negative bacterial activity.     

Structure-guided design of aminopyrimidine amides as potent, selective inhibitors of lymphocyte specific kinase: synthesis, structure-activity relationships, and inhibition of in vivo T cell activation

The lymphocyte-specific kinase (Lck), a member of the Src family of cytoplasmic tyrosine kinases, is expressed in T cells and natural killer (NK) cells. Genetic evidence, including knockout mice and human mutations, demonstrates that Lck kinase activity is critical for normal T cell development, activation, and signaling. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. With the aid of X-ray structure-based analysis, aminopyrimidine amides 2 and 3 were designed from aminoquinazolines 1, which had previously been demonstrated to exhibit potent inhibition of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminopyrimidine amides 3 possessing improved cellular potency and selectivity profiles relative to their aminoquinazoline predecessors 1. Orally bioavailable compound 13b inhibited the anti-CD3-induced production of interleukin-2 (IL-2) in mice in a dose-dependent manner (ED 50 = 9.4 mg/kg).     

New structure-activity relationships of A- and D-ring modified steroidal aromatase inhibitors: design, synthesis, and biochemical evaluation

A- and D-ring androstenedione derivatives were synthesized and tested for their abilities to inhibit aromatase. In one series, C-3 hydroxyl derivatives were studied leading to a very active compound, when the C-3 hydroxyl group assumes 3β stereochemistry (1, IC(50) = 0.18 μM). In a second series, the influence of double bonds or epoxide functions in different positions along the A-ring was studied. Among epoxides, the 3,4-epoxide 15 showed the best activity (IC(50) = 0.145 μM) revealing the possibility of the 3,4-oxiran oxygen resembling the C-3 carbonyl group of androstenedione. Among olefins, the 4,5-olefin 12 (IC(50) = 0.135 μM) revealed the best activity, pointing out the importance of planarity in the A,B-ring junction near C-5. C-4 acetoxy and acetylsalicyloxy derivatives were also studied showing that bulky substituents in C-4 diminish the activity. In addition, IFD simulations helped to explain the recognition of the C-3 hydroxyl derivatives (1 and 2) as well as 15 within the enzyme.     

Identification of potent and selective small-molecule inhibitors of caspase-3 through the use of extended tethering and structure-based drug design

The design, synthesis, and in vitro activities of a series of potent and selective small-molecule inhibitors of caspase-3 are described. From extended tethering, a salicylic acid fragment was identified as having binding affinity for the S(4) pocket of caspase-3. X-ray crystallography and molecular modeling of the initial tethering hit resulted in the synthesis of 4, which reversibly inhibited caspase-3 with a K(i) = 40 nM. Further optimization led to the identification of a series of potent and selective inhibitors with K(i) values in the 20-50 nM range. One of the most potent compounds in this series, 66b, inhibited caspase-3 with a K(i) = 20 nM and selectivity of 8-500-fold for caspase-3 vs a panel of seven caspases (1, 2, and 4-8). A high-resolution X-ray cocrystal structure of 4 and 66b supports the predicted binding modes of our compounds with caspase-3.     

Novel tricyclic poly(ADP-ribose) polymerase-1 inhibitors with potent anticancer chemopotentiating activity: design,synthesis, and X-ray cocrystal structure

A series of novel compounds have been designed that are potent inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1), and the activity and physical properties have been characterized. The new structural classes, 3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indol-6-ones and 3,4-dihydropyrrolo[4,3,2-de]isoquinolin-5-(1H)-ones, have conformationally locked benzamide cores that specifically interact with the PARP-1 protein. The compounds have been evaluated with in vitro cellular assays that measure the ability of the PARP-1 inhibitors to enhance the effect of cytotoxic agents against cancer cell lines.     

Nonpeptidic chymase inhibitors: design and structure-activity relationships of pyrimidinone derivatives based on the predicted binding mode of a peptidic inhibitor

While the biological reaction of chymase have been often studied for ten years, the pathophysiological role of chymase has not been fully elucidate due to a lack of effective inhibitors featuring potent inhibitory activity, specificity, and metabolic stability. Recently the discovery of a structurally varied range of novel nonpeptidic inhibitors presents new opportunities to explore the role of chymase under both physiological and pathophysiological conditions and to develop therapeutic agents for chymase-induced diseases. In this article the structure and the inhibitory mechanism of nonpeptidic chymase inhibitors are discussed, with special emphasis on design and structure-activity relationships of pyrimidinone derivative where inhibitory activity, protease selectivity, and pharmacokinetic profile are clarified.     

Novel potent orally active selective VEGFR-2 tyrosine kinase inhibitors: synthesis, structure-activity relationships, and antitumor activities of N-phenyl-N'-{4-(4-quinolyloxy)phenyl}ureas

N-Phenyl-N'-{4-(4-quinolyloxy)phenyl}ureas were found to be a novel class of potent inhibitors for the vascular endothelial growth factor receptor 2 (VEGFR-2) tyrosine kinase through synthetic modifications of a lead compound and structure-activity relationship studies. A representative compound 6ab, termed Ki8751, inhibited VEGFR-2 phosphorylation at an IC(50) value of 0.90 nM, and also inhibited the PDGFR family members such as PDGFRalpha and c-Kit at 67 nM and 40 nM, respectively. However, 6ab did not have any inhibitory activity against other kinases such as EGFR, HGFR, InsulinR and others even at 10000 nM. 6ab suppressed the growth of the VEGF-stimulated human umbilical vein endothelial cell (HUVEC) on a nanomolar level. 6ab showed significant antitumor activity against five human tumor xenografts such as GL07 (glioma), St-4 (stomach carcinoma), LC6 (lung carcinoma), DLD-1 (colon carcinoma) and A375 (melanoma) in nude mice and also showed complete tumor growth inhibition with the LC-6 xenograft in nude rats following oral administration once a day for 14 days at 5 mg/kg without any body weight loss.