New pyrimidine-benzoxazole/benzimidazole hybrids: Synthesis, antioxidant, cytotoxic activity, in vitro cyclooxygenase and phospholipase A2-V inhibition.

To enhance the cytotoxicity of benzimidazole and/or benzoxazole core, the benzimidazole/benzoxazole azo-pyrimidine were synthesized through diazo-coupling of 3-aminophenybenzimidazole (6a) or 3-aminophenylbenzoxazole (6b) with diethyl malonate. The new azo-molanates 6a&b mixed with urea in sodium ethoxide to afford the benzimidazolo/benzoxazolopyrimidine 7a&b. The structure elucidation of new synthesized targets was proved using spectroscopic techniques NMR, IR and elemental analysis. The cytoxicity screening had been carried out against five cancer cell lines: prostate cancer (PC-3), lung cancer (A-549), breast cancer (MCF-7), pancreas cancer (PaCa-2) and colon cancer (HT-29). Furthermore, the antioxidant activity, phospholipase A2-V and cyclooxygenases inhibitory activities of the target compounds 7a&b were evaluated and the new compounds showed potent activity (cytotoxicity IC50 range from 4.3 to 9.2 µm, antioxidant activity from 40% to 80%, COXs or LOX inhibitory activity from 1.92 µM to 8.21 µM). The docking of 7a&b was made to confirm the mechanism of action.

Antimyotoxic Activity of Synthetic Peptides Derived from Bothrops atrox Snake Gamma Phospholipase A2 Inhibitor Selected by Virtual Screening.

BACKGROUND: Several studies have aimed to identify molecules that inhibit the toxic actions of snake venom phospholipases A2 (PLA2s). Studies carried out with PLA2 inhibitors (PLIs) have been shown to be efficient in this assignment. OBJECTIVE: This work aimed to analyze the interaction of peptides derived from Bothrops atrox PLIγ (atPLIγ) with a PLA2 and to evaluate the ability of these peptides to reduce phospholipase and myotoxic activities. METHODS: Peptides were subjected to molecular docking with a homologous Lys49 PLA2 from B. atrox venom modeled by homology. Phospholipase activity neutralization assay was performed with BthTX-II and different ratios of the peptides. A catalytically active and an inactive PLA2 were purified from the B. atrox venom and used together in the in vitro myotoxic activity neutralization experiments with the peptides. RESULTS: The peptides interacted with amino acids near the PLA2 hydrophobic channel and the loop that would be bound to calcium in Asp49 PLA2. They were able to reduce phospholipase activity and peptides DFCHNV and ATHEE reached the highest reduction levels, being these two peptides the best that also interacted in the in silico experiments. The peptides reduced the myotubes cell damage with a highlight for the DFCHNV peptide, which reduced by about 65%. It has been suggested that myotoxic activity reduction is related to the sites occupied in the PLA2 structure, which could corroborate the results observed in molecular docking. CONCLUSION: This study should contribute to the investigation of the potential of PLIs to inhibit the toxic effects of PLA2s.

Design and Amberlyst-15 mediated synthesis of novel thienyl-pyrazole carboxamides that potently inhibit Phospholipase A2 by binding to an allosteric site on the enzyme.

Inflammation-mediated disorders are on the rise and hence, there is an urgent need for the design and synthesis of new anti-inflammatory drugs with higher affinity and specificity for their potential targets. The current study presents an effective and new protocol for the synthesis of thienyl-pyrazoles through 3 + 2 annulations using a recyclable heterogeneous catalyst Amberlyst-15. Chalcones 3(a-g) prepared from 3-methylthiophene-2-carbaldehyde and acetophenones by Claisen-Schmidt approach reacted with semicarbazide hydrochloride 4 in the presence of Amberlyst-15 in acetonitrile at room temperature producing thienyl-pyrazole carboxamides 5(a-h) in good yields. Alternatively, the compounds 5(a-h) were prepared by conventional method using acetic acid (30%) medium. Structures of synthesized new pyrazoles were confirmed by spectral and crystallographic studies. All the new compounds were evaluated for their in vitro inhibition of Phospholipase A2 from Vipera russelli and preliminary studies revealed that, amongst the designed series, compounds 5b, 5c and 5h showed promising inhibition. Further, the compounds exhibited linear mixed-type inhibition behavior for the sPLA2 enzyme indicating that they bind to an allosteric site distinct from either the calcium or substrate binding site on the enzyme. These kinetic conclusions were further validated by macromolecular rigid-body docking whereby compounds 5c and 5h showed binding to distinct pockets on the protein. These findings present a promising series of lead molecules that can serve as prototypes for the treatment of inflammatory related disorders.

Regioselectivity of thiouracil alkylation: Application to optimization of Darapladib synthesis.

Darapladib is one of the most potent Lp-PLA2 (Lipoprotein-associated phospholipase A2) inhibitor with an IC50 of 0.25 nM. We demonstrate that a crucial step of Darapladib synthesis was not correctly described in the literature, leading to the production of wrong regioisomers. Moreover we show that the inhibitory activity is directly linked to the position on N1 since compounds bearing alkylation on different sites have potentially less interaction within the active site of Lp-PLA2.

Synthesis of novel 2-pyrazoline analogues with potent anti-inflammatory effect mediated by inhibition of phospholipase A2: Crystallographic, in silico docking and QSAR analysis.

Oxidative-stress induces inflammatory diseases. Further, infections caused by drug-resistant microbial strains are on the rise. This necessitates the discovery of novel small-molecules for intervention therapy. A series of 3-(2,3-dichlorophenyl)-1-(aryl)prop-2-en-1-ones are synthesized as intermediates via Claisen-Schmidt reaction approach. Subsequently, these intermediates were transformed into 2-pyrazolines by their reaction with phenylhydrazine hydrochlorides in methanol and few drops of acetic acid under reflux conditions. Synthesized compounds were characterized by spectroscopic, crystallographic and elemental analyses studies and then, were evaluated for their in vitro antimicrobial and anti-inflammatory activities. Amongst the series, 3-(4-chlorophenyl)-5-(2,3-dichlorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazole (5e), 5-(2,3-dichlorophenyl)-3-(4-fluorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazole (5c) and 5-(2,3-dichlorophenyl)-3-(4-methoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazole (5h) showed significant inhibition of phospholipase A2 with IC50 values of 10.2, 11.1 and 11.9µM, respectively. Protein structure modelling and docking studies indicated that the compounds showed binding to a highly conserved calcium-binding pocket on the enzyme. Further, compounds (5e), 1-(3-chlorophenyl)-5-(2,3-dichlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole (5b), and 1-(3-chlorophenyl)-3-(4-chlorophenyl)-5-(2,3-dichlorophenyl)-4,5-dihydro-1H-pyrazole (5f) showed excellent antimicrobial activities against various bacterial and fungal strains. In conclusion, this study is a successful attempt at the synthesis and characterization of chalcone derivatives that can target phospholipase A2, an enzyme that is a prominent player in the physiological inflammatory cascade. Thus, these compounds show promise for development as next-generation nonsteroidal anti-inflammatory drugs.

Design and environmentally benign synthesis of novel thiophene appended pyrazole analogues as anti-inflammatory and radical scavenging agents: Crystallographic, in silico modeling, docking and SAR characterization.

Oxidative-stress induces inflammatory diseases and infections caused by drug-resistant microbial strains are on the rise necessitating the discovery of novel small-molecules for intervention therapy. The current study presents an effective and new green protocol for the synthesis of thiophene-appended pyrazoles through 3+2 annulations method. Chalcones 3(a-g) were prepared from 5-chloro-2-acetylthiophene and aromatic aldehydes by Claisen-Schmidt approach. The reaction of chalcones 3(a-g) with phenylhydrazine hydrochlorides 4(a-b) in acetic acid (30%) medium and also with freshly prepared citrus extract medium under reflux conditions produced the thiophene appended pyrazoles 5(a-l) in moderate yields. Structures of synthesized new pyrazoles were confirmed by spectral studies, elemental analysis and single crystal X-ray diffraction studies. Further, preliminary assessment of the anti-inflammatory properties of the compounds showed that, amongst the series, compounds 5d, 5e and 5l have excellent anti-inflammatory activities. Further, compounds 5c, 5d, 5g, and 5i exhibited excellent DPPH radical scavenging abilities in comparison with the standard ascorbic acid. Furthermore, using detailed structural modeling and docking efforts, combined with preliminary SAR, we show possible structural and chemical features on both the small-molecules and the protein that might contribute to the binding and inhibition.

2-Oxoamides based on dipeptides as selective calcium-independent phospholipase A2 inhibitors.

Calcium-independent phospholipase A2 (GVIA iPLA2) has recently attracted interest as a medicinal target. The number of known GVIA iPLA2 inhibitors is limited to a handful of synthetic compounds (bromoenol lactone and polyfluoroketones). To expand the chemical diversity, a variety of 2-oxoamides based on dipeptides and ether dipeptides were synthesized and studied for their in vitro inhibitory activity on human GVIA iPLA2 and their selectivity over the other major intracellular GIVA cPLA2 and the secreted GV sPLA2. Structure-activity relationship studies revealed the first 2-oxoamide derivative (GK317), which presents potent inhibition of GVIA iPLA2 (XI(50) value of 0.007) and at the same time significant selectivity over GIVA cPLA2 and GV sPLA2.

Synthesis, Antiphospholipase A2, Antiprotease, Antibacterial Evaluation and Molecular Docking Analysis of Certain Novel Hydrazones.

Some novel hydrazone derivatives 6a-o were synthesized from the key intermediate 4-Chloro-N-(2-hydrazinocarbonyl-phenyl)-benzamide 5 and characterized using IR, ¹H-NMR, 13C-NMR, mass spectroscopy and elemental analysis. The inhibitory potential against two secretory phospholipase A2 (sPLA2), three protease enzymes and eleven bacterial strains were evaluated. The results revealed that all compounds showed preferential inhibition towards hGIIA isoform of sPLA2 rather than DrG-IB with compounds 6l and 6e being the most active. The tested compounds exhibited excellent antiprotease activity against proteinase K and protease from Bacillus sp. with compound 6l being the most active against both enzymes. Furthermore, the maximum zones of inhibition against bacterial growth were exhibited by compounds; 6a, 6m, and 6o against P. aeruginosa; 6a, 6b, 6d, 6f, 6l, 6m, 6n, and 6o against Serratia; 6k against S. mutans; and compounds 6a, 6d, 6e, 6m, and 6n against E. feacalis. The docking simulations of hydrazones 6a-o with GIIA sPLA2, proteinase K and hydrazones 6a-e with glutamine-fructose-6-phosphate transaminase were performed to obtain information regarding the mechanism of action.

Computer-aided drug design guided by hydrogen/deuterium exchange mass spectrometry: A powerful combination for the development of potent and selective inhibitors of Group VIA calcium-independent phospholipase A2.

Potent and selective inhibitors for phospholipases A2 (PLA2) are useful for studying their intracellular functions. PLA2 enzymes liberate arachidonic acid from phospholipids activating eicosanoid pathways that involve cyclooxygenase (COX) and lipoxygenase (LOX) leading to inflammation. Anti-inflammatory drugs target COX and LOX; thus, PLA2 can also be targeted to diminish inflammation at an earlier stage in the process. This paper describes the employment of enzymatic assays, hydrogen/deuterium exchange mass spectrometry (DXMS) and computational chemistry to develop PLA2 inhibitors. Beta-thioether trifluoromethylketones (TFKs) were screened against human GVIA calcium-independent, GIVA cytosolic and GV secreted PLA2s. These compounds exhibited inhibition toward Group VIA calcium-independent PLA2 (GVIA iPLA2), with the most potent and selective inhibitor 3 (OTFP) obtaining an XI(50) of 0.0002 mole fraction (IC50 of 110nM). DXMS binding experiments in the presence of OTFP revealed the peptide regions of GVIA iPLA2 that interact with the inhibitor. Molecular docking and dynamics simulations in the presence of a membrane were guided by the DXMS data in order to identify the binding mode of OTFP. Clustering analysis showed the binding mode of OTFP that occupied 70% of the binding modes occurring during the simulation. The resulted 3D complex was used for docking studies and a structure-activity relationship (SAR) was established. This paper describes a novel multidisciplinary approach in which a 3D complex of GVIA iPLA2 with an inhibitor is reported and validated by experimental data. The SAR showed that the sulfur atom is vital for the potency of beta-thioether analogues, while the hydrophobic chain is important for selectivity. This work constitutes the foundation for further design, synthesis and inhibition studies in order to develop new beta-thioether analogues that are potent and selective for GVIA iPLA2 exclusively.

Structural and Thermodynamic Characterization of Protein-Ligand Interactions Formed between Lipoprotein-Associated Phospholipase A2 and Inhibitors.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) represents a promising therapeutic target for atherosclerosis and Alzheimer's disease. Here we reported the first crystal structures of Lp-PLA2 bound with reversible inhibitors and the thermodynamic characterization of complexes. High rigidity of Lp-PLA2 structure and similar binding modes of inhibitors with completely different scaffolds are revealed. It not only provides the molecular basis for inhibitory activity but also sheds light on the essential features of Lp-PLA2 recognition with reversible inhibitors.

Development of a potent 2-oxoamide inhibitor of secreted phospholipase A2 guided by molecular docking calculations and molecular dynamics simulations.

Inhibition of group IIA secreted phospholipase A2 (GIIA sPLA2) has been an important objective for medicinal chemists. We have previously shown that inhibitors incorporating the 2-oxoamide functionality may inhibit human and mouse GIIA sPLA2s. Herein, the development of new potent inhibitors by molecular docking calculations using the structure of the known inhibitor 7 as scaffold, are described. Synthesis and biological evaluation of the new compounds revealed that the long chain 2-oxoamide based on (S)-valine GK241 led to improved activity (IC50=143 nM and 68 nM against human and mouse GIIA sPLA2, respectively). In addition, molecular dynamics simulations were employed to shed light on GK241 potent and selective inhibitory activity.

Discovery of Potent and Orally Active Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Inhibitors as a Potential Therapy for Diabetic Macular Edema.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is considered to be a promising therapeutic target for several inflammation-associated diseases. Herein, we describe the discovery of a series of pyrimidone derivatives as Lp-PLA2 inhibitors. Systematic structural modifications led to the identification of several pyrimidone compounds with promising in vitro inhibitory potency and pharmacokinetic properties. Compound 14c, selected for in vivo evaluation, demonstrated decent pharmacokinetic profiles and robust inhibitory potency against Lp-PLA2 in Sprague-Dawley (SD) rats. Furthermore, 14c significantly inhibited retinal thickening in STZ-induced diabetic SD rats as a model of diabetic macular edema (DME) after oral dosing for 4 weeks. Taken together, these results suggested that 14c can serve as a valuable lead in the search for new Lp-PLA2 inhibitors for prevention and/or treatment of DME.

A One Pot Synthesis of Novel Bioactive Tri-Substitute-Condensed-Imidazopyridines that Targets Snake Venom Phospholipase A2.

Drugs such as necopidem, saripidem, alpidem, zolpidem, and olprinone contain nitrogen-containing bicyclic, condensed-imidazo[1,2-α]pyridines as bioactive scaffolds. In this work, we report a high-yield one pot synthesis of 1-(2-methyl-8-aryl-substitued-imidazo[1,2-α]pyridin-3-yl)ethan-1-onefor the first-time. Subsequently, we performed in silico mode-of-action analysis and predicted that the synthesized imidazopyridines targets Phospholipase A2 (PLA2). In vitro analysis confirmed the predicted target PLA2 for the novel imidazopyridine derivative1-(2-Methyl-8-naphthalen-1-yl-imidazo [1,2-α]pyridine-3-yl)-ethanone (compound 3f) showing significant inhibitory activity towards snake venom PLA2 with an IC50 value of 14.3 µM. Evidently, the molecular docking analysis suggested that imidazopyridine compound was able to bind to the active site of the PLA2 with strong affinity, whose affinity values are comparable to nimesulide. Furthermore, we estimated the potential for oral bioavailability by Lipinski's Rule of Five. Hence, it is concluded that the compound 3f could be a lead molecule against snake venom PLA2.

Regioselective π-extension of indoles with rhodium enalcarbenoids--synthesis of substituted carbazoles.

An efficient Rh(II) carboxylate and Brønsted acid catalyzed direct π-extension of indoles to 4-substituted carbazoles is developed. The reaction involves a regioselective C-3 functionalization of indole by a rhodium enalcarbenoid and a Brønsted acid assisted cyclocondensation. In addition a twofold regioselective π-extension of pyrroles to 4,8-disubstituted carbazoles has also been developed. The utility of the methodology was demonstrated with the synthesis of analogues of an hepatitis C virus replication inhibitor and a secreted phospholipase A2 (sPLA2) inhibitor.

Studies of synthetic chalcone derivatives as potential inhibitors of secretory phospholipase A2, cyclooxygenases, lipoxygenase and pro-inflammatory cytokines.

Arachidonic acid metabolism leads to the generation of key lipid mediators which play a fundamental role during inflammation. The inhibition of enzymes involved in arachidonic acid metabolism has been considered as a synergistic anti-inflammatory effect with enhanced spectrum of activity. A series of 1,3-diphenyl-2-propen-1-one derivatives were investigated for anti-inflammatory related activities involving inhibition of secretory phospholipase A2, cyclooxygenases, soybean lipoxygenase, and lipopolysaccharides-induced secretion of interleukin-6 and tumor necrosis factor-alpha in mouse RAW264.7 macrophages. The results from the above mentioned assays exhibited that the synthesized compounds were effective inhibitors of pro-inflammatory enzymes and cytokines. The results also revealed that the chalcone derivatives with 4-methlyamino ethanol substitution seem to be significant for inhibition of enzymes and cytokines. Molecular docking experiments were carried out to elucidate the molecular aspects of the observed inhibitory activities of the investigated compounds. Present findings increase the possibility that these chalcone derivatives might serve as a beneficial starting point for the design and development of improved anti-inflammatory agents.

Pharmacological evaluation and docking studies of α,ß-unsaturated carbonyl based synthetic compounds as inhibitors of secretory phospholipase A2, cyclooxygenases, lipoxygenase and proinflammatory cytokines.

Arachidonic acid and its metabolites have generated high level of interest among researchers due to their vital role in inflammation. The inhibition of enzymes involved in arachidonic acid metabolism has been considered as synergistic anti-inflammatory effect. A series of novel α,ß-unsaturated carbonyl based compounds were synthesized and evaluated for their inhibitory activity on secretory phospholipase A2 (sPLA2), cyclooxygenases (COX), soybean lipoxygenase (LOX) in addition to proinflammatory cytokines comprising IL-6 and TNF-α. Six α,ß-unsaturated carbonyl based compounds (2, 3, 4, 12, 13 and 14) exhibited strong inhibition of sPLA2 activity, with IC50 values in the range of 2.19-8.76 µM. Nine compounds 1-4 and 10-14 displayed inhibition of COX-1 with IC50 values ranging from 0.37 to 1.77 µM (lower than that of reference compound), whereas compounds 2, 10, 13 and 14 strongly inhibited the COX-2. The compounds 10-14 exhibited strong inhibitory activity against LOX enzyme. All compounds were evaluated for the inhibitory activities against LPS-induced TNF-α and IL-6 release in the macrophages. On the basis of screening results, five active compounds 3, 4, 12, 13 and 14 were found strong inhibitors of TNF-α and IL-6 release in a dose-dependent manner. Molecular docking experiments were performed to clarify the molecular aspects of the observed COX and LOX inhibitory activities of the investigated compounds. Present findings increases the possibility that these α,ß-unsaturated carbonyl based compounds might serve as beneficial starting point for the design and development of improved anti-inflammatory agents.

X-ray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases.

Structure-guided design was used to generate a series of noncovalent inhibitors with nanomolar potency against the papain-like protease (PLpro) from the SARS coronavirus (CoV). A number of inhibitors exhibit antiviral activity against SARS-CoV infected Vero E6 cells and broadened specificity toward the homologous PLP2 enzyme from the human coronavirus NL63. Selectivity and cytotoxicity studies established a more than 100-fold preference for the coronaviral enzyme over homologous human deubiquitinating enzymes (DUBs), and no significant cytotoxicity in Vero E6 and HEK293 cell lines is observed. X-ray structural analyses of inhibitor-bound crystal structures revealed subtle differences between binding modes of the initial benzodioxolane lead (15g) and the most potent analogues 3k and 3j, featuring a monofluoro substitution at para and meta positions of the benzyl ring, respectively. Finally, the less lipophilic bis(amide) 3e and methoxypyridine 5c exhibit significantly improved metabolic stability and are viable candidates for advancing to in vivo studies.

New potent and selective polyfluoroalkyl ketone inhibitors of GVIA calcium-independent phospholipase A2.

Group VIA calcium-independent phospholipase A2 (GVIA iPLA2) has recently emerged as an important pharmaceutical target. Selective and potent GVIA iPLA2 inhibitors can be used to study its role in various neurological disorders. In the current work, we explore the significance of the introduction of a substituent in previously reported potent GVIA iPLA2 inhibitors. 1,1,1,2,2-Pentafluoro-7-(4-methoxyphenyl)heptan-3-one (GK187) is the most potent and selective GVIA iPLA2 inhibitor ever reported with a XI(50) value of 0.0001, and with no significant inhibition against GIVA cPLA2 or GV sPLA2. We also compare the inhibition of two difluoromethyl ketones on GVIA iPLA2, GIVA cPLA2, and GV sPLA2.

Design and synthesis of imidazole and triazole derivatives as Lp-PLA2 inhibitors and the unexpected discovery of highly potent quaternary ammonium salts.

New Lp-PLA(2) inhibitors were synthesized by the bioisosteric replacement of the amide group of Darapladib with an imidazole or a triazole. Unfortunately, the inhibitory activities of these derivatives were lower than that of Darapladib. But interestingly, a series of quaternary ammonium salts that were isolated as by-products during this synthetic work were found with high potency. Of these by-products, compound 22c showed a similar profile to Darapladib both in vitro and in vivo.