Arylureidoaurones: Synthesis, in vitro α-glucosidase, and α-amylase inhibition activity.

Because of the colossal global burden of diabetes, there is an urgent need for more effective and safer drugs. We designed and synthesized a new series of aurone derivatives possessing phenylureido or bis-phenylureido moieties as α-glucosidase and α-amylase inhibitors. Most of the synthesized phenylureidoaurones have demonstrated superior inhibition activities (IC50s of 9.6-339.9 µM) against α-glucosidase relative to acarbose (IC50 = 750.0 µM) as the reference drug. Substitution of aurone analogues with two phenylureido substituents at the 5-position of the benzofuranone moiety and the 3' or 4' positions of the 2-phenyl ring resulted in compounds with almost 120-180 times more potent inhibitory activities than acarbose. The aurone analogue possessing two phenylureido substitutions at 5 and 4' positions (13) showed the highest inhibition activity with an IC50 of 4.2 ± 0.1 µM. Kinetic studies suggested their inhibition mode to be competitive. We also investigated the binding mode of the most potent compounds using the consensually docked 4D-QSAR methodology. Furthermore, these analogues showed weak-to-moderate non-competitive inhibitory activity against α-amylase. 5-Methyl substituted aurone with 4'-phenylureido moiety (6e) demonstrated the highest inhibition activity on α-amylase with an IC50 of 142.0 ± 1.6 µM relative to acarbose (IC50 = 108 ± 1.2 µM). Our computational studies suggested that these analogues interact with a hydrophilic allosteric site in α-amylase, located far from the enzyme active site at the N-terminal.

Antileishmanial Activities of (Z)-2-(Nitroimidazolylmethylene)-3(2H)-Benzofuranones: Synthesis, In Vitro Assessment, and Bioactivation by NTR 1 and 2.

The antileishmanial activity of a series of (Z)-2-(heteroarylmethylene)-3(2H)-benzofuranone derivatives, possessing 5-nitroimidazole or 4-nitroimidazole moieties, was investigated against Leishmania major promastigotes and some analogues exhibited prominent activities. Compounds with IC50 values lower than 20 µM were further examined against L. donovani axenic amastigotes. Evaluated analogues in 5-nitroimidazole subgroup demonstrated significantly superior activity (~17-88-folds) against L. donovani in comparison to L. major. (Z)-7-Methoxy-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5n) showed the highest L. donovani anti-axenic amastigote activity with IC50 of 0.016 µM. The cytotoxicity of these analogues was determined using PMM peritoneal mouse macrophage and THP-1 human leukemia monocytic cell lines and high selectivity indices of 26 to 431 were obtained for their anti-axenic amastigote effect over the cytotoxicity on PMM cells. Further studies on their mode of action showed that 5-nitroimidazole compounds were bioactivated predominantly by nitroreductase 1 (NTR1) and 4-nitroimidazole analogues by both NTR1 and 2. It is likely that this bioactivation results in the production of nitroso and hydroxylamine metabolites that are cytotoxic for the Leishmania parasite.

Phenylalanine and indole effects on the pathogenicity of human lysozyme amorphous aggregates.

Protein aggregation can affect the stability and function of proteins, and may lead to developing diseases, but reports on the in vivo effect of aggregates are scarce. In the current study, the effect of phenylalanine (Phe) and indole presence was first investigated on the structure and stability of human lysozyme (HLZ) and its aggregation under in vitro condition. Tm measurements, circular dichroism and spectrofluorimetric spectra, as well as and transmission electron microscopy (TEM) were performed in this stage. In the next step, pathogenicity of HLZ amorphous aggregates formed in presence or absence of the additives was investigated in vivo, by subcutaneous injection to adult male Wistar rats. Resulting inflamed tissues were studied by hematoxylin and eosin (HE), Congo red and Sudan black staining. Serum levels of liver enzymes (Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST)), specific inflammatory cytokines (Tumor Necrosis Factor Alpha (TNF-α) and Interleukin 6 (IL-6)) as well as glucose, cholesterol, and triglyceride levels were measured. Amorphous aggregates of HLZ caused inflammation and affected the number of fat cells, macrophages, cytokines, liver enzymes and glucose. Indole, that increases amorphous aggregates amount as shown with CD, fluorescence, and TEM experiments, leads into more severe inflammation. In presence of Phe, (which stabilizes HLZ structure) a markedly milder inflammatory state is observed in histological results and no increase could be detected in the inflammation-related parameters. In conclusion, amorphous aggregates of HLZ may be pathogenic in vivo, and presence of anti-aggregation compounds (such as Phe) can be effective in diminishing their deleterious manifestations.

Antimalarial Activities of (Z)-2-(Nitroheteroarylmethylene)-3(2H)-Benzofuranone Derivatives: In Vitro and In Vivo Assessment and ß-Hematin Formation Inhibition Activity.

A series of (Z)-2-(nitroheteroarylmethylene)-3(2H)-benzofuranones possessing nitroheteroaryl groups of nitroimidazole, nitrofuran, and nitrothiophene moieties was screened for antiplasmodium activity against a drug-sensitive strain (3D7 strain) and a multidrug-resistant (chloroquine [CQ] and pyrimethamine) strain (K1 strain) of Plasmodium falciparum. 5-Nitroimidazole and 4-nitroimidazole analogs were highly selective and active against resistant parasites, while 5-nitrofuran and 5-nitrothiophene derivatives were more potent against the 3D7 strain than against the K1 strain. Among the synthetic analogues, (Z)-6-chloro-2-(1-methyl-5-nitroimidazol-2-ylmethylene)-3(2H)-benzofuranone (compound 5h) exhibited the highest activity (50% inhibitory concentration [IC50], 0.654 nM) against the K1 strain and (Z)-7-methoxy-2-(5-nitrothiophen-2-ylmethylene)-3(2H)-benzofuranone (10g) showed the highest activity (IC50, 0.28 µM) against the 3D7 strain in comparison with the activities of CQ (IC50s of 3.13 and 206.3 nM against 3D7 and K1 strains, respectively). The more active compounds, with IC50s lower than 5 µg/ml (∼20 µM), were further studied for their cytotoxicity responses using KB cells. From these studies, 5-nitroimidazole, 4-nitroimidazole, and 5-nitrofuran analogues were shown to be cytotoxic against KB cells, while 5-nitrothiophene analogues were shown to have the least cytotoxic effects. To gain some insight into their potential contributing mechanisms of action, three derivatives, 10e, 10g, and 10h (from the nitrothiophene subgroup, possessing 6-methoxy, 7-methoxy, and 6,7-dimethoxy substituents, respectively, on their benzofuranone moieties), showing the least toxicity and highest selectivity indices were assessed for their ß-hematin formation inhibition activity. Compound 10g demonstrated the highest inhibition activity (IC50, 10.78 µM) in comparison with that of CQ (IC50, 2.63 µM) as the reference drug. Finally, these three analogues (10e, 10g, and 10h) were further evaluated for their in vivo activities against the Plasmodium berghei/albino mouse model (Peter's test). The tested analogues were shown to be active, reducing the percentages of erythrocytes that contained parasites by 53.4, 48.8, and 32.4%, respectively.

5-[Aryloxypyridyl (or nitrophenyl)]-4H-1,2,4-triazoles as novel flexible benzodiazepine analogues: Synthesis, receptor binding affinity and lipophilicity-dependent anti-seizure onset of action.

A new series of 5-(2-aryloxy-4-nitrophenyl)-4H-1,2,4-triazoles and 5-(2-aryloxy-3-pyridyl)-4H-1,2,4-triazoles, possessing C-3 thio or alkylthio substituents, was synthesized and evaluated for their benzodiazepine receptor affinity and anti-seizure activity. These analogues revealed similar to significantly superior affinity to GABAA/benzodiazepine receptor complex (IC50 values of 0.04-4.1 nM), relative to diazepam as the reference drug (IC50 value of 2.4 nM). To determine the onset of anti-seizure activity, the time-dependent effectiveness of i.p. administration of compounds on pentylenetetrazole induced seizure threshold was studied and a very good relationship was observed between the lipophilicity (cLogP) and onset of action of studied analogues (r2 = 0.964). The minimum effective dose of the compounds, determined at the time the analogues showed their highest activity, was demonstrated to be 0.025-0.1 mg/kg, relative to diazepam (0.025 mg/kg).

Bioassay-guided fractionation and identification of wound healing active compound from Pistacia vera L. hull extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Pistachio hull has traditionally been used to treat peptic ulcer, hemorrhoids, oral and cutaneous wounds. AIM OF THE STUDY: On the basis of its traditional uses and previous pharmacological reports, a bioassay guided fractionation procedures on pistachio (Pistacia vera L.) hulls was performed to define the fractions and bioactive compound that are responsible for wound healing activity of hulls. MATERIAL AND METHODS: A bioassay-guided fractionation of the total extract (MeOH 80%) of Pistacia vera L. hulls was carried out to evaluate wound healing activity by scratch assay on NIH/3T3 murine fibroblast cells. A combination of solvent-solvent partitioning, column chromatography, preparative thin layer chromatography and crystallization were used to obtain fractions/sub-fractions and pure compound. The wound healing potential of isolated compound was examined by fibroblasts migration and proliferation using scratch assay and CFSC dilution assay, respectively. In addition, we evaluated the gene expression of some inflammatory markers which are involved in healing process using Real Time PCR. Chemical structure of active compound was elucidated by spectrometric methods. RESULTS: Due to the higher wound healing activity of CHCl3 fraction from P. vera hulls, it was fractionated by successive chromatographic techniques to yield the active compound. 3-Epimasticadienolic acid was isolated and crystallized as a white powder. This active compound (200 µg/ml) significantly increased the fibroblast proliferation and migration, resulting in reduction of the scratch area about 45%. It showed a strong inhibitory effect on gene expression of IL-6 and TNF-α, and a stimulation effect on NF-κB gene expression at the same dose. CONCLUSION: The present study supported the traditional uses of P. vera hulls for wound-healing and 3-epimasticadienolic acid showed significantly potent on wound repair.

Characterization of arginine preventive effect on heat-induced aggregation of insulin.

Aggregation of proteins can affect their efficacy, and is especially important concerning therapeutic proteins such as insulin. Use of additives such as amino acids can counteract this deleterious process. Heat-induced aggregate formation of human insulin was kinetically studied with the use of various concentrations of the protein, at different temperatures, and in the presence of EDTA by UV-visible spectrophotometry. Effect of arginine, lysine, and histidine was then tested on the process at pH 4.8 and 45 °C. Kinetic parameters of the obtained growth curves (parameters t* and t0.5 characterizing the rate of the nucleation stage and the rate of the stage of aggregate growth respectively) were computed in all these conditions, and structure of aggregates was characterized by spectrofluorimetry, and transmission electron microscopy (TEM). Presence of high concentrations of the chelator EDTA increased aggregation. Among used additives, L-arginine (50 mM) most efficiently suppresses the heat-induced amorphous aggregation of insulin, affecting parameters t0.5 and t* presumably by preserving the protein's structure, as observed by the protein intrinsic fluorescence and CD spectra, and smaller formed aggregates in TEM images and dynamic light scattering. Docking experiment and subsequent molecular dynamics simulation indicated possible sites of interaction for arginine with the B-chain of insulin.

Monocyclic phenolic compounds stabilize human insulin and suppress its amorphous aggregation: In vitro and in vivo study.

Insulin is a small protein with 51 residues that mediates glucose uptake, and an interesting model for studying protein misfolding and aggregation. The aggregated forms of insulin undergo loss of activity and can provoke unwanted immune responses. Use of small molecules is considered to be an affordable method to counteract this aggregation process and stabilize insulin. In this study, aggregated forms of human recombinant insulin have been produced following exposure to high temperature. Aggregation process was followed over time by checking absorbance with spectrophotometry in presence and absence of various concentrations of small phenolic compounds including eugenol and epinephrine. Effects of these compounds on the structure and function of incubated insulin were evaluated by spectrofluorimetry, melting temperature (Tm) measurement and insulin tolerance test on Wistar rats. Formation of heat-induced insulin aggregation can be effectively inhibited by 1 mM eugenol and epinephrine and both compounds were found to preserve insulin activity to a considerable extent. In conclusion, simple aromatic compounds could be tailored to act as potent anti-aggregation compounds for insulin.

Amino Acids as Additives against Amorphous Aggregation: In Vitro and In Silico Study on Human Lysozyme.

The effect of 16 amino acids (AA) with various physicochemical properties was investigated on human lysozyme (HL) heat-induced amorphous aggregation. UV-Visible spectrophotometry was used to monitor the kinetics of aggregation in the absence and presence of AA, and transmission electron microscopy (TEM) images were taken from the aggregates. To conduct in silico experiments, Autodock vina was used for docking of AA into protein (via YASARA interface), and FTmap information was checked for an insight onto putative binding sites. Prediction of aggregation-prone regions of lysozyme was made by AGGRESCAN and Tango. Among all tested AA, phenylalanine had the best anti-aggregation effect, followed by lysine. In addition, based on in silico tests, Trp 109 and Val 110 of lysozyme are suggested to be of importance in the aggregation process of the enzyme. In conclusion, phenylalanine, arginine, and lysine were found to affect the nucleation phase of lysozyme aggregation and could be considered as suitable stabilizing structures for this enzyme.

Chemodiversity of Nepeta menthoides Boiss. & Bohse. essential oil from Iran and antimicrobial, acetylcholinesterase inhibitory and cytotoxic properties of 1,8-cineole chemotype.

The essential oil of Nepeta menthoides Boiss. & Bohse., from Iran, was analysed by GC/MS. Two types of multivariate analyses were done based on the chemical composition of the investigated sample in this study and 12 other samples reported in the literature to show the chemodiversity in essential oil composition. Antimicrobial, acetylcholinesterase inhibitory and cytotoxic activities of the essential oil and its major component were assessed. Twenty-one compounds were identified, representing 96.81% of the total oil and the major constituent was 1,8-cineole (70.06%). Multivariate analyses revealed two chemotypes, i.e. nepetalactone and 1,8-cineole. The essential oil of the sample investigated in this study which was a 1,8-cineole chemotype and 1,8-cineole showed moderate antimicrobial activity and significantly inhibited the activity of acetylcholinesterase enzyme. Cytotoxicity evaluation against three breast cancer cell lines showed a potent inhibitory activity. Further investigations are necessary to confirm the variety in several populations of N. menthoides.

The cellular membrane as a mediator for small molecule interaction with membrane proteins.

The cellular membrane constitutes the first element that encounters a wide variety of molecular species to which a cell might be exposed. Hosting a large number of structurally and functionally diverse proteins associated with this key metabolic compartment, the membrane not only directly controls the traffic of various molecules in and out of the cell, it also participates in such diverse and important processes as signal transduction and chemical processing of incoming molecular species. In this article, we present a number of cases where details of interaction of small molecular species such as drugs with the membrane, which are often experimentally inaccessible, have been studied using advanced molecular simulation techniques. We have selected systems in which partitioning of the small molecule with the membrane constitutes a key step for its final biological function, often binding to and interacting with a protein associated with the membrane. These examples demonstrate that membrane partitioning is not only important for the overall distribution of drugs and other small molecules into different compartments of the body, it may also play a key role in determining the efficiency and the mode of interaction of the drug with its target protein. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Synthesis, receptor affinity and effect on pentylenetetrazole-induced seizure threshold of novel benzodiazepine analogues: 3-Substituted 5-(2-phenoxybenzyl)-4H-1,2,4-triazoles and 2-amino-5-(phenoxybenzyl)-1,3,4-oxadiazoles.

The new series of 5-(2-phenoxybenzyl)-4H-1,2,4-triazoles, possessing C-3 thio, alkylthio and ethoxy substituents, and 2-amino-5-(2-phenoxybenzyl)-1,3,4-oxadiazoles were designed and synthesized as novel benzodiazepine analogues. Most of them revealed similar to superior binding affinity to the GABAA/benzodiazepine receptor complex, relative to diazepam as the reference drug. Among them, 5-(4-chloro-2-(2-fluorophenoxy)benzyl)-3-benzylthio-4H-1,2,4-triazole (8l) showed the highest affinity (IC50=0.892 nM) relative to diazepam (IC50=2.857 nM) and also showed the most increase in pentylenetetrazole-induced seizure threshold relative to diazepam as the reference drug.

Validation and uncertainty estimation of an ecofriendly and stability-indicating HPLC method for determination of diltiazem in pharmaceutical preparations.

A green, simple, and stability-indicating RP-HPLC method was developed for the determination of diltiazem in topical preparations. The separation was based on a C18 analytical column using a mobile phase consisted of ethanol: phosphoric acid solution (pH = 2.5) (35 : 65, v/v). Column temperature was set at 50°C and quantitation was achieved with UV detection at 240 nm. In forced degradation studies, the drug was subjected to oxidation, hydrolysis, photolysis, and heat. The method was validated for specificity, selectivity, linearity, precision, accuracy, and robustness. The applied procedure was found to be linear in diltiazem concentration range of 0.5-50 µ g/mL (r (2) = 0.9996). Precision was evaluated by replicate analysis in which % relative standard deviation (RSD) values for areas were found below 2.0. The recoveries obtained (99.25%-101.66%) ensured the accuracy of the developed method. The degradation products as well as the pharmaceutical excipients were well resolved from the pure drug. The expanded uncertainty (5.63%) of the method was also estimated from method validation data. Accordingly, the proposed validated and sustainable procedure was proved to be suitable for routine analyzing and stability studies of diltiazem in pharmaceutical preparations.

Benzofuranone derivatives as effective small molecules related to insulin amyloid fibrillation: a structure-function study.

Amyloids are protein fibrils of nanometer size resulting from protein self-assembly. They have been shown to be associated with a wide variety of diseases such as Alzheimer's and Parkinson's and may contribute to various other pathological conditions, known as amyloidoses. Insulin is prone to form amyloid fibrils under slightly destabilizing conditions in vitro and may form amyloid structures when subcutaneously injected into patients with diabetes. There is a great deal of interest in developing novel small molecule inhibitors of amyloidogenic processes, as potential therapeutic compounds. In this study, the effects of five new synthetic benzofuranone derivatives were investigated on the insulin amyloid formation process. Protein fibrillation was analyzed by thioflavin-T fluorescence, Congo red binding, circular dichroism, and electron microscopy. Despite high structural similarity, one of the five tested compounds was observed to enhance amyloid fibrillation, while the others inhibited the process when used at micromolar concentrations, which could make them interesting potential lead compounds for the design of therapeutic antiamyloidogenic compounds.

Synthesis of N-arylidene-2-(2-Phenoxyphenyl) Acetohydrazides as Anti-Inflammatory Agents.

Diclofenac sodium has been used for its anti-inflammatory actions for about 28 years, but since all the non-steroidal anti-inflammatory drugs (NSAIDs) suffer from the lethal gastro intestinal (GI) toxicities, diclofenac sodium is not an exception. The free -COOH group is thought to be responsible for the GI toxicity associated with all traditional NSAIDs. In the present research, the main motto was to develop new chemical entities as potential anti-inflammatory agents with no GI toxicities. A new type of 2-(2-phenoxyphenyl) acetohydrazide possessing N-arylidene substituents, was synthesized for evaluation as anti-inflammatory agents. The starting material 2-(2-Phenoxyphenyl) acetohydrazide was synthesized from 2-phenoxybenzoic acid in several steps according to the previous published method. Various substituted arylidene-2-phenoxynicotinic acid hydrazide derivatives were synthesized by the reaction of hydrazide 17 with selected aldehydes and screened for their potential anti-inflammatory activity. The structure of synthesized compounds was confirmed by different nuclear magnetic resonance technique, Fourier transform infrared spectroscopy (FTIR) and Mass-spectrometry data format. Qualitative structure-activity relationship data, acquired using the carrageenan-induced rat paw edema assay, showed that this group of arylidene-2-phenoxybenzoic acid hydrazides exhibit anti-inflammatory activity with significant reduction of rat paw edema (17-58% reduction in inflammation at different time intervals) in comparison with control group and a moderate to good activity range in comparison with diclofenac as the reference drug. Compounds 9a, 9d and 9e exhibited the most prominent and consistent anti-inflammatory activity. The compound, N-(4-Chlorobenzylidene)-2-(2-phenoxyphenyl) acetohydrazide (9d), exhibited the most in-vivo activity (32-58% reduction in inflammation) compared to the reference drug diclofenac (35-74% reduction in inflammation) in a carrageenan induced rat paw-edema assay.

Design and synthesis of 2-phenoxynicotinic acid hydrazides as anti-inflammatory and analgesic agents.

A series of 2-phenoxynicotinic acid hydrazides were synthesized and evaluated for their analgesic and anti-inflammatory activities. Several compounds having an unsubstituted phenyl/4-pyridyl or C-4 methoxy substituent on the terminal phenyl ring showed moderate to high analgesic or anti-inflammatory activity in comparison to mefenamic acid as the reference drug. The compounds with highest anti-inflammatory activity were subjected to in vitro COX-1/COX-2 inhibition assays and showed moderate to good COX-1 and weak COX-2 inhibition activities.

Dopaminergic but not glutamatergic neurotransmission is increased in the striatum after selective cyclooxygenase-2 inhibition in normal and hemiparkinsonian rats.

In the present work, we studied the effect of the selective cyclooxygenase-2 (COX-2) inhibitors, compound 11 g, celecoxib and selective COX-1 inhibitor SC-560 (intraperitoneally and acutely) on striatal glutamatergic and dopaminergic neurotransmission in normal and substantia nigra pars compacta (SNc)-lesioned rats using the microdialysis technique. We also investigated the effect of acute COX inhibition on the damaged SNc neurons. Our results indicate a significant increase in dopaminergic neurotransmission and a decrease in glutamatergic neurotransmission (P<0.05) only after selective COX-2 inhibition in the striatum of normal and hemiparkinsonian rats. Nonetheless, neither COX-1 nor COX-2 inhibitors showed any improvement in the damaged SNc neurons.

Synthesis, antibacterial activity, and quantitative structure-activity relationships of new (Z)-2-(nitroimidazolylmethylene)-3(2H)-benzofuranone derivatives.

A new series of (Z)-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranones (11a-p) and (Z)-2-(1-methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranones (12a-m) were synthesized and assayed for their antibacterial activity against Gram-positive and Gram-negative bacteria. Most of the 5-nitroimidazole analogues (11a-p) showed a remarkable inhibition of a wide spectrum of Gram-positive bacteria (Staphylococcus aureus, Streptococcus epidermidis, MRSA, and Bacillus subtilis) and Gram-negative Klebsiella pneumoniae, whereas 4-nitroimidazole analogues (12a-m) were not effective against selected bacteria. The quantitative structure-activity relationship investigations were applied to find out the correlation between the experimentally evaluated activities with various parameters of the compounds studied. The QSAR models built in this work had reasonable predictive power and could be explained by the observed trends in activities.

Design, synthesis, and biological evaluation of substituted 2-alkylthio-1,5-diarylimidazoles as selective COX-2 inhibitors.

A new type of 1-aryl-5-(4-methylsulfonylphenyl)imidazoles, possessing C-2 alkylthio (SMe or SEt) substituents, were designed and synthesized for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors with in vivo anti-inflammatory activity. The compound, 1-(4-bromophenyl)-5-(4-methylsulfonylphenyl)-2-methylthioimidazole (11g), was the most potent and selective COX-2 inhibitor (COX-2 IC50=0.43 microM with no inhibition of COX-1 up to 25 microM) relative to the reference drug celecoxib (COX-2 IC50=0.21 microM with no inhibition of COX-1 up to 25 microM) and also showed very good anti-inflammatory activity compared to celecoxib in carrageenan-induced rat paw edema assay.

Synthesis of alpha-fluorosulfonate and alpha-fluorosulfonamide analogues of a sulfated carbohydrate.

The first synthesis of sulfonamide and sulfonate analogues of a sulfated carbohydrate in which the ester oxygen of the sulfate is replaced with a CHF or CF2 group is reported. This was accomplished by electrophilic fluorination of the protected sulfonate and sulfonamide precursors. [reaction: see text].