Enzymatic redox properties of novel nitrotriazole explosives implications for their toxicity

The toxicity of conventional nitroaromatic explosives like 2,4,6-trinitrotoluene (TNT) is caused by their enzymatic free radical formation with the subsequent oxidative stress, the formation of alkylating nitroso and/or hydroxylamino metabolites, and oxyhemoglobin oxidation into methemoglobin. In order to get an insight into the mechanisms of toxicity of the novel explosives NTO (5-nitro-1,2,4-triazol-3-one) and ANTA (5-nitro-1,2,4-triazol-3-amine), we examined their reactions with the single-electron transferring flavoenzymes NADPH: cytochrome P-450 reductase and ferredoxin:NADP+ reductase, two-electron transferring flavoenzymes mammalian NAD(P)H:quinone oxidoreductase (DT-diaphorase), and Enterobacter cloacae NAD(P)H:nitroreductase, and their reactions with oxyhemoglobin. The reactivity of NTO and ANTA in the above reactions was markedly lower than that of TNT. The toxicity of NTO and ANTA in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was partly prevented by desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea. This points to the involvement of oxidative stress in their cytotoxicity, presumably to the redox cycling of free radicals. The FLK cell line cytotoxicity and the methemoglobin formation in isolated human erythrocytes of NTO and ANTA were also markedly lower than those of TNT, and similar to those of nitrobenzene. Taken together, our data demonstrate that the low toxicity of nitrotriazole explosives may be attributed to their low electron-accepting properties.     

A computational study of ANTA and NTO derivatives

This work is a study of 5-amino-3-nitro-1,2,4-triazole (ANTA), 3-nitro-1,2,4-triazol-5-one (NTO), and nitrated derivatives of ANTA and NTO. RDX and TNT were studied for comparison. ANTA and NTO are low-sensitive high explosives with detonation properties comparable to 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitroperhydro-1,3,5-triazine (RDX). We showed previously that nitrated NTO and ANTA compounds, when used in a glycidyl azide polymer (GAP) matrix in rocket propellants, could give impulses above 2600 m/s and that the oxygen balance is positive. If used in aluminized explosives, the heat of detonation may be increased to a practical level significantly above RDX/aluminum compositions. Here, we use two different methods for sensitivity and two density functional theory functionals, B3LYP and M06-2X with the 6-31G(d) basis set, together with the complete basis set method CBS-4M. Calculations indicate that most of the nitrated derivatives have nearly equal sensitivity to RDX. Significantly different bond dissociation energies in the nitrimino functional group are predicted, although most models give much the same result.     

Synthesis of 3-alkyl(aryl)-4-alkylidenamino-4,5-dihydro-1H-1,2,4-triazol-5-ones and 3-alkyl-4-alkylamino-4,5-dihydro-1H-1,2,4-triazol-5-ones as antitumor agents

A series of 3-alkyl-4-phenylethylidenamino- (8) and 3-alkyl-4-(3-phenylallylidenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones (9) was synthesized from the reaction of the corresponding 3-alkyl(aryl)-4-amino-4,5-dihydro-1H-1,2,4-triazol-5-ones (1), with phenylacetaldehyde and cinnamaldehyde. 3-Alkyl-4-(2-phenylethylamino)- (10) and 3-alkyl-4-(3-phenylpropylamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones (11) were obtained from the selective reduction of compounds (8) and (9) with NaBH₄. The in vitro antitumor activity of the novel compounds was screened and the highest inhibition of tree tumor cell lines was observed for the compounds containing phenylethylenamino and phenylethylamino groups at position 4 of 1,2,4-triazol ring.     

The synthesis and structure-activity relationships of 1,3-diaryl 1,2,4-(4H)-triazol-5-ones: a new class of calcium-dependent, large conductance, potassium (maxi-K) channel opener targeted for urge urinary incontinence

A series of 1,3-diaryl 1,2,4-(4H)-triazol-5-ones was prepared and shown by electrophysiological analysis to activate a cloned maxi-K channel mSlo (or hSlo) expressed in Xenopus laevis oocytes. The effects of these structurally novel maxi-K channel openers on bladder contractile function were studied in vitro using isolated rat bladder strips pre-contracted with carbachol. Several 1,3-diaryl 1,2,4-(4H)-triazol-5-one derivatives were found to be potent smooth muscle relaxants but this activity did not completely correlate with maxi-K channel opening.     

Pharmacokinetics and metabolism studies on (3-tert-butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy) pyrazolo[1,5-d][1,2,4]triazine, a functionally selective GABA(A) alpha5 inverse agonist for cognitive dysfunction

(3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)pyrazolo[1,5-d][1,2,4]triazine (1) was recently identified as a functionally selective, inverse agonist at the benzodiazepine site of GABA(A) alpha5 receptors and enhances performance in animal models of cognition. The routes of metabolism of this compound in vivo in rat have been well characterised, the identities of the major metabolites are confirmed by synthesis and their biological profiles were evaluated. An unusual oxidation of the pyrazolo[1,5-d][1,2,4]triazine core to the corresponding pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one scaffold by aldehyde oxidase has been observed.     

Theoretical study of adsorption of nitrogen-containing environmental contaminants on kaolinite surfaces

The adsorption of nitrogen-containing compounds (NCCs) including 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), 2,4-dinitroanisole (DNAN), and 3-nitro-1,2,4-triazol-5-one (NTO) on kaolinite surfaces was investigated. The M06-2X and M06-2X-D3 density functionals were applied with the cluster approximation. Several different positions of NCCs relative to the adsorption sites of kaolinite were examined, including NCCs in perpendicular and parallel orientation toward both surface models of kaolinite. The binding between the target molecules and kaolinite surfaces was analyzed and bond energies were calculated applying the atoms in molecules (AIM) method. All NCCs were found to prefer a parallel orientation toward both kaolinite surfaces, and were bound more strongly to the octahedral than to the tetrahedral site. TNT exhibited the strongest interaction with the octahedral surface and DNAN with the tetrahedral surface of kaolinite. Hydrogen bonding was shown to be the dominant non-covalent interaction for NCCs interacting with the octahedral surface of kaolinite with a small stabilizing effect of dispersion interactions. In the case of adsorption on the tetrahedral surface, kaolonite–NCC binding was shown to be governed by the balance between hydrogen bonds and dispersion forces. The presence of water as a solvent leads to a significant decrease in the adsorption strength for all studied NCCs interacting with both kaolinite surfaces.     

Azolylchromans as a novel scaffold for anticonvulsant activity

A series of azolylchroman derivatives were prepared as conformationally constrained analogs of (arylalkyl)azole anticonvulsants. The anticonvulsant activities of the compounds were evaluated by determining seizure latency and protective effect against pentylenetetrazole (PTZ)-induced lethal convulsions in mice at a dose of 5mg/kg. Among these compounds, 7-chloro-3-(1H-imidazol-1-yl)chroman-4-one and 3-(1H-1,2,4-triazol-1-yl)chroman-4-one exhibited significant action in delaying seizures as well as effective protection against PTZ-induced seizures and deaths.     

Metagenomic analysis of denitrifying wastewater enrichment cultures able to transform the explosive, 3-nitro-1,2,4-triazol-5-one (NTO)

Removal of 3-nitro-1,2,4-triazol-5-one (NTO) was investigated in conjunction with heterotrophic and autotrophic denitrifying growth conditions by a microbial consortium from a wastewater treatment plant. Microcosms were supplemented with molasses, methanol, or thiosulfate. Cultures were passaged twice by transferring 10 % of the culture volume to fresh media on days 11 and 21. Rates of NTO removal were 18.71 ± 0.65, 9.04 ± 2.61, and 4.34 ± 2.72 mg/L/day while rates of nitrate removal were 20.08 ± 1.13, 21.58 ± 1.20, and 24.84 ± 1.26 mg/L/day, respectively, for molasses, methanol, or thiosulfate. Metagenomic analysis showed that Proteobacteria and Firmicutes were the major phyla in the microbial communities. In molasses supplemented cultures, the community profile at the family level changed over time with Pseudomonadaceae the most abundant (67.4 %) at day 11, Clostridiaceae (65.7 %) at day 21, and Sporolactobacillaceae (35.4 %) and Clostridiaceae (41.0 %) at day 29. Pseudomonadaceae was the dominant family in methanol and thiosulfate supplemented cultures from day 21 to 29 with 76.6 and 81.6 % relative abundance, respectively.     

Diffusion of energetic compounds through biological membrane: Application of classical MD and COSMOmic approximations

Computational studies of the potential biological impact of several energetic compounds were performed. The most commonly used explosives were considered in the present studies: trinitrotoluene (TNT), 2,4-dinitrotoluene (2,4-DNT), 2,4-dinitroanisole (DNAN), and 5-Nitro-2,4-dihydro-3H-1,2,4-triazol-3-one (NTO). The effect of such factors as ionic strength and presence of DMSO in the water solution on the structure of the membrane were considered using the POPC lipid bilayer as an example. Molecular dynamics (MD) simulations revealed that, even on a short-time scale, the influence of those additives is noticeable, and therefore those factors should always be taken into account. The MD and the COSMOmic approaches were used to elucidate the ability of the energetic compounds to penetrate the living cell. Calculated free energy profiles and partitioning coefficients revealed distributions of the compounds in the lipid bilayer as well as an overall ability to enter the cell. MD in this case provides a better representation of the free energy profile, while the COSMOmic approach works better to predict log(K_lipw) values. The effect of the functional group was observed for the profiles that were obtained using the MD method.     

Study on the mutagenicity of nifurtimox and eight derivatives with the L-arabinose resistance test of Salmonella typhimurium

The mutagenicity of nifurtimox (nfx) and 8 nfx analogues has been investigated with the L-arabinose forward-mutation assay of Salmonella typhimurium. The nfx analogues tested were obtained by replacing the 3-methyl-4-yl-tetrahydro-1,4-thiazine-1,1-dioxide group of the parent compound with the following other groups: indazol-1-yl (1); pyrazol-1-yl (2); benzimidazol-1-yl (3); 1,2,4-triazol-4-yl (4); 1-methyl-3-methylthio-1,2,4-triazol-4-yl-5-thione (5); 3,5-bis(methylthio)-1,2,4-triazol-4-yl (6); 1-adamantyl (7); 4,6-diphenylpyridin-1-yl-2-one (8). The mutagenic activity of each chemical was determined by the standard plate-incorporation test, in the presence or absence of the S9 activation mixture. The 9 compounds were mutagenic and exhibited linear dose-mutagenic response relationships. They were direct-acting mutagens and showed a nearly 1000-fold range in mutagenic potency from chemical 1 to nfx. In most cases, the addition of S9 mixture to the test plates decreased the mutagenicity of compounds. This effect was particularly noticeable in the case of chemicals 1-3, 5 and 7 where a more than 70% decrease in mutagenic activity was observed in the presence of the S9 mixture. The mutagenic potency of compounds in the Ara test showed a negative linear correlation with previously reported antitrypanosomal activity. Thus, chemicals 6 and 8 with in vitro activities against Trypanosoma cruzi clearly superior to that of nfx showed 2 of the lowest mutagenic potencies in the Ara test and these were only somewhat higher than the mutagenicity of the reference drug.     

Biodegradation of insensitive munition formulations IMX101 and IMX104 in surface soils

The biodegradation potential of insensitive munition melt cast formulations IMX101 and IMX104 was investigated in two unamended training range soils under aerobic and anaerobic growth conditions. Changes in community profiles in soil microcosms were monitored via high-throughput 16S rRNA sequencing over the course of the experiments to infer key microbial phylotypes that may be linked to IMX degradation. Complete anaerobic biotransformation occurred for IMX101 and IMX104 constituents 2,4-dinitroanisole (DNAN) and 3-nitro-1,2,4-triazol-5-one during the 30-day incubation period with Camp Shelby (CS) soil. By comparison, soil from Umatilla chemical depot demonstrated incomplete DNAN degradation with reduced transformation rates for both IMX101 and IMX104. Aerobic soil microcosms for both soils demonstrated reduced transformation rates compared to anaerobic degradation for all IMX constituents with DNAN the most susceptible to biotransformation by CS soil. Overall, IMX constituents hexahydro-1,3,5-trinitro-1,3,5-triazine and 1-nitroguanidine did not undergo significant transformation. In CS soil, organisms that have been associated with explosives degradation, namely members of the Burkholderiaceae, Bacillaceae, and Paenibacillaceae phylotypes increased significantly in anaerobic treatments whereas Sphingomonadaceae increased significantly in aerobic treatments. Collectively, these data may be used to populate fate and transport models to provide more accurate estimates for assessing environmental costs associated with release of IMX101 and IMX104.     

Synthesis, in vitro evaluation and molecular docking studies of new triazole derivatives as antifungal agents

On the basis of the active site of lanosterol 14α-demethylase from Candida albicans (CACYP51), a series of 1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-1H-1,2,4-triazol-5(4H)-one derivatives were synthesized as fluconazole analogs. Results of the preliminary antifungal tests against eight human pathogenic fungi in vitro showed that these compounds exhibited activities to some extent, and some displayed excellent antifungal activities against C. albicans than reference drug fluconazole. Flexible molecular docking was used to analyze the structure-activity relationships (SARs) of the target compounds. The designed compounds interact with CACYP51 through hydrophobic, van der Waals and hydrogen-bonding interactions.     

Synthesis of Triazole Schiff’s Base Derivatives and Their Inhibitory Kinetics on Tyrosinase Activity

In the present study, new Schiff’s base derivatives: (Z)-4-amino-5-(2-(3- fluorobenzylidene)hydrazinyl)-4H-1,2,4-triazole-3-thiol (Y₁), (Z)-3-((2-(4-amino-5- mercapto-4H-1,2,4-triazol-3-yl)hydrazono)methyl)phenol (Y₂), (Z)-2-((2-(4-amino-5- mercapto-4H-1,2,4-triazol-3-yl)hydrazono)methyl)phenol (Y₃) and 3-((Z)-(2-(4- (((E)-3-hydroxybenzylidene)amino)-5-mercapto-4H-1,2,4-triazol-3-yl)hydrazono)methyl)phenol (Y₄) were synthesized and their structures were characterized by LC-MS, IR and ¹H NMR. The inhibitory effects of these compounds on tyrosinase activites were evaluated. Compounds Y₁, Y₂ and Y₃ showed potent inhibitory effects with respective IC₅₀ value of 12.5, 7.0 and 1.5 μM on the diphenolase activities. Moreover, the inhibition mechanisms were determined to be reversible and mixed types. Interactions of the compounds with tyrosinase were further analyzed by fluorescence quenching, copper interaction, and molecular simulation assays. The results together with the anti-tyrosinase activities data indicated that substitution on the second position of benzene ring showed superior ant-ityrosinase activities than that on third position, and that hydroxyl substitutes were better than fluorine substitutes. In addition, two benzene rings connecting to the triazole ring would produce larger steric hindrance, and affect the bonding between tyrosinase and inhibitors to decrease the inhibitory effects. The anti-tyrosinase effects of these compounds were in contrast to their antioxidant activities. In summary, this research will contribute to the development and design of antityrosinase agents.     

Biological activity of novel N-substituted amides of endo-3-(3-methylthio-1,2,4-triazol-5-yl)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid and N-substituted amides of 1-(5-methylthio-1,2,4-triazol-3-yl)cyclohexane-2-carboxylic acids

N-Substituted amides of endo-3-(3-methylthio-1,2,4-triazol-5-yl)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid and 1-(5-methylthio-1,2,4-triazol-3-yl)cyclohexane-2-carboxylic acid were prepared by the condensation reaction of endo-S-methyl-N1-(bicyclo[2.2.1]hept-5-ene-2,3-dicarbonyl)isothiosemicarbazide and S-methyl-N1-(cyclohexane-2,3-dicarbonyl)isothiosemicarbazide with primary amines. The synthesized compounds were screened for their microbiological and pharmacological activities.     

Synthesis and biological evaluation of novel 7-hydroxy-4-phenylchromen-2-one–linked to triazole moieties as potent cytotoxic agents

A new series of novel 7-hydroxy-4-phenylchromen-2-one (1a)–linked 1,2,4-triazoles were synthesised using a click chemistry approach. All derivatives were subjected to 3-(4,5-dimethylthiazol-yl)-diphenyl tetrazolium bromide (MTT) cytotoxicity screening against a panel of six different human cancer cell lines (AGS, MGC-803, HCT-116, A-549, HepG2, and HeLa) to assess their cytotoxic potential. Among the tested molecules, some of the analogues showed better cytotoxic activity than that shown by the 7-hydroxy-4-phenylchromen-2-one (1a). Of the synthesised 1,2,4-triazoles,the 7-((4-(4-Chlorophenyl)-4H-1,2,4-triazol-3-yl)methoxy)-4-phenyl-2H-chromen-2-one (4d) showed the best activity, with an IC₅₀ of 2.63?±?0.17?µM against AGS cells. Further flow cytometry assays demonstrated that compound 4d exerts its antiproliferative effects by arresting cells in the G2/M phase of the cell cycle and by inducing apoptosis. Collectively, our results indicate that the 1,2,4-triazole derivatives have a significantly stronger antitumour activity than 1,2,3-triazole derivatives. Most of the compounds exhibited better antitumour activity than the positive control drug 5-fluorouracil.     

Synthesis and Fungicidal Activity of 5-Aryl-1-(aryloxyacetyl)-3-(tert-butyl or phenyl)-4-(1H-1,2,4-triazol-1-yl)-4,5-Dihydropyrazole

A series of novel 5-aryl-1-(aryloxyacetyl)-3-(tert-butyl or phenyl)-4-(1H-1,2,4-triazol-1-yl)-4,5-dihydropyrazole 3a-3n were synthesized by the annulation of 2-aryloxyacetohydrazides with 3-aryl-1-t-butyl(or phenyl)-2-(1H-1,2,4-triazol-1-yl)prop-2-en-1-ones(1)in the presence of a catalytic amount of acetic acid.Compounds 2 were obtained by the Knoevenagel reactions of 1-t-butyl(or phenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone(2)with aromatic aldehydes in the presence of piperidine.Their structures were confirmed by IR,1H-NMR,ESIMS,and elemental analyses.The preliminary bioassay indicated that some compounds displayed moderate to excellent fungicidal activity.For example,compounds 31,3m,and 3n possessed100%inhibition against Cercospora arachidicola Hori at the concentration of 50 mg/L.     

Incorporation of 14C-Formaldehyde into Hexose Phosphate by Cell-free Extract of a Methanolutilizing Yeast,Candida sp.

The persistence and degradation of isoxathion ¹⁴C-labeled at the 5-position of the isoxazole ring were studied in three soil types under laboratory conditions. Persistence was influenced by soil type and moisture content; approx. half life at 30 ppmw dose level varied from 15 to 40 days in nonflooded models. In a flooded model isoxathion disappeared much faster. Isoxathion underwent biodegradation to a number of products with concomitant release of ¹⁴CO₂. 3-Hydroxy-5-phenylisoxazole, 5-phenyl-4-oxazolin-2-one, benzoylacetamide and benzoic acid were the unequivocally identified metabolites; oxon derivative of isoxathion, 3-methoxy-5-phenylisoxazole, 2-methyl-5-phenyl-4-isoxazolin-3-one, 2-acetyl-5-phenyl-4-isoxazolin-3-one, 2, 5-diphenylpyrazine and acetophenone were tentatively identified as the minor products. None of these major products was persistent in soils. 3-Hydroxy-5-phenylisoxazole, the initial metabolite or hydrolyzate of isoxathion, was adsorbed to soil to a much greater extent than isoxathion, which explains the rapid disappearance of its fungicidal activity in soil.     

Impact of tautomery of 3-(4H-1,2,4-triazol-3-ylthio)-N-phenylpropanamide on the COX-1 inhibitory mechanism

Earlier, we have reported the synthesis and anti-inflammatory evaluation of different 3-(4H-1,2,4-triazol-3-ylthio)-N-substituted propanamide. In this article, we are reporting the various tautomeric forms of the most active anti-inflammatory compound, 3-(4H-1,2,4-triazol-3-ylthio)-N-phenylpropanamide (6a) and their virtual screening by molecular docking using six principle tautomeric forms. Docking analysis suggested that compound 3-(4H-1,2,4-triazol-3-ylthio)-N-phenylpropanamide (6a) bound with COX-1 selectively and drug receptor complex was stabilized by tautomerism. Noticeably, hydroxy group formed by tautomerism appreciably improve the drug receptor interactions. It was also supervised that the compound 3-(4H-1,2,4-triazol-3-ylthio)-N-phenylpropanamide (6a) docked near the gate of COX-1 active site and might block the conversion of arachidonic acid to prostaglandin (PG) H2 in the active site of COXs. Moreover, we have carried out receptor based electrostatic analysis to clarify the electronic, steric and hydrophobic field requirement of 3-(4H-1,2,4-triazol-3-ylthio)-N-phenylpropanamide (6a) to interact with COX -1 receptor.     

Synthesis of silicon-containing azole derivatives with magnesium bromide diethyl etherate,and an investigation of their fungicidal activities

Enhancement of magnesium bromide diethyl etherate in addition reaction of trimethylsilylmethylmagnesium chloride 5 to 2-(1H-1,2,4-triazol-1-yl)acetophenones 6 allowed us to prepare silicon-containing azole derivatives, which were tested for fungicidal activity and phytotoxicity. Among them, 2-(4-fluorophenyl)-1-(1H-1,2,4-triazol-1-yl)-3-trimethylsilylpropan-2-ol 1a was determined to be the most effective and potential candidate for novel fungicide.