Convenient Preparation, Thermal Properties and X-ray Structure Determination of 2,3-Dihydro-5,6,7,8-tetranitro-1,4-benzodioxine (TNBD): A Promising High-Energy-Density Material.

2,3-dihydro-5,6,7,8-tetranitro-1,4-benzodioxine (TNBD), molecular formula = C8H4N4O10, is a completely nitrated aromatic ring 1,4-benzodioxane derivative. The convenient method of TNBD synthesis was developed (yield = 81%). The detailed structure of this compound was investigated by X-ray crystallography. The results of the thermal analysis (TG) obtained with twice re-crystallized material revealed the onset at 240 °C (partial sublimation started) and melting at 286 °C. The investigated material degraded completely at 290-329 °C. The experimental density of 1.85 g/cm3 of TNBD was determined by X-ray crystallography. The spectral properties of TNBD (NMR, FT-IR and Raman) were explored. The detonation properties of TNBD calculated by the EXPLO 5 code were slightly superior in comparison to standard high-energy material-tetryl (detonation velocity of TNBD-7727 m/s; detonation pressure-278 kbar; and tetryl-7570 m/s and 226.4 kbar at 1.614 g/cm3, or 260 kbar at higher density at 1.71 g/cm3. The obtained preliminary results might suggest TNBD can be a potential thermostable high-energy and -density material (HEDM).

Garbage in, garbage out: how reliable training data improved a virtual screening approach against SARS-CoV-2 MPro.

Introduction: The identification of chemical compounds that interfere with SARS-CoV-2 replication continues to be a priority in several academic and pharmaceutical laboratories. Computational tools and approaches have the power to integrate, process and analyze multiple data in a short time. However, these initiatives may yield unrealistic results if the applied models are not inferred from reliable data and the resulting predictions are not confirmed by experimental evidence. Methods: We undertook a drug discovery campaign against the essential major protease (MPro) from SARS-CoV-2, which relied on an in silico search strategy -performed in a large and diverse chemolibrary- complemented by experimental validation. The computational method comprises a recently reported ligand-based approach developed upon refinement/learning cycles, and structure-based approximations. Search models were applied to both retrospective (in silico) and prospective (experimentally confirmed) screening. Results: The first generation of ligand-based models were fed by data, which to a great extent, had not been published in peer-reviewed articles. The first screening campaign performed with 188 compounds (46 in silico hits and 100 analogues, and 40 unrelated compounds: flavonols and pyrazoles) yielded three hits against MPro (IC50 ≤ 25 µM): two analogues of in silico hits (one glycoside and one benzo-thiazol) and one flavonol. A second generation of ligand-based models was developed based on this negative information and newly published peer-reviewed data for MPro inhibitors. This led to 43 new hit candidates belonging to different chemical families. From 45 compounds (28 in silico hits and 17 related analogues) tested in the second screening campaign, eight inhibited MPro with IC50 = 0.12-20 µM and five of them also impaired the proliferation of SARS-CoV-2 in Vero cells (EC50 7-45 µM). Discussion: Our study provides an example of a virtuous loop between computational and experimental approaches applied to target-focused drug discovery against a major and global pathogen, reaffirming the well-known "garbage in, garbage out" machine learning principle.

An Efficient Synthesis and Preliminary Investigation of Novel 1,3-Dihydro-2H-benzimidazol-2-one Nitro and Nitramino Derivatives.

The preparation and properties of a series of novel 1,3-dihydro-2H-benzimidazol-2-one nitro and nitramino derivatives are described. A detailed crystal structure of one of the obtained compounds, 4,5,6-trinitro-1,3-dihydro-2H-benzimidazol-2-one (TriNBO), was characterized using low temperature single crystal X-ray diffraction, namely an orthorhombic yellow prism, space group 'P 2 21 21', experimental crystal density 1.767 g/cm3 (at 173 K). Methyl analog, 5-Me-TriNBO-monoclinic red plates, space group, P 21/c, crystal density 1.82 g/cm3. TriNBO contains one activated nitro group at the fifth position, which was used for the nucleophilic substitution in the aminolysis reactions with three monoalkylamines (R=CH3, C2H5, (CH2)2CH3) and ethanolamine. The 5-R-aminoderivatives were further nitrated with N2O5/ HNO3 and resulted in a new group of appropriate nitramines: 1,3-dihydro-2H-5-R-N(NO2)-4,6-dinitrobenzimidazol-2-ones. Thermal analysis (TGA) of three selected representatives was performed. The new compounds possess a high melting point (200-315 °C) and thermal stability and can find a potential application as new thermostable energetic materials. Some calculated preliminary energetic characteristics show that TriNBO, 5-Me-TriNBO, 5-methylnitramino-1,3-dihydro-2H-4,6-dinitrobenzimidazol-2-one, and 5-nitratoethylnitramino-1,3-dihydro-2H-4,6-dinitrobenzimidazol-2-one possess increased energetic characteristics in comparison with TNT and tetryl. The proposed nitrocompounds may find potential applications as thermostable high-energy materials.

Investigation of oxygen influence to the optical properties of tirapazamine.

New data on 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine) fluorescence has been obtained using the Perkin-Elmer Lambda 950 UV-Vis-NIR spectrophotometer experimental technique in combination with the extensive DFT-theory approach. Based on the results obtained, we revealed that the optical properties of the molecule under study remain significantly unchanged when the number of oxygen substitutions decreases from 2 to 0. Here we also present the results of the study of the influence of acetonitrile and ethyl acetate on the fluorescence of tirapazamine with the different number of oxygen atoms. Results of our investigation indicate the formation of anion in the case of 3-amino-1,2,4-benzotriazine 1,4-dioxide with two oxygen atoms and their transformation to tirapazamine with one oxygen atom.

Benzimidazole Derivatives as Energetic Materials: A Theoretical Study.

The explosive properties and stability of benzimidazole compounds are studied to determine the influence of substituents and their position. The results obtained reveal the conjugation of substituents as one of the crucial factors for the thermal stability of these compounds. We also found that two -CH3 substituents increase the thermal stability of the parent compound, while nitro groups decrease it. Moreover, the study clearly exhibits that the combination of an -NO2 substituent with -CH3 does not change the stability of the benzimidazole. On the other hand, nitro groups increase the chemical stability and explosive properties of the compounds under investigation, but their sensitivity could not fully satisfy the requirements of their safety and increase their toxicity. The main results of the study indicate that high thermal and chemical stability, low toxicity and sensitivity, and good explosive properties could be achieved by the precise combination of nitro, -CH3, and triazole ring substituents. These findings are very important for the design of new, effective, and non-sensitive explosives.

Aerobic Cytotoxicity of Aromatic N-Oxides: The Role of NAD(P)H:Quinone Oxidoreductase (NQO1).

Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities, which are typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the role of NAD(P)H:quinone oxidoreductase (NQO1) in ArN→O aerobic cytotoxicity. We synthesized 9 representatives of ArN→O with uncharacterized redox properties and examined their single-electron reduction by rat NADPH:cytochrome P-450 reductase (P-450R) and Plasmodium falciparum ferredoxin:NADP+ oxidoreductase (PfFNR), and by rat NQO1. NQO1 catalyzed both redox cycling and the formation of stable reduction products of ArN→O. The reactivity of ArN→O in NQO1-catalyzed reactions did not correlate with the geometric average of their activity towards P-450R- and PfFNR, which was taken for the parameter of their redox cycling efficacy. The cytotoxicity of compounds in murine hepatoma MH22a cells was decreased by antioxidants and the inhibitor of NQO1, dicoumarol. The multiparameter regression analysis of the data of this and a previous study (DOI: 10.3390/ijms20184602) shows that the cytotoxicity of ArN→O (n = 18) in MH22a and human colon carcinoma HCT-116 cells increases with the geometric average of their reactivity towards P-450R and PfFNR, and with their reactivity towards NQO1. These data demonstrate that NQO1 is a potentially important target of action of heteroaromatic N-oxides.

Reactions of Plasmodium falciparum Ferredoxin:NADP+ Oxidoreductase with Redox Cycling Xenobiotics: A Mechanistic Study.

Ferredoxin:NADP+ oxidoreductase from Plasmodium falciparum (PfFNR) catalyzes the NADPH-dependent reduction of ferredoxin (PfFd), which provides redox equivalents for the biosynthesis of isoprenoids and fatty acids in the apicoplast. Like other flavin-dependent electrontransferases, PfFNR is a potential source of free radicals of quinones and other redox cycling compounds. We report here a kinetic study of the reduction of quinones, nitroaromatic compounds and aromatic N-oxides by PfFNR. We show that all these groups of compounds are reduced in a single-electron pathway, their reactivity increasing with the increase in their single-electron reduction midpoint potential (E17). The reactivity of nitroaromatics is lower than that of quinones and aromatic N-oxides, which is in line with the differences in their electron self-exchange rate constants. Quinone reduction proceeds via a ping-pong mechanism. During the reoxidation of reduced FAD by quinones, the oxidation of FADH. to FAD is the possible rate-limiting step. The calculated electron transfer distances in the reaction of PfFNR with various electron acceptors are similar to those of Anabaena FNR, thus demonstrating their similar "intrinsic" reactivity. Ferredoxin stimulated quinone- and nitro-reductase reactions of PfFNR, evidently providing an additional reduction pathway via reduced PfFd. Based on the available data, PfFNR and possibly PfFd may play a central role in the reductive activation of quinones, nitroaromatics and aromatic N-oxides in P. falciparum, contributing to their antiplasmodial action.

Antiplasmodial Activity of Nitroaromatic Compounds: Correlation with Their Reduction Potential and Inhibitory Action on Plasmodium falciparum Glutathione Reductase.

With the aim to clarify the mechanism(s) of action of nitroaromatic compounds against the malaria parasite Plasmodium falciparum, we examined the single-electron reduction by P. falciparum ferredoxin:NADP+ oxidoreductase (PfFNR) of a series of nitrofurans and nitrobenzenes (n = 23), and their ability to inhibit P. falciparum glutathione reductase (PfGR). The reactivity of nitroaromatics in PfFNR-catalyzed reactions increased with their single-electron reduction midpoint potential (E17). Nitroaromatic compounds acted as non- or uncompetitive inhibitors towards PfGR with respect to NADPH and glutathione substrates. Using multiparameter regression analysis, we found that the in vitro activity of these compounds against P. falciparum strain FcB1 increased with their E17 values, octanol/water distribution coefficients at pH 7.0 (log D), and their activity as PfGR inhibitors. Our data demonstrate that both factors, the ease of reductive activation and the inhibition of PfGR, are important in the antiplasmodial in vitro activity of nitroaromatics. To the best of our knowledge, this is the first quantitative demonstration of this kind of relationship. No correlation between antiplasmodial activity and ability to inhibit human erythrocyte GR was detected in tested nitroaromatics. Our data suggest that the efficacy of prooxidant antiparasitic agents may be achieved through their combined action, namely inhibition of antioxidant NADPH:disulfide reductases, and the rapid reduction by single-electron transferring dehydrogenases-electrontransferases.

Kinetics of Flavoenzyme-Catalyzed Reduction of Tirapazamine Derivatives: Implications for Their Prooxidant Cytotoxicity.

Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit promising antibacterial, antiprotozoal, and tumoricidal activities. Their action is typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the mechanism(s) of aerobic mammalian cell cytotoxicity of ArN→O performing the parallel studies of their reactions with NADPH:cytochrome P-450 reductase (P-450R), adrenodoxin reductase/adrenodoxin (ADR/ADX), and NAD(P)H:quinone oxidoreductase (NQO1); we found that in P-450R and ADR/ADX-catalyzed single-electron reduction, the reactivity of ArN→O (n = 9) increased with their single-electron reduction midpoint potential (E17), and correlated with the reactivity of quinones. NQO1 reduced ArN→O at low rates with concomitant superoxide production. The cytotoxicity of ArN→O in murine hepatoma MH22a and human colon adenocarcinoma HCT-116 cells increased with their E17, being systematically higher than that of quinones. The cytotoxicity of both groups of compounds was prooxidant. Inhibitor of NQO1, dicoumarol, and inhibitors of cytochromes P-450 α-naphthoflavone, isoniazid and miconazole statistically significantly (p < 0.02) decreased the toxicity of ArN→O, and potentiated the cytotoxicity of quinones. One may conclude that in spite of similar enzymatic redox cycling rates, the cytotoxicity of ArN→O is higher than that of quinones. This is partly attributed to ArN→O activation by NQO1 and cytochromes P-450. A possible additional factor in the aerobic cytotoxicity of ArN→O is their reductive activation in oxygen-poor cell compartments, leading to the formation of DNA-damaging species similar to those forming under hypoxia.

Mechanism of Two-/Four-Electron Reduction of Nitroaromatics by Oxygen-Insensitive Nitroreductases: The Role of a Non-Enzymatic Reduction Step.

Oxygen-insensitive NAD(P)H:nitroreductases (NR) reduce nitroaromatics (Ar-NO2) into hydroxylamines (Ar-NHOH) through nitroso (Ar-NO) intermediates. Ar-NO may be reduced both enzymatically and directly by reduced nicotinamide adenine dinucleotide or its phosphate NAD(P)H, however, it is unclear which process is predominant in catalysis of NRs. We found that E. coli NR-A (NfsA) oxidizes 2 mol of NADPH per mol of 2,4,6-trinitrotoluene (TNT) and 4 mol of NADPH per mol of tetryl. Addition of ascorbate, which reduces Ar-NO into Ar-NHOH, changes the stoichiometry NADPH/Ar-NO2 into 1:1 (TNT) and 2:1 (tetryl), and decreases the rate of NADPH oxidation. Ascorbate does not interfere with the oxidation of NADPH during reduction of quinones by NfsA. Our analysis of ascorbate inhibition patterns and both enzymatic and non-enzymatic reduction of nitrosobenzene suggests that direct reduction of Ar-NO by NADPH rather than enzymatic reduction is the predominant mechanism during nitroaromatic reduction.

Flavoenzyme-mediated reduction reactions and antitumor activity of nitrogen-containing tetracyclic ortho-quinone compounds and their nitrated derivatives.

Nitrogen-based tetracyclic ortho-quinones (naphtho[1'2':4.5]imidazo[1,2-a]pyridine-5,6-diones, NPDOs) and their nitro-substituted derivatives (nitro-(P)NPDOs) were obtained by condensation of substituted 2,3-dichloro-1,4-naphthoquinones with 2-amino-pyridine and -pyrimidine and nitration at an elevated temperature. The structural features of the compounds as well as their global and regional electrophilic potency were characterized by means of DFT computation. The compounds were highly reactive substrates of single- and two-electron (hydride) - transferring P-450R (CPR; EC 1.6.2.4) and NQO-1 (DTD; EC 1.6.99.2), respectively, concomitantly producing reactive oxygen species. Their catalytic efficiency defined in terms of the apparent second-order rate constant (kcat/KM (Q)) values in P-450R- and NQO-1-mediated reactions varied in the range of 3-6 × 107 M-1 s-1 and 1.6-7.4 × 108 M-1 s-1, respectively. The cytotoxic activities of the compounds on tumor cell lines followed the concentration-dependent manner exhibiting relatively high cytotoxic potency against breast cancer MCF-7, with CL50 values of 0.08-2.02 µM L-1 and lower potency against lung cancer A-549 (CL50 = 0.28-7.66 µM L-1). 3-nitro-pyrimidino-NPDO quinone was the most active compound against MCF-7 with CL50 of 0.08 ± 0.01 µM L-1 (0.02 µg mL-1)) which was followed by 3-nitro-NPDO with CL50 of 0.12 ± 0.03 µM L-1 (0.035 µg mL-1)) and 0.28 ± 0.08 µM L-1 (0.08 µg mL-1) on A-549 and MCF-7 cells, respectively, while 1- and 4-nitro-quinoidals produced the least cytotoxic effects. Tumor cells quantified by AO/EB staining showed that the cell death induced by the compounds occurs primarily through apoptosis.

Modeling and investigation of new explosive materials based on N-(3,5-dimethyl-2,4,6-trinitrophenyl)-1H-1,2,4-triazol-3-amine.

This study presents an investigation of the effects of adding extra nitro group substituents to N-(3,5-dimethyl-2,4,6-trinitrophenyl)-1H-1,2,4-triazol-3-amine (HEM-II) on its thermal and chemical stability as well as its explosive performance. An analysis of the thermal stabilities of HEM-II and HEM-II-based molecules based on an investigation of the binding energy per atom for each molecule was performed. The values of the gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) as well as those of the chemical hardness and softness for HEM-II and the HEM-II-based molecules were studied to determine the chemical stabilities of these molecules. The detonation velocity and oxygen balance of each HEM-II-based molecule were investigated to elucidate its explosive properties. The results of our investigation show that the presence of additional nitro groups suppresses the tendency of the new HEM-II-based explosive material to react with other materials, improves its explosive properties (strength), decreases the likelihood that it will degrade, and enhances its toxicity and thermal stability. We also found that zero-point correction does not need to be performed when molecules with different substituents are investigated and compared.

Aziridinyl-substituted benzo-1,4-quinones: A preliminary investigation on the theoretical and experimental studies of their structure and spectroscopic properties.

The detailed structure, chemical and spectroscopic properties of the derivatives of the selected 2,5-bis(1-aziridinyl)-benzo-1,4-quinone conformers were studied by applying quantum chemical and experimental methods. The relationship between the structure and chemical activity of the selected 3 bifunctional bioreductive quinonic anticancer agents - aziridinyl benzoquinones (AzBQ compounds) was obtained. The results obtained showed that the position of aziridine rings influenced by the chemical activity of the investigated compound were more significant than the substitutions of the benzene ring of the AzBQ compounds. The solvents influencing this activity were obtained, too.

Identification of Novel Chemical Scaffolds Inhibiting Trypanothione Synthetase from Pathogenic Trypanosomatids.

BACKGROUND: The search for novel chemical entities targeting essential and parasite-specific pathways is considered a priority for neglected diseases such as trypanosomiasis and leishmaniasis. The thiol-dependent redox metabolism of trypanosomatids relies on bis-glutathionylspermidine [trypanothione, T(SH)2], a low molecular mass cosubstrate absent in the host. In pathogenic trypanosomatids, a single enzyme, trypanothione synthetase (TryS), catalyzes trypanothione biosynthesis, which is indispensable for parasite survival. Thus, TryS qualifies as an attractive drug target candidate. METHODOLOGY/PRINCIPAL FINDING: A library composed of 144 compounds from 7 different families and several singletons was screened against TryS from three major pathogen species (Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum). The screening conditions were adjusted to the TryS´ kinetic parameters and intracellular concentration of substrates corresponding to each trypanosomatid species, and/or to avoid assay interference. The screening assay yielded suitable Z' and signal to noise values (≥0.85 and ~3.5, respectively), and high intra-assay reproducibility. Several novel chemical scaffolds were identified as low µM and selective tri-tryp TryS inhibitors. Compounds displaying multi-TryS inhibition (N,N'-bis(3,4-substituted-benzyl) diamine derivatives) and an N5-substituted paullone (MOL2008) halted the proliferation of infective Trypanosoma brucei (EC50 in the nM range) and Leishmania infantum promastigotes (EC50 = 12 µM), respectively. A bis-benzyl diamine derivative and MOL2008 depleted intracellular trypanothione in treated parasites, which confirmed the on-target activity of these compounds. CONCLUSIONS/SIGNIFICANCE: Novel molecular scaffolds with on-target mode of action were identified as hit candidates for TryS inhibition. Due to the remarkable species-specificity exhibited by tri-tryp TryS towards the compounds, future optimization and screening campaigns should aim at designing and detecting, respectively, more potent and broad-range TryS inhibitors.

Quantitative proteomic analysis of anticancer drug RH1 resistance in liver carcinoma.

Acquired resistance of tumor cells to the therapeutic treatment is a major challenge in virtually any chemotherapy. A novel anticancer agent 2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone (RH1) is designed to be activated by NAD(P)H: quinone oxidoreductase, an enzyme expressed at high levels in many types of tumors. Here we investigated the potential mechanisms of acquired RH1 drug resistance in cancer cells by applying high-throughput differential quantitative proteomic analysis of the newly established RH1-resistant hepatoma cell lines. Over 400 proteins display significantly altered levels between drug-sensitive and drug-resistant cell lines. Differentially expressed proteins were clustered into more than 14 groups according to their functional annotation and protein-protein interactions. Bioinformatic analysis highlights the biological processes that might be responsible for acquired resistance to RH1. The level of several xenobiotic metabolism enzymes (total n=17) involved in RH1 activation and detoxification is decreased (Nqo1, catalase, Gst, Gsr), corresponding with the decrease in their catalytic activity. The altered biological processes also include the decrease of cell cycle positive regulators (n=15) and the increase of DNA repair proteins (n=5) as well as annexin family members (n=5) in the RH1-resistant cells. Drug-resistant hepatoma cell proteomes are also distinguished by the altered level of proteins involved in energy production and metabolism (n=55). Our data provide the basis for in-depth study of molecular mechanisms of tumor cell resistance to the promising anticancer drug RH1 enabling the further validation of protein biomarkers for the drug insusceptibility and of potential secondary pharmacological targets of RH1 resistant cells.

Study of Bioreductive Anticancer Agent RH-1-Induced Signals Leading the Wild-Type p53-Bearing Lung Cancer A549 Cells to Apoptosis.

Aziridinylquinone RH-1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-cyclohexa-2,5-diene-1,4-dione) is a potential anticancer agent. RH-1 action is associated with NAD(P)H: quinone oxidoreductase (NQO1) which reduces this diaziridinylbenzoquinone into DNA-alkylating hydroquinone and is overexpressed in many tumors. Another suggested mechanism of RH-1 toxicity is the formation of reactive oxygen species (ROS) arising from its redox cycling. In order to improve anticancer action of this and similar antitumor quinones, we investigated the involvement of different signaling molecules in cytotoxicity induced by RH-1 by using wild-type tumor suppressor p53 bearing nonsmall cell lung carcinoma A549 cells as a model. Gradual and prolonged increase of mitogen-activated protein kinases (MAPK) ERK, P38, and JNK phosphorylation was observed during 24-h RH-1 treatment. In parallel, activation of DNA damage-sensing ATM kinase, upregulation, and phosphorylation of TP53 (human p53) took place. Inhibition studies revealed that RH-1-induced A549 apoptosis involved the NQO1-ATM-p53 signaling pathway and ROS generation. TP53 participated in ROS- and DNA damage-induced cell death differently. Moreover, MAP kinase JNK was another TP53 activator and death inducer in A549 cells. At the same time, rapid and prolonged activation of AKT kinase during RH-1 treatment was found, and it proved to be antiapoptotic kinase in our model system. Therefore, we identified that different and opposite cell death regulating signaling pathways, which may counteract one another, are induced in cancer cells during chemotherapeutic RH-1 treatment.

Naphtho[1',2':4,5]imidazo[1,2-a]pyridine-5,6-diones: Synthesis, enzymatic reduction and cytotoxic activity.

Naphtho[1',2':4,5]imidazo[1,2-a]pyridine-5,6-diones (NPDOs), a new type of N-heterocycle-fused o-quinones, have been synthesized. They have been found to be efficient electron-accepting substrates of NADPH-dependent single-electron-transferring P-450R and two-electron transferring NQO1, generating reactive oxygen species (ROS) with a concomitant decrease in NADPH, which is consistent with redox-cycling. The reactivity of NPDOs toward P-450R (in terms of kcat/Km) varied in the range of 10(6)-10(7)M(-1)s(-1), while their reduction by NQO1 proceeded much faster, approaching the diffusion control limit (kcat/Km∼10(8)-10(9)M(-1)s(-1)). NPDOs exhibited relatively high cytotoxic activity against human lung carcinoma (A-549) and breast tumor (MCF-7) cell lines (LC50=0.1-8.3µM), while promyelocytic leukemia cells (HL-60) were less sensitive to NPDOs (LC50⩾10µM). 3-Nitro-substituted NPDO (11) revealed the highest potency against both A-549 and MCF-7 cell lines, with LC50 of 0.12±0.03µM and 0.28±0.08µM, respectively. Dicoumarol partly suppressed the activity of the compounds against A-594 and MCF-7 cell lines, suggesting that their cytotoxic action might be partially influenced by NQO1-mediated bioreductive activation.

The study of NADPH-dependent flavoenzyme-catalyzed reduction of benzo[1,2-c]1,2,5-oxadiazole N-oxides (benzofuroxans).

The enzymatic reactivity of a series of benzo[1,2-c]1,2,5-oxadiazole N-oxides (benzofuroxans; BFXs) towards mammalian single-electron transferring NADPH:cytochrome P-450 reductase (P-450R) and two-electron (hydride) transferring NAD(P)H: quinone oxidoreductase (NQO1) was examined in this work. Since the =N+ (→O)O- moiety of furoxan fragments of BFXs bears some similarity to the aromatic nitro-group, the reactivity of BFXs was compared to that of nitro-aromatic compounds (NACs) whose reduction mechanisms by these and other related flavoenzymes have been extensively investigated. The reduction of BFXs by both P-450R and NQO1 was accompanied by O2 uptake, which was much lower than the NADPH oxidation rate; except for annelated BFXs, whose reduction was followed by the production of peroxide. In order to analyze the possible quantitative structure-activity relationships (QSARs) of the enzymatic reactivity of the compounds, their electron-accepting potency and other reactivity indices were assessed by quantum mechanical methods. In P-450R-catalyzed reactions, both BFXs and NACs showed the same reactivity dependence on their electron-accepting potency which might be consistent with an "outer sphere" electron transfer mechanism. In NQO1-catalyzed two-electron (hydride) transferring reactions, BFXs acted as more efficient substrates than NACs, and the reduction efficacy of BFXs by NQO1 was in general higher than by single-electron transferring P-450R. In NQO1-catalyzed reactions, QSARs obtained showed that the reduction efficacy of BFXs, as well as that of NACs, was determined by their electron-accepting potency and could be influenced by their binding mode in the active center of NQO1 and by their global softness as their electronic characteristic. The reductive conversion of benzofuroxan by both flavoenzymes yielded the same reduction product of benzofuroxan, 2,3-diaminophenazine, with the formation of o-benzoquinone dioxime as a putative primary reductive intermediate, which undergoes a further reduction process. Overall, the data obtained show that by contrast to NACs, the flavoenzyme-catalyzed reduction of BFXs is unlikely to initiate their redox-cycling, which may argue for a minor role of the redox-cycling-type action in the cytotoxicity of BFXs.

Single-electron reduction of quinone and nitroaromatic xenobiotics by recombinant rat neuronal nitric oxide synthase.

We examined the kinetics of single-electron reduction of a large number of structurally diverse quinones and nitroaromatic compounds, including a number of antitumour and antiparasitic drugs, and nitroaromatic explosives by recombinant rat neuronal nitric oxide synthase (nNOS, EC 1.14.13.39), aiming to characterize the role of nNOS in the oxidative stress-type cytotoxicity of the above compounds. The steady-state second-order rate constants (kcat/Km) of reduction of the quinones and nitroaromatics varied from 10² M⁻¹s⁻¹ to 106 M⁻¹s⁻¹, and increased with an increase in their single-electron reduction potentials (E¹7). The presence of Ca²âº/calmodulin enhanced the reactivity of nNOS. These reactions were consistent with an 'outer sphere' electron-transfer mechanism, considering the FMNH∙/FMNH2 couple of nNOS as the most reactive reduced enzyme form. An analysis of the reactions of nNOS within the 'outer sphere' electron-transfer mechanism gave the approximate values of the distance of electron transfer, 0.39-0.47 nm, which are consistent with the crystal structure of the reductase domain of nNOS. On the other hand, at low oxygen concentrations ([O2] = 40-50 µM), nNOS performs a net two-electron reduction of quinones and nitroaromatics. This implies that NOS may in part be responsible for the bioreductive alkylation by two-electron reduced forms of antitumour aziridinyl-substituted quinones under a modest hypoxia.

Redox properties and prooxidant cytotoxicity of a neuroleptic agent 6,7-dinitrodihydroquinoxaline-2,3-dione (DNQX).

In order to characterize the possible mechanism(s) of cytotoxicity of a neuroleptic agent 6,7-dinitrodihydroquinoxaline-2,3-dione (DNQX) we examined the redox properties of DNQX, and its mononitro- (NQX) and denitro- (QX) derivatives. The irreversible electrochemical reduction of the nitro groups of DNQX was characterized by the reduction peak potentials (Ep,7) of -0.43 V and -0.72 V vs. Ag/AgCl at pH 7.0, whereas NQX was reduced at Ep,7 = -0.67 V. The reactivities of DNQX and NQX towards the single-electron transferring enzymes NADPH:cytochrome P-450 reductase and NADPH:adrenodoxin reductase/adrenodoxin complex were similar to those of model nitrobenzenes with the single-electron reduction potential (E¹7) values of -0.29 V - -0.42 V. DNQX and NQX also acted as substrates for two-electron transferring mammalian NAD(P)H:quinone oxidoreductase (DT-diaphorase). The cytotoxicity of DNQX in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was prevented by antioxidants and an inhibitor of NQO1, dicoumarol, and was enhanced by the prooxidant alkylating agent 1,3-bis(2-chloromethyl)-1-nitrosourea. A comparison with model nitrobenzene compounds shows that the cytotoxicity of DNQX and NQX reasonably agrees with the ease of their electrochemical reduction, and/or their reactivities towards the used enzymatic single-electron reducing systems. Thus, our data imply that the cytotoxicity of DNQX in FLK cells is exerted mainly through oxidative stress.