Sujata S. Shinde

Synthesis, Reduction Potentials, and Antitubercular Activity of Ring A/B Analogues of the Bioreductive Drug (6S)-2-Nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (PA-824)

The nitroimidazooxazine S-1 (PA-824) is a new class of bioreductive drug for tuberculosis. A series of related bicyclic nitroheterocycles was synthesized, designed to have a wide range of one-electron reduction potentials E(1) (from -570 to -338 mV, compared with -534 mV for S-1). The observed E(1) values closely correlated with the sigma(m) values of the heteroatom at the 4/8-position of the adjacent six-membered ring. Although the compounds spanned a range of E(1) values around that of S-1, only the nitroimidazothiazines showed significant antitubercular activity (at a similar level of potency), suggesting that E(1) is not the main driver of efficacy. Furthermore, there was a correlation between activity and the formation of imidazole ring-reduced products at the two-electron level, pointing to the potential importance of this reduction pathway, which is determined by the nature of the substituent at the 2-position of the 4-nitroimidazole ring.

Tricyclic [1,2,4]triazine 1,4-dioxides as hypoxia selective cytotoxins.

A series of novel tricyclic triazine-di- N-oxides (TTOs) related to tirapazamine have been designed and prepared. A wide range of structural arrangements with cycloalkyl, oxygen-, and nitrogen-containing saturated rings fused to the triazine core, coupled with various side chains linked to either hemisphere, resulted in TTO analogues that displayed hypoxia-selective cytotoxicity in vitro. Optimal rates of hypoxic metabolism and tissue diffusion coefficients were achieved with fused cycloalkyl rings in combination with both the 3-aminoalkyl or 3-alkyl substituents linked to weakly basic soluble amines. The selection was further refined using pharmacokinetic/pharmacodynamic model predictions of the in vivo hypoxic potency (AUC req) and selectivity (HCD) with 12 TTO analogues predicted to be active in vivo, subject to the achievement of adequate plasma pharmacokinetics.

Absence of an effect of high nitrate intake from beetroot juice on blood pressure in treated hypertensive individuals: a randomized controlled trial

BACKGROUND Dietary nitrate, which is in green leafy vegetables and beetroot, decreases blood pressure through the enterosalivary nitrate-nitrite-nitric oxide pathway in healthy individuals. Whether similar effects would occur in individuals with treated hypertension and, therefore, at increased risk of cardiovascular disease is unclear. OBJECTIVE We assessed whether increased dietary nitrate intake by using beetroot juice for 1 wk lowers blood pressure in treated hypertensive men and women. DESIGN Participants (n = 27) were recruited to a randomized, placebo-controlled, double-blind crossover trial. The effect of 1-wk intake of nitrate-rich beetroot juice was compared with 1-wk intake of nitrate-depleted beetroot juice (placebo). The primary outcome was blood pressure assessed by measuring home blood pressure during the intervention and 24-h ambulatory blood pressure on day 7 of the intervention. Other outcomes included nitrate metabolism assessed by measuring nitrate and nitrite in plasma, saliva, and urine. RESULTS Relative to the placebo, 1-wk intake of nitrate-rich beetroot juice resulted in a 3-fold increase in plasma nitrite and nitrate, a 7-fold increase in salivary nitrite, an 8-fold higher salivary nitrate, and a 4-fold increase in both urinary nitrite and nitrate (P < 0.001). However, no differences in home blood pressure and 24-h ambulatory blood pressure were observed with 1-wk intake of nitrate-rich beetroot juice in comparison with the placebo. CONCLUSION An increase in dietary nitrate intake may not be an effective short-term approach to further lower blood pressure in treated hypertensive subjects.

The nitroxide TEMPO is an efficient scavenger of protein radicals: Cellular and kinetic studies

Protein oxidation occurs during multiple human pathologies, and protein radicals are known to induce damage to other cell components. Such damage may be modulated by agents that scavenge protein radicals. In this study, the potential protective reactions of the nitroxide TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxyl radical) against Tyr- and Trp-derived radicals (TyrO./TrpN.) have been investigated. Pretreatment of macrophage cells with TEMPO provided protection against photo-oxidation-induced loss of cell viability and Tyr oxidation, with the nitroxide more effective than the hydroxylamine or parent amine. Pulse radiolysis was employed to determine rate constants, k, for the reaction of TEMPO with TyrO. and TrpN. generated on N-Ac-Tyr-amide and N-Ac-Trp-amide, with values of k~10(8) and 7×10(6)M(-1)s(-1), respectively, determined. Analogous studies with lysozyme, chymotrypsin, and pepsin yielded k for TEMPO reacting with TrpN. ranging from 1.5×10(7) (lysozyme) to 1.1×10(8) (pepsin)M(-1)s(-1). Pepsin-derived TyrO. reacted with TEMPO with k~4×10(7)M(-1)s(-1); analogous reactions for lysozyme and chymotrypsin TyrO. were much slower. These data indicate that TEMPO can inhibit secondary reactions of both TyrO. and TrpN., though this is protein dependent. Such protein radical scavenging may contribute to the positive biological effects of nitroxides.

Intermediates in the reduction of the antituberculosis drug PA-824, (6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine, in aqueous solution

The reduction chemistry of the new anti-tuberculosis drug PA-824, together with a more water-soluble analogue, have been investigated using pulse and steady-state radiolysis in aqueous solution. Stepwise reduction of these nitroimidazo-dihydrooxazine compounds through electron transfer from the CO(2) (-) species revealed that, unlike related nitroimidazoles, 2-electron addition resulted in the reduction of the imidazole ring in preference to the nitro group. In mildly acidic solution a nitrodihydroimidazo intermediate was formed, which was reduced further to the amine product. In both alkaline and neutral solution, an intermediate produced on 2-electron reduction was resistant to further reduction and reverted to parent compound on extraction or mass spectrometric analysis of the solution. The unusual reduction chemistry of these nitroimidazole compounds, exhibiting ring over nitro group reduction, is associated with alkoxy substitution in the 2-position of a 4-nitroimidazole. The unique properties of the intermediates formed on the reduction of PA-824 need to be considered as playing a possible role in its bactericidal action.

Characterization of Radicals Formed Following Enzymatic Reduction of 3-Substituted Analogues of the Hypoxia-Selective Cytotoxin 3-Amino-1,2,4-Benzotriazine 1,4-Dioxide (Tirapazamine)

The mechanism by which the 1,2,4-benzotriazine 1,4-dioxide (BTO) class of bioreductive hypoxia-selective prodrugs (HSPs) form reactive radicals that kill cancer cells has been investigated by steady-state radiolysis, pulse radiolysis (PR), electron paramagnetic resonance (EPR), and density functional theory (DFT) calculations. Tirapazamine (TPZ, 3-amino BTO, 1) and a series of 3-substituted analogues, -H (2), -methyl (3), -ethyl (4), -methoxy (5), -ethoxymethoxy (6), and -phenyl (7), were reduced in aqueous solution under anaerobic steady-state radiolysis conditions, and their radicals were found to remove the substrates by short chain reactions of different lengths in the presence of formate ions. Multiple carbon-centered radical intermediates, produced upon anaerobic incubation of the compounds with cytochrome P(450) reductase enriched microsomes, were trapped by N-tert-butyl-alpha-phenylnitrone and observed using EPR. The highly oxidizing oxymethyl radical, from compound 5, was identified, and experimental spectra obtained for compounds 1, 2, 3, and 7 were well simulated after the inclusion of aryl radicals. The identification of a range of oxidizing radicals in the metabolism of the BTO compounds gives a new insight into the mechanism by which these HSPs can cause a wide variety of damage to biological targets such as DNA.

One-Electron Reduction Potential of the Neutral Guanyl Radical in the GC Base Pair of Duplex DNA

The one-electron oxidation of guanine in the GC base pair of DNA has been investigated using pulse radiolysis combined with DFT calculations. Reaction of benzotriazinyl radicals with DNA results in the formation of the neutral guanyl radical and redox equilibria. The one-electron reduction potential, E(7), of the neutral guanyl radical in the GC base pair is determined for the first time as 1.22 +/- 0.02 V, from both absorption and kinetic data.

Development of a reference database for assessing dietary nitrate in vegetables

SCOPE Nitrate from vegetables improves vascular health with short-term intake. Whether this translates into improved long-term health outcomes has yet to be investigated. To enable reliable analysis of nitrate intake from food records, there is a strong need for a comprehensive nitrate content of vegetables database. METHODS AND RESULTS A systematic literature search (1980-2016) was performed using Medline, Agricola and Commonwealth Agricultural Bureaux abstracts databases. The nitrate content of vegetables database contains 4237 records from 255 publications with data on 178 vegetables and 22 herbs and spices. The nitrate content of individual vegetables ranged from Chinese flat cabbage (median; range: 4240; 3004-6310 mg/kg FW) to corn (median; range: 12; 5-1091 mg/kg FW). The database was applied to estimate vegetable nitrate intake using 24-h dietary recalls (24-HDRs) and food frequency questionnaires (FFQs). Significant correlations were observed between urinary nitrate excretion and 24-HDR (r = 0.4, P = 0.013), between 24-HDR and 12 month FFQs (r = 0.5, P < 0.001) as well as two 4 week FFQs administered 8 weeks apart (r = 0.86, P < 0.001). CONCLUSION This comprehensive nitrate database allows quantification of dietary nitrate from a large variety of vegetables. It can be applied to dietary records to explore the associations between nitrate intake and health outcomes in human studies.

Inhibition of radical-induced DNA strand breaks by water-soluble constituents of coffee: phenolics and caffeine metabolites

Abstract Epidemiological studies have associated coffee consumption with an inverse risk of developing Parkinsons disease, hepatocellular carcinoma and cirrhosis. The molecular mechanisms by which low concentrations of the constituents of coffee measured in human plasma can reduce the incidence of such diseases are not clear. Using an in vitro plasmid DNA system and radiolytically generated reactive oxygen species under constant radical scavenging conditions, we have shown that coffee chlorogenic acid, its derivatives and certain metabolites of caffeine reduce some of the free radical damage sustained to the DNA. A reduction in the amount of prompt DNA single-strand breaks (SSBs) was observed for all compounds whose radical one-electron reduction potential is < 1.0 V. However, except for chlorogenic acid, the compounds were found to be inactive in reducing the amount of radical damage to the DNA bases. These results support a limited antioxidant role for such compounds in their interaction with DNA radicals.

Acute effects of quercetin-3-O-glucoside on endothelial function and blood pressure: a randomized dose-response study

BACKGROUND Epidemiologic studies have suggested that a flavonoid-rich diet can reduce the risk of developing cardiovascular disease. Certain flavonoids, in particular quercetin, have been shown to ameliorate endothelial dysfunction and reduce blood pressure (BP), possibly by increasing the bioavailability of the potent vasodilator nitric oxide (NO). Several studies have indicated that improvements in measures of cardiovascular health do not occur linearly, but rather, plateau or decrease with an increasing dose of flavonoids. OBJECTIVES We determined whether the acute administration of increasing doses of a common quercetin glycoside (quercetin-3-O-glucoside) improves endothelial function and reduces BP in a dose-dependent manner. We also explored whether any effects were correlated with changes in plasma NO production. DESIGN A randomized, controlled, crossover study was performed in 15 healthy volunteers who each completed 5 visits with a minimum washout period of 1 wk between testing days. Participants received each of the following 5 interventions in a random order: 1) 0, 2) 50, 3) 100, 4) 200, or 5) 400 mg quercetin-3-O-glucoside. Endothelial function and BP were assessed before and 60 min after intervention. A blood sample was taken before and 90 min after intervention for the analysis of plasma nitrate and nitrite as markers of NO production as well as of plasma quercetin metabolites. RESULTS Although we observed a significant correlation between the dose of quercetin-3-O-glucoside and plasma concentrations of total quercetin (R(2) = 0.52, P < 0.001) and isorhamnetin (R(2) = 0.12, P = 0.005), we showed no improvements in endothelial function or BP and no changes in NO production after any dose. CONCLUSION From these results, we conclude that there are no acute changes in BP or the NO-mediated endothelium-dependent relaxation of the brachial artery with doses of quercetin ranging from 50 to 400 mg in healthy men and women. This trial was registered at www.anzctr.org.au as ACTRN12615001338550.