Mono-sulfonated tetrazolium salt based NAD(P)H detection reagents suitable for dehydrogenase and real-time cell viability assays.

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and is important for several biological processes. For GDH inhibitor screening, we developed a novel mono-sulfonated tetrazolium salt (EZMTT), which can be synthesized using H2O2 oxidation and purified easily on silica gel in large quantities. The EZMTT detection method showed linear dose responses to NAD(P)H, dehydrogenase concentration and cell numbers. In E. coli GDH assay, the EZMTT method showed excellent assay reproducibility with a Z factor of 0.9 and caused no false positives in the presence of antioxidants (such as BME). Using the EZMTT-formazan-NAD(P)H system, we showed that EGCG is a potent E. coli GDH inhibitor (IC50 45 nM) and identified that Ebselen, a multifunctional thioredoxin reductase inhibitor, inactivated E. coli GDH (IC50 213 nM). In cell-based assays at 0.5 mM tetrazolium concentration, EZMTT showed essentially no toxicity after a 3-day incubation, whereas 40% of inhibition was observed for WST-8. In conclusion, EZMTT is a novel tetrazolium salt which provides improved features that are suitable for dehydrogenases and real-time cell-based high-throughput screening (HTS).

Synthesis of angiotensin II receptor blockers by means of a catalytic system for C-H activation.

A highly efficient catalytic system for C-H activation has been worked out that involves inexpensive RuCl(3)·xH(2)O and a specific amount of PPh(3). This procedure has been successfully applied to a practical synthesis of angiotensin II receptor blockers (ARBs). The residual ruthenium that existed in the reaction mixture was thoroughly removed by treatment with properly selected metal scavengers. The new process permits ready access to the important class of drugs in a highly atom-economical and sustainable manner.

Tetrazolium salts and formazans.

The history of the tetrazolium salts and formazans goes back 100 years, to when Friese (1875) reacted benzene diazonium nitrate with nitromethane, to produce a cherry-red "Neue Verbindung". This was the first formazan. 19 years later, Von Pechmann and Runge (1894) oxidised a formazan to produce the first tetrazolium salt. Many hundreds of tetrazolium salts and formazans were prepared in the following years, but only a handful have found applications in biological research. This article has attempted to describe the properties of these compounds, and to illustrate how the tetrazolium salt-formazan reaction has been exploited to serve an extremely wide variety of functions.

Synthesis of some newer formazans and tetrazolium salts as antiviral agents.

Thirteen new formazans were prepared by the condensation of the phenylhydrazone of 3.4-dimethoxy-6-nitro-veratraldehyde with the appropriate phenyl diazonium salts. Attempts were made to oxidize these highly coloured formazans with various oxidizing agents to their corresponding tetrazolium salts. The most suitable oxidizing agent was found to be H2O2/Fe2+. Both the formazans and tetrazolium salts were screened for their antiviral activity against the Ranikhet disease virus and vaccinia virus in a stationary culture of chorioallantoic membrane of chick embryo. Among the 15 compounds tested one of the compounds namely 1-o-carboxyphenyl-3[3'.4'-dimethoxy-6'-nitrophenyl]-5-phenylformazan evinced 100% protection against the Ranikhet disease virus. Rest of the compounds showed significant protection ranging from 83 to 20%. An attempt has also been made to arrive at some structure-activity relationship.

Synthesis of some newer formazans and tetrazolium salts and their effect on Ranikhet disease virus and the vaccinia virus.

Synthesis and biological evaluation of a series of formazans and their corresponding tetrazolium salts are described. Several 1,3,5-trisubstituted formazans were prepared by condensing 4-acetoxy-3-methoxybenzaldehydephenylhydrazone and 2,4-dinitrophenylhydrazone of 2-benzimidazolecarboxaldehyde, with an appropriate phenyldiazonium salt. The formazans derived from 4-acetoxy-3-methoxybenzaldehydephenylhydrazone were further oxidized to their corresponding cyclization products, 2,3,5-trisubstituted tetrazolium salts. Oxidation of 1-aryl-3-(2'-benzimidazolyl)-5-(5-(2',4'-dinitrophenyl) formazans to their corresponding tetrazolium salt did not meet with success. Both the highly coloured formazans and water-soluble tetrazolium salts were screened for their antiviral, activity against vaccinia virus and Ranikhet disease virus in a stationary culture of chorioallantoic membrane of chick embryo. Two of the compounds were found to exhibit significant activity (87% and 83%) against the Ranikhet disease virus. However no activity against vaccinia virus could be observed.

Fluorescent redox dyes. 1. Production of fluorescent formazan by unstimulated and phorbol ester- or digitonin-stimulated Ehrlich ascites tumor cells.

The reduction of a new series of tetrazolium salts to red fluorescent formazans by Ehrlich ascites tumor cells is described. The qualitative effect on this reaction of two cell surface-active compounds and of six exogenous electron carriers was investigated by varying the incubation conditions. After incubation of Ehrlich ascites cells with the new colourless, water soluble 5-cyan-2.3-ditolyltetrazolium salts, bright red water-insoluble formazan crystals on the cell surface can be observed under fluorescence microscopy. The production of formazan is enhanced by 12-0-tetradecanoyl-phorbol-13-acetate (TPA) or digitonin (DIG), two potent stimulators of oxygen consumption or by the electron carriers phenazine methosulphate (PMS), 1-methoxy-phenazine methosulphate (MPMS), meldola blue (MB), methylene blue (MTB), and 2.6-dichlorindophenol (DCIP). These results provide further evidence for the existence of redox enzymes bound to the plasma membrane of intact ascites cells and for a free radical mechanism of tetrazolium salt reduction. The fluorescence property of the new redox dyes offers the advantage of high sensitivity. Moreover, their greater homogeneity relative to the commonly used di-tetrazolium salts lowers the chances of misinterpretations due to impurities. The possible application of these new mono-tetrazolium salts to cytochemical investigations of oxidative metabolic reactions is discussed.

Newer formazans and tetrazolium indoles as potential CNS-active agents.

Some new 2-aryl-5-(3'-indole)-3'-substituted phenyl tetrazolium bromide/iodide salts (IV) were synthesized by the oxidation of 1-aryl-3-(3'-indole)-5-substituted phenyl fromazans (III). These compounds III and IV were evaluated for their monoamine oxidase (MAO) inhibitory activity in vitro and various CNS activities in vivo. Several compounds exhibited promising CNS activities, III-b being the most active member of the present series showing marked antidepressant and MAO inhibitory activities and having low toxicity.

Tetrazolium salts and metal complexes of some formazans.

This paper presents the synthesis of some tetrazolium salts and metal complexes combinations, which are derived from aromatic and heterocyclic formazans. Elemental quantitative analyses and spectral data confirmed the structure of the new synthesized compounds. The new synthesized compounds were submitted to microbiological tests.