Fluorescently labelled thioacetazone for detecting the interaction with Mycobacterium dehydratases HadAB and HadBC.

Thioacetazone (TAC) used to be a highly affordable, bacteriostatic anti-TB drug but its use has now been restricted, owing to severe side-effects and the frequent appearance of the TAC resistant M. tuberculosis strains. In order to develop new TAC analogues with fewer side-effects, its target enzymes need to be firmly established. It is now hypothesized that TAC, after being activated by a monooxygenase EthA, binds to the dehydratase complex HadAB that finally leads to a covalent modification of HadA, the main partner involved in dehydration. Another dehydratase enzyme, namely HadC in the HadBC complex, is also thought to be a possible target for TAC, for which definitive evidence is lacking. Herein, using a recently exploited azido naphthalimide template attached to thioacetazone and adopting a photo-affinity based labelling technique, coupled with electrophoresis and in-gel visualization, we have successfully demonstrated the involvement of these enzymes including HadBC along with a possible participation of an alternate mycobacterial monooxygenase MymA. In silico studies also revealed strong interactions between the TAC-probe and the concerned enzymes.

Further Insights into the Oxidative Pathway of Thiocarbonyl-Type Antitubercular Prodrugs: Ethionamide, Thioacetazone, and Isoxyl.

A chemical activation study of the thiocarbonyl-type antitubercular prodrugs, ethionamide (ETH), thioacetazone (TAZ), and isoxyl (ISO), was performed. Biomimetic oxidation of ethionamide using H2O2 (1 equiv) led to ETH-SO as the only stable S-oxide compound, which was found to occur in solution in the preferential form of a sulfine (ETH═S═O vs the sulfenic acid tautomer ETH-S-OH), as previously observed in the crystal state. It was also demonstrated that ETH-SO is capable of reacting with amines, as the putative sulfinic derivative (ETH-SO2H) was supposed to do. Unlike ETH, oxidation of TAZ did not allow observation of the mono-oxygenated species (TAZ-SO), leading directly to the more stable sulfinic acid derivative (TAZ-SO2H), which can then lose a SOxH group after further oxidation or when placed in a basic medium. It was also noticed that the unstable TAZ-SO intermediate can lead to the carbodiimide derivative as another electrophilic species. It is suggested that TAZ-SOH, TAZ-SO2H, and the carbodiimide compound can also react with NH2-containing nucleophilic species, and therefore be involved in toxic effects. Finally, ISO showed a very complex reactivity, here assigned to the coexistence of two mono-oxygenated structures, the sulfine and sulfenic acid tautomers. The mono- and dioxygenated derivatives of ISO are also highly unstable, leading to a panel of multiple metabolites, which are still reactive and likely contribute to the toxicity of this prodrug.

Preclinical pharmacokinetics of KBF611, a new antituberculosis agent in mice and rabbits, and comparison with thiacetazone.

Thiacetazone (TAZ), one of the oldest known antituberculosis drugs, causes severe skin reactions in patients co-infected with tuberculosis and human immunodeficiency virus (HIV). KBF611 is a new fluorinated thiacetazone analogue that has shown strong antituberculosis effects. In order to provide valuable information for subsequent preclinical development, pharmacokinetics of KBF611 and its analogue (TAZ) were studied and compared in two animal species (mice and rabbits) following intravenous and oral administration, and pharmacokinetic parameters were characterized. According to the calculated parameters, KBF611 showed a more favourable pharmacokinetics profile than TAZ in terms of half-life (0.89 h compared with 0.57 in mice, p < 0.05, and 2.71 compared with 0.98 in rabbits, p < 0.001) and volume of distribution (1.45 l kg(-1) compared with 0.86 l kg(-1) in mice, p < 0.05, and 1.01 l kg(-1) compared with 0.41 l kg(-1) in rabbits, p < 0.001) for tuberculosis therapy. In rabbits, the oral bioavailability of KBF611 was markedly lower than mice (39% compared with 82%), which may be attributed to a higher presystemic metabolism in rabbit liver. The results of in vivo studies on the metabolism of KBF611, supported by liquid chromatography-mass spectrometry (LC-MS) analysis, showed that the incorporation of a fluorine atom to the TAZ structure made the molecule susceptible to N-deacetylation, a pathway not seen in TAZ metabolism. In summary, KBF611 could be considered a suitable candidate for further preclinical and clinical evaluation.

Simultaneous determination of a new antituberculosis agent KBF-611 and its de-acetylated metabolite in mouse and rabbit plasma by HPLC.

KBF-611 is a new thiosemicarbazone derivative which has demonstrated significant antituberculosis effect. A sensitive and specific HPLC method was established and validated for the determination of KBF-611 and its deacetylated metabolite (KM) in mouse and rabbit plasma. Chromatographic separation was achieved on a Eurospher-100 C8 column using acetonitrile, methanol, phosphate buffer (pH 7) and TEA (25:5:70:0.1, v/v), as mobile phase at a flow rate of 1 mL/min. KBF-611, KM and internal standard (4-acetamido-3-chlorobenzaldehyde thiosemicarbazone) were detected at the wavelength of 323 nm. The calibration curves were linear within the concentration range from 0.02-5 microg/mL and 0.02-1 microg/mL for KBF-611 and KM respectively. The limit of detection and the limit of quantitation were 6 ng/mL and 20 ng/mL respectively for both KBF-611 and KM. The relative standard deviation for intra- and inter-day precision was less than 7.5%. Average recoveries were 70.8% and 75.0% for KBF-611 and KM respectively. The established HPLC method was validated to be a simple, rapid and reliable procedure and successfully applied to study the preclinical pharmacokinetics of KBF-611 and KM in mice and rabbits.

The lipophilicity estimation of 5-arylidene derivatives of (2-thio)hydantoin with antimycobacterial activity.

The lipophilicity of antituberculotic 5-arylidene derivatives of (thio)hydantoin, thioacetazone and isoniazid has been determined by reversed-phase thin-layer chromatography (RP-TLC). Mixtures of acetone and water (with acetone content 60-85%) were used as the mobile phase. The R(M) (relative lipophilicity) of each compound (except isoniazid) decreased linearly with the increasing concentration of acetone. The partition coefficients (log P) of the compounds were calculated by the use of eight computer programs (ClogP, KowWin, XlogP, AlogPs, CAChe, Pallas, Interactive analysis and Slipper) and compared with the experimental lipophilicity (R(M0)). According to the observations the best tools for in silico predicting log P of (2-thio)hydantoins are programs KowWin and AlogPs.