Structure-activity relationships of tetramethylpiperidine-substituted phenazines against Mycobacterium leprae in vitro

Abstract

In a previous study of structure-activity relationships of selected phenazines against Mycobacterium leprae in vitro, compounds containing a 2,2,6,6-tetramethylpiperidine substitution at the imino nitrogen were most active. Therefore, the effect of substitution at the para positions of the phenyl and anilino groups in tetramethylpiperidine-substituted phenazines was assessed. As determined by radiorespirometry, activity in ascending order was observed in compounds substituted with hydrogens or fluorines, ethoxy groups, methyl groups, chlorines, and bromines and correlated with partition coefficients in octanol-water.Despite its dose-limiting gastrointestinal toxicity and doserelated skin pigmentation, clofazimine (B663) remains a firstline drug in leprosy chemotherapy (1,14). Although a wide variety of structural analogs of B663 has been synthesized (11), only a limited number of compounds have been evaluated in the 6-to 12-month mouse footpad model (13), which until recently was the only available screening system for new antileprotics. An examination of six structural analogs of clofazimine in a Mycobacterium leprae-macrophage in vitro system failed to identify compounds with superior activity (10).Using a radiorespirometric metabolic assay, we have been rapidly and inexpensively evaluating a large number of antimicrobial agents for antileprosy activity in vitro (3-8). Recently we assessed 12 phenazines which (besides not being expected to accumulate in body fat) differ from clofazimine in substitution at the imino nitrogen and in some cases in chlorination of the phenazine nucleus or anilino and phenyl rings (6). In general, activity increased with the degree of chlorination. Phenazines containing a 2,2,6,6-tetramethylpiperidine substitution (TMP) at the imino nitrogen were clearly the most active compounds in the series, with the monochloro (B4019) and dichloro (B3786) analogs displaying essentially identical activity.We describe here the in vitro assessment of substitution at the para positions of the anilino and phenyl groups of TMP, with a view toward identifying nonpigmenting compounds with high activity against M. leprae.Susceptibility testing was performed essentially as previously described (6). Briefly, footpads of M. leprae-infected, athymic nude mice were surface decontaminated, and bacilli were obtained by homogenization of the footpads in Dubosalbumin medium (pH 6.5) followed by differential centrifugation. Samples were removed for contamination checks on a variety of culture media. The remaining M. leprae suspension was held at 4°C for 2 days, during which time the culture medium was observed to confirm the absence of contamination. The suspension was then diluted in Dubos-albumin medium to a final density of 107/ml and distributed in 1-ml aliquots to 6-ml glass screw-cap vials.

Drug susceptibility testing of Mycobacterium leprae in the BACTEC 460 system

“…Overall, the results were consistent with those found by using palmitic acid oxidation in the Buddemeyer system, i.e., clofazimine being active at .1 ,ug/ml (4,5), ethionamide showing significant activity at 2 ,ug/ml (1), and clarithromycin showing activity equivalent to rifampin (3).…”

“…Among these was the use of a Buddemeyer-type detection system to measure 14CO2 production from [1-14C]palmitic acid by M. leprae in an axenic medium (1). This system is very sensitive, and its simplicity has facilitated screening of a variety of macrolides (3), clofazimine derivatives (4,5), and fluoroquinolones (S. G. Franzblau ) consists of (final concentrations) polymyxin B (50 U/ml), amphotericin B (5 pg/ml), nalidixic acid (20 pug/ml), trimethoprim (5 jig/ml), and azlocillin (10 ,ug/ml). Susceptibility testing.…”

Comparative in vitro activities of 20 fluoroquinolones against Mycobacterium leprae

“…We have employed radiorespirometry to evaluate a variety of macrolides (6) and clofazimine derivatives (7,8). In addition, we have recently demonstrated that the BACTEC 460 system can also be used in the radiorespirometric assessment of drug activity against M. Ieprae in the absence of increases in cell numbers (5).…”

Fusidic acid is highly active against extracellular and intracellular Mycobacterium leprae

“…We have previously demonstrated the utility of radiorespirometry in evaluating the in vitro activities of a variety of macrolides (9), fluoroquinolones (11), phenazines (10,12), and aminoglycosides, lincosamides, and rifamycins (7) against M. leprae in axenic media with either a Buddemeyertype detection system (5) or the BACTEC 460 instrument (6) as a means of assessing drug activity in the absence of bacterial multiplication. We and our colleagues have also used assays quantitating phenolic glycolipid-1 synthesis and adenosine triphosphate concentration (8) as indices of drug activity against both extracellular (16) and intracellular (20) M. leprae.…”

“…In vitro screening for drugs active against the uncultivable leprosy bacillus can be performed by radiorespirometric analysis of viable Mycobacterium leprae freshly harvested from nude mice (5)(6)(7)(9)(10)(11)(12). In vitro systems have many advantages over the traditional mouse footpad system (22), especially with regard to time, expense, quantity of drug required, and independence of mouse pharmacokinetics.…”