Regioisomerization of Antimalarial Drug WR99210 Explains the Inactivity of a Commercial Stock.

WR99210, a former antimalarial drug candidate now widely used for the selection of Plasmodium transfectants, selectively targets the parasite's dihydrofolate reductase thymidine synthase bifunctional enzyme (DHFR-TS) but not human DHFR, which is not fused with TS. Accordingly, WR99210 and plasmids expressing the human dhfr gene have become valued tools for the genetic modification of parasites in the laboratory. Concerns over the ineffectiveness of WR99210 from some sources encouraged us to investigate the biological and chemical differences of supplies from two different companies (compounds 1 and 2). Compound 1 proved effective at low nanomolar concentrations against Plasmodium falciparum parasites, whereas compound 2 was ineffective, even at micromolar concentrations. Intact and fragmented mass spectra indicated identical molecular formulae of the unprotonated (free base) structures of compounds 1 and 2; however, the compounds displayed differences by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and UV-visible spectroscopy, indicating important isomeric differences. Structural evaluations by 1H, 13C, and 15N nuclear magnetic resonance spectroscopy confirmed compound 1 as WR99210 and compound 2 as a dihydrotriazine regioisomer. Induced fit computational docking models showed that compound 1 binds tightly and specifically in the P. falciparum DHFR active site, whereas compound 2 fits poorly to the active site in loose and varied orientations. Stocks and concentrates of WR99210 should be monitored for the presence of regioisomer 2, particularly when they are not supplied as the hydrochloride salt or are exposed to basic conditions that may promote rearrangement. Absorption spectroscopy can serve for assays of the unrearranged and rearranged triazines.

Absolute Configurations of Stink Bug- and Plant-Produced Sesquipiperitols via Synthesis of All Stereoisomers.

Sesquipiperitol is a sesquiterpene alcohol, some stereoisomers of which were found in several plant species. The biological role of these compounds in plants and their absolute configurations have not been reported. Recently, we found that 1S,6S,7R stereoisomer of sesquipiperitol was a key precursor in the biosynthesis of the harlequin bug, Murgantia histrionica, pheromone, which consists of two stereoisomeric zingiberenol oxides. In addition, the Tibraca limbativentris stink bug was shown to produce two sesquipiperitol stereoisomers as minor components in their male-produced sex pheromone, the main constituents of which were zingiberenols. To determine absolute configurations of plant- and stink-bug-produced sesquipiperitols, we undertook syntheses of all stereoisomers of this sesquiterpene alcohol. The syntheses were based on 1,10-bisaboladien-3-ols (aka zingiberenols) with known configurations at C-6 and C-7, the oxidation of which provided sesquipiperitone precursors with retention of configurations of these stereogenic centers. The foremost challenge of the synthetic endeavor was the assignment of absolute configurations of secondary carbinol centers, which was resolved by NMR analyses of corresponding Mosher's esters. Thus, the availability of all eight diastereomers allowed us to assign sesquipiperitols from Fitzroya cupressoides and Argyranthemum adauctum spp. jacobaeifolium plants 1S,6S,7R (16) and 1R,6R,7S (14) configurations, respectively. A chiral-phase gas-chromatographic method was developed to determine 1S,6S,7R and 1R,6S,7R (15) configurations of T. limbativentris sesquipiperitol pheromone components.