Discovery of the development candidate N-tert-butyl nodulisporamide: a safe and efficacious once monthly oral agent for the control of fleas and ticks on companion animals.

Nodulisporic acid A (1) is a structurally complex fungal metabolite that exhibits systemic efficacy against fleas via modulation of an invertebrate specific glutamate-gated ion channel. In order to identify a nodulisporamide suitable for monthly oral dosing in dogs, a library of 335 nodulisporamides was examined in an artificial flea feeding system for intrinsic systemic potency as well as in a mouse/bedbug assay for systemic efficacy and safety. A cohort of 66 nodulisporamides were selected for evaluation in a dog/flea model; pharmacokinetic analysis correlated plasma levels with flea efficacy. These efforts resulted in the identification of the development candidate N-tert-butyl nodulisporamide (3) as a potent and efficacious once monthly oral agent for the control of fleas and ticks on dogs and cats which was directly compared to the topical agents fipronil and imidacloprid, with favorable results obtained. Multidose studies over 3 months confirmed the in vivo ectoparasiticidal efficacy and established that 3 lacked overt mammalian toxicity. Tissue distribution studies in mice using [(14)C]-labeled 3 indicate that adipose beds serve as ligand depots, contributing to the long terminal half-lives of these compounds.

Interpretation of high-throughput liquid chromatography mass spectrometry data for quality control analysis and analytical method development.

An approach to rapidly process and interpret high-throughput liquid chromatography mass spectrometry data is presented. This approach applies an in-house developed computer application to process LC-MS report files containing spectral and chromatographic data from four different detectors (i.e. electrospray positive ionization, electrospray negative ionization mass spectrometry, UV absorption, and evaporative light scattering detection). Properties characteristic of detection and chromatographic retention are extracted and populated into a database. Approaches to applying this analytical information database for quality control analysis of ca. 400,000 samples are presented. Compound quality assessment methods employing average purity and detection data fields are compared to methods employing multiple quality control criteria (e.g. detection, purity, retention, and signal to noise). Structural similarity searches were applied with the analytical information database to identify compounds that may be undetectable by electrospray mass spectrometry. In addition, an approach to applying the database to aid in the selection of analytical detection and chromatography conditions for rapid analytical method development is also discussed.

Nodulisporic acid side-chain modifications: access to the 2", 3", 4", and 6" registers.

Efficient routes to access the 2", 3", 4", and 6" registers of the nodulisporic acid (NsA) side chain are disclosed. A mild one-carbon, Ph(2)CdoublebondNCH(2)CtriplebondN mediated homologation of NsA's 3"-aldehyde permitted access to the 4"-register. Curtius reaction of NsA's 3"-acid yielded the corresponding 2"-aldehyde 4 from which the unnatural Delta(2",3")-olefin isomer 2b was obtained. In addition, Arndt-Eistert reactions of the parent NsA permitted a one-carbon homologation to the 6" register. These efforts identified new analogues with significant flea activity and illustrated the biological significance of unsaturation at the 1",2" register.

Side-chain homologation of nodulisporic acid: synthesis of potent new dienyl derivatives.

A series of new, diene-modified nodulisporic acid analogues (2) bearing diverse functionality at the 3"- and 4"-sites was efficiently prepared from the 3"-aldehyde 3. Biological evaluation of these synthetic nodulisporic acid analogues for systemic flea efficacy identified potent compounds and further clarified the structural requirements for ectoparasite activity.

Ivermectin and nodulisporic acid receptors in Drosophila melanogaster contain both gamma-aminobutyric acid-gated Rdl and glutamate-gated GluCl alpha chloride channel subunits.

35S-labeled derivatives of the insecticides nodulisporic acid and ivermectin were synthesized and demonstrated to bind with high affinity to a population of receptors in Drosophila head membranes that were previously shown to be associated with a glutamate-gated chloride channel. Nodulisporic acid binding was modeled as binding to a single population of receptors. Ivermectin binding was composed of at least two kinetically distinct receptor populations, only one of which was associated with nodulisporic acid binding. The binding of these two ligands was modulated by glutamate, ivermectin, and antagonists of invertebrate gamma-aminobutyric acid (GABA)ergic receptors. Because solubilized nodulisporic acid and ivermectin receptors comigrated as 230-kDa complexes by gel filtration, antisera specific for both the Drosophila glutamate-gated chloride channel subunit GluCl alpha (DmGluCl alpha) and the GABA-gated chloride channel subunit Rdl (DmRdl) proteins were generated and used to examine the possible coassembly of these two subunits within a single receptor complex. DmGluCl alpha antibodies immunoprecipitated all of the ivermectin and nodulisporic acid receptors solubilized by detergent from Drosophila head membranes. DmRdl antibodies also immunoprecipitated all solubilized nodulisporic receptors, but only approximately 70% of the ivermectin receptors. These data suggest that both DmGluCl alpha and DmRdl are components of nodulisporic acid and ivermectin receptors, and that there also exists a distinct class of ivermectin receptors that contains the DmGluCl alpha subunit but not the DmRdl subunit. This co-association of DmGluCl alpha and DmRdl represents the first biochemical and immunological evidence of coassembly of subunits from two different subclasses of ligand-gated ion channel subunits.