Drug discovery increasingly relies on the ability to rapidly identify "quality" molecules that possess the desired attributes of bioavailability, chemical tractability, selectivity, and potency. Traditional methods used to determine the pharmacokinetics (oral bioavailability, clearance, volume of distribution, and half-life) of molecules have presented a bottleneck in some drug discovery programs. We have increased throughput in in vivo pharmacokinetic screening of structurally related compounds by dosing mixtures of compounds intravenously to a single animal and using atmospheric pressure ionization (API) tandem liquid chromatography/ mass spectrometry (LC/MS/MS) for analysis. We have referred to this as N-in-one dosing where N is the number of compounds coadministered. The method was used to simultaneously determine the clearance (CL), steady-state volume of distribution (V ss ), and elimination half-life (t 1/2 ) of five R 1a receptor antagonists (compounds possessing selective R 1a antagonist properties may have potential therapeutic importance in the treatment of benign prostatic hyperplasia). 1-3 The mixture approach provides an opportunity to study the pharmacokinetics of several compounds under identical conditions while minimizing sample processing time and the number of animals required. To the best of our abilities, we found limited literature that capitialized on the advantages gained through simultaneously dosing compounds to determine pharmacokinetics and bioavailability after intravenous and intraduodenal administration, 4,5 and one of these utilized LC/MS. 6 Five R 1a receptor antagonists ( Figure 1) that had been previously studied by individual dosing (known range of CL, V ss , and t 1/2 ) were dosed intravenously as a mixture to a single dog. In the traditional individual dosing studies, plasma samples were analyzed for R 1a antagonist by reverse-phase HPLC with fluorescence detection. Plasma sample analysis of the R 1a antagonists in the mixture study relied heavily on the application of atmospheric pressure ionization (API) LC/MS methodology using a triple quadrupole instrument. LC/ MS with its inherent detection specificity, selectivity, and sensitivity enabled rapid analytical method development prior to dosing the animal as well as high throughput sample analysis.The compounds studied exhibited good mass spectrometric response in the positive ion mode using the API technique. To increase analyte specificity from the biological matrix, a reverse-phase LC/MS/MS method was developed. For this series of compounds, a characteristic neutral loss of CF 3 CH 2 OH (100 amu) was observed in the product ion mass spectra. This transition was optimized for sensitivity in the selected reaction monitoring (SRM) mode. Among the compounds studied, a pair of isobars (compounds 2 and 3 of molecular weight 638) produced the same abundant fragment ion at m/z 539. Consequently, the HPLC mobile phase conditions were optimized to resolve compounds 2 and 3, extending the analysis time to 6 min. An internal standar...

Starting from phenethanolamine aniline leads 3a and 3b, we have identified a series of functionally potent and selective beta(3) adrenergic receptor (AR) agonists containing acylsulfonamide, sulfonylsulfonamide, or sulfonylurea groups within the aniline phenethanolamine series. In beta(3), beta(2), and beta(1) AR cAMP functional assays, 3a and other right-hand side (RHS) carboxylate analogues were found to be full agonists that were modestly selective against beta(1) or beta(2) ARs, while analogues lacking RHS acid functionality were active at beta(3) AR but not selective. Replacement of the carboxylate with acylthiazole and acylmethylsulfone gave potent, but only modestly selective, compounds. Increasing the size of the RHS sulfonamide substituent with phenyl or p-toluene afforded compounds with good potency and functional selectivity (beta(3) AR pEC(50) greater than 8; beta(1) and beta(2) AR selectivity greater than 40- and 500-fold, respectively). Our SAR studies suggest that the potency and selectivity profile of the best analogues reported here is a result of both the steric bulk and acidity of the RHS sulfonamide NH group. Although all of the analogues had a pharmacokinetic half-life of less than 2 h, acylsulfonamides 43 and 44 did show moderately low clearance in dogs. These two compounds were further evaluated by thermographic imaging in mice and were found to produce a robust thermogenic response via oral administration.

The synthesis of a series of phenethanolamine aniline agonists that contain an aniline ring on the right-hand side of the molecule substituted at the meta position with a benzoic acid or a pyridyl carboxylate is described. Several of the analogues (e.g., 34, 36-38, 40, and 44) have high beta(3) adrenergic receptor (AR) potency and selectivity against beta(1) and beta(2) ARs in Chinese hamster ovary (CHO) cells expressing beta ARs. The dog pharmacokinetic profile of some of these analogues showed >25% oral bioavailability and po half-lives of at least 1.5 h. Among the compounds described herein, the 3,3'-biarylaniline carboxylate derivatives 36, 38 and the phenylpyridyl derivative 44 demonstrated outstanding in vitro properties and reasonable dog pharmacokinetic profiles. These three analogues also showed dose dependent beta(3) AR mediated responses in mice. The ease of synthesis and superior dog pharmacokinetics of compound 38 relative to that of 44 in combination with its in vitro profile led us to choose this compound as a development candidate for the treatment of type 2 diabetes.