Samuel Thomas

Rapid method development for chiral separation in drug discovery using sample pooling and supercritical fluid chromatography-mass spectrometry.

A novel strategy for rapid chiral method development has been implemented using sample pooling and supercritical fluid chromatography-mass spectrometry (SFC-MS) on four chiral stationary phases, namely Chiralpak AD and AS, and Chiralcel OJ and OD, and eight different modifier concentrations (5 to 40% methanol-0.2% isopropylamine). The screening is performed under an outlet pressure of 110 bar at 35 degrees C, and at a flow-rate of 2.5 ml/min for the initial 20 min and then ramped up to 4 ml/min and held for 4.5 min to elute all solutes from the column. The entire process is fully automated from injection to data processing, and operates unattended for 15 h overnight to obtain optimal chiral separation for multiple compounds. A unique feature of using SFC-MS to monitor chiral synthesis is the negligible interferences from achiral impurities. In addition, with SFC-MS, enantiomeric excess can be determined with much lower detection limits than UV and much shorter analysis times compared to normal-phase/reversed-phase liquid chromatography.

Two-step solution-phase synthesis of novel benzimidazoles utilizing a UDC (Ugi/de-Boc/cyclize) strategy

Abstract The novel solution-phase synthesis of an array of biologically relevant benzimidazoles in a simple two-step procedure is revealed. Transformations are carried out in excellent yield by condensation of mono-Boc protected ortho-phenylene diamine and supporting Ugi reagents. Subsequent acid treatment and evaporation affords benzimidazoles in good to excellent yield. The described protocol represents a highly attractive solution-phase procedure for the rapid generation of benzimidazole libraries.

Metabolism and Disposition of the HIV-1 Protease Inhibitor Lopinavir (ABT-378) Given in Combination with Ritonavir in Rats, Dogs, and Humans

AbstractPurpose. The objective of this study was to examine the metabolism and disposition of the HIV protease inhibitor lopinavir in humans and animal models. Methods. The plasma protein binding of [14C]lopinavir was examined in vitro via equilibrium dialysis technique. The tissue distribution of radioactivity was examined in rats dosed with [14C]lopinavir in combination with ritonavir. The metabolism and disposition of [14C]lopinavir was examined in rats, dogs, and humans given alone (in rats only) or in combination with ritonavir. Results. The plasma protein binding of lopinavir was high in all species (97.4-99.7% in human plasma), with a concentration-dependent decrease in binding. Radioactivity was extensively distributed into tissues, except brain, in rats. On oral dosing to rats, ritonavir was found to increase the exposure of lopinavir-derived radioactivity 13-fold. Radioactivity was primarily cleared via the hepato-biliary route in all species (>82% of radioactive dose excreted via fecal route), with urinary route of elimination being significant only in humans (10.4% of radioactive dose). Oxidative metabolites were the predominant components of excreted radioactivity. The predominant site of metabolism was found to be the carbon-4 of the cyclic urea moiety, with subsequent secondary metabolism occurring on the diphenyl core moiety. In all the three species examined, the primary component of plasma radioactivity was unchanged lopinavir (>88%) with small amounts of oxidative metabolites. Conclusions. Lopinavir was subject to extensive metabolism in vivo. Co-administered ritonavir markedly enhanced the pharmacokinetics of lopinavir-derived radioactivity in rats, probably due to inhibition of presystemic and systemic metabolism, leading to an increased exposure to this potent HIV protease inhibitor.

Preparative separation and analysis of the enantiomers of [3H]Abbott-69992, an HIV anti-infective nucleoside, by ligand-exchange high-performance liquid chromatography

Several chiral stationary phases (CSPs) were examined to separate the enantiomers of A-69992, a chiral HIV anti-infective nucleoside. The only CSP found to be effective was Nucleosil Chiral-1, a ligand-exchange CSP, which was used to prepare microgram amounts of the enantiomers of high optical purity. This appears to be the first separation of the enantiomers of a nucleoside by chiral high-performance liquid chromatography.

[3H]R-Terazosin binds selectively to α1-adrenoceptors over α2-adrenoceptors – comparison with racemic [3H]terazosin and [3H]prazosin

Abstract Most tissue sources for adrenoceptors contain a mixed population of α 1 - and/or α 2 -adrenoceptor subtypes; thus studies using non-specific radioligands are complicated by receptor heterogeneity. The examination of α 1 -adrenoceptor radioligand binding by radiolabeled terazosin and its enantiomers was simplified by using mouse fibroblast cells, which are thymidine kinase mutant (LTK − ), transfected with cloned α 1a -, α 1b -, α 1d -adrenoceptor subtypes. [ 3 H]Terazosin and its enantiomers were equipotent at the α 1b -adrenoceptor. [ 3 H] R -Terazosin was significantly less potent than [ 3 H]terazosin and [ 3 H] S -terazosin at the α 1a - and the α 1d -adrenoceptors. Using tissue derived α-adrenoceptors prepared in cold 25 mM glycyl-glycine buffer, [ 3 H]prazosin, [ 3 H]terazosin and [ 3 H] S -terazosin bound to two sites in the rat neonatal lung preparation consistent with the presence of both α 1 - and α 2B -adrenoceptors. The relative binding potencies of these radioligands at these two sites correlated with low affinity binding to the α 2B -adrenoceptor and high affinity binding to an α 1 -adrenoceptor. [ 3 H] R -Terazosin, on the other hand, bound to a single site in the rat neonatal lung membrane preparation, most likely an α 1 -adrenoceptor. Thus, [ 3 H] R -terazosin may be useful as a selective α 1 -adrenoceptor radioligand for establishing the functional role of adrenoceptors in tissues expressing multiple subtypes.

Preparative separation and analysis of the enantiomers of [14C]Zileuton, a 5-lipoxygenase inhibitor

Abstract Preparative resolution and quantitative methodology for the optical purity determination of [14C]Zileuton, N-[I-14C]1-(benzo-[b]thien-2-yl)ethyl-N-hydroxyurea, [14C]Abbott-64077, a 5-lipoxygenase inhibitor with potential clinical applications in the treatment of inflammatory diseases, is described. The method involves the use of a chiral stationary phase composed of amylose tris(3,5-dimethylphenylcarbamate) chemically bonded to 3-aminopropyl silica gel (ChiralPak AD) and a mobile phase consisting of n-hexane-ethanol (90:10). Optical and radiochemical purities of >99% were achieved for both.

Streamlined approach to the crude compound purification to assay process

Abstract Purification of medicinal chemistry compounds is often the slow step in the drug‐discovery process, particularly for compounds generated through parallel synthesis. To address this bottleneck we have developed a fully automated purification platform using a preparative‐LC/MS with a customized compound analysis requestor and a data tracking‐handling program. The six workstations that comprise the platform are an 8‐channel MUX, a mass‐directed Prep‐LC/MS, a liquid handling system, a balance automator, an evaporation system, and a central PC that runs the core program. The first step involved in the library purification, following the electronic submission of the dry crude synthetic material submitted in a barcoded plate, consists of automatic dissolution of the material and transfer of an aliquot for pre‐purification QC. Subsequent steps involve generation of a sequence list consisting of the methods for the analysis and processing, automatic generation of a sequence of the confirmed wells for LC/MS purification, evaporation, fraction dissolution and pooling, automatic weighing, and calculation of the amount needed to make a specified concentration. The final step involves compound distribution into a solubilization tube for the biochemical assay and plates for post‐purification QC by LC/MS and NMR. Functions incorporated into the core program were written in CSharp (C#) and Microsoft.NET to create a sample sequence list, annotate wells, track purification status, and to enable on‐line retrieval of pre‐ and post‐purification data. This platform enables a complete informatics solution from crude compound in a plate to purified compound ready for storage and assay. The processes and results for compounds purified at the multi‐milligram level by automated Prep‐LC/MS will be described.

Development of a simple recycling process for evaporated organic solvent after preparative supercritical fluid chromatography using powdered activated charcoal

In many drug discovery purification laboratories, solvent waste is often generated from the purification of compounds using preparative supercritical fluid chromatography (pSFC), which is considered a green technology. The resulting pSFC fractions containing the compounds of interest are evaporated, and distillates are usually discarded. In this study, a greener separation process was addressed by recycling the distillate using powdered activated charcoal (PAC). Adsorption behavior on PAC was studied for various classes of compounds in typical solvents used in pSFC such as methanol, ethanol, isopropanol and acetonitrile. Effects of vortexing versus flow-through methods were investigated. Results indicate that PAC adsorption is an effective method to remove most organic compounds in the aforementioned solvents. More than 99.9% of the four drug-like commercial compounds studied were removed from methanol using a ratio of 1 : 30 (compound : PAC) with the vortexing method in approximately 30 seconds. The flow-through method together with rotary evaporation using a ratio of 1 : 60, with a contact time of 10 seconds, was adequate for the removal of 49 out of a diverse set of 50 compounds. This process was successfully applied to reuse contaminated distillate from a previous pSFC purification by demonstrating the effectiveness of PAC in drug discovery labs.

Synthesis of [14C]ABT-418, a cholinergic channel activator labeled at two sites on the isoxazole ring

[ 14 C]ABT-418, (S)-3-[ 14 C]methyl-5-[N-methyl-2-pyrrolidinyl][4- 14 C]isoxazole hydrochloride, was labeled in two positions at maximum specific activity. Starting with 100 mCi of sodium [2 14 C]acetate, 14.6 mCi at 105 mCi/mmol was obtained in 8 steps including the formation of [1,3 14 C]acetone in the pyrolysis of barium (2- 14 C]acetate. The key step was the formation of the dianion of [1,3- 14 C]acetone oxime and its condensation with L-proline methyl ester.