Harold N. Weller

High Throughput Analysis and Purification in Support of Automated Parallel Synthesis

Rapid reverse-phase analytical and preparative HPLC methods havebeen developed for application to parallel synthesis libraries.Gradient methods, short columns, and high flow rates allowanalysis of over 300 compounds per day on a single system, orpurification of up to 200 compounds per day on a singlepreparative system. Hardware and software modifications allowcontinuous unattended use for maximum efficiency and throughput.

A high-throughput bioanalytical platform using automated infusion for tandem mass spectrometric method optimization and its application in a metabolic stability screen

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the bioanalytical method of choice to support plate-based, in vitro early ADME (Absorption, Distribution, Metabolism and Excretion) screens such as metabolic stability (Metstab) assessment. MS/MS method optimization has historically been the bottleneck in this environment, where samples from thousands of discrete compounds are analyzed on a monthly basis, mainly due to the lack of a high-quality commercially available platform to handle the necessary MS/MS method optimization steps for sample analysis by selected reaction monitoring (SRM) on triple quadrupole mass spectrometers. To address this challenge, we recently developed a highly automated bioanalytical platform by successfully integrating QuickQuan 2.0, a unique high-throughput solution featuring MS/MS method optimization by automated infusion, with a customized in-house software tool in support of a Metstab screen. In this platform, a dual-column setup running parallel chromatography was also implemented to reduce the bioanalytical cycle time for LC/MS/MS sample analysis. A set of 45 validation compounds was used to demonstrate the speed, quality and reproducibility of MS/MS method optimization, sample analysis, and data processing using this automated platform. Metstab results for the validation compounds in microsomes from multiple species (human, rat, mouse) showed good consistency within each batch, and also between batches conducted on different days. We have achieved and maintained a monthly throughput of 1300 compound assays representing 500 discrete compounds per instrument per month on this platform, and it has been used to generate metabolic stability data for more than 25 000 compounds to date with an overall success rate of more than 95%.

Ultrafast mass spectrometry based bioanalytical method for digoxin supporting an in vitro P‐glycoprotein (P‐gp) inhibition screen

The evaluation of interactions between drug candidates and transporters such as P-glycoprotein (P-gp) has gained considerable interest in drug discovery and development. Inhibition of P-gp can be assessed by performing bi-directional permeability studies with in vitro P-gp-expressing cellular model systems such as Caco-2 (human colon carcinoma) cells, using digoxin as a substrate probe. Existing methodologies include either assaying (3)H-digoxin with liquid scintillation counting (LSC) detection or assaying non-labeled digoxin with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis at a speed of several minutes per sample. However, it is not feasible to achieve a throughput high enough using these approaches to sustain an early liability screen that generates more than a thousand samples on a daily basis. To address this challenge, we developed an ultrafast (9 s per sample) bioanalytical method for digoxin analysis using RapidFire™, an on-line solid-phase extraction (SPE) system, with MS/MS detection. A stable isotope labeled analog, d3-digoxin, was used as internal standard to minimize potential ionization matrix effect during the RF-MS/MS analysis. The RF-MS/MS method was more than 16 times faster than the LC-MS/MS method but demonstrated similar sensitivity, selectivity, reproducibility, linearity and robustness. P-gp inhibition results of multiple validation compounds obtained with this RF-MS/MS method were in agreement with those generated by both the LC-MS/MS method and the (3)H-radiolabel assay. This method has been successfully deployed to assess P-gp inhibition potential as an important early liability screen for drug-transporter interaction.

Cassette incubation followed by bioanalysis using high-resolution MS for in vitro ADME screening assays

BACKGROUND High-resolution MS (HRMS) has recently received a considerable interest in quantitative bioanalysis using full-scan acquisition mode. The benefits include complete elimination of compound-specific MS method development, and simultaneous collection of mass spectral data on both targeted and non-targeted components. One additional advantage that has not been widely discussed is its suitability for simultaneous quantitation of, theoretically, an unlimited number of compounds, which is not possible with selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. MATERIALS & METHODS We took advantage of this unique bioanalytical capability of HRMS and developed a novel in vitro ADME workflow of cassette incubation of as many as 32 compounds, followed by quantitative bioanalysis using full-scan acquisition on an Orbitrap HRMS. The workflow was evaluated for a serum protein-binding assay and a parallel artificial membrane permeability (PAMPA) assay. RESULTS The bioanalytical assay displayed acceptable sensitivity, selectivity and linearity for all compounds in the cassettes, and the biological results obtained using this approach were similar to those from discrete incubation and analysis, demonstrating the feasibility of the workflow. Additional benefits of this platform include a saving of analysis time due to the reduced sample numbers from the cassette approach, as well as cost saving due to the reduction in the required assay reagents. CONCLUSION Cassette incubation with bioanalysis using HRMS is a feasible approach for high-throughput in vitro ADME assays evaluated in this study.

An integrated bioanalytical platform for supporting high‐throughput serum protein binding screening

Quantification of small molecules using liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a triple quadrupole mass spectrometer has become a common practice in bioanalytical support of in vitro adsorption, distribution, metabolism and excretion (ADME) screening. The bioanalysis process involves primarily three indispensable steps: MS/MS optimization for a large number of new chemical compounds undergoing various screening assays in early drug discovery, high-throughput sample analysis with LC/MS/MS for those chemically diverse compounds using the optimized MS/MS conditions, and post-acquisition data review and reporting. To improve overall efficiency of ADME bioanalysis, an integrated system was proposed featuring an automated and unattended MS/MS optimization, a staggered parallel LC/MS/MS for high-throughput sample analysis, and a sophisticated software tool for LC/MS/MS raw data review as well as biological data calculation and reporting. The integrated platform has been used in bioanalytical support of a serum protein binding screening assay with high speed, high capacity, and good robustness. In this new platform, a unique sample dilution scheme was also introduced. With this dilution design, the total number of analytical samples was reduced; therefore, the total operation time was reduced and the overall throughput was further improved. The performance of the protein binding screening assay was monitored with two controls representing high and low binding properties and an acceptable inter-assay consistency was achieved. This platform has been successfully used for the determination of serum protein binding in multiple species for more than 4000 compounds.

Purification of combinatorial libraries

In the early days of combinatorial chemistry, much attention focused on preparation of large libraries for lead discovery. Recently, though, the focus has shifted toward smaller, more focused libraries for lead optimization. These focused libraries generally consist of individual discrete compounds. Biological assay requirements often require compounds of high purity, thus development of automated high throughput purification methods has received new attention in the past several years. This paper covers automated high throughput purification methods that have been applied to libraries of discrete compounds. Literature published through February 1998 is included. Purification methods discussed include extraction methods, scavenger methods, solid phase extraction, and preparative HPLC.

Evaluation of a New Preparative Supercritical Fluid Chromatography System for Compound Library Purification: The TharSFC SFC-MS Prep-100 System

Preparative HPLC-MS is often the method of choice for purification of small amounts (<100 mg) of diverse new molecules, such as compound libraries for drug discovery. The method is robust, well proven, and widely applicable. In contrast, preparative supercritical fluid chromatography coupled with mass spectrometry (SFC-MS) has seen only slow acceptance for the same application—despite some potential scientific and economic advantages. One of the reasons for slow adoption of SFC-MS is the lack of well-proven, robust, and commercially available instrumentation. In early 2009, TharSFC (a Waters Company, Pittsburgh, PA) introduced a new fully integrated system for preparative SFC-MS: The SFC-MS Prep-100. We report herein an objective evaluation of the SFC-MS Prep-100, including tests for pump and autosampler performance, sample recovery, sample carryover, fraction triggering, detector/fraction collector synchronization, and overall robustness. Our results suggest that the SFC-MS Prep-100 represents a significant advance over previous generation instrumentation.

Benzothiadiazine dioxides: A new class of potent angiotensin-II (AT1) receptor antagonists

Abstract N-Biphenylmethyl benzothiadiazine dioxides were prepared as potential angiotensin-II receptor antagonists. Stability of the compounds is dependent upon the nature of the substituent at position 3 of the benzothiadiazine ring, while potency is dependent upon the nature of substitution in the benzo fused ring. 3-(Propylthio)-4-[2′-(2H-tetrazol-5-yl)[1,1′-biphenyl]-4-ylmethyl]-4H-1,2,4-benzothiadiazine-6-carboxylic acid, 1,1-dioxide ( 17 ) is one of the most potent examples, with functional activity (K B ) below 01 nM.

Comparison of chromatographic techniques for diastereomer separation of a diverse set of drug-like compounds.

Preparative supercritical fluid chromatography (SFC) has proven value for isocratic bulk separation of both diastereomers and enantiomers. With the recent availability of SFC equipment suitable for rapid gradient separation [Ebinger et al. JALA (2011) 16 (3) 241], we have become interested in comparing the effectiveness of traditional reverse phase high performance liquid chromatography (HPLC) with SFC using non-chiral columns for the separation of diastereomeric mixtures. The success rates for separation of a diverse set of 258 synthetic diastereomer pairs were used to determine the relative utility of the aforementioned two techniques. Our results suggest that gradient non-chiral SFC was more successful than the traditional non-chiral HPLC technique for diastereomer separations of the diverse sample set of 258 drug-like compounds.

Synthesis of N-Alkyl-1,2,4-oxadiazinones as Angiotensin-II (AT1) Receptor Antagonists

4-Alkyl-1,2,4-oxadiazinones were prepared by regiospecific alkylation of the corresponding 4H-oxadiazinones, which were synthesized by a trimethylaluminium mediated cyclization reaction. Alkylation was regiospecific and generally facile; in one example, however, an unusual fragmentation reaction occurred. A homochiral oxadiazineone was also prepared and alkylated under the described conditions. 4-Biphenylmethyl-1,2,4-oxadi-azinones were potent angiotensin-II receptor antagonists