Automation Highlights from the Literature

Laboratory Automation and High-Throughput Chemistry

Chemical Libraries via Sequential C–H Functionalization of Phenols

Phenols provide a useful template for diversification via sequential hydroarylation reactions. K. Li and J. A. Tunge report a method that begins with the hydroarylation of cinnamic acids by 3,5-dime-thoxyphenol to produce dihydrocoumarins. This activated ester undergoes facile ring-opening with amines to form a C–N bond and regenerate a phenol. The resulting phenol can be further functionalized via a second hydroarylation reaction. Thus, in 3–4 steps, a phenol is coupled with a cinnamic acid, an amine, and a cinnamic or propiolic acid (J. Comb. Chem. 2008, 10, 170–174).

Changing Requirements of Purification as Drug Discovery Programs Evolve from Hit Discovery

During the hit- and lead-finding stages of a drug discovery project, high-throughput organic synthesis and high-throughput purification play significant roles. J. Isbell describes how the needs and technologies change as a project matures into the lead optimization stages and beyond. As the chemistry becomes more focused, fewer compounds are produced and the successful isolation of each is necessary for their use in understanding the SAR. Therefore, the emphasis is on purification changes from a high-throughput method that produces acceptable purity products with a good success rate to a potentially lower-throughput, more successful one that produces highly purified compounds. The parallel, multichannel purification systems used for large-scale and focused libraries are less fully used for projects that progress into lead optimization, so multiple single-channel systems should be considered for improved flexibility (J. Comb. Chem. 2008, 10, 150–157).

PS-IIDQ: A Supported Coupling Reagent for Efficient and General Amide Bond Formation

Polystyrene-IIDQ, a polymer-supported coupling reagent, is synthesized in three steps from Merrifield resin in 86% overall conversion (Eric Valeur and Mark Bradley, Tetrahedron, 2007, 36(63), 8855–8871). This reagent efficiently couples carboxylic acids to amines in good yields and high purities, requires no pre-activation step, and is tolerant of the order of reagent addition.

PS-IIDQ is observed to be more efficient than polymer-supported carbodiimides (PSEDC and PS-DCC) and gives higher yields than HATU for general amide bond formation, including the coupling of anilines and hindered substrates. When evaluated with five carboxylic acids and nine amines (including anilines and secondary amines) PS-IIDQ gives an average isolated yield of 73%.

PS-IIDQ fails to couple 4-nitroaniline to any of the acids, which is perhaps not surprising as nitroanilines are some of the hardest amines to couple in view of the electron withdrawing nitro group. The yield is also very low when coupling aminoisobutyric acid to proline benzyl ester.

Regioselective Synthesis of 1,4-Disubstituted 1,2,3-Triazoles via Three-Component Coupling of Secondary Alcohols, TMSN₃ and Alkynes

1,4-Disubstituted 1,2,3-triazoles are obtained in excellent yields via a three-component coupling of secondary alcohols, alkynes and trimethylsilyl azide (TMSN₃) (B. Sreedhar, P. Surendra Reddy, V. Rama Krishna, Tetrahedron Lett. 2007, 33(48), 5831–5834). This one-pot reaction occurs through in situ generation of an azide from the alcohol and TMSN₃, followed by 1,3-dipolar cycloaddition of the terminal alkyne to afford the corresponding disubstituted triazoles. The presence of either Cu(II) or Cu(0) alone does not afford the desired product, hence a combination of both Cu(II) and Cu(0) catalysts is used to afford the product in good yields. The reaction conditions are optimized by varying the catalyst or the solvent. Among the various copper(II) salts screened, CuCl₂ and CuSO₄ are ineffective, whereas Cu(OTf)₂ is found to be an efficient catalyst. However, the reaction in the presence of CuI is found to be slow, giving low yields. Among the solvents screened, DMSO, methanol, and acetonitrile give the product in moderate to good yields, whereas the reaction in nitromethane gives the desired product in excellent yield.

Mechanistic Studies Leading to a New Procedure for Rapid, Microwave-Assisted Generation of Pyridine-3,5-Dicarbonitrile Libraries

Mechanistic investigations into the multicomponent synthesis of pyridine-3,5-dicarbonitriles establishes a defined reaction pathway, particularly clarifying the role of aerobic oxidation in conversion of the intermediate 1,4-dihydropyridines into the final products (K. Guo et al. Tetrahedron, 2007, 24(63), 5300–5311).

Based on such improved understanding of the reaction mechanism, optimized conditions for the preparation of compound libraries based on this core structure represent a significant improvement in yield over existing protocols. Particularly, microwave-assisted synthesis is found to provide a procedure suitable for high-throughput synthesis of pyridine-3,5-dicarbonitrile libraries.

Multicomponent Synthesis of Imidazo[1,2-a]Pyridines Using Catalytic Zinc Chloride

The novel use of zinc chloride to catalyze the one-pot, three-component synthesis of imidazo[1,2-a]pyridines from a range of substrates using either conventional heating or microwave irradiation is described by Amanda L. Rousseau et al (Tetrahedron Lett. 2007, 23(48), 4079–4082). This methodology affords a number of imidazo[1,2-a]pyridines in reasonable yields and short reaction times without any significant optimization of the reaction conditions.

In summary, they describe the novel application of zinc chloride, a cheap catalyst, for the one-pot preparation of imidazo[1,2-a]pyridines using either conventional heating or microwave irradiation. The work-up is simple and convenient for use in high-throughput synthesis. In cases where coordination is possible, as with aminated aldehydes, Montmorillonite clay K10 can be used as the catalyst.

Metal-Free Brønsted Acids Catalyzed Synthesis of Functional 1,4-Dihydropyridines

Brønsted acids catalyze the addition of β-enaminoacrylates to α,β-unsaturated aldehydes leading to substituted dihydropyridines via cascade reactions in moderate to good yields under mild conditions (Julie Moreau et al. Tetrahedron Lett. 2007, 49(48), 8647–8650). A tremendous advantage of these acids lies in the fact that they are stable, moisture insensitive, and not expensive compared with Lewis acids.

The first example of an enantioselective synthesis of a dihydropyridine is also reported. 1,4-Dihydropyridines exhibit interesting pharmacological and biological properties. They have been used as calcium channel modulators for the treatment of cardiovascular disorders, present vasodilating activity, and are NADH mimics. The best known procedure for the preparation of symmetrical 1,4-dihydropyridines is the classical Hantzch synthesis: a multicomponent condensation involving two molecules of β-ketoesters, one molecule of aldehyde, and one molecule of ammonia.

Multicomponent, Solvent-Free Synthesis of β-Aryl-β-Mercapto Ketones Using Zirconium Chloride as a Catalyst

Zirconium chloride efficiently catalyzes the one-pot, three-component reaction of an aryl aldehyde, cyclic or acyclic enolizable ketones, and thiols under solvent-free conditions at room temperature to afford the corresponding β-aryl-β-mercaptoketones via aldol–Michael addition reactions (Atul Kumar and Akanksha, Tetrahedron Lett. 2007, 49(48), 8730–8734).

This methodology affords a large number of β-aryl-β-mercapto ketone derivatives in high yields and in short reaction times. β-aryl-β-mercapto ketones are useful for the synthesis of various biologically active compounds, such as thiochromans, thiopyrans benzothiazapines, 4,5-dihydroisoxazoles, and 4,5-dihydropyrazoles. The reaction is versatile and offers several advantages, including high yields, shorter reaction times, cleaner reaction profiles, and simple experimental and work-up procedures.

Domino Knoevenagel/Diels–Alder Sequence Coupled to Suzuki Reaction: A Valuable Synthetic Platform for Chemical Biology

A small library of peculiar biphenyl- and terphenyl-containing spirocyclic triones has been synthesized in parallel by combining the organocatalytic three-component domino Knoevenagel/Diels–Alder sequence to Suzuki coupling (Daniela Pizzirani et al. Tetrahedron Lett. 2007, 40(48), 7120–7124). This methodology is fast, general, and serves as a platform to gain access to novel chemical tools to probe protein–protein interactions.

The authors demonstrate that this platform is amenable to parallel library generation, and valuable to the rapid and facile synthesis of compounds that embodies features of natural products in terms of structural and stereochemical complexity. All the synthesized compounds are tested in several biological screenings within the Broad Institute Chemical Biology Program to identify novel small-molecule probes to elucidate molecular pathways fundamental to cell and disease biology.

High-Throughput Analytics

High-Throughput Log P Determination by Ultraperformance Liquid Chromatography: A Convenient Tool for Medicinal Chemists

Accurate determinations of lipophilicity indices benefit from recent advances in chromatographic sciences, such as the launch of ultraperformance liquid chromatography (UPLC). This fast strategy, presented by P. -A. Carrupt et al, emerges as a powerful method suitable for high-throughput log P measurements of therapeutic compounds in isocratic and gradient modes. Because UPLC columns are highly stable in basic pH conditions, this approach allows a direct lipophilicity estimation of basic compounds in their neutral forms (J. Med. Chem. 2007, 51, 396–399).

Calibrant Delivery for MS

Accurate mass assignment provides improved identification confidence for MS, and is dependent on reliable internal or external calibration. Thomas R. Covey and Bradley B. Schneider from MDS SCIEX (Ontario, Canada) describe the principal use of a new independent calibrant introduction methodology for different ion sources. (J. Am. Soc. Mass Spectrom. 2007, 18, 991–996) The concept is based on the independent control of the applied ion sources and their physical separation via a curtain plate with an adjustable potential. The newly developed dual source systems for API–ES and AP–MALDI are described in detail, and the practical advances, scope, and limitations are demonstrated with several examples. A switchable internal or external calibration procedure, the key advantage, can be achieved simply by electronically decoupling the production of ions from either the analytical or the calibrant sprayer within their separate housings. Typically, the signals in external calibration mode can be eliminated within 45–100 ms and returned to its original level in a similar timeframe without the occurrence of any detrimental field effects and without any overlapping of sample and calibration ions. Further data demonstrate that it is possible to operate different ion sources of opposite polarity when using a mass spectrometer capable of rapid polarity switching.

Molecular Formula Analysis by an MS/MS/MS Technique to Expedite Dereplication of Natural Products

One of the major problems encountered in natural product research is the repeated discovery of known compounds after a tedious work of purification and structural determination. Yasuo Konishi and co-workers describe a facile and sensitive mass spectrometric method for the dereplication of natural products that provides information about the molecular formula and substructure of a precursor molecule and its fragments. (Anal. Chem. 2007, 79, 1187–1197) The elaborate procedure contains two separated MS experimental operations. First, an MS/MS experiment obtains general m/z information about product ions produced from interesting compounds. The adjacent MS/MS/MS experiments are then carried out by in-source fragmentation followed by a second fragmentation of pre-selected ions in the collision cell. The applied ESI-qTOF affords high-resolution mass separation and accurate mass measurement of nearly all precursors, fragments, and neutral products. The final analysis is based on a new data validation method with forward and reversed molecular formula analysis and computational calculations. The fragmentation pathways for the model substrate paclitaxel are discussed in detail. As a result, the exact assignment or identification of the molecular formula of minocyclin and piperacillin can be demonstrated.

Microfluidic Chip Technology and MicroReactor Technology

Dibal-H Reduction of Methyl Butyrate into Butyraldehyde Using Microreactors

The reduction of methyl butyrate into butyraldehyde with Dibal-H in microreactors is reported by L. Ducry and D. M. Roberge. Running the reaction continuously in a microreactor affords results similar to those of batch experiments, but very low temperatures are not necessary, and the reaction may be scaled-up without selectivity decrease. Different microreactors are used, and their mixing performances are compared. Increasing the reaction concentration, and thus the throughput, shows that even when working with microreactors, heat management should not be underestimated (Org. Process Res. Dev. 2008, 12, 161–167).

Kinetics and Process Development for Deoxofluorination of a Steroid

D. S. Negi et al. present investigations for developing a continuous process for the fluorination of a steroid derivative with bis(methoxyethyl)-aminosulfurtrifluoride. A kinetic model is suggested for this reaction. A simulation-based experimental design approach is implemented to find the optimum conditions for the flow reaction in a microreactor system (Org. Process Res. Dev. 2008, 12, 345–348).

Spurring Radical Reactions of Organic Halides Using Microreactors

Tributyltin hydride-mediated radical reactions of organic halides are successfully carried out by I. Ryu et al. in a continuous flow system using a microreactor. The reactions proceed within a very short period of time, coupled with quickly decomposing radical initiators. The continuous flow reaction system is applied to gram scale synthesis of a key intermediate for furofuran lignans (Org. Lett. 2008, 10, 533–536).

High-Efficiency Aminocarbonylation by Introducing CO to a Pressurized Continuous Flow Reactor

Halogenated aryl carboxylic acids are efficiently converted to the corresponding dicarboxylic acid monoamides by a one-step Pd-catalyzed aminocarbonylation in a micro/meso fluidic continuous flow reactor (X-Cube) operated at high pressure and high temperature with CO gas introduction. Reaction parameters (solvent, base, catalyst, pressure, and temperature) are rapidly optimized in the reactions, which require less than 2 min. The method reported by C. Csajagi et al. gives improved results over comparable batch techniques, and is also suited to automated parallel syntheses of compound libraries (Org. Lett. 2008, 10, 1589–1592).

Bioautomation and Screening

High-Throughput Electrophysiology: An Emerging Paradigm for Ion-Channel Screening and Physiology

Ion channels represent highly attractive targets for drug discovery and are implicated in a diverse range of disorders, particularly in the central nervous and cardiovascular systems. Assessment of cardiac ion-channel activity of new chemical entities is now an integral component of drug discovery programs to assess potential for cardiovascular side effects. Despite their attractiveness as drug discovery targets, ion channels remain an under-exploited target class, which is in large part due to the labor-intensive and low-throughput nature of patch–clamp electrophysiology. J. Dunlop et al. review the current state of the art for the various automated electrophysiology platforms that are now available, and describe their impact in terms of ion-channel screening, lead optimization, and the assessment of cardiac ion-channel safety liability (Nat. Rev. Drug Discov. 2008, 7, 358–368).

ChemBank: A Small-Molecule Screening and Cheminformatics Resource Database

ChemBank (http://chembank.broad.harvard.edu/) is a public, Web-based informatics environment developed through a collaboration between the Chemical Biology Program and Platform at the Broad Institute of Harvard and MIT. S. L. Schreiber et al. report that this knowledge environment includes freely available data derived from small molecules and small-molecule screens, and resources for studying these data. ChemBank stores an increasingly varied set of measurements derived from cells and other biological assay systems treated with small molecules. Analysis tools are available and are continuously being developed to allow relationships between small molecules, cell measurements, and cell states to be studied. Currently, ChemBank stores information on hundreds of thousands of small molecules and hundreds of biomedically relevant assays (Nucleic Acids Res. 2008, 36, D351–D359).

Discovery of Novel Targets with High-Throughput RNA Interference Screening

The identification of targets that enter HTS campaigns had been driven by basic research until the advent of genomics level data acquisition, such as sequencing and gene expression microarrays. Large-scale profiling approaches (e.g., microarrays, protein analysis by MS, and metabolite profiling) can yield vast quantities of data and important information. However, these approaches often require difficult in silico analysis and low-throughput basic research experiments to identify the function of a gene and validate the gene product as a potential therapeutic drug target. Functional genomic screening offers the promise of direct identification of genes involved in phenotypes of interest. P. D. Kassner reviews RNA interference (RNAi)-mediated loss-of-function screens and their utility in target identification. Some of the genes identified in these screens should produce similar phenotypes if their gene products are antagonized with drugs. With a carefully chosen phenotype, an understanding of the biology of RNAi, and appreciation of the limitations of RNAi screening, there is great potential for the discovery of new drug targets (Combin. Chem. High-Throughput Screen. 2008, 11, 175–184).

Impact of Novel Screening Technologies on Ion Channel Drug Discovery

Ion channels are a large superfamily of membrane proteins that pass ions across membranes. They are critical to diverse physiological functions in both excitable and nonexcitable cells, and underlie many diseases. As a result, they are an important target class that is proven to be highly “druggable.” However, for HTS, ion channels are historically difficult as a target class due to their unique molecular properties and the limitations of assay technologies that are 0-amendable. Q. Lu and W. F. An describe the background of ion channels and current status and challenges for ion-channel drug discovery, followed by an overview of both conventional and newly emerged ion-channel screening technologies. The critical impact of such new technologies on current and future ion-channel drug discovery is also discussed (Combin. Chem. High-Throughput Screen. 2008, 11, 185–194).

HTS for Orphan and Liganded GPCRs

GPCRs had significant representation in the drug discovery portfolios of most major commercial drug discovery organizations for many years. Publication of the human genome sequence in 2001 confirmed GPCRs as the largest single gene superfamily with more than 700 members, furthering the already strong appeal of addressing this target class using efficient and highly parallelized platform approaches. S. H. Xiao et al. use the GPCR research platform implemented at Amgen as a case study to review the evolution and implementation of available assays and technologies applicable to GPCR drug discovery. Particular consideration is made of the role and practice of “de-orphaning” and signaling pathway characterization as a pre-requisite to establishing effective screens. In silico and in vitro methodology developed for rapid, parallel high-throughput hit characterization and prioritization are also discussed extensively (Combin. Chem. High-Throughput Screen. 2008, 11, 195–215).

High-Content Analysis in Preclinical Drug Discovery

S. Prechtl et al. give an overview of high-content analysis (HCA) as an established tool in a wide range of academic laboratories and pharmaceutical research groups. HCA is now routinely proving to be effective in providing functionally relevant results. It is essential to select the appropriate HCA application with regard to the targeted compound's cellular function. The cellular impact and compound specificity as revealed by HCA analysis facilitates reaching definitive conclusions at an early stage in the drug discovery process. This technology, therefore, has the potential to substantially improve the efficiency of pharmaceutical research. Recent advances in fluorescent probes have significantly boosted the success of HCA. Auto-fluorescent proteins that minimally hinder the functioning of the living cell have been playing a decisive role in cell biology research. For companies, severely restricted license conditions regarding auto-fluorescent proteins hamper their general use in pharmaceutical research. This has opened the field for other solutions, such as self-labeling protein technology, which could potentially replace the well-established methods that use auto-fluorescent proteins (Combin. Chem. High-Throughput Screen. 2008, 11, 216–231).

Label-Free Technologies for Small-Molecule Screening

Small molecule HTS in drug discovery today is dominated by techniques that are dependent upon artificial labels or reporter systems. While effective, these approaches can be affected by certain experimental limitations, such as conformational restrictions imposed by the selected label or compound fluorescence or quenching. Label-free approaches potentially address many of these issues by allowing researchers to investigate more native systems without fluorescence- or luminescence-based readouts. However, due to throughput and expense constraints, label-free methods have been largely relegated to a supporting role as the basis of secondary assays. A. K. Shiau et al. describe recent improvements in impedance-based, optical biosensor-based, automated patch–clamp and MS technologies that have enhanced their ease of use and throughput, and hence, their utility for primary screening of small- to medium-sized compound libraries (Combin. Chem. High-Throughput Screen. 2008, 11, 231–237).

HTS for Neurodegeneration and Complex Disease Phenotypes

HTS for complex diseases is challenging, because complex phenotypes are difficult to adapt to rapid, high-throughput assays. B. R. Stockwell et al. describe the recent development of high-throughput and high-content screens (HCS) for neurodegenerative diseases with a focus on inherited neurodegenerative disorders, such as Huntington's disease. HTS assays based on protein aggregation, neuronal death, caspase activation, and mutant protein clearance are described (Comb. Chem. High Throughput Screen. 2008, 11, 238–248).

HTS Informatics

HTS has significantly increased the volume, complexity, and information content of datasets. Thus, lead discovery research demands a clear corporate strategy for scientific computing and subsequent establishment of robust informatics platforms that enable complicated HTS workflows, facilitate data mining, and drive effective decision making. X. B. Ling examines in a review the key elements in HTS operations and some essential data-related activities supporting or interfacing the screening process, and outlines properties that various enabling software should have (Comb. Chem. High Throughput Screen. 2008, 11, 249–257).

Recent Advances in HTS for ADME Properties

With the increasing numbers of molecules synthesized in a typical drug discovery program, there is a need for a plethora of drug metabolism and pharmacokinetic (DMPK) information to be regularly generated in discovery. Over the past decade, many in vitro and in vivo DMPK screens have been developed to generate this information in support of drug discovery efforts. Newer methods have been published recently and T. J. Carlson and M. B. Fisher summarize these advances. In particular, advances are reported for experimental approaches to metabolic clearance, CYP inhibition, in vivo exposure, and distribution, as well as in silico determinations of absorption, distribution, metabolism, and excretion (ADME) properties (Combin. Chem. High-Throughput Screen. 2008, 11, 258–264).

Stem Cells as Screening Tools in Drug Discovery

All physiologic processes operate in a cellular setting. Therefore, drug discoverers need the highest quality cells as they pursue the next generation of safe and effective medicines. Recently, investigators have begun to consider stem cells as a new source of predictive, cell-based assays in drug discovery. Stem cell technology still has hurdles to overcome before these cells are fully accepted as decision-making reagents and amenable to HTS. However, with global research interest in stem cell biology, significant advances in the application of these cells in drug discovery have been reported. These advances are aligned with three important stages of pharmaceutical research: target discovery and validation, identification of efficacious chemical leads, and drug safety pharmacology. In this review, the author describes the application of stem cells in these areas of drug discovery with emphasis on molecular screening opportunities. (J. D. McNeish, Curr. Opin. Pharmacol. 2007, 7(5), 515–520).

Cell-Based High-Content Screening of Small-Molecule Libraries

Advanced microscopy and the corresponding image analysis have developed in recent years into a powerful tool for studying molecular and morphological events in cells and tissues. Cell-based high-content screening (HCS) is an upcoming methodology for the investigation of cellular processes and their alteration by multiple chemical or genetic perturbations. Multiparametric characterization of responses to such changes can be analyzed using intact live cells as reporters. These disturbances are screened for effects on a variety of molecular and cellular targets, including subcellular localization and redistribution of proteins. In contrast to biochemical screening, they detect the responses within the context of the intercellular structural and functional networks of normal and diseased cells, respectively. As cell-based HCS of small-molecule libraries is applied to identify and characterize new therapeutic lead compounds, large pharmaceutical companies are major drivers of the technology and have already shown image-based screens using more than 100,000 compounds. (K. Korn, E. Krausz. Curr. Opin. Chem. Biol. 2007, 11(5), 503–510).