Abstract
Kerstin Thurow, Ph.D.
Hilmar Weinmann, Ph.D.
Laboratory Automation and High-Throughput Chemistry
Comprehensive Survey of Chemical Libraries for Drug Discovery and Chemical Biology: 2007
This is the 11th issue of the comprehensive survey series in combinatorial chemistry by R. E. Dolle et al. Bioactive libraries are summarized under the headings of proteases, nonproteolytic enzymes, G protein coupled receptors (GPCRs), non-GPCRs, and oncolytics/anti-infectives. Compound collections without disclosed biological activity are captured under the headings of scaffold derivatization/acyclic synthesis, monocyclic, bicyclic/spirocyclic, and polycyclic/macrocyclic synthesis. Polymer-supported reagents/scavengers/linkers and polymer-supported chiral ligands are also discussed. There are 498 total entries for 2007 (J. Comb. Chem.
Dry HCl in Parallel Synthesis of Fused Pyrimidin-4-ones
The parallel solution-phase synthesis of substituted thieno[2,3-d]pyrimidin-6-carboxylic acids is reported by S. V. Ryabukhin et al. Their strategy relies on a cyclization of 2-aminothiophen-3,5-dicarboxylates with a set of nitriles, followed by hydrolysis to construct the library of corresponding acids. A convenient procedure for use and dosage of dry HCl for the reaction is elaborated and adapted for semi-automated parallel solution-phase synthesis. With the use of another (hetero) aromatic ortho-aminocarboxylate, mini-libraries of diverse fused pyrimidin-4-ones are synthesized. The scope and limitations of the approach are discussed (J. Comb. Chem.
Microwave-Assisted Parallel Synthesis of Fused Heterocycles in a Novel Parallel Multimode Reactor
New rotor types using disposable glass vials for small-scale parallel synthesis in multimode microwave reactors are reported by A. Stadler et al. One rotor comprises 16 groups of four vials, and the second uses four silicon carbide plates with a 6 × 4 matrix to process the vials. Both rotors achieve utmost temperature homogeneity on microwave irradiation, and can be used for microwave-mediated reactions at temperatures of up to 200 °C and pressures of 20 bar. The generation of three different heterocycle libraries furnishing thiophenes, oxindoles, and benzimidazoles using the new rotor types is described (J. Comb. Chem.
Scaling-Out Pharmaceutical Reactions in an Automated Stop-Flow Microwave Reactor
Six pharmaceutically relevant reactions are assessed by J. K. Moseley and E. K. Woodmann for scale-out in an automated stop-flow microwave reactor. Daily throughputs of between 50 and 250 g are achieved at typical reaction concentrations. For more concentrated reactions, or with 24 h processing, productivity of 0.5–1.5 kg/day is achievable. This study confirms that the stop-flow approach in combination with rapid microwave heating can be equivalent to conventional continuous flow technology with comparable productivities (Organic Proc. Res. Dev.
Microfluidic Chip Technology and Microreactor Technology
Bifurcated Pathway to Thiazoles and Imidazoles Using a Modular Flow Microreactor
A scalable method for the preparation of 4,5-disubstituted thiazoles and imidazoles as distinct regioisomeric products using a modular flow microreactor has been devised by S. V. Ley et al. The process makes use of microfluidic reaction chips and packed immobilized-reagent columns to effect bifurcation of the reaction pathway (J. Comb. Chem.
Microreactor Technology and Continuous Processes in the Fine Chemical and Pharmaceutical Industry: Is the Revolution Underway?
Microreactors have shown their ability to improve chemical processes and routes. However, their integration into chemical production processes depends on more than just technical advances. Cost issues and productions logistics play a crucial role, too, and are highlighted in two different case studies by D. M. Roberge et al. Economical drivers for the pharmaceutical industry are described with emphasis on future development of microprocess engineering (Organic Proc. Res. Dev.
Microreactor Technology: Continuous Synthesis of 1H-Isochromeno[3,4-d]imidazol-5-Ones
A synthetic method is evaluated by C. V. Stevens et al. to form 1H-isochromeno[3,4-d]imidazol-5-ones by ring closure starting from 3-amino-4-(arylamino)-1H-isochromen-1-ones. The method is implemented and optimized in a microreactor environment. It is possible to synthesize these compounds in a continuous mode with an output up to 2.2 g/h (Organic Proc. Res. Dev.
An Integrated Microreactor for the Multicomponent Synthesis of α-Aminonitriles
Initial steps have been taken by C. Wiles and P. Watts to develop an integrated microreactor capable of performing multicomponent reactions consisting of both solution phase and heterogeneously catalyzed steps. Using the multicomponent Strecker reaction as a model, five α-aminonitriles are synthesized in excellent yields (>99.5%) and analytical purity under continuous flow conditions (Organic Proc. Res. Dev.
Continuous Flow Microwave-Assisted Reaction Optimization and Scale-Up Using Fluorous Spacer Technology
Microwave-assisted organic synthesis in a laboratory-scale monomodal microwave reactor is investigated for continuous flow applications using fluorous spacer technology. The benchtop continuous flow microwave described by O. Benali et al. allows sequential processing of multiple plugs using small amounts of reagents for reaction optimization, scale-up, and array synthesis. The system features online monitoring of temperature, pressure, and microwave power. Several different reactions have been scaled up, including a Suzuki–Miyaura cross-coupling reaction and nucleophilic substitutions. In all cases, it is possible to optimize the reaction conditions on a small scale (300 μL processing volume), and achieve similar conversions on an intermediate scale (30 mL), offering the potential for further scale-up without modifying the optimized conditions (Organic Proc. Res. Dev.
Investigation of the Moffatt–Swern Oxidation in a Continuous Flow Microreactor System
The Moffatt–Swern oxidation of different alcohols is performed by G. J. Kemperman et al. in a continuous flow microreactor system. The microreactor process offers significant advantages over the batch process. The process can be operated at remarkably high temperatures in comparison with a batch reaction (0–20 °C instead of ∼70 °C). In the present study, a continuous flow microreactor system is optimized using reactors of different volumes allowing modulation of the residence times of labile intermediates. The efficiency of mixing is studied using different mixing devices, and shows that the continuous flow microreactor is an ideal tool for rapid optimization of reaction parameters. Furthermore, the scaleability and reliability of the microreactor are tested by running the system for several hours. For testosterone, the system was in process for 1.5 h without any problems, resulting in a 4-androstene-3,17-dione production rate of 64 g/h (Organic Proc. Res. Dev.
Continuous Flow Nitration of Salicylic Acid
Continuous flow nitration of salicylic acid using HNO3/AcOH is studied by A. A. Kulkami et al. in a tubular microreactor. At specific reaction conditions, complete conversion of the reactant is achieved in less than 7 min. It yields only mononitro derivatives with a higher selectivity of 5-nitrosalicylic acid. Presence of the lower amount of acetic acid in the reaction mixture is seen to be detrimental, leading to precipitation of the desired product (5-nitrosalicylic acid) (Organic Proc. Res. Dev.
High-Throughput Analytics
Automated Modular Preparative HPLC-MS Purification Laboratory with Enhanced Efficiency
Automated parallel synthesis as a tool to increase productivity in chemical synthesis is well established. However, even more time-consuming than the synthesis process is the following purification of the resulting crude products. To enhance efficiency of the lead optimization process at Bayer CropScience, a HT HPLC-MS laboratory for the purification of up to 48 crude products/day in the range of 200–400 mg each in one injection per sample has been set up by K. Ilg et al. The use of Covaris technology for HPLC sample preparation, automated aliquotation during fractionation, and a novel evaporation process in combination with freeze-drying are new key technologies applied successfully for the first time in this purification unit. Together, they facilitate and achieve targeted efficiencies. The whole process is supported by a specially designed IT-landscape that covers each step of the workflow (J. Comb. Chem. 2008,
Bioautomation and Screening
Functional Cell-Based Assays in Microliter Volumes for Ultra-High-Throughput Screening
Functional cell-based assays have gained increasing importance for microplate-based high-throughput screening (HTS). The use of high-density microplates, most prominently 1536-well plates, and miniaturized assay formats allow screening of comprehensive compound collections with more than 1 million compounds at ultra-HT. Full automation with integrated robotic systems allow the realization of complex assay protocols with multiple liquid handling and signal detection steps. J. Höser et al. categorize assays in a review based on the kinetics of cellular response quantified by the readout methods used. Thus, assays measuring fast cellular responses with high temporal resolution, for example, receptor-mediated calcium signals or changes in membrane potential, are at one end of this spectrum, whereas tests quantifying cellular transcriptional responses mark the opposite end (Comb. Chem. High Throughput Screen.
Fluorescent Probes for Cellular Assays
A fluorescent probe is a fluorophore designed to localize within a specific region of a biological specimen or to respond to a specific stimulus. Fluorescent probes have been used for nearly a century to study cellular processes because of their exquisite sensitivity and selectivity. Fluorescent probes have also gained in popularity as safety and environmental concerns over the use of radioactive probes have grown. G. T. Hanson and B. G. Hanson give a broad overview of types of fluorescent probes, types of fluorescent assays, and their application in cellular assays for a number of pharmaceutically relevant target classes (Comb. Chem. High Throughput Screen.
Ion Channel Screening
Ion channels are attractive targets for drug discovery with recent estimates indicating that voltage, and ligand-gated channels account for the third and fourth largest gene families represented in company portfolios after the GPCR and nuclear hormone receptor families. A historical limitation on ion channel-targeted drug discovery in the form of the extremely low-throughput gold standard assay for assessing functional activity, patch clamp electrophysiology in mammalian cells, is overcome by the implementation of multi-well plate format cell-based screening strategies for ion channels. J. Dunlop et al. address in their review the options available for cell-based screening of ion channels. They include examples of their utility and present case studies that demonstrate the successful implementation of HTS campaigns for a ligand-gated ion channel using a fluorescent calcium indicator, and a voltage-gated ion channel using a fluorescent membrane potential sensitive dye (Comb. Chem. High Throughput Screen.
High-Content Analysis of Kinase Activity in Cells
High-content analysis (HCA) is a term used to describe techniques involving multiplexed analysis of fluorescent markers to measure multiple cellular responses to biological stimuli or drug treatment. HCA is usually based on automated microscopy or related technologies, and its value lies in providing multiparametric information on single cells within a population. During the last decade, several HCA approaches have been developed and applied to assess cellular mechanism of action of pharmacologically relevant compounds identified through biochemical screening or similar in vitro methods. With automation and instrument development, these approaches have evolved to the extent that the technique is now routinely used in screening applications, including primary HTS on compound collections. F. Gasparri et al. review the field and discuss in particular the application of HCA to the discovery of small molecule inhibitors targeting kinases that are implicated in oncology (Comb. Chem. High Throughput Screen.
The Use of Immortalized Cell Lines in GPCR Screening: The Good, Bad, and Ugly
For most membrane-bound molecular targets, including GPCRs, the optimal approach in drug discovery involves the use of cell-based HTS technologies to identify compounds that modulate target activity. Most GPCRs have been cloned and can therefore be routinely expressed in immortalized cell lines. These cells can be easily and rapidly grown in unlimited quantities, making them ideal for use in current HTS technologies. A significant advantage of this approach is that immortalized recombinant cells provide a homogenous background for expression of the target which greatly facilitates consistency in screening, thus allowing for a better understanding of the mechanism of action of the interacting compound or drug. Nonetheless, it is now evident that numerous disparities exist between the physiological environment of screening systems using recombinant cells and natural tissues. This has lead to a problem in the validity of the pharmacological data obtained using immortalized cells in as much as such cells do not always reflect the desired clinical efficacy and safety of the compounds under examination. R. M. Eglen et al. discuss in a brief review these issues, and describe how they influence the discovery of drugs using modern HTS (Comb. Chem. High Throughput Screen.
Label-Free Cell-Based Functional Assays
Label-free technologies based on electrical impedance or a refractive index are new tools for measuring a cell-based functional response. Although the technologies are relatively new to HTS cell-based applications, they are rapidly generating interest because they are able to measure a phenotypic response using cells natively expressing the target protein without using dyes or cellular extracts. In addition, one can measure the cellular response using a kinetic mode resulting in an assay potentially rich in content. L. K. Minor describes these technologies and their applications in measuring cell proliferation, cell attachment and spreading, cell apoptosis and their application in several receptor target classes, including receptor tyrosine kinases and GPCRs. The potential utility and drawbacks of these tools for HTS, directed screening, and compound profiling are also discussed (Comb. Chem. High Throughput Screen.