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Combinatorial Chemistry: Novel Strategies, Chemistry, Purification and Chemoinformatics
Drug & Market Development Publications, Oct 1999)


  • The introduction of combinatorial chemistry in the mid eighties ushered in a new paradigm for organic chemistry, and recent years have seen several reports on new leads from combinatorial libraries, as well as their successful optimization as drug candidates.
  • Although some companies still rely on the production of huge libraries to increase the chances of finding new leads, the majority of those involved in combinatorial drug design have shifted strategies. Many now attempt to reduce the effort and costs associated with synthesis and screening by carefully designing focused libraries with optimized diversity and drug-like characteristics.
  • Solid phase chemistry continues to play an important role, as large libraries of relatively pure compounds can be produced once the time-consuming reaction optimization has been performed. However, liquid phase synthesis, sometimes in combination with polymer-bound reagents or scavenger reagents, offers significant advantages; the range of applicable chemistry is much broader and most problems associated with solid-phase synthesis are not encountered.
  • For small, focused libraries, automated parallel synthesis in liquid phase is the most cost-effective strategy. Due to the increasing importance of such libraries, automated purification is now an integrated process in the production of compounds for biological testing. All steps can be automated, from the input of impure compounds in a certain format to the output of pure compounds in the same format.
  • In recent years, combinatorial chemistry has also been used to optimize organic reaction conditions, to produce materials with desired optical, electric and magnetic properties, and to discover new catalysts and polymers.
  • Two recent IBC UK ( conferences titled Combinatorial Chemistry ‘99: Novel Strategies, Chemistry, Purification & Chemoinformatics and Exploiting the Promise of Combinatorial Chemistry, held June 23-25, 1999, in London, UK, offered a forum for discussion of the latest approaches to combinatorial chemistry.

Introduction and Overview

The earliest drugs originated in folk medicine. As societies advanced, more rational approaches were adopted, but accidental discoveries still led to many valuable drugs such as chlordiazepoxide, cisplatin, penicillin, pethidine (meperidine), sulfamidochrysoidin and warfarin, to name a few. Even acetylsalicylic acid was a serendipitous discovery, as its inventor, the Bayer chemist Felix Hoffmann, did not know that this compound was not simply a prodrug of salicylic acid (as supposed by him), but a unique therapeutic in its own right; in addition to its fever-reducing and pain-killing properties, it was found to possess antiplatelet aggregation activity.

For almost a century, organic syntheses and animal experiments and trial and error governed the search for new drugs. In recent years, however, new targets from genomics, high-throughput test systems, molecular modelling and computer-aided drug design have added new dimensions to lead discovery and optimization. With the ongoing progress in protein crystallography and NMR, structure-based ligand design has become more and more important. Many successful drugs have proven the value of such rational approaches.

Combinatorial chemistry had a slow start. Although the development of the Ugi multicomponent reaction in 1962 and the Merrifield solid-phase synthesis in 1963 offered, in principle, the necessary tools to synthesize libraries of small organic compounds, the first combinatorial syntheses didn’t result until 20 years later, represented in the work of A. Furka, M. Geysen and R. Houghten on the production of peptide (later peptide and nucleic acid) libraries. From about 1990 onwards, small molecules were also synthesized, usually as multi-component mixtures. Nowadays, combinatorial chemistry is most often applied to produce libraries of single, pure compounds with drug-like properties. Correspondingly, the sizes of the libraries have become smaller and smaller and, as a consequence, the balance between the necessary effort for reaction optimization and the number of resulting compounds has become unfavorable. New synthetic procedures as well as rational design approaches needed to be developed. Combinatorial chemistry only seemingly forces chance to improve the success in drug research. The mere synthesis of enormous numbers of organic compounds, without rational design, is a waste of time and resources. Combinatorial chemistry will increase the success rate in drug research only in combination with appropriate selection procedures, such as filtering compounds by properties and/or virtual screening.

Combinatorial chemistry has been largely adopted by the pharmaceutical and biotechnology industries. From the first technological developments to the current widespread integration in discovery and development at a highly automated level, there have been questions raised regarding the ultimate extent of diversity and the leads that have been generated. Correspondingly, the most important topics at the Combinatorial Chemistry ‘99 conference (see introductory bullets) were chemoinformatics tools for rational library design, molecular diversity, sublibrary selection and virtual screening of combinatorial libraries. The shift in synthetic and design strategies was clearly reflected in a number of presentations at the conference.

Novel Strategies

Taken together, library synthesis and high-throughput screening (HTS) allow the development and screening of huge numbers of molecules. However, the futility of this approach has become apparent, as there will always be many more molecules to make than can be screened and tested. As such, several strategies for the design of appropriate molecules for lead generation and their optimization into drug-like molecules have been developed at Glaxo Wellcome (Stevenage, Hertfordshire, UK). Sublibraries are selected from virtual libraries by considering the desired properties of the final products. As illustrated by Mike Hann, Group Leader Computational Chemistry, the company is using a range of Web-based tools in this approach, including a generalized genetic algorithm (GA) approach for design against any property, a GA approach that generates diversity within a focused design, and an efficacy and efficiency score for fast monomer selection.

At SmithKline Beecham (King of Prussia, PA), high-throughput lead discovery technology uses the efficiency of split-and-mix combinatorial syntheses while still allowing the assay of individual compounds. Michael Moore, Associate Director Combinatorial and Chemical Technologies, reported on high-loading, large diameter polystyrene beads that are used as polymeric support. Single beads are arrayed and cleaved, yielding compounds in the range of 10-20 nmol per bead (i.e., about 5-10 mg), which is sufficient for multiple biological assays as well as for compound identification by LC/MS.

For successful lead optimization libraries, the library design has to be focused around a lead and biased towards "drug-like" compounds. Heuristic optimization strategies to combinatorial library design include the application of multicenter pharmacophores for the description of molecular diversity space, considering conformational flexibility of the molecules. For the design of drug-like properties and for enhancing hit-to-lead properties of lead optimization libraries, two approaches were presented by David E. Clark, Research Fellow Computer-Assisted Drug Design, Rhone-Poulenc Rorer (Dagenham, Essex, UK). The first seeks to maximize the overlap of computed physical properties (e.g., ClogP, MW, etc.) between the designed library and a collection of known drug molecules. The second considers bioavailability by using the "rule of five" and calculating polar surface areas of the compounds as a measure of their ability to permeate biological membranes. The techniques were illustrated by the optimization of a 4,5-bis-arylimidazole library with respect to Caco-2 cell permeability.

Combinatorial Chemistry and Methodology

Scavenger resins, polymer-bound reagents, catch and release methods, and resin capture have become increasingly important in the production of combinatorial libraries. Such strategies allow organic synthesis using simple, parallel product purification by filtration, avoiding silica-gel chromatography and/or extraction. Reaction procedures for parallel synthesis of libraries of tertiary amines (from diamines and polystyrene-TsCl), amides and sulfonamides (using a polystyrene-DMAP reagent), 2-aminothiazoles and 1,2,3-thiadiazoles using catch and release and resin capture techniques, and polymeric reagents for the chlorination of acids and alcohols for Mitsunobo and Wittig reactions (polystyrene-triphenylphosphine) were presented by Bernd Renneberg, Applications Chemist, Argonaut Technologies AG (Muttenz, Switzerland). New soluble polymer-supported catalysts, reagents and synthetic targets as adjuncts to solution-phase library development were described by Paul Wentworth, Jr. of The Scripps Research Institute & Skaggs Institute for Chemical Biology (La Jolla, CA). Examples are covalent scavenger reagents to remove unwanted starting materials or by-products, resin capture reagents to transfer products in solution to solid phase for further modification, or "fishing-out" reagents to remove products from a complex reaction mixture. In parallel, new resin supports for solid-phase chemistry have been developed by a novel cross-linking strategy. The physicochemical properties of PEG cross-linked polystyrene resin supports (i.e., increased mechanical stability, better swelling properties and better intra-resin diffusion in poor-swelling solvents) demonstrate the utility of this material.

The development and application of resin-bound selenium as traceless linker in solid-phase organic syntheses of small non-peptide compounds was described by Thomas Ruhland, Research Chemist, H. Lundbeck A/S (Copenhagen, Denmark). Compounds are attached by direct loading without the requirement of an auxiliary spacer, as demonstrated by the synthesis of a small-sized library of single alkyl aryl ethers by the Mitsunobu reaction. The selenium-alkyl bond that attaches the products to the resin is smoothly cleaved under radical conditions. Disadvantages of the selenium linker, however, are its incompatibility with certain reaction conditions (i.e., radical chemistry, some oxidation reactions, Pd catalysis) and the toxicity of selenium.

Anthony D. Baxter, Chief Scientific Officer, Oxford Asymmetry International (Abingdon, Oxon, UK), and Lutz Weber, Chief Scientific Officer, Morphochem AG (Martinsried, Germany), described the respective approaches of their companies for the synthesis of lead discovery libraries. Oxford Asymmetry’s building block approach to library synthesis via novel building blocks uses conformationally restricted templates, having at least three sites of diversity. These are coupled with preselected monomers. As guidelines for "drug-like" molecules, a slightly modified "rule of five" is applied. Privileged structures include biphenyls, biphenyl ethers, benzodiazepins, benzoxazins, spirohydantoins and other heterocyclic templates. Different functional groups are attached to appropriate linkers to enhance the diversity of a library that is based on one template.

The Morphochem approach towards high diversity combinatorial chemistry, on the other hand, includes the generation of libraries where substituents and backbones are varied at the same time. Once a biologically interesting molecule has been identified, rather conventional combinatorial chemistry techniques may follow. Methods that allow the generation of high diversity libraries by systematic variation of reaction types, instead of starting materials of just one reaction, have been developed. The most efficient optimization of lead structures is performed by application of intelligent selection and filter functions (e.g., ClogP, MW, Pfizer rules, etc.); in addition, affinity estimations, neural networks, genetic algorithms and pattern recognition methods are applied. However, such strategies require the synthesis of "random access" chemical libraries instead of today’s systematic combinatorial libraries.

Automation, Purification and Analytics

Michael Moore of SmithKline Beecham presented the Myriad Core System and the Irori AutoSort 10K system as equipment for medium- and large-scale array synthesis of combinatorial libraries. High-throughput purification is performed at SmithKline Beecham with a Gilson AutoPrep HPLC; 192 samples per day are purified, using LC/MS data of the crude sample and UV-triggered fractionation. Most compound fractions are collected into a single tube. Overall, this is combined to a high-throughput production process that includes high-throughput synthesis, isolation, analysis, purification, quality control and registration.

With advances in automated parallel synthesis, the construction of large combinatorial arrays has become possible; however, this has resulted in a corresponding challenge for the analyst to provide meaningful information on such large sample libraries. As illustrated by Ashley B. Sage, Project Leader LC/MS Applications, Micromass UK Ltd. (Wythenshawe, Manchester, UK), the use of LC/MS in combinatorial chemistry has the following requirements: • -Automated high throughput characterization and exact mass determination of arrays using an orthogonal acceleration time-of-flight (oa-TOF) mass spectrometer

  • The design and implementation of a multiplexed electrospray system integrated into a mass spectrometer that is capable of rapidly sampling multiple liquid streams, facilitating and increasing sample analysis throughput
  • Multi-milligram quantity purification of compounds using both reverse and normal phase HPLC conditions
  • Fraction collection by LC/MS including automated sample tracking and reporting

A high-throughput organic chemistry (HTOC) process was designed at Biotage, a division of Dyax Corporation (Charlottesville, VA), to transfer crude reaction mixtures to purified compounds of known identity and weight. The Parallex‰ HPLC, presented by Patrick Coffey, Vice President Automated Chemistry, is a four-column preparative HPLC system with deep-well microtiter plate formats for input and output. All samples and fractions are bar-coded and all information is tracked. The user can specify rules for selection, combination, dissolution and wash steps and interactively fine-tune the selections. If fractions need to be combined into smaller volumes, the corresponding plates are sent to an evaporator and redissolved to place identical fractions into the final destination sites. As a result, the input format of an impure library is reproduced as the same format of purified and analytically characterized compounds. The estimated costs of the entire process are less than $10 per 10 mg compound, which favorably compares with the significantly higher costs of synthesis of the impure samples.

Various software tools for structure verification by MS, MS/MS and NMR spectra were presented by Herbert Thiele, Manager Software Development, Bruker Daltonik GmbH (Bremen, Germany). With a corresponding increase in computational effort, NMR spectral shifts can be: (a) estimated from increment systems [e.g. SpecTool], (b) predicted [WIN-SpecEdit, SpecInfo, CSEARCH, ACD], (c) simulated [LAOCOON, WIN-DAISY], or (d) calculated by quantum mechanical treatment of the molecule [HyperNMR, NMR-Cindo, Gaussian]. Practical limitations of the empirical approaches are limited bond coverage of the topology code, through space effects, tautomers, delocalized bonds and charges, and stereochemical and solvent influences.

Chemoinformatics: Design and Virtual Screening of Libraries

The development of automatic 3D structure generators (e.g., the program CORINA) has made possible the large-scale investigation of the relationships between 3D structures and biological activity. Much controversy presently centers around 2D vs. 3D descriptors, with 2D descriptors often outperforming 3D descriptors. However, molecules are 3D objects, and biological activity is a reflection of the 3D properties of the molecules. Novel ways to code chemical structures for correlating biological activity and determining chemical diversity were presented by Johann Gasteiger, University of Erlangen-Nuremberg (Erlangen, Germany). The relationships between 3D structures and biological activities are explored by statistical analyses, pattern recognition methods and neural networks for similarity perception.

RECAP is a retrosynthetic combinatorial analysis procedure for identifying biologically privileged fragments for use in the synthesis of targeted libraries. This powerful tool, described by Duncan B. Judd, Group Leader Lead Design, Discovery Technology, Glaxo Wellcome R&D, involves the use of databases of compounds with known biological activity that are electronically "cleaved" at bonds amenable to combinatorial chemistry (e.g., amides, esters, amines, ureas, ethers, biphenyls and sulfonamides). The fragments and motifs can be readily used as building blocks to prepare combinatorial libraries containing biologically privileged substructure motifs that may be used in lead generation or lead optimization.

ChemSpace is an approach for the generation of both focused and diverse libraries. Topomeric searching of huge virtual libraries built around lead compounds facilitates the rapid and generalized definition of structure-activity relationships by guiding the synthetic chemistry program. Implementation of ChemSpace in the production of designed libraries of chemical compounds was discussed by Anthony Cooper, Managing Director, Tripos Receptor Research (Bude, Cornwall, UK). In cooperation with Bristol Myers Squibb (Princeton, NJ), ChemSpace was validated by searching angiotensin II antagonists in a virtual library of 2.6 billion compounds, from which, after several filtering processes, 425 compounds were selected and synthesized. Whereas there were 63 hits in this set, no highly active compounds were found in a control set of randomly chosen compounds.

Nick Perry, Molecular Modelling, Knoll Pharmaceuticals (Nottingham, UK), posed the question, "Does ‘more diverse’ mean ‘more informative’?" Much effort has been expended in devising methods to select structurally diverse subsets from compound libraries, with the implicit assumption that testing a diverse subset in a biological screen is more effective for lead finding than testing a random subset. MDL keys and Ghose and Crippen fingerprints were used to define dissimilarity metrics and two recursive selection methods were compared, with the Most Descriptive Compound method performing better than the Minimum Separation method. The efficacy of the selection procedure was measured as the improvement compared with random selection of compounds representing different biological activity-types. Typically, structurally diverse subsets give a better biological coverage when compared to randomly selected subsets.

Chemically related compounds most often show similar biological activities. This fundamental relationship has led to the discovery and stepwise optimization of many valuable drugs. However, pitfalls that may arise in the design of a combinatorial library were discussed by Hugo Kubinyi, BASF AG (Ludwigshafen, Germany). In many cases, presumably closely related compounds show very different modes of action and/or biological potencies, for example tricyclic antihistamines, neuroleptics and antidepressants, the female and male sex hormones, agonists and antagonists, and isosteric analoges. Other minor structural modifications result in surprisingly different binding modes.


Exploiting the Promise of Combinatorial Chemistry: Applications in Drug Research and Catalysis

Genomics and proteomics offer a host of novel therapeutic targets. Combinatorial chemistry holds the promise of decreasing overall development times by increasing the probability of success for lead discovery against "difficult" targets. John J. Baldwin, Chief Science and Technology Officer, Pharmacopeia, Inc. (Cranbury, NJ) illustrated the use of large encoded libraries in lead discovery. Success stories on micromolar and submicromolar ligands against different protein kinases and nanomolar inhibitors against the bradykinin receptor-1 (B1) were presented without providing structural details.

Combinatorial chemistry and structure-based drug design are effective methods for finding lead compounds, but both have their limitations. The PRO_SELECT methodology, discussed by Stephen C. Young, Head of Synthetic Chemistry, Proteus Molecular Design Ltd. (Macclesfield, Cheshire, UK), combines the benefits of both. A huge virtual combinatorial library is first reduced in size by applying several filters and then screened for complementarity against a receptor structure. The resulting focused sublibrary has a higher hit-per-compound ratio than a random and diverse library of related chemistry. The PRO_SELECT approach has been used to create small libraries targeted against the therapeutically important serine proteases thrombin (virtual library of 37.5 million compounds reduced to a 72-member sublibrary), trypsin, and factor Xa; a high proportion of "hits" was obtained. For factor Xa, the process was used iteratively, starting from a small template molecule. This ultimately led to a series of highly potent and selective molecules. The same approach could be used in designing slightly larger combinatorial libraries focused on targets with less clearly defined structures.

Nowadays, resistance of bacteria against most antibiotics used in the clinic is a serious problem. Thus, discovery of new medicinally useful anti-infectives against resistant gram-positive bacteria is a very important issue. A series of novel small molecules (e.g., 3,4-disubstituted 2-(indol-3-yl)-tetrahydroisoquinolines) synthesized via combinatorial methods and demonstrating very interesting in vitro and in vivo activity against resistant gram-positive infections were presented by James R. Hauske, Sr. Vice President Discovery, Sepracor, Inc. (Marlborough, MA). In this study, combinatorial methods provided both lead discovery and lead optimization libraries that were linked to in vitro ADME, toxicological and physicochemical property screens affording compounds with drug-like properties.

An overview of combinatorial catalysis and the state-of-the-art in industry was provided by Thomas Bradshaw, President, The Catalyst Group (Springhouse, PA). The high-throughput screening of either heterogeneous or homogeneous catalysts for the highest activity and yield for a specific reaction requires the optimization of multidimensional operating parameters (e.g., pH, temperature, solvent or gas phase, pressure, etc.) to maximize yield and purity of the reaction product(s). Although combinatorial catalyst development and optimization is still in its infancy, commercial commitments already exist to develop this tool further; there have been several cooperations between large companies and the combinatorial material science company, Symyx (Santa Clara, CA), and other venture capital companies. These cooperations include synthesis of catalysts using combinatorial approaches, the evaluation of catalyst activities using robotic systems, and process development for novel catalyzed reactions.

Summary and Conclusions

Drug research is an evolutionary process. In the same manner that nature developed higher organisms from more primitive forms, lead structure search and optimization follow evolutionary principles. Combinatorial chemistry will not change this. However, combinatorial chemistry does speed up drug discovery in two different ways: (1) the production of large numbers of chemically diverse libraries may increase the yield of new leads, and (2) rational lead optimization by automated parallel synthesis reduces the time needed for each evolutionary cycle.

Drug properties are more important than the chemical accessibility of a library. Thus, chemistry-driven combinatorial libraries are less attractive than rationally designed libraries. "Drug-like" properties, good oral bioavailability and metabolic stability are preconditions for valuable leads. Similarity can be better defined than diversity, or the lack of similarity. Chemically similar compounds may have very different biological activities; their "biological similarity" significantly depends on the target. On the other hand, very different compounds may have identical biological activities.

Large libraries may have a higher probability of producing hits (following a nonlinear dependence!), ease the derivation of SARs and/or QSARs, and allow a broader patent coverage. However, despite all this, large libraries are most often a waste of time and resources because small libraries need less effort for reaction optimization and production (e.g., liquid phase instead of solid phase synthesis) and generate a much higher diversity.

Structure-based and computer-assisted design of protein ligands supplement combinatorial chemistry in drug research. Virtual screening will become increasingly important in the design of combinatorial libraries. Computer-assisted, flexible, combinatorial construction of drugs within the binding pocket of the biological target using diverse building blocks will be possible in the near future. The necessary tools are already available, but scoring functions have to be improved.

Finding active compounds in libraries is no success at all. Decisive for industrial success is not "me too," but "me better." "me faster," "me first" or even "me only". Combinatorial chemistry will not replace classical chemistry. Only minor segments of chemistry and of the universe of biologically active compounds can be covered by combinatorial libraries; only simple and robust equipment is suited for automated synthesis in organic chemistry laboratories. In essence, combinatorial chemistry is a tool. It will not directly produce development candidates for clinical investigations, but a lot of important information will result that will enable the medicinal chemists to find new drugs much easier and much faster.

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