Preface Combinatorial chemistry has matured from a field where efforts initially focused on peptide-based research to become an indispensable research tool for molecular recognition, chemical-property optimization, and drug discovery. Originally used as a method to primarily generate large numbers of molecules, combinatorial chemistry has been significantly influenced and integrated with other important fields such as medicinal chemistry, analytical chemistry, syn- thetic chemistry, robotics, and computational chemistry. Even though the initial focus of attention was providing larger numbers of molecules with a ‘‘diversity’’ goal in mind, other factors came into play depending upon the problem scientists were trying to solve, such as bioactivity, solubility, permeability properties, PK, ADME, toxicity, and patentability. One can think of combinatorial chemistry and compound screening as an iterative Darwinian process of divergence and selection. Particularly in drug discovery, where time is a critical factor to success, combinatorial chemistry offers the means to test more molecule hypotheses in parallel. We will always be limited to a finite number of molecules that we can economically synthesize and evaluate. Even with all the advances in automa- tion technologies, combinatorial chemistry, and higher-throughput screens that improve our ability to rapidly confirm or disprove hypotheses, the synthesis and screening cycle remains the rate-determining process. Fortunately, we continue to make great strides forward in the quality and refinement of pre- dictive algorithms and in the breadth of the training sets amassed to aid in the drug discovery/compound optimization iterative process. Anyone who has optimized chemical reactions for combinatorial libraries or process chemistry knows first hand how much experimentation is required to identify optimal conditions. Chemical feasibility is at the heart of small mol- ecule discovery and chemotype prioritization since it essentially defines what can and cannot be analoged (i.e., analogability). Although analogability is not the only driving factor, quite often it is overlooked. For example, when com- mercially-available compounds or complex natural products are screened, the leads generated are often dropped because of the difficulty to rapidly analog them in the lead optimization stage. The desirability of a chemotype is a function of drug-likeness, potency, novelty, and analogability. A particularly attractive feature of combinatorial chemistry is that when desirable properties are identified, they can often be xiii xiv preface optimized through second-generation libraries following optimized synthetic protocols. If this process of exploring truly synthetically accessible chemical spaces could be automated, then it would open up the exciting possibility of modeling the iterative synthesis and screening cycle. Predicting, or even just mapping, synthetic feasibility is a sleeping giant; few people are looking into it, and the ramifications of a breakthrough would be revolutionary for both chemistry and drug discovery. In-roads to predicting (or even just mapping) chemical feasibility have the potential to have as large an impact on drug discovery as computational models of bioavailability and drugability. These are important questions where scientists are now starting to generate a large-enough body of information on high-throughput synthetic chemistry to begin to more globally understand what is cost-effectively pos- sible. Within the biopharmaceutical industry, significant investments in new technologies have been made in molecular biology, genomics, and proteomics. However, with the exception of combinatorial chemistry, relatively little has been done to advance the fundamental nature of chemistry in drug discovery from a conceptual perspective. Now, after having gone through the molecule-generating period where research institutions have a large historical compound collection and the pro- liferation of combinatorial chemistry services, the trend is now after making more targeted-oriented molecular entities also known as ‘‘focused libraries.’’ An important emerging question is: How can one most effectively make the best possible ‘‘focused libraries’’ to answer very specific research questions, given all the possible molecules one could theoretically synthesize? The first installment in this series (Volume 267, 1996) mostly covered peptide and peptidomimetic based research with just a few examples of small molecule libraries. In this volume we have compiled cutting-edge research in combinatorial chemistry, including divergent areas such as novel analytical techniques, microwave-assisted synthesis, novel linkers, and synthetic ap- proaches in both solid-phase and polymer-assisted synthesis of peptides, small molecules, and heterocyclic systems, as well as the application of these tech- nologies to optimize molecular properties of scientific and commercial interest. Guillermo A. Morales Barry A. Bunin METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF John N. Abelson Melvin I. Simon DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA FOUNDING EDITORS Sidney P. Colowick and Nathan O. Kaplan Contributors to Volume 369 Article numbers are in parentheses and following the names of contributors. Affiliations listed are current. Fernando Albericio (2), University Balan Chenera (24), Amgen Inc., Depart- of Barcelona, Barcelona Biomedical ment of Small Molecule Drug Discovery, Research Institute, Barcelona Science One Amgen Center Drive, Thousand Park, Josep Samitier 1, Barcelona, Oaks, California, 91320 08028, Spain James W. Christensen (5), Advanced Alessandra Bartolozzi (19), Surface ChemTech Inc., 5609 Fern Valley Road, Logix, Inc., 50 Soldiers Field Place, Louisville, Kentucky, 40228 Brighton, Massachusetts, 02135 Andrew P. Combs (12), Incyte Corpo- Hugues Bienayme´ (24), Chrysalon Mo- ration,Wilmington,Delaware,19880-0500 lecular Research, IRC, 11 Albert Einstein Avenue, Villeurbannem, 69100, France Scott M. Cowell (16), Department of Chemistry, University of Arizona, Sylvie E. Blondelle (18), Torrey Pines Tucson, Arizona, 85721 Institute for Molecular Studies, 3550 General Atomics Court, San Diego, Stefan Dahmen (7), Institut fur Orga- California, 92121 nische Chemie, RWTH Aachen, Pirlet- Str. 1, Aachen, 52074, Germany Ce´sar Boggiano (18), Torrey Pines Institute for Molecular Studies, 3550 Ninh Doan (17), Division of Hematology General Atomics Court, San Diego, and Oncology, Department of Internal California, 92121 Medicine, UC Davis Cancer Center, Uni- versity of California Davis, Sacramento, Stefan Bra¨ se (7), Institut fu¨r Organische California, 95817 Chemie, Universita¨t Karlsruhe (TH), Fritz-Haber-Weg 6, Karlsruhe, D-76131, Roland E. Dolle (8), Senior Director of Germany Chemistry, Department of Chemistry, Andrew M. Bray (3), Mimotopes Pty Adolor Corporation, 700 Pennsylvania Ltd., 11 Duerdin Street, Clayton, Vic- Drive, Exton, Pennsylvania, 19345 toria, 3168, Australia Nicholas Drinnan (14), Alchemia Pty Wolfgang K.-D. Brill (23), Discovery Ltd., Eight Mile Plains, Queensland Research Oncology, Pharmacia Italy 4113, Australia S.p.A, Viale Pasteur 10, Nerviano (MI), Amanda M. Enstrom (17), Division of I-20014, Italy Hematology and Oncology, Department Max Broadhurst (14), Alchemia Pty Ltd., of Internal Medicine, UC Davis Cancer Eight Mile Plains, Queensland 4113, Aus- Center, University of California Davis, tralia Sacramento, California, 95817 ix x contributors to volume 369 Liling Fang (1), ChemRx Division, Dis- Richard Houghten (25), Torrey Pines In- covery Partners International, 385 Oyster stitute for Molecular Studies, 3550 Gen- Point Boulevard, Suite 1, South San eral Atomics Court, Room 2-136, San Francisco, California, 94080 Diego, California, 92121 Eduard R. Felder (23), Discovery Re- Victor J. Hruby (16), Department of search Oncology, Pharmacia Italy Chemistry, University of Arizona, S.p.A., Viale Pasteur 10, Nerviano Tucson, Arizona, 85721 (MI), I-20014, Italy Christopher Hulme (24), Amgen Inc., De- A´ rpa´ d Furka (5), Eo¨tvo¨s Lora´nd Univer- partment of Small Molecule Drug Discov- sity, Department of Organic Chemistry, ery, One Amgen Center Drive, 29-1-B, P.O. Box 32, Budapest 112, H-1518, Thousand Oaks, California, 91320 Hungary Sharon A. Jackson (12), Aventis Pharma- A. Ganesan (22), University of Southamp- ceuticals, 202-206, Bridgewater, New ton, Department of Chemistry, Highfield, Jersey, 08807-0800 Southampton, SO17 1BJ,United Kingdom Ian W. James (3), Mimotopes Pty Ltd., 11 J. Gabriel Garcia (20), 4SC AG, Am Duerdin Street, Clayton, Victoria, 3168, Klopferspitz 19A, 82152, Martinsried, Australia Germany Wyeth Jones (24), Amgen Inc., Depart- Brian Glass (13), Incyte Corporation, ment of Small Molecule Drug Discovery, Wilmington, Delaware, 19880-0500 One Amgen Center Drive, 29-1-B, Thou- sand Oaks, California, 91320 Matthias Grathwohl (14), Alchemia Pty Ltd., Eight Mile Plains, Queenland 4113, Patrick Jouin (10), CNRS UPR 9023, Australia CCIPE, 141, rue de la Cardonille, Mont- pellier Cedex 05, 34094, France Michael J. Grogan (19), Surface Logix, Inc., 50 Soldiers Field Place, Brighton, C. Oliver Kappe (11), Institute of Chemis- Massachusetts, 02135 try, Karl-Franzens-University Graz, Heinrichstrasse 28, Graz, A-8010, Austria Xuyuan Gu (16), Department of Chemistry, University of Arizona, Steven A. Kates (19), Surface Logix, Inc., Tuscon, Arizona, 85721 50 Soldiers Field Place, Brighton, Massa- chusetts, 02135 Eric Healy (5), Advanced ChemTech Inc., 5609 Fern Valley Road, Louisville, Viktor Krchnˇ a´ k (6), Torviq, 3251 West Kentucky, 40228 Lambert Lane, Tuscon, Arizona, 85742 Timothy F. Herpin (4), Rhoˆne-Poulenec Kit S. Lam (15, 17), Division of Hematol- Rorer, 500 Arcola Road, Collegeville, ogy and Oncology, Department of In- Pennsylvania, 19426 ternal Medicine, UC Davis Cancer Center, University of California Davis, Cornelia E. Hoesl (25), Torrey Pines In- Sacramento, California, 95817 stitute, Room 2-136, 3550 General Atom- Alan L. Lehman (17), Division of Hema- ics Court, San Diego, California, 92121 tology and Oncology, Department of In- Christopher P. Holmes (9), Affymax Inc., ternal Medicine, UC Davis Cancer 4001 Miranda Avenue, Palo Alto, Center, University of California Davis, California, 94304 Sacramento, California, 95817 contributors to volume 369 xi Ruiwu Liu (15, 17), Division of Hematol- E.R. Palmacci (13), 77 Massachusetts ogy and Oncology, Department of In- Avenue, T18-209, Cambridge, Massachu- ternal Medicine, UC Davis Cancer setts, 02139 Center, University of California Davis, Yijun Pan (9), Affymax Inc., 4001 Mi- Sacramento, California, 95817 randa Avenue, Palo Alto, California, Matthias Lormann (7), Kekule´-Institut fu¨r 94304 Organische Chemie und Biochemie der Rheinischen, Friedrich Wilhelms Univer- Jack G. Parsons (3), Mimotopes Pty Ltd., sita¨t Bonn, Gerhard-Domagk-Strasse 1, 11 Duerdin Street, Clayton, Victoria, Bonn, D-53121, Germany 3168, Australia Jan Marik (15), Division of Hematology Robert Pascal (10), UMR 5073, Univer- and Oncology, Department of Internal site´ de Montpellier 2, CC017, place Medicine, UC Davis Cancer Center, Uni- Euge`ne Bataillon, Montpellier Cedex 05, versity of California Davis, Sacramento, F-34094, France California, 95817 Clemencia Pinilla (18), Torrey Pines In- Katia Martina (23), Discovery Research stitute for Molecular Studies and Mixture Oncology, Pharmacia Italy S.p.A., Viale Sciences, Inc., 3550 General Atomics Pasteur 10, Nerviano (MI), I-20014, Italy Court, San Diego, California, 92121 Joeseph Maxwell (17), Division of Hema- Obadiah J. Plante (13), Massachusetts tology and Oncology, Department of In- Institute of Technology, Department of ternal Medicine, UC Davis Cancer Chemistry, 77 Massachusetts Avenue, Center, University of California Davis, Cambridge, Massachusetts, 02139-4307 Sacramento, California, 95817 Gregory Qushair (2), University Wim Meutermans (14), Alchemia Pty Ltd., of Barcelona, Barcelona Biomedical 3 Hi-Tech Court, Brisbane Technology Research Institute, Barcelona Science Park, Eight Mile Plains, QLD 4113, Aus- Park, Josep Samitier 1, Barcelona, tralia 08028, Spain George C. Morton (4), Rhoˆne-Poulenc Jorg Rademann (21), Eberhard-Karls-Uni- Rorer, 500 Arcola Road, Collegeville, versity, Tu¨bingen, Institute of Organic Pennsylvania, 19426 Chemistry, Auf der Morgenstelle 18, Tu¨- Adel Nefzi (25), Torrey Pines Institute for bingen, 72076, Germany Molecular Studies, 3550 General Atomics Joseph M. Salvino (8), Director of Com- Court, San Diego, California, 92121 binational Chemistry, Adolor Corpor- Thomas Nixey (24), Amgen Inc., Depart- ation, 700 Pennsylvania Drive, Exton, ment of Small Molecule Drug Discovery, Pennsylvania, 19345 One Amgen Center Drive, 29-1-B, Thou- Peter H. Seeberger (13), Laboratorium sand Oaks, California, 91320 fuer Organische Chemie, HCI F 315, John M. Ostresh (25), Torrey Pines Insti- Wolfgang-Pauli-Str. 10, ETH-Hoengger- tute, Room 2-136, 3550 General Atomics berg, CH-8093 Zu¨rich, Switzerland Court, San Diego, California 92121 Craig S. Sheehan (3), Mimotopes Pty Vitecek Padeˇra (6), Torvic, 3251 W Lam- Ltd., 11 Duerdin Street, Clayton, Vic- bert Lane, Tucson, Arizona, 84742 toria, 3168, Australia xii contributors to volume 369 Adrian L. Smith (24), Amgen Inc., Depart- Jesus Vazquez (2), University of Barce- ment of Small Molecule Drug Discovery, lona, Barcelona Biomedical Research One Amgen Center Drive, Thousand Institute, Barcelona Science Park, Josep Oaks, California, 91320 Samitier 1, Barcelona, 08028, Spain Re´gine Sola (10), UMR 5076, Ecole Michael L. West (14), Alchemia Pty Ltd., Nationale Supe´rieure de Chimie de Eight Mile Plains, Queensland 4113, Montpellier, 8, rue Delaware l’Ecole Australia Normale, Montpellier Cedex 05, F- 34296, France Zemin Wu (3), Mimotopes Pty Ltd., 11 Aimin Song (17), University of California, Duerdin Street, Clayton, Victoria, 3168, UC Davis Cancer Center, Division of Australia Hematology and Oncology, 4501 X Street, Sacramento, California, 95817 Bing Yan (1), ChemRx Division, Discovery Partners International, 385 Oyster Point, Alexander Stadler (11), Institute of Boulevard, Suite 1, South San Francisco, Chemistry, Karl-Franzens-University California, 94080 Graz, Heinrichstrasse 28, Graz, A-8010, Austria Yongping Yu (25), Torrey Pines Institute, Paul Tempest (24), Amgen Inc., Depart- Room 2-136, 3550 General Atomics ment of Small Molecule Drug Discovery, Court, San Diego, California, 92121 One Amgen Center Drive, 29-1-B, Thou- sand Oaks, California, 91320 Florencio Zaragoza (26), Medicinal Chemistry, Novo Nordisk A/S, Novo Nor- David Tumelty (9), Affymax, Inc., disk Park, Malov, 2760, Denmark 4001 Miranda Avenue, Palo Alto, California, 94304 Jiang Zhao (1), ChemRx Division, Discov- Josef Vagner (16), Department of Chem- ery Partners International, 385 Oyster istry, University of Arizona, Tuscon, Ari- Point Boulevard, Suite 1, South San zona, 85741 Francisco, California, 94080 [1] high-throughput LC/UV/MS analysis of libraries 3 [1] High-Throughput Parallel LC/UV/MS Analysis of Combinatorial Libraries By Liling Fang, Jiang Zhao, and Bing Yan Introduction Combinatorial chemistry and high-throughput organic synthesis allow the preparation of a large number of diverse compounds in a relative short period of time in order to accelerate discovery efforts in the pharmaceut- ical and other industries. A library can comprise hundreds to thousands of compounds with the need to rapidly analyze those compounds for their identity and purity. Different compound separation and mass spectrometry (MS) techniques have been applied for the characterization of combinator- ial libraries. These include separation techniques such as liquid chromatog- raphy (LC) and capillary electrophoresis and different ionization methods 1–3 * and mass analyzers. LC/MS is the most popular technique used in com- binatorial library analysis because it combines separation, molecular weight determination, and relative purity evaluation in a single sample in- jection. However, the throughput of conventional LC/MS could not meet the need to analyze every member in a large combinatorial library in a timely fashion. Higher-throughput analysis was achieved by utilizing shorter columns 4 at higher flow rates. Supercritical fluid chromatography (SFC)/MS has 1 A. Hauser-Fang and P. Vouros, ‘‘Analytical Techniques in Combinatorial Chemistry’’ (M. E. Swartz, ed.). Marcel Dekker, New York, 2000. 2 B. Yan, ‘‘Analytical Methods in Combinatorial Chemistry.’’ Technomic, Lancaster, 2000. 3 D. G. Schmid, P. Grosche, H. Bandel, and G. Jung, Biotechnol. Bioeng. Comb. Chem. 71, 149 (2001). *Abbreviations: CLND, chemiluminescence nitrogen detection; C log P, calculated partition coefficient; ELSD, evaporative light scattering detection; ESI-MS, electrospray ionization mass spectrometry; FWHM, full width at half maximum; i.d., inner diameter; LC, HPLC, liquid chromatography, high-performance liquid chromatography; LC/MS, liquid chroma- tography – mass spectrometry; LC/MS/MS, liquid chromatography – mass spectrometry – mass spectrometry; LC/UV/MS, liquid chromatography mass spectrometry with a UV detector; LIB, compound library; log P, water/octanol partition coefficient; MUX, multiplexed; RSD, relative standard deviation; SFC, supercritical fluid chromatography; TFA, trifluoroacetic acid; TIC, total ion current; TOF, time of flight; TOFMS, time of flight mass spectrometry. 4 H. Lee, L. Li, and J. Kyranos, Proceedings of the 47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, Texas, June 13–17, 1999. Copyright 2003, Elsevier Inc. All rights reserved. METHODS IN ENZYMOLOGY, VOL. 369 0076-6879/03 $35.00 4 analytical techniques [1] been used to achieve desirable high speed taking advantage of the low vis- 5 cosity of CO2. However, the serial LC/MS approach by its nature does not match the speed of parallel synthesis. Parallel LC/MS is the method of choice to increase throughput while maintaining the separation efficiency. An eight-probe Gilson 215/889 autosampler was incorporated into a 6 quadruple mass spectrometer. This arrangement enabled the injection of eight samples (a column from a 96-well microtiter plate) simultaneously for flow-injection analysis/MS (FIA-MS) analysis to achieve a throughput 7 of 8 samples/min. A novel multiplexed electrospray interface (MUX) was developed in 1999 and became commercially available for parallel high-throughput LC/UV/MS analysis. The eight-way MUX consists of eight nebulization-assisted electrospray ionization sprayers, a desolvation gas heater probe, and a rotating aperture. It can accommodate all eight high-performance liquid chromatograph (HPLC) streams at a reduced flow rate of <100 l/min per stream and conduct electrospray ionization for all eight streams simultaneously. Ions are continuously formed at the tip of each sprayer and the MUX interface allows sprayers to be sampled sequen- tially using the rotating aperture driven by a programmable stepper motor. At any given time, only ions from one stream are admitted into the ion sampling cone, while ions from the other seven sprayers are shielded. Each liquid stream is sampled for a preset time with mass spectra acquired in full mass range into eight simultaneously open data files synchronized with the spray being sampled. With a 0.1-s acquisition time per sprayer and 0.05-s intersprayer delay time, the time-of-flight (TOF) mass analyzer can acquire a discrete data file of electrospray ion current sampled from each stream over the entire HPLC separation with a cycle time of 1.2 s. Therefore, this eight-way MUX-LCT was like having eight individual electrospray ionization (ESI)-MS systems working simultaneously. The MUX interface enables the coupling of parallel liquid chromatog- raphy to a single mass spectrometer. This technology has had a great impact in high-throughput LC/MS analysis. In drug development, a four- way MUX interface was used on a triple quadrupole mass spectrometer to simultaneously validate LC/MS/MS methods for the quantitation of 8 loratadine and its metabolite in four different biological matrixes and of 5 M. C. Ventura, W. P. Farrell, C. M. Aurigemma, and M. J. Greig, Anal. Chem. 71, 2410 (1999). 6 T. Wang, L. Zeng, T. Strader, L. Burton, and D. B. Kassel, Rapid Commun. Mass Spectrom. 12, 1123 (1998). 7 V. De Biasil, N. Haskins, A. Organ, R. Bateman, K. Giles, and S. Jarvis, Rapid Commun. Mass Spectrom. 13, 1165 (1999). 8 M. K. Bayliss, D. Little, D. M. Mallett, and R. S. Plumb, Rapid Commun. Mass Spectrom. 14, 2039 (2000). [1] high-throughput LC/UV/MS analysis of libraries 5 9 diazepam in rat liver microsomes for in vitro metabolic stability. The four- channel LC/MS/MS system was also reported for the quantification of a 10 drug in plasma on both the narrow-bore and capillary scales. By incorpor- ating divert valves into this system, aliquots of plasma could be directly analyzed without sample preparation. The four-channel LC/MS/MS has re- duced method validation time, increased sample throughput by 4-fold, and afforded adequate sensitivity, precision, and negligible intersprayer cross- 8,9 talk. In protein analysis, an eight-way MUX coupled with a TOFMS analyzer has proved to be a powerful tool to monitor the protein purifica- tion process by screening fractions from preparative ion-exchange chroma- tography with a throughput of 50 protein-containing fractions in less than 11 an hour. A high-pressure gradient parallel pumping system (JASCO PAR-1500) has been developed to conduct high-throughput parallel liquid chromatog- 12 raphy. It is a 10-pump system where two pumps are used to generate a binary gradient and eight pumps to deliver the mixed solvent to eight LC columns. Comparing this system to a conventional system with two pumps or a binary pump for LC gradient and a simple splitter to divide the gradi- ent to eight LC columns, this system can ensure uniform flow rates through each LC column. This system has been used for peptides and combinatorial 12 13 9 sample, protein analysis, and bioanalysis. We have optimized an eight-way MUX coupled to a TOFMS analyzer to carry out eight-channel parallel LC/UV/MS analysis of combinatorial 14 libraries in the past 2 years. This system has not only provided the capacity needed for library analysis, but also enabled simultaneous evalu- ation of experimental parameters to expedite the method development process. In this chapter, we discuss the optimization of this system and present a high-throughput protocol for combinatorial library analysis. We also compare the eight-channel parallel LC/UV/MS system to a conven- tional single channel LC/UV/MS system in terms of performance and operation. 9 D. Morrison, A. E. Davis, and A. P. Watt, Anal. Chem. 74, 1896 (2002). 10 L. Yang, T. D. Mann, D. Little, N. Wu, R. P. Clement, and P. J. Rudewicz, Anal. Chem. 73, 1740 (2001). 11 B. Feng, A. Patel, P. M. Keller, and J. R. Slemmon, Rapid Commun. Mass Spectrom. 15, 821 (2001). 12 D. Tolson, A. Organ, and A. Shah, Rapid Commun. Mass Spectrom. 15, 1244 (2001). 13 B. Feng, M. S. McQueney, T. M. Mezzasalma, and J. R. Slemmon, Anal. Chem. 73, 5691 (2001). 14 J. Zhao, D. Liu, J. Wheatley, L. Fang, and B. Yan, Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May, 27–31, 2001.