High Throughput Analysis for Early Drug Discovery

204 Pages·2004·5.89 MB·English

by  James Kyranos (Editor)

High Throughput Analysis
for Early Drug Discovery
This page is intentionally left blank
High Throughput Analysis
for Early Drug Discovery
Edited by
James N. Kyranos
ArQule, Inc.
Woburn, MA, USA
2004
Amsterdam–Boston–Heidelberg–London–New York–Oxford–Paris
San Diego–San Francisco–Singapore–Sydney–Tokyo
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Contents
Preface ix
List of Contributors xiii
Acknowledgments xvii
1 High Throughput Analysis of Combinatorial Libraries Encoded
with Electrophoric Molecular Tags 1
1 Introduction 1
2 Tag Decode-Assisted Single BeadLC/MS Analysis
(Library QA) 3
3 Statistical Considerations 4
4 Synthesis ofthe Encoded StatineAmide Library 5
5 Biological Screening 15
6 CorroboratingScreening Structure Activity Relationship(SAR)
Data with Library QAAnalysis 21
7 New Developments 23
8 Conclusion 33
References 33
2 Analysisof aCombinatorialLibrary Synthesized Using
a Split-and-Pool Irori MicroKan Method for Development
and Production 37
1 Introduction 38
2 Instrumentation 39
2.1 Single-Channel HPLC/UV/ELSD/MS System 39
2.2 Four-Channel Multiplexed HPLC/UV/ELSD/MS System 40
3 Data Processing 41
vi Contents
4 Automated SPE System in 96-Well Plate Format 41
5 Results and Discussion 42
6 Library DevelopmentStage 45
7 Library Production Stage 47
8 HighThroughput Purificationusing SPE 48
9 100%QC byLC/UV/ELSD/MS andAnalytical Reports 50
10 Future Trends 51
11 Conclusion 55
Acknowledgements 55
References 55
3 High ThroughputFlow Injection Analysis–Mass Spectrometry 57
1 Introduction 57
2 Open-AccessMass Spectrometry 59
3 High-Speed Flow InjectionAnalysis–Mass Spectrometry 59
3.1 Flow Rate, Injection Loops,and Transfer Tubes 61
3.2 Autosampler Overhead–Multiplexing Injection Cycle Procedures 62
3.3 Combining High Sampling Rates with Fast Analysis 65
4 Other High Speed and High ThroughputInstrumentation
for MW Measurement 67
5 Conclusion 70
Acknowledgements 70
References 70
4 High Throughput Flow Injection Analysis–Mass Spectrometry for
Combinatorial Chemistry Using Electrospray Ionization, Atmospheric
Pressure Chemical Ionization and Exact-Mass Fourier Transform
Mass Spectrometry 73
1 Introduction 74
2 HighThroughput MUX Flow-Injection ESI and APCI
Low-ResolutionMass Spectral Analysis 74
2.1 ESI Experimental Methods 75
2.2 ESI Results and Discussion 77
2.3 MUXIssues 78
2.4 APCIExperimental Methods 82
2.5 APCIResults andDiscussion 82
3 HighThroughput High-Resolution Exact-Mass FIA ESI-FTMS 84
3.1 High-Resolution Experimental Methods 85
3.2 High-Resolution Results and Discussion 86
4 Sample Preparation Methodsfor ESI-MS Analysis ofDrugs
Bound toResin Beads 88
4.1 Sample Preparation Experimental Methods 88
4.2 Sample Preparation Results and Discussion 90
Contents vii
5 Conclusions 91
Acknowledgements 92
References 92
5 Purity and Quantity Determination of ParallelSynthesis
Compound Libraries 95
1 Introduction 96
2 Library Development and Production 96
3 Analytical Process 97
4 PurityAssessment 100
5 Quantity Determination 102
6 Fast Chromatography 103
7 High ThroughputHPLC/Mass Spectrometry 105
8 Data Management 107
9 Experimental Methods 109
9.1 Sample Introduction 109
9.2 HPLC Instrumentation 109
9.3 MS Instrumentation 110
9.4 Analytical Standards 110
9.5 ELSD Quantity Determination 110
9.6 Software 111
10 Conclusion 111
References 111
6 High Throughput Parallel LC/MS/ELSD of Combinatorial Libraries
Using the Eight-ChannelLCT Systemwith MUX Technology 113
1 Introduction 113
2 Experimental 114
3 Results andDiscussion 116
4 FutureTrends 121
5 Conclusion 123
Acknowledgements 123
References 123
7 Purification and Analysisof ParallelLibraries 125
1 Introduction 126
2 Challenge 126
3 Discussion 128
3.1 Purification 128
3.2 Identification and Prioritization 130
3.3 Sorting by Library Type 134
3.4 Drying andWeighing ofCompounds 135
viii Contents
3.5 Sorting by Quantity 136
3.6 PlatingofCompounds 137
3.7 Purityand Process Check 137
3.8 CompoundTrackingSystem(CTS) 140
4 Experimental Methods 143
4.1 High Throughput PreparativeHPLC 143
4.2 Flow-Inject Mass Spectrometry 144
4.3 LiquidChromatography/MassSpectrometry (LC/MS) 144
5 Future Trends 145
5.1 Expansion ofthe DecisionTree 145
5.2 Collection of Isomers 145
6 Conclusion 146
Acknowledgements 146
References 146
8 Screening Single-Bead Combinatorial Libraries using Capillary
HPLC and MALDI-TOF-MS 147
1 Introduction 147
1.1 HPLC Separations 149
1.2 Hyphenation ofMALDI andHPLC 151
1.3 Mass Spectrometric Analysis 153
2 Future Trends 157
3 Conclusion 160
References 160
9 The Role of NMR in the Analysis ofChemical Libraries 163
1 Introduction 163
2 Information Issues inSAR Generation 164
3 Combinatorial Chemistryand SARGeneration 166
4 TheValue ofCompound Analysis 168
5 HighThroughput Analytical Characterization 169
6 NMR and CLND, How Accurate? 170
7 CaseStudy 1:Analysis ofMeta-Substituted Anilinoamides 171
8 CaseStudy 2:Analysis ofHydrazone Library 172
9 NMR Automation Technology for Sample Handling 174
10 NMR Data Analysis 175
11 Case Study 3:Analysis of96 Benzoylated Amines 176
12 Conclusion 180
Acknowledgements 181
References 181
Subject Index 183
Preface
Duringthelasttwodecades,thepharmaceuticalindustryhasbeenunderenormous
and ever-increasing pressure from a variety of interest groups to enhance the
productivity and effectiveness of drug discovery and development. Although, the
collective R&D budget for the industry has been increasing exponentially for the
pasttwentyyears,thenumbersofnewchemicalentitiesthatreachthemarkethave
remained relatively constant during this same time period. Much of the increased
investment has been in a variety of technologies focused on enhancing early drug
discovery, such as high throughput screening, combinatorial chemistry, parallel
synthesis, genomics, and automation to name just a few. Combinatorial and high
throughput parallel synthesis chemistry have been the most recent potential levers
developed to bridge the early discovery productivity gap. Although initial debate
focused on the merits and limitations of split-and-mix vs. spatially addressable
arrays or solid vs. solution phase approaches, over the last few years the emphasis
has shifted to purification and characterization support as an integral part of the
synthesisprocess.
Thetraditionalmedicinalchemistryapproachofmakingonetotencompoundsin
parallelwithsignificantquantitiesneverchallengedtheavailableanalyticalmethods
for analysis and characterization. A typical round bottom flask synthesis produced
enoughmaterialforNMRanalysis,whichisthepreferredcharacterizationtechnique
of the synthetic chemist. However, with the introduction of mix-and-split and
spatially addressable formats that now routinely generated 100 to 10,000 analogs
from a scaffold, the analytical approach became a significant issue and the
traditional medicinal chemistry approach of purifying and characterizing each
compound synthesized was challenged because the tried and true analysis
techniques like NMR could not initially be applied in a high throughput mode.
Theoverridingneedtocorrelate biological activity withmolecularstructureeither
before or after screening and use the information to follow up with synthetic
modificationstopartsofthemoleculethatappeartoregulatepotency,selectivityor
any other parameterthat needs tobe optimized has driven the industry toidentify,
developandimplementanalyticalsolutionsthatareappropriateandadaptabletothe
synthesisapproach.
Theneedtoadaptanddevelopanalyticaltechniquesandmethodsinresponseto
syntheticconstraintscanbecomparedtothetypicalevolutionaryprocessthatselects
for the most appropriate attributes in species selection. The attributes or methods