Modern Methods in Crop Protection Research

This handbook and ready reference highlights a couple of basic aspects of recently developed new methods in modern crop protection research, authored by renowned experts from major agrochemical companies. Organized into four major parts that trace the key phases of the compound development process, the first section addresses compound design, while the second covers newly developed methods for the identification of the mode of action of agrochemical compounds. The third part describes methods used in improving the bioavailability of compounds, and the final section looks at modern methods for risk assessment. As a result, the agrochemical developer will find here a valuable toolbox of advanced methods, complete with first–hand practical advice and copious examples from current industrial practice.

Preface XV List of Contributors XIX Part I Methods for the Design and Optimization of New Active Ingredients 1 1 High–Throughput Screening in Agrochemical Research 3 Mark Drewes, Klaus Tietjen, and Thomas C. Sparks 1.1 Introduction 3 1.2 Target–Based High–Throughput Screening 6 1.2.1 Targets 6 1.2.2 High–Throughput Screening Techniques 9 1.3 Other Screening Approaches 13 1.3.1 High–Throughput Virtual Screening 13 1.4 In Vivo High–Throughput Screening 13 1.4.1 Compound Sourcing and In–Silico Screening 15 1.5 Conclusions 17 Acknowledgments 18 References 18 2 Computational Approaches in Agricultural Research 21 Klaus–J¨urgen Schleifer 2.1 Introduction 21 2.2 Research Strategies 21 2.3 Ligand–Based Approaches 22 2.4 Structure–Based Approaches 26 2.5 Estimation of Adverse Effects 33 2.6 In–Silico Toxicology 34 2.7 Programs and Databases 34 2.7.1 In–Silico Toxicology Models 36 2.8 Conclusion 39 References 40 3 Quantum Chemical Methods in the Design of Agrochemicals 43 Michael Schindler 3.1 Introduction 43 3.2 Computational Quantum Chemistry: Basics, Challenges, and New Developments 44 3.3 Minimum Energy Structures and Potential Energy Surfaces 47 3.4 Physico–Chemical Properties 51 3.4.1 Electrostatic Potential, Fukui Functions, and Frontier Orbitals 53 3.4.2 Magnetic Properties 55 3.4.3 pKa Values 57 3.4.4 Solvation Free Energies 59 3.4.5 Absolute Configuration of Chiral Molecules 60 3.5 Quantitative Structure–Activity Relationships 60 3.5.1 Property Fields, Wavelets, and Multi–Resolution Analysis 61 3.5.2 The CoMFA Steroid Dataset 63 3.5.3 A Neonicotinoid Dataset 64 3.6 Outlook 66 References 67 4 The Unique Role of Halogen Substituents in the Design of Modern Crop Protection Compounds 73 Peter Jeschke 4.1 Introduction 73 4.2 The Halogen Substituent Effect 75 4.2.1 The Steric Effect 76 4.2.2 The Electronic Effect 78 4.2.2.1 Electronegativities of Halogens and Selected Elements/Groups on the Pauling Scale 78 4.2.2.2 Effect of Halogen Polarity of the C–Halogen Bond 79 4.2.2.3 Effect of Halogens on pKa Value 79 4.2.2.4 Improving Metabolic, Oxidative, and Thermal Stability with Halogens 80 4.2.3 Effect of Halogens on Physico–Chemical Properties 82 4.2.3.1 Effect of Halogens on Molecular Lipophilicity 82 4.2.3.2 Classification in the Disjoint Principle Space 84 4.2.4 Effect of Halogens on Shift of Biological Activity 84 4.3 Insecticides and Acaricides Containing Halogens 86 4.3.1 Voltage–Gated Sodium Channel (vgSCh) Modulators 86 4.3.1.1 Pyrethroids of Type A 86 4.3.1.2 Pyrethroids of Type B 89 4.3.1.3 Pyrethroids of Type C 90 4.3.2 Voltage–Gated Sodium Channel (vgSCh) Blockers 90 4.3.3 Inhibitors of the γ –Aminobutyric Acid (GABA) Receptor/Chloride Ionophore Complex 91 4.3.4 Insect Growth Regulators (IGRs) 93 4.3.5 Mitochondrial Respiratory Chain 96 4.3.5.1 Inhibitors of Mitochondrial Electron Transport at Complex I 96 4.3.5.2 Inhibitors of Qo Site of Cytochrome bc1 – Complex III 97 4.3.5.3 Inhibitors of Mitochondrial Oxidative Phosphorylation 97 4.3.6 Ryanodine Receptor (RyR) Effectors 98 4.4 Fungicides Containing Halogens 99 4.4.1 Sterol Biosynthesis Inhibitors (SBIs) and Demethylation Inhibitors (DMIs) 99 4.4.2 Mitochondrial Respiratory Chain 101 4.4.2.1 Inhibitors of Succinate Dehydrogenase (SDH) – Complex II 101 4.4.2.2 Inhibitors of Qo Site of Cytochrome bc1 – Complex III 104 4.4.2.3 NADH Inhibitors – Complex I 107 4.4.3 Fungicides Acting on Signal Transduction 107 4.5 Plant Growth Regulators (PGRs) Containing Halogens 108 4.5.1 Reduction of Internode Elongation: Inhibition of Gibberellin Biosynthesis 108 4.6 Herbicides Containing Halogens 109 4.6.1 Inhibitors of Carotenoid Biosynthesis: Phytoene Desaturase (PDS) Inhibitors 109 4.6.2 Inhibitors of Acetolactate Synthase (ALS) 111 4.6.2.1 Sulfonylurea Herbicides 111 4.6.2.2 Sulfonylaminocarbonyl–Triazolone Herbicides (SACTs) 115 4.6.2.3 Triazolopyrimidine Herbicides 116 4.6.3 Protoporphyrinogen IX Oxidase (PPO) 117 4.7 Summary and Outlook 119 References 119 Part II New Methods to Identify the Mode of Action of Active Ingredients 129 5 RNA Interference (RNAi) for Functional Genomics Studies and as a Tool for Crop Protection 131 Bernd Essigmann, Eric Paget, and Fr´ed´eric Schmitt 5.1 Introduction 131 5.2 RNA Silencing Pathways 131 5.2.1 The MicroRNA (miRNA) Pathway 133 5.2.2 The Small Interfering Pathway (siRNA) 134 5.3 RNAi as a Tool for Functional Genomics in Plants 134 5.4 RNAi as a Tool for Engineering Resistance against Fungi and Oomycetes 138 5.5 RNAi as a Tool for Engineering Insect Resistance 140 5.6 RNAi as a Tool for Engineering Nematodes Resistance 142 5.7 RNAi as a Tool for Engineering Virus Resistance 144 5.8 RNAi as a Tool for Engineering Bacteria Resistance 149 5.9 RNAi as a Tool for Engineering Parasitic Weed Resistance 150 5.10 RNAi Safety in Crop Plants 153 5.11 Summary and Outlook 153 References 153 6 Fast Identification of the Mode of Action of Herbicides by DNA Chips 161 Peter Eckes and Marco Busch 6.1 Introduction 161 6.2 Gene Expression Profiling: A Method to Measure Changes of the Complete Transcriptome 162 6.3 Classification of the Mode of Action of an Herbicide 164 6.4 Identification of Prodrugs by Gene Expression Profiling 165 6.5 Analyzing the Affected Metabolic Pathways 169 6.6 Gene Expression Profiling: Part of a Toolbox for Mode of Action Determination 171 References 172 7 Modern Approaches for Elucidating the Mode of Action of Neuromuscular Insecticides 175 Daniel Cordova 7.1 Introduction 175 7.2 Biochemical and Electrophysiological Approaches 176 7.2.1 Biochemical Studies 176 7.2.2 Electrophysiological Studies on Native and Expressed Targets 179 7.2.2.1 Whole–Cell Voltage Clamp Studies 179 7.2.2.2 Oocyte Expression Studies 180 7.2.3 Automated Two–Electrode Voltage–Clamp TEVC Recording Platforms 182 7.3 Fluorescence–Based Approaches for Mode of Action Elucidation 183 7.3.1 Calcium–Sensitive Probes 183 7.3.2 Voltage–Sensitive Probes 186 7.4 Genomic Approaches for Target Site Elucidation 187 7.4.1 Chemical–to–Gene Screening 187 7.4.2 Double–Stranded RNA Interference 190 7.4.3 Metabolomics 191 7.5 Conclusion 191 References 192 8 New Targets for Fungicides 197 Klaus Tietjen and Peter H. Schreier 8.1 Introduction: Current Fungicide Targets 197 8.2 A Retrospective Look at the Discovery of Targets for Fungicides 199 8.3 New Sources for New Fungicide Targets in the Future? 199 8.4 Methods to Identify a Novel Target for a Given Compound 200 8.4.1 Microscopy and Cellular Imaging 200 8.4.2 Cultivation on Selective Media 200 8.4.3 Incorporation of Isotopically Labeled Precursors and Metabolomics 201 8.4.4 Affinity Methods 201 8.4.5 Resistance Mutant Screening 201 8.4.6 Gene Expression Profiling and Proteomics 202 8.5 Methods of Identifying Novel Targets without Pre–Existing Inhibitors 202 8.5.1 Biochemical Ideas to Generate Novel Fungicide Targets 203 8.5.2 Genomics and Proteomics 203 8.6 Non–Protein Targets 213 8.7 Resistance Inducers 213 8.8 Beneficial Side Effects of Commercial Fungicides 214 8.9 Concluding Remarks 214 References 214 Part III New Methods to Improve the Bioavailability of Active Ingredients 217 9 New Formulation Developments 219 Rolf Pontzen and Arnoldus W.P. Vermeer 9.1 Introduction 219 9.2 Drivers for Formulation Type Decisions 223 9.3 Description of Formulation Types, Their Properties, and Problems during Development 225 9.3.1 Pesticides Dissolved in a Liquid Continuous Phase 225 9.3.2 Crystalline Pesticides in a Liquid Continuous Phase 228 9.3.3 Pesticides in a Solid Matrix 232 9.4 Bioavailability Optimization 235 9.4.1 Spray Formation and Retention 236 9.4.2 Spray Deposit Formation and Properties 238 9.4.3 Cuticular Penetration 240 9.4.3.1 Cuticular Penetration Test 242 9.4.3.2 Effect of Formulation on Cuticular Penetration 243 9.5 Conclusions and Outlook 246 References 247 10 Polymorphism and the Organic Solid State: Influence on the Optimization of Agrochemicals 249 Britta Olenik and Gerhard Thielking 10.1 Introduction 249 10.2 Theoretical Principles of Polymorphism 250 10.2.1 The Solid State 250 10.2.2 Definition of Polymorphism 251 10.2.3 Thermodynamics 251 10.2.3.1 Monotropism and Enantiotropism 251 10.2.3.2 Energy Temperature Diagrams and the Rules 252 10.2.4 Kinetics of Crystallization: Nucleation 254 10.3 Analytical Characterization of Polymorphs 255 10.3.1 Differential Thermal Analysis and Differential Scanning Calorimetry 256 10.3.2 Thermogravimetry 258 10.3.3 Hot–Stage Microscopy 259 10.3.4 IR and Raman Spectroscopies 261 10.3.5 X–Ray Analysis 265 10.4 Patentability of Polymorphs 268 10.5 Summary and Outlook 270 Acknowledgments 270 References 270 11 The Determination of Abraham Descriptors and Their Application to Crop Protection Research 273 Eric D. Clarke and Laura J. Mallon 11.1 Introduction 273 11.2 Definition of Abraham Descriptors 274 11.3 Determination of Abraham Descriptors: General Approach 275 11.3.1 V and E Descriptors 276 11.3.2 A, B, and S Descriptors 277 11.3.3 A, B, S, and L Descriptors 277 11.3.4 LSER Equations for Use in Determining Descriptors 278 11.3.5 Prediction of Abraham Descriptors 280 11.4 Determination of Abraham Descriptors: Physical Properties 281 11.5 Determination of Abraham Descriptors: Examples 283 11.5.1 Herbicides: Diuron (1) 284 11.5.2 Herbicides: Simazine (2) and Atrazine (3) 285 11.5.3 Herbicides: Acetochlor (4) and Alachlor (5) 288 11.5.4 Insecticides: Fipronil (6) 289 11.5.5 Insecticides: Imidacloprid (7) 290 11.5.6 Insecticides: Chlorantraniliprole (8) 292 11.5.7 Insecticides: Thiamethoxam (9) 293 11.5.8 Fungicides: Azoxystrobin (10) 294 11.5.9 Plant Growth Regulator: Paclobutrazol (11) 295 11.6 Application of Abraham Descriptors: Descriptor Profiles 296 11.7 Application of Abraham Descriptors: LFER Analysis 297 11.7.1 LFERs for RP–HPLC Systems 297 11.7.2 LFERs for Soil Sorption Coefficient (KOC) 299 11.7.3 LFERs for Partitioning into Plant Cuticles 300 11.7.4 LFERs for Root Concentration Factor (RCF) 300 11.7.5 LFER for Transpiration Stream Concentration Factor 301 11.8 Application of Abraham Descriptors: Generality of Approach 301 Acknowledgments 302 References 302 Part IV Modern Methods for Risk Assessment 307 12 Ecological Modeling in Pesticide Risk Assessment: Chances and Challenges 309 Walter Schmitt 12.1 Introduction 309 12.2 Ecological Models in the Regulatory Environment 311 12.2.1 Consideration of Realistic Exposure Patterns 312 12.2.2 Extrapolation to Population Level: The Link to Protection Goals 313 12.2.3 Extrapolation to Organization Levels above Populations 314 12.3 An Overview of Model Approaches 315 12.3.1 Toxicokinetic Models 316 12.3.2 Population Models 319 12.3.2.1 Differential Equation Models 319 12.3.2.2 Matrix Models 320 12.3.2.3 Individual–Based Models 322 12.3.3 Ecosystem or Food–Web Models 325 12.4 Regulatory Challenges 328 References 331 13 The Use of Metabolomics In Vivo for the Development of Agrochemical Products 335 Hennicke G. Kamp, Doerthe Ahlbory–Dieker, Eric Fabian, Michael Herold, Gerhard Krennrich, Edgar Leibold, Ralf Looser, Werner Mellert, Alexandre Prokoudine, Volker Strauss Tilmann Walk, Jan Wiemer, and Bennard van Ravenzwaay 13.1 Introduction to Metabolomics 335 13.2 MetaMap®Tox Data Base 336 13.2.1 Methods 336 13.2.1.1 Animal Treatment and Maintenance Conditions 336 13.2.1.2 Blood Sampling and Metabolite Profiling 336 13.3 Evaluation of Metabolome Data 337 13.3.1 Data Processing 337 13.3.1.1 Metabolite Profiling 337 13.3.1.2 Metabolome Patterns 337 13.3.1.3 Whole–Profile Comparison 338 13.4 Use of Metabolome Data for Development of Agrochemicals 339 13.4.1 General Applicability 339 13.4.2 Case Studies 339 13.4.2.1 Liver Enzyme Induction 339 13.4.2.2 Liver Cancer 342 13.4.3 Chemical Categories 344 13.5 Discussion 345 13.5.1 Challenges and Chances Concerning the Use of Metabolite Profiling in Toxicology 345 13.5.2 Applicability of the MetaMap®Tox Data Base 347 13.6 Concluding Remarks 347 References 348 14 Safety Evaluation of New Pesticide Active Ingredients: Enquiry–Led Approach to Data Generation 351 Paul Parsons 14.1 Background 351 14.2 What Is the Purpose of Mammalian Toxicity Studies? 354 14.3 Addressing the Knowledge Needs of Risk Assessors 358 14.4 Opportunities for Generating Data of Direct Relevance to Human Health Risk Assessment within the Existing Testing Paradigm 362 14.4.1 Dose Selection for Carcinogenicity Studies 362 14.4.2 Integrating Toxicokinetics into Toxicity Study Designs 365 14.5 Enquiry–Led Data Generation Strategies 367 14.5.1 Key Questions to Consider While Identifying Lead Molecules 369 14.5.2 Key Questions to Consider When Selecting Candidates for Full Development 370 14.5.3 Key Questions to Consider for a Compound in Full Development 371 14.6 Conclusions 371 References 378 15 Endocrine Disruption: Definition and Screening Aspects in the Light of the European Crop Protection Law 381 Susanne N. Kolle, Burkhard Flick, Tzutzuy Ramirez, Roland Buesen, Hennicke G. Kamp, and Bennard van Ravenzwaay 15.1 Introduction 381 15.2 Endocrine Disruption: Definitions 382 15.3 Current Regulatory Situation in the EU 382 15.4 US EPA Endocrine Disruptor Screening Program and OECD Conceptual Framework for the Testing and Assessment of Endocrine–Disrupting Chemicals 384 15.5 ECETOC Approach 385 15.6 Methods to Assess Endocrine Modes of Action and Endocrine–Related Adverse Effects in Screening and Regulatory Contexts 388 15.6.1 In–Vitro Assays 388 15.6.2 In–Vivo Assays 391 15.7 Proposal for Decision Criteria for EDCs: Regulatory Agencies 397 References 397 Index 401

Peter Jeschke gained his PhD in organic chemistry at the University of Halle/Wittenberg (Germany), after which he moved to Fahlberg–List Company (Germany) to pursue agrochemical research before going to the Institute of Neurobiology and Brain Research, German Academy of Sciences. In 1989 he joined Bayer as lab leader in animal health research and eight years later he took a position at the Bayer Crop Protection Business Group, where he is currently Head of Research Pest Control Chemistry 2. Since 2011, he is honorary professor at the University of Düsseldorf (Germany). Prof. Dr. Jeschke has more than 180 patent applications and scientific publications to his name. Wolfgang Krämer gained his PhD in organic chemistry from the TU Stuttgart (Germany) in 1968, after which he joined the Institute of Textile Chemistry at Stuttgart University, before moving to Bayer Plant Protection as lab leader in plant protection research in 1970. Between 1984 and 1990 he was Head of Global Chemistry Fungicides, and Head of Insecticide Chemistry thereafter. Retired since 2005, Dr. Krämer has over 250 patent applications and publications to his name. Ulrich Schirmer received his PhD in organic chemistry from Stuttgart University (Germany) in 1973, and worked subsequently postdoctoral as a researcher at Paris–Orsay (France). He joined BASF in 1974, eventually becoming Senior Vice President responsible for plant protection research for chemical synthesis, process development and biological R&D. Since 2003, he has been working as a freelance consultant to start–ups in the fields of biotechnology, chemistry and agriculture. Dr. Schirmer is author and co–author of more than 100 patent applications and scientific publications. Matthias Witschel received his PhD in organic chemistry in 1994 at the University of Erlangen–Nürnberg (Germany). After his post–doctoral stay at Stanford University, California (USA), he started in 1996 at BASF in herbicide research, where he is now Principal Scientist in the Global Research Herbicides, Agricultural Products, based in Ludwigshafen (Germany). Dr. Witschel is the author and co–author of over 160 patents and scientific publications.