Design and synthesis of molecular probes for the study of 5 ht2a

Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2009 “DESIGN AND SYNTHESIS OF MOLECULAR PROBES FOR THE STUDY OF 5-HT2A AND H1 RECEPTORS” Jitesh shah Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.edu/etd Part of the Chemicals and Drugs Commons © The Author Downloaded from https://scholarscompass.edu/etd/1869 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact libcompass@vcu. Shah 2009 All Rights Reserved DESIGN AND SYNTHESIS OF MOLECULAR PROBES FOR THE STUDY OF 5-HT2A AND H1 RECEPTORS A Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University.

SHAH MS in Pharmaceutical Sciences, University of Mumbai, India, 2003 BS in Pharmacy, University of Mumbai, India, 2001 Director: RICHARD B. WESTKAEMPER PROFESSOR, DEPARTMENT OF MEDICINAL CHEMISTRY Virginia Commonwealth University Richmond, Virginia Aug 2009 Acknowledgements First and foremost, I would like to thank my advisor Richard B. Westkaemper for his guidance and support during the entire tenure of my work. He is a great teacher and a brilliant scientist.

His broad scientific knowledge and approach towards basic research has been a true inspiration for me. I am grateful to him for his encouragement to apply for the dissertation assistantship in my final semester. I would also like to thank my committee members, Dr. Richard Glennon, Dr.

Glen Kellogg, Dr. Vladimir Sidorov and Dr. This committee has greatly contributed to my scientific knowledge and has always been available for suggestions. I would like to thank Professor Glennon for participating in some of our group meetings and giving a valuable insight into the drug design aspects.

I am thankful to Dr. Sidorov for his advice in the chemistry related to my project. I am glad to have Dr. Kellogg on my committee, who has always been there for ideas in the molecular modeling work.

I wholeheartedly thank the postdoctoral scholars Dr. Srinivas Peddi, Dr. Gajanan Dewkar and Dr. Philip Mosier in Westkaemper‟s group.

It has been a great experience to work with these talented individuals in my graduate career. Srini and Gajanan have been my mentors in laboratory. They have given me the true guidance and support in my work. Srini was always available for discussions regarding my project.

I am gratified to have Gajanan as a chemist in our lab; he has taught me some of the most valuable lessons in ii organic chemistry. I am also greatly indebted to Phil. He has guided me throughout my project and shown tremendous patience in teaching me various aspects of molecular modeling. I thank my fellow students Donna McGovern and Justin Pitts for helping me out with my work whenever needed.

Westkaemper‟s group, I would like to thank Dr. Umesh Desai, Dr. Keith Ellis and Dr. John Hackett for their support.

Dr Desai has always provided moral support and guidance when needed. Ellis and Dr. Hackett have been kind enough to guide me for the postdoctoral applications. I sincerely appreciate the guidance and support given by Arjun Raghuraman a good friend and a mentor in my graduate studies.

I wholeheartedly thank all my friends Jay, Santosh, Parthasarathy, Anil, Sagar, and Jay Patel for their support and care. Finally, I would like to thank my parents Asha and Rameshchandra Shah for their unwavering support and blessings throughout my career and years ahead. I also thank my brother Rupesh and sister-in-law, Nutan for their love and prayers. This dissertation would not be possible without the help of my friends and well-wishers whom I deeply thank.

iii Table of Contents Page Acknowledgements. ii List of Tables. viii List of Figures. ix List of Schemes ………………………………………………………………………….xi List of Abbreviations…………………………………………………………………….xv Chapter 1 INTRODUCTION .1 The Discovery of Serotonin .2 Serotonin Biosynthesis and Metabolism .3 Classification of Serotonin Receptors .4 Serotonin Receptors: Clinical Significance .5 5-HT2 Receptor Structure, Function and Localization .1 5-HT2 Receptors in the Development of Novel Antidepressant Drugs…………………………………………………………….2 5-HT2 Selective Antagonists as Potential Antipsychotic Drugs.3 5-HT2 Ligands in the Treatment of Schizophrenia…………….4 5-HT2 Ligands in the Treatment of Sleep Disorders…………….6 Clinical Relevance for Studying 5-HT2A and H1 Receptor Binding….7 5-HT2 Receptor Ligands………………………………………………17 iv 1.8 Understanding 5-HT2A Receptor-Ligand Interactions and Development of a Selective GPCR Ligand………………………………………….9 Specific Aims of the Research Project……………………………….21 2 MOLECULAR MODELING OF G PROTEIN-COUPLED RECEPTORS .2 Central Role of G Proteins .3 Common Structural Features of GPCRs .4 Challenges in GPCR Research .1 Why are there only a Few Experimental Structures of GPCRs?.5 Homology (Comparative) Modeling as a Workable Solution……….1 Bovine Rhodopsin and β2-Adrenergic Receptor Structures as Suitable Templates for Homology Modeling……………………29 2.6 Construction of Homology Models…………………………………….1 Docking and Scoring……………………………………………36 3 STRUCTURE-AFFINITY RELATIONSHIP STUDIES OF AMDA AND ITS ANALOGS AT 5-HT2A AND H1 RECEPTORS…………………………….2 Results and Discussion………………………………………………….1 Structure-Affinity Relationship Studies……….2 Modeling Receptor-Ligand Interactions: H1 and 5-HT2A Receptor Models ………………………………………………………….3 Binding Mode Analysis…………………….60 4 RING-ANNULATED AND N-SUBSTITUTED ANALOGS OF AMDA AS STRUCTURAL PROBES FOR 5-HT2A AND H1 RECEPTORS…………….2 Substituted Phenylalkylamine Analogs of AMDA as Structural Probes….3 Ring-Annulated Analogs of AMDA as Dimensional Probes …….4 Molecular Modeling Studies………………………………………………65 4.72 5 ENGINEERING A SELECTIVE NON-NITROGENOUS AMINERGIC GPCR LIGAND…………………………………………………………….2 Hypothesis Based on the Homology Modeling and Reported Site-directed Mutagenesis Studies…………………………………………………….3 Strategy for the Design of a Selective H1 Ligand……………………….4 Results and Discussion………………………………………………….1 Structure-Affinity Relationship Studies …………………….2 Molecular Modeling…………………………………………………………138 RFERENCES………………………………………………………………………….142 vii List of Tables Page Table 1: Serotonin Receptors Classification.

4 Table 2: Mental Illness where Altered Serotonin Neurotransmission has been Implicated……………………………………………………………………….6 Table 3: Common Medical Conditions in which 5-HT-Subtype-Selective Drugs have Utility……………………………………………………………………………7 Table 4: Histamine Receptors Classification………………………………………….14 Table 5: Observed Binding Affinities for 9-(aminoalkyl)-9,10-dihydroanthracene (DHA) and Diphenylalkylamine (DPA) Analogs at 5-HT2A and H1 Receptors……….43 viii List of Figures Page Figure 1: Biosynthesis and Metabolism of Serotonin. 3 Figure 2: Scaled Phylogenetic Tree Comparing Human Receptors with Bovine Rhodopsin. 5 Figure 3: Distribution of 5-HT2A and 5-HT2C Receptors on GABA-containing and Dopamine Containing Neurons in the Midbrain………………………………10 Figure 4: Serotonergic Regulation of Sleep-Wakefulness Cycle. 13 Figure 5: Structures of Representative H1 Antagonists.

15 Figure 6: Structures of Representative 5-HT2 Ligands. 19 Figure 7: Role of G Proteins. 24 Figure 8: GPCRs Structure and Receptor-Ligand Interactions. 27 Figure 9: Superimposition of the 5-HT2A GPCR Models Based on β2-AR and Rhodopsin Crystal Structures.

33 Figure 10: 3D Bar Graph Showing the Affinities at 5-HT2A and H1 for the 9-(aminoalkyl)- 9,10-dihydroanthracene (DHA) and Diphenylalkylamine (DPA) Analogs. 44 Figure 11: Alignment of the Human β2-Adrenergic, H1 and 5-HT2A Receptor Sequences. 47 Figure 12: Illustration of the Differences in Binding Site Residue Composition of the H1 and 5-HT2A Receptors within the Context of the 2AR-T4L Crystal Structure Docked Receptor-Ligand Complexes……………………………………….51 Figure 13: Docked Receptor Ligand Complex. 52 Figure 14: HINT Interaction Maps for Compound 9.

53 Figure 15: Binding Affinity Data of AMDA Analogs as Molecular Probes. 65 Figure 16: Proposed Binding Mode of Phenylalklyamine analogs of AMDA in 5HT2A and H1 Receptor……………………………………………………………………67 ix Figure 17: Proposed Binding Mode of Quaternary ammonium salt of AMDA in H1 Receptor………………………………………………………………………68 Figure 18: Proposed Binding Mode of Isomers of Ring-Annulated AMDA Analogs….69 Figure 19: Probes for the Design of Selective GPCR Ligands. …79 Figure 20: Docked Poses of Olapatadine and Compound 34. 84 Figure 21: HINT Interaction Maps for Compound 34 in H1 Binding Sites.

85 x List of Schemes Scheme 1: .89 xi List of Abbreviations Å Angstroms AC Adenylate cyclase AcOH Acetic acid AlCl3 Aluminium chloride AMDA 9-(Aminomethyl)-9,10-dihydroanthracene ASP Astex statistical potential Asp Aspartate br Broad BH3 Borane BRHO Bovine rhodopsin °C Degree Celsius Calcd Calculated CCl4 Carbon tetrachloride cAMP Cyclic 3‟, 5‟-adenosine monophosphate CDCl3 Duetereted chloroform CHCl3 Chloroform CH3OH Methanol CH3SO3H Methanesulfonic acid CH2Cl2 Methylene chloride CNS Central nervous system d Doublet DAG Diacylglycerol DIBAL-H Diisobutylaluminium hydride DMSO-d6 Deuterated Dimethyl sulfoxide DA Dopamine DHA Dihydroanthracene DPA Diphenyl amines DMF Dimethylformamide Et2O Diethyl ether ESIMS Electrospray ionization mass spectrometry EC50 Activation Concentration (half-maximal effect) EtOH Ethanol EtOAC Ethyl acetate EL Extracellular loop Fe(NO3) Ferric nitrate GABA γ-Aminobutyric acid GDP Guanine nucleotide guanosine diphosphate xii GTP Guanine nucleotide guanosine triphosphate GPCR G Protein-coupled receptors GOLD Genetic optimization for ligand docking GI Gastrointestinal HBr Hydrobromic acid HCl Hydrochloric acid 5-HT 5-Hydroxytryptamine 5-HTP 5-Hydroxy tryptophan 5-HIAA 5-Hydroxyindole acetic acid HINT Hydropathic INTeractions H1 Histamine H2SO4 Sulfuric acid HTS High throughput screening Ki Dissociation constant K Potassium KI Potassium iodide K3PO4 Potassium phosphate KCN Potassium cyanide KOH Potassium hydroxide KOR Kappa-opioid receptor liq Liquid LAH Lithium aluminium hydride LiAlH4 Lithium aluminium hydride LHMDS Lithium hexamethyldisilazide LSD Lysergic acid diethylamide MgSO4 Magnesium sulfate MeCN Acetronitrile MeOH Methanol ml Milliliter mmol Millimolar mp Melting Point MeNO2 Nitromethane m Multiplet MAO Monoamino oxidase n-BuLi n-Butyllithium NBS N-bromosuccinimide nM Nanomolar NMR Nuclear magnetic resonance Na Sodium NaOH Sodium hydroxide NaOCl Sodium hypochlorite NaClO2 Sodium chlorite NaBH4 Sodiumborohydride xiii NaBH3CN Sodium cyanoborohydride NEt3 Triethylamine NREMS Non-rapid eye movement sleep NH4OH Ammonium hydroxide NRD Nucleus raphe dorsalis Pd/C Palladium on carbon PDB Protein data bank Pd(PPh3)4 Tetrakis (triphenylphosphine) Palladium PBr3 Phosphorus tribromide PET Positron emission tomography PI Phosphatidylinosital PPA Polyphosphoric acid p-TsOH p-Toluenesulfonic acid QSAR Quantitative structure activity relationship RMSD Root mean square deviation rt Room temperature s singlet SAFIR Structure affinity relationship SERT Serotonin transporter SN Substantia niagra SWS Slow wave sleep SAFIR Structure-affinity-relationships SAR Structure activity relationship SOCl2 Thionyl chloride t Triplet TBAF Tetra-n-butylammonium fluoride TBDMS tert-Butyldimethylsilyl ether TEMPO 2,2,6,6-Tetramethylpiperidinyloxy t-BuOK Potassium tert-butoxide Ti(OiPr)4 Titanium isopropoxide THF Tetrahydrofuran TLC Thin-layer chromatography TM Transmembrane TMSCN Trimethylsilyl cyanide VTA Ventral tegmental area ZnI Zinc iodide xiv Abstract DESIGN AND SYNTHESIS OF MOLECULAR PROBES FOR THE STUDY OF 5-HT2A AND H1 RECEPTORS By Jitesh R. Shah, PhD A Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2009 Major Director: Richard B. Westkaemper Professor, Department of Medicinal Chemistry The serotonin (5-HT) receptors, with seven subtypes and at least fifteen distinct members, mediate a wide range of physiological functions both in the central nervous system and in the periphery.1,2 All members of the 5-HT family except the 5-HT3 subtype belong to the family of aminergic G protein-coupled receptors (GPCRs).3 Over the years, various molecules have been reported which act selectively at 5-HT2 receptors.4 However, there are no ligands that exhibit complete selectivity for one subpopulation of 5-HT2 receptors.

Insight into how drugs bind to 5-HT2 receptors could contribute significantly to the development of subtype-selective agents with enhanced therapeutic effects. We have xv begun to address this challenge by the combined approach of chemical synthesis and molecular modeling. 9-(Aminomethyl)-9,10-dihydroanthracene (AMDA) a novel, selective 5-HT2 antagonist that also has modest affinity for the histamine (H1) receptor has been reported by Westkaemper et al.5 A structure-affinity relationships (SAFIR) study of AMDA and its analogs was carried out by studying the effects of N-alkylation, variation of the amine-ring system linker chain length and constraint of the aromatic rings on the binding affinities of the compounds for the 5-HT2A and H1 receptors. The results of the docking studies carried out on the homology models of 5-HT2A and H1 receptors were consistent with the observed binding affinity data for both receptors.

In order to explore the additional binding site interactions of 5-HT2A receptor, synthesis and testing of the ring- annulated analogs of AMDA were carried out. A 3-methoxytetraphen analog of AMDA (26) showed high affinity (Ki = 21 nM) and selectivity (126-fold) for 5-HT2A receptor as compared to H1 receptor (Ki = 2640 nM). Further, to test the utility of our homology models, and investigate the binding site specific interaction, a compound was synthesized and tested that lacks a basic amine and contains an acidic functionality designed specifically to interact with lysine K1915.39 found in H1 but not in 5-HT2A receptor. This compound would thus be both H1-selective and demonstrate that a basic amine-D3.32 interaction is not necessary for high affinity.